Science.gov

Sample records for 16s rrna-based phylogenetic

  1. IDENTIFICATION OF ACTIVE BACTERIAL COMMUNITIES IN A MODEL DRINKING WATER BIOFILM SYSTEM USING 16S RRNA-BASED CLONE LIBRARIES

    EPA Science Inventory

    Recent phylogenetic studies have used DNA as the target molecule for the development of environmental 16S rDNA clone libraries. As DNA may persist in the environment, DNA-based libraries cannot be used to identify metabolically active bacteria in water systems. In this study, a...

  2. Phylogenetic diversity in the genus Bacillus as seen by 16S rRNA sequencing studies.

    PubMed

    Rössler, D; Ludwig, W; Schleifer, K H; Lin, C; McGill, T J; Wisotzkey, J D; Jurtshuk, P; Fox, G E

    1991-01-01

    Comparative sequence analysis of 16S ribosomal (r)RNAs or DNAs of Bacillus alvei, B. laterosporus, B. macerans, B. macquariensis, B. polymyxa and B. stearothermophilus revealed the phylogenetic diversity of the genus Bacillus. Based on the presently available data set of 16S rRNA sequences from bacilli and relatives at least four major "Bacillus clusters" can be defined: a "Bacillus subtilis cluster" including B. stearothermophilus, a "B. brevis cluster" including B. laterosporus, a "B. alvei cluster" including B. macerans, B. maquariensis and B. polymyxa and a "B. cycloheptanicus branch".

  3. Phylogenetic diversity in the genus Bacillus as seen by 16S rRNA sequencing studies

    NASA Technical Reports Server (NTRS)

    Rossler, D.; Ludwig, W.; Schleifer, K. H.; Lin, C.; McGill, T. J.; Wisotzkey, J. D.; Jurtshuk, P. Jr; Fox, G. E.

    1991-01-01

    Comparative sequence analysis of 16S ribosomal (r)RNAs or DNAs of Bacillus alvei, B. laterosporus, B. macerans, B. macquariensis, B. polymyxa and B. stearothermophilus revealed the phylogenetic diversity of the genus Bacillus. Based on the presently available data set of 16S rRNA sequences from bacilli and relatives at least four major "Bacillus clusters" can be defined: a "Bacillus subtilis cluster" including B. stearothermophilus, a "B. brevis cluster" including B. laterosporus, a "B. alvei cluster" including B. macerans, B. maquariensis and B. polymyxa and a "B. cycloheptanicus branch".

  4. 16S rRNA Phylogenetic Investigation of the Candidate Division “Korarchaeota”

    PubMed Central

    Auchtung, Thomas A.; Takacs-Vesbach, Cristina D.; Cavanaugh, Colleen M.

    2006-01-01

    The environmental distribution and phylogeny of “Korarchaeota,” a proposed ancient archaeal division, was investigated by using the 16S rRNA gene framework. Korarchaeota-specific primers were designed based on previously published sequences and used to screen a variety of environments. Korarchaeota 16S rRNA genes were amplified exclusively from high temperature Yellowstone National Park hot springs and a 9°N East Pacific Rise deep-sea hydrothermal vent. Phylogenetic analyses of these and all available sequences suggest that Korarchaeota exhibit a high level of endemicity. PMID:16820509

  5. Phylogenetic analysis of the genus Microbacterium based on 16S rRNA gene sequences.

    PubMed

    Takeuchi, M; Yokota, A

    1994-11-15

    16S rRNA gene (rDNA) studies of the six species of the genus Microbacterium, M. lacticum, M. laevaniformans, M. dextranolyticum, M. imperiale, M. arborescens and M. aurum, were performed and the primary structures were compared with those of 29 representative actinobacteria and related organisms. Phylogenetic analysis indicated that six species of the genus Microbacterium and representative four species of the genus Aureobacterium appear to be phylogenetically coherent as was suggested by Rainey et al., although the peptidoglycan types of these two genera are different (peptidoglycan type B1 or B2). Thus, the phylogenetical analyses revealed that members of actinobacteria with group B-peptidoglycan do not cluster according to their peptidoglycan types, but form compact cluster different from actinobacteria or actinomycetes with group A-peptidoglycan.

  6. Phylogenetic relationships between Bacillus species and related genera inferred from 16s rDNA sequences

    PubMed Central

    Wei Wang, Mi Sun

    2009-01-01

    Neighbor-joining, maximum-parsimony, minimum-evolution, maximum-likelihood and Bayesian trees constructed based on 16S rDNA sequences of 181 type strains of Bacillus species and related taxa manifested nine phylogenetic groups. The phylogenetic analysis showed that Bacillus was not a monophyletic group. B. subtilis was in Group 1. Group 4, 6 and 8 respectively consisted of thermophiles, halophilic or halotolerant bacilli and alkaliphilic bacilli. Group 2, 4 and 8 consisting of Bacillus species and related genera demonstrated that the current taxonomic system did not agree well with the 16S rDNA evolutionary trees. The position of Caryophanaceae and Planococcaceae in Group 2 suggested that they might be transferred into Bacillaceae, and the heterogeneity of Group 2 implied that some Bacillus species in it might belong to several new genera. Group 9 was mainly comprised of the genera (excluding Bacillus) of Bacillaceae, so some Bacillus species in Group 9: B. salarius, B. qingdaonensis and B. thermcloacae might not belong to Bacillus. Four Bacillus species, B. schlegelii, B. tusciae, B. edaphicus and B. mucilaginosus were clearly placed outside the nine groups. PMID:24031394

  7. Phylogenetic analysis of Demodex caprae based on mitochondrial 16S rDNA sequence.

    PubMed

    Zhao, Ya-E; Hu, Li; Ma, Jun-Xian

    2013-11-01

    Demodex caprae infests the hair follicles and sebaceous glands of goats worldwide, which not only seriously impairs goat farming, but also causes a big economic loss. However, there are few reports on the DNA level of D. caprae. To reveal the taxonomic position of D. caprae within the genus Demodex, the present study conducted phylogenetic analysis of D. caprae based on mt16S rDNA sequence data. D. caprae adults and eggs were obtained from a skin nodule of the goat suffering demodicidosis. The mt16S rDNA sequences of individual mite were amplified using specific primers, and then cloned, sequenced, and aligned. The sequence divergence, genetic distance, and transition/transversion rate were computed, and the phylogenetic trees in Demodex were reconstructed. Results revealed the 339-bp partial sequences of six D. caprae isolates were obtained, and the sequence identity was 100% among isolates. The pairwise divergences between D. caprae and Demodex canis or Demodex folliculorum or Demodex brevis were 22.2-24.0%, 24.0-24.9%, and 22.9-23.2%, respectively. The corresponding average genetic distances were 2.840, 2.926, and 2.665, and the average transition/transversion rates were 0.70, 0.55, and 0.54, respectively. The divergences, genetic distances, and transition/transversion rates of D. caprae versus the other three species all reached interspecies level. The five phylogenetic trees all presented that D. caprae clustered with D. brevis first, and then with D. canis, D. folliculorum, and Demodex injai in sequence. In conclusion, D. caprae is an independent species, and it is closer to D. brevis than to D. canis, D. folliculorum, or D. injai.

  8. Linear programming model to construct phylogenetic network for 16S rRNA sequences of photosynthetic organisms and influenza viruses.

    PubMed

    Mathur, Rinku; Adlakha, Neeru

    2014-06-01

    Phylogenetic trees give the information about the vertical relationships of ancestors and descendants but phylogenetic networks are used to visualize the horizontal relationships among the different organisms. In order to predict reticulate events there is a need to construct phylogenetic networks. Here, a Linear Programming (LP) model has been developed for the construction of phylogenetic network. The model is validated by using data sets of chloroplast of 16S rRNA sequences of photosynthetic organisms and Influenza A/H5N1 viruses. Results obtained are in agreement with those obtained by earlier researchers.

  9. Phylogenetic relationships among cirrate octopods (Mollusca: Cephalopoda) resolved using mitochondrial 16S ribosomal DNA sequences.

    PubMed

    Piertney, Stuart B; Hudelot, Cendrine; Hochberg, F G; Collins, Martin A

    2003-05-01

    PHYLOGENETIC RELATIONSHIPS AMONG THE CIRRATE OCTOPODS (MOLLUSCA: Cephalopoda) were investigated using partial sequences of the 16S rRNA mitochondrial gene. The derived phylogeny supports the traditional separation of cirrate families based on web form. Genera with a single web (Opisthoteuthis, Grimpoteuthis, Luteuthis, and Cirroctopus) are clearly distinct from those with an intermediate or secondary web (Cirroteuthis, Cirrothauma, and Stauroteuthis). The cirrates with a single web are separated into three groups. The first group is represented by Opisthoteuthis species, the second by Grimpoteuthis and Luteuthis, and the third by members of the genus Cirroctopus. There is no support for the isolation of Luteuthis in a separate family (Luteuthidae). There is, however, evidence of two groupings within the genus Opisthoteuthis. The data suggest the following revisions in the systematic classification of the cirrates: (1) Cirrothauma, Cirroteuthis, and Stauroteuthis be united in the Cirroteuthidae; (2) Grimpoteuthis and Luteuthis be placed in the Grimpoteuthidae; (3) Opisthoteuthis in the Opisthoteuthidae, and; (4) Cirroctopus be considered sufficiently distinct from both Opisthoteuthidae and Grimpoteuthidae to warrant placement in a new family.

  10. Phylogenetic relationships in Demodex mites (Acari: Demodicidae) based on mitochondrial 16S rDNA partial sequences.

    PubMed

    Zhao, Ya-E; Wu, Li-Ping

    2012-09-01

    To confirm phylogenetic relationships in Demodex mites based on mitochondrial 16S rDNA partial sequences, mtDNA 16S partial sequences of ten isolates of three Demodex species from China were amplified, recombined, and sequenced and then analyzed with two Demodex folliculorum isolates from Spain. Lastly, genetic distance was computed, and phylogenetic tree was reconstructed. MEGA 4.0 analysis showed high sequence identity among 16S rDNA partial sequences of three Demodex species, which were 95.85 % in D. folliculorum, 98.53 % in Demodex canis, and 99.71 % in Demodex brevis. The divergence, genetic distance, and transition/transversions of the three Demodex species reached interspecies level, whereas there was no significant difference of the divergence (1.1 %), genetic distance (0.011), and transition/transversions (3/1) of the two geographic D. folliculorum isolates (Spain and China). Phylogenetic trees reveal that the three Demodex species formed three separate branches of one clade, where D. folliculorum and D. canis gathered first, and then gathered with D. brevis. The two Spain and five China D. folliculorum isolates did not form sister clades. In conclusion, 16S mtDNA are suitable for phylogenetic relationship analysis in low taxa (genus or species), but not for intraspecies determination of Demodex. The differentiation among the three Demodex species has reached interspecies level.

  11. Mitochondrial 16S rDNA analysis of Tunisian androctonus species (Scorpions, Buthidae): phylogenetic approach.

    PubMed

    Ben Othmen, A; Said, K; Ben Alp, Z; Chatti, N; Ready, P D

    2006-01-01

    Tunisian Androctonus species, for long time discussed, were recognized on the basis of mitochondrial 16S rDNA sequences. Although the analysed nucleotide sequence is rather short (about 300 bp), the obtained phlogenetic trees revealed that A. amoreuxi and A. aeneas form two well-supported sister clades against A. australis haplotypes. Each specimen of the very rare species A. aeneas showed a specific haplotype, but together formed a well-defined clade. Some A. amoreuxi specimens highlighted unidirectional mitochondrial introgression from neighbouring A. australis population. Within A. australis, previously described, subspecies subdivision (A. a .hector and A. a. garzonii) was not supported.

  12. Distribution, hosts, 16S rDNA sequences and phylogenetic position of the Neotropical tick Amblyomma parvum (Acari: Ixodidae).

    PubMed

    Nava, S; Szabó, M P J; Mangold, A J; Guglielmone, A A

    2008-07-01

    The hosts, distribution, intraspecific genetic variation and phylogenetic position of Amblyomma parvum (Acari: Ixodidae) have recently been re-assessed. Data on this tick's hosts and distribution were obtained not only from existing literature but also from unpublished records. Sequences of the ticks' mitochondrial 16S ribosomal DNA (rDNA) were used to evaluate genetic variation among specimens of A. parvum from different localities in Argentina and Brazil, and to explore the phylogenetic relationships between this tick and other Amblyomma species. Although several species of domestic and wild mammal act as hosts for adult A. parvum, most collected adults of this species have come from cattle and goats. Caviid rodents of the subfamily Caviinae appear to be the hosts for the immature stages. So far, A. parvum has been detected in 12 Neotropical biogeographical provinces (Chaco, Cerrado, Eastern Central America, Venezuelan Coast, Pantanal, Parana Forest, Caatinga, Chiapas, Venezuelan Llanos, Monte, Western Panamanian Isthmus, and Roraima) but the Chaco province has provided significantly more specimens than any other (P<0.0001). The 16S rDNA sequences showed just 0.0%-1.1% divergence among the Argentinean A. parvum investigated and no more than 0.2% divergence among the Brazilian specimens. The observed divergence between the Argentinean and Brazilian specimens was, however, greater (3.0%-3.7%). Although there is now molecular and morphological evidence to indicate that A. parvum, A. pseudoparvum, A. auricularium and A. pseudoconcolor are members of a natural group, previous subgeneric classifications do not reflect this grouping. The subgeneric status of these tick species therefore needs to be re-evaluated. The 16S-rDNA-based evaluation of divergence indicates that the gene flow between Argentinean and Brazilian 'A. parvum' is very limited and that the Argentinean 'A. parvum' may be a different species to the Brazilian.

  13. Selective phylogenetic analysis targeting 16S rRNA genes of hyperthermophilic archaea in the deep-subsurface hot biosphere.

    PubMed

    Kimura, Hiroyuki; Ishibashi, Jun-Ichiro; Masuda, Harue; Kato, Kenji; Hanada, Satoshi

    2007-04-01

    International drilling projects for the study of microbial communities in the deep-subsurface hot biosphere have been expanded. Core samples obtained by deep drilling are commonly contaminated with mesophilic microorganisms in the drilling fluid, making it difficult to examine the microbial community by 16S rRNA gene clone library analysis. To eliminate mesophilic organism contamination, we previously developed a new method (selective phylogenetic analysis [SePA]) based on the strong correlation between the guanine-plus-cytosine (G+C) contents of the 16S rRNA genes and the optimal growth temperatures of prokaryotes, and we verified the method's effectiveness (H. Kimura, M. Sugihara, K. Kato, and S. Hanada, Appl. Environ. Microbiol. 72:21-27, 2006). In the present study we ascertained SePA's ability to eliminate contamination by archaeal rRNA genes, using deep-sea hydrothermal fluid (117 degrees C) and surface seawater (29.9 degrees C) as substitutes for deep-subsurface geothermal samples and drilling fluid, respectively. Archaeal 16S rRNA gene fragments, PCR amplified from the surface seawater, were denatured at 82 degrees C and completely digested with exonuclease I (Exo I), while gene fragments from the deep-sea hydrothermal fluid remained intact after denaturation at 84 degrees C because of their high G+C contents. An examination using mixtures of DNAs from the two environmental samples showed that denaturation at 84 degrees C and digestion with Exo I completely eliminated archaeal 16S rRNA genes from the surface seawater. Our method was quite useful for culture-independent community analysis of hyperthermophilic archaea in core samples recovered from deep-subsurface geothermal environments.

  14. Phylogenetic positions of Clostridium chauvoei and Clostridium septicum based on 16S rRNA gene sequences.

    PubMed

    Kuhnert, P; Capaul, S E; Nicolet, J; Frey, J

    1996-10-01

    The sequences of the 16S rRNA genes (rrs genes) of Clostridium chauvoei, the causative agent of blackleg in cattle, and the phenotypically related organism Clostridium septicum were determined. After amplification of 1,507-bp PCR fragments from the corresponding rrs genes, the sequences were determined in a single round of sequencing by using conserved region primers. A sequence similarity analysis of the sequences revealed the close phylogenetic relationship of C. chauvoei and C. septicum in Clostridium cluster I (M. D. Collins, P. A. Lawson, A. Willems, J. J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. E. Farrow, Int. J. Syst. Bacteriol. 44:812-826, 1994), which includes Clostridium carnis, Clostridium perfringens, Clostridium botulinum, and Clostridium tetani. We found that 99.3% of the nucleotides in the genes of C. chauvoei and C. septicum are identical.

  15. Phylogenetic relationships of chemoautotrophic bacterial symbionts of Solemya velum say (Mollusca: Bivalvia) determined by 16S rRNA gene sequence analysis.

    PubMed Central

    Eisen, J A; Smith, S W; Cavanaugh, C M

    1992-01-01

    The protobranch bivalve Solemya velum Say (Mollusca: Bivalvia) houses chemoautotrophic symbionts intracellularly within its gills. These symbionts were characterized through sequencing of polymerase chain reaction-amplified 16S rRNA coding regions and hybridization of an Escherichia coli gene probe to S. velum genomic DNA restriction fragments. The symbionts appeared to have only one copy of the 16S rRNA gene. The lack of variability in the 16S sequence and hybridization patterns within and between individual S. velum organisms suggested that one species of symbiont is dominant within and specific for this host species. Phylogenetic analysis of the 16S sequences of the symbionts indicates that they lie within the chemoautotrophic cluster of the gamma subdivision of the eubacterial group Proteobacteria. Images PMID:1577710

  16. Phylogenetic relationships of chemoautotrophic bacterial symbionts of Solemya velum say (Mollusca: Bivalvia) determined by 16S rRNA gene sequence analysis.

    PubMed

    Eisen, J A; Smith, S W; Cavanaugh, C M

    1992-05-01

    The protobranch bivalve Solemya velum Say (Mollusca: Bivalvia) houses chemoautotrophic symbionts intracellularly within its gills. These symbionts were characterized through sequencing of polymerase chain reaction-amplified 16S rRNA coding regions and hybridization of an Escherichia coli gene probe to S. velum genomic DNA restriction fragments. The symbionts appeared to have only one copy of the 16S rRNA gene. The lack of variability in the 16S sequence and hybridization patterns within and between individual S. velum organisms suggested that one species of symbiont is dominant within and specific for this host species. Phylogenetic analysis of the 16S sequences of the symbionts indicates that they lie within the chemoautotrophic cluster of the gamma subdivision of the eubacterial group Proteobacteria.

  17. Phylogenetic relationships linking Duttaphrynus (Amphibia: Anura: Bufonidae) species based on 12S and 16S rDNA sequences.

