Reclassification of Actinobacillus muris as Muribacter muris gen. nov., comb. nov.

PubMed

Nicklas, Werner; Bisgaard, Magne; Aalbæk, Bent; Kuhnert, Peter; Christensen, Henrik

2015-10-01

To reinvestigate the taxonomy of [Actinobacillus] muris, 474 strains, mainly from mice and rats, were characterized by phenotype and 130 strains selected for genotypic characterization by 16S rRNA and partial rpoB gene sequencing. The type strain was further investigated by whole-genome sequencing. Phylogenetic analysis of the DNA sequences showed one monophyletic group with intragroup similarities of 96.7 and 97.2 % for the 16S rRNA and rpoB genes, respectively. The highest 16S rRNA gene sequence similarity to a taxon with a validly published name outside the group was 95.9 %, to the type strain of [Pasteurella] pneumotropica. The closest related taxon based on rpoB sequence comparison was 'Haemophilus influenzae-murium', with 88.4 % similarity. A new genus and a new combination, Muribacter muris gen. nov., comb. nov., are proposed based on a distinct phylogenetic position based on 16S rRNA and rpoB gene sequence comparisons, with major divergence from the existing genera of the family Pasteurellaceae. The new genus has the characteristics of [A.] muris with the emendation that acid formation from ( - )-d-mannitol and hydrolysis of aesculin are variable, while the α-glucosidase test is positive. There is no requirement for exogenously supplied NAD (V factor) for the majority of strains investigated; however, one strain was found to require NAD. The major fatty acids of the type strain of Muribacter muris were C14 : 0, C14 : 0 3-OH/iso-C16 : 1 I, C16 : 1ω7c and C16 : 0, which is in line with most genera of the Pasteurellaceae. The type strain of Muribacter muris is CCUG 16938T ( = NCTC 12432T = ATCC 49577T).

Structural and sequence features of two residue turns in beta-hairpins.

PubMed

Madan, Bharat; Seo, Sung Yong; Lee, Sun-Gu

2014-09-01

Beta-turns in beta-hairpins have been implicated as important sites in protein folding. In particular, two residue β-turns, the most abundant connecting elements in beta-hairpins, have been a major target for engineering protein stability and folding. In this study, we attempted to investigate and update the structural and sequence properties of two residue turns in beta-hairpins with a large data set. For this, 3977 beta-turns were extracted from 2394 nonhomologous protein chains and analyzed. First, the distribution, dihedral angles and twists of two residue turn types were determined, and compared with previous data. The trend of turn type occurrence and most structural features of the turn types were similar to previous results, but for the first time Type II turns in beta-hairpins were identified. Second, sequence motifs for the turn types were devised based on amino acid positional potentials of two-residue turns, and their distributions were examined. From this study, we could identify code-like sequence motifs for the two residue beta-turn types. Finally, structural and sequence properties of beta-strands in the beta-hairpins were analyzed, which revealed that the beta-strands showed no specific sequence and structural patterns for turn types. The analytical results in this study are expected to be a reference in the engineering or design of beta-hairpin turn structures and sequences. © 2014 Wiley Periodicals, Inc.

An Example of Genetically Distinct HIV Type 1 Variants in Cerebrospinal Fluid and Plasma During Suppressive Therapy

PubMed Central

Dahl, Viktor; Gisslen, Magnus; Hagberg, Lars; Peterson, Julia; Shao, Wei; Spudich, Serena; Price, Richard W.; Palmer, Sarah

2014-01-01

We sequenced the genome of human immunodeficiency virus type 1 (HIV-1) recovered from 70 cerebrospinal fluid (CSF) specimens and 29 plasma samples and corresponding samples obtained before treatment initiation from 17 subjects receiving suppressive therapy. More CSF sequences than plasma sequences were hypermutants. We determined CSF sequences and plasma sequences in specimens obtained from 2 subjects after treatment initiation. In one subject, we found genetically distinct CSF and plasma sequences, indicating that they came from HIV-1 from 2 different compartments, one potentially the central nervous system, during suppressive therapy. In addition, there was little evidence of viral evolution in the CSF during therapy, suggesting that continuous virus replication is not the major cause of viral persistence in the central nervous system. PMID:24338353

An example of genetically distinct HIV type 1 variants in cerebrospinal fluid and plasma during suppressive therapy.

PubMed

Dahl, Viktor; Gisslen, Magnus; Hagberg, Lars; Peterson, Julia; Shao, Wei; Spudich, Serena; Price, Richard W; Palmer, Sarah

2014-05-15

We sequenced the genome of human immunodeficiency virus type 1 (HIV-1) recovered from 70 cerebrospinal fluid (CSF) specimens and 29 plasma samples and corresponding samples obtained before treatment initiation from 17 subjects receiving suppressive therapy. More CSF sequences than plasma sequences were hypermutants. We determined CSF sequences and plasma sequences in specimens obtained from 2 subjects after treatment initiation. In one subject, we found genetically distinct CSF and plasma sequences, indicating that they came from HIV-1 from 2 different compartments, one potentially the central nervous system, during suppressive therapy. In addition, there was little evidence of viral evolution in the CSF during therapy, suggesting that continuous virus replication is not the major cause of viral persistence in the central nervous system.

Random oligonucleotide mutagenesis: application to a large protein coding sequence of a major histocompatibility complex class I gene, H-2DP.

PubMed Central

Murray, R; Pederson, K; Prosser, H; Muller, D; Hutchison, C A; Frelinger, J A

1988-01-01

We have used random oligonucleotide mutagenesis (or saturation mutagenesis) to create a library of point mutations in the alpha 1 protein domain of a Major Histocompatibility Complex (MHC) molecule. This protein domain is critical for T cell and B cell recognition. We altered the MHC class I H-2DP gene sequence such that synthetic mutant alpha 1 exons (270 bp of coding sequence), which contain mutations identified by sequence analysis, can replace the wild type alpha 1 exon. The synthetic exons were constructed from twelve overlapping oligonucleotides which contained an average of 1.3 random point mutations per intact exon. DNA sequence analysis of mutant alpha 1 exons has shown a point mutant distribution that fits a Poisson distribution, and thus emphasizes the utility of this mutagenesis technique to "scan" a large protein sequence for important mutations. We report our use of saturation mutagenesis to scan an entire exon of the H-2DP gene, a cassette strategy to replace the wild type alpha 1 exon with individual mutant alpha 1 exons, and analysis of mutant molecules expressed on the surface of transfected mouse L cells. Images PMID:2903482

Variability of the protein sequences of lcrV between epidemic and atypical rhamnose-positive strains of Yersinia pestis.

PubMed

Anisimov, Andrey P; Panfertsev, Evgeniy A; Svetoch, Tat'yana E; Dentovskaya, Svetlana V

2007-01-01

Sequencing of lcrV genes and comparison of the deduced amino acid sequences from ten Y. pestis strains belonging mostly to the group of atypical rhamnose-positive isolates (non-pestis subspecies or pestoides group) showed that the LcrV proteins analyzed could be classified into five sequence types. This classification was based on major amino acid polymorphisms among LcrV proteins in the four "hot points" of the protein sequences. Some additional minor polymorphisms were found throughout these sequence types. The "hot points" corresponded to amino acids 18 (Lys --> Asn), 72 (Lys --> Arg), 273 (Cys --> Ser), and 324-326 (Ser-Gly-Lys --> Arg) in the LcrV sequence of the reference Y. pestis strain CO92. One possible explanation for polymorphism in amino acid sequences of LcrV among different strains is that strain-specific variation resulted from adaptation of the plague pathogen to different rodent and lagomorph hosts.

DNA-Sequence Based Typing of the Cronobacter Genus Using MLST, CRISPR-cas Array and Capsular Profiling

PubMed Central

Ogrodzki, Pauline; Forsythe, Stephen J.

2017-01-01

The Cronobacter genus is composed of seven species, within which a number of pathovars have been described. The most notable infections by Cronobacter spp. are of infants through the consumption of contaminated infant formula. The description of the genus has greatly improved in recent years through DNA sequencing techniques, and this has led to a robust means of identification. However some species are highly clonal and this limits the ability to discriminate between unrelated strains by some methods of genotyping. This article updates the application of three genotyping methods across the Cronobacter genus. The three genotyping methods were multilocus sequence typing (MLST), capsular profiling of the K-antigen and colanic acid (CA) biosynthesis regions, and CRISPR-cas array profiling. A total of 1654 MLST profiled and 286 whole genome sequenced strains, available by open access at the PubMLST Cronobacter database, were used this analysis. The predominance of C. sakazakii and C. malonaticus in clinical infections was confirmed. The majority of clinical strains being in the C. sakazakii clonal complexes (CC) 1 and 4, sequence types (ST) 8 and 12 and C. malonaticus ST7. The capsular profile K2:CA2, previously proposed as being strongly associated with C. sakazakii and C. malonaticus isolates from severe neonatal infections, was also found in C. turicensis, C. dublinensis and C. universalis. The majority of CRISPR-cas types across the genus was the I-E (Ecoli) type. Some strains of C. dublinensis and C. muytjensii encoded the I-F (Ypseudo) type, and others lacked the cas gene loci. The significance of the expanding profiling will be of benefit to researchers as well as governmental and industrial risk assessors. PMID:29033918

Polymorphisms of cytochrome b gene in Leishmania parasites and their relation to types of cutaneous leishmaniasis lesions in Pakistan.

PubMed

Myint, Chomar Kaung; Asato, Yutaka; Yamamoto, Yu-ichi; Kato, Hirotomo; Bhutto, Abdul M; Soomro, Farooq R; Memon, Muhamad Z; Matsumoto, Jun; Marco, Jorge D; Oshiro, Minoru; Katakura, Ken; Hashiguchi, Yoshihisa; Uezato, Hiroshi

2008-02-01

The exact species and/or strains of Leishmania parasites involved strongly influence the clinical and epidemiological features of leishmaniasis, and current knowledge of those influences and relationships is inadequate. We report that cytochrome b (cyt b) gene sequencing identified causal Leishmania parasites of 69 cutaneous leishmaniasis cases in Pakistan over a 3-year period. Of 21 cases in highland areas (Quetta city, Balochistan province), 16 (76.2%) were identified as Leishmania (L.) tropica and five (23.8%) as Leishmania (L.) major. Of 48 cases from lowland areas, cities/villages in Indus valley in Sindh and Balochistan provinces, 47 (97.9%) were identified as L. (L.) major and one (2.1%) as L. (L.) tropica. Statistical analysis (Fisher's exact test) revealed a significant difference (P < 0.0001) in the distribution of the two species by altitude; L. (L.) major is predominant in lowland and L. (L.) tropica at highland areas. The present result enriched our earlier finding, based on the first year's cultured parasite data, that only L. (L.) tropica was found in highland areas and only L. (L.) major in lowland areas. Among Leishmania samples analyzed, three types of cyt b polymorphism of L. (L.) major were found, including 45 (86.5%) cases of type I, six (11.5%) of type II and one (2%) of type III. We report for the first time on the presence of polymorphisms in L. (L.) major (types I, II and III) based on species identification using cyt b gene sequencing from clinical samples. Moreover, we found no correlation between clinical presentation (wet-, dry- and/or mixed-types of cutaneous lesions) and causal Leishmania parasites.

Evaluation of the class II region of the major histocompatibility complex of the greyhound with the genomic matching technique and sequence-based typing.

PubMed

Fliegner, R A; Holloway, S A; Lester, S; McLure, C A; Dawkins, R L

2008-08-01

The class II region of the major histocompatibility complex was evaluated in 25 greyhounds by sequence-based typing and the genomic matching technique (GMT). Two new DLA-DRB1 alleles were identified. Twenty-four dogs carried the DLA-DRB1*01201/DQA1*00401/DQB1*01303/DQB1*01701 haplotype, which carries two DQB1 alleles. One haplotype was identified from which DQB1 and DQA1 appeared to be deleted. The GMT enabled detection of DQB1 copy number, discrimination of the different class II haplotypes and the identification of new, possibly biologically relevant polymorphisms.

Diversity of 16S rRNA genes of new Ehrlichia strains isolated from horses with clinical signs of Potomac horse fever.

PubMed

Wen, B; Rikihisa, Y; Fuerst, P A; Chaichanasiriwithaya, W

1995-04-01

Ehrlichia risticii is the causative agent of Potomac horse fever. Variations among the major antigens of different local E. risticii strains have been detected previously. To further assess genetic variability in this species or species complex, the sequences of the 16S rRNA genes of several isolates obtained from sick horses diagnosed as having Potomac horse fever were determined. The sequences of six isolates obtained from Ohio and three isolates obtained from Kentucky were amplified by PCR. Three groups of sequences were identified. The sequences of five of the Ohio isolates were identical to the sequence of the type strain of E. risticii, the Illinois strain. The sequence of one Ohio isolate, isolate 081, was unique; this sequence differed in 10 nucleotides from the sequence of the type strain (level of similarity, 99.3%). The sequences of the three Kentucky isolates were identical to each other, but differed by five bases from the sequence of the type strain (level of similarity, 99.6%). The levels of sequence similarity of isolate 081, the Kentucky isolates, and the type strain to the next most closely related Ehrlichia sp., Ehrlichia sennetsu, were 99.3, 99.2, and 99.2%, respectively. On the basis of the distinct antigenic profiles and the levels of 16S rRNA sequence divergence, isolate 081 is as divergent from the type strain of E. risticii as E. sennetsu is. Therefore, we suggest that strain 081 and the Kentucky isolates may represent two new distinct Ehrlichia species.

Using next generation sequencing for multiplexed trait-linked markers in wheat

USDA-ARS?s Scientific Manuscript database

With the advent of next generation sequencing (NGS) technologies, single nucleotide polymorphisms (SNPs) have become the major type of marker for genotyping in many crops. However, the availability of SNP markers for important traits of bread wheat (Triticum aestivum L.) that can be effectively used...

Filovirus RefSeq Entries: Evaluation and Selection of Filovirus Type Variants, Type Sequences, and Names

PubMed Central

Kuhn, Jens H.; Andersen, Kristian G.; Bào, Yīmíng; Bavari, Sina; Becker, Stephan; Bennett, Richard S.; Bergman, Nicholas H.; Blinkova, Olga; Bradfute, Steven; Brister, J. Rodney; Bukreyev, Alexander; Chandran, Kartik; Chepurnov, Alexander A.; Davey, Robert A.; Dietzgen, Ralf G.; Doggett, Norman A.; Dolnik, Olga; Dye, John M.; Enterlein, Sven; Fenimore, Paul W.; Formenty, Pierre; Freiberg, Alexander N.; Garry, Robert F.; Garza, Nicole L.; Gire, Stephen K.; Gonzalez, Jean-Paul; Griffiths, Anthony; Happi, Christian T.; Hensley, Lisa E.; Herbert, Andrew S.; Hevey, Michael C.; Hoenen, Thomas; Honko, Anna N.; Ignatyev, Georgy M.; Jahrling, Peter B.; Johnson, Joshua C.; Johnson, Karl M.; Kindrachuk, Jason; Klenk, Hans-Dieter; Kobinger, Gary; Kochel, Tadeusz J.; Lackemeyer, Matthew G.; Lackner, Daniel F.; Leroy, Eric M.; Lever, Mark S.; Mühlberger, Elke; Netesov, Sergey V.; Olinger, Gene G.; Omilabu, Sunday A.; Palacios, Gustavo; Panchal, Rekha G.; Park, Daniel J.; Patterson, Jean L.; Paweska, Janusz T.; Peters, Clarence J.; Pettitt, James; Pitt, Louise; Radoshitzky, Sheli R.; Ryabchikova, Elena I.; Saphire, Erica Ollmann; Sabeti, Pardis C.; Sealfon, Rachel; Shestopalov, Aleksandr M.; Smither, Sophie J.; Sullivan, Nancy J.; Swanepoel, Robert; Takada, Ayato; Towner, Jonathan S.; van der Groen, Guido; Volchkov, Viktor E.; Volchkova, Valentina A.; Wahl-Jensen, Victoria; Warren, Travis K.; Warfield, Kelly L.; Weidmann, Manfred; Nichol, Stuart T.

2014-01-01

Sequence determination of complete or coding-complete genomes of viruses is becoming common practice for supporting the work of epidemiologists, ecologists, virologists, and taxonomists. Sequencing duration and costs are rapidly decreasing, sequencing hardware is under modification for use by non-experts, and software is constantly being improved to simplify sequence data management and analysis. Thus, analysis of virus disease outbreaks on the molecular level is now feasible, including characterization of the evolution of individual virus populations in single patients over time. The increasing accumulation of sequencing data creates a management problem for the curators of commonly used sequence databases and an entry retrieval problem for end users. Therefore, utilizing the data to their fullest potential will require setting nomenclature and annotation standards for virus isolates and associated genomic sequences. The National Center for Biotechnology Information’s (NCBI’s) RefSeq is a non-redundant, curated database for reference (or type) nucleotide sequence records that supplies source data to numerous other databases. Building on recently proposed templates for filovirus variant naming [ ()////-], we report consensus decisions from a majority of past and currently active filovirus experts on the eight filovirus type variants and isolates to be represented in RefSeq, their final designations, and their associated sequences. PMID:25256396

The evolution of the lithium abundances of solar-type stars. II - The Ursa Major Group

NASA Technical Reports Server (NTRS)

Soderblom, David R.; Pilachowski, Catherine A.; Fedele, Stephen B.; Jones, Burton F.

1993-01-01

We draw upon a recent study of the membership of the Ursa Major Group (UMaG) to examine lithium among 0.3 Gyr old solar-type stars. For most G and K dwarfs, Li confirms the conclusions about membership in UMaG reached on the basis of kinematics and chromospheric activity. G and K dwarfs in UMaG have less Li than comparable stars in the Pleiades. This indicates that G and K dwarfs undergo Li depletion while they are on the main sequence, in addition to any pre-main-sequence depletion they may have experienced. Moreover, the Li abundances of the Pleiades K dwarfs cannot be attributed to main-sequence depletion alone, demonstrating that pre-main-sequence depletion of Li also takes place. The sun's Li abundance implies that the main-sequence mechanism becomes less effective with age. The hottest stars in UMaG have Li abundances like those of hot stars in the Pleiades and Hyades and in T Tauris, and the two genuine UMaG members with temperatures near Boesgaard's Li chasm have Li abundances consistent with that chasm developing fully by 0.3 Gyr for stars with UMaG's metallicity. We see differences in the abundance of Li between UMaG members of the same spectral types, indicating that a real spread in the lithium abundance exists within this group.

Clonal Relatedness of Enterotoxigenic Escherichia coli (ETEC) Strains Expressing LT and CS17 Isolated from Children with Diarrhoea in La Paz, Bolivia

PubMed Central

Rodas, Claudia; Klena, John D.; Nicklasson, Matilda; Iniguez, Volga; Sjöling, Åsa

2011-01-01

Background Enterotoxigenic Escherichia coli (ETEC) is a major cause of traveller's and infantile diarrhoea in the developing world. ETEC produces two toxins, a heat-stable toxin (known as ST) and a heat-labile toxin (LT) and colonization factors that help the bacteria to attach to epithelial cells. Methodology/Principal Findings In this study, we characterized a subset of ETEC clinical isolates recovered from Bolivian children under 5 years of age using a combination of multilocus sequence typing (MLST) analysis, virulence typing, serotyping and antimicrobial resistance test patterns in order to determine the genetic background of ETEC strains circulating in Bolivia. We found that strains expressing the heat-labile (LT) enterotoxin and colonization factor CS17 were common and belonged to several MLST sequence types but mainly to sequence type-423 and sequence type-443 (Achtman scheme). To further study the LT/CS17 strains we analysed the nucleotide sequence of the CS17 operon and compared the structure to LT/CS17 ETEC isolates from Bangladesh. Sequence analysis confirmed that all sequence type-423 strains from Bolivia had a single nucleotide polymorphism; SNPbol in the CS17 operon that was also found in some other MLST sequence types from Bolivia but not in strains recovered from Bangladeshi children. The dominant ETEC clone in Bolivia (sequence type-423/SNPbol) was found to persist over multiple years and was associated with severe diarrhoea but these strains were variable with respect to antimicrobial resistance patterns. Conclusion/Significance The results showed that although the LT/CS17 phenotype is common among ETEC strains in Bolivia, multiple clones, as determined by unique MLST sequence types, populate this phenotype. Our data also appear to suggest that acquisition and loss of antimicrobial resistance in LT-expressing CS17 ETEC clones is more dynamic than acquisition or loss of virulence factors. PMID:22140423

Clonal relatedness of enterotoxigenic Escherichia coli (ETEC) strains expressing LT and CS17 isolated from children with diarrhoea in La Paz, Bolivia.

PubMed

Rodas, Claudia; Klena, John D; Nicklasson, Matilda; Iniguez, Volga; Sjöling, Asa

2011-01-01

Enterotoxigenic Escherichia coli (ETEC) is a major cause of traveller's and infantile diarrhoea in the developing world. ETEC produces two toxins, a heat-stable toxin (known as ST) and a heat-labile toxin (LT) and colonization factors that help the bacteria to attach to epithelial cells. In this study, we characterized a subset of ETEC clinical isolates recovered from Bolivian children under 5 years of age using a combination of multilocus sequence typing (MLST) analysis, virulence typing, serotyping and antimicrobial resistance test patterns in order to determine the genetic background of ETEC strains circulating in Bolivia. We found that strains expressing the heat-labile (LT) enterotoxin and colonization factor CS17 were common and belonged to several MLST sequence types but mainly to sequence type-423 and sequence type-443 (Achtman scheme). To further study the LT/CS17 strains we analysed the nucleotide sequence of the CS17 operon and compared the structure to LT/CS17 ETEC isolates from Bangladesh. Sequence analysis confirmed that all sequence type-423 strains from Bolivia had a single nucleotide polymorphism; SNP(bol) in the CS17 operon that was also found in some other MLST sequence types from Bolivia but not in strains recovered from Bangladeshi children. The dominant ETEC clone in Bolivia (sequence type-423/SNP(bol)) was found to persist over multiple years and was associated with severe diarrhoea but these strains were variable with respect to antimicrobial resistance patterns. The results showed that although the LT/CS17 phenotype is common among ETEC strains in Bolivia, multiple clones, as determined by unique MLST sequence types, populate this phenotype. Our data also appear to suggest that acquisition and loss of antimicrobial resistance in LT-expressing CS17 ETEC clones is more dynamic than acquisition or loss of virulence factors.

Molecular Typing of Legionella pneumophila Isolates in the Province of Quebec from 2005 to 2015.

PubMed

Lévesque, Simon; Lalancette, Cindy; Bernard, Kathryn; Pacheco, Ana Luisa; Dion, Réjean; Longtin, Jean; Tremblay, Cécile

2016-01-01

Legionella is found in natural and man-made aquatic environments, such as cooling towers and hot water plumbing infrastructures. Legionella pneumophila serogroup 1 (Lp1) is the most common etiological agent causing waterborne disease in the United States and Canada. This study reports the molecular characterization of Lp strains during a 10 year period. We conducted sequence-based typing (SBT) analysis on a large set of Lp isolates (n = 284) to investigate the province of Quebec sequence types (STs) distribution in order to identify dominant clusters. From 2005 to 2015, 181 clinical Lp isolates were typed by SBT (141 sporadic cases and 40 outbreak related cases). From the same period of time, 103 environmental isolates were also typed. Amongst the 108 sporadic cases of Lp1 typed, ST-62 was the most frequent (16.6%), followed by ST-213 (10.2%), ST-1 (8.3%) and ST-37 (8.3%). Amongst other serogroups (SG), ST-1327 (SG5) (27.3%) and ST-378 (SG10) (12.2%) were the most frequent. From the environmental isolates, ST-1 represent the more frequent SBT type (26.5%). Unweighted pair group method with arithmetic mean (UPGMA) dendrogram from the 108 sporadic cases of SG1 contains 4 major clusters (A to D) of related STs. Cluster B contains the majority of the strains (n = 61) and the three most frequent STs in our database (ST-62, ST-213 and ST-1). During the study period, we observed an important increase in the incidence rate in Quebec. All the community associated outbreaks, potentially or confirmed to be associated with a cooling tower were caused by Lp1 strains, by opposition to hospital associated outbreaks that were caused by serogroups of Lp other than SG1. The recent major Quebec City outbreak caused by ST-62, and the fact that this genotype is the most common in the province supports whole genome sequencing characterization of this particular sequence type in order to understand its evolution and associated virulence factors.

The genetic architecture of type 2 diabetes.

PubMed

Fuchsberger, Christian; Flannick, Jason; Teslovich, Tanya M; Mahajan, Anubha; Agarwala, Vineeta; Gaulton, Kyle J; Ma, Clement; Fontanillas, Pierre; Moutsianas, Loukas; McCarthy, Davis J; Rivas, Manuel A; Perry, John R B; Sim, Xueling; Blackwell, Thomas W; Robertson, Neil R; Rayner, N William; Cingolani, Pablo; Locke, Adam E; Tajes, Juan Fernandez; Highland, Heather M; Dupuis, Josee; Chines, Peter S; Lindgren, Cecilia M; Hartl, Christopher; Jackson, Anne U; Chen, Han; Huyghe, Jeroen R; van de Bunt, Martijn; Pearson, Richard D; Kumar, Ashish; Müller-Nurasyid, Martina; Grarup, Niels; Stringham, Heather M; Gamazon, Eric R; Lee, Jaehoon; Chen, Yuhui; Scott, Robert A; Below, Jennifer E; Chen, Peng; Huang, Jinyan; Go, Min Jin; Stitzel, Michael L; Pasko, Dorota; Parker, Stephen C J; Varga, Tibor V; Green, Todd; Beer, Nicola L; Day-Williams, Aaron G; Ferreira, Teresa; Fingerlin, Tasha; Horikoshi, Momoko; Hu, Cheng; Huh, Iksoo; Ikram, Mohammad Kamran; Kim, Bong-Jo; Kim, Yongkang; Kim, Young Jin; Kwon, Min-Seok; Lee, Juyoung; Lee, Selyeong; Lin, Keng-Han; Maxwell, Taylor J; Nagai, Yoshihiko; Wang, Xu; Welch, Ryan P; Yoon, Joon; Zhang, Weihua; Barzilai, Nir; Voight, Benjamin F; Han, Bok-Ghee; Jenkinson, Christopher P; Kuulasmaa, Teemu; Kuusisto, Johanna; Manning, Alisa; Ng, Maggie C Y; Palmer, Nicholette D; Balkau, Beverley; Stančáková, Alena; Abboud, Hanna E; Boeing, Heiner; Giedraitis, Vilmantas; Prabhakaran, Dorairaj; Gottesman, Omri; Scott, James; Carey, Jason; Kwan, Phoenix; Grant, George; Smith, Joshua D; Neale, Benjamin M; Purcell, Shaun; Butterworth, Adam S; Howson, Joanna M M; Lee, Heung Man; Lu, Yingchang; Kwak, Soo-Heon; Zhao, Wei; Danesh, John; Lam, Vincent K L; Park, Kyong Soo; Saleheen, Danish; So, Wing Yee; Tam, Claudia H T; Afzal, Uzma; Aguilar, David; Arya, Rector; Aung, Tin; Chan, Edmund; Navarro, Carmen; Cheng, Ching-Yu; Palli, Domenico; Correa, Adolfo; Curran, Joanne E; Rybin, Denis; Farook, Vidya S; Fowler, Sharon P; Freedman, Barry I; Griswold, Michael; Hale, Daniel Esten; Hicks, Pamela J; Khor, Chiea-Chuen; Kumar, Satish; Lehne, Benjamin; Thuillier, Dorothée; Lim, Wei Yen; Liu, Jianjun; van der Schouw, Yvonne T; Loh, Marie; Musani, Solomon K; Puppala, Sobha; Scott, William R; Yengo, Loïc; Tan, Sian-Tsung; Taylor, Herman A; Thameem, Farook; Wilson, Gregory; Wong, Tien Yin; Njølstad, Pål Rasmus; Levy, Jonathan C; Mangino, Massimo; Bonnycastle, Lori L; Schwarzmayr, Thomas; Fadista, João; Surdulescu, Gabriela L; Herder, Christian; Groves, Christopher J; Wieland, Thomas; Bork-Jensen, Jette; Brandslund, Ivan; Christensen, Cramer; Koistinen, Heikki A; Doney, Alex S F; Kinnunen, Leena; Esko, Tõnu; Farmer, Andrew J; Hakaste, Liisa; Hodgkiss, Dylan; Kravic, Jasmina; Lyssenko, Valeriya; Hollensted, Mette; Jørgensen, Marit E; Jørgensen, Torben; Ladenvall, Claes; Justesen, Johanne Marie; Käräjämäki, Annemari; Kriebel, Jennifer; Rathmann, Wolfgang; Lannfelt, Lars; Lauritzen, Torsten; Narisu, Narisu; Linneberg, Allan; Melander, Olle; Milani, Lili; Neville, Matt; Orho-Melander, Marju; Qi, Lu; Qi, Qibin; Roden, Michael; Rolandsson, Olov; Swift, Amy; Rosengren, Anders H; Stirrups, Kathleen; Wood, Andrew R; Mihailov, Evelin; Blancher, Christine; Carneiro, Mauricio O; Maguire, Jared; Poplin, Ryan; Shakir, Khalid; Fennell, Timothy; DePristo, Mark; de Angelis, Martin Hrabé; Deloukas, Panos; Gjesing, Anette P; Jun, Goo; Nilsson, Peter; Murphy, Jacquelyn; Onofrio, Robert; Thorand, Barbara; Hansen, Torben; Meisinger, Christa; Hu, Frank B; Isomaa, Bo; Karpe, Fredrik; Liang, Liming; Peters, Annette; Huth, Cornelia; O'Rahilly, Stephen P; Palmer, Colin N A; Pedersen, Oluf; Rauramaa, Rainer; Tuomilehto, Jaakko; Salomaa, Veikko; Watanabe, Richard M; Syvänen, Ann-Christine; Bergman, Richard N; Bharadwaj, Dwaipayan; Bottinger, Erwin P; Cho, Yoon Shin; Chandak, Giriraj R; Chan, Juliana C N; Chia, Kee Seng; Daly, Mark J; Ebrahim, Shah B; Langenberg, Claudia; Elliott, Paul; Jablonski, Kathleen A; Lehman, Donna M; Jia, Weiping; Ma, Ronald C W; Pollin, Toni I; Sandhu, Manjinder; Tandon, Nikhil; Froguel, Philippe; Barroso, Inês; Teo, Yik Ying; Zeggini, Eleftheria; Loos, Ruth J F; Small, Kerrin S; Ried, Janina S; DeFronzo, Ralph A; Grallert, Harald; Glaser, Benjamin; Metspalu, Andres; Wareham, Nicholas J; Walker, Mark; Banks, Eric; Gieger, Christian; Ingelsson, Erik; Im, Hae Kyung; Illig, Thomas; Franks, Paul W; Buck, Gemma; Trakalo, Joseph; Buck, David; Prokopenko, Inga; Mägi, Reedik; Lind, Lars; Farjoun, Yossi; Owen, Katharine R; Gloyn, Anna L; Strauch, Konstantin; Tuomi, Tiinamaija; Kooner, Jaspal Singh; Lee, Jong-Young; Park, Taesung; Donnelly, Peter; Morris, Andrew D; Hattersley, Andrew T; Bowden, Donald W; Collins, Francis S; Atzmon, Gil; Chambers, John C; Spector, Timothy D; Laakso, Markku; Strom, Tim M; Bell, Graeme I; Blangero, John; Duggirala, Ravindranath; Tai, E Shyong; McVean, Gilean; Hanis, Craig L; Wilson, James G; Seielstad, Mark; Frayling, Timothy M; Meigs, James B; Cox, Nancy J; Sladek, Rob; Lander, Eric S; Gabriel, Stacey; Burtt, Noël P; Mohlke, Karen L; Meitinger, Thomas; Groop, Leif; Abecasis, Goncalo; Florez, Jose C; Scott, Laura J; Morris, Andrew P; Kang, Hyun Min; Boehnke, Michael; Altshuler, David; McCarthy, Mark I

2016-08-04

The genetic architecture of common traits, including the number, frequency, and effect sizes of inherited variants that contribute to individual risk, has been long debated. Genome-wide association studies have identified scores of common variants associated with type 2 diabetes, but in aggregate, these explain only a fraction of the heritability of this disease. Here, to test the hypothesis that lower-frequency variants explain much of the remainder, the GoT2D and T2D-GENES consortia performed whole-genome sequencing in 2,657 European individuals with and without diabetes, and exome sequencing in 12,940 individuals from five ancestry groups. To increase statistical power, we expanded the sample size via genotyping and imputation in a further 111,548 subjects. Variants associated with type 2 diabetes after sequencing were overwhelmingly common and most fell within regions previously identified by genome-wide association studies. Comprehensive enumeration of sequence variation is necessary to identify functional alleles that provide important clues to disease pathophysiology, but large-scale sequencing does not support the idea that lower-frequency variants have a major role in predisposition to type 2 diabetes.

The genetic architecture of type 2 diabetes

PubMed Central

Ma, Clement; Fontanillas, Pierre; Moutsianas, Loukas; McCarthy, Davis J; Rivas, Manuel A; Perry, John R B; Sim, Xueling; Blackwell, Thomas W; Robertson, Neil R; Rayner, N William; Cingolani, Pablo; Locke, Adam E; Tajes, Juan Fernandez; Highland, Heather M; Dupuis, Josee; Chines, Peter S; Lindgren, Cecilia M; Hartl, Christopher; Jackson, Anne U; Chen, Han; Huyghe, Jeroen R; van de Bunt, Martijn; Pearson, Richard D; Kumar, Ashish; Müller-Nurasyid, Martina; Grarup, Niels; Stringham, Heather M; Gamazon, Eric R; Lee, Jaehoon; Chen, Yuhui; Scott, Robert A; Below, Jennifer E; Chen, Peng; Huang, Jinyan; Go, Min Jin; Stitzel, Michael L; Pasko, Dorota; Parker, Stephen C J; Varga, Tibor V; Green, Todd; Beer, Nicola L; Day-Williams, Aaron G; Ferreira, Teresa; Fingerlin, Tasha; Horikoshi, Momoko; Hu, Cheng; Huh, Iksoo; Ikram, Mohammad Kamran; Kim, Bong-Jo; Kim, Yongkang; Kim, Young Jin; Kwon, Min-Seok; Lee, Juyoung; Lee, Selyeong; Lin, Keng-Han; Maxwell, Taylor J; Nagai, Yoshihiko; Wang, Xu; Welch, Ryan P; Yoon, Joon; Zhang, Weihua; Barzilai, Nir; Voight, Benjamin F; Han, Bok-Ghee; Jenkinson, Christopher P; Kuulasmaa, Teemu; Kuusisto, Johanna; Manning, Alisa; Ng, Maggie C Y; Palmer, Nicholette D; Balkau, Beverley; Stančáková, Alena; Abboud, Hanna E; Boeing, Heiner; Giedraitis, Vilmantas; Prabhakaran, Dorairaj; Gottesman, Omri; Scott, James; Carey, Jason; Kwan, Phoenix; Grant, George; Smith, Joshua D; Neale, Benjamin M; Purcell, Shaun; Butterworth, Adam S; Howson, Joanna M M; Lee, Heung Man; Lu, Yingchang; Kwak, Soo-Heon; Zhao, Wei; Danesh, John; Lam, Vincent K L; Park, Kyong Soo; Saleheen, Danish; So, Wing Yee; Tam, Claudia H T; Afzal, Uzma; Aguilar, David; Arya, Rector; Aung, Tin; Chan, Edmund; Navarro, Carmen; Cheng, Ching-Yu; Palli, Domenico; Correa, Adolfo; Curran, Joanne E; Rybin, Denis; Farook, Vidya S; Fowler, Sharon P; Freedman, Barry I; Griswold, Michael; Hale, Daniel Esten; Hicks, Pamela J; Khor, Chiea-Chuen; Kumar, Satish; Lehne, Benjamin; Thuillier, Dorothée; Lim, Wei Yen; Liu, Jianjun; van der Schouw, Yvonne T; Loh, Marie; Musani, Solomon K; Puppala, Sobha; Scott, William R; Yengo, Loïc; Tan, Sian-Tsung; Taylor, Herman A; Thameem, Farook; Wilson, Gregory; Wong, Tien Yin; Njølstad, Pål Rasmus; Levy, Jonathan C; Mangino, Massimo; Bonnycastle, Lori L; Schwarzmayr, Thomas; Fadista, João; Surdulescu, Gabriela L; Herder, Christian; Groves, Christopher J; Wieland, Thomas; Bork-Jensen, Jette; Brandslund, Ivan; Christensen, Cramer; Koistinen, Heikki A; Doney, Alex S F; Kinnunen, Leena; Esko, Tõnu; Farmer, Andrew J; Hakaste, Liisa; Hodgkiss, Dylan; Kravic, Jasmina; Lyssenko, Valeriya; Hollensted, Mette; Jørgensen, Marit E; Jørgensen, Torben; Ladenvall, Claes; Justesen, Johanne Marie; Käräjämäki, Annemari; Kriebel, Jennifer; Rathmann, Wolfgang; Lannfelt, Lars; Lauritzen, Torsten; Narisu, Narisu; Linneberg, Allan; Melander, Olle; Milani, Lili; Neville, Matt; Orho-Melander, Marju; Qi, Lu; Qi, Qibin; Roden, Michael; Rolandsson, Olov; Swift, Amy; Rosengren, Anders H; Stirrups, Kathleen; Wood, Andrew R; Mihailov, Evelin; Blancher, Christine; Carneiro, Mauricio O; Maguire, Jared; Poplin, Ryan; Shakir, Khalid; Fennell, Timothy; DePristo, Mark; de Angelis, Martin Hrabé; Deloukas, Panos; Gjesing, Anette P; Jun, Goo; Nilsson, Peter; Murphy, Jacquelyn; Onofrio, Robert; Thorand, Barbara; Hansen, Torben; Meisinger, Christa; Hu, Frank B; Isomaa, Bo; Karpe, Fredrik; Liang, Liming; Peters, Annette; Huth, Cornelia; O'Rahilly, Stephen P; Palmer, Colin N A; Pedersen, Oluf; Rauramaa, Rainer; Tuomilehto, Jaakko; Salomaa, Veikko; Watanabe, Richard M; Syvänen, Ann-Christine; Bergman, Richard N; Bharadwaj, Dwaipayan; Bottinger, Erwin P; Cho, Yoon Shin; Chandak, Giriraj R; Chan, Juliana C N; Chia, Kee Seng; Daly, Mark J; Ebrahim, Shah B; Langenberg, Claudia; Elliott, Paul; Jablonski, Kathleen A; Lehman, Donna M; Jia, Weiping; Ma, Ronald C W; Pollin, Toni I; Sandhu, Manjinder; Tandon, Nikhil; Froguel, Philippe; Barroso, Inês; Teo, Yik Ying; Zeggini, Eleftheria; Loos, Ruth J F; Small, Kerrin S; Ried, Janina S; DeFronzo, Ralph A; Grallert, Harald; Glaser, Benjamin; Metspalu, Andres; Wareham, Nicholas J; Walker, Mark; Banks, Eric; Gieger, Christian; Ingelsson, Erik; Im, Hae Kyung; Illig, Thomas; Franks, Paul W; Buck, Gemma; Trakalo, Joseph; Buck, David; Prokopenko, Inga; Mägi, Reedik; Lind, Lars; Farjoun, Yossi; Owen, Katharine R; Gloyn, Anna L; Strauch, Konstantin; Tuomi, Tiinamaija; Kooner, Jaspal Singh; Lee, Jong-Young; Park, Taesung; Donnelly, Peter; Morris, Andrew D; Hattersley, Andrew T; Bowden, Donald W; Collins, Francis S; Atzmon, Gil; Chambers, John C; Spector, Timothy D; Laakso, Markku; Strom, Tim M; Bell, Graeme I; Blangero, John; Duggirala, Ravindranath; Tai, E Shyong; McVean, Gilean; Hanis, Craig L; Wilson, James G; Seielstad, Mark; Frayling, Timothy M; Meigs, James B; Cox, Nancy J; Sladek, Rob; Lander, Eric S; Gabriel, Stacey; Burtt, Noël P; Mohlke, Karen L; Meitinger, Thomas; Groop, Leif; Abecasis, Goncalo; Florez, Jose C; Scott, Laura J; Morris, Andrew P; Kang, Hyun Min; Boehnke, Michael; Altshuler, David; McCarthy, Mark I

2016-01-01

The genetic architecture of common traits, including the number, frequency, and effect sizes of inherited variants that contribute to individual risk, has been long debated. Genome-wide association studies have identified scores of common variants associated with type 2 diabetes, but in aggregate, these explain only a fraction of heritability. To test the hypothesis that lower-frequency variants explain much of the remainder, the GoT2D and T2D-GENES consortia performed whole genome sequencing in 2,657 Europeans with and without diabetes, and exome sequencing in a total of 12,940 subjects from five ancestral groups. To increase statistical power, we expanded sample size via genotyping and imputation in a further 111,548 subjects. Variants associated with type 2 diabetes after sequencing were overwhelmingly common and most fell within regions previously identified by genome-wide association studies. Comprehensive enumeration of sequence variation is necessary to identify functional alleles that provide important clues to disease pathophysiology, but large-scale sequencing does not support a major role for lower-frequency variants in predisposition to type 2 diabetes. PMID:27398621

Whole genome sequencing and analysis of Campylobacter coli YH502 from retail chicken reveals a plasmid-borne type VI secretion system

USDA-ARS?s Scientific Manuscript database

Campylobacter is a major cause of foodborne illnesses worldwide. Campylobacter infections, commonly caused by ingestion of undercooked poultry and meat products, can lead to gastroenteritis and chronic reactive arthritis in humans. Whole genome sequencing (WGS) is a powerful technology that provides...

A simple method for MR elastography: a gradient-echo type multi-echo sequence.

PubMed

Numano, Tomokazu; Mizuhara, Kazuyuki; Hata, Junichi; Washio, Toshikatsu; Homma, Kazuhiro

2015-01-01

To demonstrate the feasibility of a novel MR elastography (MRE) technique based on a conventional gradient-echo type multi-echo MR sequence which does not need additional bipolar magnetic field gradients (motion encoding gradient: MEG), yet is sensitive to vibration. In a gradient-echo type multi-echo MR sequence, several images are produced from each echo of the train with different echo times (TEs). If these echoes are synchronized with the vibration, each readout's gradient lobes achieve a MEG-like effect, and the later generated echo causes a greater MEG-like effect. The sequence was tested for the tissue-mimicking agarose gel phantoms and the psoas major muscles of healthy volunteers. It was confirmed that the readout gradient lobes caused an MEG-like effect and the later TE images had higher sensitivity to vibrations. The magnitude image of later generated echo suffered the T2 decay and the susceptibility artifacts, but the wave image and elastogram of later generated echo were unaffected by these effects. In in vivo experiments, this method was able to measure the mean shear modulus of the psoas major muscle. From the results of phantom experiments and volunteer studies, it was shown that this method has clinical application potential. Copyright © 2014 Elsevier Inc. All rights reserved.

Dispersion of Multidrug-Resistant Enterococcus faecium Isolates Belonging to Major Clonal Complexes in Different Portuguese Settings▿

PubMed Central

Freitas, Ana R.; Novais, Carla; Ruiz-Garbajosa, Patricia; Coque, Teresa M.; Peixe, Luísa

2009-01-01

The population structure of 56 Enterococcus faecium isolates selected from a collection of enterococci from humans, animals, and the environment in Portugal (1997 to 2007) was analyzed by multilocus sequence typing. We identified 41 sequence types clustering into CC17, CC5, CC9, CC22 and CC94, all clonal lineages comprising isolates from different hosts. Our findings highlight the role of community-associated hosts as reservoirs of enterococci able to cause human infections. PMID:19447948

First Isolate of KPC-2-Producing Klebsiella pneumonaie Sequence Type 23 from the Americas

PubMed Central

Cejas, Daniela; Fernández Canigia, Liliana; Rincón Cruz, Giovanna; Elena, Alan X.; Maldonado, Ivana; Gutkind, Gabriel O.

2014-01-01

KPC-2-producing Klebsiella pneumoniae isolates mainly correspond to clonal complex 258 (CC258); however, we describe KPC-2-producing K. pneumoniae isolates belonging to invasive sequence type 23 (ST23). KPC-2 has scarcely been reported to occur in ST23, and this report describes the first isolation of this pathogen in the Americas. Acquisition of resistant markers in virulent clones could mark an evolutionary step toward the establishment of these clones as major nosocomial pathogens. PMID:25031447

Penicillin-Binding Protein Transpeptidase Signatures for Tracking and Predicting β-Lactam Resistance Levels in Streptococcus pneumoniae

PubMed Central

Metcalf, Benjamin J.; Chochua, Sopio; Li, Zhongya; Gertz, Robert E.; Walker, Hollis; Hawkins, Paulina A.; Tran, Theresa; Whitney, Cynthia G.; McGee, Lesley; Beall, Bernard W.

2016-01-01

ABSTRACT β-Lactam antibiotics are the drugs of choice to treat pneumococcal infections. The spread of β-lactam-resistant pneumococci is a major concern in choosing an effective therapy for patients. Systematically tracking β-lactam resistance could benefit disease surveillance. Here we developed a classification system in which a pneumococcal isolate is assigned to a “PBP type” based on sequence signatures in the transpeptidase domains (TPDs) of the three critical penicillin-binding proteins (PBPs), PBP1a, PBP2b, and PBP2x. We identified 307 unique PBP types from 2,528 invasive pneumococcal isolates, which had known MICs to six β-lactams based on broth microdilution. We found that increased β-lactam MICs strongly correlated with PBP types containing divergent TPD sequences. The PBP type explained 94 to 99% of variation in MICs both before and after accounting for genomic backgrounds defined by multilocus sequence typing, indicating that genomic backgrounds made little independent contribution to β-lactam MICs at the population level. We further developed and evaluated predictive models of MICs based on PBP type. Compared to microdilution MICs, MICs predicted by PBP type showed essential agreement (MICs agree within 1 dilution) of >98%, category agreement (interpretive results agree) of >94%, a major discrepancy (sensitive isolate predicted as resistant) rate of <3%, and a very major discrepancy (resistant isolate predicted as sensitive) rate of <2% for all six β-lactams. Thus, the PBP transpeptidase signatures are robust indicators of MICs to different β-lactam antibiotics in clinical pneumococcal isolates and serve as an accurate alternative to phenotypic susceptibility testing. PMID:27302760

An improved and validated RNA HLA class I SBT approach for obtaining full length coding sequences.

PubMed

Gerritsen, K E H; Olieslagers, T I; Groeneweg, M; Voorter, C E M; Tilanus, M G J

2014-11-01

The functional relevance of human leukocyte antigen (HLA) class I allele polymorphism beyond exons 2 and 3 is difficult to address because more than 70% of the HLA class I alleles are defined by exons 2 and 3 sequences only. For routine application on clinical samples we improved and validated the HLA sequence-based typing (SBT) approach based on RNA templates, using either a single locus-specific or two overlapping group-specific polymerase chain reaction (PCR) amplifications, with three forward and three reverse sequencing reactions for full length sequencing. Locus-specific HLA typing with RNA SBT of a reference panel, representing the major antigen groups, showed identical results compared to DNA SBT typing. Alleles encountered with unknown exons in the IMGT/HLA database and three samples, two with Null and one with a Low expressed allele, have been addressed by the group-specific RNA SBT approach to obtain full length coding sequences. This RNA SBT approach has proven its value in our routine full length definition of alleles. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Molecular Evolution of a Type 1 Wild-Vaccine Poliovirus Recombinant during Widespread Circulation in China

PubMed Central

Liu, Hong-Mei; Zheng, Du-Ping; Zhang, Li-Bi; Oberste, M. Steven; Pallansch, Mark A.; Kew, Olen M.

2000-01-01

Type 1 wild-vaccine recombinant polioviruses were isolated from poliomyelitis patients in China from 1991 to 1993. We compared the sequences of 34 recombinant isolates over the 1,353-nucleotide (nt) genomic interval (nt 2480 to 3832) encoding the major capsid protein, VP1, and the protease, 2A. All recombinants had a 367-nt block of sequence (nt 3271 to 3637) derived from the Sabin 1 oral poliovirus vaccine strain spanning the 3′-terminal sequences of VP1 (115 nt) and the 5′ half of 2A (252 nt). The remaining VP1 sequences were closely (up to 99.5%) related to those of a major genotype of wild type 1 poliovirus endemic to China up to 1994. In contrast, the non-vaccine-derived sequences at the 3′ half of 2A were more distantly related (<90% nucleotide sequence match) to those of other contemporary wild polioviruses from China. The vaccine-derived sequences of the earliest (April 1991) isolates completely matched those of Sabin 1. Later isolates diverged from the early isolates primarily by accumulation of synonymous base substitutions (at a rate of ∼3.7 × 10−2 substitutions per synonymous site per year) over the entire VP1-2A interval. Distinct evolutionary lineages were found in different Chinese provinces. From the combined epidemiologic and evolutionary analyses, we propose that the recombinant virus arose during mixed infection of a single individual in northern China in early 1991 and that its progeny spread by multiple independent chains of transmission into some of the most populous areas of China within a year of the initiating infection. PMID:11070012

Distinct patterns of alteration of myc genes associated with integration of human papillomavirus type 16 or type 45 DNA in two genital tumours.

PubMed

Sastre-Garau, X; Favre, M; Couturier, J; Orth, G

2000-08-01

We previously described two genital carcinomas (IC2, IC4) containing human papillomavirus type 16 (HPV-16)- or HPV-18-related sequences integrated in chromosomal bands containing the c-myc (8q24) or N-myc (2p24) gene, respectively. The c-myc gene was rearranged and amplified in IC2 cells without evidence of overexpression. The N-myc gene was amplified and highly transcribed in IC4 cells. Here, the sequence of an 8039 bp IC4 DNA fragment containing the integrated viral sequences and the cellular junctions is reported. A 3948 bp segment of the genome of HPV-45 encompassing the upstream regulatory region and the E6 and E7 ORFs was integrated into the untranslated part of N-myc exon 3, upstream of the N-myc polyadenylation signal. Both N-myc and HPV-45 sequences were amplified 10- to 20-fold. The 3' ends of the major N-myc transcript were mapped upstream of the 5' junction. A minor N-myc/HPV-45 fusion transcript was also identified, as well as two abundant transcripts from the HPV-45 E6-E7 region. Large amounts of N-myc protein were detected in IC4 cells. A major alteration of c-myc sequences in IC2 cells involved the insertion of a non-coding sequence into the second intron and their co-amplification with the third exon, without any evidence for the integration of HPV-16 sequences within or close to the gene. Different patterns of myc gene alterations may thus be associated with integration of HPV DNA in genital tumours, including the activation of the protooncogene via a mechanism of insertional mutagenesis and/or gene amplification.

Comamonas aquatilis sp. nov., isolated from a garden pond.

PubMed

Kämpfer, Peter; Busse, Hans-Jürgen; Baars, Sophie; Wilharm, Gottfried; Glaeser, Stefanie P

2018-04-01

A beige-pigmented bacterial strain, SB30-Chr27-3 T , isolated from a garden pond, was studied for its taxonomic position. Cells of the isolate were rod-shaped and stained Gram-negative. A comparison of the 16S rRNA gene sequence with the sequences of the type strains of the most closely related species showed that the strain belongs to the genus Comamonas and showed highest sequence similarities to the type strains of Comamonas jiangduensis (97.5 %), Comamonas aquatica (97.4 %) and Comamonas phosphati (97.3 %). The 16S rRNA gene sequence similarities to all other Comamonas species were below 97.0 %. The fatty acid profile of strain SB30-Chr27-3 T consisted of the major fatty acids C16 : 0, C15 : 0iso 2-OH/ C16 : 1ω7c, C18 : 1ω7c/C18 : 1ω9c and, in a minor amount, C10 : 0 3-OH. Major compounds in the polar lipid profile were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine and diphosphatidylglycerol. The quinone system was exclusively composed of ubiquinone Q-8. The polyamine pattern contained the major compounds putrescine, cadaverine and 2-hydroxyputrescine. These data and the differentiating biochemical properties indicated that isolate SB30-CHR27-3 T represents a novel species of the genus Comamonas, for which we propose the name >Comamonas aquatilis sp. nov. with the type strain SB30-Chr27-3 T (=CIP 111491 T =CCM 8815 T ).

Class 1 integrons characterization and multilocus sequence typing of Salmonella spp. from swine production chains in Chiang Mai and Lamphun provinces, Thailand.

PubMed

Boonkhot, Phacharaporn; Tadee, Pakpoom; Yamsakul, Panuwat; Pocharoen, Chairoj; Chokesajjawatee, Nipa; Patchanee, Prapas

2015-05-01

Pigs and pork products are well known as an important source of Salmonella, one of the major zoonotic foodborne pathogens. The emergence and spread of antimicrobial resistance is becoming a major public health concern worldwide. Integrons are genetic elements known to have a role in the acquisition and expression of genes conferring antibiotic resistance. This study focuses on the prevalence of class 1 integrons-carrying Salmonella, the genetic diversity of strains of those organisms obtained from swine production chains in Chiang Mai and Lamphun provinces, Thailand, using multilocus sequence typing (MLST) and comparison of genetic diversity of sequence types of Salmonella from this study with pulsotypes identified in previous study. In 175 Salmonella strains, the overall prevalence of class 1 integrons-carrying-Salmonella was 14%. The gene cassettes array pattern "dfrA12-orfF-aadA2" was the most frequently observed. Most of the antimicrobial resistance identified was not associated with related gene cassettes harbored by Salmonella. Six sequence types were generated from 30 randomly selected strains detected by MLST. Salmonella at the human-animal-environment interface was confirmed. Linkages both in the farm to slaughterhouse contamination route and the horizontal transmission of resistance genes were demonstrated. To reduce this problem, the use of antimicrobials in livestock should be controlled by veterinarians. Education and training of food handlers as well as promotion of safe methods of food consumption are important avenues for helping prevent foodborne illness.

Prevalence of three campylobacter species, C. jejuni, C. coli, and C. lari, using multilocus sequence typing in wild birds of the Mid-Atlantic region, USA.

PubMed

Keller, Judith I; Shriver, W Gregory

2014-01-01

Campylobacter jejuni is responsible for the majority of bacterial foodborne gastroenteritis in the US, usually due to the consumption of undercooked poultry. Research on which avian species transmit the bacterium is limited, especially in the US. We sampled wild birds in three families-Anatidae, Scolopacidae, and Laridae-in eastern North America to determine the prevalence and specific strains of Campylobacter. The overall prevalence of Campylobacter spp. was 9.2% for all wild birds sampled (n = 781). Campylobacter jejuni was the most prevalent species (8.1%), while Campylobacter coli and Campylobacter lari prevalence estimates were low (1.4% and 0.3%, respectively). We used multilocus sequence typing PCR specific to C. jejuni to characterize clonal complexes and sequence types isolated from wild bird samples and detected 13 novel sequence types, along with a clonal complex previously only associated with human disease (ST-658). Wild birds share an increasing amount of habitat with humans as more landscapes become fragmented and developed for human needs. Wild birds are and will remain an important aspect of public health due to their ability to carry and disperse emerging zoonotic pathogens or their arthropod vectors. As basic information such as prevalence is limited or lacking from a majority of wild birds in the US, this study provides further insight into Campylobacter epidemiology, host preference, and strain characterization of C. jejuni.

Design and Analysis of Single-Cell Sequencing Experiments.

PubMed

Grün, Dominic; van Oudenaarden, Alexander

2015-11-05

Recent advances in single-cell sequencing hold great potential for exploring biological systems with unprecedented resolution. Sequencing the genome of individual cells can reveal somatic mutations and allows the investigation of clonal dynamics. Single-cell transcriptome sequencing can elucidate the cell type composition of a sample. However, single-cell sequencing comes with major technical challenges and yields complex data output. In this Primer, we provide an overview of available methods and discuss experimental design and single-cell data analysis. We hope that these guidelines will enable a growing number of researchers to leverage the power of single-cell sequencing. Copyright © 2015 Elsevier Inc. All rights reserved.

C-Terminal Region of EBNA-2 Determines the Superior Transforming Ability of Type 1 Epstein-Barr Virus by Enhanced Gene Regulation of LMP-1 and CXCR7

PubMed Central

Cancian, Laila; Bosshard, Rachel; Lucchesi, Walter; Karstegl, Claudio Elgueta; Farrell, Paul J.

2011-01-01

Type 1 Epstein-Barr virus (EBV) strains immortalize B lymphocytes in vitro much more efficiently than type 2 EBV, a difference previously mapped to the EBNA-2 locus. Here we demonstrate that the greater transforming activity of type 1 EBV correlates with a stronger and more rapid induction of the viral oncogene LMP-1 and the cell gene CXCR7 (which are both required for proliferation of EBV-LCLs) during infection of primary B cells with recombinant viruses. Surprisingly, although the major sequence differences between type 1 and type 2 EBNA-2 lie in N-terminal parts of the protein, the superior ability of type 1 EBNA-2 to induce proliferation of EBV-infected lymphoblasts is mostly determined by the C-terminus of EBNA-2. Substitution of the C-terminus of type 1 EBNA-2 into the type 2 protein is sufficient to confer a type 1 growth phenotype and type 1 expression levels of LMP-1 and CXCR7 in an EREB2.5 cell growth assay. Within this region, the RG, CR7 and TAD domains are the minimum type 1 sequences required. Sequencing the C-terminus of EBNA-2 from additional EBV isolates showed high sequence identity within type 1 isolates or within type 2 isolates, indicating that the functional differences mapped are typical of EBV type sequences. The results indicate that the C-terminus of EBNA-2 accounts for the greater ability of type 1 EBV to promote B cell proliferation, through mechanisms that include higher induction of genes (LMP-1 and CXCR7) required for proliferation and survival of EBV-LCLs. PMID:21857817

A novel multi-locus sequence typing (MLST) protocol for Leuconostoc lactis isolates from traditional dairy products in China and Mongolia.

PubMed

Dan, Tong; Liu, Wenjun; Sun, Zhihong; Lv, Qiang; Xu, Haiyan; Song, Yuqin; Zhang, Heping

2014-06-09

Economically, Leuconostoc lactis is one of the most important species in the genus Leuconostoc. It plays an important role in the food industry including the production of dextrans and bacteriocins. Currently, traditional molecular typing approaches for characterisation of this species at the isolate level are either unavailable or are not sufficiently reliable for practical use. Multilocus sequence typing (MLST) is a robust and reliable method for characterising bacterial and fungal species at the molecular level. In this study, a novel MLST protocol was developed for 50 L. lactis isolates from Mongolia and China. Sequences from eight targeted genes (groEL, carB, recA, pheS, murC, pyrG, rpoB and uvrC) were obtained. Sequence analysis indicated 20 different sequence types (STs), with 13 of them being represented by a single isolate. Phylogenetic analysis based on the sequences of eight MLST loci indicated that the isolates belonged to two major groups, A (34 isolates) and B (16 isolates). Linkage disequilibrium analyses indicated that recombination occurred at a low frequency in L. lactis, indicating a clonal population structure. Split-decomposition analysis indicated that intraspecies recombination played a role in generating genotypic diversity amongst isolates. Our results indicated that MLST is a valuable tool for typing L. lactis isolates that can be used for further monitoring of evolutionary changes and population genetics.

Insertion sequence ISRP10 inactivation of the oprD gene in imipenem-resistant Pseudomonas aeruginosa clinical isolates.

PubMed

Sun, Qinghui; Ba, Zhaofen; Wu, Guoying; Wang, Wei; Lin, Shuxiang; Yang, Hongjiang

2016-05-01

Carbapenem resistance mechanisms were investigated in 32 imipenem-resistant Pseudomonas aeruginosa clinical isolates recovered from hospitalised children. Sequence analysis revealed that 31 of the isolates had an insertion sequence element ISRP10 disrupting the porin gene oprD, demonstrating that ISRP10 inactivation of oprD conferred imipenem resistance in the majority of the isolates. Multilocus sequence typing (MLST) was used to discriminate the isolates. In total, 11 sequence types (STs) were identified including 3 novel STs, and 68.3% (28/41) of the tested strains were characterised as clone ST253. In combination with random amplified polymorphic DNA (RAPD) analysis, the imipenem-resistant isolates displayed a relatively high degree of genetic variability and were unlikely associated with nosocomial infections. Copyright © 2016 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Genetic diversity analysis of Leuconostoc mesenteroides from Korean vegetables and food products by multilocus sequence typing.

PubMed

Sharma, Anshul; Kaur, Jasmine; Lee, Sulhee; Park, Young-Seo

2018-06-01

In the present study, 35 Leuconostoc mesenteroides strains isolated from vegetables and food products from South Korea were studied by multilocus sequence typing (MLST) of seven housekeeping genes (atpA, groEL, gyrB, pheS, pyrG, rpoA, and uvrC). The fragment sizes of the seven amplified housekeeping genes ranged in length from 366 to 1414 bp. Sequence analysis indicated 27 different sequence types (STs) with 25 of them being represented by a single strain indicating high genetic diversity, whereas the remaining 2 were characterized by five strains each. In total, 220 polymorphic nucleotide sites were detected among seven housekeeping genes. The phylogenetic analysis based on the STs of the seven loci indicated that the 35 strains belonged to two major groups, A (28 strains) and B (7 strains). Split decomposition analysis showed that intraspecies recombination played a role in generating diversity among strains. The minimum spanning tree showed that the evolution of the STs was not correlated with food source. This study signifies that the multilocus sequence typing is a valuable tool to access the genetic diversity among L. mesenteroides strains from South Korea and can be used further to monitor the evolutionary changes.

Impact of exogenous sequences on the characteristics of an epidemic type 2 recombinant vaccine-derived poliovirus.

PubMed

Riquet, Franck B; Blanchard, Claire; Jegouic, Sophie; Balanant, Jean; Guillot, Sophie; Vibet, Marie-Anne; Rakoto-Andrianarivelo, Mala; Delpeyroux, Francis

2008-09-01

Pathogenic circulating vaccine-derived polioviruses (cVDPVs) have become a major obstacle to the successful completion of the global polio eradication program. Most cVDPVs are recombinant between the oral poliovirus vaccine (OPV) and human enterovirus species C (HEV-C). To study the role of HEV-C sequences in the phenotype of cVDPVs, we generated a series of recombinants between a Madagascar cVDPV isolate and its parental OPV type 2 strain. Results indicated that the HEV-C sequences present in this cVDPV contribute to its characteristics, including pathogenicity, suggesting that interspecific recombination contributes to the phenotypic biodiversity of polioviruses and may favor the emergence of cVDPVs.

The bacteria and bacteriophages from a Mesquite Flats site of the Death Valley desert.

PubMed

Prestel, Eric; Regeard, Christophe; Salamitou, Sylvie; Neveu, Julie; Dubow, Michael S

2013-06-01

Arid zones cover over 30 % of the Earth's continental surface. In order to better understand the role of microbes in this type of harsh environment, we isolated and characterized the bacteriophages from samples of the surface sand of the Mesquite Flats region via electron microscopy and DNA sequencing of a select number of cloned phage DNAs. An electron microscopic analysis of the recovered virus-like particles revealed at least 11 apparently different morphotypes sharing structural characteristics of the Caudoviridae family of tailed phages. We found that 36 % of the sequences contained no significant identity (e-value >10(-3)) with sequences in the databases. Pilot sequencing of cloned 16S rRNA genes identified Bacteroidetes and Proteobacteria as the major bacterial groups present in this severe environment. The majority of the 16S rDNA sequences from the total (uncultured) bacterial population displayed ≤96 % identity to 16S rRNA genes in the database, suggesting an unexplored bacterial population likely adapted to a desert environment. In addition, we also isolated and identified 38 cultivable bacterial strains, the majority of which belonged to the genus Bacillus. Mitomycin-C treatment of the cultivable bacteria demonstrated that the vast majority (84 %) contained at least one SOS-inducible prophage.

Chromosomal structures and repetitive sequences divergence in Cucumis species revealed by comparative cytogenetic mapping.

PubMed

Zhang, Yunxia; Cheng, Chunyan; Li, Ji; Yang, Shuqiong; Wang, Yunzhu; Li, Ziang; Chen, Jinfeng; Lou, Qunfeng

2015-09-25

Differentiation and copy number of repetitive sequences affect directly chromosome structure which contributes to reproductive isolation and speciation. Comparative cytogenetic mapping has been verified an efficient tool to elucidate the differentiation and distribution of repetitive sequences in genome. In present study, the distinct chromosomal structures of five Cucumis species were revealed through genomic in situ hybridization (GISH) technique and comparative cytogenetic mapping of major satellite repeats. Chromosome structures of five Cucumis species were investigated using GISH and comparative mapping of specific satellites. Southern hybridization was employed to study the proliferation of satellites, whose structural characteristics were helpful for analyzing chromosome evolution. Preferential distribution of repetitive DNAs at the subtelomeric regions was found in C. sativus, C hystrix and C. metuliferus, while majority was positioned at the pericentromeric heterochromatin regions in C. melo and C. anguria. Further, comparative GISH (cGISH) through using genomic DNA of other species as probes revealed high homology of repeats between C. sativus and C. hystrix. Specific satellites including 45S rDNA, Type I/II, Type III, Type IV, CentM and telomeric repeat were then comparatively mapped in these species. Type I/II and Type IV produced bright signals at the subtelomeric regions of C. sativus and C. hystrix simultaneously, which might explain the significance of their amplification in the divergence of Cucumis subgenus from the ancient ancestor. Unique positioning of Type III and CentM only at the centromeric domains of C. sativus and C. melo, respectively, combining with unique southern bands, revealed rapid evolutionary patterns of centromeric DNA in Cucumis. Obvious interstitial telomeric repeats were observed in chromosomes 1 and 2 of C. sativus, which might provide evidence of the fusion hypothesis of chromosome evolution from x = 12 to x = 7 in Cucumis species. Besides, the significant correlation was found between gene density along chromosome and GISH band intensity in C. sativus and C. melo. In summary, comparative cytogenetic mapping of major satellites and GISH revealed the distinct differentiation of chromosome structure during species formation. The evolution of repetitive sequences was the main force for the divergence of Cucumis species from common ancestor.

First isolate of KPC-2-producing Klebsiella pneumonaie sequence type 23 from the Americas.

PubMed

Cejas, Daniela; Fernández Canigia, Liliana; Rincón Cruz, Giovanna; Elena, Alan X; Maldonado, Ivana; Gutkind, Gabriel O; Radice, Marcela A

2014-09-01

KPC-2-producing Klebsiella pneumoniae isolates mainly correspond to clonal complex 258 (CC258); however, we describe KPC-2-producing K. pneumoniae isolates belonging to invasive sequence type 23 (ST23). KPC-2 has scarcely been reported to occur in ST23, and this report describes the first isolation of this pathogen in the Americas. Acquisition of resistant markers in virulent clones could mark an evolutionary step toward the establishment of these clones as major nosocomial pathogens. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Characteristics, stratigraphic architecture, and time framework of multi-order mixed siliciclastic and carbonate depositional sequences, outcropping Cisco Group (Late Pennsylvanian and Early Permian), Eastern Shelf, north-central Texas, USA

NASA Astrophysics Data System (ADS)

Yang, Wan; Kominz, Michelle A.

2003-01-01

The Cisco Group on the Eastern Shelf of the Midland Basin is composed of fluvial, deltaic, shelf, shelf-margin, and slope-to-basin carbonate and siliciclastic rocks. Sedimentologic and stratigraphic analyses of 181 meter-to-decimeter-scale depositional sequences exposed in the up-dip shelf indicated that the siliciclastic and carbonate parasequences in the transgressive systems tracts (TST) are thin and upward deepening, whereas those in highstand systems tracts (HST) are thick and upward shallowing. The sequences can be subdivided into five types on the basis of principal lithofacies, and exhibit variable magnitude of facies shift corresponding to variable extents of marine transgression and regression on the shelf. The sequence stacking patterns and their regional persistence suggest a three-level sequence hierarchy controlled by eustasy, whereas local and regional changes in lithology, thickness, and sequence type, magnitude, and absence were controlled by interplay of eustasy, differential shelf subsidence, depositional topography, and pattern of siliciclastic supply. The outcropping Cisco Group is highly incomplete with an estimated 6-11% stratigraphic completeness. The average duration of deposition of the major (third-order) sequences is estimated as 67-102 ka on the up-dip shelf and increases down dip, while the average duration of the major sequence boundaries (SB) is estimated as 831-1066 ka and decreases down dip. The nondepositional and erosional hiatus on the up-dip shelf was represented by lowstand deltaic systems in the basin and slope.

Differentiation of Trichophyton rubrum clinical isolates from Japanese and Chinese patients by randomly amplified polymorphic DNA and DNA sequence analysis of the non-transcribed spacer region of the rRNA gene.

PubMed

Yang, Xiumin; Sugita, Takashi; Takashima, Masako; Hiruma, Masataro; Li, Ruoyu; Sudo, Hajime; Ogawa, Hideoki; Ikeda, Shigaku

2009-04-01

Trichophyton rubrum is the most common pathogen causing dermatophytosis worldwide. Recent genetic investigations showed that the microorganism originated in Africa and then spread to Europe and North America via Asia. We investigated the intraspecific diversity of T. rubrum isolated from two closely located Asian countries, Japan and China. A total of 150 clinical isolates of T. rubrum obtained from Japanese and Chinese patients were analyzed by randomly amplified polymorphic DNA (RAPD) and DNA sequence analysis of the non-transcribed spacer (NTS) region in the rRNA gene. RAPD analysis divided the 150 strains into two major clusters, A and B. Of the Japanese isolates, 30% belonged to cluster A and 70% belonged to cluster B, whereas 91% of the Chinese isolates were in cluster A. The NTS region of the rRNA gene was divided into four major groups (I-IV) based on DNA sequencing. The majority of Japanese isolates were type IV (51%), and the majority of Chinese isolates were type III (75%). These results suggest that although Japan and China are neighboring countries, the origins of T. rubrum isolates from these countries may not be identical. These findings provide information useful for tracing the global transmission routes of T. rubrum.

SplicingTypesAnno: annotating and quantifying alternative splicing events for RNA-Seq data.

PubMed

Sun, Xiaoyong; Zuo, Fenghua; Ru, Yuanbin; Guo, Jiqiang; Yan, Xiaoyan; Sablok, Gaurav

2015-04-01

Alternative splicing plays a key role in the regulation of the central dogma. Four major types of alternative splicing have been classified as intron retention, exon skipping, alternative 5 splice sites or alternative donor sites, and alternative 3 splice sites or alternative acceptor sites. A few algorithms have been developed to detect splice junctions from RNA-Seq reads. However, there are few tools targeting at the major alternative splicing types at the exon/intron level. This type of analysis may reveal subtle, yet important events of alternative splicing, and thus help gain deeper understanding of the mechanism of alternative splicing. This paper describes a user-friendly R package, extracting, annotating and analyzing alternative splicing types for sequence alignment files from RNA-Seq. SplicingTypesAnno can: (1) provide annotation for major alternative splicing at exon/intron level. By comparing the annotation from GTF/GFF file, it identifies the novel alternative splicing sites; (2) offer a convenient two-level analysis: genome-scale annotation for users with high performance computing environment, and gene-scale annotation for users with personal computers; (3) generate a user-friendly web report and additional BED files for IGV visualization. SplicingTypesAnno is a user-friendly R package for extracting, annotating and analyzing alternative splicing types at exon/intron level for sequence alignment files from RNA-Seq. It is publically available at https://sourceforge.net/projects/splicingtypes/files/ or http://genome.sdau.edu.cn/research/software/SplicingTypesAnno.html. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Genotypes of Ciprofloxacin-Resistant Klebsiella pneumoniae in Korea and Their Characteristics According to the Genetic Lineages.

PubMed

Park, Dong Jin; Yu, Jin Kyung; Park, Kang Gyun; Park, Yeon-Joon

2015-12-01

We investigated the molecular genotypes of ciprofloxacin-resistant Klebsiella pneumoniae and their characteristics according to the genetic lineages. For 160 K. pneumoniae collected in 2013, ciprofloxacin minimum inhibitory concentrations (MICs) were determined by agar dilution method. The genotypes of ciprofloxacin-resistant K. pneumoniae isolates were determined by multilocus sequence typing (MLST) and wzi gene typing. The presence of plasmid-mediated resistance determinants [qnrA, qnrB, qnrS, aac(6')-Ib-cr, blaCTX-M, and blaSHV] was investigated. The gyrA and parC genes were sequenced. Fifty-seven isolates showed ciprofloxacin resistance. By MLST, four major sequence types (STs) or clonal complexes (CCs), that is, ST307, CC11, CC147, and ST15, were found and the two most prevalent STs were ST307 (14/57, 24.6%) and ST11 (12/57, 21.1%). By wzi gene sequencing, 46 of the 57 isolates could be differentiated. All the ST307 isolates had an identical wzi sequence and harbored qnrB. The majority of them harbored aac(6')-Ib-cr (85.7%) and CTX-M-15 (92.9%). In contrast, 12 ST11 isolates were divided into five sublineages by wzi sequence and qnrB, qnrS, and aac(6')-Ib-cr were carried by nine, seven, and three isolates, respectively. They harbored SHV-type extended-spectrum β-lactamase more frequently than CTX-M-15 (nine and four isolates, respectively). The prevalence of CTX-M-15, qnrB1, and aac(6')-Ib-cr was significantly higher in ST307 than in ST11 (p=0.003, p=0.000, and p=0.002, respectively). Both clones had identical amino acid substitution in gyrA (S83I) and parC (S80I). K. pneumoniae ST307 and ST11 were the two most common clones, and the ST307 isolates were highly homogeneous, suggesting their recent emergence.

Whole-genome sequencing reveals clonal expansion of multiresistant Staphylococcus haemolyticus in European hospitals.

PubMed

Cavanagh, Jorunn Pauline; Hjerde, Erik; Holden, Matthew T G; Kahlke, Tim; Klingenberg, Claus; Flægstad, Trond; Parkhill, Julian; Bentley, Stephen D; Sollid, Johanna U Ericson

2014-11-01

Staphylococcus haemolyticus is an emerging cause of nosocomial infections, primarily affecting immunocompromised patients. A comparative genomic analysis was performed on clinical S. haemolyticus isolates to investigate their genetic relationship and explore the coding sequences with respect to antimicrobial resistance determinants and putative hospital adaptation. Whole-genome sequencing was performed on 134 isolates of S. haemolyticus from geographically diverse origins (Belgium, 2; Germany, 10; Japan, 13; Norway, 54; Spain, 2; Switzerland, 43; UK, 9; USA, 1). Each genome was individually assembled. Protein coding sequences (CDSs) were predicted and homologous genes were categorized into three types: Type I, core genes, homologues present in all strains; Type II, unique core genes, homologues shared by only a subgroup of strains; and Type III, unique genes, strain-specific CDSs. The phylogenetic relationship between the isolates was built from variable sites in the form of single nucleotide polymorphisms (SNPs) in the core genome and used to construct a maximum likelihood phylogeny. SNPs in the genome core regions divided the isolates into one major group of 126 isolates and one minor group of isolates with highly diverse genomes. The major group was further subdivided into seven clades (A-G), of which four (A-D) encompassed isolates only from Europe. Antimicrobial multiresistance was observed in 77.7% of the collection. High levels of homologous recombination were detected in genes involved in adherence, staphylococcal host adaptation and bacterial cell communication. The presence of several successful and highly resistant clones underlines the adaptive potential of this opportunistic pathogen. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

Whole-genome sequencing reveals clonal expansion of multiresistant Staphylococcus haemolyticus in European hospitals

PubMed Central

Cavanagh, Jorunn Pauline; Hjerde, Erik; Holden, Matthew T. G.; Kahlke, Tim; Klingenberg, Claus; Flægstad, Trond; Parkhill, Julian; Bentley, Stephen D.; Sollid, Johanna U. Ericson

2014-01-01

Objectives Staphylococcus haemolyticus is an emerging cause of nosocomial infections, primarily affecting immunocompromised patients. A comparative genomic analysis was performed on clinical S. haemolyticus isolates to investigate their genetic relationship and explore the coding sequences with respect to antimicrobial resistance determinants and putative hospital adaptation. Methods Whole-genome sequencing was performed on 134 isolates of S. haemolyticus from geographically diverse origins (Belgium, 2; Germany, 10; Japan, 13; Norway, 54; Spain, 2; Switzerland, 43; UK, 9; USA, 1). Each genome was individually assembled. Protein coding sequences (CDSs) were predicted and homologous genes were categorized into three types: Type I, core genes, homologues present in all strains; Type II, unique core genes, homologues shared by only a subgroup of strains; and Type III, unique genes, strain-specific CDSs. The phylogenetic relationship between the isolates was built from variable sites in the form of single nucleotide polymorphisms (SNPs) in the core genome and used to construct a maximum likelihood phylogeny. Results SNPs in the genome core regions divided the isolates into one major group of 126 isolates and one minor group of isolates with highly diverse genomes. The major group was further subdivided into seven clades (A–G), of which four (A–D) encompassed isolates only from Europe. Antimicrobial multiresistance was observed in 77.7% of the collection. High levels of homologous recombination were detected in genes involved in adherence, staphylococcal host adaptation and bacterial cell communication. Conclusions The presence of several successful and highly resistant clones underlines the adaptive potential of this opportunistic pathogen. PMID:25038069

Virulence gene typing of methicillin-resistant Staphylococcus aureus as a complement in epidemiological typing.

PubMed

Nowrouzian, Forough L; Karami, Nahid; Welinder-Olsson, Christina; Ahrén, Christina

2013-06-01

Methicillin-resistant Staphylococcus aureus (MRSA) has widely spread to all parts of the world. For surveillance and effective infection control molecular typing is required. We have evaluated the utility of virulence gene determination as a complementary tool for epidemiological typing of MRSA in relation to spa-typing and pulsed-field gel electrophoresis (PFGE). We assessed 63 community-acquired MRSA (CA-MRSA) isolates detected in the West part of Sweden for 30 virulence factor genes (VF) and agr allele variations by serial polymerase chain reaction (PCR) assays. These isolates belonged to sequence types (ST) 8, 80, 45 and 30 as classified by multilocus sequence typing. The isolates in each spa-type and PFGE-type were examined over an extended time-period and constituted a varying number of PFGE-subtypes (5-14) and spa-types (3-11) within four major PFGE types. Each ST had a unique VF profile. For isolates within a major PFGE type showing high diversity both in PFGE subtypes and spa the VF profile varied as well in contrast to those with low diversity where no alterations were seen. Thus, the accuracy of each typing method does not only vary by the method per se but is rather dependent on the genetic repertoire of the typed strains and genes evaluated. For strains demonstrating high diversity VF typing may be a useful complement in the epidemiological investigations, and may highlight the accurate discriminatory power of spa or PFGE typing. Copyright © 2013 Elsevier B.V. All rights reserved.

Dissection of Influenza Infection In Vivo by Single-Cell RNA Sequencing.

PubMed

Steuerman, Yael; Cohen, Merav; Peshes-Yaloz, Naama; Valadarsky, Liran; Cohn, Ofir; David, Eyal; Frishberg, Amit; Mayo, Lior; Bacharach, Eran; Amit, Ido; Gat-Viks, Irit

2018-06-01

The influenza virus is a major cause of morbidity and mortality worldwide. Yet, both the impact of intracellular viral replication and the variation in host response across different cell types remain uncharacterized. Here we used single-cell RNA sequencing to investigate the heterogeneity in the response of lung tissue cells to in vivo influenza infection. Analysis of viral and host transcriptomes in the same single cell enabled us to resolve the cellular heterogeneity of bystander (exposed but uninfected) as compared with infected cells. We reveal that all major immune and non-immune cell types manifest substantial fractions of infected cells, albeit at low viral transcriptome loads relative to epithelial cells. We show that all cell types respond primarily with a robust generic transcriptional response, and we demonstrate novel markers specific for influenza-infected as opposed to bystander cells. These findings open new avenues for targeted therapy aimed exclusively at infected cells. Copyright © 2018 Elsevier Inc. All rights reserved.

New families of site-specific repetitive DNA sequences that comprise constitutive heterochromatin of the Syrian hamster (Mesocricetus auratus, Cricetinae, Rodentia).

PubMed

Yamada, Kazuhiko; Kamimura, Eikichi; Kondo, Mariko; Tsuchiya, Kimiyuki; Nishida-Umehara, Chizuko; Matsuda, Yoichi

2006-02-01

We molecularly cloned new families of site-specific repetitive DNA sequences from BglII- and EcoRI-digested genomic DNA of the Syrian hamster (Mesocricetus auratus, Cricetrinae, Rodentia) and characterized them by chromosome in situ hybridization and filter hybridization. They were classified into six different types of repetitive DNA sequence families according to chromosomal distribution and genome organization. The hybridization patterns of the sequences were consistent with the distribution of C-positive bands and/or Hoechst-stained heterochromatin. The centromeric major satellite DNA and sex chromosome-specific and telomeric region-specific repetitive sequences were conserved in the same genus (Mesocricetus) but divergent in different genera. The chromosome-2-specific sequence was conserved in two genera, Mesocricetus and Cricetulus, and a low copy number of repetitive sequences on the heterochromatic chromosome arms were conserved in the subfamily Cricetinae but not in the subfamily Calomyscinae. By contrast, the other type of repetitive sequences on the heterochromatic chromosome arms, which had sequence similarities to a LINE sequence of rodents, was conserved through the three subfamilies, Cricetinae, Calomyscinae and Murinae. The nucleotide divergence of the repetitive sequences of heterochromatin was well correlated with the phylogenetic relationships of the Cricetinae species, and each sequence has been independently amplified and diverged in the same genome.

A novel multi-locus sequence typing (MLST) protocol for Leuconostoc lactis isolates from traditional dairy products in China and Mongolia

PubMed Central

2014-01-01

Background Economically, Leuconostoc lactis is one of the most important species in the genus Leuconostoc. It plays an important role in the food industry including the production of dextrans and bacteriocins. Currently, traditional molecular typing approaches for characterisation of this species at the isolate level are either unavailable or are not sufficiently reliable for practical use. Multilocus sequence typing (MLST) is a robust and reliable method for characterising bacterial and fungal species at the molecular level. In this study, a novel MLST protocol was developed for 50 L. lactis isolates from Mongolia and China. Results Sequences from eight targeted genes (groEL, carB, recA, pheS, murC, pyrG, rpoB and uvrC) were obtained. Sequence analysis indicated 20 different sequence types (STs), with 13 of them being represented by a single isolate. Phylogenetic analysis based on the sequences of eight MLST loci indicated that the isolates belonged to two major groups, A (34 isolates) and B (16 isolates). Linkage disequilibrium analyses indicated that recombination occurred at a low frequency in L. lactis, indicating a clonal population structure. Split-decomposition analysis indicated that intraspecies recombination played a role in generating genotypic diversity amongst isolates. Conclusions Our results indicated that MLST is a valuable tool for typing L. lactis isolates that can be used for further monitoring of evolutionary changes and population genetics. PMID:24912963

Natural Variation of Epstein-Barr Virus Genes, Proteins, and Primary MicroRNA.

PubMed

Correia, Samantha; Palser, Anne; Elgueta Karstegl, Claudio; Middeldorp, Jaap M; Ramayanti, Octavia; Cohen, Jeffrey I; Hildesheim, Allan; Fellner, Maria Dolores; Wiels, Joelle; White, Robert E; Kellam, Paul; Farrell, Paul J

2017-08-01

Viral gene sequences from an enlarged set of about 200 Epstein-Barr virus (EBV) strains, including many primary isolates, have been used to investigate variation in key viral genetic regions, particularly LMP1, Zp, gp350, EBNA1, and the BART microRNA (miRNA) cluster 2. Determination of type 1 and type 2 EBV in saliva samples from people from a wide range of geographic and ethnic backgrounds demonstrates a small percentage of healthy white Caucasian British people carrying predominantly type 2 EBV. Linkage of Zp and gp350 variants to type 2 EBV is likely to be due to their genes being adjacent to the EBNA3 locus, which is one of the major determinants of the type 1/type 2 distinction. A novel classification of EBNA1 DNA binding domains, named QCIGP, results from phylogeny analysis of their protein sequences but is not linked to the type 1/type 2 classification. The BART cluster 2 miRNA region is classified into three major variants through single-nucleotide polymorphisms (SNPs) in the primary miRNA outside the mature miRNA sequences. These SNPs can result in altered levels of expression of some miRNAs from the BART variant frequently present in Chinese and Indonesian nasopharyngeal carcinoma (NPC) samples. The EBV genetic variants identified here provide a basis for future, more directed analysis of association of specific EBV variations with EBV biology and EBV-associated diseases. IMPORTANCE Incidence of diseases associated with EBV varies greatly in different parts of the world. Thus, relationships between EBV genome sequence variation and health, disease, geography, and ethnicity of the host may be important for understanding the role of EBV in diseases and for development of an effective EBV vaccine. This paper provides the most comprehensive analysis so far of variation in specific EBV genes relevant to these diseases and proposed EBV vaccines. By focusing on variation in LMP1, Zp, gp350, EBNA1, and the BART miRNA cluster 2, new relationships with the known type 1/type 2 strains are demonstrated, and a novel classification of EBNA1 and the BART miRNAs is proposed. Copyright © 2017 Correia et al.

Microbacterium agarici sp. nov., Microbacterium humi sp. nov. and Microbacterium pseudoresistens sp. nov., isolated from the base of the mushroom Agaricus blazei.

PubMed

Young, C-C; Busse, H-J; Langer, S; Chu, Jiunn-Nan; Schumann, P; Arun, A B; Shen, Fo-Ting; Rekha, P D; Kämpfer, P

2010-04-01

Three Gram-positive, rod-shaped bacteria (strains CC-SBCK-209( T), CC-12309(T) and CC-5209(T)) were isolated from the stalk of the edible mushroom Agaricus blazei grown in the laboratory. 16S rRNA gene sequence analysis indicated that all three isolates clearly belonged to the genus Microbacterium. Strains CC-SBCK-209( T) and CC-12309(T) were most related closely to the type strain of Microbacterium halotolerans (95.9 and 96.1 % 16S rRNA gene sequence similarity, respectively). These two novel strains shared 97.9 % 16S rRNA gene sequence similarity. Levels of similarity to the type strains of all other recognized Microbacterium species were lower than 95.5 %. The third strain (CC-5209( T)) showed the highest 16S rRNA gene sequence similarity to the type strain of Microbacterium resistens (97.6 %); levels of similarity to the type strains of all other recognized Microbacterium species were lower than 96 %. The quinone systems of strains CC-SBCK-209(T), CC-12309(T) and CC-5209(T) consisted of MK-11/MK-12, MK-11/MK-10 and MK-13 as major compounds, respectively. All three strains contained ornithine in their peptidoglycan. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and an unknown glycolipid. The polyamine pattern consisted of spermidine and spermine as predominant components. Fatty acid profiles (anteiso-C(15 : 0), iso-C(16 : 0) and anteiso-C(17 : 0 ) as major components) supported the affiliation of all three strains to the genus Microbacterium. The results of physiological and biochemical tests and DNA-DNA hybridization experiments allowed the clear phenotypic and genotypic differentiation of strains CC-SBCK-209(T) and CC-12309( T) from M. halotolerans and other closely related Microbacterium species. Strain CC-5209(T) could be differentiated clearly from M. resistens both genotypically and phenotypically. Based on these data, the novel strains are considered to represent three novel species of the genus Microbacterium. The names proposed for these organisms are Microbacterium agarici sp. nov. [type strain CC-SBCK-209( T) (=DSM 21798(T)=CCM 7686(T))], Microbacterium humi sp. nov. [type strain CC-12309(T) (=DSM 21799(T)=CCM 7687(T))] and Microbacterium pseudoresistens sp. nov. [type strain CC-5209(T) (=DSM 22185(T)=CCM 7688(T))].

TypeLoader: A fast and efficient automated workflow for the annotation and submission of novel full-length HLA alleles.

PubMed

Surendranath, V; Albrecht, V; Hayhurst, J D; Schöne, B; Robinson, J; Marsh, S G E; Schmidt, A H; Lange, V

2017-07-01

Recent years have seen a rapid increase in the discovery of novel allelic variants of the human leukocyte antigen (HLA) genes. Commonly, only the exons encoding the peptide binding domains of novel HLA alleles are submitted. As a result, the IPD-IMGT/HLA Database lacks sequence information outside those regions for the majority of known alleles. This has implications for the application of the new sequencing technologies, which deliver sequence data often covering the complete gene. As these technologies simplify the characterization of the complete gene regions, it is desirable for novel alleles to be submitted as full-length sequences to the database. However, the manual annotation of full-length alleles and the generation of specific formats required by the sequence repositories is prone to error and time consuming. We have developed TypeLoader to address both these facets. With only the full-length sequence as a starting point, Typeloader performs automatic sequence annotation and subsequently handles all steps involved in preparing the specific formats for submission with very little manual intervention. TypeLoader is routinely used at the DKMS Life Science Lab and has aided in the successful submission of more than 900 novel HLA alleles as full-length sequences to the European Nucleotide Archive repository and the IPD-IMGT/HLA Database with a 95% reduction in the time spent on annotation and submission when compared with handling these processes manually. TypeLoader is implemented as a web application and can be easily installed and used on a standalone Linux desktop system or within a Linux client/server architecture. TypeLoader is downloadable from http://www.github.com/DKMS-LSL/typeloader. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Food Safety in the Age of Next Generation Sequencing, Bioinformatics, and Open Data Access.

PubMed

Taboada, Eduardo N; Graham, Morag R; Carriço, João A; Van Domselaar, Gary

2017-01-01

Public health labs and food regulatory agencies globally are embracing whole genome sequencing (WGS) as a revolutionary new method that is positioned to replace numerous existing diagnostic and microbial typing technologies with a single new target: the microbial draft genome. The ability to cheaply generate large amounts of microbial genome sequence data, combined with emerging policies of food regulatory and public health institutions making their microbial sequences increasingly available and public, has served to open up the field to the general scientific community. This open data access policy shift has resulted in a proliferation of data being deposited into sequence repositories and of novel bioinformatics software designed to analyze these vast datasets. There also has been a more recent drive for improved data sharing to achieve more effective global surveillance, public health and food safety. Such developments have heightened the need for enhanced analytical systems in order to process and interpret this new type of data in a timely fashion. In this review we outline the emergence of genomics, bioinformatics and open data in the context of food safety. We also survey major efforts to translate genomics and bioinformatics technologies out of the research lab and into routine use in modern food safety labs. We conclude by discussing the challenges and opportunities that remain, including those expected to play a major role in the future of food safety science.

The evolution and population structure of Lactobacillus fermentum from different naturally fermented products as determined by multilocus sequence typing (MLST).

PubMed

Dan, Tong; Liu, Wenjun; Song, Yuqin; Xu, Haiyan; Menghe, Bilige; Zhang, Heping; Sun, Zhihong

2015-05-20

Lactobacillus fermentum is economically important in the production and preservation of fermented foods. A repeatable and discriminative typing method was devised to characterize L. fermentum at the molecular level. The multilocus sequence typing (MLST) scheme developed was based on analysis of the internal sequence of 11 housekeeping gene fragments (clpX, dnaA, dnaK, groEL, murC, murE, pepX, pyrG, recA, rpoB, and uvrC). MLST analysis of 203 isolates of L. fermentum from Mongolia and seven provinces/ autonomous regions in China identified 57 sequence types (ST), 27 of which were represented by only a single isolate, indicating high genetic diversity. Phylogenetic analyses based on the sequence of the 11 housekeeping gene fragments indicated that the L. fermentum isolates analyzed belonged to two major groups. A standardized index of association (I A (S)) indicated a weak clonal population structure in L. fermentum. Split decomposition analysis indicated that recombination played an important role in generating the genetic diversity observed in L. fermentum. The results from the minimum spanning tree strongly suggested that evolution of L. fermentum STs was not correlated with geography or food-type. The MLST scheme developed will be valuable for further studies on the evolution and population structure of L. fermentum isolates used in food products.

Streptococcus mutans clonal variation revealed by multilocus sequence typing.

PubMed

Nakano, Kazuhiko; Lapirattanakul, Jinthana; Nomura, Ryota; Nemoto, Hirotoshi; Alaluusua, Satu; Grönroos, Lisa; Vaara, Martti; Hamada, Shigeyuki; Ooshima, Takashi; Nakagawa, Ichiro

2007-08-01

Streptococcus mutans is the major pathogen of dental caries, a biofilm-dependent infectious disease, and occasionally causes infective endocarditis. S. mutans strains have been classified into four serotypes (c, e, f, and k). However, little is known about the S. mutans population, including the clonal relationships among strains of S. mutans, in relation to the particular clones that cause systemic diseases. To address this issue, we have developed a multilocus sequence typing (MLST) scheme for S. mutans. Eight housekeeping gene fragments were sequenced from each of 102 S. mutans isolates collected from the four serotypes in Japan and Finland. Between 14 and 23 alleles per locus were identified, allowing us theoretically to distinguish more than 1.2 x 10(10) sequence types. We identified 92 sequence types in these 102 isolates, indicating that S. mutans contains a diverse population. Whereas serotype c strains were widely distributed in the dendrogram, serotype e, f, and k strains were differentiated into clonal complexes. Therefore, we conclude that the ancestral strain of S. mutans was serotype c. No geographic specificity was identified. However, the distribution of the collagen-binding protein gene (cnm) and direct evidence of mother-to-child transmission were clearly evident. In conclusion, the superior discriminatory capacity of this MLST scheme for S. mutans may have important practical implications.

Paenibacillus nebraskensis sp. nov., isolated from the root surface of field-grown maize.

PubMed

Kämpfer, Peter; Busse, Hans-Jürgen; McInroy, John A; Hu, Chia-Hui; Kloepper, Joseph W; Glaeser, Stefanie P

2017-12-01

A Gram-positive-staining, aerobic, non-endospore-forming bacterial strain (JJ-59 T ), isolated from a field-grown maize plant in Dunbar, Nebraska in 2014 was studied by a polyphasic approach. Based on 16S rRNA gene sequence similarity comparisons, strain JJ-59 T was shown to be a member of the genus Paenibacillus, most closely related to the type strains of Paenibacillus aceris (98.6 % 16S rRNA gene sequence similarity) and Paenibacillus chondroitinus (97.8 %). For all other type strains of species of the genus Paenibacillus lower 16S rRNA gene sequence similarities were obtained. DNA-DNA hybridization values of strain JJ-59 T to the type strains of P. aceris and P. chondroitinus were 26 % (reciprocal, 59 %) and 52 % (reciprocal, 59 %), respectively. Chemotaxonomic characteristics such as the presence of meso-diaminopimelic acid in the peptidoglycan, the major quinone MK-7 and spermidine as the major polyamine were in agreement with the characteristics of the genus Paenibacillus. Strain JJ-59 T shared with its next related species P. aceris the major lipids diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified aminophospholipid, but the presence/absence of certain lipids was clearly distinguishable. Major fatty acids of strain JJ-59 T were anteiso-C15 : 0, iso-C15 : 0 and iso-C16 : 0, and the genomic G+C content is 47.2 mol%. Physiological and biochemical characteristics of strain JJ-59 T were clearly different from the most closely related species of the genus Paenibacillus. Thus, strain JJ-59 T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus nebraskensis sp. nov. is proposed, with JJ-59 T (=DSM 103623 T =CIP 111179 T =LMG 29764 T ) as the type strain.

Single-molecule protein sequencing through fingerprinting: computational assessment

NASA Astrophysics Data System (ADS)

Yao, Yao; Docter, Margreet; van Ginkel, Jetty; de Ridder, Dick; Joo, Chirlmin

2015-10-01

Proteins are vital in all biological systems as they constitute the main structural and functional components of cells. Recent advances in mass spectrometry have brought the promise of complete proteomics by helping draft the human proteome. Yet, this commonly used protein sequencing technique has fundamental limitations in sensitivity. Here we propose a method for single-molecule (SM) protein sequencing. A major challenge lies in the fact that proteins are composed of 20 different amino acids, which demands 20 molecular reporters. We computationally demonstrate that it suffices to measure only two types of amino acids to identify proteins and suggest an experimental scheme using SM fluorescence. When achieved, this highly sensitive approach will result in a paradigm shift in proteomics, with major impact in the biological and medical sciences.

A glow of HLA typing in organ transplantation

PubMed Central

2013-01-01

The transplant of organs and tissues is one of the greatest curative achievements of this century. In organ transplantation, the adaptive immunity is considered the main response exerted to the transplanted tissue, since the main goal of the immune response is the MHC (major histocompatibility complex) molecules expressed on the surface of donor cells. Cell surface molecules that induce an antigenic stimulus cause the rejection immune response to grafted tissue or organ. A wide variety of transplantation antigens have been described, including the major histocompatibility molecules, minor histocompatibility antigens, ABO blood group antigens and endothelial cell antigens. The sensitization to MHC antigens may be caused by transfusions, pregnancy, or failed previous grafts leading to development of anti-human leukocyte antigen (HLA) antibodies that are important factor responsible for graft rejection in solid organ transplantation and play a role in post-transfusion complication Anti-HLA Abs may be present in healthy individuals. Methods for HLA typing are described, including serological methods, molecular techniques of sequence-specific priming (SSP), sequence-specific oligonucleotide probing (SSOP), Sequence based typing (SBT) and reference strand-based conformation analysis (RSCA) method. Problems with organ transplantation are reservoir of organs and immune suppressive treatments that used to decrease rate of rejection with less side effect and complications. PMID:23432791

New insights into Trypanosoma cruzi evolution, genotyping and molecular diagnostics from satellite DNA sequence analysis.

PubMed

Ramírez, Juan C; Torres, Carolina; Curto, María de Los A; Schijman, Alejandro G

2017-12-01

Trypanosoma cruzi has been subdivided into seven Discrete Typing Units (DTUs), TcI-TcVI and Tcbat. Two major evolutionary models have been proposed to explain the origin of hybrid lineages, but while it is widely accepted that TcV and TcVI are the result of genetic exchange between TcII and TcIII strains, the origin of TcIII and TcIV is still a matter of debate. T. cruzi satellite DNA (SatDNA), comprised of 195 bp units organized in tandem repeats, from both TcV and TcVI stocks were found to have SatDNA copies type TcI and TcII; whereas contradictory results were observed for TcIII stocks and no TcIV sequence has been analyzed yet. Herein, we have gone deeper into this matter analyzing 335 distinct SatDNA sequences from 19 T. cruzi stocks representative of DTUs TcI-TcVI for phylogenetic inference. Bayesian phylogenetic tree showed that all sequences were grouped in three major clusters, which corresponded to sequences from DTUs TcI/III, TcII and TcIV; whereas TcV and TcVI stocks had two sets of sequences distributed into TcI/III and TcII clusters. As expected, the lowest genetic distances were found between TcI and TcIII, and between TcV and TcVI sequences; whereas the highest ones were observed between TcII and TcI/III, and among TcIV sequences and those from the remaining DTUs. In addition, signature patterns associated to specific T. cruzi lineages were identified and new primers that improved SatDNA-based qPCR sensitivity were designed. Our findings support the theory that TcIII is not the result of a hybridization event between TcI and TcII, and that TcIV had an independent origin from the other DTUs, contributing to clarifying the evolutionary history of T. cruzi lineages. Moreover, this work opens the possibility of typing samples from Chagas disease patients with low parasitic loads and improving molecular diagnostic methods of T. cruzi infection based on SatDNA sequence amplification.

Temporal Changes in BEXSERO® Antigen Sequence Type Associated with Genetic Lineages of Neisseria meningitidis over a 15-Year Period in Western Australia

PubMed Central

Mowlaboccus, Shakeel; Perkins, Timothy T.; Smith, Helen; Sloots, Theo; Tozer, Sarah; Prempeh, Lydia-Jessica; Tay, Chin Yen; Peters, Fanny; Speers, David; Keil, Anthony D.; Kahler, Charlene M.

2016-01-01

Neisseria meningitidis is the causative agent of invasive meningococcal disease (IMD). The BEXSERO® vaccine which is used to prevent serogroup B disease is composed of four sub-capsular protein antigens supplemented with an outer membrane vesicle. Since the sub-capsular protein antigens are variably expressed and antigenically variable amongst meningococcal isolates, vaccine coverage can be estimated by the meningococcal antigen typing system (MATS) which measures the propensity of the strain to be killed by vaccinated sera. Whole genome sequencing (WGS) which identifies the alleles of the antigens that may be recognised by the antibody response could represent, in future, an alternative estimate of coverage. In this study, WGS of 278 meningococcal isolates responsible for 62% of IMD in Western Australia from 2000–2014 were analysed for association of genetic lineage (sequence type [ST], clonal complex [cc]) with BEXSERO® antigen sequence type (BAST) and MATS to predict the annual vaccine coverage. A hyper-endemic period of IMD between 2000–05 was caused by cc41/44 with the major sequence type of ST-146 which was not predicted by MATS or BAST to be covered by the vaccine. An increase in serogroup diversity was observed between 2010–14 with the emergence of cc11 serogroup W in the adolescent population and cc23 serogroup Y in the elderly. BASTs were statistically associated with clonal complex although individual antigens underwent antigenic drift from the major type. BAST and MATS predicted an annual range of 44–91% vaccine coverage. Periods of low vaccine coverage in years post-2005 were not a result of the resurgence of cc41/44:ST-146 but were characterised by increased diversity of clonal complexes expressing BASTs which were not predicted by MATS to be covered by the vaccine. The driving force behind the diversity of the clonal complex and BAST during these periods of low vaccine coverage is unknown, but could be due to immune selection and inter-strain competition with carriage of non-disease causing meningococci. PMID:27355628

Did A Planet Survive A Post-Main Sequence Evolutionary Event?

NASA Astrophysics Data System (ADS)

Sorber, Rebecca; Jang-Condell, Hannah; Zimmerman, Mara

2018-06-01

The GL86 is star system approximately 10 pc away with a main sequence K- type ~ 0.77 M⊙ star (GL 86A) with a white dwarf ~0.49 M⊙ companion (GL86 B). The system has a ~ 18.4 AU semi-major axis, an orbital period of ~353 yrs, and an eccentricity of ~ 0.39. A 4.5 MJ planet orbits the main sequence star with a semi-major axis of 0.113 AU, an orbital period of 15.76 days, in a near circular orbit with an eccentricity of 0.046. If we assume that this planet was formed during the time when the white dwarf was a main sequence star, it would be difficult for the planet to have remained in a stable orbit during the post-main sequence evolution of GL86 B. The post-main sequence evolution with planet survival will be examined by modeling using the program Mercury (Chambers 1999). Using the model, we examine the origins of the planet: whether it formed before or after the post-main sequence evolution of GL86B. The modeling will give us insight into the dynamical evolution of, not only, the binary star system, but also the planet’s life cycle.

Molecular determinants of the V3 loop of human immunodeficiency virus type 1 glycoprotein gp120 responsible for controlling cell tropism.

PubMed

Chavda, S C; Griffin, P; Han-Liu, Z; Keys, B; Vekony, M A; Cann, A J

1994-11-01

We and others have identified the major determinant of cell tropism in human immunodeficiency virus type 1 (HIV-1) as the V3 loop of glycoprotein gp120. We have conducted a detailed study of two molecularly cloned isolates of HIV-1, HIVJR-CSF and HIVNL4-3, that differ in their tropism for immortalized CD4+ cell lines, by constructing a series of site-directed mutations within the V3 loop of HIVJR-CSF based on the sequence of HIVNL4-3. The phenotypes of these mutants fall into two classes, those which are viable and those which are not. A spontaneous mutant with significantly altered growth properties was also recovered and found to have an additional single amino acid change in the V3 loop sequence. The carboxy-terminal beta-strand part of the V3 loop is the major determinant of cell tropism. However, the results presented here indicate that the functional role of the V3 loop sequences can only be interpreted properly in the context of the original gp120 backbone from which they were derived. These findings show that over-simplistic interpretation of sequence data derived from unknown mixtures of HIV variants in infected persons may be highly misleading.

Multilocus Sequence Typing of Bartonella henselae in the United Kingdom Indicates that Only a Few, Uncommon Sequence Types Are Associated with Zoonotic Disease▿†

PubMed Central

Chaloner, Gemma L.; Harrison, Timothy G.; Coyne, Karen P.; Aanensen, David M.; Birtles, Richard J.

2011-01-01

Bartonella henselae is one of the most common zoonotic agents acquired from companion animals (cats) in industrialized countries. Nonetheless, although the prevalence of infections in cats is high, the number of human cases reported is relatively low. One hypothesis for this discrepancy is that B. henselae strains vary in their zoonotic potential. To test this hypothesis, we employed structured sampling to explore the population structure of B. henselae in the United Kingdom and to determine the distribution of strains associated with zoonotic disease within this structure. A total of 118 B. henselae strains were delineated into 12 sequence types (STs) using multilocus sequence typing. We observed that most (85%) of the zoonosis-associated strains belonged to only three genotypes, i.e., ST2, ST5, and ST8. Conversely, most (74%) of the feline isolates belonged to ST4, ST6, and ST7. The difference in host association of ST2, ST5, and ST8 (zoonosis associated) and ST6 (feline) was statistically significant (P < 0.05), indicating that a few, uncommon STs were responsible for the majority of symptomatic human infections. PMID:21471345

Large-scale genomic analyses reveal the population structure and evolutionary trends of Streptococcus agalactiae strains in Brazilian fish farms.

PubMed

Barony, Gustavo M; Tavares, Guilherme C; Pereira, Felipe L; Carvalho, Alex F; Dorella, Fernanda A; Leal, Carlos A G; Figueiredo, Henrique C P

2017-10-19

Streptococcus agalactiae is a major pathogen and a hindrance on tilapia farming worldwide. The aims of this work were to analyze the genomic evolution of Brazilian strains of S. agalactiae and to establish spatial and temporal relations between strains isolated from different outbreaks of streptococcosis. A total of 39 strains were obtained from outbreaks and their whole genomes were sequenced and annotated for comparative analysis of multilocus sequence typing, genomic similarity and whole genome multilocus sequence typing (wgMLST). The Brazilian strains presented two sequence types, including a newly described ST, and a non-typeable lineage. The use of wgMLST could differentiate each strain in a single clone and was used to establish temporal and geographical correlations among strains. Bayesian phylogenomic analysis suggests that the studied Brazilian population was co-introduced in the country with their host, approximately 60 years ago. Brazilian strains of S. agalactiae were shown to be heterogeneous in their genome sequences and were distributed in different regions of the country according to their genotype, which allowed the use of wgMLST analysis to track each outbreak event individually.

Circulation of Endemic Type 2 Vaccine-Derived Poliovirus in Egypt from 1983 to 1993

PubMed Central

Yang, Chen-Fu; Naguib, Tary; Yang, Su-Ju; Nasr, Eman; Jorba, Jaume; Ahmed, Nahed; Campagnoli, Ray; van der Avoort, Harrie; Shimizu, Hiroyuki; Yoneyama, Tetsuo; Miyamura, Tatsuo; Pallansch, Mark; Kew, Olen

2003-01-01

From 1988 to 1993, 30 cases of poliomyelitis associated with poliovirus type 2 were found in seven governorates of Egypt. Because many of the cases were geographically and temporally clustered and because the case isolates differed antigenically from the vaccine strain, it was initially assumed that the cases signaled the continued circulation of wild type 2 poliovirus. However, comparison of sequences encoding the major capsid protein, VP1 (903 nucleotides), revealed that the isolates were related (93 to 97% nucleotide sequence identity) to the Sabin type 2 oral poliovirus vaccine (OPV) strain and unrelated (<82% nucleotide sequence identity) to the wild type 2 polioviruses previously indigenous to Egypt (last known isolate: 1979) or to any contemporary wild type 2 polioviruses found elsewhere. The rate and pattern of VP1 divergence among the circulating vaccine-derived poliovirus (cVDPV) isolates suggested that all lineages were derived from a single OPV infection that occurred around 1983 and that progeny from the initiating infection circulated for approximately a decade within Egypt along several independent chains of transmission. Complete genomic sequences of an early (1988) and a late (1993) cVDPV isolate revealed that their 5′ untranslated region (5′ UTR) and noncapsid- 3′ UTR sequences were derived from other species C enteroviruses. Circulation of type 2 cVDPVs occurred at a time of low OPV coverage in the affected communities and ceased when OPV coverage rates increased. The potential for cVDPVs to circulate in populations with low immunity to poliovirus has important implications for current and future strategies to eradicate polio worldwide. PMID:12857906

Circulation of endemic type 2 vaccine-derived poliovirus in Egypt from 1983 to 1993.

PubMed

Yang, Chen-Fu; Naguib, Tary; Yang, Su-Ju; Nasr, Eman; Jorba, Jaume; Ahmed, Nahed; Campagnoli, Ray; van der Avoort, Harrie; Shimizu, Hiroyuki; Yoneyama, Tetsuo; Miyamura, Tatsuo; Pallansch, Mark; Kew, Olen

2003-08-01

From 1988 to 1993, 30 cases of poliomyelitis associated with poliovirus type 2 were found in seven governorates of Egypt. Because many of the cases were geographically and temporally clustered and because the case isolates differed antigenically from the vaccine strain, it was initially assumed that the cases signaled the continued circulation of wild type 2 poliovirus. However, comparison of sequences encoding the major capsid protein, VP1 (903 nucleotides), revealed that the isolates were related (93 to 97% nucleotide sequence identity) to the Sabin type 2 oral poliovirus vaccine (OPV) strain and unrelated (<82% nucleotide sequence identity) to the wild type 2 polioviruses previously indigenous to Egypt (last known isolate: 1979) or to any contemporary wild type 2 polioviruses found elsewhere. The rate and pattern of VP1 divergence among the circulating vaccine-derived poliovirus (cVDPV) isolates suggested that all lineages were derived from a single OPV infection that occurred around 1983 and that progeny from the initiating infection circulated for approximately a decade within Egypt along several independent chains of transmission. Complete genomic sequences of an early (1988) and a late (1993) cVDPV isolate revealed that their 5' untranslated region (5' UTR) and noncapsid- 3' UTR sequences were derived from other species C enteroviruses. Circulation of type 2 cVDPVs occurred at a time of low OPV coverage in the affected communities and ceased when OPV coverage rates increased. The potential for cVDPVs to circulate in populations with low immunity to poliovirus has important implications for current and future strategies to eradicate polio worldwide.

Are commercial providers a viable option for clinical bacterial sequencing?

PubMed

Raven, Kathy; Blane, Beth; Churcher, Carol; Parkhill, Julian; Peacock, Sharon J

2018-04-05

Bacterial whole-genome sequencing in the clinical setting has the potential to bring major improvements to infection control and clinical practice. Sequencing instruments are not currently available in the majority of routine microbiology laboratories worldwide, but an alternative is to use external sequencing providers. To foster discussion around this we investigated whether send-out services were a viable option. Four providers offering MiSeq sequencing were selected based on cost and evaluated based on the service provided and sequence data quality. DNA was prepared from five methicillin-resistant Staphylococcus aureus (MRSA) isolates, four of which were investigated during a previously published outbreak in the UK together with a reference MRSA isolate (ST22 HO 5096 0412). Cost of sequencing per isolate ranged from £155 to £342 and turnaround times from DNA postage to arrival of sequence data ranged from 12 to 63 days. Comparison of commercially generated genomes against the original sequence data demonstrated very high concordance, with no more than one single nucleotide polymorphism (SNP) difference on core genome mapping between the original sequences and the new sequence for all four providers. Multilocus sequence type could not be assigned based on assembly for the two cheapest sequence providers due to fragmented assemblies probably caused by a lower output of sequence data per isolate. Our results indicate that external providers returned highly accurate genome data, but that improvements are required in turnaround time to make this a viable option for use in clinical practice.

Diversity Analysis of Dairy and Nondairy Lactococcus lactis Isolates, Using a Novel Multilocus Sequence Analysis Scheme and (GTG)5-PCR Fingerprinting▿

PubMed Central

Rademaker, Jan L. W.; Herbet, Hélène; Starrenburg, Marjo J. C.; Naser, Sabri M.; Gevers, Dirk; Kelly, William J.; Hugenholtz, Jeroen; Swings, Jean; van Hylckama Vlieg, Johan E. T.

2007-01-01

The diversity of a collection of 102 lactococcus isolates including 91 Lactococcus lactis isolates of dairy and nondairy origin was explored using partial small subunit rRNA gene sequence analysis and limited phenotypic analyses. A subset of 89 strains of L. lactis subsp. cremoris and L. lactis subsp. lactis isolates was further analyzed by (GTG)5-PCR fingerprinting and a novel multilocus sequence analysis (MLSA) scheme. Two major genomic lineages within L. lactis were found. The L. lactis subsp. cremoris type-strain-like genotype lineage included both L. lactis subsp. cremoris and L. lactis subsp. lactis isolates. The other major lineage, with a L. lactis subsp. lactis type-strain-like genotype, comprised L. lactis subsp. lactis isolates only. A novel third genomic lineage represented two L. lactis subsp. lactis isolates of nondairy origin. The genomic lineages deviate from the subspecific classification of L. lactis that is based on a few phenotypic traits only. MLSA of six partial genes (atpA, encoding ATP synthase alpha subunit; pheS, encoding phenylalanine tRNA synthetase; rpoA, encoding RNA polymerase alpha chain; bcaT, encoding branched chain amino acid aminotransferase; pepN, encoding aminopeptidase N; and pepX, encoding X-prolyl dipeptidyl peptidase) revealed 363 polymorphic sites (total length, 1,970 bases) among 89 L. lactis subsp. cremoris and L. lactis subsp. lactis isolates with unique sequence types for most isolates. This allowed high-resolution cluster analysis in which dairy isolates form subclusters of limited diversity within the genomic lineages. The pheS DNA sequence analysis yielded two genetic groups dissimilar to the other genotyping analysis-based lineages, indicating a disparate acquisition route for this gene. PMID:17890345

Diversity analysis of dairy and nondairy Lactococcus lactis isolates, using a novel multilocus sequence analysis scheme and (GTG)5-PCR fingerprinting.

PubMed

Rademaker, Jan L W; Herbet, Hélène; Starrenburg, Marjo J C; Naser, Sabri M; Gevers, Dirk; Kelly, William J; Hugenholtz, Jeroen; Swings, Jean; van Hylckama Vlieg, Johan E T

2007-11-01

The diversity of a collection of 102 lactococcus isolates including 91 Lactococcus lactis isolates of dairy and nondairy origin was explored using partial small subunit rRNA gene sequence analysis and limited phenotypic analyses. A subset of 89 strains of L. lactis subsp. cremoris and L. lactis subsp. lactis isolates was further analyzed by (GTG)(5)-PCR fingerprinting and a novel multilocus sequence analysis (MLSA) scheme. Two major genomic lineages within L. lactis were found. The L. lactis subsp. cremoris type-strain-like genotype lineage included both L. lactis subsp. cremoris and L. lactis subsp. lactis isolates. The other major lineage, with a L. lactis subsp. lactis type-strain-like genotype, comprised L. lactis subsp. lactis isolates only. A novel third genomic lineage represented two L. lactis subsp. lactis isolates of nondairy origin. The genomic lineages deviate from the subspecific classification of L. lactis that is based on a few phenotypic traits only. MLSA of six partial genes (atpA, encoding ATP synthase alpha subunit; pheS, encoding phenylalanine tRNA synthetase; rpoA, encoding RNA polymerase alpha chain; bcaT, encoding branched chain amino acid aminotransferase; pepN, encoding aminopeptidase N; and pepX, encoding X-prolyl dipeptidyl peptidase) revealed 363 polymorphic sites (total length, 1,970 bases) among 89 L. lactis subsp. cremoris and L. lactis subsp. lactis isolates with unique sequence types for most isolates. This allowed high-resolution cluster analysis in which dairy isolates form subclusters of limited diversity within the genomic lineages. The pheS DNA sequence analysis yielded two genetic groups dissimilar to the other genotyping analysis-based lineages, indicating a disparate acquisition route for this gene.

Distilled single-cell genome sequencing and de novo assembly for sparse microbial communities.

PubMed

Taghavi, Zeinab; Movahedi, Narjes S; Draghici, Sorin; Chitsaz, Hamidreza

2013-10-01

Identification of every single genome present in a microbial sample is an important and challenging task with crucial applications. It is challenging because there are typically millions of cells in a microbial sample, the vast majority of which elude cultivation. The most accurate method to date is exhaustive single-cell sequencing using multiple displacement amplification, which is simply intractable for a large number of cells. However, there is hope for breaking this barrier, as the number of different cell types with distinct genome sequences is usually much smaller than the number of cells. Here, we present a novel divide and conquer method to sequence and de novo assemble all distinct genomes present in a microbial sample with a sequencing cost and computational complexity proportional to the number of genome types, rather than the number of cells. The method is implemented in a tool called Squeezambler. We evaluated Squeezambler on simulated data. The proposed divide and conquer method successfully reduces the cost of sequencing in comparison with the naïve exhaustive approach. Squeezambler and datasets are available at http://compbio.cs.wayne.edu/software/squeezambler/.

Comparison of Three Different Hepatitis C Virus Genotyping Methods: 5'NCR PCR-RFLP, Core Type-Specific PCR, and NS5b Sequencing in a Tertiary Care Hospital in South India.

PubMed

Daniel, Hubert D-J; David, Joel; Raghuraman, Sukanya; Gnanamony, Manu; Chandy, George M; Sridharan, Gopalan; Abraham, Priya

2017-05-01

Based on genetic heterogeneity, hepatitis C virus (HCV) is classified into seven major genotypes and 64 subtypes. In spite of the sequence heterogeneity, all genotypes share an identical complement of colinear genes within the large open reading frame. The genetic interrelationships between these genes are consistent among genotypes. Due to this property, complete sequencing of the HCV genome is not required. HCV genotypes along with subtypes are critical for planning antiviral therapy. Certain genotypes are also associated with higher progression to liver cirrhosis. In this study, 100 blood samples were collected from individuals who came for routine HCV genotype identification. These samples were used for the comparison of two different genotyping methods (5'NCR PCR-RFLP and HCV core type-specific PCR) with NS5b sequencing. Of the 100 samples genotyped using 5'NCR PCR-RFLP and HCV core type-specific PCR, 90% (κ = 0.913, P < 0.00) and 96% (κ = 0.794, P < 0.00) correlated with NS5b sequencing, respectively. Sixty percent and 75% of discordant samples by 5'NCR PCR-RFLP and HCV core type-specific PCR, respectively, belonged to genotype 6. All the HCV genotype 1 subtypes were classified accurately by both the methods. This study shows that the 5'NCR-based PCR-RFLP and the HCV core type-specific PCR-based assays correctly identified HCV genotypes except genotype 6 from this region. Direct sequencing of the HCV core region was able to identify all the genotype 6 from this region and serves as an alternative to NS5b sequencing. © 2016 Wiley Periodicals, Inc.

Molecular evolution of miraculin-like proteins in soybean Kunitz super-family.

PubMed

Selvakumar, Purushotham; Gahloth, Deepankar; Tomar, Prabhat Pratap Singh; Sharma, Nidhi; Sharma, Ashwani Kumar

2011-12-01

Miraculin-like proteins (MLPs) belong to soybean Kunitz super-family and have been characterized from many plant families like Rutaceae, Solanaceae, Rubiaceae, etc. Many of them possess trypsin inhibitory activity and are involved in plant defense. MLPs exhibit significant sequence identity (~30-95%) to native miraculin protein, also belonging to Kunitz super-family compared with a typical Kunitz family member (~30%). The sequence and structure-function comparison of MLPs with that of a classical Kunitz inhibitor have demonstrated that MLPs have evolved to form a distinct group within Kunitz super-family. Sequence analysis of new genes along with available MLP sequences in the literature revealed three major groups for these proteins. A significant feature of Rutaceae MLP type 2 sequences is the presence of phosphorylation motif. Subtle changes are seen in putative reactive loop residues among different MLPs suggesting altered specificities to specific proteases. In phylogenetic analysis, Rutaceae MLP type 1 and type 2 proteins clustered together on separate branches, whereas native miraculin along with other MLPs formed distinct clusters. Site-specific positive Darwinian selection was observed at many sites in both the groups of Rutaceae MLP sequences with most of the residues undergoing positive selection located in loop regions. The results demonstrate the sequence and thereby the structure-function divergence of MLPs as a distinct group within soybean Kunitz super-family due to biotic and abiotic stresses of local environment.

Molecular Detection, Isolation, and Physiological Characterization of Functionally Dominant Phenol-Degrading Bacteria in Activated Sludge

PubMed Central

Watanabe, Kazuya; Teramoto, Maki; Futamata, Hiroyuki; Harayama, Shigeaki

1998-01-01

DNA was isolated from phenol-digesting activated sludge, and partial fragments of the 16S ribosomal DNA (rDNA) and the gene encoding the largest subunit of multicomponent phenol hydroxylase (LmPH) were amplified by PCR. An analysis of the amplified fragments by temperature gradient gel electrophoresis (TGGE) demonstrated that two major 16S rDNA bands (bands R2 and R3) and two major LmPH gene bands (bands P2 and P3) appeared after the activated sludge became acclimated to phenol. The nucleotide sequences of these major bands were determined. In parallel, bacteria were isolated from the activated sludge by direct plating or by plating after enrichment either in batch cultures or in a chemostat culture. The bacteria isolated were classified into 27 distinct groups by a repetitive extragenic palindromic sequence PCR analysis. The partial nucleotide sequences of 16S rDNAs and LmPH genes of members of these 27 groups were then determined. A comparison of these nucleotide sequences with the sequences of the major TGGE bands indicated that the major bacterial populations, R2 and R3, possessed major LmPH genes P2 and P3, respectively. The dominant populations could be isolated either by direct plating or by chemostat culture enrichment but not by batch culture enrichment. One of the dominant strains (R3) which contained a novel type of LmPH (P3), was closely related to Valivorax paradoxus, and the result of a kinetic analysis of its phenol-oxygenating activity suggested that this strain was the principal phenol digester in the activated sludge. PMID:9797297

Duplication and concerted evolution of MiSp-encoding genes underlie the material properties of minor ampullate silks of cobweb weaving spiders.

PubMed

Vienneau-Hathaway, Jannelle M; Brassfield, Elizabeth R; Lane, Amanda Kelly; Collin, Matthew A; Correa-Garhwal, Sandra M; Clarke, Thomas H; Schwager, Evelyn E; Garb, Jessica E; Hayashi, Cheryl Y; Ayoub, Nadia A

2017-03-14

Orb-web weaving spiders and their relatives use multiple types of task-specific silks. The majority of spider silk studies have focused on the ultra-tough dragline silk synthesized in major ampullate glands, but other silk types have impressive material properties. For instance, minor ampullate silks of orb-web weaving spiders are as tough as draglines, due to their higher extensibility despite lower strength. Differences in material properties between silk types result from differences in their component proteins, particularly members of the spidroin (spider fibroin) gene family. However, the extent to which variation in material properties within a single silk type can be explained by variation in spidroin sequences is unknown. Here, we compare the minor ampullate spidroins (MiSp) of orb-weavers and cobweb weavers. Orb-web weavers use minor ampullate silk to form the auxiliary spiral of the orb-web while cobweb weavers use it to wrap prey, suggesting that selection pressures on minor ampullate spidroins (MiSp) may differ between the two groups. We report complete or nearly complete MiSp sequences from five cobweb weaving spider species and measure material properties of minor ampullate silks in a subset of these species. We also compare MiSp sequences and silk properties of our cobweb weavers to published data for orb-web weavers. We demonstrate that all our cobweb weavers possess multiple MiSp loci and that one locus is more highly expressed in at least two species. We also find that the proportion of β-spiral-forming amino acid motifs in MiSp positively correlates with minor ampullate silk extensibility across orb-web and cobweb weavers. MiSp sequences vary dramatically within and among spider species, and have likely been subject to multiple rounds of gene duplication and concerted evolution, which have contributed to the diverse material properties of minor ampullate silks. Our sequences also provide templates for recombinant silk proteins with tailored properties.

Nesterenkonia cremea sp. nov., a bacterium isolated from a soda lake.

PubMed

Sultanpuram, Vishnuvardhan Reddy; Mothe, Thirumala; Chintalapati, Sasikala; Chintalapati, Venkata Ramana

2017-06-01

A Gram-stain-positive, aerobic, non-motile, rod-shaped, non-endospore-forming bacterial strain, 10CT, was isolated from Lonar soda lake in India. Based on the 16S rRNA gene sequence analysis, this strain was identified as belonging to the genus Nesterenkonia and was most closely related to the type strains of Nesterenkonia lacusekhoensis (99.1 %, sequence similarity), Nesterenkonia aethiopica (96.9 %), Nesterenkonia flava (96.9 %) and related of the genus Nesterenkonia (<96.6 %, sequence similarity). However, the DNA-DNA relatedness of strain 10CT with N. lacusekhoensis KCTC 19283T was only 34.6±0.9. The DNA G+C content of strain 10CT was 68.6 mol%. Strain 10CT was an aerobic microbe with optimal growth at 37 °C, pH 7.5-8.0 and 5-6 % (w/v) NaCl. The cell-wall peptidoglycan of strain 10CT was of the type A4α (l-Lys-l-Glu). The major polar lipids present were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylcholine. The major isoprenoid quinones were MK-7, MK-8 and MK-9. Major fatty acids of strain 10CT were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The results of phylogenetic, chemotaxonomic and biochemical tests allowed a clear differentiation of strain 10CT, which represents a novel member of the genus Nesterenkonia for which the name Nesterenkonia cremea sp. nov. is proposed. The type strain is 10CT (=LMG 29100T=KCTC 39636T=CGMCC 1.15388T).

Development of an ELA-DRA gene typing method based on pyrosequencing technology.

PubMed

Díaz, S; Echeverría, M G; It, V; Posik, D M; Rogberg-Muñoz, A; Pena, N L; Peral-García, P; Vega-Pla, J L; Giovambattista, G

2008-11-01

The polymorphism of equine lymphocyte antigen (ELA) class II DRA gene had been detected by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) and reference strand-mediated conformation analysis. These methodologies allowed to identify 11 ELA-DRA exon 2 sequences, three of which are widely distributed among domestic horse breeds. Herein, we describe the development of a pyrosequencing-based method applicable to ELA-DRA typing, by screening samples from eight different horse breeds previously typed by PCR-SSCP. This sequence-based method would be useful in high-throughput genotyping of major histocompatibility complex genes in horses and other animal species, making this system interesting as a rapid screening method for animal genotyping of immune-related genes.

Human Babesiosis in Japan: Epizootiologic Survey of Rodent Reservoir and Isolation of New Type of Babesia microti-Like Parasite

PubMed Central

Tsuji, Masayoshi; Wei, Qiang; Zamoto, Aya; Morita, Chiharu; Arai, Satoru; Shiota, Tsunezo; Fujimagari, Masato; Itagaki, Asao; Fujita, Hiromi; Ishihara, Chiaki

2001-01-01

We have carried out epizootiologic surveys at various sites in Japan to investigate wild animals that serve as reservoirs for the agents of human babesiosis in the country. Small mammals comprising six species, Apodemus speciosus, Apodemus argenteus, Clethrionomys rufocanus, Eothenomys smithii, Crocidura dsinezumi, and Sorex unguiculatus, were trapped at various places, including Hokkaido, Chiba, Shiga, Hyogo, Shimane, and Tokushima Prefectures. Animals harboring Babesia microti-like parasites were detected in all six prefectures. Inoculation of their blood samples into hamsters gave rise to a total of 20 parasite isolates; 19 were from A. speciosus, and the other 1 was from C. rufocanus. Sequencing of the parasite small-subunit rRNA gene (rDNA) sequence revealed that 2 of the 20 isolates were classified as Kobe type because their rDNAs were identical to that of the Kobe strain (the strain from the Japanese index case). The other 18 isolates were classified as a new type, designated the Hobetsu type, because they all shared an identical rDNA sequence which differed significantly from both that of Kobe-type isolates and that of northeastern United States B. microti (U.S. type). The parasites with Kobe-, Hobetsu- and U.S.-type rDNAs were phylogenetically closely related to each other but clearly different from each other antigenically. The isolates from rodents were demonstrated to be infective for human erythrocytes by inoculation into SCID mice whose erythrocytes had been replaced with human erythrocytes. The results suggest that a new type of B. microti-like parasite, namely, the Hobetsu type, is the major one which is prevalent among Japanese wild rodents, that A. speciosus serves as a major reservoir for both Kobe- and Hobetsu-type B. microti-like parasites, and that C. rufocanus may also be an additional reservoir on Hokkaido Island. PMID:11724838

A survey of human brain transcriptome diversity at the single cell level.

PubMed

Darmanis, Spyros; Sloan, Steven A; Zhang, Ye; Enge, Martin; Caneda, Christine; Shuer, Lawrence M; Hayden Gephart, Melanie G; Barres, Ben A; Quake, Stephen R

2015-06-09

The human brain is a tissue of vast complexity in terms of the cell types it comprises. Conventional approaches to classifying cell types in the human brain at single cell resolution have been limited to exploring relatively few markers and therefore have provided a limited molecular characterization of any given cell type. We used single cell RNA sequencing on 466 cells to capture the cellular complexity of the adult and fetal human brain at a whole transcriptome level. Healthy adult temporal lobe tissue was obtained during surgical procedures where otherwise normal tissue was removed to gain access to deeper hippocampal pathology in patients with medical refractory seizures. We were able to classify individual cells into all of the major neuronal, glial, and vascular cell types in the brain. We were able to divide neurons into individual communities and show that these communities preserve the categorization of interneuron subtypes that is typically observed with the use of classic interneuron markers. We then used single cell RNA sequencing on fetal human cortical neurons to identify genes that are differentially expressed between fetal and adult neurons and those genes that display an expression gradient that reflects the transition between replicating and quiescent fetal neuronal populations. Finally, we observed the expression of major histocompatibility complex type I genes in a subset of adult neurons, but not fetal neurons. The work presented here demonstrates the applicability of single cell RNA sequencing on the study of the adult human brain and constitutes a first step toward a comprehensive cellular atlas of the human brain.

Draft genome sequence of CTX-M-type β-lactamase-producing Klebsiella quasipneumoniae subsp. similipneumoniae isolated from a Box turtle.

PubMed

Li, Chien-Feng; Tang, Hui-Ling; Chiou, Chien-Shun; Tung, Kwong-Chung; Lu, Min-Chi; Lai, Yi-Chyi

2018-03-01

Klebsiella spp. are regarded as major pathogens causing infections in humans and various animals. Here we report the draft genome sequence of a CTX-M-type β-lactamase-producing Klebsiella quasipneumoniae subsp. similipneumoniae strain CHKP0062 isolated from a Yellow-margined Box turtle. An Illumina-Solexa platform was used to sequence the genome of CHKP0062. Qualified reads were assembled de novo using Velvet. The draft genome was annotated by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The resistome and virulome of the strain were investigated. A total of 5423 protein-coding sequences, 87 tRNAs, 24 rRNAs and 12 ncRNAs were identified in the 5 699 275-bp genome. CHKP0062 was assigned to sequence type ST2131 with the K-loci type as KL67. No virulence-associated genes were identified. However, numerous antimicrobial resistance genes were present in this strain. Plasmid contigs were assembled and revealed homology to the multidrug resistance plasmids pC15-K, pCTX-M3 and pKF3-94, with the carriage of the class A β-lactamase genes bla TEM-1b and bla CTX-M-3 . The genome sequence reported in this study will be useful for comparative genomic analysis regarding the dissemination of clinically important antibiotic resistance genes among Klebsiella spp. isolated from humans and animals. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

Authentication and chemical study of isodonis herba and isodonis extracts.

PubMed

Maruyama, Takuro; Sugimoto, Naoki; Kuroyanagi, Masanori; Kim, Ik Hwi; Kamakura, Hiroyuki; Kawasaki, Takeshi; Fujita, Masao; Shimada, Hiroshi; Yamamoto, Yutaka; Tada, Atsuko; Yamazaki, Takeshi; Goda, Yukihiro

2007-11-01

Isodonis Herba is used as a Japanese dietary supplement and folk medicine. The extract of the herb (Isodonis extract) is also used as a food additive whose major compound is enmein (1). Here we compared internal transcribed spacer sequences of nuclear ribosomal DNA from Isodonis Herba available on the Japanese and Chinese crude drug markets, and found that the former derived from Isodon japonicus and Isodon trichocarpus, while the latter derived from distinct species such as Isodon eriocalyx. The liquid chromatography/mass spectrometry profiles of Isodonis Herba were classified into four chemotypes (A to D) according to the ratio of the major constituents. Types B and C contained 1 and oridonin (2) as major components, respectively. An intermediate (or mixed) form of types B and C in various ratios was designed type A. Type D contained eriocalyxin B (3) as its major component. Japanese herba were types A-C, while Chinese herba were types C and D. The commercial Isodonis extract products tested were classified as type D, suggesting that they originated from Chinese Herba. Understanding the relationship between extract constituents and DNA profiles is important for the official specification of dietary supplements and food additives of plant origin.

Relative stability of major types of beta-turns as a function of amino acid composition: a study based on Ab initio energetic and natural abundance data.

PubMed

Perczel, András; Jákli, Imre; McAllister, Michael A; Csizmadia, Imre G

2003-06-06

Folding properties of small globular proteins are determined by their amino acid sequence (primary structure). This holds both for local (secondary structure) and for global conformational features of linear polypeptides and proteins composed from natural amino acid derivatives. It thus provides the rational basis of structure prediction algorithms. The shortest secondary structure element, the beta-turn, most typically adopts either a type I or a type II form, depending on the amino acid composition. Herein we investigate the sequence-dependent folding stability of both major types of beta-turns using simple dipeptide models (-Xxx-Yyy-). Gas-phase ab initio properties of 16 carefully selected and suitably protected dipeptide models (for example Val-Ser, Ala-Gly, Ser-Ser) were studied. For each backbone fold most probable side-chain conformers were considered. Fully optimized 321G RHF molecular structures were employed in medium level [B3LYP/6-311++G(d,p)//RHF/3-21G] energy calculations to estimate relative populations of the different backbone conformers. Our results show that the preference for beta-turn forms as calculated by quantum mechanics and observed in Xray determined proteins correlates significantly.

Combining one-step Sanger sequencing with phasing probe hybridization for HLA class I typing yields rapid, G-group resolution predicting 99% of unique full length protein sequences.

PubMed

Tu, Bin; Masaberg, Carly; Hou, Lihua; Behm, Daniel; Brescia, Peter; Cha, Nuri; Kariyawasam, Kanthi; Lee, Jar How; Nong, Thoa; Sells, John; Tausch, Paul; Yang, Ruyan; Ng, Jennifer; Hurley, Carolyn Katovich

2017-02-01

Sanger-based DNA sequencing of exons 2+3 of HLA class I alleles from a heterozygote frequently results in two or more alternative genotypes. This study was undertaken to reduce the time and effort required to produce a single high resolution HLA genotype. Samples were typed in parallel by Sanger sequencing and oligonucleotide probe hybridization. This workflow, together with optimization of analysis software, was tested and refined during the typing of over 42,000 volunteers for an unrelated hematopoietic progenitor cell donor registry. Next generation DNA sequencing (NGS) was applied to over 1000 of these samples to identify the alleles present within the G group designations. Single genotypes at G level resolution were obtained for over 95% of the loci without additional assays. The vast majority of alleles identified (>99%) were the primary allele giving the G groups their name. Only 0.7% of the alleles identified encoded protein variants that were not detected by a focus on the antigen recognition domain (ARD)-encoding exons. Our combined method routinely provides biologically relevant typing resolution at the level of the ARD. It can be applied to both single samples or to large volume typing supporting either bone marrow or solid organ transplantation using technologies currently available in many HLA laboratories. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Third-Generation-Cephalosporin-Resistant Klebsiella pneumoniae Isolates from Humans and Companion Animals in Switzerland: Spread of a DHA-Producing Sequence Type 11 Clone in a Veterinary Setting

PubMed Central

Wohlwend, Nadia; Francey, Thierry

2015-01-01

Characterization of third-generation-cephalosporin-resistant Klebsiella pneumoniae isolates originating mainly from one human hospital (n = 22) and one companion animal hospital (n = 25) in Bern (Switzerland) revealed the absence of epidemiological links between human and animal isolates. Human infections were not associated with the spread of any specific clone, while the majority of animal infections were due to K. pneumoniae sequence type 11 isolates producing plasmidic DHA AmpC. This clonal dissemination within the veterinary hospital emphasizes the need for effective infection control practices. PMID:25733505

A multi-omic future for microbiome studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jansson, Janet K.; Baker, Erin S.

2016-04-26

Microbes constitute about a third of the Earth’s biomass and play critical roles in sustaining life. While results from multiple sequence-based studies have illustrated the importance of microbial communities for human health and the environment, additional technological developments are still needed to gain more insight into their functions [1]. To date, the majority of sequencing studies have focused on the 16S rRNA gene as a phylogenetic marker. This approach has enabled exploration of microbial compositions in a range of sample types, while bypassing the need for cultivation. 16S rRNA gene sequencing has also enabled a vast majority of microorganisms nevermore » previously isolated in culture to be identified and placed into a phylogenetic context [2]. These technologies have been utilized to map the locations of microbes inhabiting various locations of the body [3]. Similarly, sequencing has been used to determine the identities and distributions of microorganisms inhabiting different ecosystems [4, 5], and efforts in single cell sequencing of the microbiome have helped fill in missing branches of the phylogenetic tree [6].« less

The complete DNA sequence of lymphocystis disease virus.

PubMed

Tidona, C A; Darai, G

1997-04-14

Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease, which has been reported to occur in over 100 different fish species worldwide. LCDV is a member of the family Iridoviridae and the type species of the genus Lymphocystivirus. The virions contain a single linear double-stranded DNA molecule, which is circularly permuted, terminally redundant, and heavily methylated at cytosines in CpG sequences. The complete nucleotide sequence of LCDV-1 (flounder isolate) was determined by automated cycle sequencing and primer walking. The genome of LCDV-1 is 102.653 bp in length and contains 195 open reading frames with coding capacities ranging from 40 to 1199 amino acids. Computer-assisted analyses of the deduced amino acid sequences led to the identification of several putative gene products with significant homologies to entries in protein data banks, such as the two major subunits of the viral DNA-dependent RNA polymerase, DNA polymerase, several protein kinases, two subunits of the ribonucleoside diphosphate reductase, DNA methyltransferase, the viral major capsid protein, insulin-like growth factor, and tumor necrosis factor receptor homolog.

Distinct Circular Single-Stranded DNA Viruses Exist in Different Soil Types

PubMed Central

Swanson, Maud M.; Dawson, Lorna; Freitag, Thomas E.; Singh, Brajesh K.; Torrance, Lesley; Mushegian, Arcady R.

2015-01-01

The potential dependence of virus populations on soil types was examined by electron microscopy, and the total abundance of virus particles in four soil types was similar to that previously observed in soil samples. The four soil types examined differed in the relative abundances of four morphological groups of viruses. Machair, a unique type of coastal soil in western Scotland and Ireland, differed from the others tested in having a higher proportion of tailed bacteriophages. The other soils examined contained predominantly spherical and thin filamentous virus particles, but the Machair soil had a more even distribution of the virus types. As the first step in looking at differences in populations in detail, virus sequences from Machair and brown earth (agricultural pasture) soils were examined by metagenomic sequencing after enriching for circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) virus genomes. Sequences from the family Microviridae (icosahedral viruses mainly infecting bacteria) of CRESS-DNA viruses were predominant in both soils. Phylogenetic analysis of Microviridae major coat protein sequences from the Machair viruses showed that they spanned most of the diversity of the subfamily Gokushovirinae, whose members mainly infect obligate intracellular parasites. The brown earth soil had a higher proportion of sequences that matched the morphologically similar family Circoviridae in BLAST searches. However, analysis of putative replicase proteins that were similar to those of viruses in the Circoviridae showed that they are a novel clade of Circoviridae-related CRESS-DNA viruses distinct from known Circoviridae genera. Different soils have substantially different taxonomic biodiversities even within ssDNA viruses, which may be driven by physicochemical factors. PMID:25841004

Completing the Remedial Sequence and College-Level Credit-Bearing Math: Comparing Binary, Cumulative, and Continuation Ratio Logistic Regression Models

ERIC Educational Resources Information Center

Davidson, J. Cody

2016-01-01

Mathematics is the most common subject area of remedial need and the majority of remedial math students never pass a college-level credit-bearing math class. The majorities of studies that investigate this phenomenon are conducted at community colleges and use some type of regression model; however, none have used a continuation ratio model. The…

Myelin protein zero gene sequencing diagnoses Charcot-Marie-Tooth Type 1B disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Y.; Zhang, H.; Madrid, R.

1994-09-01

Charcot-Marie-Tooth disease (CMT), the most common genetic neuropathy, affects about 1 in 2600 people in Norway and is found worldwide. CMT Type 1 (CMT1) has slow nerve conduction with demyelinated Schwann cells. Autosomal dominant CMT Type 1B (CMT1B) results from mutations in the myelin protein zero gene which directs the synthesis of more than half of all Schwann cell protein. This gene was mapped to the chromosome 1q22-1q23.1 borderline by fluorescence in situ hybridization. The first 7 of 7 reported CMT1B mutations are unique. Thus the most effective means to identify CMT1B mutations in at-risk family members and fetuses ismore » to sequence the entire coding sequence in dominant or sporadic CMT patients without the CMT1A duplication. Of the 19 primers used in 16 pars to uniquely amplify the entire MPZ coding sequence, 6 primer pairs were used to amplify and sequence the 6 exons. The DyeDeoxy Terminator cycle sequencing method used with four different color fluorescent lables was superior to manual sequencing because it sequences more bases unambiguously from extracted genomic DNA samples within 24 hours. This protocol was used to test 28 CMT and Dejerine-Sottas patients without CMT1A gene duplication. Sequencing MPZ gene-specific amplified fragments identified 9 polymorphic sites within the 6 exons that encode the 248 amino acid MPZ protein. The large number of major CMT1B mutations identified by single strand sequencing are being verified by reverse strand sequencing and when possible, by restriction enzyme analysis. This protocol can be used to distringuish CMT1B patients from othre CMT phenotypes and to determine the CMT1B status of relatives both presymptomatically and prenatally.« less

Emergence of new types of Theileria orientalis in Australian cattle and possible cause of theileriosis outbreaks

PubMed Central

2011-01-01

Theileria parasites cause a benign infection of cattle in parts of Australia where they are endemic, but have, in recent years, been suspected of being responsible for a number of outbreaks of disease in cattle near the coast of New South Wales. The objective of this study was to identify and characterize the species of Theileria in cattle on six farms in New South Wales where disease outbreaks have occurred, and compare with Theileria from three disease-free farms in Queensland that is endemic for Theileria. Special reference was made to sub-typing of T. orientalis by type-specific PCR and sequencing of the small subunit (SSU) rRNA gene, and sequence analysis of the gene encoding a polymorphic merozoite/piroplasm surface protein (MPSP) that may be under immune selection. Nucleotide sequencing of SSU rRNA and MPSP genes revealed the presence of four Theileria genotypes: T. orientalis (buffeli), T. orientalis (ikeda), T. orientalis (chitose) and T. orientalis type 4 (MPSP) or type C (SSU rRNA). The majority of animals showed mixed infections while a few showed single infection. When MPSP nucleotide sequences were translated into amino acids, base transition did not change amino acid composition of the protein product, suggesting possible silent polymorphism. The occurrence of ikeda and type 4 (type C) previously not reported to occur and silent mutation is thought to have enhanced parasite evasion of the host immune response causing the outbreak. PMID:21338493

A polyvalent hybrid protein elicits antibodies against the diverse allelic types of block 2 in Plasmodium falciparum merozoite surface protein 1.

PubMed

Tetteh, Kevin K A; Conway, David J

2011-10-13

Merozoite surface protein 1 (MSP1) of Plasmodium falciparum has been implicated as an important target of acquired immunity, and candidate components for a vaccine include polymorphic epitopes in the N-terminal polymorphic block 2 region. We designed a polyvalent hybrid recombinant protein incorporating sequences of the three major allelic types of block 2 together with a composite repeat sequence of one of the types and N-terminal flanking T cell epitopes, and compared this with a series of recombinant proteins containing modular sub-components and similarly expressed in Escherichia coli. Immunogenicity of the full polyvalent hybrid protein was tested in both mice and rabbits, and comparative immunogenicity studies of the sub-component modules were performed in mice. The full hybrid protein induced high titre antibodies against each of the major block 2 allelic types expressed as separate recombinant proteins and against a wide range of allelic types naturally expressed by a panel of diverse P. falciparum isolates, while the sub-component modules had partial antigenic coverage as expected. This encourages further development and evaluation of the full MSP1 block 2 polyvalent hybrid protein as a candidate blood-stage component of a malaria vaccine. Copyright © 2011 Elsevier Ltd. All rights reserved.

Changes in the population structure of canine methicillin-resistant Staphylococcus pseudintermedius in Poland.

PubMed

Kizerwetter-Świda, Magdalena; Chrobak-Chmiel, Dorota; Rzewuska, Magdalena; Binek, Marian

2017-09-01

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is being reported with an increasing frequency in small animal veterinary practice. The molecular typing of MRSP isolates revealed that the dominating European multidrug-resistant lineage is the sequence type 71 (ST71), associated with staphylococcal chromosomal cassette SCCmec type II-III. However, the recent reports indicated the emergence of other clones. The study aimed to determine the genetic properties of MRSP isolates obtained from dogs in Poland over a ten-year period. A total of 42 clinical MRSP isolates were subjected to multilocus-sequence typing (MLST) and SCCmec typing. MLST typing of 42 MRSP isolates yielded six STs belonging to two major clonal complexes (CCs): CC71 and CC551, associated with SCCmec element II-III and V, respectively. CC71 comprising ST71 and its newly described single locus variant (SLV) ST680. The second dominating CC551was represented by ST551 and newly described SLV ST771. The other, ST258 and ST85 were detected in single MRSP isolates. This is the first report concerning MLST typing of MRSP isolates in Poland. The results confirmed the domination of ST71 among MRSP until 2015, and the emergence of ST551 in 2015. Furthermore, in 2016 ST551 was identified in the majority of the strains, indicating the changes in the population structure of MRSP in Poland. Polish clinical MRSP isolates showed a shift in the population structure during the period of 2007 and 2016. The dominating MRSP lineage until 2015 was multidrug-resistant ST71-SCCmecII-III. The other lineage ST551-SCCmecV emerged in Poland since 2015, and in 2016 was found in the majority of MRSP isolates. Copyright © 2017 Elsevier B.V. All rights reserved.

The toxR Gene of Vibrio (Listonella) anguillarum Controls Expression of the Major Outer Membrane Proteins but Not Virulence in a Natural Host Model

PubMed Central

Okuda, Jun; Nakai, Toshihiro; Chang, Park Se; Oh, Takanori; Nishino, Takeshi; Koitabashi, Tsutomu; Nishibuchi, Mitsuaki

2001-01-01

To examine the hypothesis that the ancestral role of the toxR gene in the family Vibrionaceae is control of the expression of outer membrane protein (OMP)-encoding genes for adaptation to environmental change, we investigated the role of the toxR gene in Vibrio anguillarum, an important fish pathogen. The toxR gene of V. angullarum (Va-toxR) was cloned from strain PT-87050 isolated from diseased ayu (Plecoglossus altivelis), and the sequence was analyzed. The toxR sequence was 63 to 51% identical to those reported for other species of the family Vibrionaceae. Distribution of the Va-toxR gene sequence in V. anguillarum strains of various serotypes was confirmed by using DNA probe and PCR methods. An isogenic toxR mutant of V. anguillarum PT-24, isolated from diseased ayu, was constructed by using an allelic exchange method. The wild-type strain and the toxR mutant did not differ in the ability to produce a protease(s) and a hemolysin(s) or in pathogenicity for ayu when examined by the intramuscular injection and immersion methods. A 35-kDa major OMP was not produced by the toxR mutant. However, a 46-kDa OMP was hardly detected in the wild-type strain but was produced as the major OMP by the toxR mutant. For the toxR mutant, the MICs of two β-lactam antibiotics were higher and the minimum bactericidal concentration of sodium dodecyl sulfate was lower than for the wild-type strain. Analysis of the N-terminal amino acid sequences of the 35- and 46-kDa OMPs indicated that these proteins are the porin-like OMPs and are related to the toxR-regulated major OMPs of the family Vibrionaceae. The results indicate that the toxR gene is not involved in virulence expression in V. anguillarum PT-24 and that toxR regulation of major OMPs is universal in the family Vibrionaceae. These results support the hypothesis that the ancestral role of the toxR gene is regulation of OMP gene expression and that only in some Vibrio species has ToxR been appropriated for the regulation of a virulence gene(s). PMID:11553547

Muricauda antarctica sp. nov., a marine member of the Flavobacteriaceae isolated from Antarctic seawater.

PubMed

Wu, Yue-Hong; Yu, Pei-Song; Zhou, Ya-Dong; Xu, Lin; Wang, Chun-Sheng; Wu, Min; Oren, Aharon; Xu, Xue-Wei

2013-09-01

A Gram-stain-negative, rod-shaped bacterium with appendages, designated Ar-22(T), was isolated from a seawater sample collected from the western part of Prydz Bay, near Cape Darnley, Antarctica. Strain Ar-22(T) grew optimally at 35 °C, at pH 7.5 and in the presence of 1-3% (w/v) NaCl. The isolate was positive for casein, gelatin and Tween 20 decomposition and negative for H2S production and indole formation. Chemotaxonomic analysis showed that MK-6 was the major isoprenoid quinone and phosphatidylethanolamine was the major polar lipid. The major fatty acids were iso-C(17:0) 3-OH, iso-C(15:1) G, iso-C(15:0) and C(16:1)ω7c/iso-C(15:0) 2OH. The genomic DNA G+C content was 44.8 mol%. Comparative 16S rRNA gene sequence analysis revealed that strain Ar-22(T) is closely related to members of the genus Muricauda, sharing 94.2-97.3% sequence similarity with the type strains of species of the genus Muricauda and being most closely related to the Muricauda aquimarina. Phylogenetic analysis based on the 16S rRNA gene sequence comparison confirmed that strain Ar-22(T) formed a deep lineage with Muricauda flavescens. Sequence similarity between strain Ar-22(T) and Muricauda ruestringensis DSM 13258(T), the type species of the genus Muricauda, was 96.9%. Strain Ar-22(T) exhibited mean DNA-DNA relatedness values of 40.1%, 49.4% and 25.7% to M. aquimarina JCM 11811(T), M. flavescens JCM 11812(T) and Muricauda lutimaris KCTC 22173(T), respectively. On the basis of phenotypic and genotypic data, strain Ar-22(T) represents a novel species of the genus Muricauda, for which the name Muricauda antarctica sp. nov. (type strain Ar-22(T) =CGMCC 1.12174(T) = JCM 18450(T)) is proposed.

Genotypes and phylogeographical relationships of infectious hematopoietic necrosis virus in California, USA

USGS Publications Warehouse

Kelley, G.O.; Bendorf, C.M.; Yun, S.C.; Kurath, G.; Hedrick, R.P.

2007-01-01

Infectious hematopoietic necrosis virus (IHNV) contains 3 major genogroups in North America with discreet geographic ranges designated as upper (U), middle (M), and lower (L). A comprehensive genotyping of 237 IHNV isolates from hatchery and wild salmonids in California revealed 25 different sequence types (a to y) all in the L genogroup; specifically, the genogroup contained 14 sequence types that were unique to individual isolates as well as 11 sequence types representing 2 or more identical isolates. The most evident trend was the phylogenetic and geographical division of the L genogroup into 2 distinct subgroups designated as LI and LII. Isolates within Subgroup LI were primarily found within waterways linked to southern Oregon and northern California coastal rivers. Isolates in Subgroup LII were concentrated within inland valley watersheds that included the Sacramento River, San Joaquin River, and their tributaries. The temporal and spatial patterns of virus occurrence suggested that infections among adult Chinook salmon in the hatchery or that spawn in the river are a major source of virus potentially infecting other migrating or resident salmonids in California. Serum neutralization results of the California isolates of IHNV corroborated a temporal trend of sequence divergence; specifically, 2 progressive shifts in which more recent virus isolates represent new serotypes. A comparison of the estimates of divergence rates for Subgroup LI (1 ?? ICT5 mutations per nucleotide site per year) indicated stasis similar to that observed in the U genogroup, while the Subgroup LII rate (1 ?? 10 3 mutations per nucleotide site per year) suggested a more active evolution similar to that of the M genogroup. ?? Inter-Research 2007.

Repetitive part of the banana (Musa acuminata) genome investigated by low-depth 454 sequencing.

PubMed

Hribová, Eva; Neumann, Pavel; Matsumoto, Takashi; Roux, Nicolas; Macas, Jirí; Dolezel, Jaroslav

2010-09-16

Bananas and plantains (Musa spp.) are grown in more than a hundred tropical and subtropical countries and provide staple food for hundreds of millions of people. They are seed-sterile crops propagated clonally and this makes them vulnerable to a rapid spread of devastating diseases and at the same time hampers breeding improved cultivars. Although the socio-economic importance of bananas and plantains cannot be overestimated, they remain outside the focus of major research programs. This slows down the study of nuclear genome and the development of molecular tools to facilitate banana improvement. In this work, we report on the first thorough characterization of the repeat component of the banana (M. acuminata cv. 'Calcutta 4') genome. Analysis of almost 100 Mb of sequence data (0.15× genome coverage) permitted partial sequence reconstruction and characterization of repetitive DNA, making up about 30% of the genome. The results showed that the banana repeats are predominantly made of various types of Ty1/copia and Ty3/gypsy retroelements representing 16 and 7% of the genome respectively. On the other hand, DNA transposons were found to be rare. In addition to new families of transposable elements, two new satellite repeats were discovered and found useful as cytogenetic markers. To help in banana sequence annotation, a specific Musa repeat database was created, and its utility was demonstrated by analyzing the repeat composition of 62 genomic BAC clones. A low-depth 454 sequencing of banana nuclear genome provided the largest amount of DNA sequence data available until now for Musa and permitted reconstruction of most of the major types of DNA repeats. The information obtained in this study improves the knowledge of the long-range organization of banana chromosomes, and provides sequence resources needed for repeat masking and annotation during the Musa genome sequencing project. It also provides sequence data for isolation of DNA markers to be used in genetic diversity studies and in marker-assisted selection.

Repetitive part of the banana (Musa acuminata) genome investigated by low-depth 454 sequencing

PubMed Central

2010-01-01

Background Bananas and plantains (Musa spp.) are grown in more than a hundred tropical and subtropical countries and provide staple food for hundreds of millions of people. They are seed-sterile crops propagated clonally and this makes them vulnerable to a rapid spread of devastating diseases and at the same time hampers breeding improved cultivars. Although the socio-economic importance of bananas and plantains cannot be overestimated, they remain outside the focus of major research programs. This slows down the study of nuclear genome and the development of molecular tools to facilitate banana improvement. Results In this work, we report on the first thorough characterization of the repeat component of the banana (M. acuminata cv. 'Calcutta 4') genome. Analysis of almost 100 Mb of sequence data (0.15× genome coverage) permitted partial sequence reconstruction and characterization of repetitive DNA, making up about 30% of the genome. The results showed that the banana repeats are predominantly made of various types of Ty1/copia and Ty3/gypsy retroelements representing 16 and 7% of the genome respectively. On the other hand, DNA transposons were found to be rare. In addition to new families of transposable elements, two new satellite repeats were discovered and found useful as cytogenetic markers. To help in banana sequence annotation, a specific Musa repeat database was created, and its utility was demonstrated by analyzing the repeat composition of 62 genomic BAC clones. Conclusion A low-depth 454 sequencing of banana nuclear genome provided the largest amount of DNA sequence data available until now for Musa and permitted reconstruction of most of the major types of DNA repeats. The information obtained in this study improves the knowledge of the long-range organization of banana chromosomes, and provides sequence resources needed for repeat masking and annotation during the Musa genome sequencing project. It also provides sequence data for isolation of DNA markers to be used in genetic diversity studies and in marker-assisted selection. PMID:20846365

Orthogonal typing methods identify genetic diversity among Belgian Campylobacter jejuni strains isolated over a decade from poultry and cases of sporadic human illness.

PubMed

Elhadidy, Mohamed; Arguello, Hector; Álvarez-Ordóñez, Avelino; Miller, William G; Duarte, Alexandra; Martiny, Delphine; Hallin, Marie; Vandenberg, Olivier; Dierick, Katelijne; Botteldoorn, Nadine

2018-06-20

Campylobacter jejuni is a zoonotic pathogen commonly associated with human gastroenteritis. Retail poultry meat is a major food-related transmission source of C. jejuni to humans. The present study investigated the genetic diversity, clonal relationship, and strain risk-analysis of 403 representative C. jejuni isolates from chicken broilers (n = 204) and sporadic cases of human diarrhea (n = 199) over a decade (2006-2015) in Belgium, using multilocus sequence typing (MLST), PCR binary typing (P-BIT), and identification of lipooligosaccharide (LOS) biosynthesis locus classes. A total of 123 distinct sequence types (STs), clustered in 28 clonal complexes (CCs) were assigned, including ten novel sequence types that were not previously documented in the international database. Sequence types ST-48, ST-21, ST-50, ST-45, ST-464, ST-2274, ST-572, ST-19, ST-257 and ST-42 were the most prevalent. Clonal complex 21 was the main clonal complex in isolates from humans and chickens. Among observed STs, a total of 35 STs that represent 72.2% (291/403) of the isolates were identified in both chicken and human isolates confirming considerable epidemiological relatedness; these 35 STs also clustered together in the most prevalent CCs. A majority of the isolates harbored sialylated LOS loci associated with potential neuropathic outcomes in humans. Although the concordance between MLST and P-BIT, determined by the adjusted Rand and Wallace coefficients, showed low congruence between both typing methods. The discriminatory power of P-BIT and MLST was similar, with Simpson's diversity indexes of 0.978 and 0.975, respectively. Furthermore, P-BIT could provide additional epidemiological information that would provide further insights regarding the potential association to human health from each strain. In addition, certain clones could be linked to specific clinical symptoms. Indeed, LOS class E was associated with less severe infections. Moreover, ST-572 was significantly associated with clinical infections occurring after travelling abroad. Ultimately, the data generated from this study will help to better understand the molecular epidemiology of C. jejuni infection. Copyright © 2018. Published by Elsevier B.V.

Persistence of Mycoplasma hyopneumoniae sequence types in spite of a control program for enzootic pneumonia in pigs.

PubMed

Overesch, Gudrun; Kuhnert, Peter

2017-09-15

Enzootic pneumonia (EP) in pigs caused by Mycoplasma (M.) hyopneumoniae has successfully been combatted in Switzerland. A control program was fully implemented in 2004 which is based on total depopulation strategies of affected fattening farms as well as partial depopulation on breeding farms. Thereby, the number of cases has dropped drastically from more than 200 in 2003 to two cases in 2013. Currently monitoring is done based on clinical observation and subsequent diagnostic of coughing pigs. Moreover, in case of more than 10% gross pathological lesions per slaughter batch laboratory confirmation for EP is compulsory. Despite these strict measures it was not possible to eliminate M. hyopneumoniae from Swiss pig production. In fact, during the last few years the number of EP cases has slightly increased. Therefore, genotyping of the involved M. hyopneumoniae strains was conducted in order to elucidate possible sources and routes of infection. All available and typeable samples from totally 22 cases during the period 2014-2016 were investigated by extended multilocus sequence typing (MLST). A total of 16 cases, including eight from 2014, five from 2015 and three from 2016 could thereby be included in the study. MLST revealed that the majority of cases in 2014/2015 were due to two major spread scenarios, i.e. two M. hyopneumoniae sequence types, each scenario involving six individual production farms in five to six different Cantons (states), respectively. Moreover, by comparison of archived sequences some sequence types were observed over ten years demonstrating their persistence over a long time and the possible partial failure of elimination measures in Switzerland. Insufficient sanitation on affected farms and subsequent animal transport of symptomless infected pigs could lead to recurrent cases. Wild boar harbor identical strains found with EP but solid data are missing to assign a role as reservoir to this wild animal. Implementing a monitoring scheme for M. hyopneumoniae in wild boar in combination with genotyping of all available samples from domestic pigs could direct responsible authorities to possible gaps and deficiencies of control measures taken for combating enzootic pneumonia. With the newly installed PubMLST database sequence types for M. hyopneumoniae are now available and allow tracing back strains on the international level. Copyright © 2017 Elsevier B.V. All rights reserved.

spa Typing and Multilocus Sequence Typing Show Comparable Performance in a Macroepidemiologic Study of Staphylococcus aureus in the United States

PubMed Central

O'Hara, F. Patrick; Suaya, Jose A.; Ray, G. Thomas; Baxter, Roger; Brown, Megan L.; Mera, Robertino M.; Close, Nicole M.; Thomas, Elizabeth

2016-01-01

A number of molecular typing methods have been developed for characterization of Staphylococcus aureus isolates. The utility of these systems depends on the nature of the investigation for which they are used. We compared two commonly used methods of molecular typing, multilocus sequence typing (MLST) (and its clustering algorithm, Based Upon Related Sequence Type [BURST]) with the staphylococcal protein A (spa) typing (and its clustering algorithm, Based Upon Repeat Pattern [BURP]), to assess the utility of these methods for macroepidemiology and evolutionary studies of S. aureus in the United States. We typed a total of 366 clinical isolates of S. aureus by these methods and evaluated indices of diversity and concordance values. Our results show that, when combined with the BURP clustering algorithm to delineate clonal lineages, spa typing produces results that are highly comparable with those produced by MLST/BURST. Therefore, spa typing is appropriate for use in macroepidemiology and evolutionary studies and, given its lower implementation cost, this method appears to be more efficient. The findings are robust and are consistent across different settings, patient ages, and specimen sources. Our results also support a model in which the methicillin-resistant S. aureus (MRSA) population in the United States comprises two major lineages (USA300 and USA100), which each consist of closely related variants. PMID:26669861

spa Typing and Multilocus Sequence Typing Show Comparable Performance in a Macroepidemiologic Study of Staphylococcus aureus in the United States.

PubMed

O'Hara, F Patrick; Suaya, Jose A; Ray, G Thomas; Baxter, Roger; Brown, Megan L; Mera, Robertino M; Close, Nicole M; Thomas, Elizabeth; Amrine-Madsen, Heather

2016-01-01

A number of molecular typing methods have been developed for characterization of Staphylococcus aureus isolates. The utility of these systems depends on the nature of the investigation for which they are used. We compared two commonly used methods of molecular typing, multilocus sequence typing (MLST) (and its clustering algorithm, Based Upon Related Sequence Type [BURST]) with the staphylococcal protein A (spa) typing (and its clustering algorithm, Based Upon Repeat Pattern [BURP]), to assess the utility of these methods for macroepidemiology and evolutionary studies of S. aureus in the United States. We typed a total of 366 clinical isolates of S. aureus by these methods and evaluated indices of diversity and concordance values. Our results show that, when combined with the BURP clustering algorithm to delineate clonal lineages, spa typing produces results that are highly comparable with those produced by MLST/BURST. Therefore, spa typing is appropriate for use in macroepidemiology and evolutionary studies and, given its lower implementation cost, this method appears to be more efficient. The findings are robust and are consistent across different settings, patient ages, and specimen sources. Our results also support a model in which the methicillin-resistant S. aureus (MRSA) population in the United States comprises two major lineages (USA300 and USA100), which each consist of closely related variants.

An Integrated Tool to Study MHC Region: Accurate SNV Detection and HLA Genes Typing in Human MHC Region Using Targeted High-Throughput Sequencing

PubMed Central

Liu, Xiao; Xu, Yinyin; Liang, Dequan; Gao, Peng; Sun, Yepeng; Gifford, Benjamin; D’Ascenzo, Mark; Liu, Xiaomin; Tellier, Laurent C. A. M.; Yang, Fang; Tong, Xin; Chen, Dan; Zheng, Jing; Li, Weiyang; Richmond, Todd; Xu, Xun; Wang, Jun; Li, Yingrui

2013-01-01

The major histocompatibility complex (MHC) is one of the most variable and gene-dense regions of the human genome. Most studies of the MHC, and associated regions, focus on minor variants and HLA typing, many of which have been demonstrated to be associated with human disease susceptibility and metabolic pathways. However, the detection of variants in the MHC region, and diagnostic HLA typing, still lacks a coherent, standardized, cost effective and high coverage protocol of clinical quality and reliability. In this paper, we presented such a method for the accurate detection of minor variants and HLA types in the human MHC region, using high-throughput, high-coverage sequencing of target regions. A probe set was designed to template upon the 8 annotated human MHC haplotypes, and to encompass the 5 megabases (Mb) of the extended MHC region. We deployed our probes upon three, genetically diverse human samples for probe set evaluation, and sequencing data show that ∼97% of the MHC region, and over 99% of the genes in MHC region, are covered with sufficient depth and good evenness. 98% of genotypes called by this capture sequencing prove consistent with established HapMap genotypes. We have concurrently developed a one-step pipeline for calling any HLA type referenced in the IMGT/HLA database from this target capture sequencing data, which shows over 96% typing accuracy when deployed at 4 digital resolution. This cost-effective and highly accurate approach for variant detection and HLA typing in the MHC region may lend further insight into immune-mediated diseases studies, and may find clinical utility in transplantation medicine research. This one-step pipeline is released for general evaluation and use by the scientific community. PMID:23894464

Close Genetic Relationship between Legionella pneumophila Serogroup 1 Isolates from Sputum Specimens and Puddles on Roads, as Determined by Sequence-Based Typing

PubMed Central

Kanatani, Jun-ichi; Isobe, Junko; Kimata, Keiko; Shima, Tomoko; Shimizu, Miwako; Kura, Fumiaki; Sata, Tetsutaro

2013-01-01

We investigated the prevalence of Legionella species isolated from puddles on asphalt roads. In addition, we carried out sequence-based typing (SBT) analysis on the genetic relationship between L. pneumophila serogroup 1 (SG 1) isolates from puddles and from stock strains previously obtained from sputum specimens and public baths. Sixty-nine water samples were collected from puddles on roads at 6 fixed locations. Legionella species were detected in 33 samples (47.8%) regardless of season. Among the 325 isolates from puddles, strains of L. pneumophila SG 1, a major causative agent of Legionnaires' disease, were the most frequently isolated (n = 62, 19.1%). Sixty-two isolates of L. pneumophila SG 1 from puddles were classified into 36 sequence types (STs) by SBT. ST120 and ST48 were identified as major STs. Environmental ST120 strains from puddles were found for the first time in this study. Among the 14 STs of the clinical isolates (n = 19), 4 STs (n = 6, 31.6%), including ST120, were also detected in isolates from puddles on roads, and the sources of infection in these cases remained unclear. The lag-1 gene, a tentative marker for clinical isolates, was prevalent in puddle isolates (61.3%). Our findings suggest that puddles on asphalt roads serve as potential reservoirs for L. pneumophila in the environment. PMID:23603681

Close genetic relationship between Legionella pneumophila serogroup 1 isolates from sputum specimens and puddles on roads, as determined by sequence-based typing.

PubMed

Kanatani, Jun-ichi; Isobe, Junko; Kimata, Keiko; Shima, Tomoko; Shimizu, Miwako; Kura, Fumiaki; Sata, Tetsutaro; Watahiki, Masanori

2013-07-01

We investigated the prevalence of Legionella species isolated from puddles on asphalt roads. In addition, we carried out sequence-based typing (SBT) analysis on the genetic relationship between L. pneumophila serogroup 1 (SG 1) isolates from puddles and from stock strains previously obtained from sputum specimens and public baths. Sixty-nine water samples were collected from puddles on roads at 6 fixed locations. Legionella species were detected in 33 samples (47.8%) regardless of season. Among the 325 isolates from puddles, strains of L. pneumophila SG 1, a major causative agent of Legionnaires' disease, were the most frequently isolated (n = 62, 19.1%). Sixty-two isolates of L. pneumophila SG 1 from puddles were classified into 36 sequence types (STs) by SBT. ST120 and ST48 were identified as major STs. Environmental ST120 strains from puddles were found for the first time in this study. Among the 14 STs of the clinical isolates (n = 19), 4 STs (n = 6, 31.6%), including ST120, were also detected in isolates from puddles on roads, and the sources of infection in these cases remained unclear. The lag-1 gene, a tentative marker for clinical isolates, was prevalent in puddle isolates (61.3%). Our findings suggest that puddles on asphalt roads serve as potential reservoirs for L. pneumophila in the environment.

Triple helix-forming oligonucleotide corresponding to the polypyrimidine sequence in the rat alpha 1(I) collagen promoter specifically inhibits factor binding and transcription.

PubMed

Kovacs, A; Kandala, J C; Weber, K T; Guntaka, R V

1996-01-19

Type I and III fibrillar collagens are the major structural proteins of the extracellular matrix found in various organs including the myocardium. Abnormal and progressive accumulation of fibrillar type I collagen in the interstitial spaces compromises organ function and therefore, the study of transcriptional regulation of this gene and specific targeting of its expression is of major interest. Transient transfection of adult cardiac fibroblasts indicate that the polypurine-polypyrimidine sequence of alpha 1(I) collagen promoter between nucleotides - 200 and -140 represents an overall positive regulatory element. DNase I footprinting and electrophoretic mobility shift assays suggest that multiple factors bind to different elements of this promoter region. We further demonstrate that the unique polypyrimidine sequence between -172 and -138 of the promoter represents a suitable target for a single-stranded polypurine oligonucleotide (TFO) to form a triple helix DNA structure. Modified electrophoretic mobility shift assays show that this TFO specifically inhibits the protein-DNA interaction within the target region. In vitro transcription assays and transient transfection experiments demonstrate that the transcriptional activity of the promoter is inhibited by this oligonucleotide. We propose that TFOs represent a therapeutic potential to specifically influence the expression of alpha 1(I) collagen gene in various disease states where abnormal type I collagen accumulation is known to occur.

Proposal of Mucilaginibacter galii sp. nov. isolated from leaves of Galium album.

PubMed

Aydogan, Ebru L; Busse, Hans-Jürgen; Moser, Gerald; Müller, Christoph; Kämpfer, Peter; Glaeser, Stefanie P

2017-05-01

A pale-pink-pigmented, Gram-stain-negative, rod-shaped, non-spore-forming bacterial strain, PP-F2F-G47T, was isolated from the phyllosphere of the herbaceous plant Galium album. Phylogenetic analysis based on the nearly full-length 16S rRNA gene sequence revealed highest sequence similarity to the type strains of Mucilaginibacter daejeonensis (96.2 %), Mucilaginibacter dorajii (95.7 %) and Mucilaginibacter phyllosphaerae (95.5 %). 16S rRNA gene sequence similarities to all other type strains were below 95.5 %. The predominant cellular fatty acids of the strain were C16 : 1ω7c/iso-C15 : 0 2-OH (measured as summed feature 3) and iso-C15 : 0. The major compound in the polyamine pattern was sym-homospermidine and major quinone was menaquinone MK-7. The polar lipid profile was composed of phosphatidylethanolamine and several unidentified aminolipipids, phospholipids, aminophospholipids and lipids without a functional group. A sphingophospholipid could not be detected but a ninhydrin-positive alkaline-stable lipid was visible. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. Based on phylogenetic, chemotaxonomic and phenotypic analyses a novel species is proposed, Mucilaginibacter galii sp. nov., with PP-F2F-G47T (=CCM 8711T=CIP 111182T=LMG 29767T) as the type strain.

Hydroxyapatite-binding peptides for bone growth and inhibition

DOEpatents

Bertozzi, Carolyn R [Berkeley, CA; Song, Jie [Shrewsbury, MA; Lee, Seung-Wuk [Walnut Creek, CA

2011-09-20

Hydroxyapatite (HA)-binding peptides are selected using combinatorial phage library display. Pseudo-repetitive consensus amino acid sequences possessing periodic hydroxyl side chains in every two or three amino acid sequences are obtained. These sequences resemble the (Gly-Pro-Hyp).sub.x repeat of human type I collagen, a major component of extracellular matrices of natural bone. A consistent presence of basic amino acid residues is also observed. The peptides are synthesized by the solid-phase synthetic method and then used for template-driven HA-mineralization. Microscopy reveal that the peptides template the growth of polycrystalline HA crystals .about.40 nm in size.

Molecular prevalence and genetic diversity of bovine Theileria orientalis in Myanmar.

PubMed

Bawm, Saw; Shimizu, Kohei; Hirota, Jun-Ichi; Tosa, Yusuke; Htun, Lat Lat; Maw, Ni Ni; Thein, Myint; Kato, Hirotomo; Sakurai, Tatsuya; Katakura, Ken

2014-08-01

Theileria orientalis is a causative agent of benign theileriosis in cattle and distributed in mainly Asian countries. In the present study, we examined the prevalence of T. orientalis infection by PCR based on the major piroplasm surface protein gene (MPSP) sequences in cattle in Myanmar, followed by phylogenetic analysis of the MPSP genes. The MPSP gene was amplified in 258 of 713 (36.2%) cattle blood DNA samples collected from five cities in different geographical regions of Myanmar. Phylogenetic analysis of MPSP sequences from 54 T. orientalis-positive DNA samples revealed the presence of six allelic genotypes, including Types 1, 3, 4, 5, 7, and N-3. Types 5 and 7 were the predominant types detected. Sequences of the MPSP genes detected in Myanmar were closely related to those from Thailand, Vietnam or Mongolia. These findings suggest that movement of animals carrying T. orientalis parasites between Southeast Asian countries could be a reason for the similar genotype distribution of the parasites in Myanmar. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Novel division level bacterial diversity in a Yellowstone hot spring.

PubMed

Hugenholtz, P; Pitulle, C; Hershberger, K L; Pace, N R

1998-01-01

A culture-independent molecular phylogenetic survey was carried out for the bacterial community in Obsidian Pool (OP), a Yellowstone National Park hot spring previously shown to contain remarkable archaeal diversity (S. M. Barns, R. E. Fundyga, M. W. Jeffries, and N. R. Page, Proc. Natl. Acad. Sci. USA 91:1609-1613, 1994). Small-subunit rRNA genes (rDNA) were amplified directly from OP sediment DNA by PCR with universally conserved or Bacteria-specific rDNA primers and cloned. Unique rDNA types among > 300 clones were identified by restriction fragment length polymorphism, and 122 representative rDNA sequences were determined. These were found to represent 54 distinct bacterial sequence types or clusters (> or = 98% identity) of sequences. A majority (70%) of the sequence types were affiliated with 14 previously recognized bacterial divisions (main phyla; kingdoms); 30% were unaffiliated with recognized bacterial divisions. The unaffiliated sequence types (represented by 38 sequences) nominally comprise 12 novel, division level lineages termed candidate divisions. Several OP sequences were nearly identical to those of cultivated chemolithotrophic thermophiles, including the hydrogen-oxidizing Calderobacterium and the sulfate reducers Thermodesulfovibrio and Thermodesulfobacterium, or belonged to monophyletic assemblages recognized for a particular type of metabolism, such as the hydrogen-oxidizing Aquificales and the sulfate-reducing delta-Proteobacteria. The occurrence of such organisms is consistent with the chemical composition of OP (high in reduced iron and sulfur) and suggests a lithotrophic base for primary productivity in this hot spring, through hydrogen oxidation and sulfate reduction. Unexpectedly, no archaeal sequences were encountered in OP clone libraries made with universal primers. Hybridization analysis of amplified OP DNA with domain-specific probes confirmed that the analyzed community rDNA from OP sediment was predominantly bacterial. These results expand substantially our knowledge of the extent of bacterial diversity and call into question the commonly held notion that Archaea dominate hydrothermal environments. Finally, the currently known extent of division level bacterial phylogenetic diversity is collated and summarized.

Conservation of the C-type lectin fold for massive sequence variation in a Treponema diversity-generating retroelement

DOE Office of Scientific and Technical Information (OSTI.GOV)

Le Coq, Johanne; Ghosh, Partho

2012-06-19

Anticipatory ligand binding through massive protein sequence variation is rare in biological systems, having been observed only in the vertebrate adaptive immune response and in a phage diversity-generating retroelement (DGR). Earlier work has demonstrated that the prototypical DGR variable protein, major tropism determinant (Mtd), meets the demands of anticipatory ligand binding by novel means through the C-type lectin (CLec) fold. However, because of the low sequence identity among DGR variable proteins, it has remained unclear whether the CLec fold is a general solution for DGRs. We have addressed this problem by determining the structure of a second DGR variable protein,more » TvpA, from the pathogenic oral spirochete Treponema denticola. Despite its weak sequence identity to Mtd ({approx}16%), TvpA was found to also have a CLec fold, with predicted variable residues exposed in a ligand-binding site. However, this site in TvpA was markedly more variable than the one in Mtd, reflecting the unprecedented approximate 10{sup 20} potential variability of TvpA. In addition, similarity between TvpA and Mtd with formylglycine-generating enzymes was detected. These results provide strong evidence for the conservation of the formylglycine-generating enzyme-type CLec fold among DGRs as a means of accommodating massive sequence variation.« less

Conservation of the C-type lectin fold for massive sequence variation in a Treponema diversity-generating retroelement

PubMed Central

Le Coq, Johanne; Ghosh, Partho

2011-01-01

Anticipatory ligand binding through massive protein sequence variation is rare in biological systems, having been observed only in the vertebrate adaptive immune response and in a phage diversity-generating retroelement (DGR). Earlier work has demonstrated that the prototypical DGR variable protein, major tropism determinant (Mtd), meets the demands of anticipatory ligand binding by novel means through the C-type lectin (CLec) fold. However, because of the low sequence identity among DGR variable proteins, it has remained unclear whether the CLec fold is a general solution for DGRs. We have addressed this problem by determining the structure of a second DGR variable protein, TvpA, from the pathogenic oral spirochete Treponema denticola. Despite its weak sequence identity to Mtd (∼16%), TvpA was found to also have a CLec fold, with predicted variable residues exposed in a ligand-binding site. However, this site in TvpA was markedly more variable than the one in Mtd, reflecting the unprecedented approximate 1020 potential variability of TvpA. In addition, similarity between TvpA and Mtd with formylglycine-generating enzymes was detected. These results provide strong evidence for the conservation of the formylglycine-generating enzyme-type CLec fold among DGRs as a means of accommodating massive sequence variation. PMID:21873231

Conservation of the C-type lectin fold for massive sequence variation in a Treponema diversity-generating retroelement.

PubMed

Le Coq, Johanne; Ghosh, Partho

2011-08-30

Anticipatory ligand binding through massive protein sequence variation is rare in biological systems, having been observed only in the vertebrate adaptive immune response and in a phage diversity-generating retroelement (DGR). Earlier work has demonstrated that the prototypical DGR variable protein, major tropism determinant (Mtd), meets the demands of anticipatory ligand binding by novel means through the C-type lectin (CLec) fold. However, because of the low sequence identity among DGR variable proteins, it has remained unclear whether the CLec fold is a general solution for DGRs. We have addressed this problem by determining the structure of a second DGR variable protein, TvpA, from the pathogenic oral spirochete Treponema denticola. Despite its weak sequence identity to Mtd (∼16%), TvpA was found to also have a CLec fold, with predicted variable residues exposed in a ligand-binding site. However, this site in TvpA was markedly more variable than the one in Mtd, reflecting the unprecedented approximate 10(20) potential variability of TvpA. In addition, similarity between TvpA and Mtd with formylglycine-generating enzymes was detected. These results provide strong evidence for the conservation of the formylglycine-generating enzyme-type CLec fold among DGRs as a means of accommodating massive sequence variation.

Whole Genome Sequencing for Genomics-Guided Investigations of Escherichia coli O157:H7 Outbreaks.

PubMed

Rusconi, Brigida; Sanjar, Fatemeh; Koenig, Sara S K; Mammel, Mark K; Tarr, Phillip I; Eppinger, Mark

2016-01-01

Multi isolate whole genome sequencing (WGS) and typing for outbreak investigations has become a reality in the post-genomics era. We applied this technology to strains from Escherichia coli O157:H7 outbreaks. These include isolates from seven North America outbreaks, as well as multiple isolates from the same patient and from different infected individuals in the same household. Customized high-resolution bioinformatics sequence typing strategies were developed to assess the core genome and mobilome plasticity. Sequence typing was performed using an in-house single nucleotide polymorphism (SNP) discovery and validation pipeline. Discriminatory power becomes of particular importance for the investigation of isolates from outbreaks in which macrogenomic techniques such as pulse-field gel electrophoresis or multiple locus variable number tandem repeat analysis do not differentiate closely related organisms. We also characterized differences in the phage inventory, allowing us to identify plasticity among outbreak strains that is not detectable at the core genome level. Our comprehensive analysis of the mobilome identified multiple plasmids that have not previously been associated with this lineage. Applied phylogenomics approaches provide strong molecular evidence for exceptionally little heterogeneity of strains within outbreaks and demonstrate the value of intra-cluster comparisons, rather than basing the analysis on archetypal reference strains. Next generation sequencing and whole genome typing strategies provide the technological foundation for genomic epidemiology outbreak investigation utilizing its significantly higher sample throughput, cost efficiency, and phylogenetic relatedness accuracy. These phylogenomics approaches have major public health relevance in translating information from the sequence-based survey to support timely and informed countermeasures. Polymorphisms identified in this work offer robust phylogenetic signals that index both short- and long-term evolution and can complement currently employed typing schemes for outbreak ex- and inclusion, diagnostics, surveillance, and forensic studies.

Whole Genome Sequencing for Genomics-Guided Investigations of Escherichia coli O157:H7 Outbreaks

PubMed Central

Rusconi, Brigida; Sanjar, Fatemeh; Koenig, Sara S. K.; Mammel, Mark K.; Tarr, Phillip I.; Eppinger, Mark

2016-01-01

Multi isolate whole genome sequencing (WGS) and typing for outbreak investigations has become a reality in the post-genomics era. We applied this technology to strains from Escherichia coli O157:H7 outbreaks. These include isolates from seven North America outbreaks, as well as multiple isolates from the same patient and from different infected individuals in the same household. Customized high-resolution bioinformatics sequence typing strategies were developed to assess the core genome and mobilome plasticity. Sequence typing was performed using an in-house single nucleotide polymorphism (SNP) discovery and validation pipeline. Discriminatory power becomes of particular importance for the investigation of isolates from outbreaks in which macrogenomic techniques such as pulse-field gel electrophoresis or multiple locus variable number tandem repeat analysis do not differentiate closely related organisms. We also characterized differences in the phage inventory, allowing us to identify plasticity among outbreak strains that is not detectable at the core genome level. Our comprehensive analysis of the mobilome identified multiple plasmids that have not previously been associated with this lineage. Applied phylogenomics approaches provide strong molecular evidence for exceptionally little heterogeneity of strains within outbreaks and demonstrate the value of intra-cluster comparisons, rather than basing the analysis on archetypal reference strains. Next generation sequencing and whole genome typing strategies provide the technological foundation for genomic epidemiology outbreak investigation utilizing its significantly higher sample throughput, cost efficiency, and phylogenetic relatedness accuracy. These phylogenomics approaches have major public health relevance in translating information from the sequence-based survey to support timely and informed countermeasures. Polymorphisms identified in this work offer robust phylogenetic signals that index both short- and long-term evolution and can complement currently employed typing schemes for outbreak ex- and inclusion, diagnostics, surveillance, and forensic studies. PMID:27446025

A Comparison of Thinking and Writing Patterns in Korea and the United States. AFS Occasional Papers in Intercultural Learning No. 12.

ERIC Educational Resources Information Center

Norton, Robert F.

Major writing style differences between Korean and U.S. essayists are examined in order to determine: (1) the types of relationships expressed in a specific essay; (2) the sequence in which related ideas are expressed; and (3) the distance between two ideas that comprise each relationship. Eighteen basic types of relationships were examined in…

iFeature: a python package and web server for features extraction and selection from protein and peptide sequences.

PubMed

Chen, Zhen; Zhao, Pei; Li, Fuyi; Leier, André; Marquez-Lago, Tatiana T; Wang, Yanan; Webb, Geoffrey I; Smith, A Ian; Daly, Roger J; Chou, Kuo-Chen; Song, Jiangning

2018-03-08

Structural and physiochemical descriptors extracted from sequence data have been widely used to represent sequences and predict structural, functional, expression and interaction profiles of proteins and peptides as well as DNAs/RNAs. Here, we present iFeature, a versatile Python-based toolkit for generating various numerical feature representation schemes for both protein and peptide sequences. iFeature is capable of calculating and extracting a comprehensive spectrum of 18 major sequence encoding schemes that encompass 53 different types of feature descriptors. It also allows users to extract specific amino acid properties from the AAindex database. Furthermore, iFeature integrates 12 different types of commonly used feature clustering, selection, and dimensionality reduction algorithms, greatly facilitating training, analysis, and benchmarking of machine-learning models. The functionality of iFeature is made freely available via an online web server and a stand-alone toolkit. http://iFeature.erc.monash.edu/; https://github.com/Superzchen/iFeature/. jiangning.song@monash.edu; kcchou@gordonlifescience.org; roger.daly@monash.edu. Supplementary data are available at Bioinformatics online.

Cumulative Axial and Torsional Fatigue: An Investigation of Load-Type Sequencing Effects

NASA Technical Reports Server (NTRS)

Kalluri, Sreeramesh; Bonacuse, Peter J.

2000-01-01

Cumulative fatigue behavior of a wrought cobalt-base superalloy, Haynes 188 was investigated at 538 C under various single-step sequences of axial and torsional loading conditions. Initially, fully-reversed, axial and torsional fatigue tests were conducted under strain control at 538 C on thin-walled tubular specimens to establish baseline fatigue life relationships. Subsequently, four sequences (axial/axial, torsional/torsional, axial/torsional, and torsional/axial) of two load-level fatigue tests were conducted to characterize both the load-order (high/low) and load-type sequencing effects. For the two load-level tests, summations of life fractions and the remaining fatigue lives at the second load-level were computed by the Miner's Linear Damage Rule (LDR) and a nonlinear Damage Curve Approach (DCA). In general, for all four cases predictions by LDR were unconservative. Predictions by the DCA were within a factor of two of the experimentally observed fatigue lives for a majority of the cumulative axial and torsional fatigue tests.

Enterobacter muelleri sp. nov., isolated from the rhizosphere of Zea mays.

PubMed

Kämpfer, Peter; McInroy, John A; Glaeser, Stefanie P

2015-11-01

A beige-pigmented, oxidase-negative bacterial strain (JM-458T), isolated from a rhizosphere sample, was studied using a polyphasic taxonomic approach. Cells of the isolate were rod-shaped and stained Gram-negative. A comparison of the 16S rRNA gene sequence of strain JM-458T with sequences of the type strains of closely related species of the genus Enterobacter showed that it shared highest sequence similarity with Enterobacter mori (98.7 %), Enterobacter hormaechei (98.3 %), Enterobacter cloacae subsp. dissolvens, Enterobacter ludwigii and Enterobacter asburiae (all 98.2 %). 16S rRNA gene sequence similarities to all other Enterobacter species were below 98 %. Multilocus sequence analysis based on concatenated partial rpoB, gyrB, infB and atpD gene sequences showed a clear distinction of strain JM-458T from its closest related type strains. The fatty acid profile of the strain consisted of C16 : 0, C17 : 0 cyclo, iso-C15 : 0 2-OH/C16 : 1ω7c and C18 : 1ω7c as major components. DNA-DNA hybridizations between strain JM-458T and the type strains of E. mori, E. hormaechei and E. ludwigii resulted in relatedness values of 29 % (reciprocal 25 %), 24 % (reciprocal 43 %) and 16 % (reciprocal 17 %), respectively. DNA-DNA hybridization results together with multilocus sequence analysis results and differential biochemical and chemotaxonomic properties showed that strain JM-458T represents a novel species of the genus Enterobacter, for which the name Enterobacter muelleri sp. nov. is proposed. The type strain is JM-458T ( = DSM 29346T = CIP 110826T = LMG 28480T = CCM 8546T).

MLST and Whole-Genome-Based Population Analysis of Cryptococcus gattii VGIII Links Clinical, Veterinary and Environmental Strains, and Reveals Divergent Serotype Specific Sub-populations and Distant Ancestors

PubMed Central

Firacative, Carolina; Roe, Chandler C.; Malik, Richard; Ferreira-Paim, Kennio; Escandón, Patricia; Sykes, Jane E.; Castañón-Olivares, Laura Rocío; Contreras-Peres, Cudberto; Samayoa, Blanca; Sorrell, Tania C.; Castañeda, Elizabeth; Lockhart, Shawn R.; Engelthaler, David M.; Meyer, Wieland

2016-01-01

The emerging pathogen Cryptococcus gattii causes life-threatening disease in immunocompetent and immunocompromised hosts. Of the four major molecular types (VGI-VGIV), the molecular type VGIII has recently emerged as cause of disease in otherwise healthy individuals, prompting a need to investigate its population genetic structure to understand if there are potential genotype-dependent characteristics in its epidemiology, environmental niche(s), host range and clinical features of disease. Multilocus sequence typing (MLST) of 122 clinical, environmental and veterinary C. gattii VGIII isolates from Australia, Colombia, Guatemala, Mexico, New Zealand, Paraguay, USA and Venezuela, and whole genome sequencing (WGS) of 60 isolates representing all established MLST types identified four divergent sub-populations. The majority of the isolates belong to two main clades, corresponding either to serotype B or C, indicating an ongoing species evolution. Both major clades included clinical, environmental and veterinary isolates. The C. gattii VGIII population was genetically highly diverse, with minor differences between countries, isolation source, serotype and mating type. Little to no recombination was found between the two major groups, serotype B and C, at the whole and mitochondrial genome level. C. gattii VGIII is widespread in the Americas, with sporadic cases occurring elsewhere, WGS revealed Mexico and USA as a likely origin of the serotype B VGIII population and Colombia as a possible origin of the serotype C VGIII population. Serotype B isolates are more virulent than serotype C isolates in a murine model of infection, causing predominantly pulmonary cryptococcosis. No specific link between genotype and virulence was observed. Antifungal susceptibility testing against six antifungal drugs revealed that serotype B isolates are more susceptible to azoles than serotype C isolates, highlighting the importance of strain typing to guide effective treatment to improve the disease outcome. PMID:27494185

Whole-organism clone tracing using single-cell sequencing.