    PubMed

    Pratihar, Suman; Bhattacharya, Manojit; Deuti, Kaushik

    2016-07-01

    Genus Duttaphrynus (Amphibia: Anura: Bufonidae) is endemic to southwestern and southern China and throughout southern Asia. Duttaphrynus phylogeny was also under debate for many years. 12S and 16S rDNAs help us to elucidate Duttaphrynus phylogeny.

  18. Application of 16S rRNA, cytochrome b and control region sequences for understanding the phylogenetic relationships in Oryx species.

    PubMed

    Khan, H A; Arif, I A; Al Homaidan, A A; Al Farhan, A H

    2008-12-16

    The present study reports the application of mitochondrial markers for the molecular phylogeny of Oryx species, including the Arabian oryx (AO), scimitar-horned oryx (SHO) and plains oryx (PO), using the Addax as an outgroup. Sequences of three molecular markers, 16S rRNA, cytochrome b and a control region, for the above four taxa were aligned and the topologies of respective phylogenetic trees were compared. All these markers clearly differentiated the genus Addax from Oryx. However, for species-level grouping, while 16S rRNA and cytochrome b produced similar phylogeny (SHO grouped with PO), the control region grouped SHO with AO. Further studies are warranted to generate more sequencing data, apply multiple bioinformatics tools and to include relevant nuclear markers for phylogenetic analysis of Oryx species.

  19. Intraspecific Genetic Variation and Phylogenetic Analysis of Dirofilaria immitis Samples from Western China Using Complete ND1 and 16S rDNA Gene Sequences

    PubMed Central

    Liu, Tianyu; Liang, Yinan; Zhong, Xiuqin; Wang, Ning; Hu, Dandan; Zhou, Xuan; Gu, Xiaobin; Peng, Xuerong; Yang, Guangyou

    2014-01-01

    Dirofilaria immitis (heartworm) is the causative agent of an important zoonotic disease that is spread by mosquitoes. In this study, molecular and phylogenetic characterization of D. immitis were performed based on complete ND1 and 16S rDNA gene sequences, which provided the foundation for more advanced molecular diagnosis, prevention, and control of heartworm diseases. The mutation rate and evolutionary divergence in adult heartworm samples from seven dogs in western China were analyzed to obtain information on genetic diversity and variability. Phylogenetic relationships were inferred using both maximum parsimony (MP) and Bayes methods based on the complete gene sequences. The results suggest that D. immitis formed an independent monophyletic group in which the 16S rDNA gene has mutated more rapidly than has ND1. PMID:24639299

  20. Insights into the phylogenetic positions of photosynthetic bacteria obtained from 5S rRNA and 16S rRNA sequence data

    NASA Technical Reports Server (NTRS)

    Fox, G. E.

    1985-01-01

    Comparisons of complete 16S ribosomal ribonucleic acid (rRNA) sequences established that the secondary structure of these molecules is highly conserved. Earlier work with 5S rRNA secondary structure revealed that when structural conservation exists the alignment of sequences is straightforward. The constancy of structure implies minimal functional change. Under these conditions a uniform evolutionary rate can be expected so that conditions are favorable for phylogenetic tree construction.

  1. [Phylogenetic characterization of endosymbionts of the hydrothermal vent mussel Bathymodiolus azoricus by analysis of the 16S rRNA, pmoL, and cbbA genes].

    PubMed

    Spiridonova, E M; Kuznetsov, B B; Pimenov, N V; Turova, T P

    2006-01-01

    In order to assess the phylogenetic diversity of the endosymbiotic microbial community of the gills of marine shellfish Bathymodiolus azoricus, total DNA was extracted from the gills. The PCR fragments corresponding to the genes encoding 16S rRNA, ribulose-bisphosphate carboxylase (cbbL), and particulate methane monooxygenase (pmoA) were amplified, cloned, and sequenced. For the 16S rDNA genes, only one phylotype was revealed; it belonged to the cluster of Mytilidae thiotrophic symbionts within the Gammaproteobacteria. For the RuBisCO genes, two phylotypes were found, both belonging to Gammaproteobacteria. One of them was closely related to the previously known mytilid symbiont, the other, to a pogonophore symbiont, presumably a methanotrophic bacterium. One phylotype of particulate methane oxygenase genes was also revealed; this finding indicated the presence of a methanotrophic symbiont. Phylogenetic analysis of the pmoA placed this endosymbiont within the Gammaproteobacteria, in a cluster including the methanotrophic bacterial genus Methylobacter and other methanotrophic Bathymodiolus gill symbionts. These results provide evidence for the existence of two types of endosymbionts (thioautotrophic and methanotrophic) in the gills of B. azoricus and demonstrate that, apart from the phylogenetic analysis of 16S rRNA genes, parallel analysis of functional genes is essential.

  2. Phylogenetic Analysis of Bacteroidales 16S rRNA Genes Unveils Sequences Specific to Diverse Swine Fecal Sources

    EPA Science Inventory

    Two of the currently available methods to assess swine fecal pollution (Bac1 and PF163) target Bacteroidales 16S rRNA genes. However, these assays have been shown to exhibit poor host-specificity and low detection limits in environmental waters, in part due to the limited number...

  3. ASSESSMENT OF FECAL POLLUTION SOURCES IN PLUM CREEK WATERSHED USING PCR AND PHYLOGENETIC ANALYSES OF BACTEROIDETES 16S RDNA

    EPA Science Inventory

    Traditional methods for assessing fecal pollution in environmental systems, such as monitoring for fecal coliforms are not capable of discriminating between different sources fecal pollution. Recently, 16S rDNA Bacteroidetes-targeted PCR assays were developed to discriminate betw...

  4. Phylogenetic analysis of Mediterranean Mugilids by allozymes and 16S mt-rRNA genes investigation: are the Mediterranean species of Liza monophyletic?

    PubMed

    Rossi, Anna Rita; Ungaro, Andrea; De Innocentiis, Sabina; Crosetti, Donatella; Sola, Luciana

    2004-10-01

    The family Mugilidae (Pisces, Mugiliformes) includes species which are present in all tropical and temperate regions. Six species, Chelon labrosus, Mugil cephalus, Liza aurata, L. ramada, L. saliens, Oedalechilus labeo, are commonly found in the Mediterranean. These species have been widely studied through morphological, biochemical, and molecular markers. However, their phylogenetic relationships, and therefore the assumed monophyly of Liza species, still remain unclear: To further investigate this topic, gene-enzyme systems and sequences of the partial 16S rRNA mitochondrial gene were analyzed in Italian samples of all six Mediterranean species. The phylogenetic reconstructions indicated M. cephalus as being the most divergent species and the existence of a main cluster including all the Mediterranean species of Liza and C. labrosus. The parametric bootstrap approach adopted to test alternative phylogenetic hypotheses indicated that the Mediterranean species of Liza do not form a monophyletic group exclusive of Chelon.

  5. Phylogenetic 16S rRNA analysis reveals the presence of complex and partly unknown bacterial communities in Tito Bustillo cave, Spain, and on its Palaeolithic paintings.

    PubMed

    Schabereiter-Gurtner, Claudia; Saiz-Jimenez, Cesareo; Piñar, Guadalupe; Lubitz, Werner; Rölleke, Sabine

    2002-07-01

    Tito Bustillo cave (Ribadesella, Spain) contains valuable Palaeolithic paintings, which date back 15 000-20 000 years. Since 1969, the cave has been open to the public. Rock wall surfaces, spelaeothems and soils are covered by apparent biofilms of phototrophic microorganisms, which develop under artificial lighting. In addition, rock surfaces present conspicuous bacterial growth in the form of round colonies of different colours and about 1-2 mm in diameter. Even the famous Paintings Panel shows some evident microbial growth. In the present study, bacterial communities on the paintings and on the rock surfaces near the paintings were analysed by culture-independent techniques, including polymerase chain reaction (PCR) amplification of bacterial 16S rRNA genes (16S rDNA), phylogenetic sequence analyses and genetic community fingerprinting by denaturing gradient gel electrophoresis (DGGE). DGGE fingerprints showed complex bacterial community patterns. Forty-one clones matching DGGE bands of the community fingerprints were sequenced, representing about 39% of DNA fragments in the DGGE patterns. Phylogenetic sequence analyses revealed a high number of phylogenetically novel 16S rDNA sequence types and a high diversity of putatively chemotrophic and heterotrophic bacteria. Sequences were phylogenetically most closely related to the Proteobacteria (20 clones), green non-sulphur bacteria (three clones), Planctomycetales order (one clone), Cytophaga-Flexibacter- Bacteroides division (one clone) and the Actinobacteria (four clones). Furthermore, we report the presence of members of the Acidobacterium division (12 clones) in a karstic hypogean environment. Members of this phylum have not so far been detected in these particular environments.

  6. Phylogenetic relationships of the endosymbionts of mealybugs (Homoptera: Pseudococcidae) based on 16S rDNA sequences.

    PubMed

    Munson, M A; Baumann, P; Moran, N A

    1992-03-01

    A portion of the gene coding for the 16S ribosomal RNA from the endosymbionts of three species of mealybugs [Pseudococcus longispinus (Targioni-Tozzetti), Pseudococcus maritimus (Ehrhorn), and Dysmicoccus neobrevipes (Beardsley)] was cloned, sequenced, and compared to a homologous fragment from bacteria representative of aphid endosymbionts as well as major subdivisions of the Proteobacteria. Parsimony analysis of the sequences indicated that the mealybug endosymbionts are related and belong to the beta-subdivision; in contrast, previous studies showed that aphid endosymbionts are part of the gamma-subdivision. These findings suggest that the endosymbiosis of mealybugs is a consequence of a single bacterial infection and indicate that this ancestor was different from the ancestor involved in aphid endosymbiosis.

  7. Assessment of fecal pollution sources in a small northern-plains watershed using PCR and phylogenetic analyses of Bacteroidetes 16S rRNA gene

    USGS Publications Warehouse

    Lamendella, R.; Domingo, J.W.S.; Oerther, D.B.; Vogel, J.R.; Stoeckel, D.M.

    2007-01-01

    We evaluated the efficacy, sensitivity, host-specificity, and spatial/temporal dynamics of human- and ruminant-specific 16S rRNA gene Bacteroidetes markers used to assess the sources of fecal pollution in a fecally impacted watershed. Phylogenetic analyses of 1271 fecal and environmental 16S rRNA gene clones were also performed to study the diversity of Bacteroidetes in this watershed. The host-specific assays indicated that ruminant feces were present in 28-54% of the water samples and in all sampling seasons, with increasing frequency in downstream sites. The human-targeted assays indicated that only 3-5% of the water samples were positive for human fecal signals, although a higher percentage of human-associated signals (19-24%) were detected in sediment samples. Phylogenetic analysis indicated that 57% of all water clones clustered with yet-to-be-cultured Bacteroidetes species associated with sequences obtained from ruminant feces, further supporting the prevalence of ruminant contamination in this watershed. However, since several clusters contained sequences from multiple sources, future studies need to consider the potential cosmopolitan nature of these bacterial populations when assessing fecal pollution sources using Bacteroidetes markers. Moreover, additional data is needed in order to understand the distribution of Bacteroidetes host-specific markers and their relationship to water quality regulatory standards. ?? 2006 Federation of European Microbiological Societies.

  8. Diversity and phylogenetic analysis of endosymbiotic bacteria from field caught Bemisia tabaci from different locations of North India based on 16S rDNA library screening.

    PubMed

    Singh, Shalini Thakur; Priya, Natarajan Gayatri; Kumar, Jitendra; Rana, Vipin Singh; Ellango, R; Joshi, Adita; Priyadarshini, Garima; Asokan, R; Rajagopal, Raman

    2012-03-01

    Bemisia tabaci is the major vector pest of agricultural crops all over the world. In this study we report the different bacterial endosymbionts associated with B. tabaci sampled from 14 different locations in North India. Using 16S rDNA clone library sequences we were able to identify Portiera, the primary endosymbiont of B. tabaci, and other secondary endosymbionts like Cardinium, Wolbachia, Rickettsia and Arsenophonus. Along with these we also detected Bacillus, Enterobacter, Paracoccus and Acinetobacter. These secondary endosymbionts were not uniformly distributed in all the locations. Phylogenetic analysis of 16S rDNA sequences of Cardinium, Wolbachia, Rickettsia and Arsenophonus showed that each of these bacteria form a separate cluster when compared to their respective counterparts from other parts of the world. MtCO1 gene based phylogenetic analysis showed the presence of Asia I and Asia II genetic groups of B. tabaci in N. India. The multiple correspondence analyses showed no correlation between the host genetic group and the endosymbiont diversity. These results suggest that the bacterial endosymbiont diversity of B. tabaci is much larger and complex than previously perceived and probably N. Indian strains of the bacterial symbionts could have evolved from some other ancestor.

  9. Phylogenetic taxonomy of the family Chlorobiaceae on the basis of 16S rRNA and fmo (Fenna-Matthews-Olson protein) gene sequences.

    PubMed

    Imhoff, Johannes F

    2003-07-01

    A new taxonomy of the green sulfur bacteria is proposed, based on phylogenetic relationships determined using the sequences of the independent 16S rRNA and fmo (Fenna-Matthews-Olson protein) genes, and supported by the DNA G + C content and sequence signatures. Comparison of the traditional classification system for these bacteria with their phylogenetic relationship yielded a confusing picture, because properties used for classification (such as cell morphology, photosynthetic pigments and substrate utilization) do not concur with their phylogeny. Using the genetic information available, strains and species assigned to the genera Chlorobium, Pelodictyon and Prosthecochloris are considered, and the following changes are proposed. Pelodictyon luteolum is transferred to the genus Chlorobium as Chlorobium luteolum comb. nov. Pelodictyon clathratiforme and Pelodictyon phaeoclathratiforme are transferred to the genus Chlorobium and combined into one species, Chlorobium clathratiforme comb. nov. The name Pelodictyon will become a synonym of Chlorobium. Strains known as Chlorobium limicola subsp. thiosulfatophilum that have a low DNA G + C content (52-52.5 mol%) are treated as strains of Chlorobium limicola; those with a high DNA G + C content (58.1 mol%) are transferred to Chlorobaculum gen. nov., as Chlorobaculum thiosulfatiphilum sp. nov. Chlorobium tepidum is transferred to Chlorobaculum tepidum comb. nov., and defined as the type species of the genus Chlorobaculum. Strains assigned to Chlorobium phaeobacteroides, but phylogenetically distant from the type strain of this species, are assigned to Chlorobium limicola and to Chlorobaculum limnaeum sp. nov. Strains known as Chlorobium vibrioforme subsp. thiosulfatophilum are transferred to Chlorobaculum parvum sp. nov. Chlorobium chlorovibrioides is transferred to 'Chlorobaculum chlorovibrioides' comb. nov. The type strain of Chlorobium vibrioforme is phylogenetically related to Prosthecochloris, and is therefore

  10. Phylogenetic position of Phthiraptera (Insecta: Paraneoptera) and elevated rate of evolution in mitochondrial 12S and 16S rDNA.

    PubMed

    Yoshizawa, Kazunori; Johnson, Kevin P

    2003-10-01

    Phthiraptera (chewing and sucking lice) and Psocoptera (booklice and barklice) are closely related to each other and compose the monophyletic taxon Psocodea. However, there are two hypotheses regarding their phylogenetic relationship: (1) monophyletic Psocoptera is the sister group of Phthiraptera or (2) Psocoptera is paraphyletic, and Liposcelididae of Psocoptera is the sister group of Phthiraptera. Each hypothesis is supported morphologically and/or embryologically, and this problem has not yet been resolved. In the present study, the phylogenetic position of Phthiraptera was examined using mitochondrial 12S and 16S rDNA sequences, with three methods of phylogenetic analysis. Results of all analyses strongly supported the close relationship between Phthiraptera and Liposcelididae. Results of the present analyses also provided some insight into the elevated rate of evolution in mitochondrial DNA (mtDNA) in Phthiraptera. An elevated substitution rate of mtDNA appears to originate in the common ancestor of Phthiraptera and Liposcelididae, and directly corresponds to an increased G+C content. Therefore, the elevated substitution rate of mtDNA in Phthiraptera and Liposcelididae appears to be directional. A high diversity of 12S rDNA secondary structure was also observed in wide range of Phthiraptera and Liposcelididae, but these structures seem to have evolved independently in different clades.

  11. Nitrifying bacterial communities in an aquaculture wastewater treatment system using fluorescence in situ hybridization (FISH), 16S rRNA gene cloning, and phylogenetic analysis.

    PubMed

    Paungfoo, Chanyarat; Prasertsan, Poonsuk; Burrell, Paul C; Intrasungkha, Nugul; Blackall, Linda L

    2007-07-01

    Aquaculture, especially shrimp farming, has played a major role in the growth of Thailand's economy in recent years, as well as in many South East Asian countries. However, the nutrient discharges from these activities have caused adverse impacts on the quality of the receiving waterways. In particular nitrogenous compounds, which may accumulate in aquaculture ponds, can be toxic to aquatic animals and cause environmental problems such as eutrophication. The mineralization process is well known, but certain aspects of the microbial ecology of nitrifiers, the microorganisms that convert ammonia to nitrate, are poorly understood. A previously reported enrichment of nitrifying bacteria (ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) from a shrimp farm inoculated in a sequencing batch reactor (SBR) was studied by molecular methods. The initial identification and partial quantification of the nitrifying bacteria (AOB and NOB) were carried out by fluorescence in situ hybridization (FISH) using previously published 16S rRNA-targeting oligonucleotide probes. The two dominant bacterial groups detected by FISH were from the Cytophaga-Flavobacterium-Bacteroides and Proteobacteria (beta subdivision) phyla. Published FISH probes for Nitrobacter and Nitrospira did not hybridize to any of the bacterial cells. Therefore it is likely that new communities of NOBs, differing from previously reported ones, exist in the enrichments. Molecular genetic techniques (cloning, sequencing, and phylogenetic analysis) targeting the 16S rRNA genes from the nitrifying enrichments were performed to identify putative AOBs and NOBs.