PubMed

Alemany, Anna; Florescu, Maria; Baron, Chloé S; Peterson-Maduro, Josi; van Oudenaarden, Alexander

2018-04-05

Embryonic development is a crucial period in the life of a multicellular organism, during which limited sets of embryonic progenitors produce all cells in the adult body. Determining which fate these progenitors acquire in adult tissues requires the simultaneous measurement of clonal history and cell identity at single-cell resolution, which has been a major challenge. Clonal history has traditionally been investigated by microscopically tracking cells during development, monitoring the heritable expression of genetically encoded fluorescent proteins and, more recently, using next-generation sequencing technologies that exploit somatic mutations, microsatellite instability, transposon tagging, viral barcoding, CRISPR-Cas9 genome editing and Cre-loxP recombination. Single-cell transcriptomics provides a powerful platform for unbiased cell-type classification. Here we present ScarTrace, a single-cell sequencing strategy that enables the simultaneous quantification of clonal history and cell type for thousands of cells obtained from different organs of the adult zebrafish. Using ScarTrace, we show that a small set of multipotent embryonic progenitors generate all haematopoietic cells in the kidney marrow, and that many progenitors produce specific cell types in the eyes and brain. In addition, we study when embryonic progenitors commit to the left or right eye. ScarTrace reveals that epidermal and mesenchymal cells in the caudal fin arise from the same progenitors, and that osteoblast-restricted precursors can produce mesenchymal cells during regeneration. Furthermore, we identify resident immune cells in the fin with a distinct clonal origin from other blood cell types. We envision that similar approaches will have major applications in other experimental systems, in which the matching of embryonic clonal origin to adult cell type will ultimately allow reconstruction of how the adult body is built from a single cell.

Genomic Encyclopedia of Type Strains, Phase I: The one thousand microbial genomes (KMG-I) project

DOE PAGES

Kyrpides, Nikos C.; Woyke, Tanja; Eisen, Jonathan A.; ...

2014-06-15

The Genomic Encyclopedia of Bacteria and Archaea (GEBA) project was launched by the JGI in 2007 as a pilot project with the objective of sequencing 250 bacterial and archaeal genomes. The two major goals of that project were (a) to test the hypothesis that there are many benefits to the use the phylogenetic diversity of organisms in the tree of life as a primary criterion for generating their genome sequence and (b) to develop the necessary framework, technology and organization for large-scale sequencing of microbial isolate genomes. While the GEBA pilot project has not yet been entirely completed, both ofmore » the original goals have already been successfully accomplished, leading the way for the next phase of the project. Here we propose taking the GEBA project to the next level, by generating high quality draft genomes for 1,000 bacterial and archaeal strains. This represents a combined 16-fold increase in both scale and speed as compared to the GEBA pilot project (250 isolate genomes in 4+ years). We will follow a similar approach for organism selection and sequencing prioritization as was done for the GEBA pilot project (i.e. phylogenetic novelty, availability and growth of cultures of type strains and DNA extraction capability), focusing on type strains as this ensures reproducibility of our results and provides the strongest linkage between genome sequences and other knowledge about each strain. In turn, this project will constitute a pilot phase of a larger effort that will target the genome sequences of all available type strains of the Bacteria and Archaea.« less

Genomic Encyclopedia of Type Strains, Phase I: The one thousand microbial genomes (KMG-I) project

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kyrpides, Nikos C.; Woyke, Tanja; Eisen, Jonathan A.

The Genomic Encyclopedia of Bacteria and Archaea (GEBA) project was launched by the JGI in 2007 as a pilot project with the objective of sequencing 250 bacterial and archaeal genomes. The two major goals of that project were (a) to test the hypothesis that there are many benefits to the use the phylogenetic diversity of organisms in the tree of life as a primary criterion for generating their genome sequence and (b) to develop the necessary framework, technology and organization for large-scale sequencing of microbial isolate genomes. While the GEBA pilot project has not yet been entirely completed, both ofmore » the original goals have already been successfully accomplished, leading the way for the next phase of the project. Here we propose taking the GEBA project to the next level, by generating high quality draft genomes for 1,000 bacterial and archaeal strains. This represents a combined 16-fold increase in both scale and speed as compared to the GEBA pilot project (250 isolate genomes in 4+ years). We will follow a similar approach for organism selection and sequencing prioritization as was done for the GEBA pilot project (i.e. phylogenetic novelty, availability and growth of cultures of type strains and DNA extraction capability), focusing on type strains as this ensures reproducibility of our results and provides the strongest linkage between genome sequences and other knowledge about each strain. In turn, this project will constitute a pilot phase of a larger effort that will target the genome sequences of all available type strains of the Bacteria and Archaea.« less

Single-nucleus analysis of accessible chromatin in developing mouse forebrain reveals cell-type-specific transcriptional regulation.

PubMed

Preissl, Sebastian; Fang, Rongxin; Huang, Hui; Zhao, Yuan; Raviram, Ramya; Gorkin, David U; Zhang, Yanxiao; Sos, Brandon C; Afzal, Veena; Dickel, Diane E; Kuan, Samantha; Visel, Axel; Pennacchio, Len A; Zhang, Kun; Ren, Bing

2018-03-01

Analysis of chromatin accessibility can reveal transcriptional regulatory sequences, but heterogeneity of primary tissues poses a significant challenge in mapping the precise chromatin landscape in specific cell types. Here we report single-nucleus ATAC-seq, a combinatorial barcoding-assisted single-cell assay for transposase-accessible chromatin that is optimized for use on flash-frozen primary tissue samples. We apply this technique to the mouse forebrain through eight developmental stages. Through analysis of more than 15,000 nuclei, we identify 20 distinct cell populations corresponding to major neuronal and non-neuronal cell types. We further define cell-type-specific transcriptional regulatory sequences, infer potential master transcriptional regulators and delineate developmental changes in forebrain cellular composition. Our results provide insight into the molecular and cellular dynamics that underlie forebrain development in the mouse and establish technical and analytical frameworks that are broadly applicable to other heterogeneous tissues.

Phylogeonomics and Ecogenomics of the Mycorrhizal Symbiosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuo, Alan; Grigoriev, Igor V.; Kohler, Annegret

Mycorrhizal fungi play critical roles in host plant health, soil community structure and chemistry, and carbon and nutrient cycling, all areas of intense interest to the US Dept. of Energy (DOE) Joint Genome Institute (JGI). To this end we are building on our earlier sequencing of the Laccaria bicolor genome by partnering with INRA-Nancy and the mycorrhizal research community in the MGI to sequence and analyze 2 dozen mycorrhizal genomes of numerous known mycorrhizal orders and several ecological types (ectomycorrhizal [ECM], ericoid, orchid, and arbuscular). JGI has developed and deployed high-throughput pipelines for genomic, transcriptomic, and re-sequencing, and platforms formore » assembly, annotation, and analysis. In the last 2 years we have sequenced 21 genomes of mycorrhizal fungi, and resequenced 6 additional strains of L. bicolor. Most of this data is publicly available on JGI MycoCosm?s Mycorrhizal Fungi Portal (http://jgi.doe.gov/Mycorrhizal_fungi/), which provides access to both the genome data and tools with which to analyze the data. These data allow us to address long-standing issues in mycorrhizal evolution and ecology. For example, a major observation of mycorrhizal evolution is that each of the major ecological types appears to have evolved independently in multiple fungal clades. Using an ecogenomic approach we provide preliminary evidence that 2 clades (Cantharellales and Sebacinales) of a single symbiotic ecotype (orchid) utilize some common regulatory (protein tyrosine kinase) and metabolic (lipase) paths, the latter of which may be the product of HGT. Using a phylogenomic approach we provide preliminary evidence that a particular ecotype (ericoid) may have evolved more than once within a major clade (Leotiomycetes).« less

Multilocus enzyme electrophoresis and cytochrome B gene sequencing-based identification of Leishmania isolates from different foci of cutaneous leishmaniasis in Pakistan.

PubMed

Marco, Jorge D; Bhutto, Abdul M; Soomro, Farooq R; Baloch, Javed H; Barroso, Paola A; Kato, Hirotomo; Uezato, Hiroshi; Katakura, Ken; Korenaga, Masataka; Nonaka, Shigeo; Hashiguchi, Yoshihisa

2006-08-01

Seventeen Leishmania stocks isolated from cutaneous lesions of Pakistani patients were studied by multilocus enzyme electrophoresis and by polymerase chain reaction amplification and sequencing of the cytochrome b (Cyt b) gene. Eleven stocks that expressed nine zymodemes were assigned to L. (Leishmania) major. All of them were isolated from patients in the lowlands of Larkana district and Sibi city in Sindh and Balochistan provinces, respectively. The remaining six, distributed in two zymodemes (five and one), isolated from the highland of Quetta city, Balochistan, were identified as L. (L.) tropica. The same result at species level was obtained by the Cyt b sequencing for all the stocks examined. No clear-cut association between the clinical features (wet or dry type lesions) and the Leishmania species involved was found. Leishmania (L.) major was highly polymorphic compared with L. (L.) tropica. This difference may be explained by the fact that humans may act as a sole reservoir of L. (L.) tropica in anthroponotic cycles; however, many wild mammals can be reservoirs of L. (L.) major in zoonotic cycles.

Evolution of early life inferred from protein and ribonucleic acid sequences

NASA Technical Reports Server (NTRS)

Dayhoff, M. O.; Schwartz, R. M.

1978-01-01

The chemical structures of ferredoxin, 5S ribosomal RNA, and c-type cytochrome sequences have been employed to construct a phylogenetic tree which connects all major photosynthesizing organisms: the three types of bacteria, blue-green algae, and chloroplasts. Anaerobic and aerobic bacteria, eukaryotic cytoplasmic components and mitochondria are also included in the phylogenetic tree. Anaerobic nonphotosynthesizing bacteria similar to Clostridium were the earliest organisms, arising more than 3.2 billion years ago. Bacterial photosynthesis evolved nearly 3.0 billion years ago, while oxygen-evolving photosynthesis, originating in the blue-green algal line, came into being about 2.0 billion years ago. The phylogenetic tree supports the symbiotic theory of the origin of eukaryotes.

Survival of antibiotic resistant bacteria following artificial solar radiation of secondary wastewater effluent.

PubMed

Glady-Croue, Julie; Niu, Xi-Zhi; Ramsay, Joshua P; Watkin, Elizabeth; Murphy, Riley J T; Croue, Jean-Philippe

2018-06-01

Urban wastewater treatment plant effluents represent one of the major emission sources of antibiotic-resistant bacteria (ARB) in natural aquatic environments. In this study, the effect of artificial solar radiation on total culturable heterotrophic bacteria and ARB (including amoxicillin-resistant, ciprofloxacin-resistant, rifampicin-resistant, sulfamethoxazole-resistant, and tetracycline-resistant bacteria) present in secondary effluent was investigated. Artificial solar radiation was effective in inactivating the majority of environmental bacteria, however, the proportion of strains with ciprofloxacin-resistance and rifampicin-resistance increased in the surviving populations. Isolates of Pseudomonas putida, Serratia marcescens, and Stenotrophomonas maltophilia nosocomial pathogens were identified as resistant to solar radiation and to at least three antibiotics. Draft genome sequencing and typing revealed isolates carrying multiple resistance genes; where S. maltophilia (resistant to all studied antibiotics) sequence type was similar to strains isolated in blood infections. Results from this study confirm that solar radiation reduces total bacterial load in secondary effluent, but may indirectly increase the relative abundance of ARB. Copyright © 2018 Elsevier B.V. All rights reserved.

Molecular Characterization of Streptococcus agalactiae Isolated from Bovine Mastitis in Eastern China

PubMed Central

Yang, Yongchun; Liu, Yinglong; Ding, Yunlei; Yi, Li; Ma, Zhe; Fan, Hongjie; Lu, Chengping

2013-01-01

One hundred and two Streptococcus agalactiae (group B streptococcus [GBS]) isolates were collected from dairy cattle with subclinical mastitis in Eastern China during 2011. Clonal groups were established by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE), respectively. Capsular polysaccharides (CPS), pilus and alpha-like-protein (Alp) family genes were also characterized by molecular techniques. MLST analysis revealed that these isolates were limited to three clonal groups and were clustered in six different lineages, i.e. ST (sequence type) 103, ST568, ST67, ST301, ST313 and ST570, of which ST568 and ST570 were new genotypes. PFGE analysis revealed this isolates were clustered in 27 PFGE types, of which, types 7, 8, 14, 15, 16, 18, 23 and 25 were the eight major types, comprising close to 70% (71/102) of all the isolates. The most prevalent sequence types were ST103 (58% isolates) and ST568 (31% isolates), comprising capsular genotype Ia isolates without any of the detected Alp genes, suggesting the appearance of novel genomic backgrounds of prevalent strains of bovine S. agalactiae. All the strains possessed the pilus island 2b (PI-2b) gene and the prevalent capsular genotypes were types Ia (89% isolates) and II (11% isolates), the conserved pilus type providing suitable data for the development of vaccines against mastitis caused by S. agalactiae. PMID:23874442

Genotypic and phenotypic evaluation of off-type grasses in hybrid Bermudagrass [Cynodon dactylon (L.) Pers. x C. transvaalensis Burtt-Davy] putting greens using genotyping-by-sequencing and morphological characterization.

PubMed

Reasor, Eric H; Brosnan, James T; Staton, Margaret E; Lane, Thomas; Trigiano, Robert N; Wadl, Phillip A; Conner, Joann A; Schwartz, Brian M

2018-01-01

Interspecific hybrid bermudagrass [ Cynodon dactylon (L.) Pers. x C. transvaalensis Burtt-Davy] is one of the most widely used grasses on golf courses, with cultivars derived from 'Tifgreen' or 'Tifdwarf' particularly used for putting greens. Many bermudagrass cultivars established for putting greens can be genetically unstable and lead to the occurrence of undesirable off-type grasses that vary in phenotype. The objective of this research was to genetically and phenotypically differentiate off-type grasses and hybrid cultivars. Beginning in 2013, off-type and desirable hybrid bermudagrass samples were collected from golf course putting greens in the southeastern United States and genetically and phenotypically characterized using genotyping-by-sequencing and morphology. Genotyping-by-sequencing determined that 11% (5) of off-type and desirable samples from putting greens were genetically divergent from standard cultivars such as Champion, MiniVerde, Tifdwarf, TifEagle, and Tifgreen. In addition, genotyping-by-sequencing was unable to genetically distinguish all standard cultivars from one another due to their similar origin and clonal propagation; however, over 90,000 potentially informative nucleotide variants were identified among the triploid hybrid cultivars. Although few genetic differences were found in this research, samples harvested from golf course putting greens had variable morphology and were clustered into three distinct phenotypic groups. The majority of off-type grasses in hybrid bermudagrass putting greens were genetically similar with variable morphological traits. Off-type grasses within golf course putting greens have the potential to compromise putting surface functionality and aesthetics.

Phylogenetic Distribution of CRISPR-Cas Systems in Antibiotic-Resistant Pseudomonas aeruginosa.

PubMed

van Belkum, Alex; Soriaga, Leah B; LaFave, Matthew C; Akella, Srividya; Veyrieras, Jean-Baptiste; Barbu, E Magda; Shortridge, Dee; Blanc, Bernadette; Hannum, Gregory; Zambardi, Gilles; Miller, Kristofer; Enright, Mark C; Mugnier, Nathalie; Brami, Daniel; Schicklin, Stéphane; Felderman, Martina; Schwartz, Ariel S; Richardson, Toby H; Peterson, Todd C; Hubby, Bolyn; Cady, Kyle C

2015-11-24

Pseudomonas aeruginosa is an antibiotic-refractory pathogen with a large genome and extensive genotypic diversity. Historically, P. aeruginosa has been a major model system for understanding the molecular mechanisms underlying type I clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas)-based bacterial immune system function. However, little information on the phylogenetic distribution and potential role of these CRISPR-Cas systems in molding the P. aeruginosa accessory genome and antibiotic resistance elements is known. Computational approaches were used to identify and characterize CRISPR-Cas systems within 672 genomes, and in the process, we identified a previously unreported and putatively mobile type I-C P. aeruginosa CRISPR-Cas system. Furthermore, genomes harboring noninhibited type I-F and I-E CRISPR-Cas systems were on average ~300 kb smaller than those without a CRISPR-Cas system. In silico analysis demonstrated that the accessory genome (n = 22,036 genes) harbored the majority of identified CRISPR-Cas targets. We also assembled a global spacer library that aided the identification of difficult-to-characterize mobile genetic elements within next-generation sequencing (NGS) data and allowed CRISPR typing of a majority of P. aeruginosa strains. In summary, our analysis demonstrated that CRISPR-Cas systems play an important role in shaping the accessory genomes of globally distributed P. aeruginosa isolates. P. aeruginosa is both an antibiotic-refractory pathogen and an important model system for type I CRISPR-Cas bacterial immune systems. By combining the genome sequences of 672 newly and previously sequenced genomes, we were able to provide a global view of the phylogenetic distribution, conservation, and potential targets of these systems. This analysis identified a new and putatively mobile P. aeruginosa CRISPR-Cas subtype, characterized the diverse distribution of known CRISPR-inhibiting genes, and provided a potential new use for CRISPR spacer libraries in accessory genome analysis. Our data demonstrated the importance of CRISPR-Cas systems in modulating the accessory genomes of globally distributed strains while also providing substantial data for subsequent genomic and experimental studies in multiple fields. Understanding why certain genotypes of P. aeruginosa are clinically prevalent and adept at horizontally acquiring virulence and antibiotic resistance elements is of major clinical and economic importance. Copyright © 2015 van Belkum et al.

Hymenobacter aquatilis sp. nov., isolated from a mesotrophic artificial lake.

PubMed

Kang, Heeyoung; Cha, Inseong; Kim, Haneul; Joh, Kiseong

2018-06-01

A novel strain, designated HMF3095 T , isolated from freshwater of a mesotrophic artificial lake in the Republic of Korea, was characterized by polyphasic taxonomy. The cells were Gram-stain-negative, aerobic, non-motile, straight rods and formed reddish colonies. Phylogenetic analysis based on its 16S rRNA gene sequence revealed that strain HMF3095 T fell within the cluster of the genus Hymenobacterand was most closely related to Hymenobacter seoulensis 16F7G T and Hymenobacter tenuis POB6 T (96.7 % sequence similarity). Sequence similarities to all other type strains were 96.3 % or less. The major fatty acids were iso-C15 : 0, C16 : 1ω5c, summed feature 4 (iso-C17 : 1 I and/or anteiso-C17 : 1 B), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and anteiso-C15 : 0. The major isoprenoid quinone was menaquinone 7. The major polar lipids were phosphatidylethanolamine, three unidentified aminophospholipids and one unidentified phospholipid. The DNA G+C content was 58.9 mol%. On the basis of the evidence presented in this study, strain HMF3095 T represents a novel species of the genus Hymenobacter, for which the name Hymenobacter aquatilis sp. nov. is proposed. The type strain is HMF3095 T (=KCTC 52398 T =NBRC 112669 T ).

BrucellaBase: Genome information resource.

PubMed

Sankarasubramanian, Jagadesan; Vishnu, Udayakumar S; Khader, L K M Abdul; Sridhar, Jayavel; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

2016-09-01

Brucella sp. causes a major zoonotic disease, brucellosis. Brucella belongs to the family Brucellaceae under the order Rhizobiales of Alphaproteobacteria. We present BrucellaBase, a web-based platform, providing features of a genome database together with unique analysis tools. We have developed a web version of the multilocus sequence typing (MLST) (Whatmore et al., 2007) and phylogenetic analysis of Brucella spp. BrucellaBase currently contains genome data of 510 Brucella strains along with the user interfaces for BLAST, VFDB, CARD, pairwise genome alignment and MLST typing. Availability of these tools will enable the researchers interested in Brucella to get meaningful information from Brucella genome sequences. BrucellaBase will regularly be updated with new genome sequences, new features along with improvements in genome annotations. BrucellaBase is available online at http://www.dbtbrucellosis.in/brucellabase.html or http://59.99.226.203/brucellabase/homepage.html. Copyright © 2016 Elsevier B.V. All rights reserved.

Quiet aircraft design and operational characteristics

NASA Technical Reports Server (NTRS)

Hodge, Charles G.

1991-01-01

The application of aircraft noise technology to the design and operation of aircraft is discussed. Areas of discussion include the setting of target airplane noise levels, operational considerations and their effect on noise, and the sequencing and timing of the design and development process. Primary emphasis is placed on commercial transport aircraft of the type operated by major airlines. Additionally, noise control engineering of other types of aircraft is briefly discussed.

Integrative analysis workflow for the structural and functional classification of C-type lectins

PubMed Central

2011-01-01

Background It is important to understand the roles of C-type lectins in the immune system due to their ubiquity and diverse range of functions in animal cells. It has been observed that currently confirmed C-type lectins share a highly conserved domain known as the C-type carbohydrate recognition domain (CRD). Using the sequence profile of the CRD, an increasing number of putative C-type lectins have been identified. Hence, it is highly needed to develop a systematic framework that enables us to elucidate their carbohydrate (glycan) recognition function, and discover their physiological and pathological roles. Results Presented herein is an integrated workflow for characterizing the sequence and structural features of novel C-type lectins. Our workflow utilizes web-based queries and available software suites to annotate features that can be found on the C-type lectin, given its amino acid sequence. At the same time, it incorporates modeling and analysis of glycans - a major class of ligands that interact with C-type lectins. Thereafter, the results are analyzed together with context-specific knowledge to filter off unlikely predictions. This allows researchers to design their subsequent experiments to confirm the functions of the C-type lectins in a systematic manner. Conclusions The efficacy and usefulness of our proposed immunoinformatics workflow was demonstrated by applying our integrated workflow to a novel C-type lectin -CLEC17A - and we report some of its possible functions that warrants further validation through wet-lab experiments. PMID:22372988

Metallo-β-lactamase-producing Pseudomonas aeruginosa in the Netherlands: the nationwide emergence of a single sequence type.

PubMed

Van der Bij, A K; Van der Zwan, D; Peirano, G; Severin, J A; Pitout, J D D; Van Westreenen, M; Goessens, W H F

2012-09-01

Recently, the first outbreak of clonally related VIM-2 metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa in a Dutch tertiary-care centre was described. Subsequently, a nationwide surveillance study was performed in 2010-2011, which identified the presence of VIM-2 MBL-producing P. aeruginosa in 11 different hospitals. Genotyping by multiple-locus variable-number tandem-repeat analysis (MLVA) showed that the majority of the 82 MBL-producing isolates found belonged to a single MLVA type (n = 70, 85%), identified as ST111 by multilocus sequence typing (MLST). As MBL-producing isolates cause serious infections that are difficult to treat, the presence of clonally related isolates in various hospitals throughout the Netherlands is of nationwide concern. © 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

SPAR: small RNA-seq portal for analysis of sequencing experiments.

PubMed

Kuksa, Pavel P; Amlie-Wolf, Alexandre; Katanic, Živadin; Valladares, Otto; Wang, Li-San; Leung, Yuk Yee

2018-05-04

The introduction of new high-throughput small RNA sequencing protocols that generate large-scale genomics datasets along with increasing evidence of the significant regulatory roles of small non-coding RNAs (sncRNAs) have highlighted the urgent need for tools to analyze and interpret large amounts of small RNA sequencing data. However, it remains challenging to systematically and comprehensively discover and characterize sncRNA genes and specifically-processed sncRNA products from these datasets. To fill this gap, we present Small RNA-seq Portal for Analysis of sequencing expeRiments (SPAR), a user-friendly web server for interactive processing, analysis, annotation and visualization of small RNA sequencing data. SPAR supports sequencing data generated from various experimental protocols, including smRNA-seq, short total RNA sequencing, microRNA-seq, and single-cell small RNA-seq. Additionally, SPAR includes publicly available reference sncRNA datasets from our DASHR database and from ENCODE across 185 human tissues and cell types to produce highly informative small RNA annotations across all major small RNA types and other features such as co-localization with various genomic features, precursor transcript cleavage patterns, and conservation. SPAR allows the user to compare the input experiment against reference ENCODE/DASHR datasets. SPAR currently supports analyses of human (hg19, hg38) and mouse (mm10) sequencing data. SPAR is freely available at https://www.lisanwanglab.org/SPAR.

Introgression from Domestic Goat Generated Variation at the Major Histocompatibility Complex of Alpine Ibex

PubMed Central

Grossen, Christine; Keller, Lukas; Biebach, Iris; Croll, Daniel

2014-01-01

The major histocompatibility complex (MHC) is a crucial component of the vertebrate immune system and shows extremely high levels of genetic polymorphism. The extraordinary genetic variation is thought to be ancient polymorphisms maintained by balancing selection. However, introgression from related species was recently proposed as an additional mechanism. Here we provide evidence for introgression at the MHC in Alpine ibex (Capra ibex ibex). At a usually very polymorphic MHC exon involved in pathogen recognition (DRB exon 2), Alpine ibex carried only two alleles. We found that one of these DRB alleles is identical to a DRB allele of domestic goats (Capra aegagrus hircus). We sequenced 2489 bp of the coding and non-coding regions of the DRB gene and found that Alpine ibex homozygous for the goat-type DRB exon 2 allele showed nearly identical sequences (99.8%) to a breed of domestic goats. Using Sanger and RAD sequencing, microsatellite and SNP chip data, we show that the chromosomal region containing the goat-type DRB allele has a signature of recent introgression in Alpine ibex. A region of approximately 750 kb including the DRB locus showed high rates of heterozygosity in individuals carrying one copy of the goat-type DRB allele. These individuals shared SNP alleles both with domestic goats and other Alpine ibex. In a survey of four Alpine ibex populations, we found that the region surrounding the DRB allele shows strong linkage disequilibria, strong sequence clustering and low diversity among haplotypes carrying the goat-type allele. Introgression at the MHC is likely adaptive and introgression critically increased MHC DRB diversity in the genetically impoverished Alpine ibex. Our finding contradicts the long-standing view that genetic variability at the MHC is solely a consequence of ancient trans-species polymorphism. Introgression is likely an underappreciated source of genetic diversity at the MHC and other loci under balancing selection. PMID:24945814

Transposon mutagenesis of type III group B Streptococcus: correlation of capsule expression with virulence.

PubMed

Rubens, C E; Wessels, M R; Heggen, L M; Kasper, D L

1987-10-01

The capsular polysaccharide of type III group B Streptococcus (GBS) is thought to be a major factor in the virulence of this organism. Transposon mutagenesis was used to obtain isogenic strains of a GBS serotype III clinical isolate (COH 31r/s) with site-specific mutations in the gene(s) responsible for capsule production. The self-conjugative transposon Tn916 was transferred to strain COH 31r/s during incubation with Streptococcus faecalis strain CG110 on membrane filters. Eleven transconjugant clones did not bind type III GBS antiserum by immunoblot. Immunofluorescence, competitive ELISA, and electron microscopy confirmed the absence of detectable GBS type III capsular polysaccharide in one of the transconjugants, COH 31-15. Southern hybridization analysis with a Tn916 probe confirmed the presence of the transposon sequence within each mutant. A 3.0-kilobase EcoRI fragment that flanked the Tn916 sequence was subcloned from mutant COH 31-15. This fragment shared homology with DNA from the other GBS serotypes, suggesting a common sequence for capsulation shared by organisms of different capsular types. Loss of capsule expression resulted in loss of virulence in a neonatal rat model. We conclude that a gene common to all capsular types of GBS is required for surface expression of the type III capsule and that inactivation of this gene by Tn916 results in the loss of virulence.

Flagellin diversity in Clostridium botulinum groups I and II: a new strategy for strain identification.

PubMed

Paul, Catherine J; Twine, Susan M; Tam, Kevin J; Mullen, James A; Kelly, John F; Austin, John W; Logan, Susan M

2007-05-01

Strains of Clostridium botulinum are traditionally identified by botulinum neurotoxin type; however, identification of an additional target for typing would improve differentiation. Isolation of flagellar filaments and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that C. botulinum produced multiple flagellin proteins. Nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis of in-gel tryptic digests identified peptides in all flagellin bands that matched two homologous tandem flagellin genes identified in the C. botulinum Hall A genome. Designated flaA1 and flaA2, these open reading frames encode the major structural flagellins of C. botulinum. Colony PCR and sequencing of flaA1/A2 variable regions classified 80 environmental and clinical strains into group I or group II and clustered isolates into 12 flagellar types. Flagellar type was distinct from neurotoxin type, and epidemiologically related isolates clustered together. Sequencing a larger PCR product, obtained during amplification of flaA1/A2 from type E strain Bennett identified a second flagellin gene, flaB. LC-MS analysis confirmed that flaB encoded a large type E-specific flagellin protein, and the predicted molecular mass for FlaB matched that observed by SDS-PAGE. In contrast, the molecular mass of FlaA was 2 to 12 kDa larger than the mass predicted by the flaA1/A2 sequence of a given strain, suggesting that FlaA is posttranslationally modified. While identification of FlaB, and the observation by SDS-PAGE of different masses of the FlaA proteins, showed the flagellin proteins of C. botulinum to be diverse, the presence of the flaA1/A2 gene in all strains examined facilitates single locus sequence typing of C. botulinum using the flagellin variable region.

Micromonospora luteifusca sp. nov. isolated from cultivated Pisum sativum.

PubMed

Carro, Lorena; Riesco, Raúl; Spröer, Cathrin; Trujillo, Martha E

2016-06-01

Three novel actinobacterial strains, GUI2(T), GUI42 and CR21 isolated from nodular tissues and the rhizosphere of a sweet pea plant collected in Cañizal, Spain were identified according to their 16S rRNA gene sequences as new members of the genus Micromonospora. The closest phylogenetic members were found to be Micromonospora saelicesensis (99.2%) "Micromonospora zeae" (99.1%), "Micromonospora jinlongensis" (99%), Micromonospora lupini (98.9%) and Micromonospora zamorensis (98.8%). To resolve their full taxonomic position, four additional genes (atpD, gyrB, recA, rpoB) were partially sequenced and compared to available Micromonospora type strain sequences. DNA-DNA hybridization, BOX-PCR and ARDRA profiles confirmed that these strains represent a novel genomic species. All strains contained meso-diaminopimelic and hydroxy-diaminopimelic acids in their cell wall. Their fatty acid profiles comprised iso-C15:0, iso-C16:0 and anteiso-C15:0 as major components. The polar lipids diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol were found in the type strain GUI2(T) which also contained MK-10(H4) as the major menaquinone. Physiological and biochemical characteristics also differentiated the new isolates. Based on the integration of the above studies, strains GUI2(T), GUI42 and CR21 represent a novel Micromonospora species and we propose the name Micromonospora luteifusca sp. nov. The type strain is GUI2(T) (=CECT 8846(T); =DSM 100204(T)). Copyright © 2016 Elsevier GmbH. All rights reserved.

Sequencing of the variable region of rpsB to discriminate between Streptococcus pneumoniae and other streptococcal species.

PubMed

Wyllie, Anne L; Pannekoek, Yvonne; Bovenkerk, Sandra; van Engelsdorp Gastelaars, Jody; Ferwerda, Bart; van de Beek, Diederik; Sanders, Elisabeth A M; Trzciński, Krzysztof; van der Ende, Arie

2017-09-01

The vast majority of streptococci colonizing the human upper respiratory tract are commensals, only sporadically implicated in disease. Of these, the most pathogenic is Mitis group member, Streptococcus pneumoniae Phenotypic and genetic similarities between streptococci can cause difficulties in species identification. Using ribosomal S2-gene sequences extracted from whole-genome sequences published from 501 streptococci, we developed a method to identify streptococcal species. We validated this method on non-pneumococcal isolates cultured from cases of severe streptococcal disease ( n = 101) and from carriage ( n = 103), and on non-typeable pneumococci from asymptomatic individuals ( n = 17) and on whole-genome sequences of 1157 pneumococcal isolates from meningitis in the Netherlands. Following this, we tested 221 streptococcal isolates in molecular assays originally assumed specific for S. pneumoniae , targeting cpsA , lytA , piaB , ply , Spn9802, zmpC and capsule-type-specific genes. Cluster analysis of S2-sequences showed grouping according to species in line with published phylogenies of streptococcal core genomes. S2-typing convincingly distinguished pneumococci from non-pneumococcal species (99.2% sensitivity, 100% specificity). Molecular assays targeting regions of lytA and piaB were 100% specific for S. pneumoniae , whereas assays targeting cpsA , ply , Spn9802, zmpC and selected serotype-specific assays (but not capsular sequence typing) showed a lack of specificity. False positive results were over-represented in species associated with carriage, although no particular confounding signal was unique for carriage isolates. © 2017 The Authors.

Sequencing of the variable region of rpsB to discriminate between Streptococcus pneumoniae and other streptococcal species

PubMed Central

Pannekoek, Yvonne; Bovenkerk, Sandra; van Engelsdorp Gastelaars, Jody; Ferwerda, Bart; van de Beek, Diederik; Sanders, Elisabeth A. M.; Trzciński, Krzysztof; van der Ende, Arie

2017-01-01

The vast majority of streptococci colonizing the human upper respiratory tract are commensals, only sporadically implicated in disease. Of these, the most pathogenic is Mitis group member, Streptococcus pneumoniae. Phenotypic and genetic similarities between streptococci can cause difficulties in species identification. Using ribosomal S2-gene sequences extracted from whole-genome sequences published from 501 streptococci, we developed a method to identify streptococcal species. We validated this method on non-pneumococcal isolates cultured from cases of severe streptococcal disease (n = 101) and from carriage (n = 103), and on non-typeable pneumococci from asymptomatic individuals (n = 17) and on whole-genome sequences of 1157 pneumococcal isolates from meningitis in the Netherlands. Following this, we tested 221 streptococcal isolates in molecular assays originally assumed specific for S. pneumoniae, targeting cpsA, lytA, piaB, ply, Spn9802, zmpC and capsule-type-specific genes. Cluster analysis of S2-sequences showed grouping according to species in line with published phylogenies of streptococcal core genomes. S2-typing convincingly distinguished pneumococci from non-pneumococcal species (99.2% sensitivity, 100% specificity). Molecular assays targeting regions of lytA and piaB were 100% specific for S. pneumoniae, whereas assays targeting cpsA, ply, Spn9802, zmpC and selected serotype-specific assays (but not capsular sequence typing) showed a lack of specificity. False positive results were over-represented in species associated with carriage, although no particular confounding signal was unique for carriage isolates. PMID:28931649

Genetic analysis of human immunodeficiency virus type 1 envelope V3 region isolates from mothers and infants after perinatal transmission.

PubMed Central

Ahmad, N; Baroudy, B M; Baker, R C; Chappey, C

1995-01-01

The human immunodeficiency virus type 1 (HIV-1) sequences from variable region 3 (V3) of the envelope gene were analyzed from seven infected mother-infant pairs following perinatal transmission. The V3 region sequences directly derived from the DNA of the uncultured peripheral blood mononuclear cells from infected mothers displayed a heterogeneous population. In contrast, the infants' sequences were less diverse than those of their mothers. In addition, the sequences from the younger infants' peripheral blood mononuclear cell DNA were more homogeneous than the older infants' sequences. All infants' sequences were different but displayed patterns similar to those seen in their mothers. In the mother-infant pair sequences analyzed, a minor genotype or subtype found in the mothers predominated in their infants. The conserved N-linked glycosylation site proximal to the first cysteine of the V3 loop was absent only in one infant's sequence set and in some variants of two other infants' sequences. Furthermore, the HIV-1 sequences of the epidemiologically linked mother-infant pairs were closer than the sequences of epidemiologically unlinked individuals, suggesting that the sequence comparison of mother-infant pairs done in order to identify genetic variants transmitted from mother to infant could be performed even in older infants. There was no evidence for transmission of a major genotype or multiple genotypes from mother to infant. In conclusion, a minor genotype of maternal virus is transmitted to the infants, and this finding could be useful in developing strategies to prevent maternal transmission of HIV-1 by means of perinatal interventions. PMID:7815476

Molecular Modeling and Structural Stability of Wild-Type and Mutant CYP51 from Leishmania major: In Vitro and In Silico Analysis of a Laboratory Strain.

PubMed

Keighobadi, Masoud; Emami, Saeed; Lagzian, Milad; Fakhar, Mahdi; Rafiei, Alireza; Valadan, Reza

2018-03-19

Cutaneous leishmaniasis is a neglected tropical disease and a major public health in the most countries. Leishmania major is the most common cause of cutaneous leishmaniasis. In the Leishmania parasites, sterol 14α-demethylase (CYP51), which is involved in the biosynthesis of sterols, has been identified as an attractive target for development of new therapeutic agents. In this study, the sequence and structure of CYP51 in a laboratory strain (MRHO/IR/75/ER) of L. major were determined and compared to the wild-type strain. The results showed 19 mutations including seven non-synonymous and 12 synonymous ones in the CYP51 sequence of strain MRHO/IR/75/ER. Importantly, an arginine to lysine substitution at position of 474 resulted in destabilization of CYP51 (ΔΔG = 1.17 kcal/mol) in the laboratory strain; however, when the overall effects of all substitutions were evaluated by 100 ns molecular dynamics simulation, the final structure did not show any significant changes ( p -value < 0.05) in stability parameter of the strain MRHO/IR/75/ER compared to the wild-type protein. The energy level for the CYP51 of wild-type and MRHO/IR/75/ER strain were -40,027.1 and -39,706.48 Kcal/mol respectively. The overall Root-mean-square deviation (RMSD) deviation between two proteins was less than 1 Å throughout the simulation and Root-mean-square fluctuation (RMSF) plot also showed no substantial differences between amino acids fluctuation of the both protein. The results also showed that, these mutations were located on the protein periphery that neither interferes with protein folding nor with substrate/inhibitor binding. Therefore, L. major strain MRHO/IR/75/ER is suggested as a suitable laboratory model for studying biological role of CYP51 and inhibitory effects of sterol 14α-demethylase inhibitors.

Preferential access to genetic information from endogenous hominin ancient DNA and accurate quantitative SNP-typing via SPEX

PubMed Central

Brotherton, Paul; Sanchez, Juan J.; Cooper, Alan; Endicott, Phillip

2010-01-01

The analysis of targeted genetic loci from ancient, forensic and clinical samples is usually built upon polymerase chain reaction (PCR)-generated sequence data. However, many studies have shown that PCR amplification from poor-quality DNA templates can create sequence artefacts at significant levels. With hominin (human and other hominid) samples, the pervasive presence of highly PCR-amplifiable human DNA contaminants in the vast majority of samples can lead to the creation of recombinant hybrids and other non-authentic artefacts. The resulting PCR-generated sequences can then be difficult, if not impossible, to authenticate. In contrast, single primer extension (SPEX)-based approaches can genotype single nucleotide polymorphisms from ancient fragments of DNA as accurately as modern DNA. A single SPEX-type assay can amplify just one of the duplex DNA strands at target loci and generate a multi-fold depth-of-coverage, with non-authentic recombinant hybrids reduced to undetectable levels. Crucially, SPEX-type approaches can preferentially access genetic information from damaged and degraded endogenous ancient DNA templates over modern human DNA contaminants. The development of SPEX-type assays offers the potential for highly accurate, quantitative genotyping from ancient hominin samples. PMID:19864251

Localization of migraine susceptibility genes in human brain by single-cell RNA sequencing.

PubMed

Renthal, William

2018-01-01

Background Migraine is a debilitating disorder characterized by severe headaches and associated neurological symptoms. A key challenge to understanding migraine has been the cellular complexity of the human brain and the multiple cell types implicated in its pathophysiology. The present study leverages recent advances in single-cell transcriptomics to localize the specific human brain cell types in which putative migraine susceptibility genes are expressed. Methods The cell-type specific expression of both familial and common migraine-associated genes was determined bioinformatically using data from 2,039 individual human brain cells across two published single-cell RNA sequencing datasets. Enrichment of migraine-associated genes was determined for each brain cell type. Results Analysis of single-brain cell RNA sequencing data from five major subtypes of cells in the human cortex (neurons, oligodendrocytes, astrocytes, microglia, and endothelial cells) indicates that over 40% of known migraine-associated genes are enriched in the expression profiles of a specific brain cell type. Further analysis of neuronal migraine-associated genes demonstrated that approximately 70% were significantly enriched in inhibitory neurons and 30% in excitatory neurons. Conclusions This study takes the next step in understanding the human brain cell types in which putative migraine susceptibility genes are expressed. Both familial and common migraine may arise from dysfunction of discrete cell types within the neurovascular unit, and localization of the affected cell type(s) in an individual patient may provide insight into to their susceptibility to migraine.

Multilocus Sequence Typing Analysis of Staphylococcus lugdunensis Implies a Clonal Population Structure

PubMed Central

Chassain, Benoît; Lemée, Ludovic; Didi, Jennifer; Thiberge, Jean-Michel; Brisse, Sylvain; Pons, Jean-Louis

2012-01-01

Staphylococcus lugdunensis is recognized as one of the major pathogenic species within the genus Staphylococcus, even though it belongs to the coagulase-negative group. A multilocus sequence typing (MLST) scheme was developed to study the genetic relationships and population structure of 87 S. lugdunensis isolates from various clinical and geographic sources by DNA sequence analysis of seven housekeeping genes (aroE, dat, ddl, gmk, ldh, recA, and yqiL). The number of alleles ranged from four (gmk and ldh) to nine (yqiL). Allelic profiles allowed the definition of 20 different sequence types (STs) and five clonal complexes. The 20 STs lacked correlation with geographic source. Isolates recovered from hematogenic infections (blood or osteoarticular isolates) or from skin and soft tissue infections did not cluster in separate lineages. Penicillin-resistant isolates clustered mainly in one clonal complex, unlike glycopeptide-tolerant isolates, which did not constitute a distinct subpopulation within S. lugdunensis. Phylogenies from the sequences of the seven individual housekeeping genes were congruent, indicating a predominantly mutational evolution of these genes. Quantitative analysis of the linkages between alleles from the seven loci revealed a significant linkage disequilibrium, thus confirming a clonal population structure for S. lugdunensis. This first MLST scheme for S. lugdunensis provides a new tool for investigating the macroepidemiology and phylogeny of this unusually virulent coagulase-negative Staphylococcus. PMID:22785196

Erwinia amylovora effector protein Eop1 suppresses PAMP-triggered immunity in Malus

USDA-ARS?s Scientific Manuscript database

Erwinia amylovora (Ea) utilizes a type three secretion system (T3SS) to deliver effector proteins into plant host cells. Several Ea effectors have been identified based on their sequence similarity to plant and animal bacterial pathogen effectors; however, the function of the majority of Ea effecto...

"Borsenberichte": A Tip for Beginning Business German.

ERIC Educational Resources Information Center

Heinemann, Paul

1994-01-01

This paper discusses the reasons stock market reports of the type appearing in major newspapers in the German-speaking countries are ideal for beginning a text/unit sequence in an introductory business German course. Suggestions are offered concerning the in-class implementation of "Borsenberichte" for third-year students of German. (JL)

Presence of Type I-F CRISPR/Cas systems is associated with antimicrobial susceptibility in Escherichia coli.

PubMed

Aydin, Seyid; Personne, Yoann; Newire, Enas; Laverick, Rebecca; Russell, Oliver; Roberts, Adam P; Enne, Virve I

2017-08-01

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated cas genes are sequence-specific DNA nuclease systems found in bacteria and archaea. CRISPR/Cas systems use RNA transcripts of previously acquired DNA (spacers) to target invading genetic elements with the same sequence, including plasmids. In this research we studied the relationship between CRISPR/Cas systems and multidrug resistance in Escherichia coli . The presence of Type I-E and Type I-F CRISPR systems was investigated among 82 antimicrobial-susceptible and 96 MDR clinical E. coli isolates by PCR and DNA sequencing. Phylogrouping and MLST were performed to determine relatedness of isolates. RT-PCR was performed to ascertain the expression of associated cas genes. Type I-F CRISPR was associated with the B2 phylogroup and was significantly overrepresented in the susceptible group (22.0%) compared with the MDR group (2.1%). The majority of CRISPR I-F-containing isolates had spacer sequences that matched IncF and IncI plasmids. RT-PCR demonstrated that Type I-F cas genes were expressed and therefore potentially functional. The CRISPR I-F system is more likely to be found in antimicrobial-susceptible E. coli . Given that the Type I-F system is expressed in WT isolates, we suggest that this difference could be due to the CRISPR system potentially interfering with the acquisition of antimicrobial resistance plasmids, maintaining susceptibility in these isolates. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Analysis of sequencing data for probing RNA secondary structures and protein-RNA binding in studying posttranscriptional regulations.

PubMed

Hu, Xihao; Wu, Yang; Lu, Zhi John; Yip, Kevin Y

2016-11-01

High-throughput sequencing has been used to study posttranscriptional regulations, where the identification of protein-RNA binding is a major and fast-developing sub-area, which is in turn benefited by the sequencing methods for whole-transcriptome probing of RNA secondary structures. In the study of RNA secondary structures using high-throughput sequencing, bases are modified or cleaved according to their structural features, which alter the resulting composition of sequencing reads. In the study of protein-RNA binding, methods have been proposed to immuno-precipitate (IP) protein-bound RNA transcripts in vitro or in vivo By sequencing these transcripts, the protein-RNA interactions and the binding locations can be identified. For both types of data, read counts are affected by a combination of confounding factors, including expression levels of transcripts, sequence biases, mapping errors and the probing or IP efficiency of the experimental protocols. Careful processing of the sequencing data and proper extraction of important features are fundamentally important to a successful analysis. Here we review and compare different experimental methods for probing RNA secondary structures and binding sites of RNA-binding proteins (RBPs), and the computational methods proposed for analyzing the corresponding sequencing data. We suggest how these two types of data should be integrated to study the structural properties of RBP binding sites as a systematic way to better understand posttranscriptional regulations. © The Author 2015. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

Comparison of variable region 3 sequences of human immunodeficiency virus type 1 from infected children with the RNA and DNA sequences of the virus populations of their mothers.

PubMed Central

Scarlatti, G; Leitner, T; Halapi, E; Wahlberg, J; Marchisio, P; Clerici-Schoeller, M A; Wigzell, H; Fenyö, E M; Albert, J; Uhlén, M

1993-01-01

We have compared the variable region 3 sequences from 10 human immunodeficiency virus type 1 (HIV-1)-infected infants to virus sequences from the corresponding mothers. The sequences were derived from DNA of uncultured peripheral blood mononuclear cells (PBMC), DNA of cultured PBMC, and RNA from serum collected at or shortly after delivery. The infected infants, in contrast to the mothers, harbored homogeneous virus populations. Comparison of sequences from the children and clones derived from DNA of the corresponding mothers showed that the transmitted virus represented either a minor or a major virus population of the mother. In contrast to an earlier study, we found no evidence of selection of minor virus variants during transmission. Furthermore, the transmitted virus variant did not show any characteristic molecular features. In some cases the transmitted virus was more related to the virus RNA population of the mother and in other cases it was more related to the virus DNA population. This suggests that either cell-free or cell-associated virus may be transmitted. These data will help AIDS researchers to understand the mechanism of transmission and to plan strategies for prevention of transmission. PMID:8446584

Aliisedimentitalea scapharcae gen. nov., sp. nov., isolated from ark shell Scapharca broughtonii.

PubMed

Kim, Young-Ok; Park, Sooyeon; Nam, Bo-Hye; Kim, Dong-Gyun; Won, Sung-Min; Park, Ji-Min; Yoon, Jung-Hoon

2015-08-01

A Gram-negative, aerobic, non-spore-forming, motile and ovoid or rod-shaped bacterial strain, designated MA2-16(T), was isolated from ark shell (Scapharca broughtonii) collected from the South Sea, South Korea. Strain MA2-16(T) was found to grow optimally at 30°C, at pH 7.0-8.0 and in the presence of 2.0% (w/v) NaCl. Neighbour-joining, maximum-likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences revealed that strain MA2-16(T) clustered with the type strain of Sedimentitalea nanhaiensis. The novel strain exhibited a 16S rRNA gene sequence similarity value of 97.1% to the type strain of S. nanhaiensis. In the neighbour-joining phylogenetic tree based on gyrB sequences, strain MA2-16(T) formed an evolutionary lineage independent of those of other taxa. Strain MA2-16(T) contained Q-10 as the predominant ubiquinone and C18:1 ω7c and 11-methyl C18:1 ω7c as the major fatty acids. The major polar lipids of strain MA2-16(T) were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, an unidentified aminolipid and an unidentified lipid. The DNA G+C content of strain MA2-16(T) was 57.7 mol% and its DNA-DNA relatedness values with the type strains of S. nanhaiensis and some phylogenetically related species of the genera Leisingera and Phaeobacter were 13-24%. On the basis of the data presented, strain MA2-16(T) is considered to represent a novel genus and novel species within the family Rhodobacteraceae, for which the name Aliisedimentitalea scapharcae gen. nov., sp. nov. is proposed. The type strain is MA2-16(T) (=KCTC 42119(T) =CECT 8598(T)).

A complementarity-determining region synthetic peptide acts as a miniantibody and neutralizes human immunodeficiency virus type 1 in vitro.

PubMed Central

Levi, M; Sällberg, M; Rudén, U; Herlyn, D; Maruyama, H; Wigzell, H; Marks, J; Wahren, B

1993-01-01

A complementarity-determining region (CDR) of the mouse monoclonal antibody (mAb) F58 was constructed with specificity to a neutralization-inducing region of human immunodeficiency virus type 1 (HIV-1). The mAb has its major reactivity to the amino acid sequence I--GPGRA in the V3 viral envelope region. All CDRs including several framework amino acids were synthesized from the sequence deduced by cloning and sequencing mAb F58 heavy- and light-chain variable domains. Peptides derived from the third heavy-chain domain (CDR-H3) alone or in combination with the other CDR sequences competed with F58 mAb for the V3 region. The CDR-H3 peptide was chemically modified by cyclization and then inhibited HIV-1 replication as well as syncytium formation by infected cells. Both the homologous IIIB viral strain to which the F58 mAb was induced and the heterologous SF2 strain were inhibited. This synthetic peptide had unexpectedly potent antiviral activity and may be a potential tool for treatment of HIV-infected persons. PMID:7685100

Human T-lymphotropic virus type 1 (HTLV-1) genetic typing in Kakeroma Island, an island at the crossroads of the ryukyuans and Wajin in Japan, providing further insights into the origin of the virus in Japan.

PubMed

Eguchi, Katsuyuki; Fujii, Hidefumi; Oshima, Kengo; Otani, Masashi; Matsuo, Toshiaki; Yamamoto, Taro

2009-08-01

Peripheral blood samples were collected from 23 human T-lymphotropic virus type-1 (HTLV-1) carriers residing in Kakeroma Island, Japan (Kagoshima Prefecture, Oshima County, Setouchi Town), one of the most highly endemic areas in Japan. The samples were subjected to amplification by PCR and sequencing of the Long Terminal Repeat in order to reconstruct a phylogenetic tree of HTLV-1 isolates. Restriction Fragment Length Polymorphism (RFLP) analysis of env region was also conducted for subgrouping of HTLV-1. Although one sample could not be amplified by PCR, and three more could not be sequenced due to the existence of conspicuous nonspecific bands or repeated sequences, the phylogenetic analysis revealed that the remaining 19 isolates obtained from Kakeroma Island belonged to either the Transcontinental or the Japanese subgroups of the Cosmopolitan subtype, one of the three major subtypes. The RFLP data corresponded closely with the typing data throughout the sequencing. The proportion of the Transcontinental subgroup among the isolates was 26.3% (5 of 19) by sequence analysis and 27.3% (6 of 22) by RFLP. Unlike in Taiwan, China and Okinawa, the Japanese subgroup was dominant in Kakeroma Island. The analysis would also suggest that the Japanese subgroup seems not to have derived from the Transcontinental subgroup, but rather that the Transcontinental subgroup came to Japan first and was followed later by the Japanese one. 2009 Wiley-Liss, Inc.

MULTILOCUS SEQUENCE TYPING OF BRUCELLA ISOLATES FROM THAILAND.

PubMed

Chawjiraphan, Wireeya; Sonthayanon, Piengchan; Chanket, Phanita; Benjathummarak, Surachet; Kerdsin, Anusak; Kalambhaheti, Thareerat

2016-11-01

Although brucellosis outbreaks in Thailand are rare, they cause abortions and infertility in animals, resulting in significant economic loss. Because Brucella spp display > 90% DNA homology, multilocus sequence typing (MLST) was employed to categorize local Brucella isolates into sequence types (STs) and to determine their genetic relatedness. Brucella samples were isolated from vaginal secretion of cows and goats, and from blood cultures of infected individuals. Brucella species were determined by multiplex PCR of eight loci, in addition to MLST based on partial DNA sequences of nine house-keeping genes. MLST analysis of 36 isolates revealed 78 distinct novel allele types and 34 novel STs, while two isolates possessed the known ST8. Sequence alignments identified polymorphic sites in each allele, ranging from 2-6%, while overall genetic diversity was 3.6%. MLST analysis of the 36 Brucella isolates classified them into three species, namely, B. melitensis, B. abortus and B. suis, in agreement with multiplex PCR results. Genetic relatedness among ST members of B. melitensis and B. abortus determined by eBURST program revealed ST2 as founder of B. abortus isolates and ST8 the founder of B. melitensis isolates. ST 36, 41 and 50 of Thai Brucella isolates were identified as single locus variants of clonal cluster (CC) 8, while the majority of STs were diverse. The genetic diversity and relatedness identified using MLST revealed hitherto unexpected diversity among Thai Brucella isolates. Genetic classification of isolates could reveal the route of brucellosis transmission among humans and farm animals and also reveal their relationship with other isolates in the region and other parts of the world.

First Description of KPC-2-Producing Escherichia coli and ST15 OXA-48-Positive Klebsiella pneumoniae in Tunisia.

PubMed

Ben Tanfous, Farah; Alonso, Carla Andrea; Achour, Wafa; Ruiz-Ripa, Laura; Torres, Carmen; Ben Hassen, Assia

2017-04-01

The aim of this study was to investigate the molecular features among Klebsiella pneumoniae and Escherichia coli strains showing a resistant/intermediate-resistant phenotype to ertapenem (R/IR-ERT), implicated in colonization/infection in patients of the Hematology and Graft Units of the National Bone Marrow Transplant Center of Tunisia (3-year period, 2011-2014). The major carbapenemase, extended-spectrum beta-lactamase, and plasmidic AmpC beta-lactamase genes were analyzed and characterized by PCR and sequencing. Genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE) using XbaI and multilocus sequencing typing. The bla OXA-48 and bla KPC carbapenemase genes were detected among R/IR-ERT isolates. All R/IR-ERT K. pneumoniae strains (n = 19) had bla OXA-48 gene, and 14/19 strains also harbored the bla CTX-M-15 gene. Eight different PFGE patterns were detected among these K. pneumoniae isolates, and they showed eight different sequences types, ST11 and ST15 being the most prevalent ones. Two out of three R/IR-ERT E. coli isolates carried bla OXA-48 and one coproduced the bla CTX-M-15 gene. One E. coli strain, ascribed to the new sequence type ST5700, harbored the bla KPC-2 gene. E. coli isolates were not clonally related and belonged to different sequence types (ST5700, ST227, and ST58). To our knowledge, this is the first report in Tunisia of either KPC-2 carbapenemase in E. coli or OXA-48 carbapenemase in K. pneumoniae of lineage ST15.

Plantmediated horizontal transmission of Wolbachia between whiteflies

PubMed Central

Li, Shao-Jian; Ahmed, Muhammad Z; Lv, Ning; Shi, Pei-Qiong; Wang, Xing-Min; Huang, Ji-Lei; Qiu, Bao-Li

2017-01-01

Maternal transmission is the main transmission pathway of facultative bacterial endosymbionts, but phylogenetically distant insect hosts harbor closely related endosymbionts, suggesting that horizontal transmission occurs in nature. Here we report the first case of plant-mediated horizontal transmission of Wolbachia between infected and uninfected Bemisia tabaci AsiaII7 whiteflies. After infected whiteflies fed on cotton leaves, Wolbachia was visualized, both in the phloem vessels and in some novel ‘reservoir' spherules along the phloem by fluorescence in situ hybridization using Wolbachia-specific 16S rRNA probes and transmission electron microscopy. Wolbachia persisted in the plant leaves for at least 50 days. When the Wolbachia-free whiteflies fed on the infected plant leaves, the majority of them became infected with the symbiont and vertically transmitted it to their progeny. Multilocus sequence typing and sequencing of the wsp (Wolbachia surface protein) gene confirmed that the sequence type of Wolbachia in the donor whiteflies, cotton phloem and the recipient whiteflies are all identical (sequence type 388). These results were replicated using cowpea and cucumber plants, suggesting that horizontal transmission is also possible through other plant species. Our findings may help explain why Wolbachia bacteria are so abundant in arthropods, and suggest that in some species, Wolbachia may be maintained in populations by horizontal transmission. PMID:27935594

Development of PCR primers specific for the amplification and direct sequencing of gyrB genes from microbacteria, order Actinomycetales.

PubMed

Richert, Kathrin; Brambilla, Evelyne; Stackebrandt, Erko

2005-01-01

PCR primer sets were developed for the specific amplification and sequence analyses encoding the gyrase subunit B (gyrB) of members of the family Microbacteriaceae, class Actinobacteria. The family contains species highly related by 16S rRNA gene sequence analyses. In order to test if the gene sequence analysis of gyrB is appropriate to discriminate between closely related species, we evaluate the 16S rRNA gene phylogeny of its members. As the published universal primer set for gyrB failed to amplify the responding gene of the majority of the 80 type strains of the family, three new primer sets were identified that generated fragments with a composite sequence length of about 900 nt. However, the amplification of all three fragments was successful only in 25% of the 80 type strains. In this study, the substitution frequencies in genes encoding gyrase and 16S rDNA were compared for 10 strains of nine genera. The frequency of gyrB nucleotide substitution is significantly higher than that of the 16S rDNA, and no linear correlation exists between the similarities of both molecules among members of the Microbacteriaceae. The phylogenetic analyses using the gyrB sequences provide higher resolution than using 16S rDNA sequences and seem able to discriminate between closely related species.

Genetic relationships and epidemiological links between wild type 1 poliovirus isolates in Pakistan and Afghanistan

PubMed Central

2012-01-01

Background/Aim Efforts have been made to eliminate wild poliovirus transmission since 1988 when the World Health Organization began its global eradication campaign. Since then, the incidence of polio has decreased significantly. However, serotype 1 and serotype 3 still circulate endemically in Pakistan and Afghanistan. Both countries constitute a single epidemiologic block representing one of the three remaining major global reservoirs of poliovirus transmission. In this study we used genetic sequence data to investigate transmission links among viruses from diverse locations during 2005-2007. Methods In order to find the origins and routes of wild type 1 poliovirus circulation, polioviruses were isolated from faecal samples of Acute Flaccid Paralysis (AFP) patients. We used viral cultures, two intratypic differentiation methods PCR, ELISA to characterize as vaccine or wild type 1 and nucleic acid sequencing of entire VP1 region of poliovirus genome to determine the genetic relatedness. Results One hundred eleven wild type 1 poliovirus isolates were subjected to nucleotide sequencing for genetic variation study. Considering the 15% divergence of the sequences from Sabin 1, Phylogenetic analysis by MEGA software revealed that active inter and intra country transmission of many genetically distinct strains of wild poliovirus type 1 belonged to genotype SOAS which is indigenous in this region. By grouping wild type 1 polioviruses according to nucleotide sequence homology, three distinct clusters A, B and C were obtained with multiple chains of transmission together with some silent circulations represented by orphan lineages. Conclusion Our results emphasize that there was a persistent transmission of wild type1 polioviruses in Pakistan and Afghanistan during 2005-2007. The epidemiologic information provided by the sequence data can contribute to the formulation of better strategies for poliomyelitis control to those critical areas, associated with high risk population groups which include migrants, internally displaced people, and refugees. The implication of this study is to maintain high quality mass immunization with oral polio vaccine (OPV) in order to interrupt chains of virus transmission in both countries to endorse substantial progress in Eastern-Mediterranean region. PMID:22353446

Serotype IV and invasive group B Streptococcus disease in neonates, Minnesota, USA, 2000-2010.

PubMed

Ferrieri, Patricia; Lynfield, Ruth; Creti, Roberta; Flores, Aurea E

2013-04-01

Group B Streptococcus (GBS) is a major cause of invasive disease in neonates in the United States. Surveillance of invasive GBS disease in Minnesota, USA, during 2000-2010 yielded 449 isolates from 449 infants; 257 had early-onset (EO) disease (by age 6 days) and 192 late-onset (LO) disease (180 at age 7-89 days, 12 at age 90-180 days). Isolates were characterized by capsular polysaccharide serotype and surface-protein profile; types III and Ia predominated. However, because previously uncommon serotype IV constitutes 5/31 EO isolates in 2010, twelve type IV isolates collected during 2000-2010 were studied further. By pulsed-field gel electrophoresis, they were classified into 3 profiles; by multilocus sequence typing, representative isolates included new sequence type 468. Resistance to clindamycin or erythromycin was detected in 4/5 serotype IV isolates. Emergence of serotype IV GBS in Minnesota highlights the need for serotype prevalence monitoring to detect trends that could affect prevention strategies.

Molecular Characterization and Phylogenetic Analysis of Pseudomonas aeruginosa Isolates Recovered from Greek Aquatic Habitats Implementing the Double-Locus Sequence Typing Scheme.

PubMed

Pappa, Olga; Beloukas, Apostolos; Vantarakis, Apostolos; Mavridou, Athena; Kefala, Anastasia-Maria; Galanis, Alex

2017-07-01

The recently described double-locus sequence typing (DLST) scheme implemented to deeply characterize the genetic profiles of 52 resistant environmental Pseudomonas aeruginosa isolates deriving from aquatic habitats of Greece. DLST scheme was able not only to assign an already known allelic profile to the majority of the isolates but also to recognize two new ones (ms217-190, ms217-191) with high discriminatory power. A third locus (oprD) was also used for the molecular typing, which has been found to be fundamental for the phylogenetic analysis of environmental isolates given the resulted increased discrimination between the isolates. Additionally, the circulation of acquired resistant mechanisms in the aquatic habitats according to their genetic profiles was proved to be more extent. Hereby, we suggest that the combination of the DLST to oprD typing can discriminate phenotypically and genetically related environmental P. aeruginosa isolates providing reliable phylogenetic analysis at a local level.

How many papillomavirus species can go undetected in papilloma lesions?

PubMed

Daudt, Cíntia; da Silva, Flavio R C; Streck, André F; Weber, Matheus N; Mayer, Fabiana Q; Cibulski, Samuel P; Canal, Cláudio W

2016-11-03

A co-infection comprising to at least seven papillomavirus (PV) types was detected by next generation sequencing (NGS) of randomly primed rolling circle amplification (RCA) products of a bovine (Bos taurus) papilloma lesion from the Brazilian Amazon region. Six putative new PV types that could not be detected by commonly used PCR protocols were identified. Their overall L1 nucleotide identities were less than 90% compared to described PV species and types. L1 nucleotide BLAST sequence hits showed that each new type was related to Beta, Gamma, Dyokappa, Dyoeta, and Xipapillomavirus, as well as two likely new unclassified genera. Our results show that the employment of NGS is relevant to the detection and characterization of distantly related PV and is of major importance in co-infection studies. This knowledge will help us understand the biology and pathogenesis of PV, as well as contribute to disease control. Moreover, we can also conclude that there are many unknown circulating PVs.

Surgical and molecular pathology of pancreatic neoplasms.

PubMed

Hackeng, Wenzel M; Hruban, Ralph H; Offerhaus, G Johan A; Brosens, Lodewijk A A

2016-06-07

Histologic characteristics have proven to be very useful for classifying different types of tumors of the pancreas. As a result, the major tumor types in the pancreas have long been classified based on their microscopic appearance. Recent advances in whole exome sequencing, gene expression profiling, and knowledge of tumorigenic pathways have deepened our understanding of the underlying biology of pancreatic neoplasia. These advances have not only confirmed the traditional histologic classification system, but also opened new doors to early diagnosis and targeted treatment. This review discusses the histopathology, genetic and epigenetic alterations and potential treatment targets of the five major malignant pancreatic tumors - pancreatic ductal adenocarcinoma, pancreatic neuroendocrine tumor, solid-pseudopapillary neoplasm, acinar cell carcinoma and pancreatoblastoma.

Photoautotrophic symbiont and geography are major factors affecting highly structured and diverse bacterial communities in the lichen microbiome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hodkinson, Brendan P; Gottel, Neil R; Schadt, Christopher Warren

2011-01-01

Although common knowledge dictates that the lichen thallus is formed solely by a fungus (mycobiont) that develops a symbiotic relationship with an alga and/or cyanobacterium (photobiont), the non-photoautotrophic bacteria found in lichen microbiomes are increasingly regarded as integral components of lichen thalli. For this study, comparative analyses were conducted on lichen-associated bacterial communities to test for effects of photobiont-types (i.e. green algal vs. cyanobacterial), mycobiont-types and large-scale spatial distances (from tropical to arctic latitudes). Amplicons of the 16S (SSU) rRNA gene were examined using both Sanger sequencing of cloned fragments and barcoded pyrosequencing. Rhizobiales is typically the most abundant andmore » taxonomically diverse order in lichen microbiomes; however, overall bacterial diversity in lichens is shown to be much higher than previously reported. Members of Acidobacteriaceae, Acetobacteraceae, Brucellaceae and sequence group LAR1 are the most commonly found groups across the phylogenetically and geographically broad array of lichens examined here. Major bacterial community trends are significantly correlated with differences in large-scale geography, photobiont-type and mycobiont-type. The lichen as a microcosm represents a structured, unique microbial habitat with greater ecological complexity and bacterial diversity than previously appreciated and can serve as a model system for studying larger ecological and evolutionary principles.« less

An Exercise in Molecular Epidemiology: Human Rhinovirus Prevalence and Genetics

ERIC Educational Resources Information Center

Albright, Catherine J.; Hall, David J.

2011-01-01

Human rhinovirus (HRV) is one of the most common human respiratory pathogens and is responsible for the majority of upper respiratory illnesses. Recently, a phylogeny was constructed from all known American Type Culture Collection (ATCC) HRV sequences. From this study, three HRV classifications (HRVA, HRVB, and HRVC) were determined and techniques…

Genotypic Characterization of Human Immunodeficiency Virus Type 1 Derived from Antiretroviral Therapy-Naive Individuals Residing in Sorong, West Papua.

PubMed

Witaningrum, Adiana Mutamsari; Kotaki, Tomohiro; Khairunisa, Siti Qamariyah; Yunifiar M, Muhammad Qushai; Indriati, Dwi Wahyu; Bramanthi, Rendra; Nasronudin; Kameoka, Masanori

2016-08-01

Papua and West Papua provinces have the highest prevalence rate of human immunodeficiency virus type 1 (HIV-1) infection in Indonesia; however, data on the molecular epidemiology of HIV-1 are limited. We conducted a genotypic study on HIV-1 genes derived from antiretroviral therapy-naive individuals residing in Sorong, West Papua. HIV-1 genomic fragments were amplified from 43 peripheral blood samples, and sequencing analysis of the genes was carried out. Of the 43 samples, 41 protease (PR), 31 reverse transcriptase (RT), 26 gag, and 25 env genes were sequenced. HIV-1 subtyping revealed that CRF01_AE (48.8%, 21/43) and subtype B (41.9%, 18/43) were the major subtypes prevalent in the region, whereas other recombinant forms were also detected. Major drug resistance-associated mutations for PR inhibitors were not detected; however, mutations for the RT inhibitors, A62V and E138A, appeared in a few samples, indicating the possible emergence of transmitted HIV-1 drug resistance in Sorong, West Papua.

Genotypic Characterization of Human Immunodeficiency Virus Type 1 Derived from Antiretroviral Drug-Treated Individuals Residing in Earthquake-Affected Areas in Nepal.

PubMed

Negi, Bharat Singh; Kotaki, Tomohiro; Joshi, Sunil Kumar; Bastola, Anup; Nakazawa, Minato; Kameoka, Masanori

2017-09-01

Molecular epidemiological data on human immunodeficiency virus type 1 (HIV-1) are limited in Nepal and have not been available in areas affected by the April 2015 earthquake. Therefore, we conducted a genotypic study on HIV-1 genes derived from individuals on antiretroviral therapy residing in 14 districts in Nepal highly affected by the earthquake. HIV-1 genomic fragments were amplified from 40 blood samples of HIV treatment-failure individuals, and a sequencing analysis was performed on these genes. In the 40 samples, 29 protease, 32 reverse transcriptase, 25 gag, and 21 env genes were sequenced. HIV-1 subtyping revealed that subtype C (84.2%, 32/38) was the major subtype prevalent in the region, while CRF01_AE (7.9%, 3/38) and other recombinant forms (7.9%, 3/38) were also detected. In addition, major drug resistance mutations were identified in 21.9% (7/32) of samples, indicating the possible emergence of HIV-1 drug resistance in earthquake-affected areas in Nepal.

Epitope design of L1 protein for vaccine production against Human Papilloma Virus types 16 and 18

PubMed Central

Baidya, Sunanda; Das, Rasel; Kabir, Md. Golam; Arifuzzaman, Md.

2017-01-01

Cervical cancer accounts for about two-thirds of all cancer cases linked etiologically to Human Papilloma Virus (HPV). 15 oncogenic HPV types can cause cervical cancer, of which HPV16 and HPV18 combinedly account for about 70% of it. So, effective epitope design for the clinically relevant HPV types 16 and 18 would be of major medical benefit. Here, a comprehensive analysis is carried out to predict the epitopes against HPV types 16 and 18 through “reverse vaccinology” approach. We attempted to identify the evolutionarily conserved regions of major capsid protein (L1) as well as minor capsid protein (L2) of HPV and designed epitopes within these regions. In this study, we analyzed about 49 and 27 sequences of HPV L2 and L1 proteins respectively. Since we found that the intertype variability of L2 is higher than for L1 proteins, our analysis was emphasized on epitopes of L1 of HPV types 16 and 18. We had selected HLA-A*0201, DRB1*1501, DQB1*0602, DRB1*0401 and DQB1*0301 alleles for the prediction of T cell epitopes of L1 of HPV 16 and 18. Finally, we reported that predicted epitope sequences EEYDLQFIFQLCKITLTA, and RHGEEYDLQFIFQLCKITLTA of L1 protein of HPV 16, and LPDPNKF, PETQRLVWAC, PVPGQYDA, YNPETQRLVWAC, DTGYGAMD, PVPGQYDATK, KQDIPKVSAYQYRVFRV, RDNVSVDYKQTQLCI and YSRHVEEYDLQFIF of L1 protein of HPV 18 could be therapeutic tools for vaccine design against HPV. PMID:28584449

Epitope design of L1 protein for vaccine production against Human Papilloma Virus types 16 and 18.

PubMed

Baidya, Sunanda; Das, Rasel; Kabir, Md Golam; Arifuzzaman, Md

2017-01-01

Cervical cancer accounts for about two-thirds of all cancer cases linked etiologically to Human Papilloma Virus (HPV). 15 oncogenic HPV types can cause cervical cancer, of which HPV16 and HPV18 combinedly account for about 70% of it. So, effective epitope design for the clinically relevant HPV types 16 and 18 would be of major medical benefit. Here, a comprehensive analysis is carried out to predict the epitopes against HPV types 16 and 18 through "reverse vaccinology" approach. We attempted to identify the evolutionarily conserved regions of major capsid protein (L1) as well as minor capsid protein (L2) of HPV and designed epitopes within these regions. In this study, we analyzed about 49 and 27 sequences of HPV L2 and L1 proteins respectively. Since we found that the intertype variability of L2 is higher than for L1 proteins, our analysis was emphasized on epitopes of L1 of HPV types 16 and 18. We had selected HLA-A*0201, DRB1*1501, DQB1*0602, DRB1*0401 and DQB1*0301 alleles for the prediction of T cell epitopes of L1 of HPV 16 and 18. Finally, we reported that predicted epitope sequences EEYDLQFIFQLCKITLTA, and RHGEEYDLQFIFQLCKITLTA of L1 protein of HPV 16, and LPDPNKF, PETQRLVWAC, PVPGQYDA, YNPETQRLVWAC, DTGYGAMD, PVPGQYDATK, KQDIPKVSAYQYRVFRV, RDNVSVDYKQTQLCI and YSRHVEEYDLQFIF of L1 protein of HPV 18 could be therapeutic tools for vaccine design against HPV.

Carbapenem Susceptibility Testing Errors Using Three Automated Systems, Disk Diffusion, Etest, and Broth Microdilution and Carbapenem Resistance Genes in Isolates of Acinetobacter baumannii-calcoaceticus Complex

DTIC Science & Technology

2011-10-01

Phoenix, and Vitek 2 systems). Discordant results were categorized as very major errors (VME), major errors (ME), and minor errors (mE). DNA sequences...01 OCT 2011 2 . REPORT TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Carbapenem Susceptibility Testing Errors Using Three Automated...FDA standards required for device approval (11). The Vitek 2 method was the only automated susceptibility method in our study that satisfied FDA

Unravelling the Molecular Epidemiology and Genetic Diversity among Burkholderia pseudomallei Isolates from South India Using Multi-Locus Sequence Typing.

PubMed

Tellapragada, Chaitanya; Kamthan, Aayushi; Shaw, Tushar; Ke, Vandana; Kumar, Subodh; Bhat, Vinod; Mukhopadhyay, Chiranjay

2016-01-01

There is a slow but steady rise in the case detection rates of melioidosis from various parts of the Indian sub-continent in the past two decades. However, the epidemiology of the disease in India and the surrounding South Asian countries remains far from well elucidated. Multi-locus sequence typing (MLST) is a useful epidemiological tool to study the genetic relatedness of bacterial isolates both with-in and across the countries. With this background, we studied the molecular epidemiology of 32 Burkholderia pseudomallei isolates (31 clinical and 1 soil isolate) obtained during 2006-2015 from various parts of south India using multi-locus sequencing typing and analysis. Of the 32 isolates included in the analysis, 30 (93.7%) had novel allelic profiles that were not reported previously. Sequence type (ST) 1368 (n = 15, 46.8%) with allelic profile (1, 4, 6, 4, 1, 1, 3) was the most common genotype observed. We did not observe a genotypic association of STs with geographical location, type of infection and year of isolation in the present study. Measure of genetic differentiation (FST) between Indian and the rest of world isolates was 0.14413. Occurrence of the same ST across three adjacent states of south India suggest the dispersion of B.pseudomallei across the south western coastal part of India with limited geographical clustering. However, majority of the STs reported from the present study remained as "outliers" on the eBURST "Population snapshot", suggesting the genetic diversity of Indian isolates from the Australasian and Southeast Asian isolates.

The evolution of energy-transducing systems: Studies with archaebacteria

NASA Technical Reports Server (NTRS)

Stan-Lotter, Helga

1993-01-01

N-ethylmaleimide (NEM) inhibits the ATPase of H. saccharovorum in a nucleotide protectable manner. The bulk of 14C-NEM is incorporated into subunit 1. Inhibition kinetics indicated a single binding site. To determine the sequence around this site, cyanogen bromide peptides of NEM-labeled ATPase enzyme were prepared and separated on Tris-Tricine gels. Autoradiography indicated that the NEM binding site is probably located in a fragment of Mr 10-12 K. This result will be confirmed by N-terminal sequencing of the peptide. Since the cysteinyl residue, to which NEM is bound, may be located at the C-terminal end, purification and proteolytic treatment of the 10 K peptide will be required. One inhibitor of V-type ATPases, fluoresceinisothiocyanate (FITC) inhibited also the ATPase of H. saccharovorum. Preliminary results indicated protection against inhibition by nucleotides. Localization of the binding sited to the major subunits is in progress. An extraction procedure for the membrane sector of the ATPase complex of H. saccharovorum yielded a preparation which was enriched in a peptide of Mr 5 500. Experiments to test the immunological crossreaction with subunit c from the Escherichia coli F-type ATPase and the labeling with 14C-DCCD are currently carried out. Polyclonal antiserum to the smaller of the major subunits of the ATPase from H. saccharovorum (subunit ll) reacts in Western blots strongly with the alpha and beta subunits of the F1 ATPase of E. coli, suggesting highly conserved regions on both types of ATPases. To elucidate further the regions of homology, cyanogen bromide peptides of the beta subunits were prepared for sequence analysis.

Investigation of Human Cancers for Retrovirus by Low-Stringency Target Enrichment and High-Throughput Sequencing.

PubMed

Vinner, Lasse; Mourier, Tobias; Friis-Nielsen, Jens; Gniadecki, Robert; Dybkaer, Karen; Rosenberg, Jacob; Langhoff, Jill Levin; Cruz, David Flores Santa; Fonager, Jannik; Izarzugaza, Jose M G; Gupta, Ramneek; Sicheritz-Ponten, Thomas; Brunak, Søren; Willerslev, Eske; Nielsen, Lars Peter; Hansen, Anders Johannes

2015-08-19

Although nearly one fifth of all human cancers have an infectious aetiology, the causes for the majority of cancers remain unexplained. Despite the enormous data output from high-throughput shotgun sequencing, viral DNA in a clinical sample typically constitutes a proportion of host DNA that is too small to be detected. Sequence variation among virus genomes complicates application of sequence-specific, and highly sensitive, PCR methods. Therefore, we aimed to develop and characterize a method that permits sensitive detection of sequences despite considerable variation. We demonstrate that our low-stringency in-solution hybridization method enables detection of <100 viral copies. Furthermore, distantly related proviral sequences may be enriched by orders of magnitude, enabling discovery of hitherto unknown viral sequences by high-throughput sequencing. The sensitivity was sufficient to detect retroviral sequences in clinical samples. We used this method to conduct an investigation for novel retrovirus in samples from three cancer types. In accordance with recent studies our investigation revealed no retroviral infections in human B-cell lymphoma cells, cutaneous T-cell lymphoma or colorectal cancer biopsies. Nonetheless, our generally applicable method makes sensitive detection possible and permits sequencing of distantly related sequences from complex material.

UCSC genome browser: deep support for molecular biomedical research.

PubMed

Mangan, Mary E; Williams, Jennifer M; Lathe, Scott M; Karolchik, Donna; Lathe, Warren C

2008-01-01

The volume and complexity of genomic sequence data, and the additional experimental data required for annotation of the genomic context, pose a major challenge for display and access for biomedical researchers. Genome browsers organize this data and make it available in various ways to extract useful information to advance research projects. The UCSC Genome Browser is one of these resources. The official sequence data for a given species forms the framework to display many other types of data such as expression, variation, cross-species comparisons, and more. Visual representations of the data are available for exploration. Data can be queried with sequences. Complex database queries are also easily achieved with the Table Browser interface. Associated tools permit additional query types or access to additional data sources such as images of in situ localizations. Support for solving researcher's issues is provided with active discussion mailing lists and by providing updated training materials. The UCSC Genome Browser provides a source of deep support for a wide range of biomedical molecular research (http://genome.ucsc.edu).

Genetic characterization of a Coxsackie A9 virus associated with aseptic meningitis in Alberta, Canada in 2010

PubMed Central

2013-01-01

Background An unusually high incidence of aseptic meningitis caused by enteroviruses was noted in Alberta, Canada between March and October 2010. Sequence based typing was performed on the enterovirus positive samples to gain a better understanding of the molecular characteristics of the Coxsackie A9 (CVA-9) strain responsible for most cases in this outbreak. Methods Molecular typing was performed by amplification and sequencing of the VP2 region. The genomic sequence of one of the 2010 outbreak isolates was compared to a CVA-9 isolate from 2003 and the prototype sequence to study genetic drift and recombination. Results Of the 4323 samples tested, 213 were positive for enteroviruses (4.93%). The majority of the positives were detected in CSF samples (n = 157, 73.71%) and 81.94% of the sequenced isolates were typed as CVA-9. The sequenced CVA-9 positives were predominantly (94.16%) detected in patients ranging in age from 15 to 29 years and the peak months for detection were between March and October. Full genome sequence comparisons revealed that the CVA-9 viruses isolated in Alberta in 2003 and 2010 were highly homologous to the prototype CVA-9 in the structural VP1, VP2 and VP3 regions but divergent in the VP4, non-structural and non-coding regions. Conclusion The increase in cases of aseptic meningitis was associated with enterovirus CVA-9. Sequence divergence between the prototype strain of CVA-9 and the Alberta isolates suggests genetic drifting and/or recombination events, however the sequence was conserved in the antigenic regions determined by the VP1, VP2 and VP3 genes. These results suggest that the increase in CVA-9 cases likely did not result from the emergence of a radically different immune escape mutant. PMID:23521862

PRISM 3: expanded prediction of natural product chemical structures from microbial genomes

PubMed Central

Skinnider, Michael A.; Merwin, Nishanth J.; Johnston, Chad W.

2017-01-01

Abstract Microbial natural products represent a rich resource of pharmaceutically and industrially important compounds. Genome sequencing has revealed that the majority of natural products remain undiscovered, and computational methods to connect biosynthetic gene clusters to their corresponding natural products therefore have the potential to revitalize natural product discovery. Previously, we described PRediction Informatics for Secondary Metabolomes (PRISM), a combinatorial approach to chemical structure prediction for genetically encoded nonribosomal peptides and type I and II polyketides. Here, we present a ground-up rewrite of the PRISM structure prediction algorithm to derive prediction of natural products arising from non-modular biosynthetic paradigms. Within this new version, PRISM 3, natural product scaffolds are modeled as chemical graphs, permitting structure prediction for aminocoumarins, antimetabolites, bisindoles and phosphonate natural products, and building upon the addition of ribosomally synthesized and post-translationally modified peptides. Further, with the addition of cluster detection for 11 new cluster types, PRISM 3 expands to detect 22 distinct natural product cluster types. Other major modifications to PRISM include improved sequence input and ORF detection, user-friendliness and output. Distribution of PRISM 3 over a 300-core server grid improves the speed and capacity of the web application. PRISM 3 is available at http://magarveylab.ca/prism/. PMID:28460067

Stratigraphic architecture and gamma ray logs of deeper ramp carbonates (Upper Jurassic, SW Germany)

NASA Astrophysics Data System (ADS)

Pawellek, T.; Aigner, T.

2003-07-01

The objective of this paper is to contribute to the development of sequence stratigraphic models for extensive epicontinental carbonate systems deposited over cratonic areas. Epicontinental carbonates of the SW German Upper Jurassic were analysed in terms of microfacies, sedimentology and sequence stratigraphy based on 2.5 km of core, 70 borehole gamma ray logs and 24 quarries. Facies analysis revealed six major facies belts across the deeper parts of the carbonate ramp, situated generally below fair-weather wave base, and mostly below average storm wave base but in the reach of occasional storm events. Observed stratigraphic patterns differ in some aspects from widely published sequence stratigraphic models: Elementary sedimentary cycles are mostly more or less symmetrical and are, thus, referred to as "genetic sequences" or "genetic units" [AAAPG Bull. 55 (1971) 1137; Frazier, D.E., 1974. Depositional episodes: their relationship to the Quaternary stratigraphic framework in the northwestern portion of the Gulf Basin. University of Texas, Austin, Bureau of Economic Geology Geologicalo Circular 71-1; AAPG Bull. 73 (1989) 125; Galloway, W.E., Hobday, D.K., 1996. Terrigenous Clastic Depositional Systems. 489 pp., Springer; Cross, T.A., Baker, M.R., Chapin, M.S., Clark, M.S., Gardner, M.H., Hanson, M.S., Lessenger, M.A., Little, L.D., McDonough, K.J., Sonnenfeld, M.D., Valasek, D.W., Williams, M.R., Witter, D.N., 1993. Applications of high-resolution sequence stratigraphy to reservoir analysis. Edition Technip 1993, 11-33; Bull. Cent. Rech. Explor. Prod. Elf-Aquitaine 16 (1992) 357; Homewood, P., Mauriaud, P., Lafont, F., 2000. Best practices in sequence stratigraphy. Elf EP Mem. 25, 81 pp.; Homewood, P., Eberli, G.P., 2000. Genetic stratigraphy on the exploration and production scales. Elf EP Mem. 24, 290 pp.], in contrast to the asymmetrical, shallowing-upward "parasequences" of the EXXON approach. Neither sequence boundaries nor maximum flooding surfaces could be clearly delineated. Cycle boundaries are generally not represented by sharp stratal surfaces but are always transitional and, thus, referred to as "turnarounds" [Nor. Pet. Soc. Spec. Publ. 8 (1998) 171]. Several types of genetic sequences were delineated. Both major types of facies and sequences show characteristic gamma ray log signatures. Based on the cycle stacking and the gamma ray patterns, a hierarchy of sequences was recognized, probably driven in part by 100,000- and 400,000-year Milankovitch signals. The cyclicity allowed regional correlations across various depositional environments such as sponge-microbial bioherms and coeval basins. The basin-wide correlation revealed evidence for a subtle clinoform-type stratigraphic architecture along very gentle slopes, rather than a so far assumed simple "layer cake" pattern.

A comparative study of retrotransposons in the centromeric regions of A and B chromosomes of maize.

PubMed

Theuri, J; Phelps-Durr, T; Mathews, S; Birchler, J

2005-01-01

Bacterial Artificial Chromosomes (BACs) derived from the B chromosome, based on homology with the B specific sequence, were subcloned and sequenced. Analysis of DNA sequence data indicated the presence of 23 common retroelements, as well as novel sequences of B chromosome origin. Generally, where the same retrotransposon type was observed in both A and B chromosomes, there were more copies per unit of sequence in the B centromeric region (the major site of B repeat) than in the A centromere, except for Huck-1. Based on previous estimates of the age of the major burst of transposition into the maize genome, the oldest retrotransposons (Ji-6 and Tekay, approximately 5.0 and 5.2 million years ago, respectively) were found in the B centromere region only, while the next two oldest (Huck-1 and Opie-1) were found in both the A and B sequences. Phylogenetic analysis of Opie retroelements from both A and B centromeres indicated that some of the B Opie centromeric sequences share a more recent common ancestor with A Opie retroelements than they do with other B Opie centromeric sequences. These results imply that the supernumerary maize B chromosome has coexisted with the A chromosomes during that period of transposition. They also support the hypothesis that the B chromosome had its origins from A chromosome elements, or that alternative origins, such as being donated to the maize genome in a wide species cross, preceded six million years ago, because the spectrum of retrotransposons in the two chromosomes is quite similar.

Repetitive sequences in plant nuclear DNA: types, distribution, evolution and function.

PubMed

Mehrotra, Shweta; Goyal, Vinod

2014-08-01

Repetitive DNA sequences are a major component of eukaryotic genomes and may account for up to 90% of the genome size. They can be divided into minisatellite, microsatellite and satellite sequences. Satellite DNA sequences are considered to be a fast-evolving component of eukaryotic genomes, comprising tandemly-arrayed, highly-repetitive and highly-conserved monomer sequences. The monomer unit of satellite DNA is 150-400 base pairs (bp) in length. Repetitive sequences may be species- or genus-specific, and may be centromeric or subtelomeric in nature. They exhibit cohesive and concerted evolution caused by molecular drive, leading to high sequence homogeneity. Repetitive sequences accumulate variations in sequence and copy number during evolution, hence they are important tools for taxonomic and phylogenetic studies, and are known as "tuning knobs" in the evolution. Therefore, knowledge of repetitive sequences assists our understanding of the organization, evolution and behavior of eukaryotic genomes. Repetitive sequences have cytoplasmic, cellular and developmental effects and play a role in chromosomal recombination. In the post-genomics era, with the introduction of next-generation sequencing technology, it is possible to evaluate complex genomes for analyzing repetitive sequences and deciphering the yet unknown functional potential of repetitive sequences. Copyright © 2014 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

Structural and functional differences in the dio1 gene in mice with inherited type 1 deiodinase deficiency.

PubMed

Maia, A L; Berry, M J; Sabbag, R; Harney, J W; Larsen, P R

1995-08-01

The type 1 deiodinase (D1) provides the major portion of the circulating T3 in vertebrates. In C3H and certain other inbred mice, liver and kidney D1 activity is 5- to 10-fold lower than in the common phenotype, C57. The lower D1 levels are paralleled by a decreased normal-sized dio1 mRNA and hyperthyroxinemia. Low activity cosegregates with a restriction fragment length variant (RFLV) in both inbred and recombinant strains, indicating it is due to differences in the dio1 gene. The exonic structure and the deduced amino acid sequences are identical for both strains and highly homologous to that of the rat. The RFLV is due to an approximately 150-base pair expansion of repetitive sequences in the second intron of the C3H gene, but this segment does not differentially affect the transient expression of a human GH gene. The promoter and 5'-flanking regions of the C3H and C57 dio1 genes are very similar and are GC rich without TATA or CCAAT boxes. However, functional assays of 1.5-kilobase 5'-flanking dio1-CAT constructs showed 2- to 3-fold higher activity of the C57-CAT constructs. Deletion mutants showed that sequences between -705 and -162 were the cause of this. In this region, the only major difference between the two genes is a 21-base pair insert containing five CTG repeats in the C3H promoter. This difference also cosegregates with low D1 activity and the intron RFLV in four other mouse strains. The correlation of the CTG repeat insert with both in vitro and in vivo expression and the absence of other significant sequence differences in the 5'-flanking region argue that this is the major explanation for the impaired expression of the dio1 gene and the resulting hyperthyroxinemia of the C3H mouse.

Molecular Epidemiology of Carbapenem-Resistant Acinetobacter baumannii Isolates in the Gulf Cooperation Council States: Dominance of OXA-23-Type Producers

PubMed Central

Sartor, Anna L.; Sidjabat, Hanna E.; Balkhy, Hanan H.; Walsh, Timothy R.; Al Johani, Sameera M.; AlJindan, Reem Y.; Alfaresi, Mubarak; Ibrahim, Emad; Al-Jardani, Amina; Al Salman, Jameela; Dashti, Ali A.; Johani, Khalid; Paterson, David L.

2015-01-01

The molecular epidemiology and mechanisms of resistance of carbapenem-resistant Acinetobacter baumannii (CRAB) were determined in hospitals in the states of the Cooperation Council for the Arab States of the Gulf (Gulf Cooperation Council [GCC]), namely, Saudi Arabia, United Arab Emirates, Oman, Qatar, Bahrain, and Kuwait. Isolates were subjected to PCR-based detection of antibiotic resistance genes and repetitive sequence-based PCR (rep-PCR) assessments of clonality. Selected isolates were subjected to multilocus sequence typing (MLST). We investigated 117 isolates resistant to carbapenem antibiotics (either imipenem or meropenem). All isolates were positive for OXA-51. The most common carbapenemases were the OXA-23-type, found in 107 isolates, followed by OXA-40-type (OXA-24-type), found in 5 isolates; 3 isolates carried the ISAba1 element upstream of blaOXA-51-type. No OXA-58-type, NDM-type, VIM-type, or IMP-type producers were detected. Multiple clones were detected with 16 clusters of clonally related CRAB. Some clusters involved hospitals in different states. MLST analysis of 15 representative isolates from different clusters identified seven different sequence types (ST195, ST208, ST229, ST436, ST450, ST452, and ST499), as well as three novel STs. The vast majority (84%) of the isolates in this study were associated with health care exposure. Awareness of multidrug-resistant organisms in GCC states has important implications for optimizing infection control practices; establishing antimicrobial stewardship programs within hospital, community, and agricultural settings; and emphasizing the need for establishing regional active surveillance systems. This will help to control the spread of CRAB in the Middle East and in hospitals accommodating transferred patients from this region. PMID:25568439

Molecular epidemiology of carbapenem-resistant Acinetobacter baumannii isolates in the Gulf Cooperation Council States: dominance of OXA-23-type producers.

PubMed

Zowawi, Hosam M; Sartor, Anna L; Sidjabat, Hanna E; Balkhy, Hanan H; Walsh, Timothy R; Al Johani, Sameera M; AlJindan, Reem Y; Alfaresi, Mubarak; Ibrahim, Emad; Al-Jardani, Amina; Al Salman, Jameela; Dashti, Ali A; Johani, Khalid; Paterson, David L

2015-03-01

The molecular epidemiology and mechanisms of resistance of carbapenem-resistant Acinetobacter baumannii (CRAB) were determined in hospitals in the states of the Cooperation Council for the Arab States of the Gulf (Gulf Cooperation Council [GCC]), namely, Saudi Arabia, United Arab Emirates, Oman, Qatar, Bahrain, and Kuwait. Isolates were subjected to PCR-based detection of antibiotic resistance genes and repetitive sequence-based PCR (rep-PCR) assessments of clonality. Selected isolates were subjected to multilocus sequence typing (MLST). We investigated 117 isolates resistant to carbapenem antibiotics (either imipenem or meropenem). All isolates were positive for OXA-51. The most common carbapenemases were the OXA-23-type, found in 107 isolates, followed by OXA-40-type (OXA-24-type), found in 5 isolates; 3 isolates carried the ISAba1 element upstream of blaOXA-51-type. No OXA-58-type, NDM-type, VIM-type, or IMP-type producers were detected. Multiple clones were detected with 16 clusters of clonally related CRAB. Some clusters involved hospitals in different states. MLST analysis of 15 representative isolates from different clusters identified seven different sequence types (ST195, ST208, ST229, ST436, ST450, ST452, and ST499), as well as three novel STs. The vast majority (84%) of the isolates in this study were associated with health care exposure. Awareness of multidrug-resistant organisms in GCC states has important implications for optimizing infection control practices; establishing antimicrobial stewardship programs within hospital, community, and agricultural settings; and emphasizing the need for establishing regional active surveillance systems. This will help to control the spread of CRAB in the Middle East and in hospitals accommodating transferred patients from this region. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Nucleotide excision repair and recombination are engaged in repair of trans-4-hydroxy-2-nonenal adducts to DNA bases in Escherichia coli.

PubMed

Janowska, Beata; Komisarski, Marek; Prorok, Paulina; Sokołowska, Beata; Kuśmierek, Jarosław; Janion, Celina; Tudek, Barbara

2009-09-23

One of the major products of lipid peroxidation is trans-4-hydroxy-2-nonenal (HNE). HNE forms highly mutagenic and genotoxic adducts to all DNA bases. Using M13 phage lacZ system, we studied the mutagenesis and repair of HNE treated phage DNA in E. coli wild-type or uvrA, recA, and mutL mutants. These studies revealed that: (i) nucleotide excision and recombination, but not mismatch repair, are engaged in repair of HNE adducts when present in phage DNA replicating in E. coli strains; (ii) in the single uvrA mutant, phage survival was drastically decreased while mutation frequency increased, and recombination events constituted 48% of all mutations; (iii) in the single recA mutant, the survival and mutation frequency of HNE-modified M13 phage was slightly elevated in comparison to that in the wild-type bacteria. The majority of mutations in recA(-) strain were G:C --> T:A transversions, occurring within the sequence which in recA(+) strains underwent RecA-mediated recombination, and the entire sequence was deleted; (iv) in the double uvrA recA mutant, phage survival was the same as in the wild-type although the mutation frequency was higher than in the wild-type and recA single mutant, but lower than in the single uvrA mutant. The majority of mutations found in the latter strain were base substitutions, with G:C --> A:T transitions prevailing. These transitions could have resulted from high reactivity of HNE with G and C, and induction of SOS-independent mutations.

Nucleotide excision repair and recombination are engaged in repair of trans-4-hydroxy-2-nonenal adducts to DNA bases in Escherichia coli

PubMed Central

Janowska, Beata; Komisarski, Marek; Prorok, Paulina; Sokołowska, Beata; Kuśmierek, Jarosław; Janion, Celina; Tudek, Barbara

2009-01-01

One of the major products of lipid peroxidation is trans-4-hydroxy-2-nonenal (HNE). HNE forms highly mutagenic and genotoxic adducts to all DNA bases. Using M13 phage lacZ system, we studied the mutagenesis and repair of HNE treated phage DNA in E. coli wild-type or uvrA, recA, and mutL mutants. These studies revealed that: (i) nucleotide excision and recombination, but not mismatch repair, are engaged in repair of HNE adducts when present in phage DNA replicating in E. coli strains; (ii) in the single uvrA mutant, phage survival was drastically decreased while mutation frequency increased, and recombination events constituted 48 % of all mutations; (iii) in the single recA mutant, the survival and mutation frequency of HNE-modified M13 phage was slightly elevated in comparison to that in the wild-type bacteria. The majority of mutations in recA- strain were G:C → T:A transversions, occurring within the sequence which in recA+ strains underwent RecA-mediated recombination, and the entire sequence was deleted; (iv) in the double uvrA recA mutant, phage survival was the same as in the wild-type although the mutation frequency was higher than in the wild-type and recA single mutant, but lower than in the single uvrA mutant. The majority of mutations found in the latter strain were base substitutions, with G:C → A:T transitions prevailing. These transitions could have resulted from high reactivity of HNE with G and C, and induction of SOS-independent mutations. PMID:19834545

Streptomyces pharmamarensis sp. nov. isolated from a marine sediment.

PubMed

Carro, Lorena; Zúñiga, Paz; de la Calle, Fernando; Trujillo, Martha E

2012-05-01

A Gram-stain-positive actinobacterium, strain PM267(T), was isolated from a marine sediment sample in the Mediterranean Sea. The novel strain produced extensively branched substrate and aerial hyphae that carried spiral spore chains. Substrate and aerial mycelia were cream-white and white, respectively. Diffusible pigments were not observed. 16S rRNA gene sequence analysis revealed that strain PM267(T) belonged to the genus Streptomyces and shared a gene sequence similarity of 97.1 % with Streptomyces artemisiae YIM 63135(T) and Streptomyces armeniacus JCM 3070(T). Values <97 % were obtained with other sequences representing members of the genus Streptomyces. The cell wall peptidoglycan contained ll-diaminopimelic acid. MK-9(H(8)) was the major menaquinone. The phospholipid pattern included phosphatidylethanolamine as diagnostic lipid (type II). Major fatty acids found were iso- and anteiso- fatty acids. The G+C content of the DNA was 71.2 mol%. The strain was halotolerant and was able to grow in the presence of 9 % (w/v) NaCl (with an optimum of 2 %). On the basis of these results and additional physiological data obtained in the present study, strain PM267(T) represents a novel species within the genus Streptomyces for which the name Streptomyces pharmamarensis sp. nov. is proposed (type strain PM267(T)  = CECT 7841(T)  = DSM 42032(T)).

Loci and candidate genes conferring resistance to soybean cyst nematode HG type 2.5.7.

PubMed

Zhao, Xue; Teng, Weili; Li, Yinghui; Liu, Dongyuan; Cao, Guanglu; Li, Dongmei; Qiu, Lijuan; Zheng, Hongkun; Han, Yingpeng; Li, Wenbin

2017-06-14

Soybean (Glycine max L. Merr.) cyst nematode (SCN, Heterodera glycines I,) is a major pest of soybean worldwide. The most effective strategy to control this pest involves the use of resistant cultivars. The aim of the present study was to investigate the genome-wide genetic architecture of resistance to SCN HG Type 2.5.7 (race 1) in landrace and elite cultivated soybeans. A total of 200 diverse soybean accessions were screened for resistance to SCN HG Type 2.5.7 and genotyped through sequencing using the Specific Locus Amplified Fragment Sequencing (SLAF-seq) approach with a 6.14-fold average sequencing depth. A total of 33,194 SNPs were identified with minor allele frequencies (MAF) over 4%, covering 97% of all the genotypes. Genome-wide association mapping (GWAS) revealed thirteen SNPs associated with resistance to SCN HG Type 2.5.7. These SNPs were distributed on five chromosomes (Chr), including Chr7, 8, 14, 15 and 18. Four SNPs were novel resistance loci and nine SNPs were located near known QTL. A total of 30 genes were identified as candidate genes underlying SCN resistance. A total of sixteen novel soybean accessions were identified with significant resistance to HG Type 2.5.7. The beneficial alleles and candidate genes identified by GWAS might be valuable for improving marker-assisted breeding efficiency and exploring the molecular mechanisms underlying SCN resistance.

Sequencing of Oligourea Foldamers by Tandem Mass Spectrometry

NASA Astrophysics Data System (ADS)

Bathany, Katell; Owens, Neil W.; Guichard, Gilles; Schmitter, Jean-Marie

2013-03-01

This study is focused on sequence analysis of peptidomimetic helical oligoureas by means of tandem mass spectrometry, to build a basis for de novo sequencing for future high-throughput combinatorial library screening of oligourea foldamers. After the evaluation of MS/MS spectra obtained for model compounds with either MALDI or ESI sources, we found that the MALDI-TOF-TOF instrument gave more satisfactory results. MS/MS spectra of oligoureas generated by decay of singly charged precursor ions show major ion series corresponding to fragmentation across both CO-NH and N'H-CO urea bonds. Oligourea backbones fragment to produce a pattern of a, x, b, and y type fragment ions. De novo decoding of spectral information is facilitated by the occurrence of low mass reporter ions, representative of constitutive monomers, in an analogous manner to the use of immonium ions for peptide sequencing.

Analysis of the type II-A CRISPR-Cas system of Streptococcus agalactiae reveals distinctive features according to genetic lineages

PubMed Central

Lier, Clément; Baticle, Elodie; Horvath, Philippe; Haguenoer, Eve; Valentin, Anne-Sophie; Glaser, Philippe; Mereghetti, Laurent; Lanotte, Philippe

2015-01-01

CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) are found in 90% of archaea and about 40% of bacteria. In this original system, CRISPR arrays comprise short, almost unique sequences called spacers that are interspersed with conserved palindromic repeats. These systems play a role in adaptive immunity and participate to fight non-self DNA such as integrative and conjugative elements, plasmids, and phages. In Streptococcus agalactiae, a bacterium implicated in colonization and infections in humans since the 1960s, two CRISPR-Cas systems have been described. A type II-A system, characterized by proteins Cas9, Cas1, Cas2, and Csn2, is ubiquitous, and a type I–C system, with the Cas8c signature protein, is present in about 20% of the isolates. Unlike type I–C, which appears to be non-functional, type II-A appears fully functional. Here we studied type II-A CRISPR-cas loci from 126 human isolates of S. agalactiae belonging to different clonal complexes that represent the diversity of the species and that have been implicated in colonization or infection. The CRISPR-cas locus was analyzed both at spacer and repeat levels. Major distinctive features were identified according to the phylogenetic lineages previously defined by multilocus sequence typing, especially for the sequence type (ST) 17, which is considered hypervirulent. Among other idiosyncrasies, ST-17 shows a significantly lower number of spacers in comparison with other lineages. This characteristic could reflect the peculiar virulence or colonization specificities of this lineage. PMID:26124774

Emergence of viral hemorrhagic septicemia virus in the North American Great Lakes region is associated with low viral genetic diversity

USGS Publications Warehouse

Thompson, T.M.; Batts, W.N.; Faisal, M.; Bowser, P.; Casey, J.W.; Phillips, K.; Garver, K.A.; Winton, J.; Kurath, G.

2011-01-01

Viral hemorrhagic septicemia virus (VHSV) is a fish rhabdovirus that causes disease in a broad range of marine and freshwater hosts. The known geographic range includes the Northern Atlantic and Pacific Oceans, and recently it has invaded the Great Lakes region of North Ame­rica. The goal of this work was to characterize genetic diversity of Great Lakes VHSV isolates at the early stage of this viral emergence by comparing a partial glycoprotein (G) gene sequence (669 nt) of 108 isolates collected from 2003 to 2009 from 31 species and at 37 sites. Phylogenetic analysis showed that all isolates fell into sub-lineage IVb within the major VHSV genetic group IV. Among these 108 isolates, genetic diversity was low, with a maximum of 1.05% within the 669 nt region. There were 11 unique sequences, designated vcG001 to vcG011. Two dominant sequence types, vcG001 and vcG002, accounted for 90% (97 of 108) of the isolates. The vcG001 isolates were most widespread. We saw no apparent association of sequence type with host or year of isolation, but we did note a spatial pattern, in which vcG002 isolates were more prevalent in the easternmost sub-regions, including inland New York state and the St. Lawrence Seaway. Different sequence types were found among isolates from single disease outbreaks, and mixtures of types were evident within 2 isolates from ­individual fish. Overall, the genetic diversity of VHSV in the Great Lakes region was found to be extremely low, consistent with an introduction of a new virus into a geographic region with ­previously naïve host populations.

Emergence of Viral hemorrhagic septicemia virus in the North American Great Lakes region is associated with low viral genetic diversity.

PubMed

Thompson, Tarin M; Batts, William N; Faisal, Mohamed; Bowser, Paul; Casey, James W; Phillips, Kenneth; Garver, Kyle A; Winton, James; Kurath, Gael

2011-08-29

Viral hemorrhagic septicemia virus (VHSV) is a fish rhabdovirus that causes disease in a broad range of marine and freshwater hosts. The known geographic range includes the Northern Atlantic and Pacific Oceans, and recently it has invaded the Great Lakes region of North America. The goal of this work was to characterize genetic diversity of Great Lakes VHSV isolates at the early stage of this viral emergence by comparing a partial glycoprotein (G) gene sequence (669 nt) of 108 isolates collected from 2003 to 2009 from 31 species and at 37 sites. Phylogenetic analysis showed that all isolates fell into sub-lineage IVb within the major VHSV genetic group IV. Among these 108 isolates, genetic diversity was low, with a maximum of 1.05% within the 669 nt region. There were 11 unique sequences, designated vcG001 to vcG011. Two dominant sequence types, vcG001 and vcG002, accounted for 90% (97 of 108) of the isolates. The vcG001 isolates were most widespread. We saw no apparent association of sequence type with host or year of isolation, but we did note a spatial pattern, in which vcG002 isolates were more prevalent in the easternmost sub-regions, including inland New York state and the St. Lawrence Seaway. Different sequence types were found among isolates from single disease outbreaks, and mixtures of types were evident within 2 isolates from individual fish. Overall, the genetic diversity of VHSV in the Great Lakes region was found to be extremely low, consistent with an introduction of a new virus into a geographic region with previously naive host populations.

Emergence of Sequence Type 779 Methicillin-Resistant Staphylococcus aureus Harboring a Novel Pseudo Staphylococcal Cassette Chromosome mec (SCCmec)-SCC-SCCCRISPR Composite Element in Irish Hospitals

PubMed Central

Kinnevey, Peter M.; Shore, Anna C.; Brennan, Grainne I.; Sullivan, Derek J.; Ehricht, Ralf; Monecke, Stefan; Slickers, Peter

2013-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) has been a major cause of nosocomial infection in Irish hospitals for 4 decades, and replacement of predominant MRSA clones has occurred several times. An MRSA isolate recovered in 2006 as part of a larger study of sporadic MRSA exhibited a rare spa (t878) and multilocus sequence (ST779) type and was nontypeable by PCR- and DNA microarray-based staphylococcal cassette chromosome mec (SCCmec) element typing. Whole-genome sequencing revealed the presence of a novel 51-kb composite island (CI) element with three distinct domains, each flanked by direct repeat and inverted repeat sequences, including (i) a pseudo SCCmec element (16.3 kb) carrying mecA with a novel mec class region, a fusidic acid resistance gene (fusC), and two copper resistance genes (copB and copC) but lacking ccr genes; (ii) an SCC element (17.5 kb) carrying a novel ccrAB4 allele; and (iii) an SCC element (17.4 kb) carrying a novel ccrC allele and a clustered regularly interspaced short palindromic repeat (CRISPR) region. The novel CI was subsequently identified by PCR in an additional 13 t878/ST779 MRSA isolates, six from bloodstream infections, recovered between 2006 and 2011 in 11 hospitals. Analysis of open reading frames (ORFs) carried by the CI showed amino acid sequence similarity of 44 to 100% to ORFs from S. aureus and coagulase-negative staphylococci (CoNS). These findings provide further evidence of genetic transfer between S. aureus and CoNS and show how this contributes to the emergence of novel SCCmec elements and MRSA strains. Ongoing surveillance of this MRSA strain is warranted and will require updating of currently used SCCmec typing methods. PMID:23147725

Multilocus sequence typing and virulence analysis of Haemophilus parasuis strains isolated in five provinces of China.

PubMed

Wang, Liyan; Ma, Lina; Liu, Yongan; Gao, Pengcheng; Li, Youquan; Li, Xuerui; Liu, Yongsheng

2016-10-01

Haemophilus parasuis is the etiological agent of Glässers disease, which causes high morbidity and mortality in swine herds. Although H. parasuis strains can be classified into 15 serovars with the Kielstein-Rapp-Gabrielson serotyping scheme, a large number of isolates cannot be classified and have been designated 'nontypeable' strains. In this study, multilocus sequence typing (MLST) of H. parasuis was used to analyze 48 H. parasuis field strains isolated in China and two strains from Australia. Twenty-six new alleles and 29 new sequence types (STs) were detected, enriching the H. parasuis MLST databases. A BURST analysis indicated that H. parasuis lacks stable population structure and is highly heterogeneous, and that there is no association between STs and geographic area. When an UPGMA dendrogram was constructed, two major clades, clade A and clade B, were defined. Animal experiments, in which guinea pigs were challenged intraperitoneally with the bacterial isolates, supported the hypothesis that the H. parasuis STs in clade A are generally avirulent or weakly virulent, whereas the STs in clade B tend to be virulent. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of ROP18 alleles in human toxoplasmosis.

PubMed

Sánchez, Víctor; de-la-Torre, Alejandra; Gómez-Marín, Jorge Enrique

2014-04-01

The role of the virulent gene ROP18 polymorphisms is not known in human toxoplasmosis. A total of 320 clinical samples were analyzed. In samples positive for ROP18 gene, we determined by an allele specific PCR, if patients got the upstream insertion positive ROP18 sequence Toxoplasma strain (mouse avirulent strain) or the upstream insertion negative ROP18 sequence Toxoplasma strain (mouse virulent strain). We designed an ELISA assay for antibodies against ROP18 derived peptides from the three major clonal lineages of Toxoplasma. 20 clinical samples were of quality for ROP18 allele analysis. In patients with ocular toxoplasmosis, a higher inflammatory reaction on eye was associated to a PCR negative result for the upstream region of ROP18. 23.3%, 33% and 16.6% of serums from individuals with ocular toxoplasmosis were positive for type I, type II and type III ROP18 derived peptides, respectively but this assay was affected by cross reaction. The absence of Toxoplasma ROP18 promoter insertion sequence in ocular toxoplasmosis was correlated with severe ocular inflammatory response. Determination of antibodies against ROP18 protein was not useful for serotyping in human toxoplasmosis. © 2013.

Genomic Epidemiology of Salmonella enterica Serotype Enteritidis based on Population Structure of Prevalent Lineages

PubMed Central

Desai, Prerak T.; den Bakker, Henk C.; Mikoleit, Matthew; Tolar, Beth; Trees, Eija; Hendriksen, Rene S.; Frye, Jonathan G.; Porwollik, Steffen; Weimer, Bart C.; Wiedmann, Martin; Weinstock, George M.; Fields, Patricia I.; McClelland, Michael

2014-01-01

Salmonella enterica serotype Enteritidis is one of the most commonly reported causes of human salmonellosis. Its low genetic diversity, measured by fingerprinting methods, has made subtyping a challenge. We used whole-genome sequencing to characterize 125 S. enterica Enteritidis and 3 S. enterica serotype Nitra strains. Single-nucleotide polymorphisms were filtered to identify 4,887 reliable loci that distinguished all isolates from each other. Our whole-genome single-nucleotide polymorphism typing approach was robust for S. enterica Enteritidis subtyping with combined data for different strains from 2 different sequencing platforms. Five major genetic lineages were recognized, which revealed possible patterns of geographic and epidemiologic distribution. Analyses on the population dynamics and evolutionary history estimated that major lineages emerged during the 17th–18th centuries and diversified during the 1920s and 1950s. PMID:25147968

How, who, and when: preferences for delivery of genome sequencing results among women diagnosed with breast cancer at a young age.

PubMed

Kaphingst, Kimberly A; Ivanovich, Jennifer; Elrick, Ashley; Dresser, Rebecca; Matsen, Cindy; Goodman, Melody S

2016-11-01

The increasing use of genome sequencing with patients raises a critical communication challenge: return of secondary findings. While the issue of what sequencing results should be returned to patients has been examined, much less attention has been paid to developing strategies to return these results in ways that meet patients' needs and preferences. To address this, we investigated delivery preferences (i.e., who, how, when) for individual genome sequencing results among women diagnosed with breast cancer at age 40 or younger. We conducted 60 semistructured, in-person individual interviews to examine preferences for the return of different types of genome sequencing results and the reasons underlying these preferences. Two coders independently coded interview transcripts; analysis was conducted using NVivo 10. The major findings from the study were that: (1) many participants wanted sequencing results as soon as possible, even at the time of breast cancer diagnosis; (2) participants wanted an opportunity for an in-person discussion of results; and (3) they put less emphasis on the type of person delivering results than on the knowledge and communicative skills of that person. Participants also emphasized the importance of a results return process tailored to a patient's individual circumstances and one that she has a voice in determining. A critical goal for future transdisciplinary research including clinicians, patients, and communication researchers may be to develop decision-making processes to help patients make decisions about how they would like various sequencing results returned.

E6 and E7 Gene Polymorphisms in Human Papillomavirus Types-58 and 33 Identified in Southwest China

PubMed Central

Wen, Qiang; Wang, Tao; Mu, Xuemei; Chenzhang, Yuwei; Cao, Man

2017-01-01

Cancer of the cervix is associated with infection by certain types of human papillomavirus (HPV). The gene variants differ in immune responses and oncogenic potential. The E6 and E7 proteins encoded by high-risk HPV play a key role in cellular transformation. HPV-33 and HPV-58 types are highly prevalent among Chinese women. To study the gene intratypic variations, polymorphisms and positive selections of HPV-33 and HPV-58 E6/E7 in southwest China, HPV-33 (E6, E7: n = 216) and HPV-58 (E6, E7: n = 405) E6 and E7 genes were sequenced and compared to others submitted to GenBank. Phylogenetic trees were constructed by Maximum-likelihood and the Kimura 2-parameters methods by MEGA 6 (Molecular Evolutionary Genetics Analysis version 6.0). The diversity of secondary structure was analyzed by PSIPred software. The selection pressures acting on the E6/E7 genes were estimated by PAML 4.8 (Phylogenetic Analyses by Maximun Likelihood version4.8) software. The positive sites of HPV-33 and HPV-58 E6/E7 were contrasted by ClustalX 2.1. Among 216 HPV-33 E6 sequences, 8 single nucleotide mutations were observed with 6/8 non-synonymous and 2/8 synonymous mutations. The 216 HPV-33 E7 sequences showed 3 single nucleotide mutations that were non-synonymous. The 405 HPV-58 E6 sequences revealed 8 single nucleotide mutations with 4/8 non-synonymous and 4/8 synonymous mutations. Among 405 HPV-58 E7 sequences, 13 single nucleotide mutations were observed with 10/13 non-synonymous mutations and 3/13 synonymous mutations. The selective pressure analysis showed that all HPV-33 and 4/6 HPV-58 E6/E7 major non-synonymous mutations were sites of positive selection. All variations were observed in sites belonging to major histocompatibility complex and/or B-cell predicted epitopes. K93N and R145 (I/N) were observed in both HPV-33 and HPV-58 E6. PMID:28141822

Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC beta-lactamase, and the foss of an outer membrane protein.

PubMed Central

Bradford, P A; Urban, C; Mariano, N; Projan, S J; Rahal, J J; Bush, K

1997-01-01

Six Escherichia coli and 12 Klebsiella pneumoniae isolates from a single hospital expressed a common beta-lactamase with a pI of approximately 9.0 and were resistant to cefoxitin and cefotetan (MIC ranges, 64 to > 128 and 16 to > 128 micrograms/ml, respectively). Seventeen of the 18 strains produced multiple beta-lactamases. Most significantly, three K. pneumoniae strains were also resistant to imipenem (MICs, 8 to 32 micrograms/ml). Spectrophotometric beta-lactamase assays with purified enzyme indicated hydrolysis of cephamycins, in addition to cephaloridine and benzylpenicillin. The 4ene encoding the pI 9.0 beta-lactamase (designated ACT-1 for AmpC type) was cloned and sequenced, which revealed an ampC-type beta-lactamase gene that originated from Enterobacter cloacae and that had 86% sequence homology to the P99 beta-lactamase and 94% homology to the partial sequence of MIR-1. Southern blotting revealed that the gene encoding ACT-1 was on a large plasmid in some of the K. pneumoniae strains as well as on the chromosomes of all of the strains, suggesting that the gene is located on an easily mobilized element. Outer membrane protein profiles of the K. pneumoniae strains revealed that the three imipenem-resistant strains were lacking a major outer membrane protein of approximately 42 kDa which was present in the imipenem-susceptible strains. ACT-1 is the first plasmid-mediated AmpC-type beta-lactamase derived from Enterobacter which has been completely sequenced. This work demonstrates that in addition to resistance to cephamycins, imipenem resistance can occur in K. pneumoniae when a high level of the ACT-1 beta-lactamase is produced in combination with the loss of a major outer membrane protein. PMID:9055993

Discovery of Genes Expressed In Basal Endosperm Transfer Cells in Maize Using 454 Transcriptome Sequencing

USDA-ARS?s Scientific Manuscript database

Basal endosperm transfer cells (BETCs) constitute one of the four cell types in an endosperm with a major role in solute acquisition and transport functions from the mother plant. The BETCs with their wall-in-growth (WIG) feature that greatly increase plasma membrane area of each cell are critical f...

DNA capture and next-generation sequencing can recover whole mitochondrial genomes from highly degraded samples for human identification

PubMed Central

2013-01-01

Background Mitochondrial DNA (mtDNA) typing can be a useful aid for identifying people from compromised samples when nuclear DNA is too damaged, degraded or below detection thresholds for routine short tandem repeat (STR)-based analysis. Standard mtDNA typing, focused on PCR amplicon sequencing of the control region (HVS I and HVS II), is limited by the resolving power of this short sequence, which misses up to 70% of the variation present in the mtDNA genome. Methods We used in-solution hybridisation-based DNA capture (using DNA capture probes prepared from modern human mtDNA) to recover mtDNA from post-mortem human remains in which the majority of DNA is both highly fragmented (<100 base pairs in length) and chemically damaged. The method ‘immortalises’ the finite quantities of DNA in valuable extracts as DNA libraries, which is followed by the targeted enrichment of endogenous mtDNA sequences and characterisation by next-generation sequencing (NGS). Results We sequenced whole mitochondrial genomes for human identification from samples where standard nuclear STR typing produced only partial profiles or demonstrably failed and/or where standard mtDNA hypervariable region sequences lacked resolving power. Multiple rounds of enrichment can substantially improve coverage and sequencing depth of mtDNA genomes from highly degraded samples. The application of this method has led to the reliable mitochondrial sequencing of human skeletal remains from unidentified World War Two (WWII) casualties approximately 70 years old and from archaeological remains (up to 2,500 years old). Conclusions This approach has potential applications in forensic science, historical human identification cases, archived medical samples, kinship analysis and population studies. In particular the methodology can be applied to any case, involving human or non-human species, where whole mitochondrial genome sequences are required to provide the highest level of maternal lineage discrimination. Multiple rounds of in-solution hybridisation-based DNA capture can retrieve whole mitochondrial genome sequences from even the most challenging samples. PMID:24289217

Flagellin Diversity in Clostridium botulinum Groups I and II: a New Strategy for Strain Identification▿

PubMed Central

Paul, Catherine J.; Twine, Susan M.; Tam, Kevin J.; Mullen, James A.; Kelly, John F.; Austin, John W.; Logan, Susan M.

2007-01-01

Strains of Clostridium botulinum are traditionally identified by botulinum neurotoxin type; however, identification of an additional target for typing would improve differentiation. Isolation of flagellar filaments and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that C. botulinum produced multiple flagellin proteins. Nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis of in-gel tryptic digests identified peptides in all flagellin bands that matched two homologous tandem flagellin genes identified in the C. botulinum Hall A genome. Designated flaA1 and flaA2, these open reading frames encode the major structural flagellins of C. botulinum. Colony PCR and sequencing of flaA1/A2 variable regions classified 80 environmental and clinical strains into group I or group II and clustered isolates into 12 flagellar types. Flagellar type was distinct from neurotoxin type, and epidemiologically related isolates clustered together. Sequencing a larger PCR product, obtained during amplification of flaA1/A2 from type E strain Bennett identified a second flagellin gene, flaB. LC-MS analysis confirmed that flaB encoded a large type E-specific flagellin protein, and the predicted molecular mass for FlaB matched that observed by SDS-PAGE. In contrast, the molecular mass of FlaA was 2 to 12 kDa larger than the mass predicted by the flaA1/A2 sequence of a given strain, suggesting that FlaA is posttranslationally modified. While identification of FlaB, and the observation by SDS-PAGE of different masses of the FlaA proteins, showed the flagellin proteins of C. botulinum to be diverse, the presence of the flaA1/A2 gene in all strains examined facilitates single locus sequence typing of C. botulinum using the flagellin variable region. PMID:17351097

Conservation of a pH-sensitive structure in the C-terminal region of spider silk extends across the entire silk gene family.

PubMed

Strickland, Michelle; Tudorica, Victor; Řezáč, Milan; Thomas, Neil R; Goodacre, Sara L

2018-06-01

Spiders produce multiple silks with different physical properties that allow them to occupy a diverse range of ecological niches, including the underwater environment. Despite this functional diversity, past molecular analyses show a high degree of amino acid sequence similarity between C-terminal regions of silk genes that appear to be independent of the physical properties of the resulting silks; instead, this domain is crucial to the formation of silk fibers. Here, we present an analysis of the C-terminal domain of all known types of spider silk and include silk sequences from the spider Argyroneta aquatica, which spins the majority of its silk underwater. Our work indicates that spiders have retained a highly conserved mechanism of silk assembly, despite the extraordinary diversification of species, silk types and applications of silk over 350 million years. Sequence analysis of the silk C-terminal domain across the entire gene family shows the conservation of two uncommon amino acids that are implicated in the formation of a salt bridge, a functional bond essential to protein assembly. This conservation extends to the novel sequences isolated from A. aquatica. This finding is relevant to research regarding the artificial synthesis of spider silk, suggesting that synthesis of all silk types will be possible using a single process.

Comparison of Campylobacter jejuni isolates from human, food, veterinary and environmental sources in Iceland using PFGE, MLST and fla-SVR sequencing.

PubMed

Magnússon, S H; Guðmundsdóttir, S; Reynisson, E; Rúnarsson, A R; Harðardóttir, H; Gunnarson, E; Georgsson, F; Reiersen, J; Marteinsson, V Th

2011-10-01

Campylobacter jejuni isolates from various sources in Iceland were genotyped with the aim of assessing the genetic diversity, population structure, source distribution and campylobacter transmission routes to humans. A collection of 584 Campylobacter isolates were collected from clinical cases, food, animals and environment in Iceland in 1999-2002, during a period of national Campylobacter epidemic in Iceland. All isolates were characterized by pulse field gel electrophoresis (PFGE), and selected subset of 52 isolates representing the diversity of the identified PFGE types was further genotyped using multilocus sequence typing (MLST) and fla-SVR sequencing to gain better insight into the population structure. The results show a substantial diversity within the Icelandic Campylobacter population. Majority of the human Campylobacter infections originated from domestic chicken and cattle isolates. MLST showed the isolates to be distributed among previously reported and common sequence type complexes in the MLST database. The genotyping of Campylobacter from various sources has not previously been reported from Iceland, and the results of the study gave a valuable insight into the population structure of Camp. jejuni in Iceland, source distribution and transmission routes to humans. The geographical isolation of Iceland in the north Atlantic provides new information on Campylobacter population dynamics on a global scale. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology No claim to Icelandic Government works.

Variation of Synechococcus Pigment Genetic Diversity Along Two Turbidity Gradients in the China Seas.

PubMed

Xia, Xiaomin; Liu, Hongbin; Choi, Donghan; Noh, Jae Hoon

2018-01-01

Synechococcus are important and widely distributed picocyanobacteria that encompass a high pigment diversity. In this study, we developed a primer set (peBF/peAR) for amplifying the cpeBA operon sequence from Synechococcus genomic DNA to study Synechococcus pigment diversity along two turbidity gradients in the China seas. Our data revealed that all previously reported pigment types occurred in the South (SCS) and East (ECS) China Seas. In addition, a novel pigment genetic type (type 3f), represented by the high phycourobilin Synechococcus sp. strain KORDI-100 (Exc495:545 = 2.35), was detected. This pigment genetic type differs from the 3c/3d types not only for a very high PUB/PEB ratio but also for a different intergenic spacer sequence and gene organization of the phycobilisome. Synechococcus of different pigment types exhibited clear niche differentiation. Type 2 dominated in the coastal waters, whereas type 3c/3d and 3f were predominant in oceanic waters of the SCS in summer. In the ECS, however, type 3a was the major pigment type throughout the transect. We suggest that in marine environment, various pigment types often co-occur but with one type dominant and PUB/PEB ratio is related to geographic distribution of Synechococcus pigment types. The two marginal seas of China have markedly different Synechococcus pigment compositions.

Streptococcal M protein extracted by nonionic detergent. III. Correlation between immunological cross-reactions and structural similarities with implications for antiphagocytosis

PubMed Central

1978-01-01

Three immunologically cross-reactive and non-cross-reactive streptococcal M proteins were analyzed by a chromatographic tryptic peptide mapping system. The results indicate that cross-reactions correlate with the extent of structural similarity among the M protein molecules analyzed. The data also reveal that free lysine is released by the action of trypsin from these three M proteins, suggesting a common lys-lys or arg-lys sequence. In addition, only one peptide has been found to be common within all three M types. This limited structural relatedness among the three M proteins examined indicates that sequence variation plays a major role in the immunological specificity of the M antigens. However, despite sequence variation, all M protein molecules have a common antiphagocytic activity. The fact that no common opsonic antibody has yet been found, even against limited M types, argues against this biological activity being solely the result of a common sequence. Based on these data, it is suggested that the antiphagocytic effect of M protein may be due to a conformationally created environment on the surface of the molecule which is selected by both immunological and biological pressure. PMID:355596

The production of Multiple Small Peptaibol Families by Single 14-Module Peptide Synthetases in Trichoderma/Hypocrea

DOE Office of Scientific and Technical Information (OSTI.GOV)

Degenkolb, Thomas; Aghchehb, Razieh Karimi; Dieckmann, Ralf

2012-03-01

The most common peptaibibiotic structures are 11-residue peptaibols found widely distributed in the genus Trichoderma/Hypocrea. Frequently associated are 14-residue peptaibols sharing partial sequence identity. Genome sequencing projects of 3 Trichoderma strains of the major clades reveal the presence of up to 3 types of nonribosomal peptide synthetases with 7, 14, or 18-20 amino acid adding modules. We here provide evidence that the 14-module NRPS type found in T. virens, T. reesei (teleomorph Hypocrea jecorina) and T. atroviride produces both 11- and 14- residue peptaibols based on the disruption of the respective NRPS gene of T. reesei, and bioinformatic analysis ofmore » their amino acid activating domains and modules. The structures of these peptides may be predicted from the gene structures and have been confirmed by analysis of families of 11- and 14-residue peptaibols from the strain 618, termed hypojecorins A (23 sequences determined, 4 new) and B (3 new sequences), and the recently established trichovirins A from T. virens. The distribution of 11- and 14-residue products is strain-specific and depends on growth conditions as well. Possible mechanisms of module skipping are discussed.« less

Repetitive DNA in the pea (Pisum sativum L.) genome: comprehensive characterization using 454 sequencing and comparison to soybean and Medicago truncatula

PubMed Central

Macas, Jiří; Neumann, Pavel; Navrátilová, Alice

2007-01-01

Background Extraordinary size variation of higher plant nuclear genomes is in large part caused by differences in accumulation of repetitive DNA. This makes repetitive DNA of great interest for studying the molecular mechanisms shaping architecture and function of complex plant genomes. However, due to methodological constraints of conventional cloning and sequencing, a global description of repeat composition is available for only a very limited number of higher plants. In order to provide further data required for investigating evolutionary patterns of repeated DNA within and between species, we used a novel approach based on massive parallel sequencing which allowed a comprehensive repeat characterization in our model species, garden pea (Pisum sativum). Results Analysis of 33.3 Mb sequence data resulted in quantification and partial sequence reconstruction of major repeat families occurring in the pea genome with at least thousands of copies. Our results showed that the pea genome is dominated by LTR-retrotransposons, estimated at 140,000 copies/1C. Ty3/gypsy elements are less diverse and accumulated to higher copy numbers than Ty1/copia. This is in part due to a large population of Ogre-like retrotransposons which alone make up over 20% of the genome. In addition to numerous types of mobile elements, we have discovered a set of novel satellite repeats and two additional variants of telomeric sequences. Comparative genome analysis revealed that there are only a few repeat sequences conserved between pea and soybean genomes. On the other hand, all major families of pea mobile elements are well represented in M. truncatula. Conclusion We have demonstrated that even in a species with a relatively large genome like pea, where a single 454-sequencing run provided only 0.77% coverage, the generated sequences were sufficient to reconstruct and analyze major repeat families corresponding to a total of 35–48% of the genome. These data provide a starting point for further investigations of legume plant genomes based on their global comparative analysis and for the development of more sophisticated approaches for data mining. PMID:18031571

Functionally conserved cis-regulatory elements of COL18A1 identified through zebrafish transgenesis.

PubMed

Kague, Erika; Bessling, Seneca L; Lee, Josephine; Hu, Gui; Passos-Bueno, Maria Rita; Fisher, Shannon

2010-01-15

Type XVIII collagen is a component of basement membranes, and expressed prominently in the eye, blood vessels, liver, and the central nervous system. Homozygous mutations in COL18A1 lead to Knobloch Syndrome, characterized by ocular defects and occipital encephalocele. However, relatively little has been described on the role of type XVIII collagen in development, and nothing is known about the regulation of its tissue-specific expression pattern. We have used zebrafish transgenesis to identify and characterize cis-regulatory sequences controlling expression of the human gene. Candidate enhancers were selected from non-coding sequence associated with COL18A1 based on sequence conservation among mammals. Although these displayed no overt conservation with orthologous zebrafish sequences, four regions nonetheless acted as tissue-specific transcriptional enhancers in the zebrafish embryo, and together recapitulated the major aspects of col18a1 expression. Additional post-hoc computational analysis on positive enhancer sequences revealed alignments between mammalian and teleost sequences, which we hypothesize predict the corresponding zebrafish enhancers; for one of these, we demonstrate functional overlap with the orthologous human enhancer sequence. Our results provide important insight into the biological function and regulation of COL18A1, and point to additional sequences that may contribute to complex diseases involving COL18A1. More generally, we show that combining functional data with targeted analyses for phylogenetic conservation can reveal conserved cis-regulatory elements in the large number of cases where computational alignment alone falls short. Copyright 2009 Elsevier Inc. All rights reserved.

Depletion of Unwanted Nucleic Acid Templates by Selective Cleavage: LNAzymes, Catalytically Active Oligonucleotides Containing Locked Nucleic Acids, Open a New Window for Detecting Rare Microbial Community Members

PubMed Central

Dolinšek, Jan; Dorninger, Christiane; Lagkouvardos, Ilias; Wagner, Michael

2013-01-01

Many studies of molecular microbial ecology rely on the characterization of microbial communities by PCR amplification, cloning, sequencing, and phylogenetic analysis of genes encoding rRNAs or functional marker enzymes. However, if the established clone libraries are dominated by one or a few sequence types, the cloned diversity is difficult to analyze by random clone sequencing. Here we present a novel approach to deplete unwanted sequence types from complex nucleic acid mixtures prior to cloning and downstream analyses. It employs catalytically active oligonucleotides containing locked nucleic acids (LNAzymes) for the specific cleavage of selected RNA targets. When combined with in vitro transcription and reverse transcriptase PCR, this LNAzyme-based technique can be used with DNA or RNA extracts from microbial communities. The simultaneous application of more than one specific LNAzyme allows the concurrent depletion of different sequence types from the same nucleic acid preparation. This new method was evaluated with defined mixtures of cloned 16S rRNA genes and then used to identify accompanying bacteria in an enrichment culture dominated by the nitrite oxidizer “Candidatus Nitrospira defluvii.” In silico analysis revealed that the majority of publicly deposited rRNA-targeted oligonucleotide probes may be used as specific LNAzymes with no or only minor sequence modifications. This efficient and cost-effective approach will greatly facilitate tasks such as the identification of microbial symbionts in nucleic acid preparations dominated by plastid or mitochondrial rRNA genes from eukaryotic hosts, the detection of contaminants in microbial cultures, and the analysis of rare organisms in microbial communities of highly uneven composition. PMID:23263968

Genetic characterization of Babesia and Theileria parasites in water buffaloes in Sri Lanka.

PubMed

Sivakumar, Thillaiampalam; Tattiyapong, Muncharee; Fukushi, Shintaro; Hayashida, Kyoko; Kothalawala, Hemal; Silva, Seekkuge Susil Priyantha; Vimalakumar, Singarayar Caniciyas; Kanagaratnam, Ratnam; Meewewa, Asela Sanjeewa; Suthaharan, Kalpana; Puvirajan, Thamotharampillai; de Silva, Weligodage Kumarawansa; Igarashi, Ikuo; Yokoyama, Naoaki

2014-02-24

Water buffaloes are thought to be the reservoir hosts for several hemoprotozoan parasites that infect cattle. In the present study, we surveyed Sri Lankan bred water buffaloes for infections with Babesia bovis, Babesia bigemina, Theileria annulata, and Theileria orientalis using parasite-specific PCR assays. When 320 blood-derived DNA samples from water buffaloes reared in three different districts (Polonnaruwa, Mannar, and Mullaitivu) of Sri Lanka were PCR screened, B. bovis, B. bigemina, and T. orientalis were detected. While T. orientalis was the predominant parasite (82.5%), low PCR-positive rates were observed for B. bovis (1.9%) and B. bigemina (1.6%). Amplicons of the gene sequences of the Rhoptry Associated Protein-1 (RAP-1) of B. bovis, the Apical Membrane Antigen-1 (AMA-1) of B. bigemina, and the Major Piroplasm Surface Protein (MPSP) of T. orientalis were compared with those characterized previously in Sri Lankan cattle. While the B. bigemina AMA-1 sequences from water buffaloes shared high identity values with those from cattle, B. bovis RAP-1 sequences from water buffaloes diverged genetically from those of cattle. For T. orientalis, none of the MPSP sequence types reported previously in Sri Lankan cattle (types 1, 3, 5, and 7) were detected in the water buffaloes, and the MPSP sequences analyzed in the present study belonged to types N1 or N2. In summary, in addition to reporting the first PCR-based survey of Babesia and Theileria parasites in water buffaloes in Sri Lanka, the present study found that the predominant variants of water buffalo-derived B. bovis RAP-1 and T. orientalis MPSP sequences were different from those previously described from cattle in this country. Copyright © 2013 Elsevier B.V. All rights reserved.

Antifungal drug susceptibility and phylogenetic diversity among Cryptococcus isolates from dogs and cats in North America.

PubMed

Singer, Lisa M; Meyer, Wieland; Firacative, Carolina; Thompson, George R; Samitz, Eileen; Sykes, Jane E

2014-06-01

Molecular types of the Cryptococcus neoformans/Cryptococcus gattii species complex that infect dogs and cats differ regionally and with host species. Antifungal drug susceptibility can vary with molecular type, but the susceptibility of Cryptococcus isolates from dogs and cats is largely unknown. Cryptococcus isolates from 15 dogs and 27 cats were typed using URA5 restriction fragment length polymorphism analysis (RFLP), PCR fingerprinting, and multilocus sequence typing (MLST). Susceptibility was determined using a microdilution assay (Sensititre YeastOne; Trek Diagnostic Systems). MICs were compared among groups. The 42 isolates studied comprised molecular types VGI (7%), VGIIa (7%), VGIIb (5%), VGIIc (5%), VGIII (38%), VGIV (2%), VNI (33%), and VNII (2%), as determined by URA5 RFLP. The VGIV isolate was more closely related to VGIII according to MLST. All VGIII isolates were from cats. All sequence types identified from veterinary isolates clustered with isolates from humans. VGIII isolates showed considerable genetic diversity compared with other Cryptococcus molecular types and could be divided into two major subgroups. Compared with C. neoformans MICs, C. gattii MICs were lower for flucytosine, and VGIII MICs were lower for flucytosine and itraconazole. For all drugs except itraconazole, C. gattii isolates exhibited a wider range of MICs than C. neoformans. MICs varied with Cryptococcus species and molecular type in dogs and cats, and MICs of VGIII isolates were most variable and may reflect phylogenetic diversity in this group. Because sequence types of dogs and cats reflect those infecting humans, these observations may also have implications for treatment of human cryptococcosis. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Description of Paenisporosarcina quisquiliarum gen. nov., sp. nov., and reclassification of Sporosarcina macmurdoensis Reddy et al. 2003 as Paenisporosarcina macmurdoensis comb. nov.

PubMed

Krishnamurthi, S; Bhattacharya, A; Mayilraj, S; Saha, P; Schumann, P; Chakrabarti, T

2009-06-01

In the course of a study of the prokaryotic diversity of a landfill site in Chandigarh, India, a strain designated SK 55(T) was isolated and characterized using a polyphasic approach. Its 16S rRNA gene sequence showed closest similarity (98.3 %) to that of Sporosarcina macmurdoensis CMS 21w(T). The sequence similarity to strains of other hitherto described species of Sporosarcina was less than 95.5 %. Strain SK 55(T) contains peptidoglycan of the A4alpha type (l-Lys-d-Asp), MK-8 and MK-7 as the major menaquinones and iso-C(15 : 0) as the major fatty acid. Strain SK 55(T), Sporosarcina macmurdoensis and Sporosarcina ureae, the type species of the genus, had some polar lipids in common (diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, a phospholipid and an unknown lipid). However, an aminolipid, an aminophospholipid and an unknown lipid found in the former two organisms are similar, though not identical, but quite different from the profile of S. ureae. The genomic DNA G+C contents of strain SK 55(T) (46.0 mol%) and S. macmurdoensis CMS 21w(T) (44.0 mol%) are higher than those reported for the majority of species of Sporosarcina (36-42 mol%). As revealed by 16S rRNA gene sequence analysis, strain SK 55(T) and S. macmurdoensis CMS 21w(T) form a clade which is distinct from the clade occupied by other species of Sporosarcina. On the basis of phenotypic characteristics including chemotaxonomic data and analysis of the 16S rRNA gene sequence, we conclude that strain SK 55(T) should be considered as a member of a novel genus and species, for which the name Paenisporosarcina quisquiliarum gen. nov., sp. nov. is proposed. The type strain of Paenisporosarcina quisquiliarum is SK 55(T) (=MTCC7604(T) =JCM 14041(T)). S. macmurdoensis CMS 21w(T) shows more similarity in its 16S rRNA gene sequence (98.3 %), DNA G+C content and polar lipid profile to strain SK 55(T) than to S. ureae DSM 2281(T). Phylogenetically, it forms a coherent cluster with strain SK 55(T) which is separate from the Sporosarcina cluster. Moreover, iso-C(15 : 0), anteiso-C(15 : 0) and C(16 : 1)omega7c alcohol are the three major fatty acids in both S. macmurdoensis CMS 21w(T) and SK 55(T). All these data suggest that S. macmurdoensis should be a member of the genus Paenisporosarcina. However, S. macmurdoensis can be differentiated from SK 55(T) in several physiological and biochemical characteristics, especially in the patterns of oxidation and acid production from carbohydrates. The genomic relatedness of S. macmurdoensis CMS 21w(T) and strain SK 55(T) was also very low (18.0 %). It is therefore logical to transfer Sporosarcina macmurdoensis to the newly created genus as Paenisporosarcina macmurdoensis comb. nov. The type strain is CMS 21w(T) (=MTCC4670(T) =DSM 15428(T)).

β-Propeller Blades as Ancestral Peptides in Protein Evolution

PubMed Central

Kopec, Klaus O.; Lupas, Andrei N.

2013-01-01

Proteins of the β-propeller fold are ubiquitous in nature and widely used as structural scaffolds for ligand binding and enzymatic activity. This fold comprises between four and twelve four-stranded β-meanders, the so called blades that are arranged circularly around a central funnel-shaped pore. Despite the large size range of β-propellers, their blades frequently show sequence similarity indicative of a common ancestry and it has been proposed that the majority of β-propellers arose divergently by amplification and diversification of an ancestral blade. Given the structural versatility of β-propellers and the hypothesis that the first folded proteins evolved from a simpler set of peptides, we investigated whether this blade may have given rise to other folds as well. Using sequence comparisons, we identified proteins of four other folds as potential homologs of β-propellers: the luminal domain of inositol-requiring enzyme 1 (IRE1-LD), type II β-prisms, β-pinwheels, and WW domains. Because, with increasing evolutionary distance and decreasing sequence length, the statistical significance of sequence comparisons becomes progressively harder to distinguish from the background of convergent similarities, we complemented our analyses with a new method that evaluates possible homology based on the correlation between sequence and structure similarity. Our results indicate a homologous relationship of IRE1-LD and type II β-prisms with β-propellers, and an analogous one for β-pinwheels and WW domains. Whereas IRE1-LD most likely originated by fold-changing mutations from a fully formed PQQ motif β-propeller, type II β-prisms originated by amplification and differentiation of a single blade, possibly also of the PQQ type. We conclude that both β-propellers and type II β-prisms arose by independent amplification of a blade-sized fragment, which represents a remnant of an ancient peptide world. PMID:24143202

Microbacterium lemovicicum sp. nov., a bacterium isolated from a natural uranium-rich soil.

PubMed

Mondani, Laure; Piette, Laurie; Christen, Richard; Bachar, Dipankar; Berthomieu, Catherine; Chapon, Virginie

2013-07-01

An actinobacterial strain, designated ViU22(T), was isolated from a natural uranium-rich soil and was studied using a polyphasic approach. Cells formed orange-pigmented colonies, were rod-shaped, Gram-positive (non-staining method), non-motile and non-spore-forming. This organism grew in 0-4.5 % (w/v) NaCl and at 15-37 °C, with optimal growth occurring in 0.5 % (w/v) NaCl and at 30 °C. Comparative 16S rRNA gene sequence analysis revealed that the strain ViU22(T) belonged to the genus Microbacterium. It exhibited highest 16S rRNA gene sequence similarity with the type strains of Microbacterium testaceum (98.14 %) and Microbacterium binotii (98.02 %). The DNA-DNA relatedness of strains ViU22(T) with the most closely related type strains Microbacterium testaceum and Microbacterium binotii DSM 19164(T) was 20.10 % (± 0.70) and 28.05 % (± 0.35), respectively. Strain ViU22(T) possessed a type B2β peptidoglycan with partial substitution of glutamic acid by 3-hydroxy glutamic acid. The major menaquinones were MK-11 and MK-12. Major polar lipids detected in the strain ViU22(T) were diphosphatidylglycerol, phosphatidylglycerol, an unknown phospholipid and unknown glycolipids. The predominant fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0, a pattern reported for other Microbacterium species. The major cell-wall sugars were galactose, xylose and mannose and the DNA G+C content was 71 mol%. Together, the DNA-DNA hybridization results and the differentiating phenotypic characteristics, showed that strain ViU22(T) should be classified as the type strain of a novel species within the genus Microbacterium, for which the name Microbacterium lemovicicum sp. nov. is proposed. The type strain is ViU22(T) ( = ATCC BAA-2396(T) = CCUG 62198(T) = DSM 25044(T)).

Molecular Epidemiology Reveals Genetic Diversity amongst Isolates of the Cryptococcus neoformans/C. gattii Species Complex in Thailand

PubMed Central

Kaocharoen, Sirada; Ngamskulrungroj, Popchai; Firacative, Carolina; Trilles, Luciana; Piyabongkarn, Dumrongdej; Banlunara, Wijit; Poonwan, Natteewan; Chaiprasert, Angkana; Meyer, Wieland; Chindamporn, Ariya

2013-01-01

To gain a more detailed picture of cryptococcosis in Thailand, a retrospective study of 498 C. neoformans and C. gattii isolates has been conducted. Among these, 386, 83 and 29 strains were from clinical, environmental and veterinary sources, respectively. A total of 485 C. neoformans and 13 C. gattii strains were studied. The majority of the strains (68.9%) were isolated from males (mean age of 37.97 years), 88.5% of C. neoformans and only 37.5% of C. gattii strains were from HIV patients. URA5-RFLP and/or M13 PCR-fingerprinting analysis revealed that the majority of the isolates were C. neoformans molecular type VNI regardless of their sources (94.8%; 94.6% of the clinical, 98.8% of the environmental and 86.2% of the veterinary isolates). In addition, the molecular types VNII (2.4%; 66.7% of the clinical and 33.3% of the veterinary isolates), VNIV (0.2%; 100% environmental isolate), VGI (0.2%; 100% clinical isolate) and VGII (2.4%; 100% clinical isolates) were found less frequently. Multilocus Sequence Type (MLST) analysis using the ISHAM consensus MLST scheme for the C. neoformans/C. gattii species complex identified a total of 20 sequence types (ST) in Thailand combining current and previous data. The Thai isolates are an integrated part of the global cryptococcal population genetic structure, with ST30 for C. gattii and ST82, ST83, ST137, ST141, ST172 and ST173 for C. neoformans being unique to Thailand. Most of the C. gattii isolates were ST7 = VGIIb, which is identical to the less virulent minor Vancouver island outbreak genotype, indicating Thailand as a stepping stone in the global spread of this outbreak strain. The current study revealed a greater genetic diversity and a wider range of major molecular types being present amongst Thai cryptococcal isolates than previously reported. PMID:23861989

Tetrahymena thermophila acidic ribosomal protein L37 contains an archaebacterial type of C-terminus.

PubMed

Hansen, T S; Andreasen, P H; Dreisig, H; Højrup, P; Nielsen, H; Engberg, J; Kristiansen, K

1991-09-15

We have cloned and characterized a Tetrahymena thermophila macronuclear gene (L37) encoding the acidic ribosomal protein (A-protein) L37. The gene contains a single intron located in the 3'-part of the coding region. Two major and three minor transcription start points (tsp) were mapped 39 to 63 nucleotides upstream from the translational start codon. The uppermost tsp mapped to the first T in a putative T. thermophila RNA polymerase II initiator element, TATAA. The coding region of L37 predicts a protein of 109 amino acid (aa) residues. A substantial part of the deduced aa sequence was verified by protein sequencing. The T. thermophila L37 clearly belongs to the P1-type family of eukaryotic A-proteins, but the C-terminal region has the hallmarks of archaebacterial A-proteins.

Using genomics for surveillance of veterinary infectious agents.

PubMed

Mathijs, E; Vandenbussche, F; Van Borm, S

2016-04-01

Factors such as globalisation, climate change and agricultural intensification can increase the risk of microbial emergence. As a result, there is a growing need for flexible laboratory-based surveillance tools to rapidly identify, characterise and monitor global (re-)emerging diseases. Although many tools are available, novel sequencing technologies have launched a new era in pathogen surveillance. Here, the authors review the potential applications of high-throughput genomic technologies for the surveillance of veterinary pathogens. They focus on the two types of surveillance that will benefit most from these new tools: hazard-specific surveillance (pathogen identification and typing) and early-warning surveillance (pathogen discovery). The paper reviews how the resulting sequencing data can be used to improve diagnosis and concludes by highlighting the major challenges that hinder the routine use of this technology in the veterinary field.

Psychromicrobium silvestre gen. nov., sp. nov., an actinobacterium isolated from alpine forest soils.

PubMed

Schumann, Peter; Zhang, De-Chao; França, Luís; Albuquerque, Luciana; da Costa, Milton S; Margesin, Rosa

2017-03-01

Two Gram-stain-variable, non-motile, catalase-positive and cytochrome c oxidase-negative bacteria, designated AK20-18 T and AM20-54, were isolated from forest soil samples collected in the Italian Alps. Growth occurred at a temperature range of 5-30 °C, at pH 6-9 and in the presence of 0-5 % (w/v) NaCl. The 16S rRNA gene sequence similarity between strains AK20-18 T and AM20-54 was 100 %. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain AK20-18 T had highest 16S rRNA gene sequence similarity with the type strain of Arthrobacter psychrochitiniphilus (96.9 %). The cell-wall peptidoglycan structure of strain AK20-18 T was of the type A3alpha l-Lys-l-Thr-l-Ala2 (A11.27). The whole-cell sugars were galactose, ribose and lesser amounts of mannose. The major respiratory quinone of the two strains was menaquinone 9(H2) [MK-9(H2)], whereas MK-10(H2) was a minor component. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and unknown glycolipids. The major cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. The genomic DNA G+C content was 59.9 mol%. Combined data of phylogenetic, phenotypic and chemotaxonomic analyses demonstrated that strains AK20-18 T and AM20-54 represent a novel genus and species, for which the name Psychromicrobium silvestre gen. nov., sp. nov. is proposed. The type strain of Psychromicrobium silvestregen. nov., sp. nov. is AK20-18 T (=DSM 102047 T =LMG 29369 T ).

Mesonia hippocampi sp. nov., isolated from the brood pouch of a diseased Barbour's Seahorse (Hippocampus barbouri).

PubMed

Kolberg, Judy; Busse, Hans-Jürgen; Wilke, Thomas; Schubert, Patrick; Kämpfer, Peter; Glaeser, Stefanie P

2015-07-01

An orange-pigmented, Gram-staining-negative, rod-shaped bacterium, designated 96_Hippo_TS_3/13(T) was isolated from the brood pouch of a diseased seahorse male of the species Hippocampus barbouri from the animal facility of the University of Giessen, Germany. Phylogenetic analyses based on the nearly full-length 16S rRNA gene sequence placed strain 96_Hippo_TS_3/13(T) into the monophyletic cluster of the genus Mesonia within the family Flavobacteriaceae. However, the strain shared only 92.2-93.8% sequence similarity to type strains of species of the genus Mesonia, with highest sequence similarity to the type strain of Mesonia aquimarina. Cellular fatty acid analysis showed a Mesonia-typical fatty acid profile including several branched and hydroxyl fatty acids with highest amounts of iso-C15 : 0 (40.9%) followed by iso-C17 : 0 3-OH (14.8%). In the polyamine pattern, sym-homospermidine was predominant. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. The quinone system contained exclusively menaquinone MK-6. The only identified compound in the polar lipid profile was phosphatidylethanolamine present in major amounts. Additionally, major amounts of an unidentified aminolipid and two unidentified lipids not containing a phosphate group, an amino group or a sugar residue were detected. The genomic G+C content of strain 96_Hippo_TS_3/13(T) was 30 mol%. Based on genotypic, chemotaxonomic and physiological characterizations we propose a novel species of the genus Mesonia, Mesonia hippocampi sp. nov., with strain 96_Hippo_TS_3/13(T) ( = CIP 110839T =  LMG 28572(T) = CCM 8557(T)) as the type strain. An emended description of the genus Mesonia is also provided.

Amphritea ceti sp. nov., isolated from faeces of Beluga whale (Delphinapterus leucas).

PubMed

Kim, Young-Ok; Park, Sooyeon; Kim, Doo Nam; Nam, Bo-Hye; Won, Sung-Min; An, Du Hae; Yoon, Jung-Hoon

2014-12-01

A Gram-stain-negative, aerobic, non-spore-forming, non-flagellated and rod-shaped or ovoid bacterial strain, designated RA1(T), was isolated from faeces collected from Beluga whale (Delphinapterus leucas) in Yeosu aquarium, South Korea. Strain RA1(T) grew optimally at 25 °C, at pH 7.0-8.0 and in the presence of 2.0 % (w/v) NaCl. Neighbour-joining, maximum-likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences revealed that strain RA1(T) joins the cluster comprising the type strains of three species of the genus Amphritea, with which it exhibited 95.8-96.0 % sequence similarity. Sequence similarities to the type strains of other recognized species were less than 94.3 %. Strain RA1(T) contained Q-8 as the predominant ubiquinone and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C18 : 1ω7c and C16 : 0 as the major fatty acids. The major polar lipids of strain RA1(T) were phosphatidylethanolamine, phosphatidylglycerol, two unidentified lipids and one unidentified aminolipid. The DNA G+C content of strain RA1(T) was 47.4 mol%. The differential phenotypic properties, together with the phylogenetic distinctiveness, revealed that strain RA1(T) is separated from other species of the genus Amphritea. On the basis of the data presented, strain RA1(T) is considered to represent a novel species of the genus Amphritea, for which the name Amphritea ceti sp. nov. is proposed. The type strain is RA1(T) ( = KCTC 42154(T) = NBRC 110551(T)). © 2014 IUMS.

Optimization of sequence alignment for simple sequence repeat regions.

PubMed

Jighly, Abdulqader; Hamwieh, Aladdin; Ogbonnaya, Francis C

2011-07-20

Microsatellites, or simple sequence repeats (SSRs), are tandemly repeated DNA sequences, including tandem copies of specific sequences no longer than six bases, that are distributed in the genome. SSR has been used as a molecular marker because it is easy to detect and is used in a range of applications, including genetic diversity, genome mapping, and marker assisted selection. It is also very mutable because of slipping in the DNA polymerase during DNA replication. This unique mutation increases the insertion/deletion (INDELs) mutation frequency to a high ratio - more than other types of molecular markers such as single nucleotide polymorphism (SNPs).SNPs are more frequent than INDELs. Therefore, all designed algorithms for sequence alignment fit the vast majority of the genomic sequence without considering microsatellite regions, as unique sequences that require special consideration. The old algorithm is limited in its application because there are many overlaps between different repeat units which result in false evolutionary relationships. To overcome the limitation of the aligning algorithm when dealing with SSR loci, a new algorithm was developed using PERL script with a Tk graphical interface. This program is based on aligning sequences after determining the repeated units first, and the last SSR nucleotides positions. This results in a shifting process according to the inserted repeated unit type.When studying the phylogenic relations before and after applying the new algorithm, many differences in the trees were obtained by increasing the SSR length and complexity. However, less distance between different linage had been observed after applying the new algorithm. The new algorithm produces better estimates for aligning SSR loci because it reflects more reliable evolutionary relations between different linages. It reduces overlapping during SSR alignment, which results in a more realistic phylogenic relationship.

Rhabdobacter roseus gen. nov., sp. nov., isolated from soil.

PubMed

Dahal, Ram Hari; Kim, Jaisoo

2016-01-01

An aerobic, Gram-stain-negative, oxidase- and catalase-positive, non-motile, non-spore-forming, rod-shaped, pink-pigmented bacterium, designated strain R49T, was isolated from soil. Flexirubin-type pigments were absent. Phylogenetic analysis based on its 16S rRNA gene sequence revealed that strain R49T formed a lineage within the family Cytophagaceae of the phylum Bacteroidetes that was distinct from the most closely related genera Dyadobacter (91.98-93.85 % sequence similarity), Persicitalea (88.69 %) and Runella (84.79-85.81 %). The major isoprenoid quinone was menaquinone-7 (MK-7) and the major polar lipid was phosphatidylethanolamine. The major cellular fatty acids were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), iso-C15 : 0, C16 : 1ω5c, C16 : 0 and iso-C17 : 0 3-OH. The DNA G+C content of strain R49T was 53.9 mol%. On the basis of phenotypic, genotypic and phylogenetic analysis, strain R49T represents a novel species of a new genus in the family Cytophagaceae, for which the name Rhabdobacter roseus gen. nov., sp. nov. is proposed. The type strain of Rhabdobacter roseus is R49T ( = KEMB 9005-318T = KACC 18395T = JCM 30685T).

Genetic diversity of pneumococcal surface protein A in invasive pneumococcal isolates from Korean children, 1991-2016.

PubMed

Yun, Ki Wook; Choi, Eun Hwa; Lee, Hoan Jong

2017-01-01

Pneumococcal surface protein A (PspA) is an important virulence factor of pneumococci and has been investigated as a primary component of a capsular serotype-independent pneumococcal vaccine. Thus, we sought to determine the genetic diversity of PspA to explore its potential as a vaccine candidate. Among the 190 invasive pneumococcal isolates collected from Korean children between 1991 and 2016, two (1.1%) isolates were found to have no pspA by multiple polymerase chain reactions. The full length pspA genes from 185 pneumococcal isolates were sequenced. The length of pspA varied, ranging from 1,719 to 2,301 base pairs with 55.7-100% nucleotide identity. Based on the sequences of the clade-defining regions, 68.7% and 49.7% were in PspA family 2 and clade 3/family 2, respectively. PspA clade types were correlated with genotypes using multilocus sequence typing and divided into several subclades based on diversity analysis of the N-terminal α-helical regions, which showed nucleotide sequence identities of 45.7-100% and amino acid sequence identities of 23.1-100%. Putative antigenicity plots were also diverse among individual clades and subclades. The differences in antigenicity patterns were concentrated within the N-terminal 120 amino acids. In conclusion, the N-terminal α-helical domain, which is known to be the major immunogenic portion of PspA, is genetically variable and should be further evaluated for antigenic differences and cross-reactivity between various PspA types from pneumococcal isolates.

Photosynthesis Is Widely Distributed among Proteobacteria as Demonstrated by the Phylogeny of PufLM Reaction Center Proteins

PubMed Central

Imhoff, Johannes F.; Rahn, Tanja; Künzel, Sven; Neulinger, Sven C.

2018-01-01

Two different photosystems for performing bacteriochlorophyll-mediated photosynthetic energy conversion are employed in different bacterial phyla. Those bacteria employing a photosystem II type of photosynthetic apparatus include the phototrophic purple bacteria (Proteobacteria), Gemmatimonas and Chloroflexus with their photosynthetic relatives. The proteins of the photosynthetic reaction center PufL and PufM are essential components and are common to all bacteria with a type-II photosynthetic apparatus, including the anaerobic as well as the aerobic phototrophic Proteobacteria. Therefore, PufL and PufM proteins and their genes are perfect tools to evaluate the phylogeny of the photosynthetic apparatus and to study the diversity of the bacteria employing this photosystem in nature. Almost complete pufLM gene sequences and the derived protein sequences from 152 type strains and 45 additional strains of phototrophic Proteobacteria employing photosystem II were compared. The results give interesting and comprehensive insights into the phylogeny of the photosynthetic apparatus and clearly define Chromatiales, Rhodobacterales, Sphingomonadales as major groups distinct from other Alphaproteobacteria, from Betaproteobacteria and from Caulobacterales (Brevundimonas subvibrioides). A special relationship exists between the PufLM sequences of those bacteria employing bacteriochlorophyll b instead of bacteriochlorophyll a. A clear phylogenetic association of aerobic phototrophic purple bacteria to anaerobic purple bacteria according to their PufLM sequences is demonstrated indicating multiple evolutionary lines from anaerobic to aerobic phototrophic purple bacteria. The impact of pufLM gene sequences for studies on the environmental diversity of phototrophic bacteria is discussed and the possibility of their identification on the species level in environmental samples is pointed out. PMID:29472894

Tracing Males From Different Continents by Genotyping JC Polyomavirus in DNA From Semen Samples.

PubMed

Rotondo, John Charles; Candian, Tommaso; Selvatici, Rita; Mazzoni, Elisa; Bonaccorsi, Gloria; Greco, Pantaleo; Tognon, Mauro; Martini, Fernanda

2017-05-01

The human JC polyomavirus (JCPyV) is an ubiquitous viral agent infecting approximately 60% of humans. Recently, JCPyV sequences have been detected in semen samples. The aim of this investigation was to test whether semen JCPyV genotyping can be employed to trace the origin continent of males. Semen DNA samples (n = 170) from males of different Continents were investigated by PCR for the polymorphic JCPyV viral capsid protein 1 (VP1) sequences, followed by DNA sequencing. JCPyV sequences were detected with an overall prevalence of 27.6% (47/170). DNA sequencing revealed that European males carried JCPyV types 1A (71.4%), 4 (11.4%), 2B (2.9%), 2D1 (2.9%), and 3A (2.9%). Asians JCPyV type 2D1 (66.7%) and Africans JCPyV types 3A (33.3%) and 1A (33.3%). In 10.6% of males, two different JCPyV genotypes were detected, suggesting that the second JCPyV genotype was acquired in the destination country. This study indicates that the majority of semen samples found to be JCPyV-positive, were infected with the JCPyV genotype found in the geographic area of male origin. Therefore, semen JCPyV genotyping could be employed to trace the origin continent of males. Our findings could be applied to forensic investigations, in case of for instance sexual crimes. Indeed, JCPyV genotyping should enable investigators to make additional detailed profiling of the offender. J. Cell. Physiol. 232: 982-985, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Lelliottia aquatilis sp. nov., isolated from drinking water.

PubMed

Kämpfer, Peter; Glaeser, Stefanie P; Packroff, Gabriele; Behringer, Katja; Exner, Martin; Chakraborty, Trinad; Schmithausen, Ricarda M; Doijad, Swapnil

2018-06-22

Five beige-pigmented, oxidase-negative bacterial isolates, 6331-17 T , 6332-17, 6333-17, 6334-17 and 9827-07, isolated either from a drinking water storage reservoir or drinking water in 2006 and 2017 in Germany, were examined in detail applying by a polyphasic taxonomic approach. Cells of the isolates were rod-shaped and Gram-stain-negative. Comparison of the 16S rRNA gene sequences of these five isolates showed highest sequence similarities to Lelliottia amnigena (99.98 %) and Lelliottia nimipressuralis (99.99 %). Multilocus sequence analyses based on concatenated partial rpoB, gyrB, infB and atpD sequences confirmed the clustering of these isolates with Lelliottia species, but also revealed a clear distinction to the closest related type strains. Analysis of the genome sequences of these isolates indicated >70 % in silico DNA-DNA hybridization and high average nucleotide identities between strains. Nevertheless, they showed only <70 and <95 % similarity to the type strains of these two Lelliottia species. The fatty acid profiles of these isolates were very similar and consisted of the major fatty acids C16:0, C17 : 0cyclo, C15 : 0iso 2-OH/C16 : 1ω7c and C18 : 1ω7c. In addition, physiological/biochemical tests revealed high phenotypic similarity to each other. These cumulative data indicate that these isolates represent a novel Lelliottia species, for which the name Lelliottia aquatilis sp. nov. is proposed, with strain 6331-17 T (=CCM 8846 T =CIP 111609 T =LMG 30560 T ) as the type strain.

Detection and characterization of carbapenemase-producing Enterobacteriaceae in wounded Syrian patients admitted to hospitals in northern Israel.

PubMed

Lerner, A; Solter, E; Rachi, E; Adler, A; Rechnitzer, H; Miron, D; Krupnick, L; Sela, S; Aga, E; Ziv, Y; Peretz, A; Labay, K; Rahav, G; Geffen, Y; Hussein, K; Eluk, O; Carmeli, Y; Schwaber, M J

2016-01-01

Since 2013, four hospitals in northern Israel have been providing care for Syrian nationals, primarily those wounded in the ongoing civil war. We analyzed carbapenemase-producing Enterobacteriaceae (CPE) isolates obtained from these patients. Isolate identification was performed using the VITEK 2 system. Polymerase chain reaction (PCR) was performed for the presence of bla KPC, bla NDM, and bla OXA-48. Susceptibility testing and genotyping were performed on selected isolates. During the study period, 595 Syrian patients were hospitalized, most of them young men. Thirty-two confirmed CPE isolates were grown from cultures taken from 30 patients. All but five isolates were identified as Klebsiella pneumoniae and Escherichia coli. Nineteen isolates produced NDM and 13 produced OXA-48. Among a further 29 isolates tested, multilocus sequence typing (MLST) showed that ST278 and ST38 were the major sequence types among the NDM-producing K. pneumoniae and OXA-48-producing E. coli isolates, respectively. Most were resistant to all three carbapenems in use in Israel and to gentamicin, but susceptible to colistin and fosfomycin. The source for bacterial acquisition could not be determined; however, some patients admitted to different medical centers were found to carry the same sequence type. CPE containing bla NDM and bla OXA-48 were prevalent among Syrian wounded hospitalized patients in northern Israel. The finding of the same sequence type among patients at different medical centers implies a common, prehospital source for these patients. These findings have implications for public health throughout the region.

Simultaneous Characterization of Somatic Events and HPV-18 Integration in a Metastatic Cervical Carcinoma Patient Using DNA and RNA Sequencing

PubMed Central

Liang, Winnie S.; Aldrich, Jessica; Nasser, Sara; Kurdoglu, Ahmet; Phillips, Lori; Reiman, Rebecca; McDonald, Jacquelyn; Izatt, Tyler; Christoforides, Alexis; Baker, Angela; Craig, Christine; Egan, Jan B.; Chase, Dana M.; Farley, John H.; Bryce, Alan H.; Stewart, A. Keith; Borad, Mitesh J.; Carpten, John D.; Craig, David W.; Monk, Bradley J.

2014-01-01

Objective Integration of carcinogenic human papillomaviruses (HPVs) into the host genome is a significant tumorigenic factor in specific cancers including cervical carcinoma. Although major strides have been made with respect to HPV diagnosis and prevention, identification and development of efficacious treatments for cervical cancer patients remains a goal and thus requires additional detailed characterization of both somatic events and HPV integration. Given this need, the goal of this study was to use the next generation sequencing to simultaneously evaluate somatic alterations and expression changes in a patient’s cervical squamous carcinoma lesion metastatic to the lung and to detect and analyze HPV infection in the same sample. Materials and Methods We performed tumor and normal exome, tumor and normal shallow whole-genome sequencing, and RNA sequencing of the patient’s lung metastasis. Results We generated over 1.2 billion mapped reads and identified 130 somatic point mutations and indels, 21 genic translocations, 16 coding regions demonstrating copy number changes, and over 36 genes demonstrating altered expression in the tumor (corrected P < 0.05). Sequencing also revealed the HPV type 18 (HPV-18) integration in the metastasis. Using both DNA and RNA reads, we pinpointed 3 major events indicating HPV-18 integration into an intronic region of chromosome 6p25.1 in the patient’s tumor and validated these events with Sanger sequencing. This integration site has not been reported for HPV-18. Conclusions We demonstrate that DNA and RNA sequencing can be used to concurrently characterize somatic alterations and expression changes in a biopsy and delineate HPV integration at base resolution in cervical cancer. Further sequencing will allow us to better understand the molecular basis of cervical cancer pathogenesis. PMID:24418928

Formation of (DNA)2-LNA triplet with recombinant base recognition: A quantum mechanical study

NASA Astrophysics Data System (ADS)

Mall, Vijaya Shri; Tiwari, Rakesh Kumar

2018-05-01

The formation of DNA triple helix offers the verity of new possibilities in molecular biology. However its applications are limited to purine and pyrimidine rich sequences recognized by forming Hoogsteen/Reverse Hoogsteen triplets in major groove sites of DNA duplex. To overcome this drawback modification in bases backbone and glucose of nucleotide unit of DNA have been proposed so that the third strand base recognized by both the bases of DNA duplex by forming Recombinant type(R-type) of bonding in mixed sequences. Here we performed Quanrum Mechanical (Hartree-Fock and DFT) methodology on natural DNA and Locked Nucleic Acids(LNA) triplets using 6-31G and some other new advance basis sets. Study suggests energetically stable conformation has been observed for recombinant triplets in order of G-C*G > A-T*A > G-C*C > T-A*T for both type of triplets. Interestingly LNA leads to more stable conformation in all set of triplets, clearly suggests an important biological tool to overcome above mentioned drawbacks.

Great expectations: patient perspectives and anticipated utility of non-diagnostic genomic-sequencing results.

PubMed

Hylind, Robyn; Smith, Maureen; Rasmussen-Torvik, Laura; Aufox, Sharon

2018-01-01

The management of secondary findings is a challenge to health-care providers relaying clinical genomic-sequencing results to patients. Understanding patients' expectations from non-diagnostic genomic sequencing could help guide this management. This study interviewed 14 individuals enrolled in the eMERGE (Electronic Medical Records and Genomics) study. Participants in eMERGE consent to undergo non-diagnostic genomic sequencing, receive results, and have results returned to their physicians. The interviews assessed expectations and intended use of results. The majority of interviewees were male (64%) and 43% identified as non-Caucasian. A unique theme identified was that many participants expressed uncertainty about the type of diseases they expected to receive results on, what results they wanted to learn about, and how they intended to use results. Participant uncertainty highlights the complex nature of deciding to undergo genomic testing and a deficiency in genomic knowledge. These results could help improve how genomic sequencing and secondary findings are discussed with patients.

Salinispirillum marinum gen. nov., sp. nov., a haloalkaliphilic bacterium in the family 'Saccharospirillaceae'.

PubMed

Shahinpei, Azadeh; Amoozegar, Mohammad Ali; Fazeli, Seyed Abolhassan Shahzadeh; Schumann, Peter; Ventosa, Antonio

2014-11-01

A novel Gram-staining-negative, motile, non-pigmented, facultatively anaerobic, spirillum-shaped, halophilic and alkaliphilic bacterium, designated strain GCWy1(T), was isolated from water of the coastal-marine wetland Gomishan in Iran. The strain was able to grow at NaCl concentrations of 1-10% (w/v) and optimal growth was achieved at 3% (w/v). The optimum pH and temperature for growth were pH 8.5 and 30 °C, while the strain was able to grow at pH 7.5-10 and 4-40 °C. Phylogenetic analysis based on the comparison of the 16S rRNA gene sequence placed the isolate within the class Gammaproteobacteria as a separate deep branch, with 92.1% or lower sequence similarity to representatives of the genera Saccharospirillum and Reinekea and less than 91.0% sequence similarity with other remotely related genera. The major cellular fatty acids of the isolate were C(18 : 1)ω7c, C(16:0) and C(17 : 0), and the major components of its polar lipid profile were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The cells of strain GCWy1(T) contained the isoprenoid quinones Q-9 and Q-8 (81% and 2%, respectively). The G+C content of the genomic DNA of this strain was 52.3 mol%. On the basis of 16S rRNA gene sequence analysis in combination with chemotaxonomic and phenotypic data, strain GCWy1(T) represents a novel species in a new genus in the family 'Saccharospirillaceae', order Oceanospirillales, for which the name Salinispirillum marinum gen. nov., sp. nov. is proposed. The type strain of the type species is GCWy1(T) ( = IBRC-M 10765(T) =CECT 8342(T)). © 2014 IUMS.

Complex Patterns of Altered MicroRNA Expression during the Adenoma-Adenocarcinoma Sequence for Microsatellite-Stable Colorectal Cancer

PubMed Central

Bartley, Angela N.; Yao, Hui; Barkoh, Bedia A.; Ivan, Cristina; Mishra, Bal M.; Rashid, Asif; Calin, George A.; Luthra, Rajyalakshmi; Hamilton, Stanley R.

2012-01-01

Purpose MicroRNAs are short noncoding RNAs that regulate gene expression and are over- or under-expressed in most tumors, including colorectal adenocarcinoma. MicroRNAs are potential biomarkers and therapeutic targets and agents, but limited information on microRNAome alterations during progression in the well-known adenoma-adenocarcinoma sequence is available to guide their usage. Experimental Design We profiled 866 human microRNAs by microarray analysis in 69 matched specimens of microsatellite-stable adenocarcinomas, adjoining precursor adenomas including areas of high- and low-grade dysplasia, and nonneoplastic mucosa. Results We found 230 microRNAs that were significantly differentially expressed during progression, including 19 not reported previously. Altered microRNAs clustered into two major patterns of early (type I) and late (type II) differential expression. The largest number (n = 108) was altered at the earliest step from mucosa to low-grade dysplasia (subtype IA) prior to major nuclear localization of β-catenin, including 36 microRNAs that had persistent differential expression throughout the entire sequence to adenocarcinoma. Twenty microRNAs were intermittently altered (subtype IB), and six were transiently altered (subtype IC). In contrast, 33 microRNAs were altered late in high-grade dysplasia and adenocarcinoma (subtype IIA), and 63 in adenocarcinoma only (subtype IIB). Predicted targets in 12 molecular pathways were identified for highly altered microRNAs, including the Wnt signaling pathway leading to low-grade dysplasia. β-catenin expression correlated with downregulated microRNAs. Conclusions Our findings suggest that numerous microRNAs play roles in the sequence of molecular events, especially early events, resulting in colorectal adenocarcinoma. The temporal patterns and complexity of microRNAome alterations during progression will influence the efficacy of microRNAs for clinical purposes. PMID:21948089

Human papillomavirus infection in females with normal cervical cytology: Genotyping and phylogenetic analysis among women in Punjab, Pakistan.

PubMed

Aziz, Hafsa; Iqbal, Huma; Mahmood, Humera; Fatima, Shazia; Faheem, Mohammad; Sattar, Areej Abdul; Tabassum, Sobia; Napper, Sanum; Batool, Syeda; Rasheed, Nuzhat

2018-01-01

Globally, cervical cancer is the fourth most common cancer in women and the seventh most common cancer overall, accounting for an estimated 300 000 annual deaths. Human papillomavirus (HPV) is the second most common cause of cervical cancer worldwide. HPV screening is not a common practice in Pakistan. The aim of this study was to determine the prevalence of HPV and HPV types in women with a normal cytology of the cervix living in the upper and lower regions of Punjab, Pakistan, and to analyze the risk factors for HPV in this region. PCR analysis was performed for 1011 female patients with a normal cytology of the cervix from various districts of Punjab Province, Pakistan. Risk factors for the acquisition of HPV were studied. High-risk HPV types (HPV16 and HPV18) were detected using the Abbott Real Time HR HPV test. To determine the genotype, partial L1 region sequences of HPV-positive samples were subjected to sequencing using MY/09/MY11 primers, and a phylogenetic tree was constructed using CLC software. The study found a 4.74% prevalence of HPV, with the most frequent HPV type found being the low-risk HPV6 (in 25% of infected individuals), followed by HPV55 (22.9%), HPV11 (20.8%), and high-risk types HPV45 (12.5%), HPV33 (8.33%), HPV18 (6.25%), and HPV16 (4.16%). Phylogenetic analysis of all HPV types in this study showed 80-99% nucleotide identity with types related to the same species. The sequences were clustered with China, India, Mexico, Iran, Slovenia, and Germany, showing the diversity in origin of the various genotypes prevalent in Pakistan. In this population with a normal cervical cytology, the prevalence of high-risk HPV types was very low. The major prevalent HPV genotype in Punjab Province of Pakistan was the low-risk HPV type 6, followed by HPV type 55. Sequencing of the partial L1 region suggested that the region was highly conserved in all reported sequences. This study highlights the need to conduct robust epidemiological studies in the region and to develop regular HPV screening so that the situation does not reach an alarming stage resulting in cervical cancer. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Genotyping-by-sequencing to re-map QTL for type II Fusarium head blight and leaf rust resistance in a wheat-tall wheatgrass introgression recombinant inbred population

USDA-ARS?s Scientific Manuscript database

Fusarium graminaerum (Fusarium head blight; FHB) and Puccinia recondita Roberge ex Desmaz. f. sp. tritici Eriks. & E. Henn (leaf rust; LR) are two major fungal pathogens threatening the wheat crop; consequently identifying resistance genes from various sources is always of importance to wheat breede...

The Sequenced Angiosperm Genomes and Genome Databases.

PubMed

Chen, Fei; Dong, Wei; Zhang, Jiawei; Guo, Xinyue; Chen, Junhao; Wang, Zhengjia; Lin, Zhenguo; Tang, Haibao; Zhang, Liangsheng

2018-01-01

Angiosperms, the flowering plants, provide the essential resources for human life, such as food, energy, oxygen, and materials. They also promoted the evolution of human, animals, and the planet earth. Despite the numerous advances in genome reports or sequencing technologies, no review covers all the released angiosperm genomes and the genome databases for data sharing. Based on the rapid advances and innovations in the database reconstruction in the last few years, here we provide a comprehensive review for three major types of angiosperm genome databases, including databases for a single species, for a specific angiosperm clade, and for multiple angiosperm species. The scope, tools, and data of each type of databases and their features are concisely discussed. The genome databases for a single species or a clade of species are especially popular for specific group of researchers, while a timely-updated comprehensive database is more powerful for address of major scientific mysteries at the genome scale. Considering the low coverage of flowering plants in any available database, we propose construction of a comprehensive database to facilitate large-scale comparative studies of angiosperm genomes and to promote the collaborative studies of important questions in plant biology.

The Sequenced Angiosperm Genomes and Genome Databases

PubMed Central

Chen, Fei; Dong, Wei; Zhang, Jiawei; Guo, Xinyue; Chen, Junhao; Wang, Zhengjia; Lin, Zhenguo; Tang, Haibao; Zhang, Liangsheng

2018-01-01

Angiosperms, the flowering plants, provide the essential resources for human life, such as food, energy, oxygen, and materials. They also promoted the evolution of human, animals, and the planet earth. Despite the numerous advances in genome reports or sequencing technologies, no review covers all the released angiosperm genomes and the genome databases for data sharing. Based on the rapid advances and innovations in the database reconstruction in the last few years, here we provide a comprehensive review for three major types of angiosperm genome databases, including databases for a single species, for a specific angiosperm clade, and for multiple angiosperm species. The scope, tools, and data of each type of databases and their features are concisely discussed. The genome databases for a single species or a clade of species are especially popular for specific group of researchers, while a timely-updated comprehensive database is more powerful for address of major scientific mysteries at the genome scale. Considering the low coverage of flowering plants in any available database, we propose construction of a comprehensive database to facilitate large-scale comparative studies of angiosperm genomes and to promote the collaborative studies of important questions in plant biology. PMID:29706973

Rubrobacter spartanus sp. nov., a moderately thermophilic oligotrophic bacterium isolated from volcanic soil.

PubMed

Norman, Jeffrey S; King, Gary M; Friesen, Maren L

2017-09-01

Bacterial strain HPK2-2T was isolated from soil adjacent to the caldera of Kilauea Volcano in Hawaii Volcanoes National Park. HPK2-2T is a chemoorganoheterotroph that shows optimal growth at 50 °C (range 45-55 °C) and pH 8.0 (range 5.0-10.0). Sequence analysis of the 16S subunit of the rRNA gene showed that HPK2-2T is most closely related to the type strain of Rubrobactertaiwanensis (ATCC BAA-406T), with which it shared 94.5 % sequence identity. The major fatty acids detected in HPK2-2T were C18 : 0 14-methyl and C16 : 0 12-methyl; internally branched fatty acids such as these are characteristic of the genus Rubrobacter. The only respiratory quinone detected was MK-8, which is the major respiratory quinone for all members of the family Rubrobacteraceae examined thus far. We propose that HPK2-2T represents a novel species of the genus Rubrobacter, for which we propose the name Rubrobacterspartanus (type strain HPK2-2T; DSM 102139T; LMG 29988T).

Fabibacter misakiensis sp. nov., a marine bacterium isolated from coastal surface water.

PubMed

Wong, Shu-Kuan; Park, Sanghwa; Lee, Jung-Sook; Lee, Keun Chul; Chiura, Hiroshi Xavier; Kogure, Kazuhiro; Hamasaki, Koji

2015-10-01

A slightly curved-rod-shaped, pink-pigmented, Gram-stain-negative, aerobic bacterial strain with gliding motility, designated SK-8T, was isolated from coastal surface water of Misaki, Japan. Phylogenetic trees generated using 16S rRNA gene sequences revealed that strain SK-8T belonged to the genus Fabibacter and showed 96.0 % sequence similarity to the type strain of the most closely related species, Fabibacter pacificus DY53T. The novel isolate was phenotypically and physiologically different from previously described strains. The major cellular fatty acids were iso-C15 : 1 G, iso-C15 : 0 and iso-C17 : 0 3-OH. Major polar lipids were phosphatidylethanolamine, two aminophospholipids and an unidentified phospholipid. The DNA G+C content was 39.1 mol% and MK-7 was the only predominant isoprenoid quinone. On the basis of this taxonomic study employing a polyphasic approach, it was suggested that strain SK-8T represents a novel species of the genus Fabibacter, with the newly proposed name Fabibacter misakiensis sp. nov. The type strain is SK-8T ( = NBRC 110216T = KCTC 32969T).

CRISPR interference and priming varies with individual spacer sequences

PubMed Central

Xue, Chaoyou; Seetharam, Arun S.; Musharova, Olga; Severinov, Konstantin; J. Brouns, Stan J.; Severin, Andrew J.; Sashital, Dipali G.

2015-01-01

CRISPR–Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems allow bacteria to adapt to infection by acquiring ‘spacer’ sequences from invader DNA into genomic CRISPR loci. Cas proteins use RNAs derived from these loci to target cognate sequences for destruction through CRISPR interference. Mutations in the protospacer adjacent motif (PAM) and seed regions block interference but promote rapid ‘primed’ adaptation. Here, we use multiple spacer sequences to reexamine the PAM and seed sequence requirements for interference and priming in the Escherichia coli Type I-E CRISPR–Cas system. Surprisingly, CRISPR interference is far more tolerant of mutations in the seed and the PAM than previously reported, and this mutational tolerance, as well as priming activity, is highly dependent on spacer sequence. We identify a large number of functional PAMs that can promote interference, priming or both activities, depending on the associated spacer sequence. Functional PAMs are preferentially acquired during unprimed ‘naïve’ adaptation, leading to a rapid priming response following infection. Our results provide numerous insights into the importance of both spacer and target sequences for interference and priming, and reveal that priming is a major pathway for adaptation during initial infection. PMID:26586800

Epidemiology of invasive group A streptococcal infections in Norway 2010-2014: A retrospective cohort study.

PubMed

Naseer, U; Steinbakk, M; Blystad, H; Caugant, D A

2016-10-01

Streptococcus pyogenes or group A streptococcus (GAS) causes mild to severe infections in humans. GAS genotype emm1 is the leading cause of invasive disease worldwide. In the Nordic countries emm28 has been the dominant type since the 1980s. Recently, a resurgence of genotype emm1 was reported from Sweden. Here we present the epidemiology of invasive GAS (iGAS) infections and their association with emm-types in Norway from 2010-2014. We retrospectively collected surveillance data on antimicrobial susceptibility, multilocus sequence type and emm-type, and linked them with demographic and clinical manifestation data to calculate age and sex distributions, major emm- and sequence types and prevalence ratios (PR) on associations between emm-types and clinical manifestations. We analysed 756 iGAS cases and corresponding isolates, with overall incidence of 3.0 per 100000, median age of 59 years (range, 0-102), and male 56 %. Most frequent clinical manifestation was sepsis (49 %) followed by necrotizing fasciitis (9 %). Fifty-two different emm-types and 67 sequence types were identified, distributed into five evolutionary clusters. The most prevalent genotype was emm1 (ST28) in all years (range, 20-33 %) followed by 15 % emm28 in 2014. All isolates were susceptible to penicillin, 15 % resistant to tetracycline and <4 % resistant to erythromycin. A PR of 4.5 (95 % CI, 2.3-8.9) was calculated for emm2 and necrotizing fasciitis. All emm22 isolates were resistant to tetracycline PR 7.5 (95 % CI, 5.8-9.9). This study documented the dominance of emm1, emergence of emm89 and probable import of tetracycline resistant emm112.2 into Norway (2010-2014). Genotype fluctuations between years suggested a mutual exclusive dominance of evolutionary clades.

Molecular characterization of edestin gene family in Cannabis sativa L.

PubMed

Docimo, Teresa; Caruso, Immacolata; Ponzoni, Elena; Mattana, Monica; Galasso, Incoronata

2014-11-01

Globulins are the predominant class of seed storage proteins in a wide variety of plants. In many plant species globulins are present in several isoforms encoded by gene families. The major seed storage protein of Cannabis sativa L. is the globulin edestin, widely known for its nutritional potential. In this work, we report the isolation of seven cDNAs encoding for edestin from the C. sativa variety Carmagnola. Southern blot hybridization is in agreement with the number of identified edestin genes. All seven sequences showed the characteristic globulin features, but they result to be divergent members/forms of two edestin types. According to their sequence similarity four forms named CsEde1A, CsEde1B, CsEde1C, CsEde1D have been assigned to the edestin type 1 and the three forms CsEde2A, CsEde2B, CsEde2C to the edestin type 2. Analysis of the coding sequences revealed a high percentage of similarity (98-99%) among the different forms belonging to the same type, which decreased significantly to approximately 64% between the forms belonging to different types. Quantitative RT-PCR analysis revealed that both edestin types are expressed in developing hemp seeds and the amount of CsEde1 was 4.44 ± 0.10 higher than CsEde2. Both edestin types exhibited a high percentage of arginine (11-12%), but CsEde2 resulted particularly rich in methionine residues (2.36%) respect to CsEde1 (0.82%). The amino acid composition determined in CsEde1 and CsEde2 types suggests that these seed proteins can be used to improve the nutritional quality of plant food-stuffs. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Integrative analysis of environmental sequences using MEGAN4.

PubMed

Huson, Daniel H; Mitra, Suparna; Ruscheweyh, Hans-Joachim; Weber, Nico; Schuster, Stephan C

2011-09-01

A major challenge in the analysis of environmental sequences is data integration. The question is how to analyze different types of data in a unified approach, addressing both the taxonomic and functional aspects. To facilitate such analyses, we have substantially extended MEGAN, a widely used taxonomic analysis program. The new program, MEGAN4, provides an integrated approach to the taxonomic and functional analysis of metagenomic, metatranscriptomic, metaproteomic, and rRNA data. While taxonomic analysis is performed based on the NCBI taxonomy, functional analysis is performed using the SEED classification of subsystems and functional roles or the KEGG classification of pathways and enzymes. A number of examples illustrate how such analyses can be performed, and show that one can also import and compare classification results obtained using others' tools. MEGAN4 is freely available for academic purposes, and installers for all three major operating systems can be downloaded from www-ab.informatik.uni-tuebingen.de/software/megan.

Clonal Transmission of Gram-Negative Bacteria with Carbapenemases NDM-1, VIM-1, and OXA-23/72 in a Bulgarian Hospital.

PubMed

Pfeifer, Yvonne; Trifonova, Angelina; Pietsch, Michael; Brunner, Magdalena; Todorova, Iva; Gergova, Ivanka; Wilharm, Gottfried; Werner, Guido; Savov, Encho

2017-04-01

We characterized 72 isolates with reduced susceptibility to carbapenems (50 Acinetobacter spp., 13 Proteus mirabilis, five Escherichia coli, one Morganella morganii, one Enterobacter cloacae, one Providencia rettgeri, and one Pseudomonas aeruginosa) from a hospital in Sofia, Bulgaria. Different β-lactamase genes were identified by polymerase chain reaction and sequencing. Bacterial strain typing was performed by enzymatic macrorestriction and pulsed-field gel electrophoresis (PFGE) typing as well as multilocus sequence typing for selected isolates. The majority of Acinetobacter baumannii (46/50) and one Acinetobacter pittii isolate harbored carbapenemase genes bla OXA-23 or bla OXA-72 ; two A. baumannii contained both genes. PFGE typing of all A. baumannii showed the presence of nine different clones belonging to eight sequence types ST350, ST208, ST436, ST437, ST449, ST231, ST502, and ST579. Molecular characterization of the remaining isolates confirmed the presence of one NDM-1-producing E. coli-ST101 clone (five isolates) and one P. mirabilis clone (13 isolates) with VIM-1 and CMY-99. Furthermore, NDM-1 was identified in P. rettgeri and M. morganii and VIM-2 in the P. aeruginosa isolate. The permanent introduction of OXA-23/72 carbapenemase-producing A. baumannii clones into the hospital and the repeated occurrence of one VIM-1-producing P. mirabilis and one NDM-1-producing E. coli-ST101 clone over a period of more than 1 year is of concern and requires intensified investigations.

Evaluation of GenoType NTM-DR Assay for Identification of Mycobacterium chimaera.

PubMed

Mok, Simone; Rogers, Thomas R; Fitzgibbon, Margaret

2017-06-01

Identification of species within the Mycobacterium avium complex (MAC) is difficult, and most current diagnostic laboratory tests cannot distinguish between species included in the complex. Differentiation of species within the MAC is important, as Mycobacterium chimaera has recently emerged as a major cause of invasive cardiovascular infections following open heart surgery. A new commercial diagnostic assay, GenoType NTM-DR ver. 1.0, is intended to differentiate between three species within the MAC, namely, Mycobacterium avium , Mycobacterium intracellulare , and Mycobacterium chimaera In this study, we investigated an archival collection of 173 MAC isolates using 16S rRNA and 16S-23S internal transcribed spacer (ITS) gene sequencing, and GenoType NTM-DR was evaluated for identifying M. chimaera and other species belonging to the MAC. Species identification of 157/173 (91%) isolates with the GenoType NTM-DR assay was in agreement with 16S rRNA and 16S-23S ITS gene sequencing results. Misidentification occurred with 16 isolates which belonged to four species included in the MAC that are rarely encountered in clinical specimens. Despite some limitations of this assay, GenoType NTM-DR had 100% specificity for identifying M. chimaera This novel assay will enable diagnostic laboratories to differentiate species belonging to the Mycobacterium avium complex and to accurately identify M. chimaera It can produce rapid results and is also more cost efficient than gene sequencing methods. Copyright © 2017 American Society for Microbiology.