  12. Comparative 16S rRNA Analysis of Lake Bacterioplankton Reveals Globally Distributed Phylogenetic Clusters Including an Abundant Group of Actinobacteria

    PubMed Central

    Glöckner, Frank Oliver; Zaichikov, Evgeny; Belkova, Natalia; Denissova, Ludmilla; Pernthaler, Jakob; Pernthaler, Annelie; Amann, Rudolf

    2000-01-01

    In a search for cosmopolitan phylogenetic clusters of freshwater bacteria, we recovered a total of 190 full and partial 16S ribosomal DNA (rDNA) sequences from three different lakes (Lake Gossenköllesee, Austria; Lake Fuchskuhle, Germany; and Lake Baikal, Russia). The phylogenetic comparison with the currently available rDNA data set showed that our sequences fall into 16 clusters, which otherwise include bacterial rDNA sequences of primarily freshwater and soil, but not marine, origin. Six of the clusters were affiliated with the α, four were affiliated with the β, and one was affiliated with the γ subclass of the Proteobacteria; four were affiliated with the Cytophaga-Flavobacterium-Bacteroides group; and one was affiliated with the class Actinobacteria (formerly known as the high-G+C gram-positive bacteria). The latter cluster (hgcI) is monophyletic and so far includes only sequences directly retrieved from aquatic environments. Fluorescence in situ hybridization (FISH) with probes specific for the hgcI cluster showed abundances of up to 1.7 × 105 cells ml−1 in Lake Gossenköllesee, with strong seasonal fluctuations, and high abundances in the two other lakes investigated. Cell size measurements revealed that Actinobacteria in Lake Gossenköllesee can account for up to 63% of the bacterioplankton biomass. A combination of phylogenetic analysis and FISH was used to reveal 16 globally distributed sequence clusters and to confirm the broad distribution, abundance, and high biomass of members of the class Actinobacteria in freshwater ecosystems. PMID:11055963

  13. Molecular phylogenetic analysis of the dragonfly genera Libellula, Ladona, and Plathemis (Odonata: Libellulidae) based on mitochondrial cytochrome oxidase I and 16S rRNA sequence data.

    PubMed

    Artiss, T; Schultz, T R; Polhemus, D A; Simon, C

    2001-03-01

    Molecular phylogenetic relationships among members of the odonate genus Libellula (Odonata: Anisoptera: Libellulidae) were examined using 735 bp of mitochondrial COI and 416 bp of 16S ribosomal RNA gene sequences. Considerable debate exists over several relationships within Libellula, as well over the status of two putative genera often placed as subgenera within Libellula: Ladona and Plathemis. Parsimony and maximum-likelihood analyses of the separate and combined data sets indicate that Plathemis is basal and monophyletic and that Ladona is the sister clade to the remainder of Libellula sensu stricto (s.s.) (all species within the genus Libellula, excluding Plathemis and Ladona). Moreover, two European taxa, Libellula fulva and L. depressa, were found to occupy a sister group relationship within the Ladona clade. Relationships within Libellula s.s. are less well resolved. However, monophyletic lineages within the genus are largely consistent with morphologically based subgeneric classifications. Although tree topologies from each analysis differed in some details, the differences were in no case statistically significant. The analysis of the combined COI and 16S data yielded trees with overall stronger support than analyses of either gene alone. Several analyses failed to support the monophyly of Libellula sensu lato due to the inclusion of one or more outgroup species. However, statistical comparisons of topologies produced by unconstrained analyses and analyses in which the monophyly of Libellula was constrained indicate that any differences are nonsignificant. Based on morphological data, we therefore reject the paraphyly of Libellula and accept the outgroup status of Orthemis ferruginea and Pachydiplax longipennis.

  14. When molecules support morphology: Phylogenetic reconstruction of the family Onuphidae (Eunicida, Annelida) based on 16S rDNA and 18S rDNA.

    PubMed

    Budaeva, Nataliya; Schepetov, Dmitry; Zanol, Joana; Neretina, Tatiana; Willassen, Endre

    2016-01-01

    Onuphid polychaetes are tubicolous marine worms commonly reported worldwide from intertidal areas to hadal depths. They often dominate in benthic communities and have economic importance in aquaculture and recreational fishing. Here we report the phylogeny of the family Onuphidae based on the combined analyses of nuclear (18S rDNA) and mitochondrial (16S rDNA) genes. Results of Bayesian and Maximum Likelihood analyses supported the monophyly of Onuphidae and its traditional subdivision into two monophyletic subfamilies: Onuphinae and Hyalinoeciinae. Ten of 22 recognized genera were monophyletic with strong node support; four more genera included in this study were either monotypic or represented by a single species. None of the genera appeared para- or polyphyletic and this indicates a strong congruence between the traditional morphology-based systematics of the family and the newly obtained molecular-based phylogenetic reconstructions. Intergeneric relationships within Hyalinoeciinae were not resolved. Two strongly supported monophyletic groups of genera were recovered within Onuphinae: ((Onuphis, Aponuphis), Diopatra, Paradiopatra) and (Hirsutonuphis, (Paxtonia, (Kinbergonuphis, Mooreonuphis))). A previously accepted hypothesis on the subdivision of Onuphinae into the Onuphis group of genera and the Diopatra group of genera was largely rejected.

  15. Phylogenetic diversity based on rrs, atpD, recA genes and 16S-23S intergenic sequence analyses of rhizobial strains isolated from Vicia faba and Pisum sativum in Peru.

    PubMed

    Santillana, Nery; Ramírez-Bahena, Martha Helena; García-Fraile, Paula; Velázquez, Encarna; Zúñiga, Doris

    2008-03-01

    In this study 17 isolates from effective nodules of Vicia faba and Pisum sativum var. macrocarpum growing in different soils from Peru were isolated and characterized. The isolates, presenting 11 different RAPD profiles, were distributed in three groups on the basis of their 16S-RFLP patterns. The 16S rRNA gene sequences of strains from 16S-RFLP groups I, II and III were closely related (identities higher than 99.5%) to Rhizobium leguminosarum bv. trifolii DSM 30141 (=ATCC 14480), R. leguminosarum bv. viciae DSM 30132(T) and Rhizobium etli CFN42(T) (=USDA 9032(T)), respectively. The analysis of the 16S-23S intergenic spacer (ITS) and two housekeeping genes, atpD and recA, confirmed the identification of strains from group I, however those from groups II and III were phylogenetically divergent to strains DSM 30132(T) and CFN42(T). These results support the fact that the 16S rRNA gene is not adequate for identification at species level within genus Rhizobium and suggest the existence of putative new species within the phylogenetic group of R. leguminosarum. They also confirm the need of a taxonomic revision of R. leguminosarum since the reference strains of the three biovars included in this study are phylogenetically divergent according to their ITS, atpD and recA gene sequences.

  16. Metagenomic and near full-length 16S rRNA sequence data in support of the phylogenetic analysis of the rumen bacterial community in steers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Next generation sequencing technologies have vastly changed the approach of sequencing of the 16S rRNA gene for studies in microbial ecology. Three distinct technologies are available for large-scale 16S sequencing. All three are subject to biases introduced by sequencing error rates, amplificatio...

  17. Evaluation of 16S Rrna amplicon sequencing using two next-generation sequencing technologies for phylogenetic analysis of the rumen bacterial community in steers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Next generation sequencing technologies have vastly changed the approach of sequencing of the 16S rRNA gene for studies in microbial ecology. Three distinct technologies are available for large-scale 16S sequencing. All three are subject to biases introduced by sequencing error rates, amplificatio...

  18. Evaluation of 16S rRNA amplicon sequencing using two next-generation sequencing technologies for phylogenetic analysis of the rumen bacterial community in steers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Next generation sequencing technologies have vastly changed the approach of sequencing of the 16S rRNA gene for studies in microbial ecology. Three distinct technologies are available for large-scale 16S sequencing. All three are subject to biases introduced by sequencing error rates, amplificatio...

  19. RNA polymerase beta subunit (rpoB) gene and the 16S-23S rRNA intergenic transcribed spacer region (ITS) as complementary molecular markers in addition to the 16S rRNA gene for phylogenetic analysis and identification of the species of the family Mycoplasmataceae.

    PubMed

    Volokhov, Dmitriy V; Simonyan, Vahan; Davidson, Maureen K; Chizhikov, Vladimir E

    2012-01-01

    Conventional classification of the species in the family Mycoplasmataceae is mainly based on phenotypic criteria, which are complicated, can be difficult to measure, and have the potential to be hampered by phenotypic deviations among the isolates. The number of biochemical reactions suitable for phenotypic characterization of the Mycoplasmataceae is also very limited and therefore the strategy for the final identification of the Mycoplasmataceae species is based on comparative serological results. However, serological testing of the Mycoplasmataceae species requires a performance panel of hyperimmune sera which contains anti-serum to each known species of the family, a high level of technical expertise, and can only be properly performed by mycoplasma-reference laboratories. In addition, the existence of uncultivated and fastidious Mycoplasmataceae species/isolates in clinical materials significantly complicates, or even makes impossible, the application of conventional bacteriological tests. The analysis of available genetic markers is an additional approach for the primary identification and phylogenetic classification of cultivable species and uncultivable or fastidious organisms in standard microbiological laboratories. The partial nucleotide sequences of the RNA polymerase β-subunit gene (rpoB) and the 16S-23S rRNA intergenic transcribed spacer (ITS) were determined for all known type strains and the available non-type strains of the Mycoplasmataceae species. In addition to the available 16S rRNA gene data, the ITS and rpoB sequences were used to infer phylogenetic relationships among these species and to enable identification of the Mycoplasmataceae isolates to the species level. The comparison of the ITS and rpoB phylogenetic trees with the 16S rRNA reference phylogenetic tree revealed a similar clustering patterns for the Mycoplasmataceae species, with minor discrepancies for a few species that demonstrated higher divergence of their ITS and rpoB in

  20. Selective Phylogenetic Analysis Targeted at 16S rRNA Genes of Thermophiles and Hyperthermophiles in Deep-Subsurface Geothermal Environments

    PubMed Central

    Kimura, Hiroyuki; Sugihara, Maki; Kato, Kenji; Hanada, Satoshi

    2006-01-01

    Deep-subsurface samples obtained by deep drilling are likely to be contaminated with mesophilic microorganisms in the drilling fluid, and this could affect determination of the community structure of the geothermal microflora using 16S rRNA gene clone library analysis. To eliminate possible contamination by PCR-amplified 16S rRNA genes from mesophiles, a combined thermal denaturation and enzyme digestion method, based on a strong correlation between the G+C content of the 16S rRNA gene and the optimum growth temperatures of most known prokaryotic cultures, was used prior to clone library construction. To validate this technique, hot spring fluid (76°C) and river water (14°C) were used to mimic a deep-subsurface sample contaminated with drilling fluid. After DNA extraction and PCR amplification of the 16S rRNA genes from individual samples separately, the amplified products from river water were observed to be denatured at 82°C and completely digested by exonuclease I (Exo I), while the amplified products from hot spring fluid remained intact after denaturation at 84°C and enzyme digestion with Exo I. DNAs extracted from the two samples were mixed and used as a template for amplification of the 16S rRNA genes. The amplified rRNA genes were denatured at 84°C and digested with Exo I before clone library construction. The results indicated that the 16S rRNA gene sequences from the river water were almost completely eliminated, whereas those from the hot spring fluid remained. PMID:16391020

  1. Selective phylogenetic analysis targeted at 16S rRNA genes of thermophiles and hyperthermophiles in deep-subsurface geothermal environments.

    PubMed

    Kimura, Hiroyuki; Sugihara, Maki; Kato, Kenji; Hanada, Satoshi

    2006-01-01

    Deep-subsurface samples obtained by deep drilling are likely to be contaminated with mesophilic microorganisms in the drilling fluid, and this could affect determination of the community structure of the geothermal microflora using 16S rRNA gene clone library analysis. To eliminate possible contamination by PCR-amplified 16S rRNA genes from mesophiles, a combined thermal denaturation and enzyme digestion method, based on a strong correlation between the G+C content of the 16S rRNA gene and the optimum growth temperatures of most known prokaryotic cultures, was used prior to clone library construction. To validate this technique, hot spring fluid (76 degrees C) and river water (14 degrees C) were used to mimic a deep-subsurface sample contaminated with drilling fluid. After DNA extraction and PCR amplification of the 16S rRNA genes from individual samples separately, the amplified products from river water were observed to be denatured at 82 degrees C and completely digested by exonuclease I (Exo I), while the amplified products from hot spring fluid remained intact after denaturation at 84 degrees C and enzyme digestion with Exo I. DNAs extracted from the two samples were mixed and used as a template for amplification of the 16S rRNA genes. The amplified rRNA genes were denatured at 84 degrees C and digested with Exo I before clone library construction. The results indicated that the 16S rRNA gene sequences from the river water were almost completely eliminated, whereas those from the hot spring fluid remained.

  2. PHYLOGENETIC ANALYSIS OF 16S RRNA GENE SEQUENCES REVEALS THE PREVALENCE OF MYCOBACTERIA SP., ALPHA-PROTEOBACTERIA, AND UNCULTURED BACTERIA IN DRINKING WATER MICROBIAL COMMUNITIES

    EPA Science Inventory

    Previous studies have shown that culture-based methods tend to underestimate the densities and diversity of bacterial populations inhabiting water distribution systems (WDS). In this study, the phylogenetic diversity of drinking water bacteria was assessed using sequence analysis...

  3. Use of 16S rRNA, 23S rRNA, and gyrB Gene Sequence Analysis To Determine Phylogenetic Relationships of Bacillus cereus Group Microorganisms

    PubMed Central

    Bavykin, Sergei G.; Lysov, Yuri P.; Zakhariev, Vladimir; Kelly, John J.; Jackman, Joany; Stahl, David A.; Cherni, Alexey

    2004-01-01

    In order to determine if variations in rRNA sequence could be used for discrimination of the members of the Bacillus cereus group, we analyzed 183 16S rRNA and 74 23S rRNA sequences for all species in the B. cereus group. We also analyzed 30 gyrB sequences for B. cereus group strains with published 16S rRNA sequences. Our findings indicated that the three most common species of the B. cereus group, B. cereus, Bacillus thuringiensis, and Bacillus mycoides, were each heterogeneous in all three gene sequences, while all analyzed strains of Bacillus anthracis were found to be homogeneous. Based on analysis of 16S and 23S rRNA sequence variations, the microorganisms within the B. cereus group were divided into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, and these seven subgroups were further organized into two distinct clusters. This classification of the B. cereus group conflicts with current taxonomic groupings, which are based on phenotypic traits. The presence of B. cereus strains in six of the seven subgroups and the presence of B. thuringiensis strains in three of the subgroups do not support the proposed unification of B. cereus and B. thuringiensis into one species. Analysis of the available phenotypic data for the strains included in this study revealed phenotypic traits that may be characteristic of several of the subgroups. Finally, our results demonstrated that rRNA and gyrB sequences may be used for discriminating B. anthracis from other microorganisms in the B. cereus group. PMID:15297521

  4. Use of 16S rRNA, 23S rRNA, and gyrB gene sequence analysis to determine phylogenetic relationships of Bacillus cereus group.

    SciTech Connect

    Bayvkin, S. G.; Lysov, Y. P.; Zakhariev, V.; Kelly, J. J.; Jackman, J.; Stahl, D. A.; Cherni, A.; Engelhardt Inst. of Molecular Biology; Loyola Univ.; Johns Hopkins Univ.; Univ. of Washington

    2004-08-01

    In order to determine if variations in rRNA sequence could be used for discrimination of the members of the Bacillus cereus group, we analyzed 183 16S rRNA and 74 23S rRNA sequences for all species in the B. cereus group. We also analyzed 30 gyrB sequences for B. cereus group strains with published 16S rRNA sequences. Our findings indicated that the three most common species of the B. cereus group, B. cereus, Bacillus thuringiensis, and Bacillus mycoides, were each heterogeneous in all three gene sequences, while all analyzed strains of Bacillus anthracis were found to be homogeneous. Based on analysis of 16S and 23S rRNA sequence variations, the microorganisms within the B. cereus group were divided into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, and these seven subgroups were further organized into two distinct clusters. This classification of the B. cereus group conflicts with current taxonomic groupings, which are based on phenotypic traits. The presence of B. cereus strains in six of the seven subgroups and the presence of B. thuringiensis strains in three of the subgroups do not support the proposed unification of B. cereus and B. thuringiensis into one species. Analysis of the available phenotypic data for the strains included in this study revealed phenotypic traits that may be characteristic of several of the subgroups. Finally, our results demonstrated that rRNA and gyrB sequences may be used for discriminating B. anthracis from other microorganisms in the B. cereus group.

  5. Identification and Phylogenetic analysis of thermophilic sulfate-reducing bacteria in oil field samples by 16S rDNA gene cloning and sequencing.

    PubMed

    Leu, J Y; McGovern-Traa, C P; Porter, A J; Harris, W J; Hamilton, W A

    1998-06-01

    Thermophilic sulfate-reducing bacteria (SRB) have been recognized as an important source of hydrogen sulfide (H2S) in hydrocarbon reservoirs and in production systems. Four thermophilic SRB enrichment cultures from three different oil field samples (sandstone core, drilling mud, and production water) were investigated using 16S rDNA sequence comparative analysis. In total, 15 different clones were identified. We found spore-forming, low G+C content, thermophilic, sulfate-reducing Desulfotomaculum-related sequences present in all oil field samples, and additionally a clone originating from sandstone core which was assigned to the mesophilic Desulfomicrobium group. Furthermore, three clones related to Gram-positive, non-sulfate-reducing Thermoanaerobacter species and four clones close to Clostridium thermocopriae were found in enrichment cultures from sandstone core and from production water, respectively. In addition, the deeply rooted lineage of two of the clones suggested previously undescribed, Gram-positive, low G+C content, thermophilic, obligately anaerobic bacteria present in production water. Such thermophilic, non-sulfate-reducing microorganisms may play an important ecological role alongside SRB in oil field environments.

  6. Phylogenetic analysis of the fecal microbial community in herbivorous land and marine iguanas of the Galápagos Islands using 16S rRNA-based pyrosequencing.

    PubMed

    Hong, Pei-Ying; Wheeler, Emily; Cann, Isaac K O; Mackie, Roderick I

    2011-09-01

    Herbivorous reptiles depend on complex gut microbial communities to effectively degrade dietary polysaccharides. The composition of these fermentative communities may vary based on dietary differences. To explore the role of diet in shaping gut microbial communities, we evaluated the fecal samples from two related host species--the algae-consuming marine iguana (Amblyrhynchus cristatus) and land iguanas (LI) (genus Conolophus) that consume terrestrial vegetation. Marine and LI fecal samples were collected from different islands in the Galápagos archipelago. High-throughput 16S rRNA-based pyrosequencing was used to provide a comparative analysis of fecal microbial diversity. At the phylum level, the fecal microbial community in iguanas was predominated by Firmicutes (69.5±7.9%) and Bacteroidetes (6.2±2.8%), as well as unclassified Bacteria (20.6±8.6%), suggesting that a large portion of iguana fecal microbiota is novel and could be involved in currently unknown functions. Host species differed in the abundance of specific bacterial groups. Bacteroides spp., Lachnospiraceae and Clostridiaceae were significantly more abundant in the marine iguanas (MI) (P-value>1E-9). In contrast, Ruminococcaceae were present at >5-fold higher abundance in the LI than MI (P-value>6E-14). Archaea were only detected in the LI. The number of operational taxonomic units (OTUs) in the LI (356-896 OTUs) was >2-fold higher than in the MI (112-567 OTUs), and this increase in OTU diversity could be related to the complexity of the resident bacterial population and their gene repertoire required to breakdown the recalcitrant polysaccharides prevalent in terrestrial plants. Our findings suggest that dietary differences contribute to gut microbial community differentiation in herbivorous lizards. Most importantly, this study provides a better understanding of the microbial diversity in the iguana gut; therefore facilitating future efforts to discover novel bacterial-associated enzymes that

  7. Phylogenetic position of Rhizobium sp. strain Or 191, a symbiont of both Medicago sativa and Phaseolus vulgaris, based on partial sequences of the 16S rRNA and nifH genes.

    PubMed Central

    Eardly, B D; Young, J P; Selander, R K

    1992-01-01

    Phenotypic and DNA sequence comparisons are presented for eight Rhizobium isolates that were cultured from field-grown alfalfa (Medicago sativa L.) in Oregon. These isolates were previously shown to nodulate both alfalfa and common bean (Phaseolus vulgaris (L.) Savi.). The objective of the present study was to determine their phylogenetic relationships to the normal symbionts of these plants, Rhizobium meliloti and Rhizobium leguminosarum biovar phaseoli, respectively. Phenotypically, the Oregon isolates more nearly resemble strains from P. vulgaris than those from M. sativa. For example, even though nitrogen fixation levels were low with both host species, the symbiotic efficiency of a representative Rhizobium isolate (Or 191) with common bean was twice that observed with alfalfa. Comparative sequencing of a 260-bp segment of the 16S rRNA gene (directly sequenced after amplification by the polymerase chain reaction) demonstrated that Or 191 is not closely related to the type strain of R. meliloti (ATCC 9930), R. leguminosarum (ATCC 10004), or Rhizobium tropici (CIAT 899). Instead, sequence comparisons of the 16S gene indicated that Or 191 belongs to a distinct and previously unrecognized taxonomic group that includes strains that have previously been called R. leguminosarum bv. phaseoli type I. Unlike type I strains, however, Or 191 has only a single copy of the nifH gene (type I strains have three), and the nucleotide sequence of this gene is substantially different from those of other rhizobial and nonrhizobial nifH genes examined thus far. Images PMID:1377901

  8. The feline oral microbiome: a provisional 16S rRNA gene based taxonomy with full-length reference sequences.

    PubMed

    Dewhirst, Floyd E; Klein, Erin A; Bennett, Marie-Louise; Croft, Julie M; Harris, Stephen J; Marshall-Jones, Zoe V

    2015-02-25

    The human oral microbiome is known to play a significant role in human health and disease. While less well studied, the feline oral microbiome is thought to play a similarly important role. To determine roles oral bacteria play in health and disease, one first has to be able to accurately identify bacterial species present. 16S rRNA gene sequence information is widely used for molecular identification of bacteria and is also useful for establishing the taxonomy of novel species. The objective of this research was to obtain full 16S rRNA gene reference sequences for feline oral bacteria, place the sequences in species-level phylotypes, and create a curated 16S rRNA based taxonomy for common feline oral bacteria. Clone libraries were produced using "universal" and phylum-selective PCR primers and DNA from pooled subgingival plaque from healthy and periodontally diseased cats. Bacteria in subgingival samples were also cultivated to obtain isolates. Full-length 16S rDNA sequences were determined for clones and isolates that represent 171 feline oral taxa. A provisional curated taxonomy was developed based on the position of each taxon in 16S rRNA phylogenetic trees. The feline oral microbiome curated taxonomy and 16S rRNA gene reference set will allow investigators to refer to precisely defined bacterial taxa. A provisional name such as "Propionibacterium sp. feline oral taxon FOT-327" is an anchor to which clone, strain or GenBank names or accession numbers can point. Future next-generation-sequencing studies of feline oral bacteria will be able to map reads to taxonomically curated full-length 16S rRNA gene sequences.

  9. Collection of small subunit (16S- and 16S-like) ribosomal RNA structures: 1994.

    PubMed Central

    Gutell, R R

    1994-01-01

    A collection of diverse 16S and 16S-like rRNA secondary structure diagrams are available. This set of rRNAs contains representative structures from all of the major phylogenetic groupings--Archaea, (eu)Bacteria, and the nucleus, mitochondrion, and chloroplast of Eucarya. Within this broad phylogenetic sampling are examples of the major forms of structural diversity currently known for this class of rRNAs. These structure diagrams are available online through our computer-network WWW server and anonymous ftp, as well as from the author in hardcopy format. PMID:7524024

  10. 16S rRNA based microarray analysis of ten periodontal bacteria in patients with different forms of periodontitis.

    PubMed

    Topcuoglu, Nursen; Kulekci, Guven

    2015-10-01

    DNA microarray analysis is a computer based technology, that a reverse capture, which targets 10 periodontal bacteria (ParoCheck) is available for rapid semi-quantitative determination. The aim of this three-year retrospective study was to display the microarray analysis results for the subgingival biofilm samples taken from patient cases diagnosed with different forms of periodontitis. A total of 84 patients with generalized aggressive periodontitis (GAP,n:29), generalized chronic periodontitis (GCP, n:25), peri-implantitis (PI,n:14), localized aggressive periodontitis (LAP,n:8) and refractory chronic periodontitis (RP,n:8) were consecutively selected from the archives of the Oral Microbiological Diagnostic Laboratory. The subgingival biofilm samples were analyzed by the microarray-based identification of 10 selected species. All the tested species were detected in the samples. The red complex bacteria were the most prevalent with very high levels in all groups. Fusobacterium nucleatum was detected in all samples at high levels. The green and blue complex bacteria were less prevalent compared with red and orange complex, except Aggregatibacter actinomycetemcomitas was detected in all LAP group. Positive correlations were found within all the red complex bacteria and between red and orange complex bacteria especially in GCP and GAP groups. Parocheck enables to monitoring of periodontal pathogens in all forms of periodontal disease and can be alternative to other guiding and reliable microbiologic tests.

  11. Phylogenetic diversity of rhizobia associated with horsegram [Macrotyloma uniflorum (Lam.) Verdc.] grown in South India based on glnII, recA and 16S-23S intergenic sequence analyses.

    PubMed

    Appunu, Chinnaswamy; Ganesan, Govindan; Kalita, Michał; Kaushik, Raghavan; Saranya, Balamurugan; Prabavathy, Vaiyapuri Ramalingam; Sudha, Nair

    2011-04-01

    Horsegram [Macrotyloma uniflorum (Lam.) Verdc.) is an important grain legume and fodder crop in India. Information on root nodule endosymbionts of this legume in India is limited. In the present study, 69 isolates from naturally occurring root nodules of horsegram collected from two agro-eco-climatic regions of South India was analyzed by generation rate, acid/alkali reaction on YMA medium, restriction fragment length polymorphism analysis of 16S-23S rDNA intergenic spacer region (IGS), and sequence analyses of IGS and housekeeping genes glnII and recA. Based on the rDNA IGS RFLP by means of three restriction enzymes rhizobia were grouped in five clusters (I-V). By sequence analysis of 16S-23S rDNA IGS identified genotypes of horsegram rhizobia were distributed into five divergent lineages of Bradyrhizobium genus which comprised (I) the IGS type IV rhizobia and valid species B. yuanmingense, (II) the strains of IGS type I and Bradyrhizobium sp. ORS 3257 isolated from Vigna sp., (III) the strains of the IGS type II and Bradyrhizobium sp. CIRADAc12 from Acacia sp., (IV) the IGS type V strains and Bradyrhizobium sp. genospecies IV, and (V) comprising genetically distinct IGS type III strains which probably represent an uncharacterized new genomic species. Nearly, 87% of indigenous horsegram isolates (IGS types I, II, III, and V) could not be related to any other species within the genus Bradyrhizobium. Phylogeny based on housekeeping glnII and recA genes confirmed those results found by the analysis of the IGS sequence. All the isolated rhizobia nodulated Macrotyloma sp. and Vigna spp., and only some of them formed nodules on Arachis hypogeae. The isolates within each IGS type varied in their ability to fix nitrogen. Selection for high symbiotic effective strains could reward horsegram production in poor soils of South India where this legume is largely cultivated.

  12. 16S rRNA Phylogeny of Sponge-Associated Cyanobacteria

    PubMed Central

    Steindler, Laura; Huchon, Dorothée; Avni, Adi; Ilan, Micha

    2005-01-01

    Phylogenetic analyses of 16S rRNA sequences of sponge-associated cyanobacteria showed them to be polyphyletic, implying that they derived from multiple independent symbiotic events. Most of the symbiont sequences were affiliated to a group of Synechococcus and Prochlorococcus species. However, other symbionts were related to different groups, such as the Oscillatoriales. PMID:16000832

  13. Intrageneric structure of the genus Gluconobacter analyzed by the 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer sequences.

    PubMed

    Takahashi, Mai; Yukphan, Pattaraporn; Yamada, Yuzo; Suzuki, Ken-ichiro; Sakane, Takeshi; Nakagawa, Yasuyoshi

    2006-06-01

    Forty-nine strains belonging to the genus Gluconobacter were re-examined with respect to their species identification based on the sequences of the 16S rDNA and 16S-23S rDNA internal transcribed spacer regions (ITS). A phylogenetic tree constructed from the 16S rDNA sequences indicated the presence of five clusters corresponding, respectively, to the major five species of the genus Gluconobacter, namely G. albidus, G. cerinus, G. frateurii, G. oxydans (type species), and G. thailandicus. The type strain of G. asaii, NBRC 3276T (T=type strain) was included in the G. cerinus cluster, which is consistent with the report that G. asaii is a junior subjective synonym of G. cerinus. Existence of the G. albidus, G. cerinus, G. frateurii, G. oxydans, and G. thailandicus clusters was also recognized by the ITS sequence analysis. Both sequence analyses revealed that the G. cerinus and G. frateurii clusters were heterogeneous. The G. cerinus cluster comprised three strains of G. cerinus and one strain of G. frateurii, while the G. frateurii cluster included ten strains of G. frateurii, three of G. cerinus, and eleven of G. oxydans. These results suggest that phenotypic differences among Gluconobacter species are ambiguous and the species definition must be re-evaluated. The 16S rDNA and ITS sequences determined in this study are valuable for the identification and phylogenetic analysis of Gluconobacter species.

  14. [16S rRNA gene sequence analysis for bacterial identification in the clinical laboratory].

    PubMed

    Matsumoto, Takehisa; Sugano, Mitsutoshi

    2013-12-01

    The traditional identification of bacteria on the basis of phenotypic characteristics is generally not as accurate as identification based on genotypic methods. For many years, sequencing of the 16S ribosomal RNA (rRNA) gene has served as an important tool for determining phylogenetic relationships between bacteria. The features of this molecular target that make it a useful phylogenetic tool also make it useful for bacterial detection and identification in the clinical laboratory. 16S rRNA gene sequence analysis can better identify poorly described, rarely isolated, or phenotypically aberrant strains, and can lead to the recognition of novel pathogens and noncultured bacteria. In clinical microbiology, molecular identification based on 16S rDNA sequencing is applied fundamentally to bacteria whose identification by means of other types of techniques is impossible or difficult. However, there are some cases in which 16S rRNA gene sequence analysis can not differentiate closely related bacteria such as Shigella spp. and Escherichia coli at the species level. Thus, it is important to understand the advantages and disadvantages of 16S rRNA gene sequence analysis.

  15. Two different 16S rRNA genes in a mycobacterial strain.

    PubMed Central

    Ninet, B; Monod, M; Emler, S; Pawlowski, J; Metral, C; Rohner, P; Auckenthaler, R; Hirschel, B

    1996-01-01

    Sequencing of the gene coding for 16S rRNA (16S rDNA) is a well-established method used to identify bacteria, particularly mycobacteria. Unique sequences allow identification of a particular genus and species. If more than one 16S rDNA is present on one mycobacterial genome, their sequences are assumed to be strictly or almost identical. We have isolated a slowly growing Mycobacterium strain, "X", identified by conventional biochemical tests as Mycobacterium terrae. Identification by amplification and direct sequencing of 16S rDNA yielded ambiguous results in two variable regions, suggesting the presence of different copies of the sequenced gene. Total DNA was digested by restriction enzymes and hybridized after Southern blotting to a probe representing about two-thirds of the 16S rDNA. Two copies of 16S rDNA were identified and cloned. By sequencing, the clones were of two different types, A and B, differing in 18 positions. Oligonucleotides specific to each copy of the 16S rDNA were used to distinguish the positions of the two genes observed in the Southern blot. We conclude that Mycobacterium strain "X" has two different copies of 16S rDNA. Variations in the sequence between two copies of 16S rDNA gene have been described in archaeobacteria, but not in mycobacteria. When placed in a phylogenetic tree together with other slowly growing mycobacteria gene A shows a common root with M. terrae, whereas gene B is placed separately. PMID:8880515

  16. Incorporating 16S gene copy number information improves estimates of microbial diversity and abundance.

    PubMed

    Kembel, Steven W; Wu, Martin; Eisen, Jonathan A; Green, Jessica L

    2012-01-01

    The abundance of different SSU rRNA ("16S") gene sequences in environmental samples is widely used in studies of microbial ecology as a measure of microbial community structure and diversity. However, the genomic copy number of the 16S gene varies greatly - from one in many species to up to 15 in some bacteria and to hundreds in some microbial eukaryotes. As a result of this variation the relative abundance of 16S genes in environmental samples can be attributed both to variation in the relative abundance of different organisms, and to variation in genomic 16S copy number among those organisms. Despite this fact, many studies assume that the abundance of 16S gene sequences is a surrogate measure of the relative abundance of the organisms containing those sequences. Here we present a method that uses data on sequences and genomic copy number of 16S genes along with phylogenetic placement and ancestral state estimation to estimate organismal abundances from environmental DNA sequence data. We use theory and simulations to demonstrate that 16S genomic copy number can be accurately estimated from the short reads typically obtained from high-throughput environmental sequencing of the 16S gene, and that organismal abundances in microbial communities are more strongly correlated with estimated abundances obtained from our method than with gene abundances. We re-analyze several published empirical data sets and demonstrate that the use of gene abundance versus estimated organismal abundance can lead to different inferences about community diversity and structure and the identity of the dominant taxa in microbial communities. Our approach will allow microbial ecologists to make more accurate inferences about microbial diversity and abundance based on 16S sequence data.

  17. Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence.

    PubMed

    Hao, Huijing; Liang, Junrong; Duan, Ran; Chen, Yuhuang; Liu, Chang; Xiao, Yuchun; Li, Xu; Su, Mingming; Jing, Huaiqi; Wang, Xin

    2016-01-01

    API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method.

  18. Aminoglycoside antibiotics: A-site specific binding to 16S

    NASA Astrophysics Data System (ADS)

    Baker, Erin Shammel; Dupuis, Nicholas F.; Bowers, Michael T.

    2009-06-01

    The A-site of 16S rRNA, which is a part of the 30S ribosomal subunit involved in prokaryotic translation, is a well known aminoglycoside binding site. Full characterization of the conformational changes undergone at the A-site upon aminoglycoside binding is essential for development of future RNA/drug complexes; however, the massiveness of 16S makes this very difficult. Recently, studies have found that a 27 base RNA construct (16S27) that comprises the A-site subdomain of 16S behaves similarly to the whole A-site domain. ESI-MS, ion mobility and molecular dynamics methods were utilized in this study to analyze the A-site of 16S27 before and after the addition of ribostamycin (R), paromomycin (P) and lividomycin (L). The ESI mass spectrum for 16S27 alone illustrated both single-stranded 16S27 and double-stranded (16S27)2 complexes. Upon aminoglycoside addition, the mass spectra showed that only one aminoglycoside binds to 16S27, while either one or two bind to (16S27)2. Ion mobility measurements and molecular dynamics calculations were utilized in determining the solvent-free structures of the 16S27 and (16S27)2 complexes. These studies found 16S27 in a hairpin conformation while (16S27)2 existed as a cruciform. Only one aminoglycoside binds to the single A-site of the 16S27 hairpin and this attachment compresses the hairpin. Since two A-sites exist for the (16S27)2 cruciform, either one or two aminoglycosides may bind. The aminoglycosides compress the A-sites causing the cruciform with just one aminoglycoside bound to be larger than the cruciform with two bound. Non-specific binding was not observed in any of the aminoglycoside/16S27 complexes.

  19. Identification of characteristic oligonucleotides in the bacterial 16S ribosomal RNA sequence dataset

    NASA Technical Reports Server (NTRS)

    Zhang, Zhengdong; Willson, Richard C.; Fox, George E.

    2002-01-01

    MOTIVATION: The phylogenetic structure of the bacterial world has been intensively studied by comparing sequences of 16S ribosomal RNA (16S rRNA). This database of sequences is now widely used to design probes for the detection of specific bacteria or groups of bacteria one at a time. The success of such methods reflects the fact that there are local sequence segments that are highly characteristic of particular organisms or groups of organisms. It is not clear, however, the extent to which such signature sequences exist in the 16S rRNA dataset. A better understanding of the numbers and distribution of highly informative oligonucleotide sequences may facilitate the design of hybridization arrays that can characterize the phylogenetic position of an unknown organism or serve as the basis for the development of novel approaches for use in bacterial identification. RESULTS: A computer-based algorithm that characterizes the extent to which any individual oligonucleotide sequence in 16S rRNA is characteristic of any particular bacterial grouping was developed. A measure of signature quality, Q(s), was formulated and subsequently calculated for every individual oligonucleotide sequence in the size range of 5-11 nucleotides and for 15mers with reference to each cluster and subcluster in a 929 organism representative phylogenetic tree. Subsequently, the perfect signature sequences were compared to the full set of 7322 sequences to see how common false positives were. The work completed here establishes beyond any doubt that highly characteristic oligonucleotides exist in the bacterial 16S rRNA sequence dataset in large numbers. Over 16,000 15mers were identified that might be useful as signatures. Signature oligonucleotides are available for over 80% of the nodes in the representative tree.