Wide spread of OXA-23-producing carbapenem-resistant Acinetobacter baumannii belonging to clonal complex II in different hospitals in Lebanon.

PubMed

Al Atrouni, Ahmad; Hamze, Monzer; Jisr, Tamima; Lemarié, Carole; Eveillard, Matthieu; Joly-Guillou, Marie-Laure; Kempf, Marie

2016-11-01

To investigate the molecular epidemiology of Acinetobacter baumannii strains isolated from different hospitals in Lebanon. A total of 119 non-duplicate Acinetobacter strains were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and partial rpoB gene sequencing. Antibiotic susceptibility testing was performed by disc diffusion method and all identified carbapenem-resistant isolates were investigated by PCR assays for the presence of the carbapenemase-encoding genes. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were used for molecular typing. Of the 119 A. baumannii isolates, 76.5% were resistant to carbapenems. The most common carbapenemase was the OXA-23-type, found in 82 isolates. The study of population structure using MLST revealed the presence of 30 sequence types (STs) including 18 new ones, with ST2 being the most commonly detected, accounting for 61% of the isolates typed. PFGE performed on all strains of ST2 identified a major cluster of 53 isolates, in addition to three other minor clusters and ten unique profiles. This study highlights the wide dissemination of highly related OXA-23-producing carbapenem-resistant A. baumannii belonging to the international clone II in Lebanon. Thus, appropriate infection control measures are recommended in order to control the geographical spread of this clone in this country. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Characterization of rumen ciliate community composition in domestic sheep, deer, and cattle, feeding on varying diets, by means of PCR-DGGE and clone libraries.

PubMed

Kittelmann, Sandra; Janssen, Peter H

2011-03-01

The structure and variability of ciliate protozoal communities in the rumens of domestic New Zealand ruminants feeding on different diets was investigated. The relative abundance of ciliates compared with bacteria was similar across all samples. However, molecular fingerprinting of communities showed ruminant-specific differences in species composition. Community compositions of cattle were significantly influenced by diet. In contrast, diet effects in deer and sheep were weaker than the animal-to-animal variation. Cloning and sequencing of almost-full-length 18S rRNA genes from representative samples revealed that New Zealand ruminants were colonized by at least nine genera of ciliates and allowed the assignment of samples to two distinct community types. Cattle contained A-type communities, with most sequences closely related to those of the genera Polyplastron and Ostracodinium. Deer and sheep (with one exception) harboured B-type communities, with the majority of sequences belonging to the genera Epidinium and Eudiplodinium. It has been suggested that species composition of ciliate communities may impact methane formation in ruminants, with the B-type producing more methane. Therefore, manipulation of ciliate communities may be a means of mitigating methane emissions from grazing sheep and deer in New Zealand. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Rhodoluna lacicola gen. nov., sp. nov., a planktonic freshwater bacterium with stream-lined genome

PubMed Central

Schmidt, Johanna; Taipale, Sami J.; Doolittle, W. Ford; Koll, Ulrike

2014-01-01

A pure culture of an actinobacterium previously described as ‘Candidatus Rhodoluna lacicola’ strain MWH-Ta8 was established and deposited in two public culture collections. Strain MWH-Ta8T represents a free-living planktonic freshwater bacterium obtained from hypertrophic Meiliang Bay, Lake Taihu, PR China. The strain was characterized by phylogenetic and taxonomic investigations, as well as by determination of its complete genome sequence. Strain MWH-Ta8T is noticeable due to its unusually low values of cell size (0.05 µm3), genome size (1.43 Mbp), and DNA G+C content (51.5 mol%). Phylogenetic analyses based on 16S rRNA gene and RpoB sequences suggested that strain MWH-Ta8T is affiliated with the family Microbacteriaceae with Pontimonas salivibrio being its closest relative among the currently described species within this family. Strain MWH-Ta8T and the type strain of Pontimonas salivibrio shared a 16S rRNA gene sequence similarity of 94.3 %. The cell-wall peptidoglycan of strain MWH-Ta8T was of type B2β (B10), containing 2,4-diaminobutyric acid as the diamino acid. The predominant cellular fatty acids were anteiso-C15 : 0 (36.5 %), iso-C16 : 0 (16.5 %), iso-C15 : 0 (15.6 %) and iso-C14 : 0 (8.9 %), and the major (>10 %) menaquinones were MK-11 and MK-12. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and two unknown glycolipids. The combined phylogenetic, phenotypic and chemotaxonomic data clearly suggest that strain MWH-Ta8T represents a novel species of a new genus in the family Microbacteriaceae, for which the name Rhodoluna lacicola gen. nov., sp. nov. is proposed. The type strain of the type species is MWH-Ta8T ( = DSM 23834T = LMG 26932T). PMID:24984700

Rhodoluna lacicola gen. nov., sp. nov., a planktonic freshwater bacterium with stream-lined genome.

PubMed

Hahn, Martin W; Schmidt, Johanna; Taipale, Sami J; Doolittle, W Ford; Koll, Ulrike

2014-09-01

A pure culture of an actinobacterium previously described as 'Candidatus Rhodoluna lacicola' strain MWH-Ta8 was established and deposited in two public culture collections. Strain MWH-Ta8(T) represents a free-living planktonic freshwater bacterium obtained from hypertrophic Meiliang Bay, Lake Taihu, PR China. The strain was characterized by phylogenetic and taxonomic investigations, as well as by determination of its complete genome sequence. Strain MWH-Ta8(T) is noticeable due to its unusually low values of cell size (0.05 µm(3)), genome size (1.43 Mbp), and DNA G+C content (51.5 mol%). Phylogenetic analyses based on 16S rRNA gene and RpoB sequences suggested that strain MWH-Ta8(T) is affiliated with the family Microbacteriaceae with Pontimonas salivibrio being its closest relative among the currently described species within this family. Strain MWH-Ta8(T) and the type strain of Pontimonas salivibrio shared a 16S rRNA gene sequence similarity of 94.3 %. The cell-wall peptidoglycan of strain MWH-Ta8(T) was of type B2β (B10), containing 2,4-diaminobutyric acid as the diamino acid. The predominant cellular fatty acids were anteiso-C15 : 0 (36.5 %), iso-C16 : 0 (16.5 %), iso-C15 : 0 (15.6 %) and iso-C14 : 0 (8.9 %), and the major (>10 %) menaquinones were MK-11 and MK-12. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and two unknown glycolipids. The combined phylogenetic, phenotypic and chemotaxonomic data clearly suggest that strain MWH-Ta8(T) represents a novel species of a new genus in the family Microbacteriaceae, for which the name Rhodoluna lacicola gen. nov., sp. nov. is proposed. The type strain of the type species is MWH-Ta8(T) ( = DSM 23834(T) = LMG 26932(T)). © 2014 IUMS.

Disruption of tetR type regulator adeN by mobile genetic element confers elevated virulence in Acinetobacter baumannii.

PubMed

Saranathan, Rajagopalan; Pagal, Sudhakar; Sawant, Ajit R; Tomar, Archana; Madhangi, M; Sah, Suresh; Satti, Annapurna; Arunkumar, K P; Prashanth, K

2017-10-03

Acinetobacter baumannii is an important human pathogen and considered as a major threat due to its extreme drug resistance. In this study, the genome of a hyper-virulent MDR strain PKAB07 of A. baumannii isolated from an Indian patient was sequenced and analyzed to understand its mechanisms of virulence, resistance and evolution. Comparative genome analysis of PKAB07 revealed virulence and resistance related genes scattered throughout the genome, instead of being organized as an island, indicating the highly mosaic nature of the genome. Many intermittent horizontal gene transfer events, insertion sequence (IS) element insertions identified were augmenting resistance machinery and elevating the SNP densities in A. baumannii eventually aiding in their swift evolution. ISAba1, the most widely distributed insertion sequence in A. baumannii was found in multiple sites in PKAB07. Out of many ISAba1 insertions, we identified novel insertions in 9 different genes wherein insertional inactivation of adeN (tetR type regulator) was significant. To assess the significance of this disruption in A. baumannii, adeN mutant and complement strains were constructed in A. baumannii ATCC 17978 strain and studied. Biofilm levels were abrogated in the adeN knockout when compared with the wild type and complemented strain of adeN knockout. Virulence of the adeN knockout mutant strain was observed to be high, which was validated by in vitro experiments and Galleria mellonella infection model. The overexpression of adeJ, a major component of AdeIJK efflux pump observed in adeN knockout strain could be the possible reason for the elevated virulence in adeN mutant and PKB07 strain. Knocking out of adeN in ATCC strain led to increased resistance and virulence at par with the PKAB07. Disruption of tetR type regulator adeN by ISAba1 consequently has led to elevated virulence in this pathogen.

Longitudinal Monitoring of Successive Commercial Layer Flocks for Salmonella enterica Serovar Enteritidis.

PubMed

Denagamage, Thomas N; Patterson, Paul; Wallner-Pendleton, Eva; Trampel, Darrell; Shariat, Nikki; Dudley, Edward G; Jayarao, Bhushan M; Kariyawasam, Subhashinie

2016-11-01

The Pennsylvania Egg Quality Assurance Program (EQAP) provided the framework for Salmonella Enteritidis (SE) control programs, including the Food and Drug Administration (FDA) mandated Final Egg Rule, for commercial layer facilities throughout the United States. Although flocks with ≥3000 birds must comply with the FDA Final Egg Rule, smaller flocks are exempted from the rule. As a result, eggs produced by small layer flocks may pose a greater public health risk than those from larger flocks. It is also unknown if the EQAPs developed with large flocks in mind are suitable for small- and medium-sized flocks. Therefore, a study was performed to evaluate the effectiveness of best management practices included in EQAPs in reducing SE contamination of small- and medium-sized flocks by longitudinal monitoring of their environment and eggs. A total of 59 medium-sized (3000 to 50,000 birds) and small-sized (<3000 birds) flocks from two major layer production states of the United States were enrolled and monitored for SE by culturing different types of environmental samples and shell eggs for two consecutive flock cycles. Isolated SE was characterized by phage typing, pulsed-field gel electrophoresis (PFGE), and clustered regularly interspaced short palindromic repeats-multi-virulence-locus sequence typing (CRISPR-MVLST). Fifty-four Salmonella isolates belonging to 17 serovars, 22 of which were SE, were isolated from multiple sample types. Typing revealed that SE isolates belonged to three phage types (PTs), three PFGE fingerprint patterns, and three CRISPR-MVLST SE Sequence Types (ESTs). The PT8 and JEGX01.0004 PFGE pattern, the most predominant SE types associated with foodborne illness in the United States, were represented by a majority (91%) of SE. Of the three ESTs observed, 85% SE were typed as EST4. The proportion of SE-positive hen house environment during flock cycle 2 was significantly less than the flock cycle 1, demonstrating that current EQAP practices were effective in reducing SE contamination of medium and small layer flocks.

PRISM 3: expanded prediction of natural product chemical structures from microbial genomes.

PubMed

Skinnider, Michael A; Merwin, Nishanth J; Johnston, Chad W; Magarvey, Nathan A

2017-07-03

Microbial natural products represent a rich resource of pharmaceutically and industrially important compounds. Genome sequencing has revealed that the majority of natural products remain undiscovered, and computational methods to connect biosynthetic gene clusters to their corresponding natural products therefore have the potential to revitalize natural product discovery. Previously, we described PRediction Informatics for Secondary Metabolomes (PRISM), a combinatorial approach to chemical structure prediction for genetically encoded nonribosomal peptides and type I and II polyketides. Here, we present a ground-up rewrite of the PRISM structure prediction algorithm to derive prediction of natural products arising from non-modular biosynthetic paradigms. Within this new version, PRISM 3, natural product scaffolds are modeled as chemical graphs, permitting structure prediction for aminocoumarins, antimetabolites, bisindoles and phosphonate natural products, and building upon the addition of ribosomally synthesized and post-translationally modified peptides. Further, with the addition of cluster detection for 11 new cluster types, PRISM 3 expands to detect 22 distinct natural product cluster types. Other major modifications to PRISM include improved sequence input and ORF detection, user-friendliness and output. Distribution of PRISM 3 over a 300-core server grid improves the speed and capacity of the web application. PRISM 3 is available at http://magarveylab.ca/prism/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Next-generation sequencing of cancer genomes: back to the future

PubMed Central

Walter, Matthew J; Graubert, Timothy A; DiPersio, John F; Mardis, Elaine R; Wilson, Richard K; Ley, Timothy J

2010-01-01

The systematic karyotyping of bone marrow cells was the first genomic approach used to personalize therapy for patients with leukemia. The paradigm established by cytogenetic studies in leukemia (from gene discovery to therapeutic intervention) now has the potential to be rapidly extended with the use of whole-genome sequencing approaches for cancer, which are now possible. We are now entering a period of exponential growth in cancer gene discovery that will provide many novel therapeutic targets for a large number of cancer types. Establishing the pathogenetic relevance of individual mutations is a major challenge that must be solved. However, after thousands of cancer genomes have been sequenced, the genetic rules of cancer will become known and new approaches for diagnosis, risk stratification and individualized treatment of cancer patients will surely follow. PMID:20161678

Insertion sequence typing of Mycobacterium tuberculosis: characterization of a widespread subtype with a single copy of IS6110.

PubMed

Fomukong, N G; Tang, T H; al-Maamary, S; Ibrahim, W A; Ramayah, S; Yates, M; Zainuddin, Z F; Dale, J W

1994-12-01

DNA fingerprinting with the insertion sequence IS6110 (also known as IS986) has become established as a major tool for investigating the spread of tuberculosis. Most strains of Mycobacterium tuberculosis have multiple copies of IS6110, but a small minority carry a single copy only. We have examined selected strains from Malaysia, Tanzania and Oman, in comparison with M. bovis isolates and BCG strains carrying one or two copies of IS6110. The insertion sequence appears to be present in the same position in all these strains, which suggests that in these organisms the element is defective in transposition and that the loss of transposability may have occurred at an early stage in the evolution of the M. tuberculosis complex.

Use of the LUS in sequence allele designations to facilitate probabilistic genotyping of NGS-based STR typing results.

PubMed

Just, Rebecca S; Irwin, Jodi A

2018-05-01

Some of the expected advantages of next generation sequencing (NGS) for short tandem repeat (STR) typing include enhanced mixture detection and genotype resolution via sequence variation among non-homologous alleles of the same length. However, at the same time that NGS methods for forensic DNA typing have advanced in recent years, many caseworking laboratories have implemented or are transitioning to probabilistic genotyping to assist the interpretation of complex autosomal STR typing results. Current probabilistic software programs are designed for length-based data, and were not intended to accommodate sequence strings as the product input. Yet to leverage the benefits of NGS for enhanced genotyping and mixture deconvolution, the sequence variation among same-length products must be utilized in some form. Here, we propose use of the longest uninterrupted stretch (LUS) in allele designations as a simple method to represent sequence variation within the STR repeat regions and facilitate - in the nearterm - probabilistic interpretation of NGS-based typing results. An examination of published population data indicated that a reference LUS region is straightforward to define for most autosomal STR loci, and that using repeat unit plus LUS length as the allele designator can represent greater than 80% of the alleles detected by sequencing. A proof of concept study performed using a freely available probabilistic software demonstrated that the LUS length can be used in allele designations when a program does not require alleles to be integers, and that utilizing sequence information improves interpretation of both single-source and mixed contributor STR typing results as compared to using repeat unit information alone. The LUS concept for allele designation maintains the repeat-based allele nomenclature that will permit backward compatibility to extant STR databases, and the LUS lengths themselves will be concordant regardless of the NGS assay or analysis tools employed. Further, these biologically based, easy-to-derive designations uphold clear relationships between parent alleles and their stutter products, enabling analysis in fully continuous probabilistic programs that model stutter while avoiding the algorithmic complexities that come with string based searches. Though using repeat unit plus LUS length as the allele designator does not capture variation that occurs outside of the core repeat regions, this straightforward approach would permit the large majority of known STR sequence variation to be used for mixture deconvolution and, in turn, result in more informative mixture statistics in the near term. Ultimately, the method could bridge the gap from current length-based probabilistic systems to facilitate broader adoption of NGS by forensic DNA testing laboratories. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Early antiretroviral treatment (eART) limits viral diversity over time in a long-term HIV viral suppressed perinatally infected child.

PubMed

Palma, Paolo; Zangari, Paola; Alteri, Claudia; Tchidjou, Hyppolite K; Manno, Emma Concetta; Liuzzi, Giuseppina; Perno, Carlo Federico; Rossi, Paolo; Bertoli, Ada; Bernardi, Stefania

2016-12-09

HIV genetic diversity implicates major challenges for the control of viral infection by the immune system and for the identification of an effective immunotherapeutic strategy. With the present case report we underline as HIV evolution could be effectively halted by early antiretroviral treatment (eART). Few cases supported this evidence due to the difficulty of performing amplification and sequencing analysis in long-term viral suppressed patients. Here, we reported the case of limited HIV-1 viral evolution over time in a successful early treated child. A perinatally HIV-1 infected infant was treated within 7 weeks of age with zidovudine, lamivudine, nevirapine and lopinavir/ritonavir. At antiretroviral treatment (ART) initiation HIV-1 viral load (VL) and CD4 percentage were >500,000 copies/ml and 35%, respectively. Plasma genotypic resistance test showed a wild-type virus. The child reached VL undetectability after 33 weeks of combination antiretroviral therapy (cART) since he maintained a stable VL <40copies/ml. After 116 weeks on ART we were able to perform amplification and sequencing assay on the plasma virus. At this time VL was <40 copies/ml and CD4 percentage was 40%. Again the genotypic resistance test revealed a wild-type virus. The phylogenetic analysis performed on the HIV-1 pol sequences of the mother and the child revealed that sequences clustered with C subtype reference strains and formed a monophyletic cluster distinct from the other C sequences included in the analysis (bootstrap value >90%). Any major evolutionary divergence was detected. eART limits the viral evolution avoiding the emergence of new viral variants. This result may have important implications in host immune control and may sustain the challenge search of new personalized immunotherapeutic approaches to achieve a prolonged viral remission.

Brevibacillus sediminis sp. nov., isolated from a hot spring.

PubMed

Xian, Wen-Dong; Yin, Yi-Rui; Liu, Lan; Yuan, Chang-Guo; Hussain, Firasat; Khan, Inamullah; Habib, Neeli; Zhou, En-Min; Li, Wen-Jun

2016-02-01

Strain YIM 78300 T , a novel Gram-stain-positive, moderately thermophilic, endospore-forming, rod-shaped, motile bacterium, was recovered from the sediment of a hot spring in the Tagejia Geothermal Field, Angren, Tibet province, western China. Optimum growth was observed at 50-55 °C, at pH 7.0 and with 0-1.5 % (w/v) NaCl. Phylogenetic analysis of the 16S rRNA gene sequence of strain YIM 78300 T indicated that it belongs to the genus Brevibacillus . Similarity levels between the 16S rRNA gene sequences of the new isolate and those of the type strains of Brevibacillus members were 96.9-96.3 %; highest sequence similarity was with Brevibacillus thermoruber DSM 7064 T . The predominant menaquinone was MK-7 and the major cellular fatty acids were iso-C 15 : 0 and iso-C 17 : 0 . The major polar lipids were phosphatidyl- N -methylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, two unidentified phospholipids, an unidentified aminophospholipid and two unidentified polar lipids. The G+C content of the genomic DNA of strain YIM 78300 T was 57.9 mol%. Based on phylogenetic analyses, and physiological and biochemical characteristics, strain YIM 78300 T is considered to represent a novel species of the genus Brevibacillus , for which the name Brevibacillus sediminis sp. nov. is proposed. The type strain is YIM 78300 T ( = DSM 29928 T  = CPCC 100738 T ).

Rarimicrobium hominis gen. nov., sp. nov., representing the fifth genus in the phylum Synergistetes that includes human clinical isolates.

PubMed

Jumas-Bilak, Estelle; Bouvet, Philippe; Allen-Vercoe, Emma; Aujoulat, Fabien; Lawson, Paul A; Jean-Pierre, Hélène; Marchandin, Hélène

2015-11-01

Five human clinical isolates of an unknown, strictly anaerobic, slow-growing, Gram-stain-negative, rod-shaped micro-organism were subjected to a polyphasic taxonomic study. Comparative 16S rRNA gene sequence-based phylogeny showed that the isolates grouped in a clade that included members of the genera Pyramidobacter, Jonquetella, and Dethiosulfovibrio; the type strain of Pyramidobacter piscolens was the closest relative with 91.5-91.7 % 16S rRNA gene sequence similarity. The novel strains were mainly asaccharolytic and unreactive in most conventional biochemical tests. Major metabolic end products in trypticase/glucose/yeast extract broth were acetic acid and propionic acid and the major cellular fatty acids were C13 : 0 and C16 : 0, each of which could be used to differentiate the strains from P. piscolens. The DNA G+C content based on whole genome sequencing for the reference strain 22-5-S 12D6FAA was 57 mol%. Based on these data, a new genus, Rarimicrobium gen. nov., is proposed with one novel species, Rarimicrobium hominis sp. nov., named after the exclusive and rare finding of the taxon in human samples. Rarimicrobium is the fifth genus of the 14 currently characterized in the phylum Synergistetes and the third one in subdivision B that includes human isolates. The type strain of Rarimicrobium hominis is ADV70T ( = LMG 28163T = CCUG 65426T).

Method for identifying and quantifying nucleic acid sequence aberrations

DOEpatents

Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

1998-01-01

A method for detecting nucleic acid sequence aberrations by detecting nucleic acid sequences having both a first and a second nucleic acid sequence type, the presence of the first and second sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. The method uses a first hybridization probe which includes a nucleic acid sequence that is complementary to a first sequence type and a first complexing agent capable of attaching to a second complexing agent and a second hybridization probe which includes a nucleic acid sequence that selectively hybridizes to the second nucleic acid sequence type over the first sequence type and includes a detectable marker for detecting the second hybridization probe.

Method for identifying and quantifying nucleic acid sequence aberrations

DOEpatents

Lucas, J.N.; Straume, T.; Bogen, K.T.

1998-07-21

A method is disclosed for detecting nucleic acid sequence aberrations by detecting nucleic acid sequences having both a first and a second nucleic acid sequence type, the presence of the first and second sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. The method uses a first hybridization probe which includes a nucleic acid sequence that is complementary to a first sequence type and a first complexing agent capable of attaching to a second complexing agent and a second hybridization probe which includes a nucleic acid sequence that selectively hybridizes to the second nucleic acid sequence type over the first sequence type and includes a detectable marker for detecting the second hybridization probe. 11 figs.

Molecular characterization of muscle-parasitizing didymozoid from a chub mackerel, Scomber japonicus.

PubMed

Abe, Niichiro; Okamoto, Mitsuru

2015-09-01

Didymozoids found in the muscles of marine fish are almost always damaged because they are usually found after being sliced. Therefore, identifying muscle-parasitizing didymozoids is difficult because of the difficulty in collecting non-damaged worms and observing their organs as key points for morphological identification. Moreover, muscle-parasitizing didymozoids are not easily found because they parasitize at the trunk muscles. Therefore, muscle-parasitizing didymozoid classification has not progressed because there are few opportunities to detect them. Our recent report was the first to describe the usefulness of sequencing analysis for discrimination among muscle-parasitizing didymozoids. Recently, we found a didymozoid in the trunk muscle of a chub mackerel Scomber japonicus. The present study genetically compares the present isolate with other muscle-parasitizing didymozoids. The present isolate differs markedly from the previously unidentified didymozoid from an Atlantic mackerel S. scombrus by phylogenetic analysis of 18S rDNA. It also differs from other muscle-parasitizing didymozoids from other host species based on phylogenetic analyses of 18S, 28S rDNAs, and coxI loci. These results suggest that sequencing analysis is useful for the discrimination of muscle-parasitizing didymozoids. Combining the present data with earlier data for sequencing analysis, muscle-parasitizing didymozoids from seven marine fish species were classified as seven species. We proposed appellations for six distinct muscle-parasitizing didymozoids for future analysis: sweetlips fish type from Diagramma pictum and Plectorhinchus cinctus, red sea bream type from Pagrus major, flying fish type from Cypselurus heterurus, Atlantic mackerel type from Scomber scombrus, chub mackerel type from S. japonicus, and purple rockcod type from Epinephelus cyanopodus.

Genotypic characterization of CRF01_AE env genes derived from human immunodeficiency virus type 1-infected patients residing in central Thailand.

PubMed

Utachee, Piraporn; Jinnopat, Piyamat; Isarangkura-Na-Ayuthaya, Panasda; de Silva, Udayanga Chandimal; Nakamura, Shota; Siripanyaphinyo, Uamporn; Wichukchinda, Nuanjun; Tokunaga, Kenzo; Yasunaga, Teruo; Sawanpanyalert, Pathom; Ikuta, Kazuyoshi; Auwanit, Wattana; Kameoka, Masanori

2009-02-01

CRF01_AE is a major subtype of human immunodeficiency virus type 1 (HIV-1) circulating in Southeast Asia, including Thailand. HIV-1 env genes were amplified by polymerase chain reaction from blood samples of HIV-1-infected patients residing in Thailand in 2006, and cloned into the pNL4-3-derived reporter viral construct. Generated envelope protein (Env)-recombinant virus was examined for its infectivity, and then 35 infectious CRF01_AE Env-recombinant viruses were selected. Sequencing analysis revealed that the interclone variation of the deduced amino acid sequences was higher in CRF01_AE env genes isolated in 2006 than in those isolated in the early 1990s, suggesting that env gene variation has been increasing gradually among CRF01_AE viruses prevalent in Thailand. We also examined the characteristics of the deduced amino acid sequences of 35 CRF01_AE env genes. Our results may provide useful information to help in better understanding the genotype of env genes of CRF01_AE viruses currently circulating in Thailand.

Isolation and molecular characterization of Chikungunya virus from the Andaman and Nicobar archipelago, India: evidence of an East, Central, and South African genotype.

PubMed

Muruganandam, N; Chaaithanya, I K; Senthil, G S; Shriram, A N; Bhattacharya, D; Jeevabharathi, G S; Sudeep, A B; Pradeepkumar, N; Vijayachari, P

2011-12-01

Chikungunya virus (CHIKV) is an Alphavirus belonging to the family Togaviridae. In 2006, CHIKV infection struck the Andaman and Nicobar archipelago, with an attack rate of 60%. There were more than 10 cases with acute flaccid paralysis simulating the Guillian Barre Syndrome. The majority of the patients presented severe joint pain. The cause for such an explosive nature of the outbreak with increased morbidity was not known. The isolation of CHIKV was attempted and succeeded from nine subjects presenting clinical symptoms of Chikungunya fever. The cDNA of all the isolates was sequenced for partial E1 and nsP1 genes. Sequences were aligned based on the double locus sequence typing concept. The phylogenetic analysis shows that sequences of Andaman isolates grouped with the East, Central, and South African genotype of virus isolates from India, Sri Lanka, and Réunion. The genetic distance between Andaman isolates and the Réunion isolates was very small. The phylogenetic analysis confirmed the origin of the isolates responsible for the first ever confirmed CHIKV outbreak in these islands to be the East, Central, and South African genotype. In this manuscript, we discuss the involvement of the East, Central, and South African strain with the Chikungunya fever outbreak in this archipelago and double locus sequence typing as a first time approach.

Molecular characterization of influenza viruses collected from young children in Uberlandia, Brazil - from 2001 to 2010.

PubMed

de Mattos Silva Oliveira, Thelma Fátima; Yokosawa, Jonny; Motta, Fernando Couto; Siqueira, Marilda Mendonça; da Silveira, Hélio Lopes; Queiróz, Divina Aparecida Oliveira

2015-02-18

Influenza remains a major health problem due to the seasonal epidemics that occur every year caused by the emergence of new influenza virus strains. Hemagglutinin (HA) and neuraminidase (NA) glycoproteins are under selective pressure and subjected to frequent changes by antigenic drift. Therefore, our main objective was to investigate the influenza cases in Uberlândia city, Midwestern Brazil, in order to monitor the appearance of new viral strains, despite the availability of a prophylactic vaccine. Nasopharyngeal samples were collected from 605 children less than five years of age presenting with acute respiratory disease and tested by immunofluorescence assay (IFA) for detection of adenovirus, respiratory syncytial virus, parainfluenza virus types 1, 2, and 3 and influenza virus types A and B. A reverse transcription-PCR (RT-PCR) for influenza viruses A and B was carried out to amplify partial segments of the HA and NA genes. The nucleotide sequences were analyzed and compared with sequences of the virus strains of the vaccine available in the same year of sample collection. Forty samples (6.6%) were tested positive for influenza virus by IFA and RT-PCR, with 39 samples containing virus of type A and one of type B. By RT-PCR, the type A viruses were further characterized in subtypes H3N2, H1N2 and H1N1 (41.0%, 17.9%, and 2.6%, respectively). Deduced amino acid sequence analysis of the partial hemagglutinin sequence compared to sequences from vaccine strains, revealed that all strains found in Uberlândia had variations in the antigenic sites. The sequences of the receptor binding sites were preserved, although substitutions with similar amino acids were observed in few cases. The neuraminidase sequences did not show significant changes. All the H3 isolates detected in the 2001-2003 period had drifted from vaccine strain, unlike the isolates of the 2004-2007 period. These results suggest that the seasonal influenza vaccine effectiveness could be reduced because of A H3N2 variants that circulated in 2001-2003 years. Thus, an early monitoring of variants circulating in the country or in a region may provide important information about the probable efficacy of the vaccine that will be administered in an influenza season.

Hepatitis C: a possible etiology for cryoglobulinaemia type II.

PubMed Central

Pechère-Bertschi, A; Perrin, L; de Saussure, P; Widmann, J J; Giostra, E; Schifferli, J A

1992-01-01

Out of 15 successive patients with mixed essential cryoglobulinaemia type II (monoclonal IgM kappa/IgG), 13 had serological evidence for hepatitis C infection as shown by specific enzyme immunoassays and immunoblot. RNA was purified from the serum of seven patients and hepatitis C sequences were identified in five following reverse transcription and DNA amplification. The liver histology showed chronic active hepatitis with or without cirrhosis in the 12 patients with hepatitis C who had a liver biopsy. The two patients without serological evidence of hepatitis C suffered from haematological malignancies. Hepatitis C may be a major etiological agent of cryoglobulinaemia type II. PMID:1381302

Diversity and Evolutionary Analysis of Iron-Containing (Type-III) Alcohol Dehydrogenases in Eukaryotes

PubMed Central

Gaona-López, Carlos; Julián-Sánchez, Adriana

2016-01-01

Background Alcohol dehydrogenase (ADH) activity is widely distributed in the three domains of life. Currently, there are three non-homologous NAD(P)+-dependent ADH families reported: Type I ADH comprises Zn-dependent ADHs; type II ADH comprises short-chain ADHs described first in Drosophila; and, type III ADH comprises iron-containing ADHs (FeADHs). These three families arose independently throughout evolution and possess different structures and mechanisms of reaction. While types I and II ADHs have been extensively studied, analyses about the evolution and diversity of (type III) FeADHs have not been published yet. Therefore in this work, a phylogenetic analysis of FeADHs was performed to get insights into the evolution of this protein family, as well as explore the diversity of FeADHs in eukaryotes. Principal Findings Results showed that FeADHs from eukaryotes are distributed in thirteen protein subfamilies, eight of them possessing protein sequences distributed in the three domains of life. Interestingly, none of these protein subfamilies possess protein sequences found simultaneously in animals, plants and fungi. Many FeADHs are activated by or contain Fe2+, but many others bind to a variety of metals, or even lack of metal cofactor. Animal FeADHs are found in just one protein subfamily, the hydroxyacid-oxoacid transhydrogenase (HOT) subfamily, which includes protein sequences widely distributed in fungi, but not in plants), and in several taxa from lower eukaryotes, bacteria and archaea. Fungi FeADHs are found mainly in two subfamilies: HOT and maleylacetate reductase (MAR), but some can be found also in other three different protein subfamilies. Plant FeADHs are found only in chlorophyta but not in higher plants, and are distributed in three different protein subfamilies. Conclusions/Significance FeADHs are a diverse and ancient protein family that shares a common 3D scaffold with a patchy distribution in eukaryotes. The majority of sequenced FeADHs from eukaryotes are distributed in just two subfamilies, HOT and MAR (found mainly in animals and fungi). These two subfamilies comprise almost 85% of all sequenced FeADHs in eukaryotes. PMID:27893862

Characterization of Staphylococcus aureus isolates from buffalo, bovine, ovine, and caprine milk samples collected in Rio de Janeiro State, Brazil.

PubMed

Aires-de-Sousa, Marta; Parente, Carlos E S R; Vieira-da-Motta, Olney; Bonna, Isabel C F; Silva, Denise A; de Lencastre, Hermínia

2007-06-01

Eighty-four staphylococcal isolates were obtained from milk samples from cows, sheep, goats, and buffalo with subclinical mastitis and from colonization samples from ostriches. The animals were hosted in 18 small dairy herds and an ostrich breeding located in 10 municipalities of the state of Rio de Janeiro, Brazil. Thirty isolates were identified as Staphylococcus aureus by biochemical and molecular techniques and were comparatively characterized by phenotypic and genotypic methods. The molecular characterization by pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST) revealed five clonal types (PFGE A, spa type t359, sequence type 747 [ST747]; PFGE B, spa type t1180, ST750; PFGE C, spa type t605, ST126; PFGE D, spa type t127, ST751; and PFGE F, spa type t002, ST5). None of the isolates harbored the Panton-Valentine leukocidin or exfoliative toxin D gene. The detection of major clone A (in 63% of the isolates) in different herds, among all animal species studied, and in infection and colonization samples evidenced its geographical spread among Rio de Janeiro State and no host preference among the animal species. Comparison with S. aureus from a human origin suggested that all but one clone found in the present study might be animal specific.

Vibrio cholerae Serogroup O139: Isolation from Cholera Patients and Asymptomatic Household Family Members in Bangladesh between 2013 and 2014.

PubMed

Chowdhury, Fahima; Mather, Alison E; Begum, Yasmin Ara; Asaduzzaman, Muhammad; Baby, Nabilah; Sharmin, Salma; Biswas, Rajib; Uddin, Muhammad Ikhtear; LaRocque, Regina C; Harris, Jason B; Calderwood, Stephen B; Ryan, Edward T; Clemens, John D; Thomson, Nicholas R; Qadri, Firdausi

2015-11-01

Cholera is endemic in Bangladesh, with outbreaks reported annually. Currently, the majority of epidemic cholera reported globally is El Tor biotype Vibrio cholerae isolates of the serogroup O1. However, in Bangladesh, outbreaks attributed to V. cholerae serogroup O139 isolates, which fall within the same phylogenetic lineage as the O1 serogroup isolates, were seen between 1992 and 1993 and in 2002 to 2005. Since then, V. cholerae serogroup O139 has only been sporadically isolated in Bangladesh and is now rarely isolated elsewhere. Here, we present case histories of four cholera patients infected with V. cholerae serogroup O139 in 2013 and 2014 in Bangladesh. We comprehensively typed these isolates using conventional approaches, as well as by whole genome sequencing. Phenotypic typing and PCR confirmed all four isolates belonging to the O139 serogroup. Whole genome sequencing revealed that three of the isolates were phylogenetically closely related to previously sequenced El Tor biotype, pandemic 7, toxigenic V. cholerae O139 isolates originating from Bangladesh and elsewhere. The fourth isolate was a non-toxigenic V. cholerae that, by conventional approaches, typed as O139 serogroup but was genetically divergent from previously sequenced pandemic 7 V. cholerae lineages belonging to the O139 or O1 serogroups. These results suggest that previously observed lineages of V. cholerae O139 persist in Bangladesh and can cause clinical disease and that a novel disease-causing non-toxigenic O139 isolate also occurs.

Prevotella timonensis sp. nov., isolated from a human breast abscess.

PubMed

Glazunova, Olga O; Launay, Thierry; Raoult, Didier; Roux, Véronique

2007-04-01

Gram-negative anaerobic rods were isolated from a human breast abscess. Based on genotypic and phenotypic characteristics, the novel strain belonged to the genus Prevotella. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that it was closely related to Prevotella buccalis (94 % 16S rRNA gene sequence similarity), Prevotella salivae (90 %) and Prevotella oris (89.1 %). The major cellular fatty acid was C(14 : 0) (19.5 %). The new isolate represents a novel species in the genus Prevotella, for which the name Prevotella timonensis sp. nov. is proposed. The type strain is strain 4401737(T) (=CIP 108522(T)=CCUG 50105(T)).

Rhizobium etli asparaginase II

PubMed Central

Huerta-Saquero, Alejandro; Evangelista-Martínez, Zahaed; Moreno-Enriquez, Angélica; Perez-Rueda, Ernesto

2013-01-01

Bacterial l-asparaginase has been a universal component of therapies for childhood acute lymphoblastic leukemia since the 1970s. Two principal enzymes derived from Escherichia coli and Erwinia chrysanthemi are the only options clinically approved to date. We recently reported a study of recombinant l-asparaginase (AnsA) from Rhizobium etli and described an increasing type of AnsA family members. Sequence analysis revealed four conserved motifs with notable differences with respect to the conserved regions of amino acid sequences of type I and type II l-asparaginases, particularly in comparison with therapeutic enzymes from E. coli and E. chrysanthemi. These differences suggested a distinct immunological specificity. Here, we report an in silico analysis that revealed immunogenic determinants of AnsA. Also, we used an extensive approach to compare the crystal structures of E. coli and E. chrysantemi asparaginases with a computational model of AnsA and identified immunogenic epitopes. A three-dimensional model of AsnA revealed, as expected based on sequence dissimilarities, completely different folding and different immunogenic epitopes. This approach could be very useful in transcending the problem of immunogenicity in two major ways: by chemical modifications of epitopes to reduce drug immunogenicity, and by site-directed mutagenesis of amino acid residues to diminish immunogenicity without reduction of enzymatic activity. PMID:22895060

Rhizobium etli asparaginase II: an alternative for acute lymphoblastic leukemia (ALL) treatment.

PubMed

Huerta-Saquero, Alejandro; Evangelista-Martínez, Zahaed; Moreno-Enriquez, Angélica; Perez-Rueda, Ernesto

2013-01-01

Bacterial L-asparaginase has been a universal component of therapies for childhood acute lymphoblastic leukemia since the 1970s. Two principal enzymes derived from Escherichia coli and Erwinia chrysanthemi are the only options clinically approved to date. We recently reported a study of recombinant L-asparaginase (AnsA) from Rhizobium etli and described an increasing type of AnsA family members. Sequence analysis revealed four conserved motifs with notable differences with respect to the conserved regions of amino acid sequences of type I and type II L-asparaginases, particularly in comparison with therapeutic enzymes from E. coli and E. chrysanthemi. These differences suggested a distinct immunological specificity. Here, we report an in silico analysis that revealed immunogenic determinants of AnsA. Also, we used an extensive approach to compare the crystal structures of E. coli and E. chrysantemi asparaginases with a computational model of AnsA and identified immunogenic epitopes. A three-dimensional model of AsnA revealed, as expected based on sequence dissimilarities, completely different folding and different immunogenic epitopes. This approach could be very useful in transcending the problem of immunogenicity in two major ways: by chemical modifications of epitopes to reduce drug immunogenicity, and by site-directed mutagenesis of amino acid residues to diminish immunogenicity without reduction of enzymatic activity.

Human germline and pan-cancer variomes and their distinct functional profiles

PubMed Central

Pan, Yang; Karagiannis, Konstantinos; Zhang, Haichen; Dingerdissen, Hayley; Shamsaddini, Amirhossein; Wan, Quan; Simonyan, Vahan; Mazumder, Raja

2014-01-01

Identification of non-synonymous single nucleotide variations (nsSNVs) has exponentially increased due to advances in Next-Generation Sequencing technologies. The functional impacts of these variations have been difficult to ascertain because the corresponding knowledge about sequence functional sites is quite fragmented. It is clear that mapping of variations to sequence functional features can help us better understand the pathophysiological role of variations. In this study, we investigated the effect of nsSNVs on more than 17 common types of post-translational modification (PTM) sites, active sites and binding sites. Out of 1 705 285 distinct nsSNVs on 259 216 functional sites we identified 38 549 variations that significantly affect 10 major functional sites. Furthermore, we found distinct patterns of site disruptions due to germline and somatic nsSNVs. Pan-cancer analysis across 12 different cancer types led to the identification of 51 genes with 106 nsSNV affected functional sites found in 3 or more cancer types. 13 of the 51 genes overlap with previously identified Significantly Mutated Genes (Nature. 2013 Oct 17;502(7471)). 62 mutations in these 13 genes affecting functional sites such as DNA, ATP binding and various PTM sites occur across several cancers and can be prioritized for additional validation and investigations. PMID:25232094

Direct typing of Canine parvovirus (CPV) from infected dog faeces by rapid mini sequencing technique.

PubMed

V, Pavana Jyothi; S, Akila; Selvan, Malini K; Naidu, Hariprasad; Raghunathan, Shwethaa; Kota, Sathish; Sundaram, R C Raja; Rana, Samir Kumar; Raj, G Dhinakar; Srinivasan, V A; Mohana Subramanian, B

2016-12-01

Canine parvovirus (CPV) is a non-enveloped single stranded DNA virus with an icosahedral capsid. Mini-sequencing based CPV typing was developed earlier to detect and differentiate all the CPV types and FPV in a single reaction. This technique was further evaluated in the present study by performing the mini-sequencing directly from fecal samples which avoided tedious virus isolation steps by cell culture system. Fecal swab samples were collected from 84 dogs with enteritis symptoms, suggestive of parvoviral infection from different locations across India. Seventy six of these samples were positive by PCR; the subsequent mini-sequencing reaction typed 74 of them as type 2a virus, and 2 samples as type 2b. Additionally, 25 of the positive samples were typed by cycle sequencing of PCR products. Direct CPV typing from fecal samples using mini-sequencing showed 100% correlation with CPV typing by cycle sequencing. Moreover, CPV typing was achieved by mini-sequencing even with faintly positive PCR amplicons which was not possible by cycle sequencing. Therefore, the mini-sequencing technique is recommended for regular epidemiological follow up of CPV types, since the technique is rapid, highly sensitive and high capacity method for CPV typing. Copyright © 2016. Published by Elsevier B.V.

Microbiological Features of KPC-Producing Enterobacter Isolates Identified in a U.S. Hospital System

PubMed Central

Ahn, Chulsoo; Syed, Alveena; Hu, Fupin; O’Hara, Jessica A.; Rivera, Jesabel I.; Doi, Yohei

2014-01-01

Microbiological data regarding KPC-producing Enterobacter spp. are scarce. In this study, 11 unique KPC-producing Enterobacter isolates were identified among 44 ertapenem-non-susceptible Enterobacter isolates collected between 2009 and 2013 at a hospital system in Western Pennsylvania. All cases were healthcare-associated and occurred in medically complex patients. While pulsed-field gel electrophoresis (PFGE) showed diverse restriction patterns overall, multilocus sequence typing (MLST) identified Enterobacter cloacae isolates with sequence types (STs) 93 and 171 from two hospitals each. The levels of carbapenem minimum inhibitory concentrations were highly variable. All isolates remained susceptible to colistin, tigecycline, and the majority to amikacin and doxycycline. A blaKPC-carrying IncN plasmid conferring trimethoprim-sulfamethoxazole resistance was identified in three of the isolates. Spread of blaKPC in Enterobacter spp. appears to be due to a combination of plasmid-mediated and clonal processes. PMID:25053203

Secondary structure and phylogeny of Staphylococcus and Micrococcus 5S rRNAs.

PubMed Central

Dekio, S; Yamasaki, R; Jidoi, J; Hori, H; Osawa, S

1984-01-01

Nucleotide sequences of 5S rRNAs from four bacteria, Staphylococcus aureus Smith (diffuse), Staphylococcus epidermidis ATCC 14990, Micrococcus luteus ATCC 9341 and Micrococcus luteus ATCC 4698, were determined. The secondary structural models of S. aureus and S. epidermidis sequences showed characteristics of the gram-positive bacterial 5S rRNA (116-N type [H. Hori and S. Osawa, Proc. Natl. Acad. Sci. U.S.A. 76:381-385, 1979]). Those of M. luteus ATCC 9341 and M. luteus ATCC 4698 together with that of Streptomyces griseus (A. Simoncsits, Nucleic Acids Res. 8:4111-4124, 1980) showed intermediary characteristics between the gram-positive and gram-negative (120-N type [H. Hori and S. Osawa, 1979]) 5S rRNAs. This and previous studies revealed that there exist at least three major groups of eubacteria having distinct 5S rRNA and belonging to different stems in the 5S rRNA phylogenic tree. PMID:6735981

Preparation of metagenomic libraries from naturally occurring marine viruses.

PubMed

Solonenko, Sergei A; Sullivan, Matthew B

2013-01-01

Microbes are now well recognized as major drivers of the biogeochemical cycling that fuels the Earth, and their viruses (phages) are known to be abundant and important in microbial mortality, horizontal gene transfer, and modulating microbial metabolic output. Investigation of environmental phages has been frustrated by an inability to culture the vast majority of naturally occurring diversity coupled with the lack of robust, quantitative, culture-independent methods for studying this uncultured majority. However, for double-stranded DNA phages, a quantitative viral metagenomic sample-to-sequence workflow now exists. Here, we review these advances with special emphasis on the technical details of preparing DNA sequencing libraries for metagenomic sequencing from environmentally relevant low-input DNA samples. Library preparation steps broadly involve manipulating the sample DNA by fragmentation, end repair and adaptor ligation, size fractionation, and amplification. One critical area of future research and development is parallel advances for alternate nucleic acid types such as single-stranded DNA and RNA viruses that are also abundant in nature. Combinations of recent advances in fragmentation (e.g., acoustic shearing and tagmentation), ligation reactions (adaptor-to-template ratio reference table availability), size fractionation (non-gel-sizing), and amplification (linear amplification for deep sequencing and linker amplification protocols) enhance our ability to generate quantitatively representative metagenomic datasets from low-input DNA samples. Such datasets are already providing new insights into the role of viruses in marine systems and will continue to do so as new environments are explored and synergies and paradigms emerge from large-scale comparative analyses. © 2013 Elsevier Inc. All rights reserved.

A Bacterial Analysis Platform: An Integrated System for Analysing Bacterial Whole Genome Sequencing Data for Clinical Diagnostics and Surveillance.

PubMed

Thomsen, Martin Christen Frølund; Ahrenfeldt, Johanne; Cisneros, Jose Luis Bellod; Jurtz, Vanessa; Larsen, Mette Voldby; Hasman, Henrik; Aarestrup, Frank Møller; Lund, Ole

2016-01-01

Recent advances in whole genome sequencing have made the technology available for routine use in microbiological laboratories. However, a major obstacle for using this technology is the availability of simple and automatic bioinformatics tools. Based on previously published and already available web-based tools we developed a single pipeline for batch uploading of whole genome sequencing data from multiple bacterial isolates. The pipeline will automatically identify the bacterial species and, if applicable, assemble the genome, identify the multilocus sequence type, plasmids, virulence genes and antimicrobial resistance genes. A short printable report for each sample will be provided and an Excel spreadsheet containing all the metadata and a summary of the results for all submitted samples can be downloaded. The pipeline was benchmarked using datasets previously used to test the individual services. The reported results enable a rapid overview of the major results, and comparing that to the previously found results showed that the platform is reliable and able to correctly predict the species and find most of the expected genes automatically. In conclusion, a combined bioinformatics platform was developed and made publicly available, providing easy-to-use automated analysis of bacterial whole genome sequencing data. The platform may be of immediate relevance as a guide for investigators using whole genome sequencing for clinical diagnostics and surveillance. The platform is freely available at: https://cge.cbs.dtu.dk/services/CGEpipeline-1.1 and it is the intention that it will continue to be expanded with new features as these become available.

Piscine reovirus: Genomic and molecular phylogenetic analysis from farmed and wild salmonids collected on the Canada/US Pacific Coast

USGS Publications Warehouse

Siah, Ahmed; Morrison, Diane B.; Fringuelli, Elena; Savage, Paul S.; Richmond, Zina; Purcell, Maureen K.; Johns, Robert; Johnson, Stewart C.; Sakasida, Sonja M.

2015-01-01

Piscine reovirus (PRV) is a double stranded non-enveloped RNA virus detected in farmed and wild salmonids. This study examined the phylogenetic relationships among different PRV sequence types present in samples from salmonids in Western Canada and the US, including Alaska (US), British Columbia (Canada) and Washington State (US). Tissues testing positive for PRV were partially sequenced for segment S1, producing 71 sequences that grouped into 10 unique sequence types. Sequence analysis revealed no identifiable geographical or temporal variation among the sequence types. Identical sequence types were found in fish sampled in 2001, 2005 and 2014. In addition, PRV positive samples from fish derived from Alaska, British Columbia and Washington State share identical sequence types. Comparative analysis of the phylogenetic tree indicated that Canada/US Pacific Northwest sequences formed a subgroup with some Norwegian sequence types (group II), distinct from other Norwegian and Chilean sequences (groups I, III and IV). Representative PRV positive samples from farmed and wild fish in British Columbia and Washington State were subjected to genome sequencing using next generation sequencing methods. Individual analysis of each of the 10 partial segments indicated that the Canadian and US PRV sequence types clustered separately from available whole genome sequences of some Norwegian and Chilean sequences for all segments except the segment S4. In summary, PRV was genetically homogenous over a large geographic distance (Alaska to Washington State), and the sequence types were relatively stable over a 13 year period.

Piscine Reovirus: Genomic and Molecular Phylogenetic Analysis from Farmed and Wild Salmonids Collected on the Canada/US Pacific Coast

PubMed Central

Siah, Ahmed; Morrison, Diane B.; Fringuelli, Elena; Savage, Paul; Richmond, Zina; Johns, Robert; Purcell, Maureen K.; Johnson, Stewart C.; Saksida, Sonja M.

2015-01-01

Piscine reovirus (PRV) is a double stranded non-enveloped RNA virus detected in farmed and wild salmonids. This study examined the phylogenetic relationships among different PRV sequence types present in samples from salmonids in Western Canada and the US, including Alaska (US), British Columbia (Canada) and Washington State (US). Tissues testing positive for PRV were partially sequenced for segment S1, producing 71 sequences that grouped into 10 unique sequence types. Sequence analysis revealed no identifiable geographical or temporal variation among the sequence types. Identical sequence types were found in fish sampled in 2001, 2005 and 2014. In addition, PRV positive samples from fish derived from Alaska, British Columbia and Washington State share identical sequence types. Comparative analysis of the phylogenetic tree indicated that Canada/US Pacific Northwest sequences formed a subgroup with some Norwegian sequence types (group II), distinct from other Norwegian and Chilean sequences (groups I, III and IV). Representative PRV positive samples from farmed and wild fish in British Columbia and Washington State were subjected to genome sequencing using next generation sequencing methods. Individual analysis of each of the 10 partial segments indicated that the Canadian and US PRV sequence types clustered separately from available whole genome sequences of some Norwegian and Chilean sequences for all segments except the segment S4. In summary, PRV was genetically homogenous over a large geographic distance (Alaska to Washington State), and the sequence types were relatively stable over a 13 year period. PMID:26536673

Roseomonas aerofrigidensis sp. nov., isolated from an air conditioner.

PubMed

Hyeon, Jong Woo; Jeon, Che Ok

2017-10-01

A Gram-stain-negative, strictly aerobic bacterium, designated HC1 T , was isolated from an air conditioner in South Korea. Cells were orange, non-motile cocci with oxidase- and catalase-positive activities and did not contain bacteriochlorophyll a. Growth of strain HC1 T was observed at 10-45 °C (optimum, 30 °C), pH 4.5-9.5 (optimum, pH 7.0) and 0-3 % (w/v) NaCl (optimum, 0 %). Strain HC1 T contained summed feature 8 (comprising C18 : 1ω7c/C18 : 1ω6c), C16 : 0 and cyclo-C19 : 0ω8c as the major fatty acids and ubiquinone-10 as the sole isoprenoid quinone. Phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and an unknown aminolipid were detected as the major polar lipids. The major carotenoid was hydroxyspirilloxanthin. The G+C content of the genomic DNA was 70.1 mol%. Phylogenetic analysis, based on 16S rRNA gene sequences, showed that strain HC1 T formed a phylogenetic lineage within the genus Roseomonas. Strain HC1 T was most closely related to the type strains of Roseomonas oryzae, Roseomonas rubra, Roseomonas aestuarii and Roseomonas rhizosphaerae with 98.1, 97.9, 97.6 and 96.8 % 16S rRNA gene sequence similarities, respectively, but the DNA-DNA relatedness values between strain HC1 T and closely related type strains were less than 70 %. Based on phenotypic, chemotaxonomic and molecular properties, strain HC1 T represents a novel species of the genus Roseomonas, for which the name Roseomonas aerofrigidensis sp. nov. is proposed. The type strain is HC1 T (=KACC 19097 T =JCM 31878 T ).

“Epidemic Clones” of Listeria monocytogenes Are Widespread and Ancient Clonal Groups

PubMed Central

Cantinelli, Thomas; Chenal-Francisque, Viviane; Diancourt, Laure; Frezal, Lise; Leclercq, Alexandre; Wirth, Thierry

2013-01-01

The food-borne pathogen Listeria monocytogenes is genetically heterogeneous. Although some clonal groups have been implicated in multiple outbreaks, there is currently no consensus on how “epidemic clones” should be defined. The objectives of this work were to compare the patterns of sequence diversity on two sets of genes that have been widely used to define L. monocytogenes clonal groups: multilocus sequence typing (MLST) and multi-virulence-locus sequence typing (MvLST). Further, we evaluated the diversity within clonal groups by pulsed-field gel electrophoresis (PFGE). Based on 125 isolates of diverse temporal, geographical, and source origins, MLST and MvLST genes (i) had similar patterns of sequence polymorphisms, recombination, and selection, (ii) provided concordant phylogenetic clustering, and (iii) had similar discriminatory power, which was not improved when we combined both data sets. Inclusion of representative strains of previous outbreaks demonstrated the correspondence of epidemic clones with previously recognized MLST clonal complexes. PFGE analysis demonstrated heterogeneity within major clones, most of which were isolated decades before their involvement in outbreaks. We conclude that the “epidemic clone” denominations represent a redundant but largely incomplete nomenclature system for MLST-defined clones, which must be regarded as successful genetic groups that are widely distributed across time and space. PMID:24006010

Molecular epidemiology demonstrated three emerging clusters of human immunodeficiency virus type 1 subtype B infection in Hong Kong.

PubMed

Leung, Tommy W C; Mak, Darwin; Wong, K H; Wang, Y; Song, Y H; Tsang, D N C; Wong, C; Shao, Y M; Lim, W L

2008-07-01

We conducted a molecular epidemiological study on newly diagnosed human immunodeficiency virus type 1 (HIV-1)-infected patients in Hong Kong to identify the epidemiological linkage of HIV-1 infection in the locality. Reverse transcription polymerase chain reaction (RT-PCR) for HIV-1 was performed on newly diagnosed HIV-1-positive sera collected from January 2002 to December 2006. PCR products correspond to the env C2V3V4 region and gag p17/p24 junction of the HIV-1 genome were nucleotide sequenced. Phylogenetic analyses performed on the acquired nucleotide sequences revealed that CRF01_AE and subtype B were the two dominant HIV-1 subtypes. Analyses also demonstrated the presence of three emerging HIV-1 clusters among the subtype B sequences in Hong Kong. Individual cluster possesses a unique cluster-specific amino acid signature for identification. Data show that one of the clusters (Cluster I) is rapidly expanding. In addition to the unique cluster-specific amino acid signature, the majority of sequences in Cluster I harbor a 6-amino acid insertion at the gag p17/p24 junction in a region that is thought to be closely associated with HIV-1 infectivity.

AmericaPlex26: A SNaPshot Multiplex System for Genotyping the Main Human Mitochondrial Founder Lineages of the Americas

PubMed Central

Coutinho, Alexandra; Valverde, Guido; Fehren-Schmitz, Lars; Cooper, Alan; Barreto Romero, Maria Inés; Espinoza, Isabel Flores; Llamas, Bastien; Haak, Wolfgang

2014-01-01

Phylogeographic studies have described a reduced genetic diversity in Native American populations, indicative of one or more bottleneck events during the peopling and prehistory of the Americas. Classical sequencing approaches targeting the mitochondrial diversity have reported the presence of five major haplogroups, namely A, B, C, D and X, whereas the advent of complete mitochondrial genome sequencing has recently refined the number of founder lineages within the given diversity to 15 sub-haplogroups. We developed and optimized a SNaPshot assay to study the mitochondrial diversity in pre-Columbian Native American populations by simultaneous typing of 26 single nucleotide polymorphisms (SNPs) characterising Native American sub-haplogroups. Our assay proved to be highly sensitive with respect to starting concentrations of target DNA and could be applied successfully to a range of ancient human skeletal material from South America from various time periods. The AmericaPlex26 is a powerful assay with enhanced phylogenetic resolution that allows time- and cost-efficient mitochondrial DNA sub-typing from valuable ancient specimens. It can be applied in addition or alternative to standard sequencing of the D-loop region in forensics, ancestry testing, and population studies, or where full-resolution mitochondrial genome sequencing is not feasible. PMID:24671218

AmericaPlex26: a SNaPshot multiplex system for genotyping the main human mitochondrial founder lineages of the Americas.

PubMed

Coutinho, Alexandra; Valverde, Guido; Fehren-Schmitz, Lars; Cooper, Alan; Barreto Romero, Maria Inés; Espinoza, Isabel Flores; Llamas, Bastien; Haak, Wolfgang

2014-01-01

Phylogeographic studies have described a reduced genetic diversity in Native American populations, indicative of one or more bottleneck events during the peopling and prehistory of the Americas. Classical sequencing approaches targeting the mitochondrial diversity have reported the presence of five major haplogroups, namely A, B, C, D and X, whereas the advent of complete mitochondrial genome sequencing has recently refined the number of founder lineages within the given diversity to 15 sub-haplogroups. We developed and optimized a SNaPshot assay to study the mitochondrial diversity in pre-Columbian Native American populations by simultaneous typing of 26 single nucleotide polymorphisms (SNPs) characterising Native American sub-haplogroups. Our assay proved to be highly sensitive with respect to starting concentrations of target DNA and could be applied successfully to a range of ancient human skeletal material from South America from various time periods. The AmericaPlex26 is a powerful assay with enhanced phylogenetic resolution that allows time- and cost-efficient mitochondrial DNA sub-typing from valuable ancient specimens. It can be applied in addition or alternative to standard sequencing of the D-loop region in forensics, ancestry testing, and population studies, or where full-resolution mitochondrial genome sequencing is not feasible.

GALACTIC ANGULAR MOMENTUM IN THE ILLUSTRIS SIMULATION: FEEDBACK AND THE HUBBLE SEQUENCE

DOE Office of Scientific and Technical Information (OSTI.GOV)

Genel, Shy; Fall, S. Michael; Snyder, Gregory F.

We study the stellar angular momentum of thousands of galaxies in the Illustris cosmological simulation, which captures gravitational and gas dynamics within galaxies, as well as feedback from stars and black holes. We find that the angular momentum of the simulated galaxies matches observations well, and in particular two distinct relations are found for late-type versus early-type galaxies. The relation for late-type galaxies corresponds to the value expected from full conservation of the specific angular momentum generated by cosmological tidal torques. The relation for early-type galaxies corresponds to retention of only ∼30% of that, but we find that those early-typemore » galaxies with low angular momentum at z = 0 nevertheless reside at high redshift on the late-type relation. Some of them abruptly lose angular momentum during major mergers. To gain further insight, we explore the scaling relations in simulations where the galaxy formation physics is modified with respect to the fiducial model. We find that galactic winds with high mass-loading factors are essential for obtaining the high angular momentum relation typical for late-type galaxies, while active galactic nucleus feedback largely operates in the opposite direction. Hence, feedback controls the stellar angular momentum of galaxies, and appears to be instrumental for establishing the Hubble sequence.« less

Staphylococcus aureus activates type I IFN signaling in mice and humans through the Xr repeated sequences of protein A

PubMed Central

Martin, Francis J.; Gomez, Marisa I.; Wetzel, Dawn M.; Memmi, Guido; O’Seaghdha, Maghnus; Soong, Grace; Schindler, Christian; Prince, Alice

2009-01-01

The activation of type I IFN signaling is a major component of host defense against viral infection, but it is not typically associated with immune responses to extracellular bacterial pathogens. Using mouse and human airway epithelial cells, we have demonstrated that Staphylococcus aureus activates type I IFN signaling, which contributes to its virulence as a respiratory pathogen. This response was dependent on the expression of protein A and, more specifically, the Xr domain, a short sequence–repeat region encoded by DNA that consists of repeated 24-bp sequences that are the basis of an internationally used epidemiological typing scheme. Protein A was endocytosed by airway epithelial cells and subsequently induced IFN-β expression, JAK-STAT signaling, and IL-6 production. Mice lacking IFN-α/β receptor 1 (IFNAR-deficient mice), which are incapable of responding to type I IFNs, were substantially protected against lethal S. aureus pneumonia compared with wild-type control mice. The profound immunological consequences of IFN-β signaling, particularly in the lung, may help to explain the conservation of multiple copies of the Xr domain of protein A in S. aureus strains and the importance of protein A as a virulence factor in the pathogenesis of staphylococcal pneumonia. PMID:19603548

Aestuariispira insulae gen. nov., sp. nov., a lipolytic bacterium isolated from a tidal flat.

PubMed

Park, Sooyeon; Park, Ji-Min; Kang, Chul-Hyung; Yoon, Jung-Hoon

2014-06-01

A Gram-stain-negative, non-motile, aerobic, curved-to-spiral-rod-shaped bacterium, designated AH-MY2(T), was isolated from a tidal flat on Aphae island in the sea to the south-west of South Korea, and its taxonomic position was investigated using a polyphasic taxonomic approach. Strain AH-MY2(T) grew optimally at 30 °C, at pH 7.0-8.0 and in the presence of 2.0% (w/v) NaCl. Neighbour-joining, maximum-likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences showed that strain AH-MY2(T) clustered with the type strain of Terasakiella pusilla and that this cluster joined the clade comprising the type strains of species of the genus Thalassospira. Strain AH-MY2(T) exhibited 16S rRNA gene sequence similarity values of 90.6% to the type strain of Terasakiella pusilla and of less than 91.0% to the type strains of other species with validly published names. Strain AH-MY2(T) contained Q-10 as the predominant ubiquinone and C(18 : 1)ω7c as the major fatty acid. The major polar lipids detected in strain AH-MY2(T) were phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminolipids and one unidentified glycolipid. The DNA G+C content of strain AH-MY2(T) was 56.0 mol%. The phylogenetic data and differential chemotaxonomic and other phenotypic properties revealed that strain AH-MY2(T) represented a novel genus and species within the family Rhodospirillaceae of the class Alphaproteobacteria, for which the name Aestuariispira insulae gen. nov., sp. nov. is proposed. The type strain of Aestuariispira insulae is AH-MY2(T) ( = KCTC 32577(T) = CECT 8488(T)). © 2014 IUMS.

DeepGene: an advanced cancer type classifier based on deep learning and somatic point mutations.

PubMed

Yuan, Yuchen; Shi, Yi; Li, Changyang; Kim, Jinman; Cai, Weidong; Han, Zeguang; Feng, David Dagan

2016-12-23

With the developments of DNA sequencing technology, large amounts of sequencing data have become available in recent years and provide unprecedented opportunities for advanced association studies between somatic point mutations and cancer types/subtypes, which may contribute to more accurate somatic point mutation based cancer classification (SMCC). However in existing SMCC methods, issues like high data sparsity, small volume of sample size, and the application of simple linear classifiers, are major obstacles in improving the classification performance. To address the obstacles in existing SMCC studies, we propose DeepGene, an advanced deep neural network (DNN) based classifier, that consists of three steps: firstly, the clustered gene filtering (CGF) concentrates the gene data by mutation occurrence frequency, filtering out the majority of irrelevant genes; secondly, the indexed sparsity reduction (ISR) converts the gene data into indexes of its non-zero elements, thereby significantly suppressing the impact of data sparsity; finally, the data after CGF and ISR is fed into a DNN classifier, which extracts high-level features for accurate classification. Experimental results on our curated TCGA-DeepGene dataset, which is a reformulated subset of the TCGA dataset containing 12 selected types of cancer, show that CGF, ISR and DNN all contribute in improving the overall classification performance. We further compare DeepGene with three widely adopted classifiers and demonstrate that DeepGene has at least 24% performance improvement in terms of testing accuracy. Based on deep learning and somatic point mutation data, we devise DeepGene, an advanced cancer type classifier, which addresses the obstacles in existing SMCC studies. Experiments indicate that DeepGene outperforms three widely adopted existing classifiers, which is mainly attributed to its deep learning module that is able to extract the high level features between combinatorial somatic point mutations and cancer types.

Livestock-Associated Methicillin Resistant and Methicillin Susceptible Staphylococcus aureus Sequence Type (CC)1 in European Farmed Animals: High Genetic Relatedness of Isolates from Italian Cattle Herds and Humans.

PubMed

Alba, Patricia; Feltrin, Fabiola; Cordaro, Gessica; Porrero, María Concepción; Kraushaar, Britta; Argudín, María Angeles; Nykäsenoja, Suvi; Monaco, Monica; Stegger, Marc; Aarestrup, Frank M; Butaye, Patrick; Franco, Alessia; Battisti, Antonio

2015-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) Sequence Type (ST)1, Clonal Complex(CC)1, SCCmec V is one of the major Livestock-Associated (LA-) lineages in pig farming industry in Italy and is associated with pigs in other European countries. Recently, it has been increasingly detected in Italian dairy cattle herds. The aim of this study was to analyse the differences between ST1 MRSA and methicillin-susceptible S. aureus (MSSA) from cattle and pig herds in Italy and Europe and human isolates. Sixty-tree animal isolates from different holdings and 20 human isolates were characterized by pulsed-field gel electrophoresis (PFGE), spa-typing, SCCmec typing, and by micro-array analysis for several virulence, antimicrobial resistance, and strain/host-specific marker genes. Three major PFGE clusters were detected. The bovine isolates shared a high (≥90% to 100%) similarity with human isolates and carried the same SCCmec type IVa. They often showed genetic features typical of human adaptation or present in human-associated CC1: Immune evasion cluster (IEC) genes sak and scn, or sea; sat and aphA3-mediated aminoglycoside resistance. Contrary, typical markers of porcine origin in Italy and Spain, like erm(A) mediated macrolide-lincosamide-streptograminB, and of vga(A)-mediated pleuromutilin resistance were always absent in human and bovine isolates. Most of ST(CC)1 MRSA from dairy cattle were multidrug-resistant and contained virulence and immunomodulatory genes associated with full capability of colonizing humans. As such, these strains may represent a greater human hazard than the porcine strains. The zoonotic capacity of CC1 LA-MRSA from livestock must be taken seriously and measures should be implemented at farm-level to prevent spill-over.

Cross-Laboratory Analysis of Brain Cell Type Transcriptomes with Applications to Interpretation of Bulk Tissue Data

PubMed Central

Toker, Lilah; Rocco, Brad; Sibille, Etienne

2017-01-01

Establishing the molecular diversity of cell types is crucial for the study of the nervous system. We compiled a cross-laboratory database of mouse brain cell type-specific transcriptomes from 36 major cell types from across the mammalian brain using rigorously curated published data from pooled cell type microarray and single-cell RNA-sequencing (RNA-seq) studies. We used these data to identify cell type-specific marker genes, discovering a substantial number of novel markers, many of which we validated using computational and experimental approaches. We further demonstrate that summarized expression of marker gene sets (MGSs) in bulk tissue data can be used to estimate the relative cell type abundance across samples. To facilitate use of this expanding resource, we provide a user-friendly web interface at www.neuroexpresso.org. PMID:29204516

Jointly characterizing epigenetic dynamics across multiple human cell types

PubMed Central

An, Lin; Yue, Feng; Hardison, Ross C

2016-01-01

Advanced sequencing technologies have generated a plethora of data for many chromatin marks in multiple tissues and cell types, yet there is lack of a generalized tool for optimal utility of those data. A major challenge is to quantitatively model the epigenetic dynamics across both the genome and many cell types for understanding their impacts on differential gene regulation and disease. We introduce IDEAS, an integrative and discriminative epigenome annotation system, for jointly characterizing epigenetic landscapes in many cell types and detecting differential regulatory regions. A key distinction between our method and existing state-of-the-art algorithms is that IDEAS integrates epigenomes of many cell types simultaneously in a way that preserves the position-dependent and cell type-specific information at fine scales, thereby greatly improving segmentation accuracy and producing comparable annotations across cell types. PMID:27095202

Mipartoxin-I, a novel three-finger toxin, is the major neurotoxic component in the venom of the redtail coral snake Micrurus mipartitus (Elapidae).

PubMed

Rey-Suárez, Paola; Floriano, Rafael Stuani; Rostelato-Ferreira, Sandro; Saldarriaga-Córdoba, Mónica; Núñez, Vitelbina; Rodrigues-Simioni, Léa; Lomonte, Bruno

2012-10-01

The major venom component of Micrurus mipartitus, a coral snake distributed from Nicaragua to northern South America, was characterized biochemically and functionally. This protein, named mipartoxin-I, is a novel member of the three-finger toxin superfamily, presenting the characteristic cysteine signature and amino acid sequence length of the short-chain, type-I, α-neurotoxins. Nevertheless, it varies considerably from related toxins, with a sequence identity not higher than 70% in a multiple alignment of 67 proteins within this family. Its observed molecular mass (7030.0) matches the value predicted by its amino acid sequence, indicating lack of post-translational modifications. Mipartoxin-I showed a potent lethal effect in mice (intraperitoneal median lethal dose: 0.06 μg/g body weight), and caused a clear neuromuscular blockade on both avian and mouse nerve-muscle preparations, presenting a post-synaptic action through the cholinergic nicotinic receptor. Since mipartoxin-I is the most abundant (28%) protein in M. mipartitus venom, it should play a major role in its toxicity, and therefore represents an important target for developing a therapeutic antivenom, which is very scarce or even unavailable in the regions where this snake inhabits. The structural information here provided might help in the preparation of a synthetic or recombinant immunogen to overcome the limited venom availability. Copyright © 2012 Elsevier Ltd. All rights reserved.

Serratia aquatilis sp. nov., isolated from drinking water systems.

PubMed

Kämpfer, Peter; Glaeser, Stefanie P

2016-01-01

A cream-white-pigmented, oxidase-negative bacterium (strain 2015-2462-01T), isolated from a drinking water system, was investigated in detail to determine its taxonomic position. Cells of the isolate were rod-shaped and stained Gram-negative. A comparison of the 16S rRNA gene sequence of strain 2015-2462-01T with sequences of the type strains of closely related species of the genus Serratia revealed highest similarity to Serratia fonticola (98.4 %), Serratia proteamaculans (97.8 %), Serratia liquefaciens and Serratia grimesii (both 97.7 %). 16S rRNA gene sequence similarities to all other Serratia species were below 97.4 %. Multilocus sequence analysis (MLSA) on the basis of concatenated partial gyrB, rpoB, infB and atpD gene sequences showed a clear distinction of strain 2015-2462-01T from the type strains of the closest related Serratia species. The fatty acid profile of the strain consisted of C16 : 1 ω7c, C16 : 0; C14 : 0 and C14 : 0 3-OH/iso-C16 : 1 I as major components. DNA-DNA hybridizations between 2015-2462-01T and S. fonticola ATCC 29844T resulted in a relatedness value of 27 % (reciprocal 20 %). This DNA-DNA hybridization result in combination with the MLSA results and the differential biochemical properties indicated that strain 2015-2462-01T represents a novel species of the genus Serratia, for which the name Serratia aquatilis sp. nov. is proposed. The type strain is 2015-2462-01T ( = LMG 29119T = CCM 8626T).

Prevalence, Molecular Characterization, and Antibiotic Susceptibility of Cronobacter sakazakii Isolates from Powdered Infant Formula Collected from Chinese Retail Markets

PubMed Central

Fei, Peng; Jiang, Yichao; Jiang, Yan; Yuan, Xiujuan; Yang, Tongxiang; Chen, Junliang; Wang, Ziyuan; Kang, Huaibin; Forsythe, Stephen J.

2017-01-01

Cronobacter sakazakii is an opportunistic pathogen that causes severe infections in neonates and infants through contaminated powdered infant formula (PIF). Therefore, the aim of this study was a large-scale study on determine the prevalence, molecular characterization and antibiotic susceptibility of C. sakazakii isolates from PIF purchased from Chinese retail markets. Two thousand and twenty PIF samples were collected from different institutions. Fifty-six C. sakazakii strains were isolated, and identified using fusA sequencing analysis, giving a contamination rate of 2.8%. Multilocus sequence typing (MLST) was more discriminatory than other genotyping methods. The C. sakazakii isolates were divided into 14 sequence types (STs) by MLST, compared with only seven clusters by ompA and rpoB sequence analysis, and four C. sakazakii serotypes by PCR-based O-antigen serotyping. C. sakazakii ST4 (19/56, 33.9%), ST1 (12/56, 21.4%), and ST64 (11/56, 16.1%) were the dominant sequence types isolated. C. sakazakii serotype O2 (34/56, 60.7%) was the primary serotype, along with ompA6 and rpoB1 as the main allele profiles, respectively. Antibiotic susceptibility testing indicated that all C. sakazakii isolates were susceptible to ampicillin-sulbactam, cefotaxime, ciprofloxacin, meropenem, tetracycline, piperacillin-tazobactam, and trimethoprim-sulfamethoxazole. The majority of C. sakazakii strains were susceptible to chloramphenicol and gentamicin (87.5 and 92.9%, respectively). In contrast, 55.4% C. sakazakii strains were resistant to cephalothin. In conclusion, this large-scale study revealed the prevalence and characteristics of C. sakazakii from PIF in Chinese retail markets, demonstrating a potential risk for neonates and infants, and provide a guided to effective control the contamination of C. sakazakii in production process. PMID:29089940

Phylogenetic and environmental diversity of DsrAB-type dissimilatory (bi)sulfite reductases

PubMed Central

Müller, Albert Leopold; Kjeldsen, Kasper Urup; Rattei, Thomas; Pester, Michael; Loy, Alexander

2015-01-01

The energy metabolism of essential microbial guilds in the biogeochemical sulfur cycle is based on a DsrAB-type dissimilatory (bi)sulfite reductase that either catalyzes the reduction of sulfite to sulfide during anaerobic respiration of sulfate, sulfite and organosulfonates, or acts in reverse during sulfur oxidation. Common use of dsrAB as a functional marker showed that dsrAB richness in many environments is dominated by novel sequence variants and collectively represents an extensive, largely uncharted sequence assemblage. Here, we established a comprehensive, manually curated dsrAB/DsrAB database and used it to categorize the known dsrAB diversity, reanalyze the evolutionary history of dsrAB and evaluate the coverage of published dsrAB-targeted primers. Based on a DsrAB consensus phylogeny, we introduce an operational classification system for environmental dsrAB sequences that integrates established taxonomic groups with operational taxonomic units (OTUs) at multiple phylogenetic levels, ranging from DsrAB enzyme families that reflect reductive or oxidative DsrAB types of bacterial or archaeal origin, superclusters, uncultured family-level lineages to species-level OTUs. Environmental dsrAB sequences constituted at least 13 stable family-level lineages without any cultivated representatives, suggesting that major taxa of sulfite/sulfate-reducing microorganisms have not yet been identified. Three of these uncultured lineages occur mainly in marine environments, while specific habitat preferences are not evident for members of the other 10 uncultured lineages. In summary, our publically available dsrAB/DsrAB database, the phylogenetic framework, the multilevel classification system and a set of recommended primers provide a necessary foundation for large-scale dsrAB ecology studies with next-generation sequencing methods. PMID:25343514

Identification and Characterization of 30 K Protein Genes Found in Bombyx mori (Lepidoptera: Bombycidae) Transcriptome

PubMed Central

Shi, Xiao-Feng; Li, Yi-Nü; Yi, Yong-Zhu; Xiao, Xing-Guo; Zhang, Zhi-Fang

2015-01-01

The 30 K proteins, the major group of hemolymph proteins in the silkworm, Bombyx mori (Lepidoptera: Bombycidae), are structurally related with molecular masses of ∼30 kDa and are involved in various physiological processes, e.g., energy storage, embryonic development, and immune responses. For this report, known 30 K protein gene sequences were used as Blastn queries against sequences in the B. mori transcriptome (SilkTransDB). Twenty-nine cDNAs (Bm30K-1–29) were retrieved, including four being previously unidentified in the Lipoprotein_11 family. The genomic structures of the 29 genes were analyzed and they were mapped to their corresponding chromosomes. Furthermore, phylogenetic analysis revealed that the 29 genes encode three types of 30 K proteins. The members increased in each type is mainly a result of gene duplication with the appearance of each type preceding the differentiation of each species included in the tree. Real-Time Quantitative Polymerase Chain Reaction (Q-PCR) confirmed that the genes could be expressed, and that the three types have different temporal expression patterns. Proteins from the hemolymph was separated by SDS-PAGE, and those with molecular mass of ∼30 kDa were isolated and identified by mass spectrometry sequencing in combination with searches of various databases containing B. mori 30K protein sequences. Of the 34 proteins identified, 13 are members of the 30 K protein family, with one that had not been found in the SilkTransDB, although it had been found in the B. mori genome. Taken together, our results indicate that the 30 K protein family contains many members with various functions. Other methods will be required to find more members of the family. PMID:26078299

Presence and mechanisms of acquired antimicrobial resistance in Belgian Brachyspira hyodysenteriae isolates belonging to different clonal complexes.

PubMed

Mahu, M; Pasmans, F; Vranckx, K; De Pauw, N; Vande Maele, L; Vyt, Philip; Vandersmissen, Tamara; Martel, A; Haesebrouck, F; Boyen, F

2017-08-01

Swine dysentery (SD) is an economically important disease for which antimicrobial treatment still occupies an important place to control outbreaks. However, acquired antimicrobial resistance is increasingly observed in Brachyspira hyodysenteriae. In this study, the Minimal Inhibitory Concentrations (MIC) of six antimicrobial compounds for 30 recent Belgian B. hyodysenteriae isolates were determined using a broth microdilution method. In addition, relevant regions of the 16S rRNA, 23S rRNA and the L3 protein encoding genes were sequenced to reveal mutations associated with acquired resistance. Finally, a phylogeny was reconstructed using minimal spanning tree analysis of multi locus sequence typing of the isolates. For lincomycin, doxycycline, tylosin and tylvalosin, at least 70% of the isolates did not belong to the wild-type population and were considered to have acquired resistance. For valnemulin and tiamulin, this was over 50%. In all isolates with acquired resistance to doxycycline, the G1058C mutation was present in their 16S rRNA gene. All isolates showing acquired resistance to lincomycin and both macrolides displayed the A2058T mutation in their 23S rRNA gene. Other mutations in this gene and the N148S mutation in the L3 protein were present in both wild-type isolates and isolates considered to have acquired resistance. Multi locus sequence analysis revealed a previously undescribed clonal complex, with 4 novel sequence types in which the majority of isolates showed acquired resistance to all tested antimicrobial products. In conclusion, acquired antimicrobial resistance is widespread among Belgian B. hyodysenteriae isolates. The emergence of multi-resistant clonal complexes can pose a threat to swine industry. Copyright © 2017 Elsevier B.V. All rights reserved.

Frequency of papillary tubal hyperplasia (PTH), salpingoliths and transition from adenoma to borderline ovarian tumors (BOT): A systematic analysis of 74 BOT with different histologic types.

PubMed

Horn, Lars-Christian; Angermann, Karolin; Hentschel, Bettina; Einenkel, Jens; Höhn, Anne Kathrin

2017-04-01

Borderline ovarian tumors (BOT) arise from cystadenomas and represent a transition step within the development of low-grade ovarian carcinomas (Type I tumors). That pathway mirrors the adenoma-to-carcinoma sequence known for colorectal cancer. It has been suggested that papillary tubal hyperplasia (PTH) and salpingoliths may be associated with the development of BOT. To evaluate the frequency of the presence of benign cystadenoma and its transition to BOT in a given patient as well as the presence of PTH and salpingoliths we re-valuated in 74 consecutive cases of BOT with different histologic types. The majority of cases represented serous-BOT (60.8%), followed by mucinous BOT (25.7%), other histologic types were rare. 86.5% showed an adenoma-BOT sequence, which was seen in all mucinous BOT but was missed in 15.6% of serous BOT. Two cases had salpingoliths without associated PTH. PTH was seen in four out of the 74 (5.4%) BOT and occurred only in cases with serous histology. The vast majority of BOT represent a transition from benign cystadenoma to BOT in cases with mucinous and serous histology. Salpingoliths are rarely seen in association with BOT and occurred exclusively in BOT with serous histology. PTH may represent a distinct lesion but is rarely seen in association with BOT, especially in those with non-serous histology. Further studies are needed to evaluate the frequency and pathogenetic association of PTH with BOT. Copyright © 2017 Elsevier GmbH. All rights reserved.

Complete genome sequence of DSM 30083(T), the type strain (U5/41(T)) of Escherichia coli, and a proposal for delineating subspecies in microbial taxonomy.

PubMed

Meier-Kolthoff, Jan P; Hahnke, Richard L; Petersen, Jörn; Scheuner, Carmen; Michael, Victoria; Fiebig, Anne; Rohde, Christine; Rohde, Manfred; Fartmann, Berthold; Goodwin, Lynne A; Chertkov, Olga; Reddy, Tbk; Pati, Amrita; Ivanova, Natalia N; Markowitz, Victor; Kyrpides, Nikos C; Woyke, Tanja; Göker, Markus; Klenk, Hans-Peter

2014-01-01

Although Escherichia coli is the most widely studied bacterial model organism and often considered to be the model bacterium per se, its type strain was until now forgotten from microbial genomics. As a part of the G enomic E ncyclopedia of B acteria and A rchaea project, we here describe the features of E. coli DSM 30083(T) together with its genome sequence and annotation as well as novel aspects of its phenotype. The 5,038,133 bp containing genome sequence includes 4,762 protein-coding genes and 175 RNA genes as well as a single plasmid. Affiliation of a set of 250 genome-sequenced E. coli strains, Shigella and outgroup strains to the type strain of E. coli was investigated using digital DNA:DNA-hybridization (dDDH) similarities and differences in genomic G+C content. As in the majority of previous studies, results show Shigella spp. embedded within E. coli and in most cases forming a single subgroup of it. Phylogenomic trees also recover the proposed E. coli phylotypes as monophyla with minor exceptions and place DSM 30083(T) in phylotype B2 with E. coli S88 as its closest neighbor. The widely used lab strain K-12 is not only genomically but also physiologically strongly different from the type strain. The phylotypes do not express a uniform level of character divergence as measured using dDDH, however, thus an alternative arrangement is proposed and discussed in the context of bacterial subspecies. Analyses of the genome sequences of a large number of E. coli strains and of strains from > 100 other bacterial genera indicate a value of 79-80% dDDH as the most promising threshold for delineating subspecies, which in turn suggests the presence of five subspecies within E. coli.

Efficient and Accurate Algorithm for Cleaved Fragments Prediction (CFPA) in Protein Sequences Dataset Based on Consensus and Its Variants: A Novel Degradomics Prediction Application.

PubMed

El-Assaad, Atlal; Dawy, Zaher; Nemer, Georges; Hajj, Hazem; Kobeissy, Firas H

2017-01-01

Degradomics is a novel discipline that involves determination of the proteases/substrate fragmentation profile, called the substrate degradome, and has been recently applied in different disciplines. A major application of degradomics is its utility in the field of biomarkers where the breakdown products (BDPs) of different protease have been investigated. Among the major proteases assessed, calpain and caspase proteases have been associated with the execution phases of the pro-apoptotic and pro-necrotic cell death, generating caspase/calpain-specific cleaved fragments. The distinction between calpain and caspase protein fragments has been applied to distinguish injury mechanisms. Advanced proteomics technology has been used to identify these BDPs experimentally. However, it has been a challenge to identify these BDPs with high precision and efficiency, especially if we are targeting a number of proteins at one time. In this chapter, we present a novel bioinfromatic detection method that identifies BDPs accurately and efficiently with validation against experimental data. This method aims at predicting the consensus sequence occurrences and their variants in a large set of experimentally detected protein sequences based on state-of-the-art sequence matching and alignment algorithms. After detection, the method generates all the potential cleaved fragments by a specific protease. This space and time-efficient algorithm is flexible to handle the different orientations that the consensus sequence and the protein sequence can take before cleaving. It is O(mn) in space complexity and O(Nmn) in time complexity, with N number of protein sequences, m length of the consensus sequence, and n length of each protein sequence. Ultimately, this knowledge will subsequently feed into the development of a novel tool for researchers to detect diverse types of selected BDPs as putative disease markers, contributing to the diagnosis and treatment of related disorders.

DNA extraction for streamlined metagenomics of diverse environmental samples.

PubMed

Marotz, Clarisse; Amir, Amnon; Humphrey, Greg; Gaffney, James; Gogul, Grant; Knight, Rob

2017-06-01

A major bottleneck for metagenomic sequencing is rapid and efficient DNA extraction. Here, we compare the extraction efficiencies of three magnetic bead-based platforms (KingFisher, epMotion, and Tecan) to a standardized column-based extraction platform across a variety of sample types, including feces, oral, skin, soil, and water. Replicate sample plates were extracted and prepared for 16S rRNA gene amplicon sequencing in parallel to assess extraction bias and DNA quality. The data demonstrate that any effect of extraction method on sequencing results was small compared with the variability across samples; however, the KingFisher platform produced the largest number of high-quality reads in the shortest amount of time. Based on these results, we have identified an extraction pipeline that dramatically reduces sample processing time without sacrificing bacterial taxonomic or abundance information.

The Three Major Spanish Clones of Penicillin-Resistant Streptococcus pneumoniae Are the Most Common Clones Recovered in Recent Cases of Meningitis in Spain

PubMed Central

Enright, Mark C.; Fenoll, Asunción; Griffiths, David; Spratt, Brian G.

1999-01-01

One hundred six isolates of Streptococcus pneumoniae recovered in Spain from patients with meningitis in 1997 and 1998 were characterized by multilocus sequence typing. A heterogeneous collection of genotypes was associated with meningitis in Spain: 65 different sequence types were resolved and, even at a genetic distance of 0.43, there were 37 distinct lineages. Thirty-eight percent of the isolates, including all isolates of serotypes 6B, 9V, 14, and 23F, were resistant to penicillin, and 24% of the isolates were members of the three major Spanish penicillin-resistant or multidrug-resistant clones of serotypes 6B, 9V, and 23F or serotype variants of these clones. These three clones (MICs, 1 to 2 μg of penicillin/ml) were the most common clones associated with pneumococcal meningitis in Spain during 1997 and 1998. Only two of the other clones associated with meningitis were penicillin resistant (MICs, 0.12 to 0.5 μg/ml). One of the two most prevalent penicillin-susceptible clones causing meningitis (serotype 3) has not been detected outside of Spain, whereas the other (serotype 18C) has been recovered from patients with meningitis in the United Kingdom, The Netherlands, and Denmark. The prevalence of meningitis caused by isolates of the three major Spanish penicillin-resistant or multiply antibiotic-resistant clones, which are now globally distributed, is disturbing and clearly establishes their ability to cause life-threatening disease. PMID:10488179

Degradation of methyl bromide and methyl chloride in soil microcosms: Use of stable C isotope fractionation and stable isotope probing to identify reactions and the responsible microorganisms

USGS Publications Warehouse

Miller, L.G.; Warner, K.L.; Baesman, S.M.; Oremland, R.S.; McDonald, I.R.; Radajewski, S.; Murrell, J.C.

2004-01-01

Bacteria in soil microcosm experiments oxidized elevated levels of methyl chloride (MeCl) and methyl bromide (MeBr), the former compound more rapidly than the latter. MeBr was also removed by chemical reactions while MeCl was not. Chemical degradation dominated the early removal of MeBr and accounted for more than half of its total loss. Fractionation of stable carbon isotopes during chemical degradation of MeBr resulted in a kinetic isotope effect (KIE) of 59 ?? 7???. Soil bacterial oxidation dominated the later removal of MeBr and MeCl and was characterized by different KIEs for each compound. The KIE for MeBr oxidation was 69 ?? 9??? and the KIE for MeCl oxidation was 49 ?? 3???. Stable isotope probing revealed that different populations of soil bacteria assimilated added 13C-labeled MeBr and MeCl. The identity of the active MeBr and MeCl degrading bacteria in soil was determined by analysis of 16S rRNA gene sequences amplified from 13C-DNA fractions, which identified a number of sequences from organisms not previously thought to be involved in methyl halide degradation. These included Burkholderia , the major clone type in the 13C-MeBr fraction, and Rhodobacter, Lysobacter and Nocardioides the major clone types in the 13C-MeCl fraction. None of the 16S rRNA gene sequences for methyl halide oxidizing bacteria currently in culture (including Aminobacter strain IMB-1 isolated from fumigated soil) were identified. Functional gene clone types closely related to Aminobacter spp. were identified in libraries containing the sequences for the cmuA gene, which codes for the enzyme known to catalyze the initial step in the oxidation of MeBr and MeCl. The cmuA gene was limited to members of the alpha-Proteobacteria whereas the greater diversity demonstrated by the 16S rRNA gene may indicate that other enzymes catalyze methyl halide oxidation in different groups of bacteria. Copyright ?? 2004 Elsevier Ltd.

Francisella guangzhouensis sp. nov., isolated from air-conditioning systems.

PubMed

Qu, Ping-Hua; Chen, Shou-Yi; Scholz, Holger C; Busse, Hans-Jürgen; Gu, Quan; Kämpfer, Peter; Foster, Jeffrey T; Glaeser, Stefanie P; Chen, Cha; Yang, Zhi-Chong

2013-10-01

Four strains (08HL01032(T), 09HG994, 10HP82-6 and 10HL1960) were isolated from water of air-conditioning systems of various cooling towers in Guangzhou city, China. Cells were Gram-stain-negative coccobacilli without flagella, catalase-positive and oxidase-negative, showing no reduction of nitrate, no hydrolysis of urea and no production of H2S. Growth was characteristically enhanced in the presence of l-cysteine, which was consistent with the properties of members of the genus Francisella. The quinone system was composed of ubiquinone Q-8 with minor amounts of Q-9. The polar lipid profile consisted of the predominant lipids phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, two unidentified phospholipids (PL2, PL3), an unidentified aminophospholipid and an unidentified glycolipid (GL2). The polyamine pattern consisted of the major compounds spermidine, cadaverine and spermine. The major cellular fatty acids were C10 : 0, C14 : 0, C16 : 0, C18 : 1ω9c and C18 : 1 3-OH. A draft whole-genome sequence of the proposed type strain 08HL01032(T) was generated. Comparative sequence analysis of the complete 16S and 23S rRNA genes confirmed affiliation to the genus Francisella, with 95 % sequence identity to the closest relatives in the database, the type strains of Francisella philomiragia and Francisella noatunensis subsp. orientalis. Full-length deduced amino acid sequences of various housekeeping genes, recA, gyrB, groEL, dnaK, rpoA, rpoB, rpoD, rpoH, fopA and sdhA, exhibited similarities of 67-92 % to strains of other species of the genus Francisella. Strains 08HL01032(T), 09HG994, 10HP82-6 and 10HL1960 exhibited highly similar pan-genome PCR profiles. Both the phenotypic and molecular data support the conclusion that the four strains belong to the genus Francisella but exhibit considerable divergence from all recognized Francisella species. Therefore, we propose the name Francisella guangzhouensis sp. nov., with the type strain 08HL01032(T) ( = CCUG 60119(T) = NCTC 13503(T)).

Degradation of methyl bromide and methyl chloride in soil microcosms: Use of stable C isotope fractionation and stable isotope probing to identify reactions and the responsible microorganisms

NASA Astrophysics Data System (ADS)

Miller, Laurence G.; Warner, Karen L.; Baesman, Shaun M.; Oremland, Ronald S.; McDonald, Ian R.; Radajewski, Stefan; Murrell, J. Colin

2004-08-01

Bacteria in soil microcosm experiments oxidized elevated levels of methyl chloride (MeCl) and methyl bromide (MeBr), the former compound more rapidly than the latter. MeBr was also removed by chemical reactions while MeCl was not. Chemical degradation dominated the early removal of MeBr and accounted for more than half of its total loss. Fractionation of stable carbon isotopes during chemical degradation of MeBr resulted in a kinetic isotope effect (KIE) of 59 ± 7‰. Soil bacterial oxidation dominated the later removal of MeBr and MeCl and was characterized by different KIEs for each compound. The KIE for MeBr oxidation was 69 ± 9‰ and the KIE for MeCl oxidation was 49 ± 3‰. Stable isotope probing revealed that different populations of soil bacteria assimilated added 13C-labeled MeBr and MeCl. The identity of the active MeBr and MeCl degrading bacteria in soil was determined by analysis of 16S rRNA gene sequences amplified from 13C-DNA fractions, which identified a number of sequences from organisms not previously thought to be involved in methyl halide degradation. These included Burkholderia, the major clone type in the 13C-MeBr fraction, and Rhodobacter, Lysobacter and Nocardioides the major clone types in the 13C-MeCl fraction. None of the 16S rRNA gene sequences for methyl halide oxidizing bacteria currently in culture (including Aminobacter strain IMB-1 isolated from fumigated soil) were identified. Functional gene clone types closely related to Aminobacter spp. were identified in libraries containing the sequences for the cmuA gene, which codes for the enzyme known to catalyze the initial step in the oxidation of MeBr and MeCl. The cmuA gene was limited to members of the alpha-Proteobacteria whereas the greater diversity demonstrated by the 16S rRNA gene may indicate that other enzymes catalyze methyl halide oxidation in different groups of bacteria.

Multiple-locus, variable number of tandem repeat analysis (MLVA) of the fish-pathogen Francisella noatunensis

PubMed Central

2011-01-01

Background Since Francisella noatunensis was first isolated from cultured Atlantic cod in 2004, it has emerged as a global fish pathogen causing disease in both warm and cold water species. Outbreaks of francisellosis occur in several important cultured fish species making a correct management of this disease a matter of major importance. Currently there are no vaccines or treatments available. A strain typing system for use in studies of F. noatunensis epizootics would be an important tool for disease management. However, the high genetic similarity within the Francisella spp. makes strain typing difficult, but such typing of the related human pathogen Francisella tullarensis has been performed successfully by targeting loci with higher genetic variation than the traditional signature sequences. These loci are known as Variable Numbers of Tandem Repeat (VNTR). The aim of this study is to identify possible useful VNTRs in the genome of F. noatunensis. Results Seven polymorphic VNTR loci were identified in the preliminary genome sequence of F. noatunensis ssp. noatunensis GM2212 isolate. These VNTR-loci were sequenced in F. noatunensis isolates collected from Atlantic cod (Gadus morhua) from Norway (n = 21), Three-line grunt (Parapristipoma trilineatum) from Japan (n = 1), Tilapia (Oreochromis spp.) from Indonesia (n = 3) and Atlantic salmon (Salmo salar) from Chile (n = 1). The Norwegian isolates presented in this study show both nine allelic profiles and clades, and that the majority of the farmed isolates belong in two clades only, while the allelic profiles from wild cod are unique. Conclusions VNTRs can be used to separate isolates belonging to both subspecies of F. noatunensis. Low allelic diversity in F. noatunensis isolates from outbreaks in cod culture compared to isolates wild cod, indicate that transmission of these isolates may be a result of human activity. The sequence based MLVA system presented in this study should provide a good starting point for further development of a genotyping system that can be used in studies of epizootics and disease management of francisellosis. PMID:21261955

Structure-based design of broadly protective group a streptococcal M protein-based vaccines.