  20. Distinct Genetic Lineages of Bactrocera caudata (Insecta: Tephritidae) Revealed by COI and 16S DNA Sequences

    PubMed Central

    Lim, Phaik-Eem; Tan, Ji; Suana, I. Wayan; Eamsobhana, Praphathip; Yong, Hoi Sen

    2012-01-01

    The fruit fly Bactrocera caudata is a pest species of economic importance in Asia. Its larvae feed on the flowers of Cucurbitaceae such as Cucurbita moschata. To-date it is distinguished from related species based on morphological characters. Specimens of B. caudata from Peninsular Malaysia and Indonesia (Bali and Lombok) were analysed using the partial DNA sequences of cytochrome c oxidase subunit I (COI) and 16S rRNA genes. Both gene sequences revealed that B. caudata from Peninsular Malaysia was distinctly different from B. caudata of Bali and Lombok, without common haplotype between them. Phylogenetic analysis revealed two distinct clades, indicating distinct genetic lineage. The uncorrected ‘p’ distance for COI sequences between B. caudata of Malaysia-Thailand-China and B. caudata of Bali-Lombok was 5.65%, for 16S sequences from 2.76 to 2.99%, and for combined COI and 16S sequences 4.45 to 4.46%. The ‘p’ values are distinctly different from intraspecific ‘p’ distance (0–0.23%). Both the B. caudata lineages are distinctly separated from related species in the subgenus Zeugodacus – B. ascita, B. scutellata, B. ishigakiensis, B. diaphora, B. tau, B. cucurbitae, and B. depressa. Molecular phylogenetic analysis indicates that the B. caudata lineages are closely related to B. ascita sp. B, and form a clade with B. scutellata, B. ishigakiensis, B. diaphora and B. ascita sp. A. This study provides additional baseline for the phylogenetic relationships of Bactrocera fruit flies of the subgenus Zeugodacus. Both the COI and 16S genes could be useful markers for the molecular differentiation and phylogenetic analysis of tephritid fruit flies. PMID:22615962

  1. Distinct genetic lineages of Bactrocera caudata (Insecta: Tephritidae) revealed by COI and 16S DNA sequences.

    PubMed

    Lim, Phaik-Eem; Tan, Ji; Suana, I Wayan; Eamsobhana, Praphathip; Yong, Hoi Sen

    2012-01-01

    The fruit fly Bactrocera caudata is a pest species of economic importance in Asia. Its larvae feed on the flowers of Cucurbitaceae such as Cucurbita moschata. To-date it is distinguished from related species based on morphological characters. Specimens of B. caudata from Peninsular Malaysia and Indonesia (Bali and Lombok) were analysed using the partial DNA sequences of cytochrome c oxidase subunit I (COI) and 16S rRNA genes. Both gene sequences revealed that B. caudata from Peninsular Malaysia was distinctly different from B. caudata of Bali and Lombok, without common haplotype between them. Phylogenetic analysis revealed two distinct clades, indicating distinct genetic lineage. The uncorrected 'p' distance for COI sequences between B. caudata of Malaysia-Thailand-China and B. caudata of Bali-Lombok was 5.65%, for 16S sequences from 2.76 to 2.99%, and for combined COI and 16S sequences 4.45 to 4.46%. The 'p' values are distinctly different from intraspecific 'p' distance (0-0.23%). Both the B. caudata lineages are distinctly separated from related species in the subgenus Zeugodacus - B. ascita, B. scutellata, B. ishigakiensis, B. diaphora, B. tau, B. cucurbitae, and B. depressa. Molecular phylogenetic analysis indicates that the B. caudata lineages are closely related to B. ascita sp. B, and form a clade with B. scutellata, B. ishigakiensis, B. diaphora and B. ascita sp. A. This study provides additional baseline for the phylogenetic relationships of Bactrocera fruit flies of the subgenus Zeugodacus. Both the COI and 16S genes could be useful markers for the molecular differentiation and phylogenetic analysis of tephritid fruit flies.

  2. Comparative analysis of bacteria associated with different mosses by 16S rRNA and 16S rDNA sequencing.

    PubMed

    Tian, Yang; Li, Yan Hong

    2017-01-01

    To understand the differences of the bacteria associated with different mosses, a phylogenetic study of bacterial communities in three mosses was carried out based on 16S rDNA and 16S rRNA sequencing. The mosses used were Hygroamblystegium noterophilum, Entodon compressus and Grimmia montana, representing hygrophyte, shady plant and xerophyte, respectively. In total, the operational taxonomic units (OTUs), richness and diversity were different regardless of the moss species and the library level. All the examined 1183 clones were assigned to 248 OTUs, 56 genera were assigned in rDNA libraries and 23 genera were determined at the rRNA level. Proteobacteria and Bacteroidetes were considered as the most dominant phyla in all the libraries, whereas abundant Actinobacteria and Acidobacteria were detected in the rDNA library of Entodon compressus and approximately 24.7% clones were assigned to Candidate division TM7 in Grimmia montana at rRNA level. The heatmap showed the bacterial profiles derived from rRNA and rDNA were partly overlapping. However, the principle component analysis of all the profiles derived from rDNA showed sharper differences between the different mosses than that of rRNA-based profiles. This suggests that the metabolically active bacterial compositions in different mosses were more phylogenetically similar and the differences of the bacteria associated with different mosses were mainly detected at the rDNA level. Obtained results clearly demonstrate that combination of 16S rDNA and 16S rRNA sequencing is preferred approach to have a good understanding on the constitution of the microbial communities in mosses.

  3. Effects of 16S rDNA sampling on estimates of the number of endosymbiont lineages in sucking lice

    PubMed Central

    Burleigh, J. Gordon; Light, Jessica E.; Reed, David L.

    2016-01-01

    Phylogenetic trees can reveal the origins of endosymbiotic lineages of bacteria and detect patterns of co-evolution with their hosts. Although taxon sampling can greatly affect phylogenetic and co-evolutionary inference, most hypotheses of endosymbiont relationships are based on few available bacterial sequences. Here we examined how different sampling strategies of Gammaproteobacteria sequences affect estimates of the number of endosymbiont lineages in parasitic sucking lice (Insecta: Phthirapatera: Anoplura). We estimated the number of louse endosymbiont lineages using both newly obtained and previously sequenced 16S rDNA bacterial sequences and more than 42,000 16S rDNA sequences from other Gammaproteobacteria. We also performed parametric and nonparametric bootstrapping experiments to examine the effects of phylogenetic error and uncertainty on these estimates. Sampling of 16S rDNA sequences affects the estimates of endosymbiont diversity in sucking lice until we reach a threshold of genetic diversity, the size of which depends on the sampling strategy. Sampling by maximizing the diversity of 16S rDNA sequences is more efficient than randomly sampling available 16S rDNA sequences. Although simulation results validate estimates of multiple endosymbiont lineages in sucking lice, the bootstrap results suggest that the precise number of endosymbiont origins is still uncertain. PMID:27547523

  4. C16S - a Hidden Markov Model based algorithm for taxonomic classification of 16S rRNA gene sequences.

    PubMed

    Ghosh, Tarini Shankar; Gajjalla, Purnachander; Mohammed, Monzoorul Haque; Mande, Sharmila S

    2012-04-01

    Recent advances in high throughput sequencing technologies and concurrent refinements in 16S rDNA isolation techniques have facilitated the rapid extraction and sequencing of 16S rDNA content of microbial communities. The taxonomic affiliation of these 16S rDNA fragments is subsequently obtained using either BLAST-based or word frequency based approaches. However, the classification accuracy of such methods is observed to be limited in typical metagenomic scenarios, wherein a majority of organisms are hitherto unknown. In this study, we present a 16S rDNA classification algorithm, called C16S, that uses genus-specific Hidden Markov Models for taxonomic classification of 16S rDNA sequences. Results obtained using C16S have been compared with the widely used RDP classifier. The performance of C16S algorithm was observed to be consistently higher than the RDP classifier. In some scenarios, this increase in accuracy is as high as 34%. A web-server for the C16S algorithm is available at http://metagenomics.atc.tcs.com/C16S/.

  5. Molecular phylogenetics before sequences

    PubMed Central

    Ragan, Mark A; Bernard, Guillaume; Chan, Cheong Xin

    2014-01-01

    From 1971 to 1985, Carl Woese and colleagues generated oligonucleotide catalogs of 16S/18S rRNAs from more than 400 organisms. Using these incomplete and imperfect data, Carl and his colleagues developed unprecedented insights into the structure, function, and evolution of the large RNA components of the translational apparatus. They recognized a third domain of life, revealed the phylogenetic backbone of bacteria (and its limitations), delineated taxa, and explored the tempo and mode of microbial evolution. For these discoveries to have stood the test of time, oligonucleotide catalogs must carry significant phylogenetic signal; they thus bear re-examination in view of the current interest in alignment-free phylogenetics based on k-mers. Here we consider the aims, successes, and limitations of this early phase of molecular phylogenetics. We computationally generate oligonucleotide sets (e-catalogs) from 16S/18S rRNA sequences, calculate pairwise distances between them based on D2 statistics, compute distance trees, and compare their performance against alignment-based and k-mer trees. Although the catalogs themselves were superseded by full-length sequences, this stage in the development of computational molecular biology remains instructive for us today. PMID:24572375

  6. Molecular systematics of the genus Troglophilus (Rhaphidophoridae, Orthoptera) in Turkey: mitochondrial 16S rDNA evidences

    PubMed Central

    Taylan, Mehmet Sait; Russo, Claudio Di; Rampini, Mauro; Ketmaier, Valerio

    2013-01-01

    Abstract This study focuses on the evolutionary relationships among Turkish species of the cave cricket genus Troglophilus.Fifteen populations were studied for sequence variation in a fragment (543 base pairs) of the mitochondrial DNA (mtDNA) 16S rDNA gene (16S) to reconstruct their phylogenetic relationships and biogeographic history. Genetic data retrieved three main clades and at least three divergent lineages that could not be attributed to any of the taxa known for the area. Molecular time estimates suggest that the diversification of the group took place between the Messinian and the Plio-Pleistocene. PMID:23653493

  7. PHYLOGENETIC AFFILIATION OF WATER DISTRIBUTION SYSTEM BACTERIAL ISOLATES USING 16S RDNA SEQUENCE ANALYSIS

    EPA Science Inventory

    In a previously described study, only 15% of the bacterial strains isolated from a water distribution system (WDS) grown on R2A agar were identifiable using fatty acid methyl esthers (FAME) profiling. The lack of success was attributed to the use of fatty acid databases of bacter...

  8. Structure of E. coli 16S RNA elucidated by psoralen crosslinking

    SciTech Connect

    Thompson, J.F.; Hearst, J.E.

    1983-04-01

    E. coli 16S RNA in solution was photoreacted with hydroxymethyltrimethylpsoralen and long wave ultraviolet light. Positions of crosslinks were determined to high resolution by partially digesting the RNA with T/sub 1/ RNase, separating the crosslinked fragments by two-dimensional gel electrophoresis, reversing the crosslink, and sequencing the separated fragments. This method yielded the locations of crosslinks to +/-15 nucleotides. Even finer placement has been made on the basis of our knowledge of psoralen reactivity. Thirteen unique crosslinks were mapped. Seven crosslinks confirmed regions of secondary structure which had been predicted in published phylogenetic models, three crosslinks discriminated between phylogenetic models, and three proved the existence of new structures. The new structures were all long-range interactions which appear to be in dynamic equilibrium with local secondary structure. Because this technique yields direct information about the secondary structure of large RNAs, it should prove invaluable in studying the structure of other RNAs of all sizes.

  9. Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences

    PubMed Central

    Langille, Morgan G. I.; Zaneveld, Jesse; Caporaso, J. Gregory; McDonald, Daniel; Knights, Dan; Reyes, Joshua A.; Clemente, Jose C.; Burkepile, Deron E.; Vega Thurber, Rebecca L.; Knight, Rob; Beiko, Robert G.; Huttenhower, Curtis

    2013-01-01

    Profiling phylogenetic marker genes, such as the 16S rRNA gene, is a key tool for studies of microbial communities but does not provide direct evidence of a community’s functional capabilities. Here we describe PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States), a computational approach to predict the functional composition of a metagenome using marker gene data and a database of reference genomes. PICRUSt uses an extended ancestral-state reconstruction algorithm to predict which gene families are present and then combines gene families to estimate the composite metagenome. Using 16S information, PICRUSt recaptures key findings from the Human Microbiome Project and accurately predicts the abundance of gene families in host-associated and environmental communities, with quantifiable uncertainty. Our results demonstrate that phylogeny and function are sufficiently linked that this ‘predictive metagenomic’ approach should provide useful insights into the thousands of uncultivated microbial communities for which only marker gene surveys are currently available. PMID:23975157

  10. Identification and characterization of rhizospheric microbial diversity by 16S ribosomal RNA gene sequencing

    PubMed Central

    Naveed, Muhammad; Mubeen, Samavia; khan, SamiUllah; Ahmed, Iftikhar; Khalid, Nauman; Suleria, Hafiz Ansar Rasul; Bano, Asghari; Mumtaz, Abdul Samad

    2014-01-01

    In the present study, samples of rhizosphere and root nodules were collected from different areas of Pakistan to isolate plant growth promoting rhizobacteria. Identification of bacterial isolates was made by 16S rRNA gene sequence analysis and taxonomical confirmation on EzTaxon Server. The identified bacterial strains were belonged to 5 genera i.e. Ensifer, Bacillus, Pseudomona, Leclercia and Rhizobium. Phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relationship of bacterial strains with the respective genera. Based on phylogenetic analysis, some candidate novel species were also identified. The bacterial strains were also characterized for morphological, physiological, biochemical tests and glucose dehydrogenase (gdh) gene that involved in the phosphate solublization using cofactor pyrroloquinolone quinone (PQQ). Seven rhizoshperic and 3 root nodulating stains are positive for gdh gene. Furthermore, this study confirms a novel association between microbes and their hosts like field grown crops, leguminous and non-leguminous plants. It was concluded that a diverse group of bacterial population exist in the rhizosphere and root nodules that might be useful in evaluating the mechanisms behind plant microbial interactions and strains QAU-63 and QAU-68 have sequence similarity of 97 and 95% which might be declared as novel after further taxonomic characterization. PMID:25477935

  11. Phylogenetic placement of the Spirosomaceae

    NASA Technical Reports Server (NTRS)

    Woese, C. R.; Maloy, S.; Mandelco, L.; Raj, H. D.

    1990-01-01

    Comparative analysis of 16S rRNA sequences shows that the family Spirosomaceae belongs within the eubacterial phylum defined by the flavobacteria and bacteriodes. Its constituent genera, Spirosoma, Flectobacillus, and Runella form a monophyletic grouping therein. The phylogenetic assignment is based not only upon evolutionary distance analysis, but also upon sequence signatures and higher order structural synapomorphies in 16S rRNA. Another genus peripherally associated with the Spirosomaceae, Ancylobacter ("Microcyclus"), does not cluster with the flavobacteria and their relatives, but rather belongs to the alpha subdivision of the purple bacteria.

  12. Diagnosis of Bacterial Bloodstream Infections: A 16S Metagenomics Approach

    PubMed Central

    Van Puyvelde, Sandra; De Block, Tessa; Maltha, Jessica; Palpouguini, Lompo; Tahita, Marc; Tinto, Halidou; Jacobs, Jan; Deborggraeve, Stijn

    2016-01-01

    Background Bacterial bloodstream infection (bBSI) is one of the leading causes of death in critically ill patients and accurate diagnosis is therefore crucial. We here report a 16S metagenomics approach for diagnosing and understanding bBSI. Methodology/Principal Findings The proof-of-concept was delivered in 75 children (median age 15 months) with severe febrile illness in Burkina Faso. Standard blood culture and malaria testing were conducted at the time of hospital admission. 16S metagenomics testing was done retrospectively and in duplicate on the blood of all patients. Total DNA was extracted from the blood and the V3–V4 regions of the bacterial 16S rRNA genes were amplified by PCR and deep sequenced on an Illumina MiSeq sequencer. Paired reads were curated, taxonomically labeled, and filtered. Blood culture diagnosed bBSI in 12 patients, but this number increased to 22 patients when combining blood culture and 16S metagenomics results. In addition to superior sensitivity compared to standard blood culture, 16S metagenomics revealed important novel insights into the nature of bBSI. Patients with acute malaria or recovering from malaria had a 7-fold higher risk of presenting polymicrobial bloodstream infections compared to patients with no recent malaria diagnosis (p-value = 0.046). Malaria is known to affect epithelial gut function and may thus facilitate bacterial translocation from the intestinal lumen to the blood. Importantly, patients with such polymicrobial blood infections showed a 9-fold higher risk factor for not surviving their febrile illness (p-value = 0.030). Conclusions/Significance Our data demonstrate that 16S metagenomics is a powerful approach for the diagnosis and understanding of bBSI. This proof-of-concept study also showed that appropriate control samples are crucial to detect background signals due to environmental contamination. PMID:26927306

  13. Diversity and abundance of Crenarchaeota in terrestrial habitats studied by 16S RNA surveys and real time PCR.

    PubMed

    Ochsenreiter, Torsten; Selezi, Drazenka; Quaiser, Achim; Bonch-Osmolovskaya, Liza; Schleper, Christa

    2003-09-01

    Novel phylogenetic lineages of as yet uncultivated crenarchaeota have been frequently detected in low to moderate-temperature, marine and terrestrial environments. In order to gain a more comprehensive view on the distribution and diversity of Crenarchaeota in moderate habitats, we have studied 18 different terrestrial and freshwater samples by 16S rDNA-based phylogenetic surveys. In seven different soil samples of diverse geographic areas in Europe (forest, grassland, ruderal) and Asia (permafrost, ruderal) as well as in two microbial mats, we have consistently found one particular lineage of crenarchaeota. The diversity of Crenarchaeota in freshwater sediments was considerably higher with respresentative 16S rDNA sequences distributed over four different groups within the moderate crenarchaeota. Systematic analysis of a 16S rDNA universal library from a sandy ecosystem containing 800 clones exclusively revealed the presence of the soil-specific crenarchaeotal cluster. With primers specific for non-thermophilic crenarchaeota we established a rapid method to quantify archaeal 16S rDNA in real time PCR. The relative abundance of crenarchaeotal rDNA was 0.5-3% in the bulk soil sample and only 0.16% in the rhizosphere of the sandy ecosystem. A nearby agricultural setting yielded a relative abundance of 0.17% crenarchaeotal rDNA. In total our data suggest that soil crenarchaeota represent a stable and specific component of the microbiota in terrestrial habitats.

  14. [Sequence analysis of 16S rDNA gene of endosymbiont of Acanthamoeba sp. CB/S1 isolated from soil].

    PubMed

    Xuan, Ying-hua; Cui, Chun-quan; Zheng, Shan-zi

    2011-04-30

    The endosymbiont of Acanthamoeba sp. CB/SI was identified by orcein-carmine staining and 16S rDNA sequence analysis. The endosymbiont bacteria were rod-shaped and darkly stained, and irregularly localized within the cytoplasm. The length of the 16S rDNA was 1534 bp and its DNA sequence was closely related to those of Candidatus Amoebophilus asiaticus and Acanthamoeba sp. KA/E21 with 98% homology. Phylogenetic analysis showed that the endosymbiont of CB/SI, the endosymbiont of KA/E21, Candidatus Amoebophilus asiaticus, the endosymbiont of Ixodes scapularis, and the endosymbiont of Encarsia pergandiella constitute a monophyletic lineage in phylogenetic tree.