PubMed

Dale, James B; Smeesters, Pierre R; Courtney, Harry S; Penfound, Thomas A; Hohn, Claudia M; Smith, Jeremy C; Baudry, Jerome Y

2017-01-03

A major obstacle to the development of broadly protective M protein-based group A streptococcal (GAS) vaccines is the variability within the N-terminal epitopes that evoke potent bactericidal antibodies. The concept of M type-specific protective immune responses has recently been challenged based on the observation that multivalent M protein vaccines elicited cross-reactive bactericidal antibodies against a number of non-vaccine M types of GAS. Additionally, a new "cluster-based" typing system of 175M proteins identified a limited number of clusters containing closely related M proteins. In the current study, we used the emm cluster typing system, in combination with computational structure-based peptide modeling, as a novel approach to the design of potentially broadly protective M protein-based vaccines. M protein sequences (AA 16-50) from the E4 cluster containing 17 emm types of GAS were analyzed using de novo 3-D structure prediction tools and the resulting structures subjected to chemical diversity analysis to identify sequences that were the most representative of the 3-D physicochemical properties of the M peptides in the cluster. Five peptides that spanned the range of physicochemical attributes of all 17 peptides were used to formulate synthetic and recombinant vaccines. Rabbit antisera were assayed for antibodies that cross-reacted with E4 peptides and whole bacteria by ELISA and for bactericidal activity against all E4GAS. The synthetic vaccine rabbit antisera reacted with all 17 E4M peptides and demonstrated bactericidal activity against 15/17 E4GAS. A recombinant hybrid vaccine containing the same E4 peptides also elicited antibodies that cross-reacted with all E4M peptides. Comprehensive studies using structure-based design may result in a broadly protective M peptide vaccine that will elicit cluster-specific and emm type-specific antibody responses against the majority of clinically relevant emm types of GAS. Copyright © 2016 Elsevier Ltd. All rights reserved.

Alu expression in human cell lines and their retrotranspositional potential.

PubMed

Oler, Andrew J; Traina-Dorge, Stephen; Derbes, Rebecca S; Canella, Donatella; Cairns, Brad R; Roy-Engel, Astrid M

2012-06-20

The vast majority of the 1.1 million Alu elements are retrotranspositionally inactive, where only a few loci referred to as 'source elements' can generate new Alu insertions. The first step in identifying the active Alu sources is to determine the loci transcribed by RNA polymerase III (pol III). Previous genome-wide analyses from normal and transformed cell lines identified multiple Alu loci occupied by pol III factors, making them candidate source elements. Analysis of the data from these genome-wide studies determined that the majority of pol III-bound Alus belonged to the older subfamilies Alu S and Alu J, which varied between cell lines from 62.5% to 98.7% of the identified loci. The pol III-bound Alus were further scored for estimated retrotransposition potential (ERP) based on the absence or presence of selected sequence features associated with Alu retrotransposition capability. Our analyses indicate that most of the pol III-bound Alu loci candidates identified lack the sequence characteristics important for retrotransposition. These data suggest that Alu expression likely varies by cell type, growth conditions and transformation state. This variation could extend to where the same cell lines in different laboratories present different Alu expression patterns. The vast majority of Alu loci potentially transcribed by RNA pol III lack important sequence features for retrotransposition and the majority of potentially active Alu loci in the genome (scored high ERP) belong to young Alu subfamilies. Our observations suggest that in an in vivo scenario, the contribution of Alu activity on somatic genetic damage may significantly vary between individuals and tissues.

Deciphering the biodiversity of Listeria monocytogenes lineage III strains by polyphasic approaches.

PubMed

Zhao, Hanxin; Chen, Jianshun; Fang, Chun; Xia, Ye; Cheng, Changyong; Jiang, Lingli; Fang, Weihuan

2011-10-01

Listeria monocytogenes is a foodborne pathogen of humans and animals. The majority of human listeriosis cases are caused by strains of lineages I and II, while lineage III strains are rare and seldom implicated in human listeriosis. We revealed by 16S rRNA sequencing the special evolutionary status of L. monocytogenes lineage III, which falls between lineages I and II strains of L. monocytogenes and the non-pathogenic species L. innocua and L. marthii in the dendrogram. Thirteen lineage III strains were then characterized by polyphasic approaches. Biochemical reactions demonstrated 8 biotypes, internalin profiling identified 10 internal-in types clustered in 4 groups, and multilocus sequence typing differentiated 12 sequence types. These typing schemes show that lineage III strains represent the most diverse population of L. monocytogenes, and comprise at least four subpopulations IIIA-1, IIIA-2, HIB, and IIIC. The in vitro and in vivo virulence assessments showed that two lineage IIIA-2 strains had reduced pathogenicity, while the other lineage III strains had comparable virulence to lineages I and II. The HIB strains are phylogenetically distinct from other sub-populations, providing additional evidence that this sublineage represents a novel lineage. The two biochemical reactions L-rhamnose and L-lactate alkalinization, and 10 internalins were identified as potential markers for lineage III subpopulations. This study provides new insights into the biodiversity and population structure of lineage III strains, which are important for understanding the evolution of the L. mono-cytogenes-L. innocua clade.

Variability and repertoire size of T-cell receptor V alpha gene segments.

PubMed

Becker, D M; Pattern, P; Chien, Y; Yokota, T; Eshhar, Z; Giedlin, M; Gascoigne, N R; Goodnow, C; Wolf, R; Arai, K

The immune system of higher organisms is composed largely of two distinct cell types, B lymphocytes and T lymphocytes, each of which is independently capable of recognizing an enormous number of distinct entities through their antigen receptors; surface immunoglobulin in the case of the former, and the T-cell receptor (TCR) in the case of the latter. In both cell types, the genes encoding the antigen receptors consist of multiple gene segments which recombine during maturation to produce many possible peptides. One striking difference between B- and T-cell recognition that has not yet been resolved by the structural data is the fact that T cells generally require a major histocompatibility determinant together with an antigen whereas, in most cases, antibodies recognize antigen alone. Recently, we and others have found that a series of TCR V beta gene sequences show conservation of many of the same residues that are conserved between heavy- and light-chain immunoglobulin V regions, and these V beta sequences are predicted to have an immunoglobulin-like secondary structure. To extend these studies, we have isolated and sequenced eight additional alpha-chain complementary cDNA clones and compared them with published sequences. Analyses of these sequences, reported here, indicate that V alpha regions have many of the characteristics of V beta gene segments but differ in that they almost always occur as cross-hybridizing gene families. We conclude that there may be very different selective pressures operating on V alpha and V beta sequences and that the V alpha repertoire may be considerably larger than that of V beta.

Immune Selection In Vitro Reveals Human Immunodeficiency Virus Type 1 Nef Sequence Motifs Important for Its Immune Evasion Function In Vivo

PubMed Central

Lee, Patricia; Ng, Hwee L.; Yang, Otto O.

2012-01-01

Human immunodeficiency virus type 1 (HIV-1) Nef downregulates major histocompatibility complex class I (MHC-I), impairing the clearance of infected cells by CD8+ cytotoxic T lymphocytes (CTLs). While sequence motifs mediating this function have been determined by in vitro mutagenesis studies of laboratory-adapted HIV-1 molecular clones, it is unclear whether the highly variable Nef sequences of primary isolates in vivo rely on the same sequence motifs. To address this issue, nef quasispecies from nine chronically HIV-1-infected persons were examined for sequence evolution and altered MHC-I downregulatory function under Gag-specific CTL immune pressure in vitro. This selection resulted in decreased nef diversity and strong purifying selection. Site-by-site analysis identified 13 codons undergoing purifying selection and 1 undergoing positive selection. Of the former, only 6 have been reported to have roles in Nef function, including 4 associated with MHC-I downregulation. Functional testing of naturally occurring in vivo polymorphisms at the 7 sites with no previously known functional role revealed 3 mutations (A84D, Y135F, and G140R) that ablated MHC-I downregulation and 3 (N52A, S169I, and V180E) that partially impaired MHC-I downregulation. Globally, the CTL pressure in vitro selected functional Nef from the in vivo quasispecies mixtures that predominately lacked MHC-I downregulatory function at the baseline. Overall, these data demonstrate that CTL pressure exerts a strong purifying selective pressure for MHC-I downregulation and identifies novel functional motifs present in Nef sequences in vivo. PMID:22553319

Translocation and gross deletion breakpoints in human inherited disease and cancer II: Potential involvement of repetitive sequence elements in secondary structure formation between DNA ends.

PubMed

Chuzhanova, Nadia; Abeysinghe, Shaun S; Krawczak, Michael; Cooper, David N

2003-09-01

Translocations and gross deletions are responsible for a significant proportion of both cancer and inherited disease. Although such gene rearrangements are nonuniformly distributed in the human genome, the underlying mutational mechanisms remain unclear. We have studied the potential involvement of various types of repetitive sequence elements in the formation of secondary structure intermediates between the single-stranded DNA ends that recombine during rearrangements. Complexity analysis was used to assess the potential of these ends to form secondary structures, the maximum decrease in complexity consequent to a gross rearrangement being used as an indicator of the type of repeat and the specific DNA ends involved. A total of 175 pairs of deletion/translocation breakpoint junction sequences available from the Gross Rearrangement Breakpoint Database [GRaBD; www.uwcm.ac.uk/uwcm/mg/grabd/grabd.html] were analyzed. Potential secondary structure was noted between the 5' flanking sequence of the first breakpoint and the 3' flanking sequence of the second breakpoint in 49% of rearrangements and between the 5' flanking sequence of the second breakpoint and the 3' flanking sequence of the first breakpoint in 36% of rearrangements. Inverted repeats, inversions of inverted repeats, and symmetric elements were found in association with gross rearrangements at approximately the same frequency. However, inverted repeats and inversions of inverted repeats accounted for the vast majority (83%) of deletions plus small insertions, symmetric elements for one-half of all antigen receptor-mediated translocations, while direct repeats appear only to be involved in mediating simple deletions. These findings extend our understanding of illegitimate recombination by highlighting the importance of secondary structure formation between single-stranded DNA ends at breakpoint junctions. Copyright 2003 Wiley-Liss, Inc.

Analysis of Typing Methods for Epidemiological Surveillance of both Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Strains▿ †

PubMed Central

Faria, Nuno A.; Carrico, João A.; Oliveira, Duarte C.; Ramirez, Mário; de Lencastre, Hermínia

2008-01-01

Sequence-based methods for typing Staphylococcus aureus, such as multilocus sequence typing (MLST) and spa typing, have increased interlaboratory reproducibility, portability, and speed in obtaining results, but pulsed-field gel electrophoresis (PFGE), remains the method of choice in many laboratories due to the extensive experience with this methodology and the large body of data accumulated using the technique. Comparisons between typing methods have been overwhelmingly based on a qualitative assessment of the overall agreement of results and the relative discriminatory indexes. In this study, we quantitatively assess the congruence of the major typing methods for S. aureus, using a diverse collection of 198 S. aureus strains previously characterized by PFGE, spa typing, MLST, and, in the case of methicillin-resistant S. aureus (MRSA), SCCmec typing in order to establish the quantitative congruence between the typing methods. The results of most typing methods agree in that MRSA and methicillin-susceptible S. aureus (MSSA) differ in terms of diversity of genetic backgrounds, with MSSA being more diverse. Our results show that spa typing has a very good predictive power over the clonal relationships defined by eBURST, while PFGE is less accurate for that purpose but nevertheless provides better typeability and discriminatory power. The combination of PFGE and spa typing provided even better results. Based on these observations, we suggest the use of the conjugation of spa typing and PFGE typing for epidemiological surveillance studies, since this combination provides the ability to infer long-term relationships while maintaining the discriminatory power and typeability needed in short-term studies. PMID:17989188

Most Uv-Induced Reciprocal Translocations in SORDARIA MACROSPORA Occur in or near Centromere Regions

PubMed Central

Leblon, G.; Zickler, D.; Lebilcot, S.

1986-01-01

In fungi, translocations can be identified and classified by the patterns of ascospore abortion in asci from crosses of rearrangement x normal sequence. Previous studies of UV-induced rearrangements in Sordaria macrospora revealed that a major class (called type III) appeared to be reciprocal translocations that were anomalous in producing an unexpected class of asci with four aborted ascospores in bbbbaaaa linear sequence (b = black; a = abortive). The present study shows that the anomalous type III rearrangements are, in fact, reciprocal translocations having both breakpoints within or adjacent to centromeres and that bbbbaaaa asci result from 3:1 disjunction from the translocation quadrivalent.—Electron microscopic observations of synaptonemal complexes enable centromeres to be visualized. Lengths of synaptonemal complexes lateral elements in translocation quadrivalents accurately reflect chromosome arm lengths, enabling breakpoints to be located reliably in centromere regions. All genetic data are consistent with the behavior expected of translocations with breakpoints at centromeres.—Two-thirds of the UV-induced reciprocal translocations are of this type. Certain centromere regions are involved preferentially. Among 73 type-III translocations, there were but 13 of the 21 possible chromosome combinations and 20 of the 42 possible combinations of chromosome arms. PMID:17246312

Most Uv-Induced Reciprocal Translocations in SORDARIA MACROSPORA Occur in or near Centromere Regions.

PubMed

Leblon, G; Zickler, D; Lebilcot, S

1986-02-01

In fungi, translocations can be identified and classified by the patterns of ascospore abortion in asci from crosses of rearrangement x normal sequence. Previous studies of UV-induced rearrangements in Sordaria macrospora revealed that a major class (called type III) appeared to be reciprocal translocations that were anomalous in producing an unexpected class of asci with four aborted ascospores in bbbbaaaa linear sequence (b = black; a = abortive). The present study shows that the anomalous type III rearrangements are, in fact, reciprocal translocations having both breakpoints within or adjacent to centromeres and that bbbbaaaa asci result from 3:1 disjunction from the translocation quadrivalent.-Electron microscopic observations of synaptonemal complexes enable centromeres to be visualized. Lengths of synaptonemal complexes lateral elements in translocation quadrivalents accurately reflect chromosome arm lengths, enabling breakpoints to be located reliably in centromere regions. All genetic data are consistent with the behavior expected of translocations with breakpoints at centromeres.-Two-thirds of the UV-induced reciprocal translocations are of this type. Certain centromere regions are involved preferentially. Among 73 type-III translocations, there were but 13 of the 21 possible chromosome combinations and 20 of the 42 possible combinations of chromosome arms.

Pathogenesis of Helicobacter pylori-Related Gastroduodenal Diseases from Molecular Epidemiological Studies.

PubMed

Yamaoka, Yoshio

2012-01-01

Helicobacter pylori is a major human pathogen that infects the stomach and produces inflammation that is responsible for various gastroduodenal diseases. Despite the high prevalence of H. pylori infections in Africa and South Asia, the incidence of gastric cancer in these areas is much lower than in other countries. The incidence of gastric cancer also tends to decrease from north to south in East Asia. Data from molecular epidemiological studies show that this variation in different geographic areas could be explained in part by different types of H. pylori virulence factors, especially CagA, VacA, and OipA. H. pylori infection is thought to be involved in both gastric cancer and duodenal ulcer, which are at opposite ends of the disease spectrum. This discrepancy can also be explained in part by another H. pylori factor, DupA, as well as by CagA typing (East Asian type versus Western type). H. pylori has a genome of approximately 1,600 genes; therefore, there might be other novel virulence factors. Because genome wide analyses using whole-genome sequencing technology give a broad view of the genome of H. pylori, we hope that next-generation sequencers will enable us to efficiently investigate novel virulence factors.

Species delimitation of common reef corals in the genus Pocillopora using nucleotide sequence phylogenies, population genetics and symbiosis ecology.

PubMed

Pinzón, Jorge H; LaJeunesse, Todd C

2011-01-01

Stony corals in the genus Pocillopora are among the most common and widely distributed of Indo-Pacific corals and, as such, are often the subject of physiological and ecological research. In the far Tropical Eastern Pacific (TEP), they are major constituents of shallow coral communities, exhibiting considerable variability in colony shape and branch morphology and marked differences in response to thermal stress. Numerous intermediates occur between morphospecies that may relate to extensive hybridization. The diversity of the Pocillopora genus in the TEP was analysed genetically using nuclear ribosomal (ITS2) and mitochondrial (ORF) sequences, and population genetic markers (seven microsatellite loci). The resident dinoflagellate endosymbiont (Symbiodinium sp.) in each sample was also characterized using sequences of the internal transcribed spacer 2 (ITS2) rDNA and the noncoding region of the chloroplast psbA minicircle. From these analyses, three symbiotically distinct, reproductively isolated, nonhybridizing, evolutionarily divergent animal lineages were identified. Designated types 1, 2 and 3, these groupings were incongruent with traditional morphospecies classification. Type 1 was abundant and widespread throughout the TEP; type 2 was restricted to the Clipperton Atoll; and type 3 was found only in Panama and the Galapagos Islands. Each type harboured a different Symbiodinium'species lineage' in Clade C, and only type 1 associated with the 'stress-tolerant'Symbiodinium glynni (D1). The accurate delineation of species and implementation of a proper taxonomy may profoundly improve our assessment of Pocillopora's reproductive biology, biogeographic distributions, and resilience to climate warming, information that must be considered when planning for the conservation of reef corals. © 2010 Blackwell Publishing Ltd.

Species identification and molecular typing of human Brucella isolates from Kuwait.

PubMed

Mustafa, Abu S; Habibi, Nazima; Osman, Amr; Shaheed, Faraz; Khan, Mohd W

2017-01-01

Brucellosis is a zoonotic disease of major concern in Kuwait and the Middle East. Human brucellosis can be caused by several Brucella species with varying degree of pathogenesis, and relapses are common after apparently successful therapy. The classical biochemical methods for identification of Brucella are time-consuming, cumbersome, and provide information limited to the species level only. In contrast, molecular methods are rapid and provide differentiation at intra-species level. In this study, four molecular methods [16S rRNA gene sequencing, real-time PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus variable-number tandem-repeat analysis (MLVA)-8, MLVA-11 and MLVA-16 were evaluated for the identification and typing of 75 strains of Brucella isolated in Kuwait. 16S rRNA gene sequencing of all isolates showed 90-99% sequence identity with B. melitensis and real-time PCR with genus- and species- specific primers identified all isolates as B. melitensis. The results of ERIC-PCR suggested the existence of 75 ERIC genotypes of B. melitensis with a discriminatory index of 0.997. Cluster classification of these genotypes divided them into two clusters, A and B, diverging at ~25%. The maximum number of genotypes (n = 51) were found in cluster B5. MLVA-8 analysis identified all isolates as B. melitensis, and MLVA-8, MLVA-11 and MLVA-16 typing divided the isolates into 10, 32 and 71 MLVA types, respectively. Furthermore, the combined minimum spanning tree analysis demonstrated that, compared to MLVA types discovered all over the world, the Kuwaiti isolates were a distinct group of MLVA-11 and MLVA-16 types in the East Mediterranean Region.

Species identification and molecular typing of human Brucella isolates from Kuwait

PubMed Central

Osman, Amr; Shaheed, Faraz; Khan, Mohd W.

2017-01-01

Brucellosis is a zoonotic disease of major concern in Kuwait and the Middle East. Human brucellosis can be caused by several Brucella species with varying degree of pathogenesis, and relapses are common after apparently successful therapy. The classical biochemical methods for identification of Brucella are time-consuming, cumbersome, and provide information limited to the species level only. In contrast, molecular methods are rapid and provide differentiation at intra-species level. In this study, four molecular methods [16S rRNA gene sequencing, real-time PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus variable-number tandem-repeat analysis (MLVA)-8, MLVA-11 and MLVA-16 were evaluated for the identification and typing of 75 strains of Brucella isolated in Kuwait. 16S rRNA gene sequencing of all isolates showed 90–99% sequence identity with B. melitensis and real-time PCR with genus- and species- specific primers identified all isolates as B. melitensis. The results of ERIC-PCR suggested the existence of 75 ERIC genotypes of B. melitensis with a discriminatory index of 0.997. Cluster classification of these genotypes divided them into two clusters, A and B, diverging at ~25%. The maximum number of genotypes (n = 51) were found in cluster B5. MLVA-8 analysis identified all isolates as B. melitensis, and MLVA-8, MLVA-11 and MLVA-16 typing divided the isolates into 10, 32 and 71 MLVA types, respectively. Furthermore, the combined minimum spanning tree analysis demonstrated that, compared to MLVA types discovered all over the world, the Kuwaiti isolates were a distinct group of MLVA-11 and MLVA-16 types in the East Mediterranean Region. PMID:28800594

High-Mg basalts as a Signal of Magma System Replenishment at Lopevi Island, Vanuatu

NASA Astrophysics Data System (ADS)

Stewart, R. B.; Smith, I. E.; Turner, M. B.; Cronin, S. J.

2007-05-01

Lopevi is is a basalt to basaltic andesite island stratovolcano in central Vanuatu and is part of a long-lived, mature Island Arc chain. Central Vanuatu is tectonically influenced by the subduction of the D'Entrecasteaux zone. Primitive rock types that have been identified from the arc include picrites, ankaramites and high MgO basalts. High MgO rocks are generally considered to be a relatively rare component of arc-type magma suites but as detailed sequence sampling of individual volcanoes occurs, they have been identified more often. Here we report on the occurrence of high-Mg basalts in a sequence of lavas erupted in the last 100 years from Lopevi volcano. Activity at Lopevi is characteristically intermittent with eruptive sequences occurring over a c. 6 year period, separated by longer periods of repose. A major eruptive episode in 1939 caused evacuation of the island and the next eruptive episode in the 1960's also led to evacuation. The 1960's cycle of activity ended in 1982. The most recent phase of activity commenced in 1998 with a return to eruption of more siliceous, high alumina basaltic andesite. Geochemical data show that the 1960's lavas were different from those erupted earlier and later. They are olivine basalts with up to 9 wt percent MgO, 70 ppm Ni and 300 ppm Cr; Al2O3 content is about 12 wt percent. The 2003 lavas and pre-1960's lavas, in contrast, are basaltic andesites with c. 4 wt percent MgO, less than 25 ppm Ni, less than 100 ppm Cr and c. 20 wt percent Al2O3. The 1960's Lopevi sequence of eruptions represents an injection of a more primitive, high MgO magma at the end of a 21 year quiescent period after the major eruptions of 1939. Injection of small batches of more primitive magmas over decadal time periods at Lopevi marks the initiation of a new magmatic cycle. The occurrence of high MgO magmas as part of a cycle that includes typically low MgO arc type rocks demonstrates a consanguineous relationship and shows that high MgO arc type rocks are part of a genetically linked suite rather than a distinct magma type. Their comparative scarcity in many subduction related associations is probably a function of tectonic environment rather than of fundamental petrological factors.

Echoviruses are a major cause of aseptic meningitis in infants and young children in Kuwait.

PubMed

Dalwai, Ajmal; Ahmad, Suhail; Al-Nakib, Widad

2010-09-16

The etiologic agents of aseptic meningitis (AM) often include human enteroviruses. The role of enteroviruses causing AM in young children was investigated during a 3-year period in Kuwait. Enteroviral RNA was detected in cerebrospinal fluid (CSF) by reverse transcription-PCR and specific genotypes of enteroviruses were identified by direct DNA sequencing of VP4-VP2 region. Enteroviral RNA was detected in 92 of 387 (24%) suspected AM cases and the results were confirmed by hybridization of amplicons with an internal, enterovirus-specific probe. The CSF samples from 75 of 281 (27%) children < 2 years old but only from 3 of 38 (8%) 4-12 year-old children were positive for enteroviral RNA (p = 0.011). Majority of infections in children < 2 years old (49 of 75, 65%) were due to three echoviruses; echovirus type 9 (E9), E11 and E30. Only three other enteroviruses, namely coxsackievirus type B4, coxsackievirus type B5 and enterovirus 71 were detected among AM cases in Kuwait. Our data show that three types of echoviruses (E9, E11 and E30) are associated with the majority of AM cases in Kuwait. To the best of our knowledge, this is the first report to characterize different enterovirus genotypes associated with AM in the Arabian Gulf region.

The Arabidopsis lyrata genome sequence and the basis of rapid genome size change

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Tina T.; Pattyn, Pedro; Bakker, Erica G.

2011-04-29

In our manuscript, we present a high-quality genome sequence of the Arabidopsis thaliana relative, Arabidopsis lyrata, produced by dideoxy sequencing. We have performed the usual types of genome analysis (gene annotation, dN/dS studies etc. etc.), but this is relegated to the Supporting Information. Instead, we focus on what was a major motivation for sequencing this genome, namely to understand how A. thaliana lost half its genome in a few million years and lived to tell the tale. The rather surprising conclusion is that there is not a single genomic feature that accounts for the reduced genome, but that every aspectmore » centromeres, intergenic regions, transposable elements, gene family number is affected through hundreds of thousands of cuts. This strongly suggests that overall genome size in itself is what has been under selection, a suggestion that is strongly supported by our demonstration (using population genetics data from A. thaliana) that new deletions seem to be driven to fixation.« less

Equus caballus Major Histocompatibility Complex Class I Is an Entry Receptor for Equine Herpesvirus Type 1▿

PubMed Central

Kurtz, Brian M.; Singletary, Lauren B.; Kelly, Sean D.; Frampton, Arthur R.

2010-01-01

In this study, Equus caballus major histocompatibility complex class I (MHC-I) was identified as a cellular entry receptor for the alphaherpesvirus equine herpesvirus type 1 (EHV-1). This novel EHV-1 receptor was discovered using a cDNA library from equine macrophages. cDNAs from this EHV-1-susceptible cell type were inserted into EHV-1-resistant B78H1 murine melanoma cells, these cells were infected with an EHV-1 lacZ reporter virus, and cells that supported virus infection were identified by X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) staining. Positive cells were subjected to several rounds of purification to obtain homogeneous cell populations that were shown to be uniformly infected with EHV-1. cDNAs from these cell populations were amplified by PCR and then sequenced. The sequence data revealed that the EHV-1-susceptible cells had acquired an E. caballus MHC-I cDNA. Cell surface expression of this receptor was verified by confocal immunofluorescence microscopy. The MHC-I cDNA was cloned into a mammalian expression vector, and stable B78H1 cell lines were generated that express this receptor. These cell lines were susceptible to EHV-1 infection while the parental B78H1 cells remained resistant to infection. In addition, EHV-1 infection of the B78H1 MHC-I-expressing cell lines was inhibited in a dose-dependent manner by an anti-MHC-I antibody. PMID:20610718

Proposal of Mucilaginibacter phyllosphaerae sp. nov. isolated from the phyllosphere of Galium album.

PubMed

Aydogan, Ebru L; Busse, Hans-Jürgen; Moser, Gerald; Müller, Christoph; Kämpfer, Peter; Glaeser, Stefanie P

2016-10-01

A pink-pigmented, Gram-stain-negative, rod-shaped, non-spore-forming bacterial strain, PP-F2F-G21T, was isolated from the phyllosphere of Galium album. Phylogenetic analysis of the nearly full-length 16S rRNA gene sequence of strain PP-F2F-G21T showed the closest relationship to type strains of the species Mucilaginibacter lutimaris (97.7 %), Mucilaginibacter soli (97.3 %) and Mucilaginibacter rigui (97.1 %). Sequence similarities to all other type strains were below 97 %. The predominant cellular fatty acids of strain PP-F2F-G21T are C16 : 1 ω7c/iso-C15 : 0 2-OH (measured as summed feature 3 fatty acids) and iso-C15 : 0 followed by iso-C17 : 0 3-OH, C16 : 1 ω5c and C16 : 0. The major compound in the polyamine pattern was sym-homospermidine and the diamino acid of the peptidoglycan was meso-diaminopimelic acid. The quinone system was exclusively composed of menaquinone MK-7. The polar lipid profile contained the major lipid phosphatidylethanolamine and in addition 18 unidentified lipids. Based on phylogenetic, chemotaxonomic and phenotypic analyses, we propose a novel species of the genus Mucilaginibacter named Mucilaginibacter phyllosphaeraesp. nov. The type strain is PP-F2F-G21T (=CCM 8625T=CIP 110921T=LMG 29118T).

Delftia rhizosphaerae sp. nov. isolated from the rhizosphere of Cistus ladanifer.

PubMed

Carro, Lorena; Mulas, Rebeca; Pastor-Bueis, Raquel; Blanco, Daniel; Terrón, Arsenio; González-Andrés, Fernando; Peix, Alvaro; Velázquez, Encarna

2017-06-01

A bacterial strain, designated RA6T, was isolated from the rhizosphere of Cistus ladanifer. Phylogenetic analyses based on 16S rRNA gene sequence placed the isolate into the genus Delftia within a cluster encompassing the type strains of Delftia lacustris, Delftia tsuruhatensis, Delftia acidovorans and Delftia litopenaei, which presented greater than 97 % sequence similarity with respect to strain RA6T. DNA-DNA hybridization studies showed average relatedness ranging from of 11 to 18 % between these species of the genus Delftia and strain RA6T. Catalase and oxidase were positive. Casein was hydrolysed but gelatin and starch were not. Ubiquinone 8 was the major respiratory quinone detected in strain RA6T together with low amounts of ubiquinones 7 and 9. The major fatty acids were those from summed feature 3 (C16 : 1ω7c/C16 : 1 ω6c) and C16 : 0. The predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain RA6T should be considered as a representative of a novel species of genus Delftia, for which the name Delftia rhizosphaerae sp. nov. is proposed. The type strain is RA6T (=LMG 29737T= CECT 9171T).

Hyperendemic Campylobacter jejuni in guinea pigs (Cavia porcellus) raised for food in a semi-rural community of Quito, Ecuador.

PubMed

Graham, Jay P; Vasco, Karla; Trueba, Gabriel

2016-06-01

Domestic animals and animal products are the source of pathogenic Campylobacter jejuni and C. coli in industrialized countries, yet little is known about the transmission of these bacteria in developing countries. Guinea pigs (Cavia porcellus) are commonly raised for food in the Andean region of South America, however, limited research has characterized this rodent as a reservoir of zoonotic enteric pathogens. In this study, we examined the prevalence of Campylobacter spp. in 203 fecal samples from domestic animals of 59 households in a semi-rural parish of Quito, Ecuador. Of the twelve animal species studied, guinea pigs showed the highest prevalence of C. jejuni (n = 39/40; 97.5%). Multilocus sequence typing (MLST) was used to characterize the genetic relationship of C. jejuni from domestic animals and 21 sequence types (STs) were identified. The majority of STs from guinea pigs appeared to form new clonal complexes that were not related to STs of C. jejuni isolated from other animal species and shared only a few alleles with other C. jejuni previously characterized. The study identifies guinea pigs as a major reservoir of C. jejuni and suggests that some C. jejuni strains are adapted to this animal species. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Actinomyces haliotis sp. nov., a bacterium isolated from the gut of an abalone, Haliotis discus hannai.

PubMed

Hyun, Dong-Wook; Shin, Na-Ri; Kim, Min-Soo; Kim, Pil Soo; Kim, Joon Yong; Whon, Tae Woong; Bae, Jin-Woo

2014-02-01

A novel, Gram-staining-positive, facultatively anaerobic, non-motile and coccus-shaped bacterium, strain WL80(T), was isolated from the gut of an abalone, Haliotis discus hannai, collected from the northern coast of Jeju in Korea. Optimal growth occurred at 30 °C, pH 7-8 and with 1% (w/v) NaCl. Phylogenetic analyses based on the 16S rRNA gene sequence revealed that strain WL80(T) fell within the cluster of the genus Actinomyces, with highest sequence similarity to the type strains of Actinomyces radicidentis (98.8% similarity) and Actinomyces urogenitalis (97.0% similarity). The major cellular fatty acids were C18 : 1ω9c and C16 : 0. Menaquinone-10 (H4) was the major respiratory quinone. The genomic DNA G+C content of the isolate was 70.4 mol%. DNA-DNA hybridization values with closely related strains indicated less than 7.6% genomic relatedness. The results of physiological, biochemical, chemotaxonomic and genotypic analyses indicated that strain WL80(T) represents a novel species of the genus Actinomyces, for which the name Actinomyces haliotis sp. nov. is proposed. The type strain is WL80(T) ( = KACC 17211(T) = JCM 18848(T)).

A major stylar matrix polypeptide (sp41) is a member of the pathogenesis-related proteins superclass.

PubMed Central

Ori, N; Sessa, G; Lotan, T; Himmelhoch, S; Fluhr, R

1990-01-01

A novel stylar-specific glycosylated protein, sp41, was characterized. Sp41 constitutes greater than 12% of the transmitting tract tissue soluble proteins and is mainly localized in the extracellular matrix. Two cDNA clones corresponding to sp41 mRNA were isolated and sequenced. The decoded sequences are, respectively, 80% and 49% homologous to acidic and basic pathogen-induced (1-3)-beta-glucanases of the leaf. Thus a subfamily of (1-3)-beta-glucanase pathogenesis-related (PR) proteins constitutes one of the major stylar matrix proteins. The accumulation of sp41 transcripts in normally developing and elicitor-treated styles and leaves was followed using an RNase protection assay. During development sp41 transcript accumulation starts well after carpel differentiation. It is first detected in styles at 8 days before anthesis. The maximal level of accumulation is reached during anthesis. Elicitor-treated styles do not accumulate the leaf-type (1-3)-beta-glucanase transcript, although they retain the capacity to synthesize leaf-type pathogenesis-related proteins such as the pathogen-induced acidic chitinase. The developmental regulation of sp41 expression points to a role for them in the normal processes of flowering and reproductive physiology. Images Fig.1 Fig.2 Fig.5 Fig.6 Fig.7 Fig.8 Fig.9 PMID:2120041

Paenibacillus beijingensis sp. nov., a novel nitrogen-fixing species isolated from jujube garden soil.

PubMed

Gao, Miao; Xie, Lin-qi; Wang, Ya-xiong; Chen, Jian; Xu, Jing; Zhang, Xiao-xia; Sui, Xin-hua; Gao, Jun-lian; Sun, Jian-guang

2012-11-01

A novel Gram-positive, rod-shaped, motile, spore-forming, nitrogen-fixing bacterium, designated strain 7188(T), was isolated from jujube rhizosphere soil in Beijing, China. The strain grew at 4-40 °C and pH 6-12, with an optimum of 30 °C and pH 7.0, respectively. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain 7188(T) is a member of the genus Paenibacillus. Levels of 16S rRNA gene sequence similarities between strain 7188(T) and the type strains of all recognized members of the genus Paenibacillus were below 96 %. The major cellular fatty acids were anteiso-C(15:0), anteiso-C(17:0) and C(16:0). The predominant menaquinone was MK-7. The DNA G+C content of strain 7188(T) was 60.3 mol%. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and unknown aminophospholipids. The diamino acid in the cell wall peptidoglycan is meso-diaminopimelic acid. On the basis of these results, strain 7188(T) is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus beijingensis sp. nov. is proposed. The type strain is 7188(T) (=ACCC 03082(T) = DSM 24997(T)).

Massively parallel sequencing of 32 forensic markers using the Precision ID GlobalFiler™ NGS STR Panel and the Ion PGM™ System.

PubMed

Wang, Zheng; Zhou, Di; Wang, Hui; Jia, Zhenjun; Liu, Jing; Qian, Xiaoqin; Li, Chengtao; Hou, Yiping

2017-11-01

Massively parallel sequencing (MPS) technologies have proved capable of sequencing the majority of the key forensic STR markers. By MPS, not only the repeat-length size but also sequence variations could be detected. Recently, Thermo Fisher Scientific has designed an advanced MPS 32-plex panel, named the Precision ID GlobalFiler™ NGS STR Panel, where the primer set has been designed specifically for the purpose of MPS technologies and the data analysis are supported by a new version HID STR Genotyper Plugin (V4.0). In this study, a series of experiments that evaluated concordance, reliability, sensitivity of detection, mixture analysis, and the ability to analyze case-type and challenged samples were conducted. In addition, 106 unrelated Han individuals were sequenced to perform genetic analyses of allelic diversity. As expected, MPS detected broader allele variations and gained higher power of discrimination and exclusion rate. MPS results were found to be concordant with current capillary electrophoresis methods, and single source complete profiles could be obtained stably using as little as 100pg of input DNA. Moreover, this MPS panel could be adapted to case-type samples and partial STR genotypes of the minor contributor could be detected up to 19:1 mixture. Aforementioned results indicate that the Precision ID GlobalFiler™ NGS STR Panel is reliable, robust and reproducible and have the potential to be used as a tool for human forensics. Copyright © 2017 Elsevier B.V. All rights reserved.

Molecular characterization of the major membrane skeletal protein in the ciliate Tetrahymena pyriformis suggests n-plication of an early evolutionary intermediate filament protein subdomain.

PubMed

Bouchard, P; Chomilier, J; Ravet, V; Mornon, J P; Viguès, B

2001-01-01

Epiplasmin C is the major protein component of the membrane skeleton in the ciliate Tetrahymena pyriformis. Cloning and analysis of the gene encoding epiplasmin C showed this protein to be a previously unrecognized protein. In particular, epiplasmin C was shown to lack the canonical features of already known epiplasmic proteins in ciliates and flagellates. By means of hydrophobic cluster analysis (HCA), it has been shown that epiplasmin C is constituted of a repeat of 25 domains of 40 residues each. These domains are related and can be grouped in two families called types I and types II. Connections between types I and types II present rules that can be evidenced in the sequence itself, thus enforcing the validity of the splitting of the domains. Using these repeated domains as queries, significant structural similarities were demonstrated with an extra six heptads shared by nuclear lamins and invertebrate cytoplasmic intermediate filament proteins and deleted in the cytoplasmic intermediate filament protein lineage at the protostome-deuterostome branching in the eukaryotic phylogenetic tree.

Channel-Opening Kinetic Mechanism of Wild-Type GluK1 Kainate Receptors and a C-Terminal Mutant

PubMed Central

Han, Yan; Wang, Congzhou; Park, Jae Seon; Niu, Li

2012-01-01

GluK1 is a kainate receptor subunit in the ionotropic glutamate receptor family and can form functional channels when expressed, for instance, in HEK-293 cells. However, the channel-opening mechanism of GluK1 is poorly understood. One major challenge to studying the GluK1 channel is its apparent low surface expression, which results in a low whole-cell current response even to a saturating concentration of agonist. The low surface expression is thought to be contributed by an endoplasmic reticulum (ER) retention signal sequence. When this sequence motif is present as in the wild-type GluK1-2b C-terminus, the receptor is significantly retained in the ER. Conversely, when this sequence is lacking, as in wild-type GluK1-2a (i.e., a different alternatively spliced isoform at the C-terminus) and in a GluK1-2b mutant (i.e., R896A, R897A, R900A and K901A) that disrupts the ER retention signal, there is higher surface expression and greater whole-cell current response. Here we characterize the channel-opening kinetic mechanism for these three GluK1 receptors expressed in HEK-293 cells by using a laser-pulse photolysis technique. Our results show that the wild-type GluK1-2a, wild-type GluK1-2b and the mutant GluK1-2b have identical channel-opening and channel-closing rate constants. These results indicate that the C-terminal ER retention signal sequence, which affects receptor trafficking/expression, does not affect channel-gating properties. Furthermore, as compared with the GluK2 kainate receptor, the GluK1 channel is faster to open, close, and desensitize by at least two-fold, yet the EC50 value of GluK1 is similar to that of GluK2. PMID:22191429

Population structure and genetic diversity of the parasite Trichomonas vaginalis in Bristol, UK.

PubMed

Hawksworth, Joseph; Levy, Max; Smale, Chloe; Cheung, Dean; Whittle, Alice; Longhurst, Denise; Muir, Peter; Gibson, Wendy

2015-08-01

The protozoan parasite Trichomonas vaginalis is the causative agent of trichomoniasis, an extremely common, but non-life-threatening, sexually-transmitted disease throughout the world. Recent population genetics studies of T. vaginalis have detected high genetic diversity and revealed a two-type population structure, associated with phenotypic differences in sensitivity to metronidazole, the drug commonly used for treatment, and presence of T. vaginalis virus. There is currently a lack of data on UK isolates; most isolates examined to date are from the US. Here we used a recently described system for multilocus sequence typing (MLST) of T. vaginalis to study diversity of clinical isolates from Bristol, UK. We used MLST to characterise 23 clinical isolates of T. vaginalis collected from female patients during 2013. Seven housekeeping genes were PCR-amplified for each isolate and sequenced. The concatenated sequences were then compared with data from other MLST-characterised isolates available from http://tvaginalis.mlst.net/ to analyse the population structure and construct phylogenetic trees. Among the 23 isolates from the Bristol population of T. vaginalis, we found 23 polymorphic nucleotide sites, 25 different alleles and 19 sequence types (genotypes). Most isolates had a unique genotype, in agreement with the high levels of heterogeneity observed elsewhere in the world. A two-type population structure was evident from population genetic analysis and phylogenetic reconstruction split the isolates into two major clades. Tests for recombination in the Bristol population of T. vaginalis gave conflicting results, suggesting overall a clonal pattern of reproduction. We conclude that the Bristol population of T. vaginalis parasites conforms to the two-type population structure found in most other regions of the world. We found the MLST scheme to be an efficient genotyping method. The online MLST database provides a useful repository and resource that will prove invaluable in future studies linking the genetics of T. vaginalis with the clinical manifestation of trichomoniasis. Copyright © 2015 Elsevier B.V. All rights reserved.

Epidemiological study of erythromycin-resistant Streptococcus pyogenes from Korea and Japan by emm genotyping and multilocus sequence typing.

PubMed

Takahashi, Takashi; Arai, Kazuaki; Lee, Dong Hyun; Koh, Eun Ha; Yoshida, Haruno; Yano, Hisakazu; Kaku, Mitsuo; Kim, Sunjoo

2016-01-01

We determined the epidemiological characteristics of erythromycin (EM)-resistant Streptococcus pyogenes (group A streptococci, GAS) strains isolated from Korea and Japan, using emm genotyping and multilocus sequence typing (MLST). Clinical isolates of GAS had been collected from 1992 to 2012 in Korea and from 2004 to 2009 in Japan. EM resistance was determined by the microdilution method, and resistance genotypes were assessed by PCR. The emm genotyping and MLST were performed by DNA sequencing. The emm genotypes and sequence types (STs) were concordant in 143 (85.1%) of 168 EM-resistant GAS strains from Korea. ST36/emm12 (35.1%), ST52/emm28 (22.6%), and ST49/emm75 (16.1%) were the most common types. Most of the ST36 (93.9%) and ST52 (95.8%) strains harbored erm(B), whereas strains ST49, ST42, and ST15 contained mef(A). The concordance between emm genotypes and STs was 41 (93.2%) among 44 EM-resistant GAS strains from Japan. ST36/emm12 (34.1%), ST49/emm75 (18.2%), and ST28/emm1 (15.9%) were the major types. ST36 isolates harbored either erm(B) (56.3%) or mef(A) (37.5%), whereas isolates ST28, ST49, and ST38 carried only mef(A). The proportion of erm(B) and mef(A) was 66.1% and 33.3% in Korea and 22.7% and 68.2% in Japan, respectively. The common STs in Korea and Japan were ST36 and ST49, whereas ST52 was present only in Korea and ST28 only in Japan. Genotype erm(B) was predominant in Korea, whereas mef(A) was frequent in Japan. There were differences between Korea and Japan regarding the frequencies of emm genotypes, STs, and EM resistance genes among the EM-resistant GAS.

Clinical Epidemiology and Molecular Analysis of Extended-Spectrum-β-Lactamase-Producing Escherichia coli in Nepal: Characteristics of Sequence Types 131 and 648

PubMed Central

Sherchan, Jatan Bahadur; Miyoshi-Akiyama, Tohru; Ohmagari, Norio; Kirikae, Teruo; Nagamatsu, Maki; Tojo, Masayoshi; Ohara, Hiroshi; Sherchand, Jeevan B.; Tandukar, Sarmila

2015-01-01

Recently, CTX-M-type extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli strains have emerged worldwide. In particular, E. coli with O antigen type 25 (O25) and sequence type 131 (ST131), which is often associated with the CTX-M-15 ESBL, has been increasingly reported globally; however, epidemiology reports on ESBL-producing E. coli in Asia are limited. Patients with clinical isolates of ESBL-producing E. coli in the Tribhuvan University teaching hospital in Kathmandu, Nepal, were included in this study. Whole-genome sequencing of the isolates was conducted to analyze multilocus sequence types, phylotypes, virulence genotypes, O25b-ST131 clones, and distribution of acquired drug resistance genes. During the study period, 105 patients with ESBL-producing E. coli isolation were identified, and the majority (90%) of these isolates were CTX-M-15 positive. The most dominant ST was ST131 (n = 54; 51.4%), followed by ST648 (n = 15; 14.3%). All ST131 isolates were identified as O25b-ST131 clones, subclone H30-Rx. Three ST groups (ST131, ST648, and non-ST131/648) were compared in further analyses. ST648 isolates had a proportionally higher resistance to non-β-lactam antibiotics and featured drug-resistant genes more frequently than ST131 or non-ST131/648 isolates. ST131 possessed the most virulence genes, followed by ST648. The clinical characteristics were similar among groups. More than 38% of ESBL-producing E. coli isolates were from the outpatient clinic, and pregnant patients comprised 24% of ESBL-producing E. coli cases. We revealed that the high resistance of ESBL-producing E. coli to multiple classes of antibiotics in Nepal is driven mainly by CTX-M-producing ST131 and ST648. Their immense prevalence in the communities is a matter of great concern. PMID:25824221

G to A substitution in 5{prime} donor splice site of introns 18 and 48 of COL1A1 gene of type I collagen results in different splicing alternatives in osteogenesis imperfecta type I cell strains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Willing, M.; Deschenes, S.

We have identified a G to A substitution in the 5{prime} donor splice site of intron 18 of one COL1A1 allele in two unrelated families with osteogenesis imperfecta (OI) type I. A third OI type I family has a G to A substitution at the identical position in intron 48 of one COL1A1 allele. Both mutations abolish normal splicing and lead to reduced steady-state levels of mRNA from the mutant COL1A1 allele. The intron 18 mutation leads to both exon 18 skipping in the mRNA and to utilization of a single alternative splice site near the 3{prime} end of exonmore » 18. The latter results in deletion of the last 8 nucleotides of exon 18 from the mRNA, a shift in the translational reading-frame, and the creation of a premature termination codon in exon 19. Of the potential alternative 5{prime} splice sites in exon 18 and intron 18, the one utilized has a surrounding nucleotide sequence which most closely resembles that of the natural splice site. Although a G to A mutation was detected at the identical position in intron 48 of one COL1A1 allele in another OI type I family, nine complex alternative splicing patterns were identified by sequence analysis of cDNA clones derived from fibroblast mRNA from this cell strain. All result in partial or complete skipping of exon 48, with in-frame deletions of portions of exons 47 and/or 49. The different patterns of RNA splicing were not explained by their sequence homology with naturally occuring 5{prime} splice sites, but rather by recombination between highly homologous exon sequences, suggesting that we may not have identified the major splicing alternative(s) in this cell strain. Both G to A mutations result in decreased production of type I collagen, the common biochemical correlate of OI type I.« less

Molecular Epidemiology and Phylogeny Reveal Complex Spatial Dynamics in Areas Where Canine Parvovirus Is Endemic ▿†

PubMed Central

Clegg, S. R.; Coyne, K. P.; Parker, J.; Dawson, S.; Godsall, S. A.; Pinchbeck, G.; Cripps, P. J.; Gaskell, R. M.; Radford, A. D.

2011-01-01

Canine parvovirus type 2 (CPV-2) is a severe enteric pathogen of dogs, causing high mortality in unvaccinated dogs. After emerging, CPV-2 spread rapidly worldwide. However, there is now some evidence to suggest that international transmission appears to be more restricted. In order to investigate the transmission and evolution of CPV-2 both nationally and in relation to the global situation, we have used a long-range PCR to amplify and sequence the full VP2 gene of 150 canine parvoviruses obtained from a large cross-sectional sample of dogs presenting with severe diarrhea to veterinarians in the United Kingdom, over a 2-year period. Among these 150 strains, 50 different DNA sequence types (S) were identified, and apart from one case, all appeared unique to the United Kingdom. Phylogenetic analysis provided clear evidence for spatial clustering at the international level and for the first time also at the national level, with the geographical range of some sequence types appearing to be highly restricted within the United Kingdom. Evolution of the VP2 gene in this data set was associated with a lack of positive selection. In addition, the majority of predicted amino acid sequences were identical to those found elsewhere in the world, suggesting that CPV VP2 has evolved a highly fit conformation. Based on typing systems using key amino acid mutations, 43% of viruses were CPV-2a, and 57% CPV-2b, with no type 2 or 2c found. However, phylogenetic analysis suggested complex antigenic evolution of this virus, with both type 2a and 2b viruses appearing polyphyletic. As such, typing based on specific amino acid mutations may not reflect the true epidemiology of this virus. The geographical restriction that we observed both within the United Kingdom and between the United Kingdom and other countries, together with the lack of CPV-2c in this population, strongly suggests the spread of CPV within its population may be heterogeneously subject to limiting factors. This cross-sectional study of national and global CPV phylogeographic segregation reveals a substantially more complex epidemic structure than previously described. PMID:21593180

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baraibar, Martin A.; Muhoberac, Barry B.; Garringer, Holly J.

Mutations in the coding sequence of the ferritin light chain (FTL) gene cause a neurodegenerative disease known as neuroferritinopathy or hereditary ferritinopathy, which is characterized by the presence of intracellular inclusion bodies containing the mutant FTL polypeptide and by abnormal accumulation of iron in the brain. Here, we describe the x-ray crystallographic structure and report functional studies of ferritin homopolymers formed from the mutant FTL polypeptide p.Phe167SerfsX26, which has a C terminus that is altered in amino acid sequence and length. The structure was determined and refined to 2.85 {angstrom} resolution and was very similar to the wild type betweenmore » residues Ile-5 and Arg-154. However, instead of the E-helices normally present in wild type ferritin, the C-terminal sequences of all 24 mutant subunits showed substantial amounts of disorder, leading to multiple C-terminal polypeptide conformations and a large disruption of the normally tiny 4-fold axis pores. Functional studies underscored the importance of the mutant C-terminal sequence in iron-induced precipitation and revealed iron mishandling by soluble mutant FTL homopolymers in that only wild type incorporated iron when in direct competition in solution with mutant ferritin. Even without competition, the amount of iron incorporation over the first few minutes differed severalfold. Our data suggest that disruption at the 4-fold pores may lead to direct iron mishandling through attenuated iron incorporation by the soluble form of mutant ferritin and that the disordered C-terminal polypeptides may play a major role in iron-induced precipitation and formation of ferritin inclusion bodies in hereditary ferritinopathy.« less

Diversity of mitochondrial large subunit rDNA haplotypes of Glomus intraradices in two agricultural field experiments and two semi-natural grasslands.

PubMed

Börstler, Boris; Thiéry, Odile; Sýkorová, Zuzana; Berner, Alfred; Redecker, Dirk

2010-04-01

Glomus intraradices, an arbuscular mycorrhizal fungus (AMF), is frequently found in a surprisingly wide range of ecosystems all over the world. It is used as model organism for AMF and its genome is being sequenced. Despite the ecological importance of AMF, little has been known about their population structure, because no adequate molecular markers have been available. In the present study we analyse for the first time the intraspecific genetic structure of an AMF directly from colonized roots in the field. A recently developed PCR-RFLP approach for the mitochondrial rRNA large subunit gene (mtLSU) of these obligate symbionts was used and complemented by sequencing and primers specific for a particularly frequent mtLSU haplotype. We analysed root samples from two agricultural field experiments in Switzerland and two semi-natural grasslands in France and Switzerland. RFLP type composition of G. intraradices (phylogroup GLOM A-1) differed strongly between agricultural and semi-natural sites and the G. intraradices populations of the two agricultural sites were significantly differentiated. RFLP type richness was higher in the agricultural sites compared with the grasslands. Detailed sequence analyses which resolved multiple sequence haplotypes within some RFLP types even revealed that there was no overlap of haplotypes among any of the study sites except between the two grasslands. Our results demonstrate a surprisingly high differentiation among semi-natural and agricultural field sites for G. intraradices. These findings will have major implications on our views of processes of adaptation and specialization in these plant/fungus associations.

CRISPR regulation of intraspecies diversification by limiting IS transposition and intercellular recombination.

PubMed

Watanabe, Takayasu; Nozawa, Takashi; Aikawa, Chihiro; Amano, Atsuo; Maruyama, Fumito; Nakagawa, Ichiro

2013-01-01

Mobile genetic elements (MGEs) and genetic rearrangement are considered as major driving forces of bacterial diversification. Previous comparative genome analysis of Porphyromonas gingivalis, a pathogen related to periodontitis, implied such an important relationship. As a counterpart system to MGEs, clustered regularly interspaced short palindromic repeats (CRISPRs) in bacteria may be useful for genetic typing. We found that CRISPR typing could be a reasonable alternative to conventional methods for characterizing phylogenetic relationships among 60 highly diverse P. gingivalis isolates. Examination of genetic recombination along with multilocus sequence typing suggests the importance of such events between different isolates. MGEs appear to be strategically located at the breakpoint gaps of complicated genome rearrangements. Of these MGEs, insertion sequences (ISs) were found most frequently. CRISPR analysis identified 2,150 spacers that were clustered into 1,187 unique ones. Most of these spacers exhibited no significant nucleotide similarity to known sequences (97.6%: 1,158/1,187). Surprisingly, CRISPR spacers exhibiting high nucleotide similarity to regions of P. gingivalis genomes including ISs were predominant. The proportion of such spacers to all the unique spacers (1.6%: 19/1,187) was the highest among previous studies, suggesting novel functions for these CRISPRs. These results indicate that P. gingivalis is a bacterium with high intraspecies diversity caused by frequent insertion sequence (IS) transposition, whereas both the introduction of foreign DNA, primarily from other P. gingivalis cells, and IS transposition are limited by CRISPR interference. It is suggested that P. gingivalis CRISPRs could be an important source for understanding the role of CRISPRs in the development of bacterial diversity.

Evidence for two transferrin loci in the Salmo trutta genome.

PubMed

Rozman, T; Dovc, P; Marić, S; Kokalj-Vokac, N; Erjavec-Skerget, A; Rab, P; Snoj, A

2008-12-01

To determine the organization of transferrin (TF) locus in the Salmo trutta genome, partial DNA and cDNA sequencing, fluorescent in situ hybridization (FISH) and Salmo salar BAC analysis were performed. TF expression levels and copy number prediction were assessed using real-time PCR. In addition to two previously reported DNA TF variant sequences of S. trutta and Salmo marmoratus (TF1), two novel variant sequences (TF2) were revealed in both species. Variant-specific sequence tags, characterizing two variants for each TF type (TF1 and TF2), were identified in genomic clones from each of the F1 hybrids between S. trutta and S. marmoratus. These clearly documented double heterozygote status at the TF loci. The real-time PCR data showed that each of the two TF types (TF1 and TF2) existed in one copy only and that the transcription of TF2 was considerably lower compared with TF1. Using FISH, hybridization signals were observed on two medium-sized acrocentric chromosomes of S. trutta karyotype. A TF type-specific PCR followed by a restriction analysis revealed the presence of two TF loci in the majority of analysed BAC clones. It was concluded that the TF gene is duplicated in the genome of S. trutta, and that the two TF loci are located adjacent to one another on the same chromosome. The differing transcription levels of TF1 and TF2 appear to depend on the corresponding promoter activity, which at least for TF2 seems to vary between different Salmo congeners.

Analysis of the major epitope of the alpha2 chain of bovine type I collagen in children with bovine gelatin allergy.

PubMed

Hori, Hisae; Hattori, Shunji; Inouye, Sakae; Kimura, Akinori; Irie, Shinkichi; Miyazawa, Hiroshi; Sakaguchi, Masahiro

2002-10-01

Anaphylaxis to measles, mumps, and rubella vaccines has been reported. It has been found that most of these reactions to live vaccines are caused by type I allergy with the bovine gelatin present in the vaccines as an allergen. Gelatin mainly includes denatured type I collagen, which consists of alpha1 and alpha2 chains. We previously reported that allergic reactions to gelatin are caused by the type I collagen alpha2 (alpha2[I]) chain. To aid in the development of gelatin that has little or no allergenicity in human subjects, we investigated epitopes of bovine alpha2(I) chain with use of IgE in gelatin-sensitive children. Serum samples were collected from 15 patients who had systemic allergic reactions to vaccines and high levels of specific IgE to bovine gelatin. Eleven overlapping recombinant proteins that cover bovine alpha2(I) were prepared with a bacterial expression vector. We examined IgE reactivity to these recombinant proteins by means of ELISA. Fifteen peptides covering a major reactive recombinant protein were synthesized. The IgE-reacting epitope was identified by means of IgE-ELISA inhibition with these synthetic peptides and pooled serum from the patients. We found that of the 15 patients, 13 showed IgE reactivity to a recombinant protein (no. 3) spanning the central region of the collagenous domain ((418)Gly-(662)Pro). Furthermore, all 13 patients showed IgE reactivity to the 4-kd recombinant protein (no. 3a) spanning the region from (461)Pro to (500)Glu. In IgE-ELISA inhibition we found that a minimum IgE epitope of gelatin allergen was composed of the 10-amino-acid sequence (485)Ile-Pro-Gly-Glu-Phe-Gly-Leu-Pro-Gly-Pro(494). This sequence is not observed in the human type I collagen alpha1 and alpha2 chains, nor is it found in the bovine type I collagen alpha1 chain. We found that Ile-Pro-Gly-Glu-Phe-Gly-Leu-Pro-Gly-Pro is a major IgE epitope of the alpha2 chain of bovine type I collagen in patients with gelatin allergy. The degree of anaphylaxis to gelatin in vaccines might be reduced by digestion of this IgE-binding site in gelatin.

Mitogenic effect contributes to increased virulence of Streptococcus suis sequence type 7 to cause streptococcal toxic shock-like syndrome.

PubMed

Zheng, H; Ye, C; Segura, M; Gottschalk, M; Xu, J

2008-09-01

Streptococcus suis serotype 2 sequence type 7 strains emerged in 1996 and caused a streptococcal toxic shock-like syndrome in 1998 and 2005 in China. Evidence indicated that the virulence of S. suis sequence type 7 had increased, but the mechanism was unknown. The sequence type 7 strain SC84, isolated from a patient with streptococcal toxic shock-like syndrome during the Sichuan outbreak, and the sequence type 1 strain 31533, a typical highly pathogenic strain isolated from a diseased pig, were used in comparative studies. In this study we show the mechanisms underlying cytokine production differed between the two types of strains. The S. suis sequence type 7 strain SC84 possesses a stronger capacity to stimulate T cells, naive T cells and peripheral blood mononuclear cell proliferation than does S. suis sequence type 1 strain 31533. The T cell response to both strains was dependent upon the presence of antigen-presenting cells. Histo-incompatible antigen-presenting cells were sufficient to provide the accessory signals to naive T cell stimulated by the two strains, indicating that both sequence type 7 and 1 strains possess mitogens; however, the mitogenic effect was different. Therefore, we propose that the difference in the mitogenic effect of sequence type 7 strain SC84 compared with the sequence type 1 strain 31533 of S. suis may be associated with the clinical, epidemiological and microbiological difference, where the ST 7 strains have a larger mitogenic effect.

Mitogenic effect contributes to increased virulence of Streptococcus suis sequence type 7 to cause streptococcal toxic shock-like syndrome

PubMed Central

Zheng, H; Ye, C; Segura, M; Gottschalk, M; Xu, J

2008-01-01

Streptococcus suis serotype 2 sequence type 7 strains emerged in 1996 and caused a streptococcal toxic shock-like syndrome in 1998 and 2005 in China. Evidence indicated that the virulence of S. suis sequence type 7 had increased, but the mechanism was unknown. The sequence type 7 strain SC84, isolated from a patient with streptococcal toxic shock-like syndrome during the Sichuan outbreak, and the sequence type 1 strain 31533, a typical highly pathogenic strain isolated from a diseased pig, were used in comparative studies. In this study we show the mechanisms underlying cytokine production differed between the two types of strains. The S. suis sequence type 7 strain SC84 possesses a stronger capacity to stimulate T cells, naive T cells and peripheral blood mononuclear cell proliferation than does S. suis sequence type 1 strain 31533. The T cell response to both strains was dependent upon the presence of antigen-presenting cells. Histo-incompatible antigen-presenting cells were sufficient to provide the accessory signals to naive T cell stimulated by the two strains, indicating that both sequence type 7 and 1 strains possess mitogens; however, the mitogenic effect was different. Therefore, we propose that the difference in the mitogenic effect of sequence type 7 strain SC84 compared with the sequence type 1 strain 31533 of S. suis may be associated with the clinical, epidemiological and microbiological difference, where the ST 7 strains have a larger mitogenic effect. PMID:18803762

Influence of Oxidation and Multimerization on the Immunogenicity of a Thioredoxin-L2 Prophylactic Papillomavirus Vaccine

PubMed Central

Seitz, Hanna; Dantheny, Tatiana; Burkart, Frank; Ottonello, Simone

2013-01-01

Current commercial prophylactic human papillomavirus (HPV) vaccines are based on virus-like particles assembled from the major capsid protein L1 and show excellent safety and efficacy profiles. Still, a major limitation is their rather narrow range of protection against different HPV types. In contrast, the minor capsid protein L2 contains a so-called major cross-neutralizing epitope that can induce broad-range protective responses against multiple HPV types. This epitope is conserved among different papillomaviruses (PV) and contains two cysteine residues that are present in the L2 proteins of all known PV types. The main challenge in developing L2-directed vaccines is to overcome the intrinsically low immunogenicity of the L2 protein. Previously, we developed a recombinant L2-based prototype vaccine by inserting peptide epitopes spanning the cross-neutralizing L2 sequence into a bacterial thioredoxin (Trx) scaffold. These antigens induced high-titer neutralizing antibodies in mice. Here, we address the question of whether Trx scaffold multimerization may further enhance the immunogenicity of the TrxL2 vaccine. We also demonstrate that the oxidation state of the conserved cysteine residues is not essential for vaccine functionality, but it contributes to immunogenicity. PMID:23677323

Complex alternative splicing of acetylcholinesterase transcripts in Torpedo electric organ; primary structure of the precursor of the glycolipid-anchored dimeric form.

PubMed Central

Sikorav, J L; Duval, N; Anselmet, A; Bon, S; Krejci, E; Legay, C; Osterlund, M; Reimund, B; Massoulié, J

1988-01-01

In this paper, we show the existence of alternative splicing in the 3' region of the coding sequence of Torpedo acetylcholinesterase (AChE). We describe two cDNA structures which both diverge from the previously described coding sequence of the catalytic subunit of asymmetric (A) forms (Schumacher et al., 1986; Sikorav et al., 1987). They both contain a coding sequence followed by a non-coding sequence and a poly(A) stretch. Both of these structures were shown to exist in poly(A)+ RNAs, by S1 mapping experiments. The divergent region encoded by the first sequence corresponds to the precursor of the globular dimeric form (G2a), since it contains the expected C-terminal amino acids, Ala-Cys. These amino acids are followed by a 29 amino acid extension which contains a hydrophobic segment and must be replaced by a glycolipid in the mature protein. Analyses of intact G2a AChE showed that the common domain of the protein contains intersubunit disulphide bonds. The divergent region of the second type of cDNA consists of an adjacent genomic sequence, which is removed as an intron in A and Ga mRNAs, but may encode a distinct, less abundant catalytic subunit. The structures of the cDNA clones indicate that they are derived from minor mRNAs, shorter than the three major transcripts which have been described previously (14.5, 10.5 and 5.5 kb). Oligonucleotide probes specific for the asymmetric and globular terminal regions hybridize with the three major transcripts, indicating that their size is determined by 3'-untranslated regions which are not related to the differential splicing leading to A and Ga forms. Images PMID:3181125

A knockout mutation in the lignin biosynthesis gene CCR1 explains a major QTL for acid detergent lignin content in Brassica napus seeds.

PubMed

Liu, Liezhao; Stein, Anna; Wittkop, Benjamin; Sarvari, Pouya; Li, Jiana; Yan, Xingying; Dreyer, Felix; Frauen, Martin; Friedt, Wolfgang; Snowdon, Rod J

2012-05-01

Seed coat phenolic compounds represent important antinutritive fibre components that cause a considerable reduction in value of seed meals from oilseed rape (Brassica napus). The nutritionally most important fibre compound is acid detergent lignin (ADL), to which a significant contribution is made by phenylpropanoid-derived lignin precursors. In this study, we used bulked-segregant analysis in a population of recombinant inbred lines (RILs) from a cross of the Chinese oilseed rape lines GH06 (yellow seed, low ADL) and P174 (black seed, high ADL) to identify markers with tight linkage to a major quantitative trait locus (QTL) for seed ADL content. Fine mapping of the QTL was performed in a backcross population comprising 872 BC(1)F(2) plants from a cross of an F(7) RIL from the above-mentioned population, which was heterozygous for this major QTL and P174. A 3:1 phenotypic segregation for seed ADL content indicated that a single, dominant, major locus causes a substantial reduction in ADL. This locus was successively narrowed to 0.75 cM using in silico markers derived from a homologous Brassica rapa sequence contig spanning the QTL. Subsequently, we located a B. rapa orthologue of the key lignin biosynthesis gene CINNAMOYL CO-A REDUCTASE 1 (CCR1) only 600 kbp (0.75 cM) upstream of the nearest linked marker. Sequencing of PCR amplicons, covering the full-length coding sequences of Bna.CCR1 homologues, revealed a locus in P174 whose sequence corresponds to the Brassica oleracea wild-type allele from chromosome C8. In GH06, however, this allele is replaced by a homologue derived from chromosome A9 that contains a loss-of-function frameshift mutation in exon 1. Genetic and physical map data infer that this loss-of-function allele has replaced a functional Bna.CCR1 locus on chromosome C8 in GH06 by homoeologous non-reciprocal translocation.

Genetic mutation analysis of human gastric adenocarcinomas using ion torrent sequencing platform.

PubMed

Xu, Zhi; Huo, Xinying; Ye, Hua; Tang, Chuanning; Nandakumar, Vijayalakshmi; Lou, Feng; Zhang, Dandan; Dong, Haichao; Sun, Hong; Jiang, Shouwen; Zhang, Guangchun; Liu, Zhiyuan; Dong, Zhishou; Guo, Baishuai; He, Yan; Yan, Chaowei; Wang, Lu; Su, Ziyi; Li, Yangyang; Gu, Dongying; Zhang, Xiaojing; Wu, Xiaomin; Wei, Xiaowei; Hong, Lingzhi; Zhang, Yangmei; Yang, Jinsong; Gong, Yonglin; Tang, Cuiju; Jones, Lindsey; Huang, Xue F; Chen, Si-Yi; Chen, Jinfei

2014-01-01

Gastric cancer is the one of the major causes of cancer-related death, especially in Asia. Gastric adenocarcinoma, the most common type of gastric cancer, is heterogeneous and its incidence and cause varies widely with geographical regions, gender, ethnicity, and diet. Since unique mutations have been observed in individual human cancer samples, identification and characterization of the molecular alterations underlying individual gastric adenocarcinomas is a critical step for developing more effective, personalized therapies. Until recently, identifying genetic mutations on an individual basis by DNA sequencing remained a daunting task. Recent advances in new next-generation DNA sequencing technologies, such as the semiconductor-based Ion Torrent sequencing platform, makes DNA sequencing cheaper, faster, and more reliable. In this study, we aim to identify genetic mutations in the genes which are targeted by drugs in clinical use or are under development in individual human gastric adenocarcinoma samples using Ion Torrent sequencing. We sequenced 737 loci from 45 cancer-related genes in 238 human gastric adenocarcinoma samples using the Ion Torrent Ampliseq Cancer Panel. The sequencing analysis revealed a high occurrence of mutations along the TP53 locus (9.7%) in our sample set. Thus, this study indicates the utility of a cost and time efficient tool such as Ion Torrent sequencing to screen cancer mutations for the development of personalized cancer therapy.

Free-Living and Particle-Associated Bacterioplankton in Large Rivers of the Mississippi River Basin Demonstrate Biogeographic Patterns

PubMed Central

Millar, Justin J.; Payne, Jason T.; Ochs, Clifford A.

2014-01-01

The different drainage basins of large rivers such as the Mississippi River represent interesting systems in which to study patterns in freshwater microbial biogeography. Spatial variability in bacterioplankton communities in six major rivers (the Upper Mississippi, Missouri, Illinois, Ohio, Tennessee, and Arkansas) of the Mississippi River Basin was characterized using Ion Torrent 16S rRNA amplicon sequencing. When all systems were combined, particle-associated (>3 μm) bacterial assemblages were found to be different from free-living bacterioplankton in terms of overall community structure, partly because of differences in the proportional abundance of sequences affiliated with major bacterial lineages (Alphaproteobacteria, Cyanobacteria, and Planctomycetes). Both particle-associated and free-living communities ordinated by river system, a pattern that was apparent even after rare sequences or those affiliated with Cyanobacteria were removed from the analyses. Ordination of samples by river system correlated with environmental characteristics of each river, such as nutrient status and turbidity. Communities in the Upper Mississippi and the Missouri and in the Ohio and the Tennessee, pairs of rivers that join each other, contained similar taxa in terms of presence-absence data but differed in the proportional abundance of major lineages. The most common sequence types detected in particle-associated communities were picocyanobacteria in the Synechococcus/Prochlorococcus/Cyanobium (Syn/Pro) clade, while free-living communities also contained a high proportion of LD12 (SAR11/Pelagibacter)-like Alphaproteobacteria. This research shows that while different tributaries of large river systems such as the Mississippi River harbor distinct bacterioplankton communities, there is also microhabitat variation such as that between free-living and particle-associated assemblages. PMID:25217018

Mitochondrial Genomes of Kinorhyncha: trnM Duplication and New Gene Orders within Animals.

PubMed

Popova, Olga V; Mikhailov, Kirill V; Nikitin, Mikhail A; Logacheva, Maria D; Penin, Aleksey A; Muntyan, Maria S; Kedrova, Olga S; Petrov, Nikolai B; Panchin, Yuri V; Aleoshin, Vladimir V

2016-01-01

Many features of mitochondrial genomes of animals, such as patterns of gene arrangement, nucleotide content and substitution rate variation are extensively used in evolutionary and phylogenetic studies. Nearly 6,000 mitochondrial genomes of animals have already been sequenced, covering the majority of animal phyla. One of the groups that escaped mitogenome sequencing is phylum Kinorhyncha-an isolated taxon of microscopic worm-like ecdysozoans. The kinorhynchs are thought to be one of the early-branching lineages of Ecdysozoa, and their mitochondrial genomes may be important for resolving evolutionary relations between major animal taxa. Here we present the results of sequencing and analysis of mitochondrial genomes from two members of Kinorhyncha, Echinoderes svetlanae (Cyclorhagida) and Pycnophyes kielensis (Allomalorhagida). Their mitochondrial genomes are circular molecules approximately 15 Kbp in size. The kinorhynch mitochondrial gene sequences are highly divergent, which precludes accurate phylogenetic inference. The mitogenomes of both species encode a typical metazoan complement of 37 genes, which are all positioned on the major strand, but the gene order is distinct and unique among Ecdysozoa or animals as a whole. We predict four types of start codons for protein-coding genes in E. svetlanae and five in P. kielensis with a consensus DTD in single letter code. The mitochondrial genomes of E. svetlanae and P. kielensis encode duplicated methionine tRNA genes that display compensatory nucleotide substitutions. Two distant species of Kinorhyncha demonstrate similar patterns of gene arrangements in their mitogenomes. Both genomes have duplicated methionine tRNA genes; the duplication predates the divergence of two species. The kinorhynchs share a few features pertaining to gene order that align them with Priapulida. Gene order analysis reveals that gene arrangement specific of Priapulida may be ancestral for Scalidophora, Ecdysozoa, and even Protostomia.

Mitochondrial Genomes of Kinorhyncha: trnM Duplication and New Gene Orders within Animals

PubMed Central

Popova, Olga V.; Mikhailov, Kirill V.; Nikitin, Mikhail A.; Logacheva, Maria D.; Penin, Aleksey A.; Muntyan, Maria S.; Kedrova, Olga S.; Petrov, Nikolai B.; Panchin, Yuri V.

2016-01-01

Many features of mitochondrial genomes of animals, such as patterns of gene arrangement, nucleotide content and substitution rate variation are extensively used in evolutionary and phylogenetic studies. Nearly 6,000 mitochondrial genomes of animals have already been sequenced, covering the majority of animal phyla. One of the groups that escaped mitogenome sequencing is phylum Kinorhyncha—an isolated taxon of microscopic worm-like ecdysozoans. The kinorhynchs are thought to be one of the early-branching lineages of Ecdysozoa, and their mitochondrial genomes may be important for resolving evolutionary relations between major animal taxa. Here we present the results of sequencing and analysis of mitochondrial genomes from two members of Kinorhyncha, Echinoderes svetlanae (Cyclorhagida) and Pycnophyes kielensis (Allomalorhagida). Their mitochondrial genomes are circular molecules approximately 15 Kbp in size. The kinorhynch mitochondrial gene sequences are highly divergent, which precludes accurate phylogenetic inference. The mitogenomes of both species encode a typical metazoan complement of 37 genes, which are all positioned on the major strand, but the gene order is distinct and unique among Ecdysozoa or animals as a whole. We predict four types of start codons for protein-coding genes in E. svetlanae and five in P. kielensis with a consensus DTD in single letter code. The mitochondrial genomes of E. svetlanae and P. kielensis encode duplicated methionine tRNA genes that display compensatory nucleotide substitutions. Two distant species of Kinorhyncha demonstrate similar patterns of gene arrangements in their mitogenomes. Both genomes have duplicated methionine tRNA genes; the duplication predates the divergence of two species. The kinorhynchs share a few features pertaining to gene order that align them with Priapulida. Gene order analysis reveals that gene arrangement specific of Priapulida may be ancestral for Scalidophora, Ecdysozoa, and even Protostomia. PMID:27755612

A genetic linkage map for the apicomplexan protozoan parasite Eimeria maxima and comparison with Eimeria tenella.

PubMed

Blake, Damer P; Oakes, Richard; Smith, Adrian L

2011-02-01

Eimeria maxima is one of the seven Eimeria spp. that infect the chicken and cause the disease coccidiosis. The well characterised immunogenicity and genetic diversity associated with E. maxima promote its use in genetics-led studies on avian coccidiosis. The development of a genetic map for E. maxima, presented here based upon 647 amplified fragment length polymorphism markers typed from 22 clonal hybrid lines and assembled into 13 major linkage groups, is a major new resource for work with this parasite. Comparison with genetic maps produced for other coccidial parasites indicates relatively high levels of genetic recombination. Conversion of ∼14% of the markers representing the major linkage groups to sequence characterised amplified region markers can provide a scaffold for the assembly of future genomic sequences as well as providing a foundation for more detailed genetic maps. Comparison with the Eimeria tenella genetic map produced 10years ago has revealed a less biased marker distribution, with no more than nine markers mapped within any unresolved heritable unit. Nonetheless, preliminary bioinformatic characterisation of the three largest publicly available genomic E. maxima sequences suggest that the feature-poor/feature-rich structure which has previously been found to define the first sequenced E. tenella chromosome also defines the E. maxima genome. The significance of such a segmented genome and the apparent potential for variation in genetic recombination will be relevant to haplotype stability and the longevity of future anticoccidial strategies based upon multiple loci targeted by novel chemotherapeutic drugs or recombinant subunit vaccines. Copyright © 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Advances in Molecular Serotyping and Subtyping of Escherichia coli.

PubMed

Fratamico, Pina M; DebRoy, Chitrita; Liu, Yanhong; Needleman, David S; Baranzoni, Gian Marco; Feng, Peter

2016-01-01

Escherichia coli plays an important role as a member of the gut microbiota; however, pathogenic strains also exist, including various diarrheagenic E. coli pathotypes and extraintestinal pathogenic E. coli that cause illness outside of the GI-tract. E. coli have traditionally been serotyped using antisera against the ca. 186 O-antigens and 53 H-flagellar antigens. Phenotypic methods, including bacteriophage typing and O- and H- serotyping for differentiating and characterizing E. coli have been used for many years; however, these methods are generally time consuming and not always accurate. Advances in next generation sequencing technologies have made it possible to develop genetic-based subtyping and molecular serotyping methods for E. coli, which are more discriminatory compared to phenotypic typing methods. Furthermore, whole genome sequencing (WGS) of E. coli is replacing established subtyping methods such as pulsed-field gel electrophoresis, providing a major advancement in the ability to investigate food-borne disease outbreaks and for trace-back to sources. A variety of sequence analysis tools and bioinformatic pipelines are being developed to analyze the vast amount of data generated by WGS and to obtain specific information such as O- and H-group determination and the presence of virulence genes and other genetic markers.

Lentibacillus kapialis sp. nov., from fermented shrimp paste in Thailand.

PubMed

Pakdeeto, Amnat; Tanasupawat, Somboon; Thawai, Chitti; Moonmangmee, Somporn; Kudo, Takuji; Itoh, Takashi

2007-02-01

Two strains of strictly aerobic, moderately halophilic Gram-positive rods were isolated from fermented shrimp paste ('ka-pi') produced in Thailand. They produced a red pigment and grew optimally in the presence of 5-30 % NaCl. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The predominant menaquinone was MK-7. The major cellular fatty acid was anteiso-C(15 : 0). Phosphatidylglycerol, diphosphatidylglycerol and two unidentified glycolipids were found to be the major polar lipid components. The DNA G+C content was 41.2-41.6 mol%. Comparative 16S rRNA gene sequence analyses showed that strain PN7-6T was most closely related to Lentibacillus salarius KCTC 3911T with 96.5 % sequence similarity. On the basis of phenotypic and molecular properties, the two isolates represent a novel species of the genus Lentibacillus, for which the name Lentibacillus kapialis sp. nov. is proposed. The type strain is PN7-6T (=JCM 12580T=PCU 259T=TISTR 1551T).

Repetitive sequences based on genotyping of Candida albicans isolates obtained from Iranian patients with human immunodeficiency virus

PubMed Central

Tamai, Iradj Ashrafi; Salehi, Taghi Zahraei; Sharifzadeh, Aghil; Shokri, Hojjatollah; Khosravi, Ali Reza

2014-01-01

Objective(s): Candidiasis infection caused by Candida albicans has been known as a major problem in patients with immune disorders. The objective of this study was to genotype the C. albicans isolates obtained from oral cavity of patients with positive human immunodeficiency virus (HIV+) with or/and without oropharyngeal candidiasis (OPC). Materials and Methods: A total of 100 C. albicans isolates from Iranian HIV+patients were genotyped using specific PCR primers of the 25S rDNA and RPS genes. Results: The frequencies of genotypes A, B and C which were achieved using 25S rDNA , were 66, 24 and 10 percent, respectively. In addition, genotypes D and E were not found in this study. Each C. albicans genotype was further classified into four subtypes (types 2, 3, 2/3 and 3/4) by PCR amplification targeting RPS sequence. Conclusion: In general, genotype A3 constituted the majority of understudy clinical isolates obtained from oral cavity of Iranian HIV+ patients. PMID:25691923

Histoimmunogenetics Markup Language 1.0: Reporting next generation sequencing-based HLA and KIR genotyping.

PubMed

Milius, Robert P; Heuer, Michael; Valiga, Daniel; Doroschak, Kathryn J; Kennedy, Caleb J; Bolon, Yung-Tsi; Schneider, Joel; Pollack, Jane; Kim, Hwa Ran; Cereb, Nezih; Hollenbach, Jill A; Mack, Steven J; Maiers, Martin

2015-12-01

We present an electronic format for exchanging data for HLA and KIR genotyping with extensions for next-generation sequencing (NGS). This format addresses NGS data exchange by refining the Histoimmunogenetics Markup Language (HML) to conform to the proposed Minimum Information for Reporting Immunogenomic NGS Genotyping (MIRING) reporting guidelines (miring.immunogenomics.org). Our refinements of HML include two major additions. First, NGS is supported by new XML structures to capture additional NGS data and metadata required to produce a genotyping result, including analysis-dependent (dynamic) and method-dependent (static) components. A full genotype, consensus sequence, and the surrounding metadata are included directly, while the raw sequence reads and platform documentation are externally referenced. Second, genotype ambiguity is fully represented by integrating Genotype List Strings, which use a hierarchical set of delimiters to represent allele and genotype ambiguity in a complete and accurate fashion. HML also continues to enable the transmission of legacy methods (e.g. site-specific oligonucleotide, sequence-specific priming, and Sequence Based Typing (SBT)), adding features such as allowing multiple group-specific sequencing primers, and fully leveraging techniques that combine multiple methods to obtain a single result, such as SBT integrated with NGS. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Herbaspirillum robiniae sp. nov., isolated from root nodules of Robinia pseudoacacia in a lead-zinc mine.

PubMed

Fan, Miao-Chun; Guo, Yan-Qing; Zhang, Li-Ping; Zhu, Ya-Min; Chen, Wei-Min; Lin, Yan-Bing; Wei, Ge-Hong

2018-04-01

A novel endophytic bacterium, designated strain HZ10 T , was isolated from root nodules of Robinia pseudoacacia growing in a lead-zinc mine in Mianxian County, Shaanxi Province, China. The bacterium was Gram-stain-negative, aerobic, motile, slightly curved- and rod-shaped, methyl red-negative, catalase-positive, and did not produce H2S. Strain HZ10 T grew at 4-45 °C (optimum, 25-30 °C), pH 5-9 (optimum, pH 7-8) and 0-1 % (w/v) NaCl. The major fatty acids were identified as C16 : 0, summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), and the quinone type was Q-8. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content of the genomic DNA was 64.9 mol% based on the whole genome sequence. According to the 16S rRNA gene sequence analysis, the closest phylogenetic relative to strain HZ10 T is Herbaspirillum chlorophenolicum CPW301 T (98.72 % sequence identity). Genome relatedness of the type strains H. chlorophenolicum CPW301 T , Herbaspirillum seropedicae Z67 T and Herbaspirillum aquaticum IEH 4430 T , was quantified by using the average nucleotide identity (86.9-88.0 %) and a genome-to-genome distance analysis (26.6 %-29.3 %), with both strongly supporting the notion that strain HZ10 T belongs to the genus Herbaspirillum as a novel species. Based on the results from phylogenetic, chemotaxonomic and physiological analyses, strain HZ10 T represents a novel Herbaspirillum species, for which the name Herbaspirillum robiniae sp. nov. is proposed. The type strain is HZ10 T (=JCM 31754 T =CCTCC AB 2014352 T ).

Cellulophaga geojensis sp. nov., a member of the family Flavobacteriaceae isolated from marine sand.

PubMed

Park, Sooyeon; Oh, Ki-Hoon; Lee, Soo-Young; Oh, Tae-Kwang; Yoon, Jung-Hoon

2012-06-01

A Gram-stain-negative, aerobic, non-flagellated, non-spore-forming, motile (by gliding) bacterial strain, designated M-M6(T), was isolated from marine sand of Geoje island, Korea. Strain M-M6(T) grew optimally at 25 °C, at pH 7.0-8.0 and in the presence of 2 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain M-M6(T) fell within the clade comprising Cellulophaga species, forming a coherent cluster with Cellulophaga lytica ATCC 23178(T) and Cellulophaga fucicola NN015860(T), with which it shared 16S rRNA gene sequence similarities of 98.1 and 98.2 %, respectively. Sequence similarities between strain M-M6(T) and the type strains of other recognized Cellulophaga species were in the range 92.4-93.8 %. Strain M-M6(T) contained MK-6 as the predominant menaquinone and iso-C(15:0), iso-C(15:1) G, iso-C(17:0) 3-OH, and C(16:1)ω7c and/or iso-C(15:0) 2-OH as the major fatty acids. The major polar lipids detected in strain M-M6(T) and the type strains of C. lytica and C. fucicola were two unidentified lipids, one unidentified aminolipid and one unidentified aminophospholipid. The DNA G+C content of strain M-M6(T) was 35.4 mol%. Levels of DNA-DNA relatedness between strain M-M6(T) and C. lytica JCM 8516(T) and C. fucicola JCM 21778(T) were 33 and 35 %, respectively. Differential phenotypic properties and phylogenetic and genetic distinctiveness distinguished strain M-M6(T) from all recognized Cellulophaga species. On the basis of the data presented, strain M-M6(T) is considered to represent a novel species of the genus Cellulophaga, for which the name Cellulophaga geojensis sp. nov. is proposed. The type strain is M-M6(T) ( = KCTC 23498(T) = CCUG 60801(T)).

Recombination Is a Major Driving Force of Genetic Diversity in the Anaplasmataceae Ehrlichia ruminantium.

PubMed

Cangi, Nídia; Gordon, Jonathan L; Bournez, Laure; Pinarello, Valérie; Aprelon, Rosalie; Huber, Karine; Lefrançois, Thierry; Neves, Luís; Meyer, Damien F; Vachiéry, Nathalie

2016-01-01

The disease, Heartwater, caused by the Anaplasmataceae E. ruminantium , represents a major problem for tropical livestock and wild ruminants. Up to now, no effective vaccine has been available due to a limited cross protection of vaccinal strains on field strains and a high genetic diversity of Ehrlichia ruminantium within geographical locations. To address this issue, we inferred the genetic diversity and population structure of 194 E. ruminantium isolates circulating worldwide using Multilocus Sequence Typing based on lipA, lipB, secY, sodB , and sucA genes . Phylogenetic trees and networks were generated using BEAST and SplitsTree, respectively, and recombination between the different genetic groups was tested using the PHI test for recombination. Our study reveals the repeated occurrence of recombination between E. ruminantium strains, suggesting that it may occur frequently in the genome and has likely played an important role in the maintenance of genetic diversity and the evolution of E. ruminantium . Despite the unclear phylogeny and phylogeography, E. ruminantium isolates are clustered into two main groups: Group 1 (West Africa) and a Group 2 (worldwide) which is represented by West, East, and Southern Africa, Indian Ocean, and Caribbean strains. Some sequence types are common between West Africa and Caribbean and between Southern Africa and Indian Ocean strains. These common sequence types highlight two main introduction events due to the movement of cattle: from West Africa to Caribbean and from Southern Africa to the Indian Ocean islands. Due to the long branch lengths between Group 1 and Group 2, and the propensity for recombination between these groups, it seems that the West African clusters of Subgroup 2 arrived there more recently than the original divergence of the two groups, possibly with the original waves of domesticated ruminants that spread across the African continent several thousand years ago.

Question 7: Comparative Genomics and Early Cell Evolution: A Cautionary Methodological Note

NASA Astrophysics Data System (ADS)

Islas, Sara; Hernández-Morales, Ricardo; Lazcano, Antonio

2007-10-01

Inventories of the gene content of the last common ancestor (LCA), i.e., the cenancestor, include sequences that may have undergone horizontal transfer events, as well as sequences that have originated in different pre-cenancestral epochs. However, the universal distribution of highly conserved genes involved in RNA metabolism provide insights into early stages of cell evolution during which RNA played a much more conspicuous biological role, and is consistent with the hypothesis that extant living systems were preceded by an RNA/protein world. Insights into the traits of primitive entities from which the LCA evolved may be derived from the analysis of paralogous gene families, including those formed by sequences that resulted from internal elongation events. Three major types of paralogous gene families can be recognized. The importance of this grouping for understanding the traits of early cells is discussed.

Isolation and sequence analysis of a canine distemper virus from a raccoon dog in Jilin Province, China.

PubMed

Cheng, Yuening; Wang, Jianke; Zhang, Miao; Zhao, Jianjun; Shao, Xiqun; Ma, Zengjun; Zhao, Hang; Lin, Peng; Wu, Hua

2015-10-01

Canine distemper virus (CDV) is a major pathogen not only in raccoon dogs but also in a variety of carnivorous animals, including domesticated animals, particularly if they have not been vaccinated. In this study, a wild-type strain of CDV was isolated from lung tissue from a raccoon dog kept at a fur farm in Jilin Province, China. Cytopathic effects typical of CDV infection were observed after three blind passages in Vero cells, yielding a virus titer of 10(4.6) TCID50/mL. Virus identification was carried out by RT-PCR, immunofluorescence, electron microscopy, and genome sequencing. The results showed that the isolated virus, termed the SY strain, corresponded to the Asia-1 genotype of CDV and has a genome of 15,690 nucleotides. This represents the first complete nucleotide sequence of a CDV strain circulating in raccoon dogs in China.

Rhizocola hellebori gen. nov., sp. nov., an actinomycete of the family Micromonosporaceae containing 3,4-dihydroxydiaminopimelic acid in the cell-wall peptidoglycan.

PubMed

Matsumoto, Atsuko; Kawaguchi, Yoko; Nakashima, Takuji; Iwatsuki, Masato; Ōmura, Satoshi; Takahashi, Yōko

2014-08-01

An actinomycete strain, K12-0602(T), was isolated from the root of a Helleborus orientalis plant in Japan. The 16S rRNA gene sequence of strain K12-0602(T) showed that it had a close relationship with members of the family Micromonosporaceae and the 16S rRNA gene sequence similarity values between strain K12-0602(T) and type strains of type species of 27 genera belonging to the family Micromonosporaceae were below 96.2%. MK-9 (H4) and MK-9 (H6) were detected as major menaquinones, and galactose, xylose, mannose and ribose were present in the whole-cell hydrolysate. The acyl type of the peptidoglycan was glycolyl. Major fatty acids were iso-C(15 : 0), iso-C(16 : 0), C(17 : 1)ω9c and anteiso-C(17 : 0). Phosphatidylethanolamine was detected as the phospholipid corresponding to phospholipid type II. The G+C content of the genomic DNA was 67 mol%. Analyses of the cell-wall peptidoglycan by TLC and LC/MS showed that it was composed of alanine, glycine, hydroxylglutamic acid and an unknown amino acid, which was subsequently determined to be 3,4-dihydroxydiaminopimelic acid using instrumental analyses, including NMR and mass spectrometry. On the basis of the phylogenetic analysis and chemotaxonomic characteristics, strain K12-0602(T) represents a novel species of a new genus in the family Micromonosporaceae, for which the name Rhizocola hellebori gen. nov., sp. nov. is proposed. The type strain of the type species is K12-0602(T) ( = NBRC 109834(T) = DSM 45988(T)). This is the first report, to our knowledge, of 3,4-dihydroxydiaminopimelic acid being found as a diamino acid in bacterial cell-wall peptidoglycan. © 2014 IUMS.

Metagenomic and metabolic profiling of nonlithifying and lithifying stromatolitic mats of Highborne Cay, The Bahamas.

PubMed

Khodadad, Christina L M; Foster, Jamie S

2012-01-01

Stromatolites are laminated carbonate build-ups formed by the metabolic activity of microbial mats and represent one of the oldest known ecosystems on Earth. In this study, we examined a living stromatolite located within the Exuma Sound, The Bahamas and profiled the metagenome and metabolic potential underlying these complex microbial communities. The metagenomes of the two dominant stromatolitic mat types, a nonlithifying (Type 1) and lithifying (Type 3) microbial mat, were partially sequenced and compared. This deep-sequencing approach was complemented by profiling the substrate utilization patterns of the mats using metabolic microarrays. Taxonomic assessment of the protein-encoding genes confirmed previous SSU rRNA analyses that bacteria dominate the metagenome of both mat types. Eukaryotes comprised less than 13% of the metagenomes and were rich in sequences associated with nematodes and heterotrophic protists. Comparative genomic analyses of the functional genes revealed extensive similarities in most of the subsystems between the nonlithifying and lithifying mat types. The one exception was an increase in the relative abundance of certain genes associated with carbohydrate metabolism in the lithifying Type 3 mats. Specifically, genes associated with the degradation of carbohydrates commonly found in exopolymeric substances, such as hexoses, deoxy- and acidic sugars were found. The genetic differences in carbohydrate metabolisms between the two mat types were confirmed using metabolic microarrays. Lithifying mats had a significant increase in diversity and utilization of carbon, nitrogen, phosphorus and sulfur substrates. The two stromatolitic mat types retained similar microbial communities, functional diversity and many genetic components within their metagenomes. However, there were major differences detected in the activity and genetic pathways of organic carbon utilization. These differences provide a strong link between the metagenome and the physiology of the mats, as well as new insights into the biological processes associated with carbonate precipitation in modern marine stromatolites.

Animal selection for whole genome sequencing by quantifying the unique contribution of homozygous haplotypes sequenced

USDA-ARS?s Scientific Manuscript database

Major whole genome sequencing projects promise to identify rare and causal variants within livestock species; however, the efficient selection of animals for sequencing remains a major problem within these surveys. The goal of this project was to develop a library of high accuracy genetic variants f...

Veillonella infantium sp. nov., an anaerobic, Gram-stain-negative coccus isolated from tongue biofilm of a Thai child.

PubMed

Mashima, Izumi; Liao, Yu-Chieh; Miyakawa, Hiroshi; Theodorea, Citra F; Thawboon, Boonyanit; Thaweboon, Sroisiri; Scannapieco, Frank A; Nakazawa, Futoshi

2018-04-01

A strain of a novel anaerobic, Gram-stain-negative coccus was isolated from the tongue biofilm of a Thai child. This strain was shown, at the phenotypic level and based on 16S rRNA gene sequencing, to be a member of the genus Veillonella. Comparative analysis of the 16S rRNA, dnaK and rpoB gene sequences indicated that phylogenetically the strain comprised a distinct novel branch within the genus Veillonella. The novel strain showed 99.8, 95.1 and 95.9 % similarity to partial 16S rRNA, dnaK and rpoB gene sequences, respectively, to the type strains of the two most closely related species, Veillonelladispar ATCC 17748 T and Veillonellatobetsuensis ATCC BAA-2400 T . The novel strain could be discriminated from previously reported species of the genus Veillonella based on partial dnaK and rpoB gene sequencing and average nucleotide identity values. The major acid end-product produced by this strain was acetic acid under anaerobic conditions in trypticase-yeast extract-haemin with 1 % (w/v) glucose or fructose medium. Lactate was fermented to acetic acid and propionic acid. Based on these observations, this strain represents a novel species, for which the name Veillonella infantium sp. nov. is proposed. The type strain is T11011-4 T (=JCM 31738 T =TSD-88 T ).

Methylobacterium phyllosphaerae sp. nov., a pink-pigmented, facultative methylotroph from the phyllosphere of rice.

PubMed

Madhaiyan, Munusamy; Poonguzhali, Selvaraj; Kwon, Soon-Wo; Sa, Tong-Min

2009-01-01

A pink-pigmented, aerobic, facultatively methylotrophic bacterial strain, CBMB27T, isolated from leaf tissues of rice (Oryza sativa L. 'Dong-Jin'), was analysed using a polyphasic taxonomic approach. Comparative 16S rRNA gene sequence-based phylogenetic analysis placed the strain in a clade with the species Methylobacterium oryzae, Methylobacterium fujisawaense and Methylobacterium mesophilicum; strain CBMB27T showed sequence similarities of 98.3, 98.5 and 97.3 %, respectively, to the type strains of these three species. DNA-DNA hybridization experiments revealed low levels (<38 %) of DNA-DNA relatedness between strain CBMB27T and its closest relatives. The sequence of the 1-aminocyclopropane-1-carboxylate deaminase gene (acdS) in strain CBMB27T differed from those of close relatives. The major fatty acid of the isolate was C(18 : 1)omega7c and the G+C content of the genomic DNA was 66.8 mol%. Based on the results of 16S rRNA gene sequence analysis, DNA-DNA hybridization, and physiological and biochemical characterization, which enabled the isolate to be differentiated from all recognized species of the genus Methylobacterium, it was concluded that strain CBMB27T represents a novel species in the genus Methylobacterium for which the name Methylobacterium phyllosphaerae sp. nov. is proposed (type strain CBMB27T =LMG 24361T =KACC 11716T =DSM 19779T).

Reclassification of Bacillus beijingensis Qiu et al. 2009 and Bacillus ginsengi Qiu et al. 2009 as Bhargavaea beijingensis comb. nov. and Bhargavaea ginsengi comb. nov. and emended description of the genus Bhargavaea.

PubMed

Verma, Pankaj; Pandey, Prashant Kumar; Gupta, Arvind Kumar; Seong, Chi Nam; Park, Seong Chan; Choe, Han Na; Baik, Keun Sik; Patole, Milind Shivaji; Shouche, Yogesh Shreepad

2012-10-01

We have carried out a polyphasic taxonomic characterization of Bacillus beijingensis DSM 19037(T) and Bacillus ginsengi DSM 19038(T), which are closely related phylogenetically to Bhargavaea cecembensis LMG 24411(T). All three strains are Gram-stain-positive, non-motile, moderately halotolerant and non-spore-forming. 16S rRNA gene sequence analyses showed that the strains constituted a coherent cluster, with sequence similarities between 99.7 and 98.7 %. The percentage similarity on the basis of amino acid sequences deduced from partial gyrB gene nucleotide sequences of these three type strains was 96.1-92.7 %. Phylogenetic trees based on the 16S rRNA gene and GyrB amino acid sequences, obtained by using three different algorithms, were consistent and showed that these three species constituted a deeply rooted cluster separated from the clades represented by the genera Bacillus, Planococcus, Planomicrobium, Sporosarcina, Lysinibacillus, Viridibacillus, Kurthia and Geobacillus, supporting their placement in the genus Bhargavaea. All three type strains have menaquinone MK-8 as the major respiratory quinone and showed similar fatty acid profiles. The main polar lipids present in the three type strains were diphosphatidylglycerol and phosphatidylglycerol, and the three strains showed peptidoglycan type A4α with L-lysine as the diagnostic diamino acid. The DNA G+C contents of Bacillus beijingensis DSM 19037(T), Bacillus ginsengi DSM 19038(T) and Bhargavaea cecembensis LMG 24411(T) were 53.1, 50.2 and 53.7 mol%, respectively. The level of DNA-DNA hybridization among the three strains was 57-39 %, indicating that they are members of different species of the genus Bhargavaea. The phenotypic data are consistent with the placement of these three species in a single genus and support their differentiation at the species level. On the basis of these data, we have emended the description of the genus Bhargavaea and propose the reclassification of Bacillus beijingensis and Bacillus ginsengi to the genus Bhargavaea, as Bhargavaea beijingensis comb. nov. (type strain ge10(T)  = DSM 19037(T)  = CGMCC 1.6762(T)) and Bhargavaea ginsengi comb. nov. (type strain ge14(T)  = DSM 19038(T)  = CGMCC 1.6763(T)).

Characterization of avian paramyxovirus type 6 isolated from a Eurasian teal in the intersection of migratory flyways in Russia.

PubMed

Sobolev, Ivan A; Sharshov, Kirill; Yurchenko, Kseniya; Korneev, Denis; Glushchenko, Alexandra; Alikina, Tatyana; Kabilov, Marsel; Bi, Yuhai; Liu, Wenjun; Gubanova, Natalia; Shestopalov, Alexander

2016-11-01

The complete genome sequence was determined for avian paramyxovirus (APMV-6) serotype 6 strain teal/Chany/455/2009, isolated from a teal (Anas crecca) in Siberia. Siberia is crossed by four major migration flyways and represents the major breeding area for many wild bird species in the Palearctic. Strain teal/Chany/455/2009 is genetically closely related to Kazakh and Chinese strains and belongs to the genetic group of duck/Hong Kong/18/199/77-like APMV-6 viruses. We show that the virus has low pathogenic potential according to genetic markers and animal model experiments.

Clonal spread of carbapenem-resistant Acinetobacter baumannii across a community hospital and its affiliated long-term care facilities: A cross sectional study.

PubMed

Chen, Chang-Hua; Kuo, Han-Yueh; Hsu, Po-Jui; Chang, Chien-Min; Chen, Jiann-Yuan; Lu, Henry Horng-Shing; Chen, Hsin-Yao; Liou, Ming-Li

2018-06-01

The global spread of carbapenem-resistant Acinetobacter baumannii (CRAB) is now a public health problem. In Taiwan, the relationship of the CRAB circulation between long-term care facilities (LTCFs) and acute care hospitals remains unclear. Here, we use molecular epidemiologic methods to describe the transmission of CRAB isolates between a community hospital and its affiliated LTCFs. Subjects localized in eight LTCFs who were not admitted acute care hospitals in recent a year were enrolled in this study. CRAB isolates were collected during June 1, 2015 and December 31, 2015. DNA fingerprinting was performed by repetitive extragenic palindromic sequence-based polymerase chain reaction (Rep-PCR) and multilocus sequence typing (MLST). Multiplex-PCR amplification for the detection of bla OXA genes and beta-lactamase genes was performed. Twenty one subjects were enrolled. The major hospital admission diagnoses among the 21 subjects were pneumonia (71.4%). Genotyping of CRAB isolates by Rep-PCR revealed that a major clone, designated as type III, comprised fifteen of 21 (71.4%) isolates taken from 5 LTCFs and one study hospital. The isolates with type III were subtyped by PubMLST into 4 ST types. The most prevalent bla OXA genes in these isolates were bla OXA-23 -like (85.70%, 18/21). Twenty isolates carried bla SHV. CONCLUSION: Clonal spread of bla OxA-23 -carrying CRABs was found around LTCFs and the affiliated hospital. In Taiwan, it is important for the government to focus attention on the importance of identifying and tracing CRAB infections in LTCFs. Copyright © 2017. Published by Elsevier B.V.

Identification of random nucleic acid sequence aberrations using dual capture probes which hybridize to different chromosome regions

DOEpatents

Lucas, J.N.; Straume, T.; Bogen, K.T.

1998-03-24

A method is provided for detecting nucleic acid sequence aberrations using two immobilization steps. According to the method, a nucleic acid sequence aberration is detected by detecting nucleic acid sequences having both a first nucleic acid sequence type (e.g., from a first chromosome) and a second nucleic acid sequence type (e.g., from a second chromosome), the presence of the first and the second nucleic acid sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. In the method, immobilization of a first hybridization probe is used to isolate a first set of nucleic acids in the sample which contain the first nucleic acid sequence type. Immobilization of a second hybridization probe is then used to isolate a second set of nucleic acids from within the first set of nucleic acids which contain the second nucleic acid sequence type. The second set of nucleic acids are then detected, their presence indicating the presence of a nucleic acid sequence aberration. 14 figs.

Identification of random nucleic acid sequence aberrations using dual capture probes which hybridize to different chromosome regions

DOEpatents

Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

1998-01-01

A method is provided for detecting nucleic acid sequence aberrations using two immobilization steps. According to the method, a nucleic acid sequence aberration is detected by detecting nucleic acid sequences having both a first nucleic acid sequence type (e.g., from a first chromosome) and a second nucleic acid sequence type (e.g., from a second chromosome), the presence of the first and the second nucleic acid sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. In the method, immobilization of a first hybridization probe is used to isolate a first set of nucleic acids in the sample which contain the first nucleic acid sequence type. Immobilization of a second hybridization probe is then used to isolate a second set of nucleic acids from within the first set of nucleic acids which contain the second nucleic acid sequence type. The second set of nucleic acids are then detected, their presence indicating the presence of a nucleic acid sequence aberration.

Genomic insights from whole genome sequencing of four clonal outbreak Campylobacter jejuni assessed within the global C. jejuni population.

PubMed

Clark, Clifford G; Berry, Chrystal; Walker, Matthew; Petkau, Aaron; Barker, Dillon O R; Guan, Cai; Reimer, Aleisha; Taboada, Eduardo N

2016-12-03

Whole genome sequencing (WGS) is useful for determining clusters of human cases, investigating outbreaks, and defining the population genetics of bacteria. It also provides information about other aspects of bacterial biology, including classical typing results, virulence, and adaptive strategies of the organism. Cell culture invasion and protein expression patterns of four related multilocus sequence type 21 (ST21) C. jejuni isolates from a significant Canadian water-borne outbreak were previously associated with the presence of a CJIE1 prophage. Whole genome sequencing was used to examine the genetic diversity among these isolates and confirm that previous observations could be attributed to differential prophage carriage. Moreover, we sought to determine the presence of genome sequences that could be used as surrogate markers to delineate outbreak-associated isolates. Differential carriage of the CJIE1 prophage was identified as the major genetic difference among the four outbreak isolates. High quality single-nucleotide variant (hqSNV) and core genome multilocus sequence typing (cgMLST) clustered these isolates within expanded datasets consisting of additional C. jejuni strains. The number and location of homopolymeric tract regions was identical in all four outbreak isolates but differed from all other C. jejuni examined. Comparative genomics and PCR amplification enabled the identification of large chromosomal inversions of approximately 93 kb and 388 kb within the outbreak isolates associated with transducer-like proteins containing long nucleotide repeat sequences. The 93-kb inversion was characteristic of the outbreak-associated isolates, and the gene content of this inverted region displayed high synteny with the reference strain. The four outbreak isolates were clonally derived and differed mainly in the presence of the CJIE1 prophage, validating earlier findings linking the prophage to phenotypic differences in virulence assays and protein expression. The identification of large, genetically syntenous chromosomal inversions in the genomes of outbreak-associated isolates provided a unique method for discriminating outbreak isolates from the background population. Transducer-like proteins appear to be associated with the chromosomal inversions. CgMLST and hqSNV analysis also effectively delineated the outbreak isolates within the larger C. jejuni population structure.

Multiple mitochondrial introgression events and heteroplasmy in trypanosoma cruzi revealed by maxicircle MLST and next generation sequencing.

PubMed

Messenger, Louisa A; Llewellyn, Martin S; Bhattacharyya, Tapan; Franzén, Oscar; Lewis, Michael D; Ramírez, Juan David; Carrasco, Hernan J; Andersson, Björn; Miles, Michael A

2012-01-01

Mitochondrial DNA is a valuable taxonomic marker due to its relatively fast rate of evolution. In Trypanosoma cruzi, the causative agent of Chagas disease, the mitochondrial genome has a unique structural organization consisting of 20-50 maxicircles (∼20 kb) and thousands of minicircles (0.5-10 kb). T. cruzi is an early diverging protist displaying remarkable genetic heterogeneity and is recognized as a complex of six discrete typing units (DTUs). The majority of infected humans are asymptomatic for life while 30-35% develop potentially fatal cardiac and/or digestive syndromes. However, the relationship between specific clinical outcomes and T. cruzi genotype remains elusive. The availability of whole genome sequences has driven advances in high resolution genotyping techniques and re-invigorated interest in exploring the diversity present within the various DTUs. To describe intra-DTU diversity, we developed a highly resolutive maxicircle multilocus sequence typing (mtMLST) scheme based on ten gene fragments. A panel of 32 TcI isolates was genotyped using the mtMLST scheme, GPI, mini-exon and 25 microsatellite loci. Comparison of nuclear and mitochondrial data revealed clearly incongruent phylogenetic histories among different geographical populations as well as major DTUs. In parallel, we exploited read depth data, generated by Illumina sequencing of the maxicircle genome from the TcI reference strain Sylvio X10/1, to provide the first evidence of mitochondrial heteroplasmy (heterogeneous mitochondrial genomes in an individual cell) in T. cruzi. mtMLST provides a powerful approach to genotyping at the sub-DTU level. This strategy will facilitate attempts to resolve phenotypic variation in T. cruzi and to address epidemiologically important hypotheses in conjunction with intensive spatio-temporal sampling. The observations of both general and specific incidences of nuclear-mitochondrial phylogenetic incongruence indicate that genetic recombination is geographically widespread and continues to influence the natural population structure of TcI, a conclusion which challenges the traditional paradigm of clonality in T. cruzi.

Characterization of rpoB mutations in rifampin-resistant clinical Mycobacterium tuberculosis isolates from Kuwait and Dubai.

PubMed

Ahmad, Suhail; Mokaddas, Eiman; Fares, Esther

2002-11-01

Mutations conferring resistance to rifampin in rifampin-resistant clinical Mycobacterium tuberculosis isolates occur mostly in the 81 bp rifampin-resistance-determining region (RRDR) of the rpoB gene. In this study, 29 rifampin-resistant and 12 -susceptible clinical M. tuberculosis isolates were tested for characterization of mutations in the rpoB gene by line probe (INNO-LiPA Rif. TB) assay and the results were confirmed and extended by DNA sequencing of the PCR amplified target DNA. The line probe assay identified all 12 susceptible strains as rifampin-sensitive and the DNA sequence of RRDR in the amplified rpoB gene from two isolates matched perfectly with the wild-type sequence. The line probe assay identified 28 resistant isolates as rifampin-resistant with specific detection of mutation in 22 isolates including one isolate that exhibited hetro-resistance containing both the wild-type pattern as well as a specific mutation within RRDR while one of the rifampin-resistant strain was identified as rifampin-susceptible. DNA sequencing confirmed these results and, in addition, led to the specific detection of mutations in 5 rifampin-resistant isolates in which specific base changes within RRDR could not be determined by the line probe assay. These analyses identified 8 different mutations within RRDR of the rpoB gene including one novel mutation (S522W) that has not been reported so far. The genotyping performed on the isolates carrying similar mutations showed that majority of these isolates were unique as they exhibited varying DNA banding patterns. Correlating the ethnic origin of the infected TB patients with the occurrence of specific mutations at three main codon positions (516, 526 and 531) in the rpoB gene showed that most patients (11 of 15) from South Asian region contained mutations at codon 526 while majority of isolates from patients (6 of 11) of Middle Eastern origin contained mutations at codon 531.

Amino acid and structural variability of Yersinia pestis LcrV protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anisimov, A P; Dentovskaya, S V; Panfertsev, E A

2009-11-09

The LcrV protein is a multifunctional virulence factor and protective antigen of the plague bacterium which is generally conserved between the epidemic strains of Yersinia pestis. They investigated the diversity in the LcrV sequences among non-epidemic Y. pestis strains which have a limited virulence in selected animal models and for humans. Sequencing of lcrV genes from ten Y. pestis strains belonging to different phylogenetic groups (subspecies) showed that the LcrV proteins possess four major variable hotspots at positions 18, 72, 273, and 324-326. These major variations, together with other minor substitutions in amino acid sequences, allowed them to classify themore » LcrV alleles into five sequence types (A-E). They observed that the strains of different Y. pestis subspecies can have the same typ of LcrV, and different types of LcrV can exist within the same natural plague focus. The LcrV polymorphisms were structurally analyzed by comparing the modeled structures of LcrV from all available strains. All changes except one occurred either in flexible regions or on the surface of the protein, but local chemical properties (i.e. those of a hydrophobic, hydrophilic, amphipathic, or charged nature) were conserved across all of the strains. Polymorphisms in flexible and surface regions are likely subject to less selective pressure, and have a limited impact on the structure. In contrast, the substitution of tryptophan at position 113 with either glutamic acid or glycine likely has a serious influence on the regional structure of the protein, and these mutations might have an effect on the function of LcrV. The polymorphisms at positions 18, 72 and 273 were accountable for differences in oligomerization of LcrV. The importance of the latter property in emergence of epidemic strains of Y. pestis during evolution of this pathogen will need to be further investigated.« less

Contrasting patterns of evolution of 45S and 5S rDNA families uncover new aspects in the genome constitution of the agronomically important grass Thinopyrum intermedium (Triticeae).

PubMed

Mahelka, Václav; Kopecky, David; Baum, Bernard R

2013-09-01

We employed sequencing of clones and in situ hybridization (genomic and fluorescent in situ hybridization [GISH and rDNA-FISH]) to characterize both the sequence variation and genomic organization of 45S (herein ITS1-5.8S-ITS2 region) and 5S (5S gene + nontranscribed spacer) ribosomal DNA (rDNA) families in the allohexaploid grass Thinopyrum intermedium. Both rDNA families are organized within several rDNA loci within all three subgenomes of the allohexaploid species. Both families have undergone different patterns of evolution. The 45S rDNA family has evolved in a concerted manner: internal transcribed spacer (ITS) sequences residing within the arrays of two subgenomes out of three got homogenized toward one major ribotype, whereas the third subgenome contained a minor proportion of distinct unhomogenized copies. Homogenization mechanisms such as unequal crossover and/or gene conversion were coupled with the loss of certain 45S rDNA loci. Unlike in the 45S family, the data suggest that neither interlocus homogenization among homeologous chromosomes nor locus loss occurred in 5S rDNA. Consistently with other Triticeae, the 5S rDNA family in intermediate wheatgrass comprised two distinct array types-the long- and short-spacer unit classes. Within the long and short units, we distinguished five and three different types, respectively, likely representing homeologous unit classes donated by putative parental species. Although the major ITS ribotype corresponds in our phylogenetic analysis to the E-genome species, the minor ribotype corresponds to Dasypyrum. 5S sequences suggested the contributions from Pseudoroegneria, Dasypyrum, and Aegilops. The contribution from Aegilops to the intermediate wheatgrass' genome is a new finding with implications in wheat improvement. We discuss rDNA evolution and potential origin of intermediate wheatgrass.

Impact of cultivation on characterisation of species composition of soil bacterial communities.

PubMed

McCaig, A E.; Grayston, S J.; Prosser, J I.; Glover, L A.

2001-03-01

The species composition of culturable bacteria in Scottish grassland soils was investigated using a combination of Biolog and 16S rDNA analysis for characterisation of isolates. The inclusion of a molecular approach allowed direct comparison of sequences from culturable bacteria with sequences obtained during analysis of DNA extracted directly from the same soil samples. Bacterial strains were isolated on Pseudomonas isolation agar (PIA), a selective medium, and on tryptone soya agar (TSA), a general laboratory medium. In total, 12 and 21 morphologically different bacterial cultures were isolated on PIA and TSA, respectively. Biolog and sequencing placed PIA isolates in the same taxonomic groups, the majority of cultures belonging to the Pseudomonas (sensu stricto) group. However, analysis of 16S rDNA sequences proved more efficient than Biolog for characterising TSA isolates due to limitations of the Microlog database for identifying environmental bacteria. In general, 16S rDNA sequences from TSA isolates showed high similarities to cultured species represented in sequence databases, although TSA-8 showed only 92.5% similarity to the nearest relative, Bacillus insolitus. In general, there was very little overlap between the culturable and uncultured bacterial communities, although two sequences, PIA-2 and TSA-13, showed >99% similarity to soil clones. A cloning step was included prior to sequence analysis of two isolates, TSA-5 and TSA-14, and analysis of several clones confirmed that these cultures comprised at least four and three sequence types, respectively. All isolate clones were most closely related to uncultured bacteria, with clone TSA-5.1 showing 99.8% similarity to a sequence amplified directly from the same soil sample. Interestingly, one clone, TSA-5.4, clustered within a novel group comprising only uncultured sequences. This group, which is associated with the novel, deep-branching Acidobacterium capsulatum lineage, also included clones isolated during direct analysis of the same soil and from a wide range of other sample types studied elsewhere. The study demonstrates the value of fine-scale molecular analysis for identification of laboratory isolates and indicates the culturability of approximately 1% of the total population but under a restricted range of media and cultivation conditions.

Ogataea pignaliae sp. nov., the teleomorph of Candida pignaliae.

PubMed

Péter, Gábor; Tornai-Lehoczki, Judit; Dlauchy, Dénes

2010-10-01

Six ascosporulating Candida pignaliae strains were isolated from epigeal plant parts in Hungary. They share identical D1/D2 LSU rRNA gene sequences with the type strain of C. pignaliae, and the physiological characteristics investigated are also very similar to that of the type strain. The only substantial difference compared to the type strain of C. pignaliae is their ability to assimilate β-glucosides (cellobiose, salicin and arbutin). The majority of the isolation sources of the strains reported in this study have the common feature of containing tannic acid, while the type strain of C. pignaliae was recovered from tanning fluid. We were able to induce ascosporulation also in the type strain of C. pignaliae. Therefore, Ogataea pignaliae Péter, Tornai-Lehoczki & Dlauchy sp. nov. is proposed as the teleomorph of C. pignaliae (F. H. Jacob) S. A. Meyer & Yarrow. The type strain is CBS 6071(T).

tRNAs as Therapeutic Agents of Breast Cancer

DTIC Science & Technology

2013-07-01

their anticodon sequence. Wild-type tRNA reads codon for serine, Suppressor (Sup) tRNA for amber stop, and killer tRNA for isoleucine . Figure 6...endoplasmic reticulum (ER) is a eukaryotic organelle that performs the major functions of synthesizing and packaging pro- teins. Overloading of...anticodons tested in HeLa, tRNASer with the AAU anticodon (tRNASer(AAU)) leads to the substitution of isoleucine with serine within the proteome and

NOSS altimeter algorithm specifications

NASA Technical Reports Server (NTRS)

Hancock, D. W.; Forsythe, R. G.; Mcmillan, J. D.

1982-01-01

A description of all algorithms required for altimeter processing is given. Each description includes title, description, inputs/outputs, general algebraic sequences and data volume. All required input/output data files are described and the computer resources required for the entire altimeter processing system were estimated. The majority of the data processing requirements for any radar altimeter of the Seasat-1 type are scoped. Additions and deletions could be made for the specific altimeter products required by other projects.

Molecular analysis and antimicrobial susceptibility of methicillin resistant Staphylococcus aureus in one of the hospitals of Tehran University of Medical Sciences: high prevalence of sequence type 239 (ST239) clone.

PubMed

Shahsavan, Shadi; Jabalameli, Leila; Maleknejad, Parviz; Aligholi, Marzieh; Imaneini, Hossein; Jabalameli, Fereshteh; Halimi, Shahnaz; Taherikalani, Morovat; Khoramian, Babak; Eslampour, Mohammad Amin; Feizabadi, Mohammad Mehdi; Emaneini, Mohammad

2011-03-01

Methicillin-resistant Staphylococcus aureus (MRSA), particularly the multidrug-resistant clones, is an increasing worldwide problem. The average incidence rate of MRSA in Tehran was found to be over 40%. A total of 140 MRSA isolates obtained from patients attending a teaching hospital in Tehran, from May 2009 to December 2009, were included in this study. The antimicrobial susceptibility profile of MRSA isolates was determined by the agar disk diffusion method. Molecular analysis of MRSA strains was accomplished by Pulsed-Field Gel Electrophoresis (PFGE) and Multi-locus sequence typing (MLST). Detection of mecA gene was used to confirm resistance to methicillin among the MRSA isolates. All the MRSA isolates were susceptible to chloramphenicol, teicoplanin, tigecycline and vancomycin. All MRSAisolates were resistant to oxacillin, whilst 139 strains showed resistance against ciprofloxacin, erythromycin, gentamicin, tetracycline and trimethoprim-sulfamethoxazole. PFGE analysis of all the 140 MRSA isolates produced five distinct pulsotypes designated as pulsotypes A-E. Most of the isolates (n=132) were clustered into pulsotype A. The most prevalent sequence type (ST) was ST 239 (pulsotype A) found in 82% (37/45) of the tested isolates. The second most prevalent type was ST 1238 (pulsotypes B, C and D) found in 15% (7/45) of the isolates. The remaining type, ST 8 (pulsotype E) was found in a single isolate. The results of this study indicated that the MRSA clone ST 239 was a major clone in the selected university hospital of Tehran and that it was widely spread among the different wards as well as all the age groups of patients.

Molecular Epidemiology and Clinical Impact of Acinetobacter calcoaceticus-baumannii Complex in a Belgian Burn Wound Center

PubMed Central

Bilocq, Florence; Jennes, Serge; Verbeken, Gilbert; Rose, Thomas; Keersebilck, Elkana; Bosmans, Petra; Pieters, Thierry; Hing, Mony; Heuninckx, Walter; De Pauw, Frank; Soentjens, Patrick; Merabishvili, Maia; Deschaght, Pieter; Vaneechoutte, Mario; Bogaerts, Pierre; Glupczynski, Youri; Pot, Bruno; van der Reijden, Tanny J.; Dijkshoorn, Lenie

2016-01-01

Multidrug resistant Acinetobacter baumannii and its closely related species A. pittii and A. nosocomialis, all members of the Acinetobacter calcoaceticus-baumannii (Acb) complex, are a major cause of hospital acquired infection. In the burn wound center of the Queen Astrid military hospital in Brussels, 48 patients were colonized or infected with Acb complex over a 52-month period. We report the molecular epidemiology of these organisms, their clinical impact and infection control measures taken. A representative set of 157 Acb complex isolates was analyzed using repetitive sequence-based PCR (rep-PCR) (DiversiLab) and a multiplex PCR targeting OXA-51-like and OXA-23-like genes. We identified 31 rep-PCR genotypes (strains). Representatives of each rep-type were identified to species by rpoB sequence analysis: 13 types to A. baumannii, 10 to A. pittii, and 3 to A. nosocomialis. It was assumed that isolates that belonged to the same rep-type also belonged to the same species. Thus, 83.4% of all isolates were identified to A. baumannii, 9.6% to A. pittii and 4.5% to A. nosocomialis. We observed 12 extensively drug resistant Acb strains (10 A. baumannii and 2 A. nosocomialis), all carbapenem-non-susceptible/colistin-susceptible and imported into the burn wound center through patients injured in North Africa. The two most prevalent rep-types 12 and 13 harbored an OXA-23-like gene. Multilocus sequence typing allocated them to clonal complex 1 corresponding to EU (international) clone I. Both strains caused consecutive outbreaks, interspersed with periods of apparent eradication. Patients infected with carbapenem resistant A. baumannii were successfully treated with colistin/rifampicin. Extensive infection control measures were required to eradicate the organisms. Acinetobacter infection and colonization was not associated with increased attributable mortality. PMID:27223476

Antimicrobial Resistance Mechanisms and Genetic Diversity of Multidrug-Resistant Acinetobacter baumannii Isolated from a Teaching Hospital in Malaysia.

PubMed

Biglari, Shirin; Hanafiah, Alfizah; Mohd Puzi, Shaliawani; Ramli, Ramliza; Rahman, Mostafizur; Lopes, Bruno Silvester

2017-07-01

Multidrug-resistant (MDR) Acinetobacter baumannii has increasingly emerged as an important nosocomial pathogen. The aim of this study was to determine the resistance profiles and genetic diversity in A. baumannii clinical isolates in a tertiary medical center in Malaysia. The minimum inhibitory concentrations of carbapenems (imipenem and meropenem), cephalosporins (ceftazidime and cefepime), and ciprofloxacin were determined by E-test. PCR and sequencing were carried out for the detection of antibiotic resistance genes and mutations. Clonal relatedness among A. baumannii isolates was determined by REP-PCR. Sequence-based typing of OXA-51 and multilocus sequence typing were performed. One hundred twenty-five of 162 (77.2%) A. baumannii isolates had MDR phenotype. From the 162 A. baumannii isolates, 20 strain types were identified and majority of A. baumannii isolates (66%, n = 107) were classified as strain type 1 and were positive for ISAba1-bla OXA-23 and ISAba1-bla ADC and had mutations in both gyrA and parC genes at positions, 83 and 80, resulting in serine-to-leucine conversion. REP-PCR analysis showed 129 REP types that generated 31 clones with a 90% similarity cutoff value. OXA-66 variant of the bla OXA-51-like genes was predominantly detected among our A. baumannii clinical isolates belonging to ST195 (found in six clones: 1, 8, 9, 19, 27, and 30) and ST208 (found in clone 21). The study helps us in understanding the genetic diversity of A. baumannii isolates in our setting and confirms that international clone II is the most widely distributed clone in Universiti Kebangsaan Malaysia Medical Centre, Malaysia.

Prevalence, antimicrobial resistance and genetic diversity of Campylobacter coli and Campylobacter jejuni in Ecuadorian broilers at slaughter age

PubMed Central

Vinueza-Burgos, Christian; Wautier, Magali; Martiny, Delphine; Cisneros, Marco; Van Damme, Inge; De Zutter, Lieven

2017-01-01

Abstract Thermotolerant Campylobacter spp. are a major cause of foodborne gastrointestinal infections worldwide. The linkage of human campylobacteriosis and poultry has been widely described. In this study we aimed to investigate the prevalence, antimicrobial resistance and genetic diversity of C. coli and C. jejuni in broilers from Ecuador. Caecal content from 379 randomly selected broiler batches originating from 115 farms were collected from 6 slaughterhouses located in the province of Pichincha during 1 year. Microbiological isolation was performed by direct plating on mCCDA agar. Identification of Campylobacter species was done by PCR. Minimum inhibitory concentration (MIC) values for gentamicin, ciprofloxacin, nalidixic acid, tetracycline, streptomycin, and erythromycin were obtained. Genetic variation was assessed by RFLP-flaA typing and Multilocus Sequence Typing (MLST) of selected isolates. Prevalence at batch level was 64.1%. Of the positive batches 68.7% were positive for C. coli, 18.9% for C. jejuni, and 12.4% for C. coli and C. jejuni. Resistance rates above 67% were shown for tetracycline, ciprofloxacin, and nalidixic acid. The resistance pattern tetracycline, ciprofloxin, and nalidixic acid was the dominant one in both Campylobacter species. RFLP-flaA typing analysis showed that C. coli and C. jejuni strains belonged to 38 and 26 profiles respectively. On the other hand MLST typing revealed that C. coli except one strain belonged to CC-828, while C. jejuni except 2 strains belonged to 12 assigned clonal complexes (CCs). Furthermore 4 new sequence types (STs) for both species were described, whereby 2 new STs for C. coli were based on new allele sequences. Further research is necessary to estimate the impact of the slaughter of Campylobacter positive broiler batches on the contamination level of carcasses in slaughterhouses and at retail in Ecuador. PMID:28339716

Molecular Epidemiology and Clinical Impact of Acinetobacter calcoaceticus-baumannii Complex in a Belgian Burn Wound Center.

PubMed

De Vos, Daniel; Pirnay, Jean-Paul; Bilocq, Florence; Jennes, Serge; Verbeken, Gilbert; Rose, Thomas; Keersebilck, Elkana; Bosmans, Petra; Pieters, Thierry; Hing, Mony; Heuninckx, Walter; De Pauw, Frank; Soentjens, Patrick; Merabishvili, Maia; Deschaght, Pieter; Vaneechoutte, Mario; Bogaerts, Pierre; Glupczynski, Youri; Pot, Bruno; van der Reijden, Tanny J; Dijkshoorn, Lenie

2016-01-01

Multidrug resistant Acinetobacter baumannii and its closely related species A. pittii and A. nosocomialis, all members of the Acinetobacter calcoaceticus-baumannii (Acb) complex, are a major cause of hospital acquired infection. In the burn wound center of the Queen Astrid military hospital in Brussels, 48 patients were colonized or infected with Acb complex over a 52-month period. We report the molecular epidemiology of these organisms, their clinical impact and infection control measures taken. A representative set of 157 Acb complex isolates was analyzed using repetitive sequence-based PCR (rep-PCR) (DiversiLab) and a multiplex PCR targeting OXA-51-like and OXA-23-like genes. We identified 31 rep-PCR genotypes (strains). Representatives of each rep-type were identified to species by rpoB sequence analysis: 13 types to A. baumannii, 10 to A. pittii, and 3 to A. nosocomialis. It was assumed that isolates that belonged to the same rep-type also belonged to the same species. Thus, 83.4% of all isolates were identified to A. baumannii, 9.6% to A. pittii and 4.5% to A. nosocomialis. We observed 12 extensively drug resistant Acb strains (10 A. baumannii and 2 A. nosocomialis), all carbapenem-non-susceptible/colistin-susceptible and imported into the burn wound center through patients injured in North Africa. The two most prevalent rep-types 12 and 13 harbored an OXA-23-like gene. Multilocus sequence typing allocated them to clonal complex 1 corresponding to EU (international) clone I. Both strains caused consecutive outbreaks, interspersed with periods of apparent eradication. Patients infected with carbapenem resistant A. baumannii were successfully treated with colistin/rifampicin. Extensive infection control measures were required to eradicate the organisms. Acinetobacter infection and colonization was not associated with increased attributable mortality.

Evidence of genotypic diversity among Candida auris isolates by multilocus sequence typing, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and amplified fragment length polymorphism.

PubMed

Prakash, A; Sharma, C; Singh, A; Kumar Singh, P; Kumar, A; Hagen, F; Govender, N P; Colombo, A L; Meis, J F; Chowdhary, A

2016-03-01

Candida auris is a multidrug-resistant nosocomial bloodstream pathogen that has been reported from Asian countries and South Africa. Herein, we studied the population structure and genetic relatedness among 104 global C. auris isolates from India, South Africa and Brazil using multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) fingerprinting and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). RPB1, RPB2 and internal transcribed spacer (ITS) and D1/D2 regions of the ribosomal DNA were sequenced for MLST. Further, genetic variation and proteomic assessment was carried out using AFLP and MALDI-TOF MS, respectively. Both MLST and AFLP typing clearly demarcated two major clusters comprising Indian and Brazilian isolates. However, the South African isolates were randomly distributed, suggesting different genotypes. MALDI-TOF MS spectral profiling also revealed evidence of geographical clustering but did not correlate fully with the genotyping methods. Notably, overall the population structure of C. auris showed evidence of geographical clustering by all the three techniques analysed. Antifungal susceptibility testing by the CLSI microbroth dilution method revealed that fluconazole had limited activity against 87% of isolates (MIC90, 64 mg/L). Also, MIC90 of AMB was 4 mg/L. Candida auris is emerging as an important yeast pathogen globally and requires reproducible laboratory methods for identification and typing. Evaluation of MALDI-TOF MS as a typing method for this yeast is warranted. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Identification of Variable-Number Tandem-Repeat (VNTR) Sequences in Acinetobacter baumannii and Interlaboratory Validation of an Optimized Multiple-Locus VNTR Analysis Typing Scheme▿†

PubMed Central

Pourcel, Christine; Minandri, Fabrizia; Hauck, Yolande; D'Arezzo, Silvia; Imperi, Francesco; Vergnaud, Gilles; Visca, Paolo

2011-01-01

Acinetobacter baumannii is an important opportunistic pathogen responsible for nosocomial outbreaks, mostly occurring in intensive care units. Due to the multiplicity of infection sources, reliable molecular fingerprinting techniques are needed to establish epidemiological correlations among A. baumannii isolates. Multiple-locus variable-number tandem-repeat analysis (MLVA) has proven to be a fast, reliable, and cost-effective typing method for several bacterial species. In this study, an MLVA assay compatible with simple PCR- and agarose gel-based electrophoresis steps as well as with high-throughput automated methods was developed for A. baumannii typing. Preliminarily, 10 potential polymorphic variable-number tandem repeats (VNTRs) were identified upon bioinformatic screening of six annotated genome sequences of A. baumannii. A collection of 7 reference strains plus 18 well-characterized isolates, including unique types and representatives of the three international A. baumannii lineages, was then evaluated in a two-center study aimed at validating the MLVA assay and comparing it with other genotyping assays, namely, macrorestriction analysis with pulsed-field gel electrophoresis (PFGE) and PCR-based sequence group (SG) profiling. The results showed that MLVA can discriminate between isolates with identical PFGE types and SG profiles. A panel of eight VNTR markers was selected, all showing the ability to be amplified and good amounts of polymorphism in the majority of strains. Independently generated MLVA profiles, composed of an ordered string of allele numbers corresponding to the number of repeats at each VNTR locus, were concordant between centers. Typeability, reproducibility, stability, discriminatory power, and epidemiological concordance were excellent. A database containing information and MLVA profiles for several A. baumannii strains is available from http://mlva.u-psud.fr/. PMID:21147956

An automated genotyping tool for enteroviruses and noroviruses.

PubMed

Kroneman, A; Vennema, H; Deforche, K; v d Avoort, H; Peñaranda, S; Oberste, M S; Vinjé, J; Koopmans, M

2011-06-01

Molecular techniques are established as routine in virological laboratories and virus typing through (partial) sequence analysis is increasingly common. Quality assurance for the use of typing data requires harmonization of genotype nomenclature, and agreement on target genes, depending on the level of resolution required, and robustness of methods. To develop and validate web-based open-access typing-tools for enteroviruses and noroviruses. An automated web-based typing algorithm was developed, starting with BLAST analysis of the query sequence against a reference set of sequences from viruses in the family Picornaviridae or Caliciviridae. The second step is phylogenetic analysis of the query sequence and a sub-set of the reference sequences, to assign the enterovirus type or norovirus genotype and/or variant, with profile alignment, construction of phylogenetic trees and bootstrap validation. Typing is performed on VP1 sequences of Human enterovirus A to D, and ORF1 and ORF2 sequences of genogroup I and II noroviruses. For validation, we used the tools to automatically type sequences in the RIVM and CDC enterovirus databases and the FBVE norovirus database. Using the typing-tools, 785(99%) of 795 Enterovirus VP1 sequences, and 8154(98.5%) of 8342 norovirus sequences were typed in accordance with previously used methods. Subtyping into variants was achieved for 4439(78.4%) of 5838 NoV GII.4 sequences. The online typing-tools reliably assign genotypes for enteroviruses and noroviruses. The use of phylogenetic methods makes these tools robust to ongoing evolution. This should facilitate standardized genotyping and nomenclature in clinical and public health laboratories, thus supporting inter-laboratory comparisons. Copyright © 2011 Elsevier B.V. All rights reserved.

Genomic deletions of OFD1 account for 23% of oral-facial-digital type 1 syndrome after negative DNA sequencing.

PubMed

Thauvin-Robinet, Christel; Franco, Brunella; Saugier-Veber, Pascale; Aral, Bernard; Gigot, Nadège; Donzel, Anne; Van Maldergem, Lionel; Bieth, Eric; Layet, Valérie; Mathieu, Michèle; Teebi, Ahmad; Lespinasse, James; Callier, Patrick; Mugneret, Francine; Masurel-Paulet, Alice; Gautier, Elodie; Huet, Frédéric; Teyssier, Jean-Raymond; Tosi, Mario; Frébourg, Thierry; Faivre, Laurence

2009-02-01

Oral-facial-digital type I syndrome (OFDI) is characterised by an X-linked dominant mode of inheritance with lethality in males. Clinical features include facial dysmorphism with oral, dental and distal abnormalities, polycystic kidney disease and central nervous system malformations. Considerable allelic heterogeneity has been reported within the OFD1 gene, but DNA bi-directional sequencing of the exons and intron-exon boundaries of the OFD1 gene remains negative in more than 20% of cases. We hypothesized that genomic rearrangements could account for the majority of the remaining undiagnosed cases. Thus, we took advantage of two independent available series of patients with OFDI syndrome and negative DNA bi-directional sequencing of the exons and intron-exon boundaries of the OFD1 gene from two different European labs: 13/36 cases from the French lab; 13/95 from the Italian lab. All patients were screened by a semiquantitative fluorescent multiplex method (QFMPSF) and relative quantification by real-time PCR (qPCR). Six OFD1 genomic deletions (exon 5, exons 1-8, exons 1-14, exons 10-11, exons 13-23 and exon 17) were identified, accounting for 5% of OFDI patients and for 23% of patients with negative mutation screening by DNA sequencing. The association of DNA direct sequencing, QFMPSF and qPCR detects OFD1 alteration in up to 85% of patients with a phenotype suggestive of OFDI syndrome. Given the average percentage of large genomic rearrangements (5%), we suggest that dosage methods should be performed in addition to DNA direct sequencing analysis to exclude the involvement of the OFD1 transcript when there are genetic counselling issues. (c) 2008 Wiley-Liss, Inc.

Polynucleobacter meluiroseus sp. nov., a bacterium isolated from a lake located in the mountains of the Mediterranean island of Corsica.

PubMed

Pitt, Alexandra; Schmidt, Johanna; Lang, Elke; Whitman, William B; Woyke, Tanja; Hahn, Martin W

2018-06-01

Strain AP-Melu-1000-B4 was isolated from a lake located in the mountains of the Mediterranean island of Corsica (France). Phenotypic, chemotaxonomic and genomic traits were investigated. Phylogenetic analyses based on 16S rRNA gene sequencing referred the strain to the cryptic species complex PnecC within the genus Polynucleobacter. The strain encoded genes for biosynthesis of proteorhodopsin and retinal. When pelleted by centrifugation the strain showed an intense rose colouring. Major fatty acids were C16 : 1ω7c, C16 : 0, C18 : 1ω7c and summed feature 2 (C16 : 1 isoI and C14 : 0-3OH). The sequence of the 16S rRNA gene contained an indel which was not present in any previously described Polynucleobacter species. Genome sequencing revealed a genome size of 1.89 Mbp and a G+C content of 46.6 mol%. In order to resolve the phylogenetic position of the new strain within subcluster PnecC, its phylogeny was reconstructed from sequences of 319 shared genes. To represent all currently described Polynucleobacter species by whole genome sequences, three type strains were additionally sequenced. Our phylogenetic analysis revealed that strain AP-Melu-100-B4 occupied a basal position compared with previously described PnecC strains. Pairwise determined whole genome average nucleotide identity (gANI) values suggested that strain AP-Melu-1000-B4 represents a new species, for which we propose the name Polynucleobacter meluiroseus sp. nov. with the type strain AP-Melu-1000-B4 T (=DSM 103591 T =CIP 111329 T ).

[Study on the genetic difference of SEO type Hantaviruses].

PubMed

Zhang, X; Zhou, S; Wang, H; Hu, J; Guan, Z; Liu, H

2000-10-01

To understand the genetic type of Hantaviruses and the difference between them caused by rodents in Beijing and to furhter explore the source of the infectious factors. Hantavirus RNA, isolated from lungs of rodents captured in Beijing and positive with Hantavirus antigens with frozen sectioning and Immunofluorescent assay, were reverse-transcribed and amplified with PCR with Hantavirus-specific primers. Five of the PCR amplifications were discovered and sequenced with 300 bp sequence data of M segments (from 2003 - 2302nt according cDNA of seoul 8039 strain). Nucleotide sequence homology showed that they were sequences of SEO-type Hantavirus. Compared with SEO type Hantavirus, the nucleotide sequence homology of these samples was more than 94% while the homology of amonia acid sequence was more than 98%. When compared with HNT type Hantavirus, the homology of nucleotide sequence became less than 72% with the homology of amonia acid sequence less than 81%. Similar to other Hantavirus of SEO type, their nucleotide sequences and deduced amino acid sequences were highly preserved. Phylogenetic tree analysis showed that the five viruses could be divided into at least 4 branches. It was quite likely that there were at least two sub-type SEO viruses with 4 branches that were circulating in Beijing.

What you learn is more than what you see: what can sequencing effects tell us about inductive category learning?

PubMed Central

Carvalho, Paulo F.; Goldstone, Robert L.

2015-01-01

Inductive category learning takes place across time. As such, it is not surprising that the sequence in which information is studied has an impact in what is learned and how efficient learning is. In this paper we review research on different learning sequences and how this impacts learning. We analyze different aspects of interleaved (frequent alternation between categories during study) and blocked study (infrequent alternation between categories during study) that might explain how and when one sequence of study results in improved learning. While these different sequences of study differ in the amount of temporal spacing and temporal juxtaposition between items of different categories, these aspects do not seem to account for the majority of the results available in the literature. However, differences in the type of category being studied and the duration of the retention interval between study and test may play an important role. We conclude that there is no single aspect that is able to account for all the evidence available. Understanding learning as a process of sequential comparisons in time and how different sequences fundamentally alter the statistics of this experience offers a promising framework for understanding sequencing effects in category learning. We use this framework to present novel predictions and hypotheses for future research on sequencing effects in inductive category learning. PMID:25983699

VWF mutations and new sequence variations identified in healthy controls are more frequent in the African-American population.

PubMed

Bellissimo, Daniel B; Christopherson, Pamela A; Flood, Veronica H; Gill, Joan Cox; Friedman, Kenneth D; Haberichter, Sandra L; Shapiro, Amy D; Abshire, Thomas C; Leissinger, Cindy; Hoots, W Keith; Lusher, Jeanne M; Ragni, Margaret V; Montgomery, Robert R

2012-03-01

Diagnosis and classification of VWD is aided by molecular analysis of the VWF gene. Because VWF polymorphisms have not been fully characterized, we performed VWF laboratory testing and gene sequencing of 184 healthy controls with a negative bleeding history. The controls included 66 (35.9%) African Americans (AAs). We identified 21 new sequence variations, 13 (62%) of which occurred exclusively in AAs and 2 (G967D, T2666M) that were found in 10%-15% of the AA samples, suggesting they are polymorphisms. We identified 14 sequence variations reported previously as VWF mutations, the majority of which were type 1 mutations. These controls had VWF Ag levels within the normal range, suggesting that these sequence variations might not always reduce plasma VWF levels. Eleven mutations were found in AAs, and the frequency of M740I, H817Q, and R2185Q was 15%-18%. Ten AA controls had the 2N mutation H817Q; 1 was homozygous. The average factor VIII level in this group was 99 IU/dL, suggesting that this variation may confer little or no clinical symptoms. This study emphasizes the importance of sequencing healthy controls to understand ethnic-specific sequence variations so that asymptomatic sequence variations are not misidentified as mutations in other ethnic or racial groups.

Increasing diversity of Human Immunodeficiency Virus type 1 subtypes circulating in Australia.

PubMed

Chibo, Doris; Birch, Chris

2012-06-01

Characterization of HIV subtypes can provide a more comprehensive understanding of the epidemic within a distinct region, and when combined with notification data, may also be helpful in enhancing current HIV prevention strategies. In this study, we characterized 1056 HIV-positive individuals (948 males and 108 females) living in Victoria and whose infection was detected for the first time between 2005 and 2010 inclusive. HIV-1 strains were subtyped based on pol gene sequence. Phylogenetic analysis was performed on all non-B subtype sequences identified. Of the 1056 sequences analyzed, 825 were subtype B and 231 were non-B. Overall 6 HIV-1 subtypes, 6 circulating recombinant forms (CRFs), and 12 unique recombinant forms (URFs) were identified. Regardless of gender, the majority of individuals were infected with a subtype B virus (78%). Subtype B was dominant in males (n=806, 85%). In contrast, the majority of females were infected with non-B subtypes (n=89, 82%), in particular subtype C (n=48, 45%). Phylogenetic analysis of the non-B subtypes revealed that the majority of clustering, and thereby transmission, occurred with CRF01_AE strains. Despite the relatively high numbers identified in females there was very little clustering of subtype C viruses. Subtypes C and A1 both historically associated with heterosexual transmission, and CRF01_AE often associated with IVDU, were also associated with transmission within the MSM population, demonstrating the potential for non-B subtypes to expand into the MSM population. The observation of increasing numbers of females and heterosexual males infected with non-subtype B viruses, the majority imported through migration and travel to countries where there is a high prevalence of HIV, suggests a targeted public health message may be required to prevent further increases within these two groups.

Submarine fans: Characteristics, models, classification, and reservoir potential

NASA Astrophysics Data System (ADS)

Shanmugam, G.; Moiola, R. J.

1988-02-01

Submarine-fan sequences are important hydrocarbon reservoirs throughout the world. Submarine-fan sequences may be interpreted from bed-thickness trends, turbidite facies associations, log motifs, and seismic-reflection profiles. Turbidites occurring predominantly in channels and lobes (or sheet sands) constitute the major portion of submarine-fan sequences. Thinning- and thickening-upward trends are suggestive of channel and lobe deposition, respectively. Mounded seismic reflections are commonly indicative of lower-fan depositional lobes. Fan models are discussed in terms of modern and ancient fans, attached and detached lobes, highly efficient and poorly efficient systems, and transverse and longitudinal fans. In general, depositional lobes are considered to be attached to feeder channels. Submarine fans can be classified into four types based on their tectonic settings: (1) immature passive-margin fans (North Sea type); (2) mature passive-margin fans (Atlantic type); (3) active-margin fans (Pacific type); and (4) mixed-setting fans. Immature passive-margin fans (e.g., Balder, North Sea), and active-margin fans (e.g., Navy, Pacific Ocean) are usually small, sand-rich, and possess well developed lobes. Mature passive-margin fans (e.g., Amazon, Atlantic Ocean) are large, mud-rich, and do not develop typical lobes. However, sheet sands are common in the lower-fan regions of mature passive-margin fans. Mixed-setting fans display characteristics of either Atlantic type (e.g., Bengal, Bay of Bengal), or Pacific type (Orinoco, Caribbean), or both. Conventional channel-lobe models may not be applicable to fans associated with mature passive margins. Submarine fans develop primarily during periods of low sea level on both active- and passive-margin settings. Consequently, hydrocarbon-bearing fan sequences are associated generally with global lowstands of sea level. Channel-fill sandstones in most tectonic settings are potential reservoirs. Lobes exhibit the most favorable reservoir quality in terms of sand content, lateral continuity, and porosity development. Lower-fan sheet sands may also make good reservoirs. Quartz-rich sandstones of mature passive-margin fans are most likely to preserve depositional porosity, whereas lithic sandstones of active-margin fans may not.

Phylogeny of 54 representative strains of species in the family Pasteurellaceae as determined by comparison of 16S rRNA sequences.

PubMed Central

Dewhirst, F E; Paster, B J; Olsen, I; Fraser, G J

1992-01-01

Virtually complete 16S rRNA sequences were determined for 54 representative strains of species in the family Pasteurellaceae. Of these strains, 15 were Pasteurella, 16 were Actinobacillus, and 23 were Haemophilus. A phylogenetic tree was constructed based on sequence similarity, using the Neighbor-Joining method. Fifty-three of the strains fell within four large clusters. The first cluster included the type strains of Haemophilus influenzae, H. aegyptius, H. aphrophilus, H. haemolyticus, H. paraphrophilus, H. segnis, and Actinobacillus actinomycetemcomitans. This cluster also contained A. actinomycetemcomitans FDC Y4, ATCC 29522, ATCC 29523, and ATCC 29524 and H. aphrophilus NCTC 7901. The second cluster included the type strains of A. seminis and Pasteurella aerogenes and H. somnus OVCG 43826. The third cluster was composed of the type strains of Pasteurella multocida, P. anatis, P. avium, P. canis, P. dagmatis, P. gallinarum, P. langaa, P. stomatis, P. volantium, H. haemoglobinophilus, H. parasuis, H. paracuniculus, H. paragallinarum, and A. capsulatus. This cluster also contained Pasteurella species A CCUG 18782, Pasteurella species B CCUG 19974, Haemophilus taxon C CAPM 5111, H. parasuis type 5 Nagasaki, P. volantium (H. parainfluenzae) NCTC 4101, and P. trehalosi NCTC 10624. The fourth cluster included the type strains of Actinobacillus lignieresii, A. equuli, A. pleuropneumoniae, A. suis, A. ureae, H. parahaemolyticus, H. parainfluenzae, H. paraphrohaemolyticus, H. ducreyi, and P. haemolytica. This cluster also contained Actinobacillus species strain CCUG 19799 (Bisgaard taxon 11), A. suis ATCC 15557, H. ducreyi ATCC 27722 and HD 35000, Haemophilus minor group strain 202, and H. parainfluenzae ATCC 29242. The type strain of P. pneumotropica branched alone to form a fifth group. The branching of the Pasteurellaceae family tree was quite complex. The four major clusters contained multiple subclusters. The clusters contained both rapidly and slowly evolving strains (indicated by differing numbers of base changes incorporated into the 16S rRNA sequence relative to outgroup organisms). While the results presented a clear picture of the phylogenetic relationships, the complexity of the branching will make division of the family into genera a difficult and somewhat subjective task. We do not suggest any taxonomic changes at this time. PMID:1548238

Sulfur-doped Graphene Nanoribbons with a Sequence of Distinct Band Gaps

NASA Astrophysics Data System (ADS)

Du, Shi-Xuan; Zhang, Yan-Fang; Zhang, Yi; Berger, Reinhard; Feng, Xinliang; Mullen, Klaus; Lin, Xiao; Zhang, Yu-Yang; Pantelides, Sokrates T.; Gao, Hong-Jun

Unlike free-standing graphene, graphene nanoribbons (GNRs) can possess semiconducting band gap. However, achieving such control has been a major challenge in the fabrication of GNRs. Chevron-type GNRs were recently achieved by surface-assisted polymerization of pristine or N-substituted oligophenylene monomers. By mixing two different monomers, GNR heterojunctions can in principle be fabricated. Here we report fabrication and characterization of chevron-type GNRs by using sulfur-substituted oligophenylene monomers to achieve GNRs and related heterostructures for the first time. Importantly, our first-principles calculations show that the band gaps of GNRs can be tailored by different S configurations in cyclodehydrogenated isomers through debromination and intramolecular cyclodehydrogenation. This feature should open up new avenues to create multiple GNR heterojunctions by engineering the sulfur configurations. These predictions have been confirmed by Scanning Tunneling Microscopy (STM) and Scanning Tunneling Spectroscopy (STS). The unusual sequence of intraribbon heterojunctions may be useful for nanoscale optoelectronic applications based on quantum dots

Multidrug-resistant Escherichia coli in Asia: epidemiology and management.

PubMed

Sidjabat, Hanna E; Paterson, David L

2015-05-01

Escherichia coli has become multiresistant by way of production of a variety of β-lactamases. The prevalence of CTX-M-producing E. coli has reached 60-79% in certain parts of Asia. The acquisition of CTX-M plasmids by E. coli sequence type 131, a successful clone of E. coli, has caused further dissemination of CTX-M-producing E. coli. The prevalence of carbapenemase-producing E. coli, especially Klebsiella pneumoniae carbapenemase, and New Delhi metallo-β-lactamase (NDM)-producing E. coli has been increasing in Asia. K. pneumoniae carbapenemase and NDM have now been found in E. coli sequence type 131. The occurrence of NDM-producing E. coli is a major concern particularly in the Indian subcontinent, but now elsewhere in Asia as well. There are multiple reasons why antibiotic resistance in E. coli in Asia has reached such extreme levels. Approaches beyond antibiotic therapy, such as prevention of antibiotic resistance by antibiotic stewardship and protecting natural microbiome, are strategies to avoid further spread of antibiotic resistance.

Pseudomonas aeruginosa Type III Secretory Toxin ExoU and Its Predicted Homologs.

PubMed

Sawa, Teiji; Hamaoka, Saeko; Kinoshita, Mao; Kainuma, Atsushi; Naito, Yoshifumi; Akiyama, Koichi; Kato, Hideya

2016-10-26

Pseudomonas aeruginosa ExoU, a type III secretory toxin and major virulence factor with patatin-like phospholipase activity, is responsible for acute lung injury and sepsis in immunocompromised patients. Through use of a recently updated bacterial genome database, protein sequences predicted to be homologous to Ps. aeruginosa ExoU were identified in 17 other Pseudomonas species ( Ps. fluorescens , Ps. lundensis , Ps. weihenstephanensis , Ps. marginalis, Ps. rhodesiae, Ps. synxantha , Ps. libanensis , Ps. extremaustralis , Ps. veronii , Ps. simiae , Ps. trivialis , Ps. tolaasii , Ps. orientalis , Ps. taetrolens , Ps. syringae , Ps. viridiflava , and Ps. cannabina ) and 8 Gram-negative bacteria from three other genera ( Photorhabdus , Aeromonas , and Paludibacterium ). In the alignment of the predicted primary amino acid sequences used for the phylogenetic analyses, both highly conserved and nonconserved parts of the toxin were discovered among the various species. Further comparative studies of the predicted ExoU homologs should provide us with more detailed information about the unique characteristics of the Ps. aeruginosa ExoU toxin.

A model of EcoRII restriction endonuclease action: the active complex is most likely formed by one protein subunit and one DNA recognition site

NASA Technical Reports Server (NTRS)

Karpova, E. A.; Kubareva, E. A.; Shabarova, Z. A.

1999-01-01

To elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNA-duplexes containing the modified or canonical recognition sequence 5'-d(CCA/TGG)-3'. All binding substrate or substrate analogues tested could be divided into two major groups: (i) duplexes that, at the interaction with endonuclease EcoRII, form two types of stable complexes on native gel in the absence of Mg2+ cofactor; (ii) duplexes that form only one type of complex, observed both in the presence and absence of Mg2+. Unlike the latter, duplexes under the first group can be hydrolyzed by endonuclease. Data obtained suggest that the active complex is most likely formed by one protein subunit and one DNA recognition sequence. A model of EcoRII endonuclease action is presented.

A sequence of basic residues in the porcine circovirus type 2 capsid protein is crucial for its co-expression and co-localization with the replication protein.

PubMed

Huang, Liping; Van Renne, Nicolaas; Liu, Changming; Nauwynck, Hans J

2015-12-01

Porcine circovirus type 2 (PCV2) encodes two major proteins: the replication protein (Rep) and the capsid protein (Cap). Cap displays a conserved stretch of basic residues situated on the inside of the capsid, whose role is so far unknown. We used a reverse-genetics approach to investigate its function and found that mutations in these amino acids hindered Cap mRNA translation and hampered Cap/Rep co-localization, yielding unfit viruses. Intriguingly, co-transfection with a WT PCV2 of a different genotype partially rescued mutant Cap expression, showing the importance of this basic pattern for efficient translation of Cap mRNA into protein. Our results show that Cap and Rep are expressed independently of each other, and that this amino acid sequence of Cap is vital for virus propagation. This study provides a method for studying unfit PCV2 virions and offers new insights into the intracellular modus vivendi of PCV2.

Phylogenetic analysis of members of the Phycodnaviridae virus family, using amplified fragments of the major capsid protein gene.

PubMed

Larsen, J B; Larsen, A; Bratbak, G; Sandaa, R-A

2008-05-01

Algal viruses are considered ecologically important by affecting host population dynamics and nutrient flow in aquatic food webs. Members of the family Phycodnaviridae are also interesting due to their extraordinary genome size. Few algal viruses in the Phycodnaviridae family have been sequenced, and those that have been have few genes in common and low gene homology. It has hence been difficult to design general PCR primers that allow further studies of their ecology and diversity. In this study, we screened the nine type I core genes of the nucleocytoplasmic large DNA viruses for sequences suitable for designing a general set of primers. Sequence comparison between members of the Phycodnaviridae family, including three partly sequenced viruses infecting the prymnesiophyte Pyramimonas orientalis and the haptophytes Phaeocystis pouchetii and Chrysochromulina ericina (Pyramimonas orientalis virus 01B [PoV-01B], Phaeocystis pouchetii virus 01 [PpV-01], and Chrysochromulina ericina virus 01B [CeV-01B], respectively), revealed eight conserved regions in the major capsid protein (MCP). Two of these regions also showed conservation at the nucleotide level, and this allowed us to design degenerate PCR primers. The primers produced 347- to 518-bp amplicons when applied to lysates from algal viruses kept in culture and from natural viral communities. The aim of this work was to use the MCP as a proxy to infer phylogenetic relationships and genetic diversity among members of the Phycodnaviridae family and to determine the occurrence and diversity of this gene in natural viral communities. The results support the current legitimate genera in the Phycodnaviridae based on alga host species. However, while placing the mimivirus in close proximity to the type species, PBCV-1, of Phycodnaviridae along with the three new viruses assigned to the family (PoV-01B, PpV-01, and CeV-01B), the results also indicate that the coccolithoviruses and phaeoviruses are more diverged from this group. Phylogenetic analysis of amplicons from virus assemblages from Norwegian coastal waters as well as from isolated algal viruses revealed a cluster of viruses infecting members of the prymnesiophyte and prasinophyte alga divisions. Other distinct clusters were also identified, containing amplicons from this study as well as sequences retrieved from the Sargasso Sea metagenome. This shows that closely related sequences of this family are present at geographically distant locations within the marine environment.

Petroleum source-rock potentials of the cretaceous transgressive-regressive sedimentary sequences of the Cauvery Basin

NASA Astrophysics Data System (ADS)

Chandra, Kuldeep; Philip, P. C.; Sridharan, P.; Chopra, V. S.; Rao, Brahmaji; Saha, P. K.

The present work is an attempt to contribute to knowledge on the petroleum source-rock potentials of the marine claystones and shales of basins associated with passive continental margins where the source-rock developments are known to have been associated with the anoxic events in the Mesozoic era. Data on three key exploratory wells from three major depressions Ariyallur-Pondicherry, Thanjavur and Nagapattinam of the Cauvery Basin are described and discussed. The average total organic carbon contents of the transgressive Pre-Albian-Cinomanian and Coniacian/Santonian claystones/shales range from 1.44 and 1.16%, respectively. The transgressive/regressive Campanian/Maastrichtian claystones contain average total organic carbon varying from 0.62 to 1.19%. The kerogens in all the studied stratigraphic sequences are classified as type-III with Rock-Eval hydrogen indices varying from 30 to 275. The nearness of land masses to the depositional basin and the mainly clastic sedimentation resulted in accumulation and preservation of dominantly type-III kerogens. The Pre-Albian to Cinomanian sequences of peak transgressive zone deposited in deep marine environments have kerogens with a relatively greater proportion of type-II components with likely greater contribution of planktonic organic matters. The global anoxic event associated with the Albian-Cinomanian marine transgression, like in many other parts of the world, has pervaded the Cauvery Basin and favoured development of good source-rocks with type-III kerogens. The Coniacian-Campanian-Maastrichtian transgressive/regressive phase is identified to be relatively of lesser significance for development of good quality source-rocks.

Expression of Sme efflux pumps and multilocus sequence typing in clinical isolates of Stenotrophomonas maltophilia.

PubMed

Cho, Hye Hyun; Sung, Ji Youn; Kwon, Kye Chul; Koo, Sun Hoe

2012-01-01

Stenotrophomonas maltophilia has emerged as an important opportunistic pathogen, which causes infections that are often difficult to manage because of the inherent resistance of the pathogen to a variety of antimicrobial agents. In this study, we analyzed the expressions of smeABC and smeDEF and their correlation with antimicrobial susceptibility. We also evaluated the genetic relatedness and epidemiological links among 33 isolates of S. maltophilia. In total, 33 S. maltophilia strains were isolated from patients in a tertiary hospital in Daejeon. Minimum inhibitory concentrations (MICs) of 11 antimicrobial agents were determined by using agar dilution method and E-test (BioMérieux, France). Real-time PCR analysis was performed to evaluate the expression of the Sme efflux systems in the S. maltophilia isolates. Additionally, an epidemiological investigation was performed using multilocus sequence typing (MLST) assays. The findings of susceptibility testing showed that the majority of the S. maltophilia isolates were resistant to β-lactams and aminoglycosides. Twenty-one clinical isolates overexpressed smeABC and showed high resistance to ciprofloxacin. Moreover, a high degree of genetic diversity was observed among the S. maltophilia isolates; 3 sequence types (STs) and 23 allelic profiles were observed. The smeABC efflux pump was associated with multidrug resistance in clinical isolates of S. maltophilia. In particular, smeABC efflux pumps appear to perform an important role in ciprofloxacin resistance of S. maltophilia. The MLST scheme for S. maltophilia represents a discriminatory typing method with stable markers and is appropriate for studying population structures.

Divergent nuclear 18S rDNA paralogs in a turkey coccidium, Eimeria meleagrimitis, complicate molecular systematics and identification.

PubMed

El-Sherry, Shiem; Ogedengbe, Mosun E; Hafeez, Mian A; Barta, John R

2013-07-01

Multiple 18S rDNA sequences were obtained from two single-oocyst-derived lines of each of Eimeria meleagrimitis and Eimeria adenoeides. After analysing the 15 new 18S rDNA sequences from two lines of E. meleagrimitis and 17 new sequences from two lines of E. adenoeides, there were clear indications that divergent, paralogous 18S rDNA copies existed within the nuclear genome of E. meleagrimitis. In contrast, mitochondrial cytochrome c oxidase subunit I (COI) partial sequences from all lines of a particular Eimeria sp. were identical and, in phylogenetic analyses, COI sequences clustered unambiguously in monophyletic and highly-supported clades specific to individual Eimeria sp. Phylogenetic analysis of the new 18S rDNA sequences from E. meleagrimitis showed that they formed two distinct clades: Type A with four new sequences; and Type B with nine new sequences; both Types A and B sequences were obtained from each of the single-oocyst-derived lines of E. meleagrimitis. Together these rDNA types formed a well-supported E. meleagrimitis clade. Types A and B 18S rDNA sequences from E. meleagrimitis had a mean sequence identity of only 97.4% whereas mean sequence identity within types was 99.1-99.3%. The observed intraspecific sequence divergence among E. meleagrimitis 18S rDNA sequence types was even higher (approximately 2.6%) than the interspecific sequence divergence present between some well-recognized species such as Eimeria tenella and Eimeria necatrix (1.1%). Our observations suggest that, unlike COI sequences, 18S rDNA sequences are not reliable molecular markers to be used alone for species identification with coccidia, although 18S rDNA sequences have clear utility for phylogenetic reconstruction of apicomplexan parasites at the genus and higher taxonomic ranks. Copyright © 2013. Published by Elsevier Ltd.

Prevalence and characterization of methicillin-resistant Staphylococcus aureus isolates from retail meat and humans in Georgia.

PubMed

Jackson, Charlene R; Davis, Johnnie A; Barrett, John B

2013-04-01

There is increasing interest in the presence of Staphylococcus aureus, specifically methicillin-resistant S. aureus (MRSA), on retail meat products. In this study, staphylococci were isolated from retail pork and retail beef in Georgia, and MRSA from the products was compared to human MRSA from the same geographic area using broth microdilution antimicrobial susceptibility testing, multilocus sequence typing (MLST), spa typing, SCCmec typing, and pulsed-field gel electrophoresis (PFGE). S. aureus was isolated from 45% (45/100) of pork products and 63% (63/100) of beef products; mecA was detected in S. aureus from both pork (3/100; 3%) and beef (4/100; 4%). Fifty percent (50/100) of human S. aureus also contained mecA. Multidrug resistance was detected among MRSA from all sources. All MRSA (n = 57) was SCCmec type IV, and nine different spa types were present among the isolates (t002, t008, t012, t024, t179, t337, t548, t681, and t1062). Four sequence types (ST5, ST8, ST9, and ST30) were detected using MLST; the majority of MRSA isolates belonged to ST8, followed by ST5. One retail beef MRSA isolate belonged to ST8, while the remaining three were ST5. In retail pork MRSA, ST5, ST9, and ST30 were observed. The majority of human MRSA isolates belonged to ST8. Thirty-seven MRSA isolates, one of which was a retail beef MRSA isolate, were pvl(+). Using PFGE, MLST, and spa typing, three retail beef MRSA isolates were found to be identical in PFGE pattern, ST, and spa type to two human clonal MRSA isolates (USA100 and USA300). One additional retail beef MRSA isolate had a PFGE pattern similar to that of a human MRSA isolate, whereas none of the retail pork MRSA isolates had PFGE patterns similar to those of human MRSA isolates. These data suggest that the retail beef samples were contaminated by a human source, possibly during processing of the meat, and may present a source of MRSA for consumers and others who handle raw meat.

Prevalence and Characterization of Methicillin-Resistant Staphylococcus aureus Isolates from Retail Meat and Humans in Georgia

PubMed Central

Davis, Johnnie A.; Barrett, John B.

2013-01-01

There is increasing interest in the presence of Staphylococcus aureus, specifically methicillin-resistant S. aureus (MRSA), on retail meat products. In this study, staphylococci were isolated from retail pork and retail beef in Georgia, and MRSA from the products was compared to human MRSA from the same geographic area using broth microdilution antimicrobial susceptibility testing, multilocus sequence typing (MLST), spa typing, SCCmec typing, and pulsed-field gel electrophoresis (PFGE). S. aureus was isolated from 45% (45/100) of pork products and 63% (63/100) of beef products; mecA was detected in S. aureus from both pork (3/100; 3%) and beef (4/100; 4%). Fifty percent (50/100) of human S. aureus also contained mecA. Multidrug resistance was detected among MRSA from all sources. All MRSA (n = 57) was SCCmec type IV, and nine different spa types were present among the isolates (t002, t008, t012, t024, t179, t337, t548, t681, and t1062). Four sequence types (ST5, ST8, ST9, and ST30) were detected using MLST; the majority of MRSA isolates belonged to ST8, followed by ST5. One retail beef MRSA isolate belonged to ST8, while the remaining three were ST5. In retail pork MRSA, ST5, ST9, and ST30 were observed. The majority of human MRSA isolates belonged to ST8. Thirty-seven MRSA isolates, one of which was a retail beef MRSA isolate, were pvl+. Using PFGE, MLST, and spa typing, three retail beef MRSA isolates were found to be identical in PFGE pattern, ST, and spa type to two human clonal MRSA isolates (USA100 and USA300). One additional retail beef MRSA isolate had a PFGE pattern similar to that of a human MRSA isolate, whereas none of the retail pork MRSA isolates had PFGE patterns similar to those of human MRSA isolates. These data suggest that the retail beef samples were contaminated by a human source, possibly during processing of the meat, and may present a source of MRSA for consumers and others who handle raw meat. PMID:23363837

Pathotypic and molecular characterization of a fowl adenovirus associated with inclusion body hepatitis in Saskatchewan chickens.

PubMed

Dar, Arshud; Gomis, Susantha; Shirley, Ian; Mutwiri, George; Brownlie, Robert; Potter, Andrew; Gerdts, Volker; Tikoo, Suresh K

2012-03-01

Inclusion body hepatitis (IBH) is one of the major global disease problems, causing significant economic losses to poultry industry of the United States and Canada. The disease is characterized by its sudden onset and high mortalities. Amongst different serotypes of fowl adenoviruses (FAdVs) associated with IBH, serotype 8 of group I FAdV has been isolated from majority of IBH cases. In present studies, we isolated a FAdV from morbid liver of a 17-day-old broiler from a Saskatchewan broiler farm. This newly isolated virus was designated as IBHV(SK). However, based on the sequence analysis of the L1 region of the hexon gene, the IBHV(SK) may be classified as FAdV 8b strain 764. These studies describe for the first time the complete hexon gene sequence of FAdV serotype 8b. Experimental infection of 2-day-old (n = 48) and 2-wk-old (n = 56) chicks caused 83% and 43% mortalities, respectively. Determination of the complete hexon gene sequence of IBHV(SK) with establishment of a disease model in chickens will facilitate the development of type-specific diagnostic reagents and assays for the evaluation of potential experimental vaccines against pathogenic FAdV infections.

Nioella nitratireducens gen. nov., sp. nov., a novel member of the family Rhodobacteraceae isolated from Azorean Island.

PubMed

Rajasabapathy, Raju; Mohandass, Chellandi; Yoon, Jung-Hoon; Dastager, Syed Gulam; Liu, Qing; Khieu, Thi-Nhan; Son, Chu Ky; Li, Wen-Jun; Colaco, Ana

2015-02-01

A novel Gram-negative, non-spore forming, rod-shaped aerobic bacterium, designated SSW136(T), was isolated from a surface seawater sample collected at Espalamaca (in Faial Island), Azores. Growth was found to occur from 10 to 37 °C, pH 6.0-8.0, and with 2-11 % of NaCl. 16S rRNA gene sequence indicated that the strain SSW136(T) belongs to the family Rhodobacteraceae. Strain SSW136(T) exhibited 96.3, 95.9, 95.7 and 95.5 sequence similarity to the type strains Oceanicola litoreus M-M22(T), Roseovarius aestuarii SMK-122(T), Marivita geojedonensis DPG-138(T), and Pseudoruegeria aquimaris SW-255(T) respectively. Neighbour-joining and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences revealed that strain SSW136(T) was affiliated to the family Rhodobacteraceae and formed a separate branch. The G+C content was 63.5 mol%. The major respiratory quinone was found to be Q-10. The polar lipids of strain SSW136(T) consisted of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids and three unidentified phospholipids. The major fatty acids were C18:1 ω7c (46.5 %), Cyclo-C19:0 ω8c (16.0 %) and C16:0 (12.8 %). On the basis of the morphological, genotypic, chemotaxonomic characteristics and low DNA-DNA relatedness, strain SSW136(T) is proposed to represent a novel genus and novel species, Nioella nitratireducens gen. nov., sp. nov., in the family Rhodobacteraceae. The type strain is SSW136(T) (=KCTC 32417(T) = NCIM 5499(T)).

Geothermomicrobium terrae gen. nov., sp. nov., a novel member of the family Thermoactinomycetaceae.

PubMed

Zhou, En-Min; Yu, Tian-Tian; Liu, Lan; Ming, Hong; Yin, Yi-Rui; Dong, Lei; Tseng, Min; Nie, Guo-Xing; Li, Wen-Jun

2014-09-01

Strains YIM 77562(T) and YIM 77580, two novel Gram-staining-positive, filamentous bacterial isolates, were recovered from the Rehai geothermal field, Tengchong, Yunnan province, south-west China. Good growth was observed at 50-55 °C and pH 7.0. Aerial mycelium was absent on all media tested. Substrate mycelium was well-developed, long and moderately flexuous, and formed abundant, single, warty, ornamented endospores. Phylogenetic analysis of the 16S rRNA gene sequences of the two strains indicated that they belong to the family Thermoactinomycetaceae. Similarity levels between the 16S rRNA gene sequences of the two strains and those of type strains of members of the Thermoactinomycetaceae were 88.33-93.24 %; the highest sequence similarity was with Hazenella coriacea DSM 45707(T). In both strains, the predominant menaquinone was MK-7, the diagnostic diamino acid was meso-diaminopimelic acid and the major cellular fatty acids were iso-C14 : 0, iso-C15 : 0 and iso-C16 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylmethylethanolamine, unidentified polar lipids and unidentified phospholipids. The genomic DNA G+C contents of strains YIM 77562(T) and YIM 77580 were 45.5 and 44.2 mol%, respectively. DNA-DNA relatedness data suggest that the two isolates represent a single species. Based on phylogenetic analyses and physiological and biochemical characteristics, it is proposed that the two strains represent a single novel species in a new genus, Geothermomicrobium terrae gen. nov., sp. nov. The type strain of Geothermomicrobium terrae is YIM 77562(T) ( = CCTCC AA 2011022(T) = JCM 18057(T)). © 2014 IUMS.

Emergence of endemic MLST non-typeable vancomycin-resistant Enterococcus faecium.

PubMed

Carter, Glen P; Buultjens, Andrew H; Ballard, Susan A; Baines, Sarah L; Tomita, Takehiro; Strachan, Janet; Johnson, Paul D R; Ferguson, John K; Seemann, Torsten; Stinear, Timothy P; Howden, Benjamin P

2016-12-01

Enterococcus faecium is a major nosocomial pathogen causing significant morbidity and mortality worldwide. Assessment of E. faecium using MLST to understand the spread of this organism is an important component of hospital infection control measures. Recent studies, however, suggest that MLST might be inadequate for E. faecium surveillance. To use WGS to characterize recently identified vancomycin-resistant E. faecium (VREfm) isolates non-typeable by MLST that appear to be causing a multi-jurisdictional outbreak in Australia. Illumina NextSeq and Pacific Biosciences SMRT sequencing platforms were used to determine the genome sequences of 66 non-typeable E. faecium (NTEfm) isolates. Phylogenetic and bioinformatics analyses were subsequently performed using a number of in silico tools. Sixty-six E. faecium isolates were identified by WGS from multiple health jurisdictions in Australia that could not be typed by MLST due to a missing pstS allele. SMRT sequencing and complete genome assembly revealed a large chromosomal rearrangement in representative strain DMG1500801, which likely facilitated the deletion of the pstS region. Phylogenomic analysis of this population suggests that deletion of pstS within E. faecium has arisen independently on at least three occasions. Importantly, the majority of these isolates displayed a vancomycin-resistant genotype. We have identified NTEfm isolates that appear to be causing a multi-jurisdictional outbreak in Australia. Identification of these isolates has important implications for MLST-based typing activities designed to monitor the spread of VREfm and provides further evidence supporting the use of WGS for hospital surveillance of E. faecium. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types.

PubMed

Kilian, A; Bowtell, D D; Abud, H E; Hime, G R; Venter, D J; Keese, P K; Duncan, E L; Reddel, R R; Jefferson, R A

1997-11-01

Telomerase is a multicomponent reverse transcriptase enzyme that adds DNA repeats to the ends of chromosomes using its RNA component as a template for synthesis. Telomerase activity is detected in the germline as well as the majority of tumors and immortal cell lines, and at low levels in several types of normal cells. We have cloned a human gene homologous to a protein from Saccharomyces cerevisiae and Euplotes aediculatus that has reverse transcriptase motifs and is thought to be the catalytic subunit of telomerase in those species. This gene is present in the human genome as a single copy sequence with a dominant transcript of approximately 4 kb in a human colon cancer cell line, LIM1215. The cDNA sequence was determined using clones from a LIM1215 cDNA library and by RT-PCR, cRACE and 3'RACE on mRNA from the same source. We show that the gene is expressed in several normal tissues, telomerase-positive post-crisis (immortal) cell lines and various tumors but is not expressed in the majority of normal tissues analyzed, pre-crisis (non-immortal) cells and telomerase-negative immortal (ALT) cell lines. Multiple products were identified by RT-PCR using primers within the reverse transcriptase domain. Sequencing of these products suggests that they arise by alternative splicing. Strikingly, various tumors, cell lines and even normal tissues (colonic crypt and testis) showed considerable differences in the splicing patterns. Alternative splicing of the telomerase catalytic subunit transcript may be important for the regulation of telomerase activity and may give rise to proteins with different biochemical functions.

Mucilaginibacter antarcticus sp. nov., isolated from tundra soil.

PubMed

Zheng, Ruichen; Zhao, Yiming; Wang, Liqiu; Chang, Xulu; Zhang, Yumin; Da, Xuyang; Peng, Fang

2016-12-01

The novel, pale yellow bacterial strain, designated S14-88T, was isolated from a tundra soil near Antarctic Peninsula, South Shetland Islands, and its taxonomic position was investigated by a genotypic and phenotypic analysis. Cells were facultatively anaerobic, Gram-stain-negative, non-motile and rod-shaped. Growth occurred at 4-28 °C (optimum at 15 °C), at pH 7.0-8.0 (optimum at 7.0) and with 0-0.6 % (w/v) NaCl (optimum, no NaCl). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain S14-88T formed a lineage within the genus Mucilaginibacter. The 16S rRNA gene sequence similarity between strain S14-88T and the type strains of related species ranged from 92.2 to 96.5 %, and the 16S rRNA gene sequence of S14-88T showed highest similarity of 96.5 % to Mucilaginibacter soyangensis HME6664T. The major cellular fatty acids of strain S14-88T were iso-C15 : 0 and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The major respiratory quinone was menaquinone MK-7, and the main polar lipid was phosphatidylethanolamine. The DNA G+C content of strain S14-88T was 42.3 mol%. On the basis of the evidence presented in this study, strain S14-88T is considered to represent a novel species of the genus Mucilaginibacter, for which the name Mucilaginibacter antarcticus sp. nov. is proposed. The type strain is S14-88T (=CCTCC AB 2015321T=KCTC 52232T).

Genome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites.

PubMed

Omura, S; Ikeda, H; Ishikawa, J; Hanamoto, A; Takahashi, C; Shinose, M; Takahashi, Y; Horikawa, H; Nakazawa, H; Osonoe, T; Kikuchi, H; Shiba, T; Sakaki, Y; Hattori, M

2001-10-09

Streptomyces avermitilis is a soil bacterium that carries out not only a complex morphological differentiation but also the production of secondary metabolites, one of which, avermectin, is commercially important in human and veterinary medicine. The major interest in this genus Streptomyces is the diversity of its production of secondary metabolites as an industrial microorganism. A major factor in its prominence as a producer of the variety of secondary metabolites is its possession of several metabolic pathways for biosynthesis. Here we report sequence analysis of S. avermitilis, covering 99% of its genome. At least 8.7 million base pairs exist in the linear chromosome; this is the largest bacterial genome sequence, and it provides insights into the intrinsic diversity of the production of the secondary metabolites of Streptomyces. Twenty-five kinds of secondary metabolite gene clusters were found in the genome of S. avermitilis. Four of them are concerned with the biosyntheses of melanin pigments, in which two clusters encode tyrosinase and its cofactor, another two encode an ochronotic pigment derived from homogentiginic acid, and another polyketide-derived melanin. The gene clusters for carotenoid and siderophore biosyntheses are composed of seven and five genes, respectively. There are eight kinds of gene clusters for type-I polyketide compound biosyntheses, and two clusters are involved in the biosyntheses of type-II polyketide-derived compounds. Furthermore, a polyketide synthase that resembles phloroglucinol synthase was detected. Eight clusters are involved in the biosyntheses of peptide compounds that are synthesized by nonribosomal peptide synthetases. These secondary metabolite clusters are widely located in the genome but half of them are near both ends of the genome. The total length of these clusters occupies about 6.4% of the genome.

The Swedish new variant of Chlamydia trachomatis: genome sequence, morphology, cell tropism and phenotypic characterization

PubMed Central

Unemo, Magnus; Seth-Smith, Helena M. B.; Cutcliffe, Lesley T.; Skilton, Rachel J.; Barlow, David; Goulding, David; Persson, Kenneth; Harris, Simon R.; Kelly, Anne; Bjartling, Carina; Fredlund, Hans; Olcén, Per; Thomson, Nicholas R.; Clarke, Ian N.

2010-01-01

Chlamydia trachomatis is a major cause of bacterial sexually transmitted infections worldwide. In 2006, a new variant of C. trachomatis (nvCT), carrying a 377 bp deletion within the plasmid, was reported in Sweden. This deletion included the targets used by the commercial diagnostic systems from Roche and Abbott. The nvCT is clonal (serovar/genovar E) and it spread rapidly in Sweden, undiagnosed by these systems. The degree of spread may also indicate an increased biological fitness of nvCT. The aims of this study were to describe the genome of nvCT, to compare the nvCT genome to all available C. trachomatis genome sequences and to investigate the biological properties of nvCT. An early nvCT isolate (Sweden2) was analysed by genome sequencing, growth kinetics, microscopy, cell tropism assay and antimicrobial susceptibility testing. It was compared with relevant C. trachomatis isolates, including a similar serovar E C. trachomatis wild-type strain that circulated in Sweden prior to the initially undetected expansion of nvCT. The nvCT genome does not contain any major genetic polymorphisms – the genes for central metabolism, development cycle and virulence are conserved – or phenotypic characteristics that indicate any altered biological fitness. This is supported by the observations that the nvCT and wild-type C. trachomatis infections are very similar in terms of epidemiological distribution, and that differences in clinical signs are only described, in one study, in women. In conclusion, the nvCT does not appear to have any altered biological fitness. Therefore, the rapid transmission of nvCT in Sweden was due to the strong diagnostic selective advantage and its introduction into a high-frequency transmitting population. PMID:20093289

Chryseobacterium chaponense sp. nov., isolated from farmed Atlantic salmon (Salmo salar).

PubMed

Kämpfer, Peter; Fallschissel, Kerstin; Avendaño-Herrera, Ruben

2011-03-01

Two bacterial strains, designated Sa 1147-06(T) and Sa 1143-06, were isolated from Atlantic salmon (Salmo salar) farmed in Lake Chapo, Chile, and were studied using a polyphasic approach. Both isolates were very similar; cells were rod-shaped, formed yellow-pigmented colonies and were Gram-reaction-negative. Based on 16S rRNA gene sequence analysis, strains Sa 1147-06(T) and Sa 1143-06 shared 100  % sequence similarity and showed 98.9 and 97.5 % sequence similarity to Chryseobacterium jeonii AT1047(T) and Chryseobacterium antarcticum AT1013(T), respectively. Sequence similarities to all other members of the genus Chryseobacterium were below 97.3  %. The major fatty acids of strain Sa 1147-06(T) were iso-C₁₃:₀, iso-C₁₅:₀, anteiso-C₁₅:₀ and iso-C₁₇:₁ω9c, with iso-C₁₅:₀ 3-OH, iso-C₁₆:₀ 3-OH and iso-C₁₇:₀ 3-OH constituting the major hydroxylated fatty acids. DNA-DNA hybridizations with C. jeonii JMSNU 14049(T) and C. antarcticum JMNSU 14040(T) gave relatedness values of 20.7  % (reciprocal 15.1  %) and 15.7 % (reciprocal 25.7  %), respectively. Together, the DNA-DNA hybridization results and differentiating biochemical properties showed that strains Sa 1147-06(T) and Sa 1143-06 represent a novel species, for which the name Chryseobacterium chaponense sp. nov. is proposed. The type strain is Sa 1147-06(T) (=DSM 23145(T) =CCM 7737(T)).

Comparison of double-locus sequence typing (DLST) and multilocus sequence typing (MLST) for the investigation of Pseudomonas aeruginosa populations.

PubMed

Cholley, Pascal; Stojanov, Milos; Hocquet, Didier; Thouverez, Michelle; Bertrand, Xavier; Blanc, Dominique S

2015-08-01

Reliable molecular typing methods are necessary to investigate the epidemiology of bacterial pathogens. Reference methods such as multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) are costly and time consuming. Here, we compared our newly developed double-locus sequence typing (DLST) method for Pseudomonas aeruginosa to MLST and PFGE on a collection of 281 isolates. DLST was as discriminatory as MLST and was able to recognize "high-risk" epidemic clones. Both methods were highly congruent. Not surprisingly, a higher discriminatory power was observed with PFGE. In conclusion, being a simple method (single-strand sequencing of only 2 loci), DLST is valuable as a first-line typing tool for epidemiological investigations of P. aeruginosa. Coupled to a more discriminant method like PFGE or whole genome sequencing, it might represent an efficient typing strategy to investigate or prevent outbreaks. Copyright © 2015 Elsevier Inc. All rights reserved.

Sequence analysis of chloroplast chlB gene of medicinal Ephedra species and its application to authentication of Ephedra Herb.

PubMed

Guo, Yahong; Tsuruga, Ayako; Yamaguchi, Shigeharu; Oba, Koji; Iwai, Kasumi; Sekita, Setsuko; Mizukami, Hajime

2006-06-01

Chloroplast chlB gene encoding subunit B of light-independent protochlorophyllide reductase was amplified from herbarium and crude drug specimens of Ephedra sinica, E. intermedia, E. equisetina, and E. przewalskii. Sequence comparison of the chlB gene indicated that all the E. sinica specimens have the same sequence type (Type S) distinctive from other species, while there are two sequence types (Type E1 and Type E2) in E. equisetina. E. intermedia and E. prezewalskii revealed an identical sequence type (Type IP). E. sinica was also identified by digesting the chlB fragment with Bcl I. A novel method for DNA authentication of Ephedra Herb based on the sequences of the chloroplast chlB gene and internal transcribed spacer of nuclear rRNA genes was developed and successfully applied for identification of the crude drugs obtained in the Chinese market.

Phylogenetic Distribution of CRISPR-Cas Systems in Antibiotic-Resistant Pseudomonas aeruginosa

PubMed Central

van Belkum, Alex; Soriaga, Leah B.; LaFave, Matthew C.; Akella, Srividya; Veyrieras, Jean-Baptiste; Barbu, E. Magda; Shortridge, Dee; Blanc, Bernadette; Hannum, Gregory; Zambardi, Gilles; Miller, Kristofer; Enright, Mark C.; Mugnier, Nathalie; Brami, Daniel; Schicklin, Stéphane; Felderman, Martina; Schwartz, Ariel S.; Richardson, Toby H.; Peterson, Todd C.; Hubby, Bolyn

2015-01-01

ABSTRACT Pseudomonas aeruginosa is an antibiotic-refractory pathogen with a large genome and extensive genotypic diversity. Historically, P. aeruginosa has been a major model system for understanding the molecular mechanisms underlying type I clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas)-based bacterial immune system function. However, little information on the phylogenetic distribution and potential role of these CRISPR-Cas systems in molding the P. aeruginosa accessory genome and antibiotic resistance elements is known. Computational approaches were used to identify and characterize CRISPR-Cas systems within 672 genomes, and in the process, we identified a previously unreported and putatively mobile type I-C P. aeruginosa CRISPR-Cas system. Furthermore, genomes harboring noninhibited type I-F and I-E CRISPR-Cas systems were on average ~300 kb smaller than those without a CRISPR-Cas system. In silico analysis demonstrated that the accessory genome (n = 22,036 genes) harbored the majority of identified CRISPR-Cas targets. We also assembled a global spacer library that aided the identification of difficult-to-characterize mobile genetic elements within next-generation sequencing (NGS) data and allowed CRISPR typing of a majority of P. aeruginosa strains. In summary, our analysis demonstrated that CRISPR-Cas systems play an important role in shaping the accessory genomes of globally distributed P. aeruginosa isolates. PMID:26604259

Sub-typing of extended-spectrum-β-lactamase-producing isolates from a nosocomial outbreak: application of a 10-loci generic Escherichia coli multi-locus variable number tandem repeat analysis.

PubMed

Karami, Nahid; Helldal, Lisa; Welinder-Olsson, Christina; Ahrén, Christina; Moore, Edward R B

2013-01-01

Extended-spectrum β-lactamase producing Escherichia coli (ESBL-E. coli) were isolated from infants hospitalized in a neonatal, post-surgery ward during a four-month-long nosocomial outbreak and six-month follow-up period. A multi-locus variable number tandem repeat analysis (MLVA), using 10 loci (GECM-10), for 'generic' (i.e., non-STEC) E. coli was applied for sub-species-level (i.e., sub-typing) delineation and characterization of the bacterial isolates. Ten distinct GECM-10 types were detected among 50 isolates, correlating with the types defined by pulsed-field gel electrophoresis (PFGE), which is recognized to be the 'gold-standard' method for clinical epidemiological analyses. Multi-locus sequence typing (MLST), multiplex PCR genotyping of bla CTX-M, bla TEM, bla OXA and bla SHV genes and antibiotic resistance profiling, as well as a PCR assay specific for detecting isolates of the pandemic O25b-ST131 strain, further characterized the outbreak isolates. Two clusters of isolates with distinct GECM-10 types (G06-04 and G07-02), corresponding to two major PFGE types and the MLST-based sequence types (STs) 131 and 1444, respectively, were confirmed to be responsible for the outbreak. The application of GECM-10 sub-typing provided reliable, rapid and cost-effective epidemiological characterizations of the ESBL-producing isolates from a nosocomial outbreak that correlated with and may be used to replace the laborious PFGE protocol for analyzing generic E. coli.

Sabulilitoribacter multivorans gen. nov., sp. nov., a polysaccharide-degrading bacterium of the family Flavobacteriaceae isolated from seashore sand.

PubMed

Park, Sooyeon; Jung, Yong-Taek; Lee, Jung-Sook; Lee, Kenu-Chul; Yoon, Jung-Hoon

2013-12-01

A Gram-negative, aerobic, non-flagellated, non-gliding and rod-shaped bacterial strain, designated M-M16(T), was isolated from seashore sand around a seaweed farm on the South Sea, South Korea, and its taxonomic position was investigated by using a polyphasic study. Strain M-M16(T) grew optimally at 30 °C, at pH 7.0-8.0 and in the presence of 2 % (w/v) NaCl. Strain M-M16(T) exhibited the highest 16S rRNA gene sequence similarity values to the type strains of Gaetbulibacter lutimaris (96.5 %) and Flaviramulus basaltis (95.8 %). Neighbour-joining and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences revealed that strain M-M16(T) clustered with the type strains of Gaetbulibacter species and F. basaltis. Strain M-M16(T) contained MK-6 as the predominant menaquinone and iso-C15:1 G, iso-C15:0 and iso-C17:0 3-OH as the major fatty acids. The major polar lipids detected in strain M-M16(T) were phosphatidylethanolamine and one unidentified lipid. The DNA G+C content of strain M-M16(T) was 37.4 mol%. The phylogenetic and chemotaxonomic data and other phenotypic properties revealed that strain M-M16(T) represents a novel genus and species within the family Flavobacteriaceae, for which the name Sabulilitoribacter multivorans gen. nov., sp. nov. is proposed. The type strain of S. multivorans is M-M16(T) (= KCTC 32326(T) = CCUG 63831(T)).

Sequence Elements Upstream of the Core Promoter Are Necessary for Full Transcription of the Capsule Gene Operon in Streptococcus pneumoniae Strain D39

PubMed Central

Wen, Zhensong; Sertil, Odeniel; Cheng, Yongxin; Zhang, Shanshan; Liu, Xue; Wang, Wen-Ching

2015-01-01

Streptococcus pneumoniae is a major bacterial pathogen in humans. Its polysaccharide capsule is a key virulence factor that promotes bacterial evasion of human phagocytic killing. While S. pneumoniae produces at least 94 antigenically different types of capsule, the genes for biosynthesis of almost all capsular types are arranged in the same locus. The transcription of the capsular polysaccharide (cps) locus is not well understood. This study determined the transcriptional features of the cps locus in the type 2 virulent strain D39. The initial analysis revealed that the cps genes are cotranscribed from a major transcription start site at the −25 nucleotide (G) upstream of cps2A, the first gene in the locus. Using unmarked chromosomal truncations and a luciferase-based transcriptional reporter, we showed that the full transcription of the cps genes not only depends on the core promoter immediately upstream of cps2A, but also requires additional elements upstream of the core promoter, particularly a 59-bp sequence immediately upstream of the core promoter. Unmarked deletions of these promoter elements in the D39 genome also led to significant reduction in CPS production and virulence in mice. Lastly, common cps gene (cps2ABCD) mutants did not show significant abnormality in cps transcription, although they produced significantly less CPS, indicating that the CpsABCD proteins are involved in the encapsulation of S. pneumoniae in a posttranscriptional manner. This study has yielded important information on the transcriptional characteristics of the cps locus in S. pneumoniae. PMID:25733517

Naumannella halotolerans gen. nov., sp. nov., a Gram-positive coccus of the family Propionibacteriaceae isolated from a pharmaceutical clean room and from food.