  15. Intragenomic heterogeneity of the 16S rRNA gene in strain UFO1 caused by a 100-bp insertion in helix 6

    SciTech Connect

    Allison E. Ray; Stephanie A. Connon; Peter P. Sheridan; Jeremy Gilbreath; Malcolm S. Shields; Deborah T. Newby; Yoshiko Fujita; Timothy S. Magnuson

    2010-06-01

    The determination of variation in 16S rRNA gene sequences is perhaps the most common method for assessing microbial community diversity. However, the occurrence of multiple copies of 16S rRNA genes within some organisms can bias estimates of microbial diversity. During phylogenetic characterization of a metal-transforming, fermentative bacterium (strain UFO1) isolated from the Field Research Center (FRC) in Oak Ridge, TN, we detected an apparent 16S rRNA pseudogene. The putative 16S rRNA pseudogene was first detected in clone libraries constructed with 16S rRNA genes amplified from UFO1 genomic DNA. Sequencing revealed two distinct 16S rRNA genes, with one differing from the other by a 100 bp insert near the 5’ end. Ribosomal RNA was extracted from strain UFO1 and analyzed by RT-qPCR with insert and non-insert specific primers; however, only the non-insert 16S rRNA sequence was expressed. Reverse-transcribed rRNA from strain UFO1 was also used to construct a cDNA library. Of 190 clones screened by PCR, none contained the 16S rRNA gene with the 100 bp insert. Examination of GenBank 16S rRNA gene sequences revealed that the same insert sequence was present in other clones, including those from an environmental library constructed from FRC enrichments. These findings demonstrate the existence of widely disparate copies of the 16S rRNA gene in the same species and a putative 16S rRNA pseudogene, which may confound 16S rRNA-based methods for assessments of microbial diversity in environmental samples.

  16. Phylogenetic diversity of nonmarine picocyanobacteria.

    PubMed

    Callieri, Cristiana; Coci, Manuela; Corno, Gianluca; Macek, Miroslav; Modenutti, Beatriz; Balseiro, Esteban; Bertoni, Roberto

    2013-08-01

    We studied the phylogenetic diversity of nonmarine picocyanobacteria broadening the sequence data set with 43 new sequences of the 16S rRNA gene. The sequences were derived from monoclonal strains isolated from four volcanic high-altitude athalassohaline lakes in Mexico, five glacial ultraoligotrophic North Patagonian lakes and six Italian lakes of glacial, volcanic and morenic origin. The new sequences fall into a number of both novel and previously described clades within the phylogenetic tree of 16S rRNA gene. The new cluster of Lake Nahuel Huapi (North Patagonia) forms a sister clade to the subalpine cluster II and the marine Synechococcus subcluster 5.2. Our finding of the novel clade of 'halotolerants' close to the marine subcluster 5.3 (Synechococcus RCC307) constitutes an important demonstration that euryhaline and marine strains affiliate closely. The intriguing results obtained shed new light on the importance of the nonmarine halotolerants in the phylogenesis of picocyanobacteria.

  17. A framework for establishing predictive relationships between specific bacterial 16S rRNA sequence abundances and biotransformation rates.

    PubMed

    Helbling, Damian E; Johnson, David R; Lee, Tae Kwon; Scheidegger, Andreas; Fenner, Kathrin

    2015-03-01

    The rates at which wastewater treatment plant (WWTP) microbial communities biotransform specific substrates can differ by orders of magnitude among WWTP communities. Differences in taxonomic compositions among WWTP communities may predict differences in the rates of some types of biotransformations. In this work, we present a novel framework for establishing predictive relationships between specific bacterial 16S rRNA sequence abundances and biotransformation rates. We selected ten WWTPs with substantial variation in their environmental and operational metrics and measured the in situ ammonia biotransformation rate constants in nine of them. We isolated total RNA from samples from each WWTP and analyzed 16S rRNA sequence reads. We then developed multivariate models between the measured abundances of specific bacterial 16S rRNA sequence reads and the ammonia biotransformation rate constants. We constructed model scenarios that systematically explored the effects of model regularization, model linearity and non-linearity, and aggregation of 16S rRNA sequences into operational taxonomic units (OTUs) as a function of sequence dissimilarity threshold (SDT). A large percentage (greater than 80%) of model scenarios resulted in well-performing and significant models at intermediate SDTs of 0.13-0.14 and 0.26. The 16S rRNA sequences consistently selected into the well-performing and significant models at those SDTs were classified as Nitrosomonas and Nitrospira groups. We then extend the framework by applying it to the biotransformation rate constants of ten micropollutants measured in batch reactors seeded with the ten WWTP communities. We identified phylogenetic groups that were robustly selected into all well-performing and significant models constructed with biotransformation rates of isoproturon, propachlor, ranitidine, and venlafaxine. These phylogenetic groups can be used as predictive biomarkers of WWTP microbial community activity towards these specific

  18. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development - Poster

    EPA Science Inventory

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  19. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  20. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

    The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based ...

  1. 16S-23S rDNA internal transcribed spacer regions in four Proteus species.

    PubMed

    Cao, Boyang; Wang, Min; Liu, Lei; Zhou, Zhemin; Wen, Shaoping; Rozalski, Antoni; Wang, Lei

    2009-04-01

    Proteus is a Gram-negative, facultative anaerobic bacterium. In this study, 813 Proteus 16S-23S rDNA internal transcribed spacer (ITS) sequences were determined from 46 Proteus strains, including 388 ITS from 22 P. mirabilis strains, 211 ITS from 12 P. vulgaris strains, 169 ITS from 10 P. penneri strains, and 45 ITS from 2 P. myxofaciens strains. The Proteus strains carry mainly two types of ITS, ITS(Glu) (containing tRNA(Glu (UUC)) gene) and ITS(Ile+Ala) (containing tRNA(Ile (GAU)) and tRNA(Ala (UGC)) gene), and are in the forms of 28 variants with 25 genomic origins. The ITS sequences are a mosaic-like structure consisting of three conservative regions and two variable regions. The nucleotide identity of ITS subtypes in strains of the same species ranges from 96.2% to 100%. The divergence of Proteus ITS divergence was most likely due to intraspecies recombinations or horizontal transfers of sequence blocks. The phylogenetic relationship deduced from the second variable region of ITS sequences of the three facultative human pathogenic species P. mirabilis, P. vulgaris and P. penneri is similar with that based on 16S rDNA sequences, but has higher resolution to differentiate closely related P. vulgaris and P. penneri. This study is the first comprehensive study of ITS in four Proteus species and laid solid foundation for the development of high-throughput technology for quick and accurate identification of the important foodborne and nosocomial pathogens.

  2. Identification of grass-associated and toluene-degrading diazotrophs, Axoarcus spp., by analyses of partial 16S ribosomal DNA sequences

    SciTech Connect

    Hurek, T.; Reinhold-Hurek, B.

    1995-06-01

    The genus Azoarcus includes nitrogen-fixing, grass-associated strains as well as denitrifying toluene degraders. In order to identify and group members of the genus Azoarcus, phylogenetic analysis based on partial sequences of 16S rRNA genes (16S rDNAs) is proposed. 16S rRNA-targeted PCR using specific primers to exclude amplification in the majority of other members of the beta subclass of the class Proteobacteria was combined with direct sequencing of the PCR products. Tree inference from comparisons of 446-bp rDNA fragments yielded similar results for the three known Azoarcus spp. sequences and for analysis of the complete 16S rDNA sequence. These three species formed a phylogenetically coherent group with representatives of two other Azoarcus species which were subjected to 16S rRNA sequencing in this study. This group was related to Rhodocyclus purpureus and Thaurea selenatis. New isolates and also sequences of so far uncultured bacteria from roots of Kallar grass were assigned to the genus Azoarcus as well. Also, strains degrading monoaromatic hydrocarbons anaerobically in the presence of nitrate clustered within this genus, albeit not with grass-associated isolates. All representative members of the five species harboring rhizospheric bacteria were able to form N{sub 2}O from nitrate and showed anaerobic growth on malic acid with nitrate but not on toluene. In order to visualize different Azoarcus spp. by whole-cell in situ hybridizations, we generated 16S rRNA-targeted, fluorescent probes by in vitro transcription directly from PCR products which spanned the variable region V2. Hybridization was species specific for Azoarcus communis and Azoarcus indigens. The proposed scheme of phylogenetic analysis of PCR-generated 16S rDNA segements will facilitate studies on ecological distribution, host range, and diversity of Azoarcus spp. and may even allow rapid identification of unc ultured strains from environmental DNAs. 30 refs., 3 figs.

  3. Comprehensive Analysis of Bacterial Flora in Postoperative Maxillary Cyst Fluid by 16S rRNA Gene and Culture Methods

    PubMed Central

    Sano, Naoto; Yamashita, Yoshio; Fukuda, Kazumasa; Taniguchi, Hatsumi; Goto, Masaaki; Miyamoto, Hiroshi

    2012-01-01

    Intracystic fluid was aseptically collected from 11 patients with postoperative maxillary cyst (POMC), and DNA was extracted from the POMC fluid. Bacterial species were identified by sequencing after cloning of approximately 580 bp of the 16S rRNA gene. Identification of pathogenic bacteria was also performed by culture methods. The phylogenetic identity was determined by sequencing 517–596 bp in each of the 1139 16S rRNA gene clones. A total of 1114 clones were classified while the remaining 25 clones were unclassified. A total of 103 bacterial species belonging to 42 genera were identified in POMC fluid samples by 16S rRNA gene analysis. Species of Prevotella (91%), Neisseria (73%), Fusobacterium (73%), Porphyromonas (73%), and Propionibacterium (73%) were found to be highly prevalent in all patients. Streptococcus mitis (64%), Fusobacterium nucleatum (55%), Propionibacterium acnes (55%), Staphylococcus capitis (55%), and Streptococcus salivarius (55%) were detected in more than 6 of the 11 patients. The results obtained by the culture method were different from those obtained by 16S rRNA gene analysis, but both approaches may be necessary for the identification of pathogens, especially of bacteria that are difficult to detect by culture methods, and the development of rational treatments for patients with POMC. PMID:22685668

  4. Analysis of transduction in wastewater bacterial populations by targeting the phage-derived 16S rRNA gene sequences.

    PubMed

    Del Casale, Antonio; Flanagan, Paul V; Larkin, Michael J; Allen, Christopher C R; Kulakov, Leonid A

    2011-04-01

    Bacterial 16S rRNA genes transduced by bacteriophages were identified and analyzed in order to estimate the extent of the bacteriophage-mediated horizontal gene transfer in the wastewater environment. For this purpose, phage and bacterial DNA was isolated from the oxidation tank of a municipal wastewater treatment plant. Phylogenetic analysis of the 16S rRNA gene sequences cloned from a phage metagenome revealed that bacteriophages transduce genetic material in several major groups of bacteria. The groups identified were as follows: Betaproteobacteria, Gammaproteobacteria, Alphaproteobacteria, Actinomycetales and Firmicutes. Analysis of the 16S rRNA gene sequences in the total bacterial DNA from the same sample revealed that several bacterial groups found in the oxidation tank were not present in the phage metagenome (e.g. Deltaproteobacteria, Nitrospira, Planctomycetes and many Actinobacteria genera). These results suggest that transduction in a wastewater environment occurs in several bacterial groups; however, not all species are equally involved into this process. The data also showed that a number of distinctive bacterial strains participate in transduction-mediated gene transfer within identified bacterial groupings. Denaturing gradient gel electrophoresis analysis confirmed that profiles of the transduced 16S rRNA gene sequences and those present in the whole microbial community show significant differences.

  5. Characterization of viable bacteria from Siberian permafrost by 16S rDNA sequencing

    NASA Technical Reports Server (NTRS)

    Shi, T.; Reeves, R. H.; Gilichinsky, D. A.; Friedmann, E. I.

    1997-01-01

    Viable bacteria were found in permafrost core samples from the Kolyma-Indigirka lowland of northeast Siberia. The samples were obtained at different depths; the deepest was about 3 million years old. The average temperature of the permafrost is -10 degrees C. Twenty-nine bacterial isolates were characterized by 16S rDNA sequencing and phylogenetic analysis, cell morphology, Gram staining, endospore formation, and growth at 30 degrees C. The majority of the bacterial isolates were rod shaped and grew well at 30 degrees C; but two of them did not grow at or above 28 degrees C, and had optimum growth temperatures around 20 degrees C. Thirty percent of the isolates could form endospores. Phylogenetic analysis revealed that the isolates fell into four categories: high-GC Gram-positive bacteria, beta-proteobacteria, gamma-proteobacteria, and low-GC Gram-positive bacteria. Most high-GC Gram-positive bacteria and beta-proteobacteria, and all gamma-proteobacteria, came from samples with an estimated age of 1.8-3.0 million years (Olyor suite). Most low-GC Gram-positive bacteria came from samples with an estimated age of 5,000-8,000 years (Alas suite).

  6. Comparison of the small 16S to 23S intergenic spacer region (ISR) of the rRNA operons of some Escherichia coli strains of the ECOR collection and E. coli K-12.

    PubMed

    García-Martínez, J; Martínez-Murcia, A; Antón, A I; Rodríguez-Valera, F

    1996-11-01

    Several 16S to 23S spacers of 354 bp have been sequenced from six Escherichia coli strains belonging to the ECOR collection. Four phylogenetically informative variable sites were identified. The results of their comparison confirm the existence of two major phylogenetic branches in this species, as previously reported. Remarkable intercistronic heterogeneity was found in strain ECOR35 and its closest relatives, in which at least one of the operons has suffered a major mutagenic event or has an independent phylogenetic origin.

  7. Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing.

    PubMed Central

    Schmidt, T M; DeLong, E F; Pace, N R

    1991-01-01

    The phylogenetic diversity of an oligotrophic marine picoplankton community was examined by analyzing the sequences of cloned ribosomal genes. This strategy does not rely on cultivation of the resident microorganisms. Bulk genomic DNA was isolated from picoplankton collected in the north central Pacific Ocean by tangential flow filtration. The mixed-population DNA was fragmented, size fractionated, and cloned into bacteriophage lambda. Thirty-eight clones containing 16S rRNA genes were identified in a screen of 3.2 x 10(4) recombinant phage, and portions of the rRNA gene were amplified by polymerase chain reaction and sequenced. The resulting sequences were used to establish the identities of the picoplankton by comparison with an established data base of rRNA sequences. Fifteen unique eubacterial sequences were obtained, including four from cyanobacteria and eleven from proteobacteria. A single eucaryote related to dinoflagellates was identified; no archaebacterial sequences were detected. The cyanobacterial sequences are all closely related to sequences from cultivated marine Synechococcus strains and with cyanobacterial sequences obtained from the Atlantic Ocean (Sargasso Sea). Several sequences were related to common marine isolates of the gamma subdivision of proteobacteria. In addition to sequences closely related to those of described bacteria, sequences were obtained from two phylogenetic groups of organisms that are not closely related to any known rRNA sequences from cultivated organisms. Both of these novel phylogenetic clusters are proteobacteria, one group within the alpha subdivision and the other distinct from known proteobacterial subdivisions. The rRNA sequences of the alpha-related group are nearly identical to those of some Sargasso Sea picoplankton, suggesting a global distribution of these organisms. Images PMID:2066334

  8. Avoidance and Potential Remedy Solutions of Chimeras in Reconstructing the Phylogeny of Aphids Using the 16S rRNA Gene of Buchnera: A Case in Lachninae (Hemiptera).

    PubMed

    Chen, Rui; Wang, Zhe; Chen, Jing; Qiao, Ge-Xia

    2015-08-25

    It is known that PCR amplification of highly homologous genes from complex DNA mixtures can generate a significant proportion of chimeric sequences. The 16S rRNA gene is not only widely used in estimating the species diversity of endosymbionts in aphids but also used to explore the co-diversification of aphids and their endosymbionts. Thus, chimeric sequences may lead to the discovery of non-existent endosymbiont species and mislead Buchnera-based phylogenetic analysis that lead to false conclusions. In this study, a high probability (6.49%) of chimeric sequence occurrence was found in the amplified 16S rRNA gene sequences of endosymbionts from aphid species in the subfamily Lachninae. These chimeras are hybrid products of multiple parent sequences from the dominant species of endosymbionts in each corresponding host. It is difficult to identify the chimeric sequences of a new or unidentified species due to the high variability of their main parent, Buchnera aphidicola, and because the chimeric sequences can confuse the phylogenetic analysis of 16S rRNA gene sequences. These chimeras present a challenge to Buchnera-based phylogenetic research in aphids. Thus, our study strongly suggests that using appropriate methods to detect chimeric 16S rRNA sequences may avoid some false conclusions in endosymbiont-based aphid research.

  9. A comprehensive benchmarking study of protocols and sequencing platforms for 16S rRNA community profiling

    SciTech Connect

    Podar, Mircea; Shakya, Migun; D'Amore, Rosalinda; Ijaz, Umer Zeeshan; Schirmer, Melanie; Kenny, John G.; Gregory, Richard; Darby, Alistair C.; Quince, Christopher; Hall, Neil

    2016-01-14

    In the last 5 years, the rapid pace of innovations and improvements in sequencing technologies has completely changed the landscape of metagenomic and metagenetic experiments. Therefore, it is critical to benchmark the various methodologies for interrogating the composition of microbial communities, so that we can assess their strengths and limitations. Here, the most common phylogenetic marker for microbial community diversity studies is the 16S ribosomal RNA gene and in the last 10 years the field has moved from sequencing a small number of amplicons and samples to more complex studies where thousands of samples and multiple different gene regions are interrogated.

  10. A comprehensive benchmarking study of protocols and sequencing platforms for 16S rRNA community profiling

    DOE PAGES

    Podar, Mircea; Shakya, Migun; D'Amore, Rosalinda; ...

    2016-01-14

    In the last 5 years, the rapid pace of innovations and improvements in sequencing technologies has completely changed the landscape of metagenomic and metagenetic experiments. Therefore, it is critical to benchmark the various methodologies for interrogating the composition of microbial communities, so that we can assess their strengths and limitations. Here, the most common phylogenetic marker for microbial community diversity studies is the 16S ribosomal RNA gene and in the last 10 years the field has moved from sequencing a small number of amplicons and samples to more complex studies where thousands of samples and multiple different gene regions aremore » interrogated.« less

  11. Plastid 16S rRNA gene diversity among eukaryotic picophytoplankton sorted by flow cytometry from the South Pacific Ocean.

    PubMed

    Shi, Xiao Li; Lepère, Cécile; Scanlan, David J; Vaulot, Daniel

    2011-04-28

    The genetic diversity of photosynthetic picoeukaryotes was investigated in the South East Pacific Ocean. Genetic libraries of the plastid 16S rRNA gene were constructed on picoeukaryote populations sorted by flow cytometry, using two different primer sets, OXY107F/OXY1313R commonly used to amplify oxygenic organisms, and PLA491F/OXY1313R, biased towards plastids of marine algae. Surprisingly, the two sets revealed quite different photosynthetic picoeukaryote diversity patterns, which were moreover different from what we previously reported using the 18S rRNA nuclear gene as a marker. The first 16S primer set revealed many sequences related to Pelagophyceae and Dictyochophyceae, the second 16S primer set was heavily biased toward Prymnesiophyceae, while 18S sequences were dominated by Prasinophyceae, Chrysophyceae and Haptophyta. Primer mismatches with major algal lineages is probably one reason behind this discrepancy. However, other reasons, such as DNA accessibility or gene copy numbers, may be also critical. Based on plastid 16S rRNA gene sequences, the structure of photosynthetic picoeukaryotes varied along the BIOSOPE transect vertically and horizontally. In oligotrophic regions, Pelagophyceae, Chrysophyceae, and Prymnesiophyceae dominated. Pelagophyceae were prevalent at the DCM depth and Chrysophyceae at the surface. In mesotrophic regions Pelagophyceae were still important but Chlorophyta contribution increased. Phylogenetic analysis revealed a new clade of Prasinophyceae (clade 16S-IX), which seems to be restricted to hyper-oligotrophic stations. Our data suggest that a single gene marker, even as widely used as 18S rRNA, provides a biased view of eukaryotic communities and that the use of several markers is necessary to obtain a complete image.

  12. Different organisms associated with heartwater as shown by analysis of 16S ribosomal RNA gene sequences.

    PubMed

    Allsopp, M; Visser, E S; du Plessis, J L; Vogel, S W; Allsopp, B A

    1997-08-01

    Cowdria ruminantium is a rickettsial parasite which causes heartwater, a economically important disease of domestic and wild ruminants in tropical and subtropical Africa and parts of the Caribbean. Because existing diagnostic methods are unreliable, we investigated the small-subunit ribosomal RNA (srRNA) gene from heartwater-infected material to characterise the organisms present and to develop specific oligonucleotide probes for polymerase chain reaction (PCR) based diagnosis. DNA was obtained from ticks and ruminants from heartwater-free and heartwater-endemic areas from Cowdria in tissue culture. PCR was carried out using primers designed to amplify only rickettsial srRNA genes, the target region being the highly variable V1 loop. Amplicons were cloned and sequenced; 51% were C. ruminantium sequences corresponding to four genotypes, two of which were identical to previously reported C. ruminantium sequences while the other two were new. The four different Cowdria genotypes can be correlated with different phenotypes. Tissue-culture samples yielded only Cowdria genotype sequences, but an extraordinary heterogeneity of 16S sequences was obtained from field samples. In addition to Cowdria genotypes we found sequences from previously unknown Ehrlichia spp., sequences showing homology to other Rickettsiales and a variety of Pseudomonadaceae. One Ehrlichia sequence was phylogenetically closely related to Ehrlichia platys (Group II Ehrlichia) and one to Ehrlichia canis (Group III Ehrlichia). This latter sequence was from an isolate (Germishuys) made from a naturally infected sheep which, from brain smear examination and pathology, appeared to be suffering from heartwater; nevertheless no Cowdria genotype sequences were found in this isolate. In addition no Cowdria sequences were obtained from uninfected ticks. Complete 16S rRNA gene sequences were determined for two C. ruminantium genotypes and for two previously uncharacterised heartwater-associated Ehrlichia spp

  13. Microbial community of salt crystals processed from Mediterranean seawater based on 16S rRNA analysis.

    PubMed

    Baati, Houda; Guermazi, Sonda; Gharsallah, Neji; Sghir, Abdelghani; Ammar, Emna

    2010-01-01

    Phylogenetic analysis of 16S rRNA was used to investigate for the first time the structure of the microbial community that inhabits salt crystals retrieved from the bottom of a solar saltern, located in the coastal area of the Mediterranean Sea (Sfax, Tunisia). This community lives in an extremely salty environment of 250-310 g/L total dissolved salt. A total of 78 bacterial 16S rRNA clone sequences making up to 21 operational taxonomic units (OTUs), determined by the DOTUR program to 97% sequence similarity, was analyzed. These OTUs were affiliated to Bacteroidetes (71.4% of OTUs), and gamma-Proteobacteria and alpha-Proteobacteria (equally represented by 14.2% of the OTUs observed). The archaeal community composition appeared more diverse with 68 clones, resulting in 44 OTUs, all affiliated with the Euryarchaeota phylum. Of the bacterial and archaeal clones showing <97% 16S rRNA sequence identity with sequences in public databases, 47.6% and 84.1% respectively were novel clones. Both rarefaction curves and diversity measurements (Simpson, Shannon-Weaver, Chao) showed a more diverse archaeal than bacterial community at the Tunisian solar saltern pond. The analysis of an increasing clone's number may reveal additional local diversity.

  14. Microbial diversity in the sputum of a cystic fibrosis patient studied with 16S rDNA pyrosequencing.

    PubMed

    Armougom, F; Bittar, F; Stremler, N; Rolain, J-M; Robert, C; Dubus, J-C; Sarles, J; Raoult, D; La Scola, B

    2009-09-01

    Recent studies using 16S rRNA gene amplification followed by clonal Sanger sequencing in cystic fibrosis demonstrated that cultured microorganisms are only part of the infecting flora. The purpose of this paper was to compare pyrosequencing and clonal Sanger sequencing on sputum. The sputum of a patient with cystic fibrosis was analysed by culture, Sanger clone sequencing and pyrosequencing after 16S rRNA gene amplification. A total of 4,499 sequencing reads were obtained, which could be attributed to six consensus sequences, but the length of reads leads to fastidious data analysis. Compared to clonal Sanger sequencing and to cultivation results, pyrosequencing recovers greater species richness and gives a more reliable estimate of the relative abundance of bacterial species. The 16S pyrosequencing approach expands our knowledge of the microbial diversity of cystic fibrosis sputum. The current lack of phylogenetic resolution at the species level for the GS 20 sequencing reads will be overcome with the next generation of pyrosequencing apparatus.

  15. New Screening Software Shows that Most Recent Large 16S rRNA Gene Clone Libraries Contain Chimeras†

    PubMed Central

    Ashelford, Kevin E.; Chuzhanova, Nadia A.; Fry, John C.; Jones, Antonia J.; Weightman, Andrew J.

    2006-01-01

    A new computer program, called Mallard, is presented for screening entire 16S rRNA gene libraries of up to 1,000 sequences for chimeras and other artifacts. Written in the Java computer language and capable of running on all major operating systems, the program provides a novel graphical approach for visualizing phylogenetic relationships among 16S rRNA gene sequences. To illustrate its use, we analyzed most of the large libraries of cloned bacterial 16S rRNA gene sequences submitted to the public repository during 2005. Defining a large library as one containing 100 or more sequences of 1,200 bases or greater, we screened 25 of the 28 libraries and found that all but three contained substantial anomalies. Overall, 543 anomalous sequences were found. The average anomaly content per clone library was 9.0%, 4% higher than that previously estimated for the public repository overall. In addition, 90.8% of anomalies had characteristic chimeric patterns, a rise of 25.4% over that found previously. One library alone was found to contain 54 chimeras, representing 45.8% of its content. These figures far exceed previous estimates of artifacts within public repositories and further highlight the urgent need for all researchers to adequately screen their libraries prior to submission. Mallard is freely available from our website at http://www.cardiff.ac.uk/biosi/research/biosoft/. PMID:16957188

  16. Diversity of thermophiles in a Malaysian hot spring determined using 16S rRNA and shotgun metagenome sequencing.

    PubMed

    Chan, Chia Sing; Chan, Kok-Gan; Tay, Yea-Ling; Chua, Yi-Heng; Goh, Kian Mau

    2015-01-01

    The Sungai Klah (SK) hot spring is the second hottest geothermal spring in Malaysia. This hot spring is a shallow, 150-m-long, fast-flowing stream, with temperatures varying from 50 to 110°C and a pH range of 7.0-9.0. Hidden within a wooded area, the SK hot spring is continually fed by plant litter, resulting in a relatively high degree of total organic content (TOC). In this study, a sample taken from the middle of the stream was analyzed at the 16S rRNA V3-V4 region by amplicon metagenome sequencing. Over 35 phyla were detected by analyzing the 16S rRNA data. Firmicutes and Proteobacteria represented approximately 57% of the microbiome. Approximately 70% of the detected thermophiles were strict anaerobes; however, Hydrogenobacter spp., obligate chemolithotrophic thermophiles, represented one of the major taxa. Several thermophilic photosynthetic microorganisms and acidothermophiles were also detected. Most of the phyla identified by 16S rRNA were also found using the shotgun metagenome approaches. The carbon, sulfur, and nitrogen metabolism within the SK hot spring community were evaluated by shotgun metagenome sequencing, and the data revealed diversity in terms of metabolic activity and dynamics. This hot spring has a rich diversified phylogenetic community partly due to its natural environment (plant litter, high TOC, and a shallow stream) and geochemical parameters (broad temperature and pH range). It is speculated that symbiotic relationships occur between the members of the community.

  17. Comparison of 16S rDNA analysis and rep-PCR genomic fingerprinting for molecular identification of Yersinia pseudotuberculosis.

    PubMed

    Kim, Wonyong; Song, Mi-Ok; Song, Wonkeun; Kim, Ki-Jung; Chung, Sang-In; Choi, Chul-Soon; Park, Yong-Ha

    2003-01-01

    16S rDNA sequence analysis and repetitive element sequence-based PCR (rep-PCR) genomic fingerprinting were evaluated on 11 type strains of the genus Yersinia and 17 recognized serotype strains of Y. pseudotuberculosis to investigate their genetic relatedness and to establish the value of techniques for the identification of Y. pseudotuberculosis. A phylogenetic tree constructed from 16S rDNA sequences showed that the type strains of Yersinia species formed distinct clusters with the exception of Y. pestis and Y. pseudotuberculosis. Moreover, Y. pestis NCTC 5923T was found to be closely related to Y. pseudotuberculosis serotypes 1b, 3, and 7. Dendrograms generated from REP-PCR, and ERIC-PCR data revealed that members of the genus Yersinia differed from each other with the degree of similarity 62% and 58%, respectively. However, the BOX-PCR results showed that Y. pestis 5923T clustered with the Y. pseudotuberculosis group with a degree of similarity 74%. According to these findings, 16S rDNA sequence analysis was unable to reliably discriminate Y. pseudotuberculosis from Y. pestis. However, REP-PCR and especially ERIC-PCR provided an effective means of differentiating between members of the taxa.

  18. Flow Cytometry-assisted Cloning of Specific Sequence Motifs fromComplex 16S ribosomal RNA Gene Libraries.

    SciTech Connect

    Nielsen, J.L.; Schramm, A.; Bernhard, A.E.; van den Engh, G.J.; Stahl, D.A.

    2004-07-21

    A flow cytometry method was developed for rapid screeningand recovery of cloned DNA containing common sequence motifs. Thisapproach, termed fluorescence-activated cell sorting-assisted cloning,was used to recover sequences affiliated with a unique lineage within theBacteroidetes not abundant in a clone library of environmental 16S rRNAgenes. Retrieval and sequence analysis of phylogenetically informativegenes has become a standard cultivation-independent technique toinvestigate microbial diversity in nature (7, 18). Genes encoding the 16SrRNA, because of the relative ease of their selective amplification, havebeen most frequently employed for general diversity surveys (16).Environmental studies have also focused on specific subpopulationsaffiliated with a phylogenetic group or identified by genes encodingspecific metabolic functions (e.g., ammonia oxidation, sulfaterespiration, and nitrate reduction) (8,15,20). However, specificpopulations may be of low abundance (1,23), or the genes encodingspecific metabolic functions may be insufficiently conserved to providepriming sites for general PCR amplification. Three general approacheshave been used to obtain 16S rRNA sequence information from low-abundancepopulations: screening hundreds to thousands of clones in a general 16SrRNA gene library (21), flow cytometric sorting of a subpopulation ofenvironmentally derived cells labeled by fluorescent in situhybridization (FISH) (27), or selective PCR amplification using primersspecific for the subpopulation (2,23). While the first approach is simplytime-consuming and tedious, the second has been restricted to fairlylarge and strongly fluorescent cells from aquatic samples (5, 27). Thethird approach often generates fragments of only a few hundred bases dueto the limited number of specific priming sites. Partial sequenceinformation often degrades analysis, obscuring or distorting thephylogenetic placement of the new sequences (11, 20). A more robustcharacterization of environ

  19. Analysis of the unexplored features of rrs (16S rDNA) of the Genus Clostridium

    PubMed Central

    2011-01-01

    Background Bacterial taxonomy and phylogeny based on rrs (16S rDNA) sequencing is being vigorously pursued. In fact, it has been stated that novel biological findings are driven by comparison and integration of massive data sets. In spite of a large reservoir of rrs sequencing data of 1,237,963 entries, this analysis invariably needs supplementation with other genes. The need is to divide the genetic variability within a taxa or genus at their rrs phylogenetic boundaries and to discover those fundamental features, which will enable the bacteria to naturally fall within them. Within the large bacterial community, Clostridium represents a large genus of around 110 species of significant biotechnological and medical importance. Certain Clostridium strains produce some of the deadliest toxins, which cause heavy economic losses. We have targeted this genus because of its high genetic diversity, which does not allow accurate typing with the available molecular methods. Results Seven hundred sixty five rrs sequences (> 1200 nucleotides, nts) belonging to 110 Clostridium species were analyzed. On the basis of 404 rrs sequences belonging to 15 Clostridium species, we have developed species specific: (i) phylogenetic framework, (ii) signatures (30 nts) and (iii) in silico restriction enzyme (14 Type II REs) digestion patterns. These tools allowed: (i) species level identification of 95 Clostridium sp. which are presently classified up to genus level, (ii) identification of 84 novel Clostridium spp. and (iii) potential reduction in the number of Clostridium species represented by small populations. Conclusions This integrated approach is quite sensitive and can be easily extended as a molecular tool for diagnostic and taxonomic identification of any microbe of importance to food industries and health services. Since rapid and correct identification allows quicker diagnosis and consequently treatment as well, it is likely to lead to reduction in economic losses and mortality

  20. DECIPHER, a search-based approach to chimera identification for 16S rRNA sequences.

    PubMed

    Wright, Erik S; Yilmaz, L Safak; Noguera, Daniel R

    2012-02-01

    DECIPHER is a new method for finding 16S rRNA chimeric sequences by the use of a search-based approach. The method is based upon detecting short fragments that are uncommon in the phylogenetic group where a query sequence is classified but frequently found in another phylogenetic group. The algorithm was calibrated for full sequences (fs_DECIPHER) and short sequences (ss_DECIPHER) and benchmarked against WigeoN (Pintail), ChimeraSlayer, and Uchime using artificially generated chimeras. Overall, ss_DECIPHER and Uchime provided the highest chimera detection for sequences 100 to 600 nucleotides long (79% and 81%, respectively), but Uchime's performance deteriorated for longer sequences, while ss_DECIPHER maintained a high detection rate (89%). Both methods had low false-positive rates (1.3% and 1.6%). The more conservative fs_DECIPHER, benchmarked only for sequences longer than 600 nucleotides, had an overall detection rate lower than that of ss_DECIPHER (75%) but higher than those of the other programs. In addition, fs_DECIPHER had the lowest false-positive rate among all the benchmarked programs (<0.20%). DECIPHER was outperformed only by ChimeraSlayer and Uchime when chimeras were formed from closely related parents (less than 10% divergence). Given the differences in the programs, it was possible to detect over 89% of all chimeras with just the combination of ss_DECIPHER and Uchime. Using fs_DECIPHER, we detected between 1% and 2% additional chimeras in the RDP, SILVA, and Greengenes databases from which chimeras had already been removed with Pintail or Bellerophon. DECIPHER was implemented in the R programming language and is directly accessible through a webpage or by downloading the program as an R package (http://DECIPHER.cee.wisc.edu).

  1. Microbial Dark Matter: Unusual intervening sequences in 16S rRNA genes of candidate phyla from the deep subsurface

    SciTech Connect

    Jarett, Jessica; Stepanauskas, Ramunas; Kieft, Thomas; Onstott, Tullis; Woyke, Tanja

    2014-03-17

    The Microbial Dark Matter project has sequenced genomes from over 200 single cells from candidate phyla, greatly expanding our knowledge of the ecology, inferred metabolism, and evolution of these widely distributed, yet poorly understood lineages. The second phase of this project aims to sequence an additional 800 single cells from known as well as potentially novel candidate phyla derived from a variety of environments. In order to identify whole genome amplified single cells, screening based on phylogenetic placement of 16S rRNA gene sequences is being conducted. Briefly, derived 16S rRNA gene sequences are aligned to a custom version of the Greengenes reference database and added to a reference tree in ARB using parsimony. In multiple samples from deep subsurface habitats but not from other habitats, a large number of sequences proved difficult to align and therefore to place in the tree. Based on comparisons to reference sequences and structural alignments using SSU-ALIGN, many of these ?difficult? sequences appear to originate from candidate phyla, and contain intervening sequences (IVSs) within the 16S rRNA genes. These IVSs are short (39 - 79 nt) and do not appear to be self-splicing or to contain open reading frames. IVSs were found in the loop regions of stem-loop structures in several different taxonomic groups. Phylogenetic placement of sequences is strongly affected by IVSs; two out of three groups investigated were classified as different phyla after their removal. Based on data from samples screened in this project, IVSs appear to be more common in microbes occurring in deep subsurface habitats, although the reasons for this remain elusive.

  2. Differentiation of acetic acid bacteria based on sequence analysis of 16S-23S rRNA gene internal transcribed spacer sequences.

    PubMed

    González, Angel; Mas, Albert

    2011-06-30

    The 16S-23S gene internal transcribed spacer sequence of sixty-four strains belonging to different acetic acid bacteria genera were analyzed, and phylogenetic trees were generated for each genera. The topologies of the different trees were in accordance with the 16S rRNA gene trees, although the similarity percentages obtained between the species was shown to be much lower. These values suggest the usefulness of including the 16S-23S gene internal transcribed spacer region as a part of the polyphasic approach required for the further classification of acetic acid bacteria. Furthermore, the region could be a good target for primer and probe design. It has also been validated for use in the identification of unknown samples of this bacterial group from wine vinegar and fruit condiments.

  3. Leuconostoc pseudomesenteroides WCFur3 partial 16S rRNA gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study used a partial 535 base pair 16S rRNA gene sequence to identify a bacterial isolate. Fatty acid profiles are consistent with the 16S rRNA gene sequence identification of this bacterium. The isolate was obtained from a compost bin in Fort Collins, Colorado, USA. The 16S rRNA gene sequen...