PubMed

Rieser, Gernot; Scherer, Siegfried; Wenning, Mareike

2012-12-01

Four Gram-stain-positive, aerobic bacterial strains isolated from a pharmaceutical clean room (strain WS4616(T)), a dessert milk product (strain WS4617) and from raw milk (strains WS4623 and WS4624) were characterized using a polyphasic approach. Phylogenetic analyses based on 16S rRNA and recA gene sequences showed that they formed a distinct lineage within the family Propionibacteriaceae. Similarity values between 16S rRNA gene sequences of the four novel strains and the type species of all genera belonging to the family Propionibacteriaceae were 89.2-94.1%. The major cellular fatty acid was anteiso-C(15:0) and the major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. Respiratory quinones were MK-8(H(4)) and MK-9(H(4)). The cell-wall peptidoglycan of type A3γ contained ll-diaminopimelic acid, alanine, glycine and glutamic acid. The G+C content of the genomic DNA of strain WS4616(T) was 67.7 mol%. The whole-cell sugar pattern contained ribose, mannose, arabinose, glucose and galactose. On the basis of phenotypic and genetic data, strains WS4616(T), WS4617, WS4623 and WS4624 are classified as members of a novel species in a new genus of the family Propionibacteriaceae, for which the name Naumannella halotolerans gen. nov., sp. nov. is proposed. The type strain is WS4616(T) ( = DSM 24323(T) = LMG 26184(T)) and three additional strains are WS4617, WS4623 and WS4624.

Color differences among feral pigeons (Columba livia) are not attributable to sequence variation in the coding region of the melanocortin-1 receptor gene (MC1R)

PubMed Central

2013-01-01

Background Genetic variation at the melanocortin-1 receptor (MC1R) gene is correlated with melanin color variation in many birds. Feral pigeons (Columba livia) show two major melanin-based colorations: a red coloration due to pheomelanic pigment and a black coloration due to eumelanic pigment. Furthermore, within each color type, feral pigeons display continuous variation in the amount of melanin pigment present in the feathers, with individuals varying from pure white to a full dark melanic color. Coloration is highly heritable and it has been suggested that it is under natural or sexual selection, or both. Our objective was to investigate whether MC1R allelic variants are associated with plumage color in feral pigeons. Findings We sequenced 888 bp of the coding sequence of MC1R among pigeons varying both in the type, eumelanin or pheomelanin, and the amount of melanin in their feathers. We detected 10 non-synonymous substitutions and 2 synonymous substitution but none of them were associated with a plumage type. It remains possible that non-synonymous substitutions that influence coloration are present in the short MC1R fragment that we did not sequence but this seems unlikely because we analyzed the entire functionally important region of the gene. Conclusions Our results show that color differences among feral pigeons are probably not attributable to amino acid variation at the MC1R locus. Therefore, variation in regulatory regions of MC1R or variation in other genes may be responsible for the color polymorphism of feral pigeons. PMID:23915680

A Single Multilocus Sequence Typing (MLST) Scheme for Seven Pathogenic Leptospira Species

PubMed Central

Amornchai, Premjit; Wuthiekanun, Vanaporn; Bailey, Mark S.; Holden, Matthew T. G.; Zhang, Cuicai; Jiang, Xiugao; Koizumi, Nobuo; Taylor, Kyle; Galloway, Renee; Hoffmaster, Alex R.; Craig, Scott; Smythe, Lee D.; Hartskeerl, Rudy A.; Day, Nicholas P.; Chantratita, Narisara; Feil, Edward J.; Aanensen, David M.; Spratt, Brian G.; Peacock, Sharon J.

2013-01-01

Background The available Leptospira multilocus sequence typing (MLST) scheme supported by a MLST website is limited to L. interrogans and L. kirschneri. Our aim was to broaden the utility of this scheme to incorporate a total of seven pathogenic species. Methodology and Findings We modified the existing scheme by replacing one of the seven MLST loci (fadD was changed to caiB), as the former gene did not appear to be present in some pathogenic species. Comparison of the original and modified schemes using data for L. interrogans and L. kirschneri demonstrated that the discriminatory power of the two schemes was not significantly different. The modified scheme was used to further characterize 325 isolates (L. alexanderi [n = 5], L. borgpetersenii [n = 34], L. interrogans [n = 222], L. kirschneri [n = 29], L. noguchii [n = 9], L. santarosai [n = 10], and L. weilii [n = 16]). Phylogenetic analysis using concatenated sequences of the 7 loci demonstrated that each species corresponded to a discrete clade, and that no strains were misclassified at the species level. Comparison between genotype and serovar was possible for 254 isolates. Of the 31 sequence types (STs) represented by at least two isolates, 18 STs included isolates assigned to two or three different serovars. Conversely, 14 serovars were identified that contained between 2 to 10 different STs. New observations were made on the global phylogeography of Leptospira spp., and the utility of MLST in making associations between human disease and specific maintenance hosts was demonstrated. Conclusion The new MLST scheme, supported by an updated MLST website, allows the characterization and species assignment of isolates of the seven major pathogenic species associated with leptospirosis. PMID:23359622

Fontibacillus aquaticus gen. nov., sp. nov., isolated from a warm spring.

PubMed

Saha, P; Krishnamurthi, S; Bhattacharya, A; Sharma, R; Chakrabarti, T

2010-02-01

A novel facultatively anaerobic strain, designated GPTSA 19(T), was isolated from a warm spring and characterized using a polyphasic approach. The strain behaved as Gram-negative in the Gram staining procedure but showed a Gram-positive reaction in the aminopeptidase test. The novel strain was a mesophilic rod with ellipsoidal endospores. On the basis of 16S rRNA gene sequence analysis, the strain showed closest similarity (96.0 %) with Paenibacillus motobuensis MC10(T). The gene sequence similarity of the novel strain with other species of the genus Paenibacillus was <95.8 %. The novel strain also had PAEN 515F and 682F signature sequence stretches in the 16S rRNA gene that are usually found in most species of the genus Paenibacillus. The strain possessed anteiso-C(15 : 0) as the major fatty acid and MK-7 as the predominant menaquinone. Polar lipids included diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), six unknown phospholipids (PLs), one aminophospholipid (PN), three glycolipids (GLs), two aminolipids (ALs), one aminophosphoglycolipid (APGL) and three unknown lipids (ULs). The polar lipid profile of the novel strain, especially as regards ALs, GLs and PLs, distinguished it from the recognized type species of the genus Paenibacillus, Paenibacillus polymyxa, as well as from its closest relative P. motobuensis. Based on phenotypic and chemotaxonomic characteristics and analysis of the 16S rRNA gene sequence, the new strain merits the rank of a novel genus for which the name Fontibacillus gen. nov. is proposed. The type species of the new genus is Fontibacillus aquaticus gen. nov., sp. nov. with the type strain GPTSA 19(T) (=MTCC 7155(T)=DSM 17643(T)).

The Poultry-Associated Microbiome: Network Analysis and Farm-to-Fork Characterizations

PubMed Central

Oakley, Brian B.; Morales, Cesar A.; Line, J.; Berrang, Mark E.; Meinersmann, Richard J.; Tillman, Glenn E.; Wise, Mark G.; Siragusa, Gregory R.; Hiett, Kelli L.; Seal, Bruce S.

2013-01-01

Microbial communities associated with agricultural animals are important for animal health, food safety, and public health. Here we combine high-throughput sequencing (HTS), quantitative-PCR assays, and network analysis to profile the poultry-associated microbiome and important pathogens at various stages of commercial poultry production from the farm to the consumer. Analysis of longitudinal data following two flocks from the farm through processing showed a core microbiome containing multiple sequence types most closely related to genera known to be pathogenic for animals and/or humans, including Campylobacter, Clostridium, and Shigella. After the final stage of commercial poultry processing, taxonomic richness was ca. 2–4 times lower than the richness of fecal samples from the same flocks and Campylobacter abundance was significantly reduced. Interestingly, however, carcasses sampled at 48 hr after processing harboured the greatest proportion of unique taxa (those not encountered in other samples), significantly more than expected by chance. Among these were anaerobes such as Prevotella, Veillonella, Leptrotrichia, and multiple Campylobacter sequence types. Retail products were dominated by Pseudomonas, but also contained 27 other genera, most of which were potentially metabolically active and encountered in on-farm samples. Network analysis was focused on the foodborne pathogen Campylobacter and revealed a majority of sequence types with no significant interactions with other taxa, perhaps explaining the limited efficacy of previous attempts at competitive exclusion of Campylobacter. These data represent the first use of HTS to characterize the poultry microbiome across a series of farm-to-fork samples and demonstrate the utility of HTS in monitoring the food supply chain and identifying sources of potential zoonoses and interactions among taxa in complex communities. PMID:23468931

Unraveling Core Functional Microbiota in Traditional Solid-State Fermentation by High-Throughput Amplicons and Metatranscriptomics Sequencing.

PubMed

Song, Zhewei; Du, Hai; Zhang, Yan; Xu, Yan

2017-01-01

Fermentation microbiota is specific microorganisms that generate different types of metabolites in many productions. In traditional solid-state fermentation, the structural composition and functional capacity of the core microbiota determine the quality and quantity of products. As a typical example of food fermentation, Chinese Maotai-flavor liquor production involves a complex of various microorganisms and a wide variety of metabolites. However, the microbial succession and functional shift of the core microbiota in this traditional food fermentation remain unclear. Here, high-throughput amplicons (16S rRNA gene amplicon sequencing and internal transcribed space amplicon sequencing) and metatranscriptomics sequencing technologies were combined to reveal the structure and function of the core microbiota in Chinese soy sauce aroma type liquor production. In addition, ultra-performance liquid chromatography and headspace-solid phase microextraction-gas chromatography-mass spectrometry were employed to provide qualitative and quantitative analysis of the major flavor metabolites. A total of 10 fungal and 11 bacterial genera were identified as the core microbiota. In addition, metatranscriptomic analysis revealed pyruvate metabolism in yeasts (genera Pichia, Schizosaccharomyces, Saccharomyces , and Zygosaccharomyces ) and lactic acid bacteria (genus Lactobacillus ) classified into two stages in the production of flavor components. Stage I involved high-level alcohol (ethanol) production, with the genus Schizosaccharomyces serving as the core functional microorganism. Stage II involved high-level acid (lactic acid and acetic acid) production, with the genus Lactobacillus serving as the core functional microorganism. The functional shift from the genus Schizosaccharomyces to the genus Lactobacillus drives flavor component conversion from alcohol (ethanol) to acid (lactic acid and acetic acid) in Chinese Maotai-flavor liquor production. Our findings provide insight into the effects of the core functional microbiota in soy sauce aroma type liquor production and the characteristics of the fermentation microbiota under different environmental conditions.

Single-cell RNA-sequencing reveals a distinct population of proglucagon-expressing cells specific to the mouse upper small intestine.

PubMed

Glass, Leslie L; Calero-Nieto, Fernando J; Jawaid, Wajid; Larraufie, Pierre; Kay, Richard G; Göttgens, Berthold; Reimann, Frank; Gribble, Fiona M

2017-10-01

To identify sub-populations of intestinal preproglucagon-expressing (PPG) cells producing Glucagon-like Peptide-1, and their associated expression profiles of sensory receptors, thereby enabling the discovery of therapeutic strategies that target these cell populations for the treatment of diabetes and obesity. We performed single cell RNA sequencing of PPG-cells purified by flow cytometry from the upper small intestine of 3 GLU-Venus mice. Cells from 2 mice were sequenced at low depth, and from the third mouse at high depth. High quality sequencing data from 234 PPG-cells were used to identify clusters by tSNE analysis. qPCR was performed to compare the longitudinal and crypt/villus locations of cluster-specific genes. Immunofluorescence and mass spectrometry were used to confirm protein expression. PPG-cells formed 3 major clusters: a group with typical characteristics of classical L-cells, including high expression of Gcg and Pyy (comprising 51% of all PPG-cells); a cell type overlapping with Gip-expressing K-cells (14%); and a unique cluster expressing Tph1 and Pzp that was predominantly located in proximal small intestine villi and co-produced 5-HT (35%). Expression of G-protein coupled receptors differed between clusters, suggesting the cell types are differentially regulated and would be differentially targetable. Our findings support the emerging concept that many enteroendocrine cell populations are highly overlapping, with individual cells producing a range of peptides previously assigned to distinct cell types. Different receptor expression profiles across the clusters highlight potential drug targets to increase gut hormone secretion for the treatment of diabetes and obesity. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

Disruption of predicted dengue virus type 3 major outbreak cycle coincided with switching of the dominant circulating virus genotype.

PubMed

Tan, Kim-Kee; Zulkifle, Nurul-Izzani; Abd-Jamil, Juraina; Sulaiman, Syuhaida; Yaacob, Che Norainon; Azizan, Noor Syahida; Che Mat Seri, Nurul Asma Anati; Samsudin, Nur Izyan; Mahfodz, Nur Hidayana; AbuBakar, Sazaly

2017-10-01

Dengue is hyperendemic in most of Southeast Asia. In this region, all four dengue virus serotypes are persistently present. Major dengue outbreak cycle occurs in a cyclical pattern involving the different dengue virus serotypes. In Malaysia, since the 1980s, the major outbreak cycles have involved dengue virus type 3 (DENV3), dengue virus type 1 (DENV1) and dengue virus type 2 (DENV2), occurring in that order (DENV3/DENV1/DENV2). Only limited information on the DENV3 cycles, however, have been described. In the current study, we examined the major outbreak cycle involving DENV3 using data from 1985 to 2016. We examined the genetic diversity of DENV3 isolates obtained during the period when DENV3 was the dominant serotype and during the inter-dominant transmission period. Results obtained suggest that the typical DENV3/DENV1/DENV2 cyclical outbreak cycle in Malaysia has recently been disrupted. The last recorded major outbreak cycle involving DENV3 occurred in 2002, and the expected major outbreak cycle involving DENV3 in 2006-2012 did not materialize. DENV genome analyses revealed that DENV3 genotype II (DENV3/II) was the predominant DENV3 genotype (67%-100%) recovered between 1987 and 2002. DENV3 genotype I (DENV3/I) emerged in 2002 followed by the introduction of DENV3 genotype III (DENV3/III) in 2008. These newly emerged DENV3 genotypes replaced DENV3/II, but there was no major upsurge of DENV3 cases that accompanied the emergence of these viruses. DENV3 remained in the background of DENV1 and DENV2 until now. Virus genome sequence analysis suggested that intrinsic differences within the different dengue virus genotypes could have influenced the transmission efficiency of DENV3. Further studies and continuous monitoring of the virus are needed for better understanding of the DENV transmission dynamics in hyperendemic regions. Copyright © 2017 Elsevier B.V. All rights reserved.

Pathogenesis of Helicobacter pylori-Related Gastroduodenal Diseases from Molecular Epidemiological Studies

PubMed Central

Yamaoka, Yoshio

2012-01-01

Helicobacter pylori is a major human pathogen that infects the stomach and produces inflammation that is responsible for various gastroduodenal diseases. Despite the high prevalence of H. pylori infections in Africa and South Asia, the incidence of gastric cancer in these areas is much lower than in other countries. The incidence of gastric cancer also tends to decrease from north to south in East Asia. Data from molecular epidemiological studies show that this variation in different geographic areas could be explained in part by different types of H. pylori virulence factors, especially CagA, VacA, and OipA. H. pylori infection is thought to be involved in both gastric cancer and duodenal ulcer, which are at opposite ends of the disease spectrum. This discrepancy can also be explained in part by another H. pylori factor, DupA, as well as by CagA typing (East Asian type versus Western type). H. pylori has a genome of approximately 1,600 genes; therefore, there might be other novel virulence factors. Because genome wide analyses using whole-genome sequencing technology give a broad view of the genome of H. pylori, we hope that next-generation sequencers will enable us to efficiently investigate novel virulence factors. PMID:22829807

Comparison of Genotypes and Enterotoxin Genes Between Staphylococcus aureus Isolates from Blood and Nasal Colonizers in a Korean Hospital

PubMed Central

Peck, Kyong Ran; Baek, Jin Yang; Song, Jae-Hoon

2009-01-01

In this study, we investigated the genetic background of 70 Staphylococcus aureus isolates (36 methicillin-resistant S. aureus [MRSA] and 34 methicillin-susceptible S. aureus [MSSA]) obtained from blood at a Korean tertiary-care hospital, using spa typing, multilocus sequence typing, and SCCmec typing. In addition, the prevalence of enterotoxin (sea, seb, sec, sed, see, seg, seh, sei, and sek), tst, and pvl genes among the samples was assessed via polymerase chain reaction, and the results were compared with those of 95 isolates of S. aureus obtained from nasal swabs. All MRSA isolates from blood, except one, belonged to three major clones: sequence type (ST)5-MRSA-II, ST72-MRSA-II (or IVA), and ST239-MRSA-III, among which ST5-MRSA-II was the predominant clone. The prevalence of enterotoxin genes in the S. aureus isolates obtained from blood differed significantly from those from the nasal swabs for the sea, seb, sec, and seh gene. In particular, the seb and sec genes were detected exclusively in the MRSA isolates of ST5 or spa-CC002, thereby suggesting the co-adaptation of virulence genes with the genetic background and their contribution to biological fitness. PMID:19654937

Virgibacillus halophilus sp. nov., spore-forming bacteria isolated from soil in Japan.

PubMed

An, Sun-Young; Asahara, Mika; Goto, Keiichi; Kasai, Hiroaki; Yokota, Akira

2007-07-01

Two Gram-positive, round-spore-forming, rod-shaped, halophilic bacterial strains, 5B73C(T) and 5B133E, were isolated from field soil in Kakegawa, Shizuoka, Japan, and were characterized taxonomically using a polyphasic approach. These two strains were found to comprise strictly aerobic, motile rods that formed subterminal endospores. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains 5B73C(T) and 5B133E are phylogenetically affiliated to the genus Virgibacillus, exhibiting sequence similarities of 94.1-96.4 % with respect to the type strains of Virgibacillus species. The DNA G+C contents of strains 5B73C(T) and 5B133E were 42.6 and 42.3 mol%, respectively. The cell-wall peptidoglycan type (meso-diaminopimelic acid), the major cellular fatty acids (anteiso-C(15 : 0), iso-C(15 : 0), anteiso-C(17 : 0) and iso-C(16 : 0)) and the quinone type (MK-7) of the isolates support their affiliation to the genus Virgibacillus. On the basis of their genotypic and phenotypic characteristics, the isolates represent a novel species of the genus Virgibacillus, for which the name Virgibacillus halophilus sp. nov. is proposed. The type strain is 5B73C(T) (=IAM 15308(T)=KCTC 13935(T)).

Whole-Genome Sequencing and Variant Analysis of Human Papillomavirus 16 Infections.

PubMed

van der Weele, Pascal; Meijer, Chris J L M; King, Audrey J

2017-10-01

Human papillomavirus (HPV) is a strongly conserved DNA virus, high-risk types of which can cause cervical cancer in persistent infections. The most common type found in HPV-attributable cancer is HPV16, which can be subdivided into four lineages (A to D) with different carcinogenic properties. Studies have shown HPV16 sequence diversity in different geographical areas, but only limited information is available regarding HPV16 diversity within a population, especially at the whole-genome level. We analyzed HPV16 major variant diversity and conservation in persistent infections and performed a single nucleotide polymorphism (SNP) comparison between persistent and clearing infections. Materials were obtained in the Netherlands from a cohort study with longitudinal follow-up for up to 3 years. Our analysis shows a remarkably large variant diversity in the population. Whole-genome sequences were obtained for 57 persistent and 59 clearing HPV16 infections, resulting in 109 unique variants. Interestingly, persistent infections were completely conserved through time. One reinfection event was identified where the initial and follow-up samples clustered differently. Non-A1/A2 variants seemed to clear preferentially ( P = 0.02). Our analysis shows that population-wide HPV16 sequence diversity is very large. In persistent infections, the HPV16 sequence was fully conserved. Sequencing can identify HPV16 reinfections, although occurrence is rare. SNP comparison identified no strongly acting effect of the viral genome affecting HPV16 infection clearance or persistence in up to 3 years of follow-up. These findings suggest the progression of an early HPV16 infection could be host related. IMPORTANCE Human papillomavirus 16 (HPV16) is the predominant type found in cervical cancer. Progression of initial infection to cervical cancer has been linked to sequence properties; however, knowledge of variants circulating in European populations, especially with longitudinal follow-up, is limited. By sequencing a number of infections with known follow-up for up to 3 years, we gained initial insights into the genetic diversity of HPV16 and the effects of the viral genome on the persistence of infections. A SNP comparison between sequences obtained from clearing and persistent infections did not identify strongly acting DNA variations responsible for these infection outcomes. In addition, we identified an HPV16 reinfection event where sequencing of initial and follow-up samples showed different HPV16 variants. Based on conventional genotyping, this infection would incorrectly be considered a persistent HPV16 infection. In the context of vaccine efficacy and monitoring studies, such infections could potentially cause reduced reported efficacy or efficiency. Copyright © 2017 van der Weele et al.

The Evolution of Vp1 Gene in Enterovirus C Species Sub-Group That Contains Types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99

PubMed Central

Smura, Teemu; Blomqvist, Soile; Vuorinen, Tytti; Ivanova, Olga; Samoilovich, Elena; Al-Hello, Haider; Savolainen-Kopra, Carita; Hovi, Tapani; Roivainen, Merja

2014-01-01

Genus Enterovirus (Family Picornaviridae,) consists of twelve species divided into genetically diverse types by their capsid protein VP1 coding sequences. Each enterovirus type can further be divided into intra-typic sub-clusters (genotypes). The aim of this study was to elucidate what leads to the emergence of novel enterovirus clades (types and genotypes). An evolutionary analysis was conducted for a sub-group of Enterovirus C species that contains types Coxsackievirus A21 (CVA-21), CVA-24, Enterovirus C95 (EV-C95), EV-C96 and EV-C99. VP1 gene datasets were collected and analysed to infer the phylogeny, rate of evolution, nucleotide and amino acid substitution patterns and signs of selection. In VP1 coding gene, high intra-typic sequence diversities and robust grouping into distinct genotypes within each type were detected. Within each type the majority of nucleotide substitutions were synonymous and the non-synonymous substitutions tended to cluster in distinct highly polymorphic sites. Signs of positive selection were detected in some of these highly polymorphic sites, while strong negative selection was indicated in most of the codons. Despite robust clustering to intra-typic genotypes, only few genotype-specific ‘signature’ amino acids were detected. In contrast, when different enterovirus types were compared, there was a clear tendency towards fixation of type-specific ‘signature’ amino acids. The results suggest that permanent fixation of type-specific amino acids is a hallmark associated with evolution of different enterovirus types, whereas neutral evolution and/or (frequency-dependent) positive selection in few highly polymorphic amino acid sites are the dominant forms of evolution when strains within an enterovirus type are compared. PMID:24695547

The evolution of Vp1 gene in enterovirus C species sub-group that contains types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99.

PubMed

Smura, Teemu; Blomqvist, Soile; Vuorinen, Tytti; Ivanova, Olga; Samoilovich, Elena; Al-Hello, Haider; Savolainen-Kopra, Carita; Hovi, Tapani; Roivainen, Merja

2014-01-01

Genus Enterovirus (Family Picornaviridae,) consists of twelve species divided into genetically diverse types by their capsid protein VP1 coding sequences. Each enterovirus type can further be divided into intra-typic sub-clusters (genotypes). The aim of this study was to elucidate what leads to the emergence of novel enterovirus clades (types and genotypes). An evolutionary analysis was conducted for a sub-group of Enterovirus C species that contains types Coxsackievirus A21 (CVA-21), CVA-24, Enterovirus C95 (EV-C95), EV-C96 and EV-C99. VP1 gene datasets were collected and analysed to infer the phylogeny, rate of evolution, nucleotide and amino acid substitution patterns and signs of selection. In VP1 coding gene, high intra-typic sequence diversities and robust grouping into distinct genotypes within each type were detected. Within each type the majority of nucleotide substitutions were synonymous and the non-synonymous substitutions tended to cluster in distinct highly polymorphic sites. Signs of positive selection were detected in some of these highly polymorphic sites, while strong negative selection was indicated in most of the codons. Despite robust clustering to intra-typic genotypes, only few genotype-specific 'signature' amino acids were detected. In contrast, when different enterovirus types were compared, there was a clear tendency towards fixation of type-specific 'signature' amino acids. The results suggest that permanent fixation of type-specific amino acids is a hallmark associated with evolution of different enterovirus types, whereas neutral evolution and/or (frequency-dependent) positive selection in few highly polymorphic amino acid sites are the dominant forms of evolution when strains within an enterovirus type are compared.

Association between Helicobacter pylori Virulence Factors and Gastroduodenal Diseases in Okinawa, Japan

PubMed Central

Matsunari, Osamu; Shiota, Seiji; Suzuki, Rumiko; Watada, Masahide; Kinjo, Nagisa; Murakami, Kazunari; Fujioka, Toshio; Kinjo, Fukunori

2012-01-01

The incidence of gastric cancer in Okinawa is lowest in Japan. Some previous reports using small number of strains suggested that the high prevalence of Helicobacter pylori with Western-type cagA in Okinawa compared to other areas in Japan might contribute to the low incidence of gastric cancer. It has still not been confirmed why the prevalence of Western-type cagA strains is high in Okinawa. We examined the association between the virulence factors of H. pylori and gastroduodenal diseases in Okinawa. The genotypes of cagA and vacA of 337 H. pylori strains were determined by PCR and gene sequencing. The genealogy of these Western-type cagA strains in Okinawa was analyzed by multilocus sequence typing (MLST). Overall, 86.4% of the strains possessed cagA: 70.3% were East-Asian type and 16.0% were Western type. After adjustment by age and sex, the presence of East-Asian-type cagA/vacA s1m1 genotypes was significantly associated with gastric cancer compared to gastritis (odds ratio = 6.68, 95% confidence interval = 1.73 to 25.8). The structure of Western-type CagA in Okinawa was different from that of typical Western-type CagA found in Western countries. Intriguingly, MLST analysis revealed that the majority of Western-type cagA strains formed individual clusters but not hpEurope. Overall, low prevalence of gastric cancer in Okinawa may result from the high prevalence of non-East-Asian-type cagA strains. The origin of Western-type cagA strains in Okinawa may be different from those of Western countries. PMID:22189111

Development of a Single Locus Sequence Typing (SLST) Scheme for Typing Bacterial Species Directly from Complex Communities.

PubMed

Scholz, Christian F P; Jensen, Anders

2017-01-01

The protocol describes a computational method to develop a Single Locus Sequence Typing (SLST) scheme for typing bacterial species. The resulting scheme can be used to type bacterial isolates as well as bacterial species directly from complex communities using next-generation sequencing technologies.

Concordance of the ForenSeq™ system and characterisation of sequence-specific autosomal STR alleles across two major population groups.

PubMed

Devesse, Laurence; Ballard, David; Davenport, Lucinda; Riethorst, Immy; Mason-Buck, Gabriella; Syndercombe Court, Denise

2018-05-01

By using sequencing technology to genotype loci of forensic interest it is possible to simultaneously target autosomal, X and Y STRs as well as identity, ancestry and phenotypic informative SNPs, resulting in a breadth of data obtained from a single run that is considerable when compared to that generated with standard technologies. It is important however that this information aligns with the genotype data currently obtained using commercially available kits for CE-based investigations such that results are compatible with existing databases and hence can be of use to the forensic community. In this work, 400 samples were typed using commercially available STR kits and CE, as well as using the Ilumina ForenSeq™ DNA Signature Prep Kit and MiSeq ® FGx to assess concordance of autosomal STRs and population variability. Results show a concordance rate between the two technologies exceeding 99.98% while numerous novel sequence based alleles are described. In order to make use of the sequence variation observed, sequence specific allele frequencies were generated for White British and British Chinese populations. Copyright © 2017 Elsevier B.V. All rights reserved.

Navigating highly homologous genes in a molecular diagnostic setting: a resource for clinical next-generation sequencing.

PubMed

Mandelker, Diana; Schmidt, Ryan J; Ankala, Arunkanth; McDonald Gibson, Kristin; Bowser, Mark; Sharma, Himanshu; Duffy, Elizabeth; Hegde, Madhuri; Santani, Avni; Lebo, Matthew; Funke, Birgit

2016-12-01

Next-generation sequencing (NGS) is now routinely used to interrogate large sets of genes in a diagnostic setting. Regions of high sequence homology continue to be a major challenge for short-read technologies and can lead to false-positive and false-negative diagnostic errors. At the scale of whole-exome sequencing (WES), laboratories may be limited in their knowledge of genes and regions that pose technical hurdles due to high homology. We have created an exome-wide resource that catalogs highly homologous regions that is tailored toward diagnostic applications. This resource was developed using a mappability-based approach tailored to current Sanger and NGS protocols. Gene-level and exon-level lists delineate regions that are difficult or impossible to analyze via standard NGS. These regions are ranked by degree of affectedness, annotated for medical relevance, and classified by the type of homology (within-gene, different functional gene, known pseudogene, uncharacterized noncoding region). Additionally, we provide a list of exons that cannot be analyzed by short-amplicon Sanger sequencing. This resource can help guide clinical test design, supplemental assay implementation, and results interpretation in the context of high homology.Genet Med 18 12, 1282-1289.

Longitudinal Metagenomic Analysis of Hospital Air Identifies Clinically Relevant Microbes.

PubMed

King, Paula; Pham, Long K; Waltz, Shannon; Sphar, Dan; Yamamoto, Robert T; Conrad, Douglas; Taplitz, Randy; Torriani, Francesca; Forsyth, R Allyn

2016-01-01

We describe the sampling of sixty-three uncultured hospital air samples collected over a six-month period and analysis using shotgun metagenomic sequencing. Our primary goals were to determine the longitudinal metagenomic variability of this environment, identify and characterize genomes of potential pathogens and determine whether they are atypical to the hospital airborne metagenome. Air samples were collected from eight locations which included patient wards, the main lobby and outside. The resulting DNA libraries produced 972 million sequences representing 51 gigabases. Hierarchical clustering of samples by the most abundant 50 microbial orders generated three major nodes which primarily clustered by type of location. Because the indoor locations were longitudinally consistent, episodic relative increases in microbial genomic signatures related to the opportunistic pathogens Aspergillus, Penicillium and Stenotrophomonas were identified as outliers at specific locations. Further analysis of microbial reads specific for Stenotrophomonas maltophilia indicated homology to a sequenced multi-drug resistant clinical strain and we observed broad sequence coverage of resistance genes. We demonstrate that a shotgun metagenomic sequencing approach can be used to characterize the resistance determinants of pathogen genomes that are uncharacteristic for an otherwise consistent hospital air microbial metagenomic profile.

CtGEM typing: Discrimination of Chlamydia trachomatis ocular and urogenital strains and major evolutionary lineages by high resolution melting analysis of two amplified DNA fragments.

PubMed

Giffard, Philip M; Andersson, Patiyan; Wilson, Judith; Buckley, Cameron; Lilliebridge, Rachael; Harris, Tegan M; Kleinecke, Mariana; O'Grady, Kerry-Ann F; Huston, Wilhelmina M; Lambert, Stephen B; Whiley, David M; Holt, Deborah C

2018-01-01

Chlamydia trachomatis infects the urogenital tract (UGT) and eyes. Anatomical tropism is correlated with variation in the major outer membrane protein encoded by ompA. Strains possessing the ocular ompA variants A, B, Ba and C are typically found within the phylogenetically coherent "classical ocular lineage". However, variants B, Ba and C have also been found within three distinct strains in Australia, all associated with ocular disease in children and outside the classical ocular lineage. CtGEM genotyping is a method for detecting and discriminating ocular strains and also the major phylogenetic lineages. The rationale was facilitation of surveillance to inform responses to C. trachomatis detection in UGT specimens from young children. CtGEM typing is based on high resolution melting analysis (HRMA) of two PCR amplified fragments with high combinatorial resolving power, as defined by computerised comparison of 65 whole genomes. One fragment is from the hypothetical gene defined by Jali-1891 in the C. trachomatis B_Jali20 genome, while the other is from ompA. Twenty combinatorial CtGEM types have been shown to exist, and these encompass unique genotypes for all known ocular strains, and also delineate the TI and T2 major phylogenetic lineages, identify LGV strains and provide additional resolution beyond this. CtGEM typing and Sanger sequencing were compared with 42 C. trachomatis positive clinical specimens, and there were no disjunctions. CtGEM typing is a highly efficient method designed and tested using large scale comparative genomics. It divides C. trachomatis into clinically and biologically meaningful groups, and may have broad application in surveillance.

The role of gut microbiota in the development of type 1, type 2 diabetes mellitus and obesity.

PubMed

Tai, Ningwen; Wong, F Susan; Wen, Li

2015-03-01

Diabetes is a group of metabolic disorders characterized by persistent hyperglycemia and has become a major public health concern. Autoimmune type 1 diabetes (T1D) and insulin resistant type 2 diabetes (T2D) are the two main types. A combination of genetic and environmental factors contributes to the development of these diseases. Gut microbiota have emerged recently as an essential player in the development of T1D, T2D and obesity. Altered gut microbiota have been strongly linked to disease in both rodent models and humans. Both classic 16S rRNA sequencing and shot-gun metagenomic pyrosequencing analysis have been successfully applied to explore the gut microbiota composition and functionality. This review focuses on the association between gut microbiota and diabetes and discusses the potential mechanisms by which gut microbiota regulate disease development in T1D, T2D and obesity.

Genotype and biotype of invasive Anopheles stephensi in Mannar Island of Sri Lanka.

PubMed

Surendran, Sinnathamby N; Sivabalakrishnan, Kokila; Gajapathy, Kanapathy; Arthiyan, Sivasingham; Jayadas, Tibutius T P; Karvannan, Kalingarajah; Raveendran, Selvarajah; Parakrama Karunaratne, S H P; Ramasamy, Ranjan

2018-01-03

Anopheles stephensi, the major vector of urban malaria in India, was recently detected for the first time in Sri Lanka in Mannar Island on the northwestern coast. Since there are different biotypes of An. stephensi with different vector capacities in India, a study was undertaken to further characterise the genotype and biotype of An. stephensi in Mannar Island. Mosquito larvae were collected in Pesalai village in Mannar and maintained in the insectary until adulthood. Adult An. stephensi were identified morphologically using published keys. Identified adult An. stephensi were molecularly characterized using two mitochondrial (cox1 and cytb) and one nuclear (ITS2) markers. Their PCR-amplified target fragments were sequenced and checked against available sequences in GenBank for phylogenetic analysis. The average spiracular and thoracic lengths and the spiracular index were determined to identify biotypes based on corresponding indices for Indian An. stephensi. All DNA sequences for the Mannar samples matched reported sequences for An. stephensi from the Middle East and India. However, a single nucleotide variation in the cox1 sequence suggested an amino acid change from valine to methionine in the cox1 protein in Sri Lankan An. stephensi. Morphological data was consistent with the presence of the Indian urban vector An. stephensi type-form in Sri Lanka. The present study provides a more detailed molecular characterization of An. stephensi and suggests the presence of the type-form of the vector for the first time in Sri Lanka. The single mutation in the cox1 gene may be indicative of a founder effect causing the initial diversification of An. stephensi in Sri Lanka from the Indian form. The distribution of the potent urban vector An. stephensi type-form needs to be established by studies throughout the island as its spread adds to the challenge of maintaining the country's malaria-free status.

An epidemiological survey of bovine Babesia and Theileria parasites in cattle, buffaloes, and sheep in Egypt.

PubMed

Elsify, Ahmed; Sivakumar, Thillaiampalam; Nayel, Mohammed; Salama, Akram; Elkhtam, Ahmed; Rizk, Mohamed; Mosaab, Omar; Sultan, Khaled; Elsayed, Shimaa; Igarashi, Ikuo; Yokoyama, Naoaki

2015-02-01

Cattle, buffaloes, and sheep are the main sources of meat and milk in Egypt, but their productivity is thought to be greatly reduced by hemoprotozoan parasitic diseases. In this study, we analyzed the infection rates of Babesia bovis, Babesia bigemina, Theileria annulata, and Theileria orientalis, using parasite-specific PCR assays in blood-DNA samples sourced from cattle (n=439), buffaloes (n=50), and sheep (n=105) reared in Menoufia, Behera, Giza, and Sohag provinces of Egypt. In cattle, the positive rates of B. bovis, B. bigemina, T. annulata, and T. orientalis were 3.18%, 7.97%, 9.56%, and 0.68%, respectively. On the other hand, B. bovis and T. orientalis were the only parasites detected in buffaloes and each of these parasites was only found in two individual DNA samples (both 2%), while one (0.95%) and two (1.90%) of the sheep samples were positive for B. bovis and B. bigemina, respectively. Sequence analysis showed that the B. bovis Rhoptry Associated Protein-1 and the B. bigemina Apical Membrane Antigen-1 genes were highly conserved among the samples, with 99.3-100% and 95.3-100% sequence identity values, respectively. In contrast, the Egyptian T. annulata merozoite surface antigen-1 gene sequences were relatively diverse (87.8-100% identity values), dispersing themselves across several clades in the phylogenetic tree containing sequences from other countries. Additionally, the T. orientalis Major Piroplasm Surface Protein (MPSP) gene sequences were classified as types 1 and 2. This is the first report of T. orientalis in Egypt, and of type 2 MPSP in buffaloes. Detection of MPSP type 2, which is considered a relatively virulent genotype, suggests that T. orientalis infection may have veterinary and economic significance in Egypt. In conclusion, the present study, which analyzed multiple species of Babesia and Theileria parasites in different livestock animals, may shed an additional light on the epidemiology of hemoprotozoan parasites in Egypt. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

First detection of canine parvovirus type 2b from diarrheic dogs in Himachal Pradesh.

PubMed

Sharma, Shalini; Dhar, Prasenjit; Thakur, Aneesh; Sharma, Vivek; Sharma, Mandeep

2016-09-01

The present study was conducted to detect the presence of canine parvovirus (CPV) among diarrheic dogs in Himachal Pradesh and to identify the most prevalent antigenic variant of CPV based on molecular typing and sequence analysis of VP2 gene. A total of 102 fecal samples were collected from clinical cases of diarrhea or hemorrhagic gastroenteritis from CPV vaccinated or non-vaccinated dogs. Samples were tested using CPV-specific polymerase chain reaction (PCR) targeting VP2 gene, multiplex PCR for detection of CPV-2a and CPV-2b antigenic variants, and a PCR for the detection of CPV-2c. CPV-2b isolate was cultured on Madin-Darby canine kidney (MDCK) cell lines and sequenced using VP2 structural protein gene. Multiple alignment and phylogenetic analysis was done using ClustalW and MEGA6 and inferred using the Neighbor-Joining method. No sample was found positive for the original CPV strain usually present in the vaccine. However, about 50% (52 out of 102) of the samples were found to be positive with CPV-2ab PCR assay that detects newer variants of CPV circulating in the field. In addition, multiplex PCR assay that identifies both CPV-2ab and CPV-2b revealed that CPV-2b was the major antigenic variant present in the affected dogs. A PCR positive isolate of CPV-2b was adapted to grow in MDCK cells and produced characteristic cytopathic effect after 5 th passage. Multiple sequence alignment of VP2 structural gene of CPV-2b isolate (Accession number HG004610) used in the study was found to be similar to other sequenced isolates in NCBI sequence database and showed 98-99% homology. This study reports the first detection of CPV-2b in dogs with hemorrhagic gastroenteritis in Himachal Pradesh and absence of other antigenic types of CPV. Further, CPV-specific PCR assay can be used for rapid confirmation of circulating virus strains under field conditions.

Using Next Generation Sequencing for Multiplexed Trait-Linked Markers in Wheat

PubMed Central

Bernardo, Amy; Wang, Shan; St. Amand, Paul; Bai, Guihua

2015-01-01

With the advent of next generation sequencing (NGS) technologies, single nucleotide polymorphisms (SNPs) have become the major type of marker for genotyping in many crops. However, the availability of SNP markers for important traits of bread wheat ( Triticum aestivum L.) that can be effectively used in marker-assisted selection (MAS) is still limited and SNP assays for MAS are usually uniplex. A shift from uniplex to multiplex assays will allow the simultaneous analysis of multiple markers and increase MAS efficiency. We designed 33 locus-specific markers from SNP or indel-based marker sequences that linked to 20 different quantitative trait loci (QTL) or genes of agronomic importance in wheat and analyzed the amplicon sequences using an Ion Torrent Proton Sequencer and a custom allele detection pipeline to determine the genotypes of 24 selected germplasm accessions. Among the 33 markers, 27 were successfully multiplexed and 23 had 100% SNP call rates. Results from analysis of "kompetitive allele-specific PCR" (KASP) and sequence tagged site (STS) markers developed from the same loci fully verified the genotype calls of 23 markers. The NGS-based multiplexed assay developed in this study is suitable for rapid and high-throughput screening of SNPs and some indel-based markers in wheat. PMID:26625271

Extraction of High Molecular Weight DNA from Fungal Rust Spores for Long Read Sequencing.

PubMed

Schwessinger, Benjamin; Rathjen, John P

2017-01-01

Wheat rust fungi are complex organisms with a complete life cycle that involves two different host plants and five different spore types. During the asexual infection cycle on wheat, rusts produce massive amounts of dikaryotic urediniospores. These spores are dikaryotic (two nuclei) with each nucleus containing one haploid genome. This dikaryotic state is likely to contribute to their evolutionary success, making them some of the major wheat pathogens globally. Despite this, most published wheat rust genomes are highly fragmented and contain very little haplotype-specific sequence information. Current long-read sequencing technologies hold great promise to provide more contiguous and haplotype-phased genome assemblies. Long reads are able to span repetitive regions and phase structural differences between the haplomes. This increased genome resolution enables the identification of complex loci and the study of genome evolution beyond simple nucleotide polymorphisms. Long-read technologies require pure high molecular weight DNA as an input for sequencing. Here, we describe a DNA extraction protocol for rust spores that yields pure double-stranded DNA molecules with molecular weight of >50 kilo-base pairs (kbp). The isolated DNA is of sufficient purity for PacBio long-read sequencing, but may require additional purification for other sequencing technologies such as Nanopore and 10× Genomics.

Global epidemiology of capsular group W meningococcal disease (1970-2015): Multifocal emergence and persistence of hypervirulent sequence type (ST)-11 clonal complex.

PubMed

Mustapha, Mustapha M; Marsh, Jane W; Harrison, Lee H

2016-03-18

Following an outbreak in Mecca Saudi Arabia in 2000, meningococcal strains expressing capsular group W (W) emerged as a major cause of invasive meningococcal disease (IMD) worldwide. The Saudi Arabian outbreak strain (Hajj clone) belonging to the ST-11 clonal complex (cc11) is similar to W cc11 causing occasional sporadic disease before 2000. Since 2000, W cc11 has caused large meningococcal disease epidemics in the African meningitis belt and endemic disease in South America, Europe and China. Traditional molecular epidemiologic typing suggested that a majority of current W cc11 burden represented global spread of the Hajj clone. However, recent whole genome sequencing (WGS) analyses revealed significant genetic heterogeneity among global W cc11 strains. While continued spread of the Hajj clone occurs in the Middle East, the meningitis belt and South Africa have co-circulation of the Hajj clone and other unrelated W cc11 strains. Notably, South America, the UK, and France share a genetically distinct W cc11 strain. Other W lineages persist in low numbers in Europe, North America and the meningitis belt. In summary, WGS is helping to unravel the complex genomic epidemiology of group W meningococcal strains. Wider application of WGS and strengthening of global IMD surveillance is necessary to monitor the continued evolution of group W lineages. Copyright © 2016 Elsevier Ltd. All rights reserved.

Jannaschia seohaensis sp. nov., isolated from a tidal flat sediment.

PubMed

Yoon, Jung-Hoon; Kang, So-Jung; Park, Sooyeon; Oh, Ki-Hoon; Oh, Tae-Kwang

2010-01-01

A Gram-negative, motile and pleomorphic bacterial strain, SMK-146(T), was isolated from a tidal flat sediment of the Yellow Sea, Korea, and its taxonomic position was investigated. Strain SMK-146(T) grew optimally at pH 7.0-8.0 and 30 degrees C. It contained Q-10 as the predominant ubiquinone and C(18 : 1)omega7c and 11-methyl C(18 : 1)omega7c as the major fatty acids. The major polar lipids were phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content was 68.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain SMK-146(T) belongs to the genus Jannaschia. Strain SMK-146(T) exhibited 16S rRNA gene sequence similarity values of 95.3-97.0 % to the type strains of the five recognized Jannaschia species. The mean DNA-DNA relatedness value between strain SMK-146(T) and Jannaschia seosinensis KCCM 42114(T), the closest phylogenetic neighbour, was 17 %. Differential phenotypic properties also revealed that strain SMK-146(T) differs from the recognized Jannaschia species. On the basis of phenotypic, phylogenetic and genetic data, strain SMK-146(T) represents a novel species of the genus Jannaschia, for which the name Jannaschia seohaensis sp. nov. is proposed. The type strain is SMK-146(T) (=KCTC 22172(T) =CCUG 55326(T)).

Actinomyces vulturis sp. nov., isolated from Gyps himalayensis.

PubMed

Meng, Xiangli; Lu, Shan; Wang, Yiting; Lai, Xin-He; Wen, Yumeng; Jin, Dong; Yang, Jing; Bai, Xiangning; Zhang, Gui; Pu, Ji; Lan, Ruiting; Xu, Jianguo

2017-06-01

Two strains of Gram-stain-positive, facultatively anaerobic, non-spore-forming short rods (VUL7T and VUL8) were isolated from rectal swabs of Old World vultures, namely Gyps himalayensis, in Tibet-Qinghai Plateau, China. Optimal growth occurred at 37 °C, pH 6-7, with 1 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences classified the two strains to the genus Actinomyces, with highest 16S rRNA gene sequence similarity (95 %) to type strains of Actinomyces haliotis, Actinomyces radicidentis and Actinomyces urogenitalis. The major cellular fatty acids were C18 : 1ω9c and C16 : 0. MK-10(H4) was the major respiratory quinone. The genomic DNA G+C content of the isolate was 54.4 mol%. DNA-DNA hybridization values with the most closely related species ofthe genusActinomyces was 24.6 %. The two strains can be differentiated from the most closely related species such as A. haliotis, A. radicidentis, A. graevenitzii and A. urogenitalis by a list of carbohydrate fermentations and enzyme activities. On the basis of physiological, biochemical and phylogenetic analysis, strains VUL7T and VUL8 represent novel species of the genus Actinomyces, for which the name Actinomyces vulturis sp. nov. is proposed. The type strain is VUL7T (=CGMCC 4.7366T=DSM 103437T).

Difference in drug resistance patterns between minor HIV-1 populations in cerebrospinal fluid and plasma.

PubMed

Bergroth, T; Ekici, H; Gisslén, M; Hagberg, L; Sönnerborg, A

2009-02-01

The aim of the study was to determine to what extent unique drug resistance patterns appear in minor and major HIV-1 quasispecies in cerebrospinal fluid (CSF) as compared with blood. Forty-four plasma and CSF samples from 13 multi-treatment-experienced patients, seven of whom provided longitudinal samples, were included in the study. The subjects had failed antiretroviral therapy including lamivudine. The reverse transcriptase (RT) gene was examined by selective real-time polymerase chain reaction (SPCR), which can detect M184I/V mutants down to 0.2% of the viral population. SPCR revealed differences at amino acid position 184 in the plasma/CSF populations in 12 paired samples from eight patients. One plasma sample was positive by SPCR where direct sequencing showed wild-type M184. The other 11 paired samples showed quantitative differences in the mixed populations of the mutant or wild-type M184 quasispecies. Differences in other resistance-associated mutations between plasma and CSF viruses were also found by direct sequencing. In multi-treatment-experienced patients with therapy failure, differences in drug resistance patterns were found frequently between plasma and CSF in both minor and major viral populations. To what extent this was a true biological phenomenon remains to be established, and the clinical relevance of these findings is yet to be determined.

Gracilibacillus aidingensis sp. nov., a novel moderately halophilic bacterium isolated from Aiding salt lake.

PubMed

Guan, Tong-Wei; Tian, Lei; Li, En-Yuan; Tang, Shu-Kun; Zhang, Xiao-Ping

2017-11-01

A novel Gram-positive, aerobe, moderately halophilic bacterium was isolated from saline soil of Aiding lake in Xinjiang, north-west of China, designated strain YIM 98001 T . Cells were rod-shaped, motile and grew at 5-20% (w/v) NaCl (optimum 10%), pH 6-10 (optimum pH 7.0) and 4-45 °C (optimum 37 °C). The major cellular fatty acids were anteiso C 15:0 , anteiso C 17:0 , iso C 15:0 . The predominant respiratory quinone was MK-7. Diphosphatidylglycerol, phosphatidylglycerol, phosphoglycolipid were the major polar lipids. Meso-diaminopimelic acid was the diagnostic diamino acid of the cell-wall peptidoglycan. The G+C content was 36.46 mol%. 16S rRNA gene sequence analysis showed that the strain belongs to the family Bacillaceae, with the highest sequence similarity to the type strain Gracilibacillus thailandensis TP2-8 T (96.84%), followed by Gracilibacillus saliphilus YIM 91119 T (96.78%) and Gracilibacillus ureilyticus MF38 T (96.57%), thus confirming the affiliation of strain YIM 98001 T to the genus Gracilibacillus. The polyphasic approach indicates that strain YIM 98001 T represents a novel species of the genus Gracilibacillus, for which the name Gracilibacillus aidingensis is proposed. The type strain is YIM 98001 T (=KCTC 42683 T  = DSMZ 104330 T ).

Prevalence and Antimicrobial Resistance of Salmonella Isolates Recovered from Retail Pork in Major Village Markets in Tai'an Region, China.

PubMed

Miao, Zengmin; Li, Song; Qin, Kun; Zhou, Yufa

2017-10-01

The current study was undertaken to evaluate Salmonella contamination in retail pork at major village markets of the Tai'an region, China. In total, 200 retail pork samples were collected from four village markets between June 2015 and February 2016, of which 69 samples (34.5%) were determined to be positive for Salmonella. Eleven serotypes were identified from the 69 Salmonella isolates, and Salmonella Derby was the most common (18 of 69, 26.1%), followed by Typhimurium (17 of 69, 24.6%) and Meleagridis (11 of 69, 15.9%). Antimicrobial susceptibility testing showed that antimicrobial resistance against tetracycline was the most prevalent (42 of 69, 60.9%), but antimicrobial resistance against both ceftriaxone and cefotaxime was 1.4% (1 of 69) and 2.9% (2 of 69), respectively. Multilocus sequence typing revealed that the 69 Salmonella isolates were divided into 11 sequence types (STs), among which ST40 (18 of 69, 26.1%) was the most common, followed by ST34 (15 of 69, 21.7%) and ST64 (13 of 69, 18.8%). Collectively, retail pork at village markets in the Tai'an region has a high Salmonella contamination rate, and these isolates exhibit broad-spectrum antimicrobial resistance. However, the absence of a dominant ST demonstrates that the Salmonella isolates from retail pork may be of diverse origins.

Advances in computational approaches for prioritizing driver mutations and significantly mutated genes in cancer genomes.

PubMed

Cheng, Feixiong; Zhao, Junfei; Zhao, Zhongming

2016-07-01

Cancer is often driven by the accumulation of genetic alterations, including single nucleotide variants, small insertions or deletions, gene fusions, copy-number variations, and large chromosomal rearrangements. Recent advances in next-generation sequencing technologies have helped investigators generate massive amounts of cancer genomic data and catalog somatic mutations in both common and rare cancer types. So far, the somatic mutation landscapes and signatures of >10 major cancer types have been reported; however, pinpointing driver mutations and cancer genes from millions of available cancer somatic mutations remains a monumental challenge. To tackle this important task, many methods and computational tools have been developed during the past several years and, thus, a review of its advances is urgently needed. Here, we first summarize the main features of these methods and tools for whole-exome, whole-genome and whole-transcriptome sequencing data. Then, we discuss major challenges like tumor intra-heterogeneity, tumor sample saturation and functionality of synonymous mutations in cancer, all of which may result in false-positive discoveries. Finally, we highlight new directions in studying regulatory roles of noncoding somatic mutations and quantitatively measuring circulating tumor DNA in cancer. This review may help investigators find an appropriate tool for detecting potential driver or actionable mutations in rapidly emerging precision cancer medicine. © The Author 2015. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

Micrococcus lactis sp. nov., isolated from dairy industry waste.

PubMed

Chittpurna; Singh, Pradip K; Verma, Dipti; Pinnaka, Anil Kumar; Mayilraj, Shanmugam; Korpole, Suresh

2011-12-01

A Gram-positive, yellow-pigmented, actinobacterial strain, DW152(T), was isolated from a dairy industry effluent treatment plant. 16S rRNA gene sequence analysis indicated that strain DW152(T) exhibited low similarity with many species with validly published names belonging to the genera Micrococcus and Arthrobacter. However, phenotypic properties including chemotaxonomic markers affiliated strain DW152(T) to the genus Micrococcus. Strain DW152(T) had ai-C(15:0) and i-C(15:0) as major cellular fatty acids, and MK-8(H(2)) as the major menaquinone. The cell-wall peptidoglycan of strain DW152(T) had l-lysine as the diagnostic amino acid and the type was A4α. The DNA G+C content of strain DW152(T) was 68.0 mol%. In 16S rRNA gene sequence analysis, strain DW152(T) exhibited significant similarity with Micrococcus terreus NBRC 104258(T), but the mean value of DNA-DNA relatedness between these strains was only 42.3%. Moreover, strain DW152(T) differed in biochemical and chemotaxonomic characteristics from M. terreus and other species of the genus Micrococcus. Based on the above differences, we conclude that strain DW152(T) should be treated as a novel species of the genus Micrococcus, for which the name Micrococcus lactis sp. nov. is proposed. The type strain of Micrococcus lactis sp. nov. is DW152(T) (=MTCC10523(T) =DSM 23694(T)).

Mitochondrial control-region sequence variation in aboriginal Australians.

PubMed Central

van Holst Pellekaan, S; Frommer, M; Sved, J; Boettcher, B

1998-01-01

The mitochondrial D-loop hypervariable segment 1 (mt HVS1) between nucleotides 15997 and 16377 has been examined in aboriginal Australian people from the Darling River region of New South Wales (riverine) and from Yuendumu in central Australia (desert). Forty-seven unique HVS1 types were identified, varying at 49 nucleotide positions. Pairwise analysis by calculation of BEPPI (between population proportion index) reveals statistically significant structure in the populations, although some identical HVS1 types are seen in the two contrasting regions. mt HVS1 types may reflect more-ancient distributions than do linguistic diversity and other culturally distinguishing attributes. Comparison with sequences from five published global studies reveals that these Australians demonstrate greatest divergence from some Africans, least from Papua New Guinea highlanders, and only slightly more from some Pacific groups (Indonesian, Asian, Samoan, and coastal Papua New Guinea), although the HVS1 types vary at different nucleotide sites. Construction of a median network, displaying three main groups, suggests that several hypervariable nucleotide sites within the HVS1 are likely to have undergone mutation independently, making phylogenetic comparison with global samples by conventional methods difficult. Specific nucleotide-site variants are major separators in median networks constructed from Australian HVS1 types alone and for one global selection. The distribution of these, requiring extended study, suggests that they may be signatures of different groups of prehistoric colonizers into Australia, for which the time of colonization remains elusive. PMID:9463317

Competitive amplification of differentially melting amplicons (CADMA) enables sensitive and direct detection of all mutation types by high-resolution melting analysis.

PubMed

Kristensen, Lasse S; Andersen, Gitte B; Hager, Henrik; Hansen, Lise Lotte

2012-01-01

Sensitive and specific mutation detection is of particular importance in cancer diagnostics, prognostics, and individualized patient treatment. However, the majority of molecular methodologies that have been developed with the aim of increasing the sensitivity of mutation testing have drawbacks in terms of specificity, convenience, or costs. Here, we have established a new method, Competitive Amplification of Differentially Melting Amplicons (CADMA), which allows very sensitive and specific detection of all mutation types. The principle of the method is to amplify wild-type and mutated sequences simultaneously using a three-primer system. A mutation-specific primer is designed to introduce melting temperature decreasing mutations in the resulting mutated amplicon, while a second overlapping primer is designed to amplify both wild-type and mutated sequences. When combined with a third common primer very sensitive mutation detection becomes possible, when using high-resolution melting (HRM) as detection platform. The introduction of melting temperature decreasing mutations in the mutated amplicon also allows for further mutation enrichment by fast coamplification at lower denaturation temperature PCR (COLD-PCR). For proof-of-concept, we have designed CADMA assays for clinically relevant BRAF, EGFR, KRAS, and PIK3CA mutations, which are sensitive to, between 0.025% and 0.25%, mutated alleles in a wild-type background. In conclusion, CADMA enables highly sensitive and specific mutation detection by HRM analysis. © 2011 Wiley Periodicals, Inc.

Reads2Type: a web application for rapid microbial taxonomy identification.

PubMed

Saputra, Dhany; Rasmussen, Simon; Larsen, Mette V; Haddad, Nizar; Sperotto, Maria Maddalena; Aarestrup, Frank M; Lund, Ole; Sicheritz-Pontén, Thomas

2015-11-25

Identification of bacteria may be based on sequencing and molecular analysis of a specific locus such as 16S rRNA, or a set of loci such as in multilocus sequence typing. In the near future, healthcare institutions and routine diagnostic microbiology laboratories may need to sequence the entire genome of microbial isolates. Therefore we have developed Reads2Type, a web-based tool for taxonomy identification based on whole bacterial genome sequence data. Raw sequencing data provided by the user are mapped against a set of marker probes that are derived from currently available bacteria complete genomes. Using a dataset of 1003 whole genome sequenced bacteria from various sequencing platforms, Reads2Type was able to identify the species with 99.5 % accuracy and on the minutes time scale. In comparison with other tools, Reads2Type offers the advantage of not needing to transfer sequencing files, as the entire computational analysis is done on the computer of whom utilizes the web application. This also prevents data privacy issues to arise. The Reads2Type tool is available at http://www.cbs.dtu.dk/~dhany/reads2type.html.

Method for isolating chromosomal DNA in preparation for hybridization in suspension

DOEpatents

Lucas, Joe N.

2000-01-01

A method is provided for detecting nucleic acid sequence aberrations using two immobilization steps. According to the method, a nucleic acid sequence aberration is detected by detecting nucleic acid sequences having both a first nucleic acid sequence type (e.g., from a first chromosome) and a second nucleic acid sequence type (e.g., from a second chromosome), the presence of the first and the second nucleic acid sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. In the method, immobilization of a first hybridization probe is used to isolate a first set of nucleic acids in the sample which contain the first nucleic acid sequence type. Immobilization of a second hybridization probe is then used to isolate a second set of nucleic acids from within the first set of nucleic acids which contain the second nucleic acid sequence type. The second set of nucleic acids are then detected, their presence indicating the presence of a nucleic acid sequence aberration. Chromosomal DNA in a sample containing cell debris is prepared for hybridization in suspension by treating the mixture with RNase. The treated DNA can also be fixed prior to hybridization.

A novel high-resolution multilocus sequence typing of Giardia intestinalis Assemblage A isolates reveals zoonotic transmission, clonal outbreaks and recombination.

PubMed

Ankarklev, Johan; Lebbad, Marianne; Einarsson, Elin; Franzén, Oscar; Ahola, Harri; Troell, Karin; Svärd, Staffan G

2018-06-01

Molecular epidemiology and genotyping studies of the parasitic protozoan Giardia intestinalis have proven difficult due to multiple factors, such as low discriminatory power in the commonly used genotyping loci, which has hampered molecular analyses of outbreak sources, zoonotic transmission and virulence types. Here we have focused on assemblage A Giardia and developed a high-resolution assemblage-specific multilocus sequence typing (MLST) method. Analyses of sequenced G. intestinalis assemblage A genomes from different sub-assemblages identified a set of six genetic loci with high genetic variability. DNA samples from both humans (n = 44) and animals (n = 18) that harbored Giardia assemblage A infections, were PCR amplified (557-700 bp products) and sequenced at the six novel genetic loci. Bioinformatic analyses showed five to ten-fold higher levels of polymorphic sites than what was previously found among assemblage A samples using the classic genotyping loci. Phylogenetically, a division of two major clusters in assemblage A became apparent, separating samples of human and animal origin. A subset of human samples (n = 9) from a documented Giardia outbreak in a Swedish day-care center, showed full complementarity at nine genetic loci (the six new and the standard BG, TPI and GDH loci), strongly suggesting one source of infection. Furthermore, three samples of human origin displayed MLST profiles that were phylogenetically more closely related to MLST profiles from animal derived samples, suggesting zoonotic transmission. These new genotyping loci enabled us to detect events of recombination between different assemblage A isolates but also between assemblage A and E isolates. In summary, we present a novel and expanded MLST strategy with significantly improved sensitivity for molecular analyses of virulence types, zoonotic potential and source tracking for assemblage A Giardia. Copyright © 2018. Published by Elsevier B.V.

Multiple ESBL-Producing Escherichia coli Sequence Types Carrying Quinolone and Aminoglycoside Resistance Genes Circulating in Companion and Domestic Farm Animals in Mwanza, Tanzania, Harbor Commonly Occurring Plasmids

PubMed Central

Seni, Jeremiah; Falgenhauer, Linda; Simeo, Nabina; Mirambo, Mariam M.; Imirzalioglu, Can; Matee, Mecky; Rweyemamu, Mark; Chakraborty, Trinad; Mshana, Stephen E.

2016-01-01

The increased presence of extended-spectrum beta-lactamase (ESBL)-producing bacteria in humans, animals, and their surrounding environments is of global concern. Currently there is limited information on ESBL presence in rural farming communities worldwide. We performed a cross-sectional study in Mwanza, Tanzania, involving 600 companion and domestic farm animals between August/September 2014. Rectal swab/cloaca specimens were processed to identify ESBL-producing Enterobacteriaceae. We detected 130 (21.7%) animals carrying ESBL-producing bacteria, the highest carriage being among dogs and pigs [39.2% (51/130) and 33.1% (43/130), respectively]. The majority of isolates were Escherichia coli [93.3% (125/134)] and exotic breed type [OR (95%CI) = 2.372 (1.460–3.854), p-value < 0.001] was found to be a predictor of ESBL carriage among animals. Whole-genome sequences of 25 ESBL-producing E. coli were analyzed for phylogenetic relationships using multi-locus sequence typing (MLST) and core genome comparisons. Fourteen different sequence types were detected of which ST617 (7/25), ST2852 (3/25), ST1303 (3/25) were the most abundant. All isolates harbored the blaCTX-M-15 allele, 22/25 carried strA and strB, 12/25 aac(6′)-lb-cr, and 11/25 qnrS1. Antibiotic resistance was associated with IncF, IncY, as well as non-typable plasmids. Eleven isolates carried pPGRT46-related plasmids, previously reported from isolates in Nigeria. Five isolates had plasmids exhibiting 85–99% homology to pCA28, previously detected in isolates from the US. Our findings indicate a pan-species distribution of ESBL-producing E. coli clonal groups in farming communities and provide evidence for plasmids harboring antibiotic resistances of regional and international impact. PMID:26904015

Parapusillimonas granuli gen. nov., sp. nov., isolated from granules from a wastewater-treatment bioreactor.

PubMed

Kim, Yeon-Ju; Kim, Myung Kyum; Im, Wan-Taek; Srinivasan, Sathiyaraj; Yang, Deok-Chun

2010-06-01

A novel betaproteobacterium, designated strain Ch07(T), was isolated from granules from the wastewater-treatment bioreactor of an alcohol fermentation factory in South Korea. In order to determine its taxonomic position, the novel strain was characterized using a polyphasic approach. The new strain was Gram-negative, facultatively anaerobic, non-spore-forming, motile and short rod-shaped. 16S rRNA gene sequence analysis revealed that strain Ch07(T) belonged to the class Betaproteobacteria, being related to Pusillimonas noertemannii BN9(T) (gene sequence similarity 97.30 %), Achromobacter xylosoxidans subsp. xylosoxidans DSM 10346(T) (97.09 %), Bordetella pertussis DSM 5571(T) (97.01 %), Pigmentiphaga kullae DSM 13608(T) (96.68 %) and Castellaniella defragrans DSM 1214(T) (96.47 %). The results of DNA-DNA hybridization tests showed that reassociation values were less than 62 % with respect to these closely related type strains. Chemotaxonomic data showed that strain Ch07(T) possessed ubiquinone Q-8. The G+C content of the genomic DNA was 67.9+/-0.1 mol%. The major polyamine of strain Ch07(T) was putrescine. The major polar lipids of strain Ch07(T) were phosphatidylethanolamine, followed by diphosphatidylglycerol and phosphatidylglycerol. When strain Ch07(T) was incubated on tryptic soy agar, the major cellular fatty acids were C(16 : 0), C(17 : 0) cyclo, summed feature 3 (C(16 : 1)omega7c/iso C(15 : 0) 2-OH) and summed feature 5 (C(18 : 1)omega7c/omega9t/omega12t). The results of DNA-DNA hybridizations, in combination with the chemotaxonomic and physiological data, demonstrated that strain Ch07(T) represents a novel species of a new genus, for which the name Parapusillimonas granuli gen. nov., sp. nov. is proposed. The type strain of the type species is Ch07(T) (=KCTC 12668(T)=LMG 24012(T)).

The association between Staphylococcus aureus nasal colonization and symptomatic infection in children in Korea where ST72 is the major genotype: A prospective observational study.

PubMed

Kang, Sunghan; Lee, Jina; Kim, Mina

2017-08-01

This study was performed to investigate the concordance in terms of molecular characteristics and antimicrobial susceptibility between colonizing and clinical Staphylococcus aureus isolates obtained from children in Korea, where ST72 is the major genotype.This was a prospective observational descriptive study of culture-confirmed S aureus infections obtained from children ≤18 years old admitted to Asan Medical Center Children's Hospital in Seoul, Korea, from March 2014 to April 2015. Molecular studies including multilocus sequence typing (MLST), SCCmec typing, polymerase chain reaction amplification of the Panton-Valentine leukocidin (PVL) genes, and antibiotic susceptibility tests were performed on S aureus isolates obtained from nares and clinical specimens.During the study period, 126 clinically significant S aureus infections were identified. Nasal swab cultures were made from 113 of the 126 children, and 46.0% (52/113) showed S aureus colonization. The overall concordance between colonizing and clinical isolates by methicillin susceptibility was 94.2% (49/52); all 3 discordant cases were HA-MSSA cases with nasal MRSA. Among the 37 pairs of colonizing and clinical S aureus isolates included in the genotyping analysis, ST72-SCCmec type IV was the most prevalent clone and the PVL genes were positive in 2 patients. Among the 31 pairs of healthcare-associated cases, concordance rates by methicillin susceptibility and sequence type (ST) were 90.3% (28/31) and 84% (26/31), respectively. For the 6 pairs of community-associated (CA) S aureus including 3 CA-MRSA cases, 100% concordance was observed by methicillin susceptibility and ST.The concordance between isolates obtained from children who required medical services was relatively high in Korean children where ST72-SCCmec type IV is the predominant clone as the colonizer and the pathogen. It is suggested that decolonization and continuous care to prevent transmission could be effective in managing and preventing both HA- and CA-SA infections in our setting.

A STELLAR-MASS-DEPENDENT DROP IN PLANET OCCURRENCE RATES

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mulders, Gijs D.; Pascucci, Ilaria; Apai, Dániel

2015-01-10

The Kepler spacecraft has discovered a large number of planets with up to one-year periods and down to terrestrial sizes. While the majority of the target stars are main-sequence dwarfs of spectral type F, G, and K, Kepler covers stars with effective temperatures as low as 2500 K, which corresponds to M stars. These cooler stars allow characterization of small planets near the habitable zone, yet it is not clear if this population is representative of that around FGK stars. In this paper, we calculate the occurrence of planets around stars of different spectral types as a function of planetmore » radius and distance from the star and show that they are significantly different from each other. We further identify two trends. First, the occurrence of Earth- to Neptune-sized planets (1-4 R {sub ⊕}) is successively higher toward later spectral types at all orbital periods probed by Kepler; planets around M stars occur twice as frequently as around G stars, and thrice as frequently as around F stars. Second, a drop in planet occurrence is evident at all spectral types inward of a ∼10 day orbital period, with a plateau further out. By assigning to each spectral type a median stellar mass, we show that the distance from the star where this drop occurs is stellar mass dependent, and scales with semi-major axis as the cube root of stellar mass. By comparing different mechanisms of planet formation, trapping, and destruction, we find that this scaling best matches the location of the pre-main-sequence co-rotation radius, indicating efficient trapping of migrating planets or planetary building blocks close to the star. These results demonstrate the stellar-mass dependence of the planet population, both in terms of occurrence rate and of orbital distribution. The prominent stellar-mass dependence of the inner boundary of the planet population shows that the formation or migration of planets is sensitive to the stellar parameters.« less

Proposals of Sphingomonas paucimobilis gen. nov. and comb. nov., Sphingomonas parapaucimobilis sp. nov., Sphingomonas yanoikuyae sp. nov., Sphingomonas adhaesiva sp. nov., Sphingomonas capsulata comb. nov., and two genospecies of the genus Sphingomonas.

PubMed

Yabuuchi, E; Yano, I; Oyaizu, H; Hashimoto, Y; Ezaki, T; Yamamoto, H

1990-01-01

Based on the partial nucleotide sequence analysis of 16S ribosomal ribonucleic acid (rRNA), presence of unique sphingoglycolipids in cellular lipid, and the major type of ubiquinone (Q10), we propose Sphingomonas gen. nov. with the type species Sphingomonas paucimobilis (Holmes et al, 1977) comb. nov. From the homology values of deoxyribonucleic acid-deoxyribonucleic acid hybridization and the phenotypic characteristics, three new species, Sphingomonas parapaucimobilis, Sphingomonas yanoikuyae, Sphingomonas adhaesiva, and one new combination, Sphingomonas capsulata, are described. S. parapaucimobilis JCM 7510 (= GIFU 11387), S. yanoikuyae JCM 7371 (= GIFU 9882), and S. adhaesiva JCM 7370 (= GIFU 11458) are designated as the type strains of the three new species. Emended description of the type strain of S. capsulata is presented.

Phyllobacterium sophorae sp. nov., a symbiotic bacterium isolated from root nodules of Sophora flavescens.

PubMed

Jiao, Yin Shan; Yan, Hui; Ji, Zhao Jun; Liu, Yuan Hui; Sui, Xin Hua; Zhang, Xiao Xia; Wang, En Tao; Chen, Wen Xin; Chen, Wen Feng

2015-02-01

Two novel Gram-stain-negative strains (CCBAU 03422(T) and CCBAU 03415) isolated from root nodules of Sophora flavescens were classified phylogenetically into the genus Phyllobacterium based on the comparative analysis of 16S rRNA and atpD genes. They showed 99.8 % rRNA gene sequence similarities to Phyllobacterium brassicacearum LMG 22836(T), and strain CCBAU 03422(T) showed 91.2 and 88.6 % atpD gene sequence similarities to strains Phyllobacterium endophyticum LMG 26470(T) and Phyllobacterium brassicacearum LMG 22836(T), respectively. Strain CCBAU 03422(T) contained Q-10 as its major quinone and showed a cellular fatty acid profile, carbon source utilization and other phenotypic characteristics differing from type strains of related species. DNA-DNA relatedness (lower than 48.8 %) further confirmed the differences between the novel strains and the type strains of related species. Strain CCBAU 03422(T) could nodulate and fix nitrogen effectively on its original host plant, Sophora flavescens. Based upon the results mentioned above, a novel species named Phyllobacterium sophorae is proposed and the type strain is CCBAU 03422(T) ( = A-6-3(T) = LMG 27899(T) = HAMBI 3508(T)). © 2015 IUMS.

Methylobacterium pseudosasicola sp. nov. and Methylobacterium phyllostachyos sp. nov., isolated from bamboo leaf surfaces.

PubMed

Madhaiyan, Munusamy; Poonguzhali, Selvaraj

2014-07-01

Two strains of Gram-negative, methylotrophic bacteria, isolated because of their abilities to promote plant growth, were subjected to a polyphasic taxonomic study. The isolates were strictly aerobic, motile, pink-pigmented, facultatively methylotrophic, non-spore-forming rods. The chemotaxonomic characteristics of the isolates included the presence of C18 : 1ω7c as the major cellular fatty acid. The DNA G+C contents of strains BL36(T) and BL47(T) were 69.4 and 69.8 mol%, respectively. 16S rRNA gene sequence analysis of strains BL36(T) and BL47(T) placed them under the genus Methylobacterium, with the pairwise sequence similarity between them and the type strains of closely related species ranging from 97.2 to 99.0%. On the basis of their phenotypic and phylogenetic distinctiveness and the results of DNA-DNA hybridization analysis, the isolates represent two novel species within the genus Methylobacterium, for which the names Methylobacterium pseudosasicola sp. nov. (type strain BL36(T) = NBRC 105203(T) = ICMP 17621(T)) and Methylobacterium phyllostachyos sp. nov. (type strain BL47(T) = NBRC 105206(T) = ICMP 17619(T)) are proposed. © 2014 IUMS.

Characterization of Methicillin Resistant Staphylococcus aureus isolated from human and animal samples in Egypt.

PubMed

Bendary, M M; Solyman, S M; Azab, M M; Mahmoud, N F; Hanora, A M

2016-02-29

Staphylococcus aureus (S. aureus) has been one of the most problematic pathogens. Methicillin Resistant S. aureus (MRSA) has emerged as a major concern for both human and animal. Antibiotic resistance genes dissemination might be possible between human and animal bacteria. The aim of this study is to show phenotypic and genotypic diversity of human and animal MRSA isolates. Antibiogram typing and biofilm production were used as a primary phenotypic typing tool for the characterization of (40) animal and (38) human MRSA isolates. Genetic typing based on sequencing of 16S rRNA gene and virulence gene profiles were done. Antimicrobial resistance profiles of the animal isolates showed little evidence of widespread of resistance, although this was seen in many human isolates. The biofilm production was detected in higher percentage among animal isolates. Based on the genetic typing and multiple antibiotic resistance (MAR) index, the majority of animal isolates clustered into lineages that were not found in human isolates. Animal and human MRSA isolates showed diversity in antibiotic resistance and virulence gene profiles may be due to host adaptation or chances for contamination between the two hosts were not present in our study.

Sensorineural Deafness, Distinctive Facial Features and Abnormal Cranial Bones

PubMed Central

Gad, Alona; Laurino, Mercy; Maravilla, Kenneth R.; Matsushita, Mark; Raskind, Wendy H.

2008-01-01

The Waardenburg syndromes (WS) account for approximately 2% of congenital sensorineural deafness. This heterogeneous group of diseases currently can be categorized into four major subtypes (WS types 1-4) on the basis of characteristic clinical features. Multiple genes have been implicated in WS, and mutations in some genes can cause more than one WS subtype. In addition to eye, hair and skin pigmentary abnormalities, dystopia canthorum and broad nasal bridge are seen in WS type 1. Mutations in the PAX3 gene are responsible for the condition in the majority of these patients. In addition, mutations in PAX3 have been found in WS type 3 that is distinguished by musculoskeletal abnormalities, and in a family with a rare subtype of WS, craniofacial-deafness-hand syndrome (CDHS), characterized by dysmorphic facial features, hand abnormalities, and absent or hypoplastic nasal and wrist bones. Here we describe a woman who shares some, but not all features of WS type 3 and CDHS, and who also has abnormal cranial bones. All sinuses were hypoplastic, and the cochlea were small. No sequence alteration in PAX3 was found. These observations broaden the clinical range of WS and suggest there may be genetic heterogeneity even within the CDHS subtype. PMID:18553554

Molecular epidemiology of type 1 and 2 dengue viruses in Brazil from 1988 to 2001.

PubMed

Pires Neto, R J; Lima, D M; de Paula, S O; Lima, C M; Rocco, I M; Fonseca, B A L

2005-06-01

Dengue is a mosquito-borne viral infection that in recent decades has become a major international public health concern. Epidemic dengue fever reemerged in Brazil in 1981. Since 1990 more than one dengue virus serotype has been circulating in this tropical country and increasing rates of dengue hemorrhagic fever and dengue shock syndrome have been detected every year. Some evidence supports the association between the introduction of a new serotype and/or genotype in a region and the appearance of dengue hemorrhagic fever. In order to study the evolutionary relationships and possible detection of the introduction of new dengue virus genotypes in Brazil in the last years, we analyzed partial nucleotide sequences of 52 Brazilian samples of both dengue type 1 and dengue type 2 isolated from 1988 to 2001 from highly endemic regions. A 240-nucleotide-long sequence from the envelope/nonstructural protein 1 gene junction was used for phylogenetic analysis. After comparing the nucleotide sequences originally obtained in this study to those previously studied by others, and analyzing the phylogenetic trees, we conclude that, after the initial introduction of the currently circulating dengue-1 and dengue-2 genotypes in Brazil, there has been no evidence of introduction of new genotypes since 1988. The increasing number of dengue hemorrhagic fever cases seen in Brazil in the last years is probably associated with secondary infections or with the introduction of new serotypes but not with the introduction of new genotypes.

Methods for MHC genotyping in non-model vertebrates.

PubMed

Babik, W

2010-03-01

Genes of the major histocompatibility complex (MHC) are considered a paradigm of adaptive evolution at the molecular level and as such are frequently investigated by evolutionary biologists and ecologists. Accurate genotyping is essential for understanding of the role that MHC variation plays in natural populations, but may be extremely challenging. Here, I discuss the DNA-based methods currently used for genotyping MHC in non-model vertebrates, as well as techniques likely to find widespread use in the future. I also highlight the aspects of MHC structure that are relevant for genotyping, and detail the challenges posed by the complex genomic organization and high sequence variation of MHC loci. Special emphasis is placed on designing appropriate PCR primers, accounting for artefacts and the problem of genotyping alleles from multiple, co-amplifying loci, a strategy which is frequently necessary due to the structure of the MHC. The suitability of typing techniques is compared in various research situations, strategies for efficient genotyping are discussed and areas of likely progress in future are identified. This review addresses the well established typing methods such as the Single Strand Conformation Polymorphism (SSCP), Denaturing Gradient Gel Electrophoresis (DGGE), Reference Strand Conformational Analysis (RSCA) and cloning of PCR products. In addition, it includes the intriguing possibility of direct amplicon sequencing followed by the computational inference of alleles and also next generation sequencing (NGS) technologies; the latter technique may, in the future, find widespread use in typing complex multilocus MHC systems. © 2009 Blackwell Publishing Ltd.

Influence of geochemical processes on hydrochemistry and irrigation suitability of groundwater in part of semi-arid Deccan Plateau, India

NASA Astrophysics Data System (ADS)

Vasu, Duraisamy; Singh, Surendra Kumar; Tiwary, Pramod; Sahu, Nisha; Ray, Sanjay Kumar; Butte, Pravin; Duraisami, Veppangadu Perumal

2017-11-01

Major ion geochemistry was used to characterise the chemical composition of groundwater in part of semi-arid Deccan plateau region to understand the geochemical evolution and to evaluate the groundwater quality for irrigation. The study area comprises peninsular gneissic complex of Archean age, younger granites and basaltic alluvium. Forty-nine georeferenced groundwater samples were collected and analysed for major ions. The ionic sequence based on relative proportions was Na+ > Mg2+ > Ca2+ > SO4 2- > HCO3 - > Cl- > CO3 2- > BO3 3- > K+. High Na+, Mg2+ and Ca2+ were generally associated with basaltic alluvial formation, whereas pH, electrical conductivity (EC) and total dissolved salts (TDS) were found to be higher in granitic formations. High standard deviation for EC, TDS, Na+, Ca2+ and Mg2+ indicated the dispersion of ionic concentration throughout the study area. Four major hydrochemical facies identified were Na-Mg-HCO3 type; Mg-Na-HCO3 type; Na-Mg-Ca-SO4 and Mg-Na-Ca-SO4 type. The graphical plots indicated that the groundwater chemistry was influenced by rock-water interaction, silicate weathering and reverse ion exchange. Sodium-dominated waters might have impeded the hydraulic properties of soils as a result of long-term irrigation.

Few Highly Abundant Operational Taxonomic Units Dominate within Rumen Methanogenic Archaeal Species in New Zealand Sheep and Cattle

PubMed Central

Seedorf, Henning; Kittelmann, Sandra

2014-01-01

Sequencing and analyses of 16S rRNA gene amplicons were performed to estimate the composition of the rumen methanogen community in 252 samples from eight cohorts of sheep and cattle, separated into 16 different sample groups by diet, and to determine which methanogens are most prominent in the rumens of farmed New Zealand ruminants. Methanobacteriales (relative abundance ± standard deviation, 89.6% ± 9.8%) and Methanomassiliicoccales (10.4% ± 9.8%) were the two major orders and contributed 99.98% (±0.1%) to the rumen methanogen communities in the samples. Sequences from Methanobacteriales were almost entirely from only four different species (or clades of very closely related species). Each was detectable in at least 89% of the samples. These four species or clades were the Methanobrevibacter gottschalkii clade and Methanobrevibacter ruminantium clade with a mean abundance of 42.4% (±19.5% standard deviation) and 32.9% (±18.8%), respectively, and Methanosphaera sp. ISO3-F5 (8.2% ± 6.7%) and Methanosphaera sp. group5 (5.6% ± 5.7%). These four species or clades appeared to be primarily represented by only one or, in one case, two dominant sequence types per species or clade when the sequences were grouped into operational taxonomic units (OTUs) at 99% sequence identity. The mean relative abundance of Methanomassiliicoccales in the samples was relatively low but exceeded 40% in some of the treatment groups. Animal feed affected the apparent methanogen community structure of both orders, as evident from differences in relative abundances of the major OTUs in animals under different feeding regimens. PMID:25416771

Genomic investigation of a suspected outbreak of Legionella pneumophila ST82 reveals undetected heterogeneity by the present gold-standard methods, Denmark, July to November 2014

PubMed Central

Schjørring, Susanne; Stegger, Marc; Kjelsø, Charlotte; Lilje, Berit; Bangsborg, Jette M; Petersen, Randi F; David, Sophia; Uldum, Søren A

2017-01-01

Between July and November 2014, 15 community-acquired cases of Legionnaires´ disease (LD), including four with Legionella pneumophila serogroup 1 sequence type (ST) 82, were diagnosed in Northern Zealand, Denmark. An outbreak was suspected. No ST82 isolates were found in environmental samples and no external source was established. Four putative-outbreak ST82 isolates were retrospectively subjected to whole genome sequencing (WGS) followed by phylogenetic analyses with epidemiologically unrelated ST82 sequences. The four putative-outbreak ST82 sequences fell into two clades, the two clades were separated by ca 1,700 single nt polymorphisms (SNP)s when recombination regions were included but only by 12 to 21 SNPs when these were removed. A single putative-outbreak ST82 isolate sequence segregated in the first clade. The other three clustered in the second clade, where all included sequences had < 5 SNP differences between them. Intriguingly, this clade also comprised epidemiologically unrelated isolate sequences from the UK and Denmark dating back as early as 2011. The study confirms that recombination plays a major role in L. pneumophila evolution. On the other hand, strains belonging to the same ST can have only few SNP differences despite being sampled over both large timespans and geographic distances. These are two important factors to consider in outbreak investigations. PMID:28662761

Whole Genome Sequencing Demonstrates Limited Transmission within Identified Mycobacterium tuberculosis Clusters in New South Wales, Australia

PubMed Central

Gurjav, Ulziijargal; Outhred, Alexander C.; Jelfs, Peter; McCallum, Nadine; Wang, Qinning; Hill-Cawthorne, Grant A.; Marais, Ben J.; Sintchenko, Vitali

2016-01-01

Australia has a low tuberculosis incidence rate with most cases occurring among recent immigrants. Given suboptimal cluster resolution achieved with 24-locus mycobacterium interspersed repetitive unit (MIRU-24) genotyping, the added value of whole genome sequencing was explored. MIRU-24 profiles of all Mycobacterium tuberculosis culture-confirmed tuberculosis cases diagnosed between 2009 and 2013 in New South Wales (NSW), Australia, were examined and clusters identified. The relatedness of cases within the largest MIRU-24 clusters was assessed using whole genome sequencing and phylogenetic analyses. Of 1841 culture-confirmed TB cases, 91.9% (1692/1841) had complete demographic and genotyping data. East-African Indian (474; 28.0%) and Beijing (470; 27.8%) lineage strains predominated. The overall rate of MIRU-24 clustering was 20.1% (340/1692) and was highest among Beijing lineage strains (35.7%; 168/470). One Beijing and three East-African Indian (EAI) clonal complexes were responsible for the majority of observed clusters. Whole genome sequencing of the 4 largest clusters (30 isolates) demonstrated diverse single nucleotide polymorphisms (SNPs) within identified clusters. All sequenced EAI strains and 70% of Beijing lineage strains clustered by MIRU-24 typing demonstrated distinct SNP profiles. The superior resolution provided by whole genome sequencing demonstrated limited M. tuberculosis transmission within NSW, even within identified MIRU-24 clusters. Routine whole genome sequencing could provide valuable public health guidance in low burden settings. PMID:27737005

Whole genome sequencing identifies influenza A H3N2 transmission and offers superior resolution to classical typing methods.

PubMed

Meinel, Dominik M; Heinzinger, Susanne; Eberle, Ute; Ackermann, Nikolaus; Schönberger, Katharina; Sing, Andreas

2018-02-01

Influenza with its annual epidemic waves is a major cause of morbidity and mortality worldwide. However, only little whole genome data are available regarding the molecular epidemiology promoting our understanding of viral spread in human populations. We implemented a RT-PCR strategy starting from patient material to generate influenza A whole genome sequences for molecular epidemiological surveillance. Samples were obtained within the Bavarian Influenza Sentinel. The complete influenza virus genome was amplified by a one-tube multiplex RT-PCR and sequenced on an Illumina MiSeq. We report whole genomic sequences for 50 influenza A H3N2 viruses, which was the predominating virus in the season 2014/15, directly from patient specimens. The dataset included random samples from Bavaria (Germany) throughout the influenza season and samples from three suspected transmission clusters. We identified the outbreak samples based on sequence identity. Whole genome sequencing (WGS) was superior in resolution compared to analysis of single segments or partial segment analysis. Additionally, we detected manifestation of substantial amounts of viral quasispecies in several patients, carrying mutations varying from the dominant virus in each patient. Our rapid whole genome sequencing approach for influenza A virus shows that WGS can effectively be used to detect and understand outbreaks in large communities. Additionally, the genomic data provide in-depth details about the circulating virus within one season.

Rapid Classification and Identification of Microcystis aeruginosa Strains Using MALDI-TOF MS and Polygenetic Analysis.

PubMed

Sun, Li-Wei; Jiang, Wen-Jing; Sato, Hiroaki; Kawachi, Masanobu; Lu, Xi-Wu

2016-01-01

Matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was used to establish a rapid, simple, and accurate method to differentiate among strains of Microcystis aeruginosa, one of the most prevalent types of bloom-forming cyanobacteria. M. aeruginosa NIES-843, for which a complete genome has been sequenced, was used to characterize ribosomal proteins as biomarkers and to optimize conditions for observing ribosomal proteins as major peaks in a given mass spectrum. Thirty-one of 52 ribosomal subunit proteins were detected and identified along the mass spectrum. Fifty-five strains of M. aeruginosa from different habitats were analyzed using MALDI-TOF MS; among these samples, different ribosomal protein types were observed. A polygenetic analysis was performed using an unweighted pair-group method with arithmetic means and different ribosomal protein types to classify the strains into five major clades. Two clades primarily contained toxic strains, and the other three clades contained exclusively non-toxic strains. This is the first study to differentiate cyanobacterial strains using MALDI-TOF MS.

High-Resolution Melting Analysis for Rapid Detection of Sequence Type 131 Escherichia coli.

PubMed

Harrison, Lucas B; Hanson, Nancy D

2017-06-01

Escherichia coli isolates belonging to the sequence type 131 (ST131) clonal complex have been associated with the global distribution of fluoroquinolone and β-lactam resistance. Whole-genome sequencing and multilocus sequence typing identify sequence type but are expensive when evaluating large numbers of samples. This study was designed to develop a cost-effective screening tool using high-resolution melting (HRM) analysis to differentiate ST131 from non-ST131 E. coli in large sample populations in the absence of sequence analysis. The method was optimized using DNA from 12 E. coli isolates. Singleplex PCR was performed using 10 ng of DNA, Type-it HRM buffer, and multilocus sequence typing primers and was followed by multiplex PCR. The amplicon sizes ranged from 630 to 737 bp. Melt temperature peaks were determined by performing HRM analysis at 0.1°C resolution from 50 to 95°C on a Rotor-Gene Q 5-plex HRM system. Derivative melt curves were compared between sequence types and analyzed by principal component analysis. A blinded study of 191 E. coli isolates of ST131 and unknown sequence types validated this methodology. This methodology returned 99.2% specificity (124 true negatives and 1 false positive) and 100% sensitivity (66 true positives and 0 false negatives). This HRM methodology distinguishes ST131 from non-ST131 E. coli without sequence analysis. The analysis can be accomplished in about 3 h in any laboratory with an HRM-capable instrument and principal component analysis software. Therefore, this assay is a fast and cost-effective alternative to sequencing-based ST131 identification. Copyright © 2017 Harrison and Hanson.

Paenibacillus profundus sp. nov., a deep sediment bacterium that produces isocoumarin and peptide antibiotics.

PubMed

Romanenko, Lyudmila A; Tanaka, Naoto; Svetashev, Vassilii I; Kalinovskaya, Natalia I

2013-04-01

A novel bacterial strain Sl 79(T) was isolated from a deep surface sediment sample obtained from the Sea of Japan and investigated by phenotypic and molecular methods. The bacterium Sl 79(T) was Gram-positive, facultatively anaerobic, spore-forming, motile and able to form two different types of colonies. It contained the major menaquinone MK-7 and anteiso-C(15:0) followed by iso-C(15:0) as predominant fatty acids. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Sl 79(T) belonged to the genus Paenibacillus where it clustered to Paenibacillus apiarius NRRL NRS-1438(T) with a sequence similarity of 97.7 % and sharing sequence similarities below than 96.7 % to other validly named Paenibacillus species. Strain Sl 79(T) was found to possess a remarkable inhibitory activity against indicatory microorganisms. On the basis of combined spectral analyses, strain Paenibacillus sp. Sl 79(T) was established to produce isocoumarin and novel peptide antibiotics. On the basis of DNA-DNA relatedness, phenotypic and phylogenetic data obtained, it was concluded that strain Sl 79(T) represents a novel species, Paenibacillus profundus sp. nov. with the type strain Sl 79(T) = KMM 9420(T) = NRIC 0885(T).

Advances in molecular serotyping and subtyping of Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fratamico, Pina M.; DebRoy, Chitrita; Liu, Yanhong

Escherichia coli plays an important role as a member of the gut microbiota; however, pathogenic strains also exist, including various diarrheagenic E. coli pathotypes and extraintestinal pathogenic E. coli that cause illness outside of the GI-tract. E. coli have traditionally been serotyped using antisera against the ca. 186 O-antigens and 53 H-flagellar antigens. Phenotypic methods, including bacteriophage typing and O- and H- serotyping for differentiating and characterizing E. coli have been used for many years; however, these methods are generally time consuming and not always accurate. Advances in next generation sequencing technologies have made it possible to develop genetic-based subtypingmore » and molecular serotyping methods for E. coli, which are more discriminatory compared to phenotypic typing methods. Furthermore, whole genome sequencing (WGS) of E. coli is replacing established subtyping methods such as pulsedfield gel electrophoresis, providing a major advancement in the ability to investigate food-borne disease outbreaks and for trace-back to sources. Furthermore, a variety of sequence analysis tools and bioinformatic pipelines are being developed to analyze the vast amount of data generated by WGS and to obtain specific information such as O- and H-group determination and the presence of virulence genes and other genetic markers.« less

Advances in molecular serotyping and subtyping of Escherichia coli

DOE PAGES

Fratamico, Pina M.; DebRoy, Chitrita; Liu, Yanhong; ...

2016-05-03

Escherichia coli plays an important role as a member of the gut microbiota; however, pathogenic strains also exist, including various diarrheagenic E. coli pathotypes and extraintestinal pathogenic E. coli that cause illness outside of the GI-tract. E. coli have traditionally been serotyped using antisera against the ca. 186 O-antigens and 53 H-flagellar antigens. Phenotypic methods, including bacteriophage typing and O- and H- serotyping for differentiating and characterizing E. coli have been used for many years; however, these methods are generally time consuming and not always accurate. Advances in next generation sequencing technologies have made it possible to develop genetic-based subtypingmore » and molecular serotyping methods for E. coli, which are more discriminatory compared to phenotypic typing methods. Furthermore, whole genome sequencing (WGS) of E. coli is replacing established subtyping methods such as pulsedfield gel electrophoresis, providing a major advancement in the ability to investigate food-borne disease outbreaks and for trace-back to sources. Furthermore, a variety of sequence analysis tools and bioinformatic pipelines are being developed to analyze the vast amount of data generated by WGS and to obtain specific information such as O- and H-group determination and the presence of virulence genes and other genetic markers.« less

Aspergillus Section Fumigati Typing by PCR-Restriction Fragment Polymorphism▿

PubMed Central

Staab, Janet F.; Balajee, S. Arunmozhi; Marr, Kieren A.

2009-01-01

Recent studies have shown that there are multiple clinically important members of the Aspergillus section Fumigati that are difficult to distinguish on the basis of morphological features (e.g., Aspergillus fumigatus, A. lentulus, and Neosartorya udagawae). Identification of these organisms may be clinically important, as some species vary in their susceptibilities to antifungal agents. In a prior study, we utilized multilocus sequence typing to describe A. lentulus as a species distinct from A. fumigatus. The sequence data show that the gene encoding β-tubulin, benA, has high interspecies variability at intronic regions but is conserved among isolates of the same species. These data were used to develop a PCR-restriction fragment length polymorphism (PCR-RFLP) method that rapidly and accurately distinguishes A. fumigatus, A. lentulus, and N. udagawae, three major species within the section Fumigati that have previously been implicated in disease. Digestion of the benA amplicon with BccI generated unique banding patterns; the results were validated by screening a collection of clinical strains and by in silico analysis of the benA sequences of Aspergillus spp. deposited in the GenBank database. PCR-RFLP of benA is a simple method for the identification of clinically important, similar morphotypes of Aspergillus spp. within the section Fumigati. PMID:19403766