  4. In silico analysis of the 16S rRNA gene of endophytic bacteria, isolated from the aerial parts and seeds of important agricultural crops.

    PubMed

    Bredow, C; Azevedo, J L; Pamphile, J A; Mangolin, C A; Rhoden, S A

    2015-08-19

    Because of human population growth, increased food production and alternatives to conventional methods of biocontrol and development of plants such as the use of endophytic bacteria and fungi are required. One of the methods used to study microorganism diversity is sequencing of the 16S rRNA gene, which has several advantages, including universality, size, and availability of databases for comparison. The objective of this study was to analyze endophytic bacterial diversity in agricultural crops using published papers, sequence databases, and phylogenetic analysis. Fourteen papers were selected in which the ribosomal 16S rRNA gene was used to identify endophytic bacteria, in important agricultural crops, such as coffee, sugar cane, beans, corn, soybean, tomatoes, and grapes, located in different geographical regions (America, Europe, and Asia). The corresponding 16S rRNA gene sequences were selected from the NCBI database, aligned using the Mega 5.2 program, and phylogenetic analysis was undertaken. The most common orders present in the analyzed cultures were Bacillales, Enterobacteriales, and Actinomycetales and the most frequently observed genera were Bacillus, Pseudomonas, and Microbacterium. Phylogenetic analysis showed that only approximately 1.56% of the total sequences were not properly grouped, demonstrating reliability in the identification of microorganisms. This study identified the main genera found in endophytic bacterial cultures from plants, providing data for future studies on improving plant agriculture, biotechnology, endophytic bacterium prospecting, and to help understand relationships between endophytic bacteria and their interactions with plants.

  5. STITCH: algorithm to splice, trim, identify, track, and capture the uniqueness of 16S rRNAs sequence pairs using public or in-house database.

    PubMed

    Zhu, Dianhui; Vaishampayan, Parag A; Venkateswaran, Kasthuri; Fox, George E

    2011-04-01

    A comparison of variable regions within the 16S rRNA gene is widely used to characterize relationships between bacteria and to identify phylogenetic affiliation of unknown bacteria. In environmental studies, polymerase chain reaction amplification of 16S rRNA followed by cloning and sequencing of numerous individual clones is an extensively used molecular method for elucidating microbial diversity. The sequencing process typically utilizes a forward and reverse primer pair to produce two partial reads (~700 to 800 base pairs each) that overlap and in total cover a large region of the full 16S rRNA sequence (~1.5 k base). In a typical application, this approach rapidly generates very large numbers of 16S rRNA datasets that can overwhelm manual processing efforts leading to both delays and errors. In particular, the approach presents two computational challenges: (1) the assembly of a composite sequence from the two partial reads and (2) the subsequent appropriate identification of the organism represented by the newly sequenced clones. Herein, we describe a software package, search, trim, identify, track, and capture the uniqueness of 16S rRNAs using public and in-house database (STITCH), which offers automated sequence pair splicing and genetic identification, thus simplifying the computationally intensive analysis of large sequencing libraries. The STITCH software is freely accessible over the Internet at: http://prion.bchs.uh.edu/stitch/.

  6. High-resolution phylogenetic microbial community profiling

    SciTech Connect

    Singer, Esther; Coleman-Derr, Devin; Bowman, Brett; Schwientek, Patrick; Clum, Alicia; Copeland, Alex; Ciobanu, Doina; Cheng, Jan-Fang; Gies, Esther; Hallam, Steve; Tringe, Susannah; Woyke, Tanja

    2014-03-17

    The representation of bacterial and archaeal genome sequences is strongly biased towards cultivated organisms, which belong to merely four phylogenetic groups. Functional information and inter-phylum level relationships are still largely underexplored for candidate phyla, which are often referred to as microbial dark matter. Furthermore, a large portion of the 16S rRNA gene records in the GenBank database are labeled as environmental samples and unclassified, which is in part due to low read accuracy, potential chimeric sequences produced during PCR amplifications and the low resolution of short amplicons. In order to improve the phylogenetic classification of novel species and advance our knowledge of the ecosystem function of uncultivated microorganisms, high-throughput full length 16S rRNA gene sequencing methodologies with reduced biases are needed. We evaluated the performance of PacBio single-molecule real-time (SMRT) sequencing in high-resolution phylogenetic microbial community profiling. For this purpose, we compared PacBio and Illumina metagenomic shotgun and 16S rRNA gene sequencing of a mock community as well as of an environmental sample from Sakinaw Lake, British Columbia. Sakinaw Lake is known to contain a large age of microbial species from candidate phyla. Sequencing results show that community structure based on PacBio shotgun and 16S rRNA gene sequences is highly similar in both the mock and the environmental communities. Resolution power and community representation accuracy from SMRT sequencing data appeared to be independent of GC content of microbial genomes and was higher when compared to Illumina-based metagenome shotgun and 16S rRNA gene (iTag) sequences, e.g. full-length sequencing resolved all 23 OTUs in the mock community, while iTags did not resolve closely related species. SMRT sequencing hence offers various potential benefits when characterizing uncharted microbial communities.

  7. Contraception for the under 16s: better safe than sorry.

    PubMed

    Cook, A

    1981-09-16

    acceptible if the couple was engaged, and 5.4% were totally against it, 9) 62% felt abortion was the right of every woman and 31.1% felt it was acceptible if the physical or mental well being of the mother was at risk, 10) 40.9% agreed with the British Medical Association policy on teenage contraception which advises doctors to encourage under 16's to tell their parents, but if they refuse, the doctor can still prescribe the pill, 11) 22.7% wanted contraception unconditionally available, 18.2% felt it should be dependent on parental knowledge, and 17% said it should not be available, 12) there was a trend for opinions to become less liberal as age increased, and 13) young girls appear to be less conscientious in using contraception than older women.

  8. High-resolution phylogenetic microbial community profiling

    PubMed Central

    Singer, Esther; Bushnell, Brian; Coleman-Derr, Devin; Bowman, Brett; Bowers, Robert M; Levy, Asaf; Gies, Esther A; Cheng, Jan-Fang; Copeland, Alex; Klenk, Hans-Peter; Hallam, Steven J; Hugenholtz, Philip; Tringe, Susannah G; Woyke, Tanja

    2016-01-01

    Over the past decade, high-throughput short-read 16S rRNA gene amplicon sequencing has eclipsed clone-dependent long-read Sanger sequencing for microbial community profiling. The transition to new technologies has provided more quantitative information at the expense of taxonomic resolution with implications for inferring metabolic traits in various ecosystems. We applied single-molecule real-time sequencing for microbial community profiling, generating full-length 16S rRNA gene sequences at high throughput, which we propose to name PhyloTags. We benchmarked and validated this approach using a defined microbial community. When further applied to samples from the water column of meromictic Sakinaw Lake, we show that while community structures at the phylum level are comparable between PhyloTags and Illumina V4 16S rRNA gene sequences (iTags), variance increases with community complexity at greater water depths. PhyloTags moreover allowed less ambiguous classification. Last, a platform-independent comparison of PhyloTags and in silico generated partial 16S rRNA gene sequences demonstrated significant differences in community structure and phylogenetic resolution across multiple taxonomic levels, including a severe underestimation in the abundance of specific microbial genera involved in nitrogen and methane cycling across the Lake's water column. Thus, PhyloTags provide a reliable adjunct or alternative to cost-effective iTags, enabling more accurate phylogenetic resolution of microbial communities and predictions on their metabolic potential. PMID:26859772

  9. Phylogenetic analysis of the kenaf fiber microbial retting community by semiconductor sequencing of 16S rDNA amplicons

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Kenaf, hemp, and jute have been used for cordage and fiber production since prehistory. To obtain the fibers, harvested plants are soaked in ponds where indigenous microflora digests pectins and other heteropolysaccharides, releasing fibers in a process called retting. Renewed interest in “green” ...

  10. Identification of Bacillus Probiotics Isolated from Soil Rhizosphere Using 16S rRNA, recA, rpoB Gene Sequencing and RAPD-PCR.

    PubMed

    Mohkam, Milad; Nezafat, Navid; Berenjian, Aydin; Mobasher, Mohammad Ali; Ghasemi, Younes

    2016-03-01

    Some Bacillus species, especially Bacillus subtilis and Bacillus pumilus groups, have highly similar 16S rRNA gene sequences, which are hard to identify based on 16S rDNA sequence analysis. To conquer this drawback, rpoB, recA sequence analysis along with randomly amplified polymorphic (RAPD) fingerprinting was examined as an alternative method for differentiating Bacillus species. The 16S rRNA, rpoB and recA genes were amplified via a polymerase chain reaction using their specific primers. The resulted PCR amplicons were sequenced, and phylogenetic analysis was employed by MEGA 6 software. Identification based on 16S rRNA gene sequencing was underpinned by rpoB and recA gene sequencing as well as RAPD-PCR technique. Subsequently, concatenation and phylogenetic analysis showed that extent of diversity and similarity were better obtained by rpoB and recA primers, which are also reinforced by RAPD-PCR methods. However, in one case, these approaches failed to identify one isolate, which in combination with the phenotypical method offsets this issue. Overall, RAPD fingerprinting, rpoB and recA along with concatenated genes sequence analysis discriminated closely related Bacillus species, which highlights the significance of the multigenic method in more precisely distinguishing Bacillus strains. This research emphasizes the benefit of RAPD fingerprinting, rpoB and recA sequence analysis superior to 16S rRNA gene sequence analysis for suitable and effective identification of Bacillus species as recommended for probiotic products.

  11. Empirical testing of 16S rRNA gene PCR primer pairs reveals variance in target specificity and efficacy not suggested by in silico analysis.

    PubMed

    Morales, Sergio E; Holben, William E

    2009-05-01

    Phylogenetic and "fingerprinting" analyses of the 16S rRNA genes of prokaryotes have been a mainstay of microbial ecology during the last two decades. However, many methods and results from studies that rely on the 16S rRNA gene for detection and quantification of specific microbial taxa have seemingly received only cursory or even no validation. To directly examine the efficacy and specificity of 16S rRNA gene-based primers for phylum-, class-, and operational taxonomic unit-specific target amplification in quantitative PCR, we created a collection of primers based solely on an extensive soil bacterial 16S rRNA gene clone library containing approximately 5,000 sequences from a single soil sample (i.e., a closed site-specific library was used to create PCR primers for use at this site). These primers were initially tested in silico prior to empirical testing by PCR amplification of known target sequences and of controls based on disparate phylogenetic groups. Although all primers were highly specific according to the in silico analysis, the empirical analyses clearly exhibited a high degree of nonspecificity for many of the phyla or classes, while other primers proved to be highly specific. These findings suggest that significant care must be taken when interpreting studies whose results were obtained with target specific primers that were not adequately validated, especially where population densities or dynamics have been inferred from the data. Further, we suggest that the reliability of quantification of specific target abundance using 16S rRNA-based quantitative PCR is case specific and must be determined through rigorous empirical testing rather than solely in silico.

  12. 16S ribosomal RNA and phylograms: Characterizing student reasoning to learning outcomes from the American Society for Microbiology Curriculum

    NASA Astrophysics Data System (ADS)

    Grassie, Chelsey Lee

    The American Society for Microbiology (ASM) has established a suggested curriculum for introductory microbiology courses that includes a focus on evolution. However, no data is published to describe how proficiently students address the learning outcomes, in part because validated assessments do not exist. Thus, the goal of this project was to develop assessment prompts that capture student understanding about fundamental statement five under the core concept of evolution. In total, 167 written responses were collected from upper-division microbiology courses, with pre-pharmacy and microbiology majors comprising the majority of students (74.6%). Two coders coded all written responses, and five student interviews were conducted. Results indicate that students have not retained instruction on 16S rRNA, or have not been exposed to it in their classes. Additionally, most students have not been exposed to phylograms, and are unfamiliar with genetic distance being represented on a phylogenetic tree. Emergent reasoning techniques are described.

  13. Microbial diversity in an in situ reactor system treating monochlorobenzene contaminated groundwater as revealed by 16S ribosomal DNA analysis.

    PubMed

    Alfreider, Albin; Vogt, Carsten; Babel, Wolfgang

    2002-08-01

    A molecular approach based on the construction of 16S ribosomal DNA clone libraries was used to investigate the microbial diversity of an underground in situ reactor system filled with the original aquifer sediments. After chemical steady state was reached in the monochlorobenzene concentration between the original inflowing groundwater and the reactor outflow, samples from different reactor locations and from inflowing and outflowing groundwater were taken for DNA extraction. Small-subunit rRNA genes were PCR-amplified with primers specific for Bacteria, subsequently cloned and screened for variation by restriction fragment length polymorphism (RFLP). A total of 87 bacterial 16S rDNA genes were sequenced and subjected to phylogenetic analysis. The original groundwater was found to be dominated by a bacterial consortium affiliated with various members of the class of Proteobacteria, by phylotypes not affiliated with currently recognized bacterial phyla, and also by sporulating and non-sporulating sulfate-reducing bacteria. The most occurring clone types obtained from the sediment samples of the reactor were related to the beta-Proteobacteria, dominated by sequences almost identical to the widespread bacterium Alcaligenes faecalis, to low G+C gram-positive bacteria and to Acidithiobacillus ferrooxidans (formerly Thiobacillus ferrooxidans) within the gamma subclass of Proteobacteria in the upper reactor sector. Although bacterial phylotypes originating from the groundwater outflow of the reactors also grouped within different subdivisions of Proteobacteria and low G+C gram-positive bacteria, most of the 16S rDNA sequences were not associated with the sequence types observed in the reactor samples. Our results suggest that the different environments were inhabited by distinct microbial communities in respect to their taxonomic diversity, particular pronounced between sediment attached microbial communities from the reactor samples and free-living bacteria from the

  14. Diversity of thermophiles in a Malaysian hot spring determined using 16S rRNA and shotgun metagenome sequencing

    PubMed Central

    Chan, Chia Sing; Chan, Kok-Gan; Tay, Yea-Ling; Chua, Yi-Heng; Goh, Kian Mau

    2015-01-01

    The Sungai Klah (SK) hot spring is the second hottest geothermal spring in Malaysia. This hot spring is a shallow, 150-m-long, fast-flowing stream, with temperatures varying from 50 to 110°C and a pH range of 7.0–9.0. Hidden within a wooded area, the SK hot spring is continually fed by plant litter, resulting in a relatively high degree of total organic content (TOC). In this study, a sample taken from the middle of the stream was analyzed at the 16S rRNA V3-V4 region by amplicon metagenome sequencing. Over 35 phyla were detected by analyzing the 16S rRNA data. Firmicutes and Proteobacteria represented approximately 57% of the microbiome. Approximately 70% of the detected thermophiles were strict anaerobes; however, Hydrogenobacter spp., obligate chemolithotrophic thermophiles, represented one of the major taxa. Several thermophilic photosynthetic microorganisms and acidothermophiles were also detected. Most of the phyla identified by 16S rRNA were also found using the shotgun metagenome approaches. The carbon, sulfur, and nitrogen metabolism within the SK hot spring community were evaluated by shotgun metagenome sequencing, and the data revealed diversity in terms of metabolic activity and dynamics. This hot spring has a rich diversified phylogenetic community partly due to its natural environment (plant litter, high TOC, and a shallow stream) and geochemical parameters (broad temperature and pH range). It is speculated that symbiotic relationships occur between the members of the community. PMID:25798135

  15. Molecular identification of four phenotypes of human Demodex mites (Acari: Demodicidae) based on mitochondrial 16S rDNA.

    PubMed

    Zhao, Ya-E; Hu, Li; Ma, Jun-Xian

    2013-11-01

    Classification of Demodex mites has long depended on hosts and morphological characteristics. However, the fact that two species coexist in the same host and phenotype is easily influenced by environment causes difficulty and indeterminacy in traditional classification. Genotype, which directly reflects the molecular structure characteristics, is relatively stable. In this study, species identification of four phenotypes of human Demodex mites was conducted. Mites were morphologically classified into four phenotypes: long- and short-bodied Demodex folliculorum with finger-like terminus and Demodex brevis with finger- or cone-like terminus. The mitochondrial 16S ribosomal DNA (rDNA) fragment of individual mite was amplified, cloned, sequenced, and aligned. Sequence divergences, genetic distances, transition/transversion rates, and phylogenetic trees were analyzed. The results demonstrated that the 16S rDNA sequence of three phenotypes with finger-like terminus was 337 bp, and that of phenotype with cone-like terminus was 342 bp. The divergences, genetic distances, and transition/transversion rates among the three phenotypes with finger-like terminus were 0.0-2.7%, 0.000-0.029, and 5.0-7/0 (5/1-7/0), respectively, indicating an intraspecific variation. Yet, those between these three phenotypes and the one with cone-like terminus were 21.6-22.8%, 2.510-2.589, and 0.47-0.59 (22/47-27/46), respectively, suggesting an interspecific variation. The five phylogenetic trees showed that the three phenotypes with finger-like terminus clustered into one branch, while the phenotype with cone-like terminus clustered into another. In conclusion, terminus is a major morphological characteristic for the identification of human Demodex species. The three phenotypes with finger-like terminus belong to D. folliculorum, while the phenotype with cone-like terminus belongs to D. brevis. Molecular identification can verify and replenish morphological identification.

  16. Identification of single nucleotide polymorphisms (SNPs) in the 16S rRNA gene of foodborne Bacillus spp.

    PubMed

    Fernández-No, I C; Böhme, K; Caamaño-Antelo, S; Barros-Velázquez, J; Calo-Mata, P

    2015-04-01

    The main goal of this work was the identification of single nucleotide polymorphisms (SNPs) in the 16S rRNA gene of foodborne Bacillus spp. that may be useful for typing purposes. These species include, among others, Bacillus cereus, an important pathogenic species involved in food poisoning, and Bacillus licheniformis, Bacillus subtilis and Bacillus pumilus, which are causative agents of food spoilage described as responsible for foodborne disease outbreaks. With this purpose in mind, 52 Bacillus strains isolated from culture collections and fresh and processed food were considered. SNP type "Y" at sites 212 and 476 appeared in the majority of B. licheniformis studied strains. SNP type "R" at site 278 was detected in many strains of the B. subtilis/Bacillus amyloliquefaciens group, while polymorphism "Y" at site 173 was characteristic of the majority of strains of B. cereus/Bacillus thuringiensis group. The analysis of SNPs provided more intra-specific information than phylogenetic analysis in the cases of B. cereus and B. subtilis. Moreover, this study describes novel SNPs that should be considered when designing 16S rRNA-based primers and probes for multiplex-PCR, Real-Time PCR and microarray systems for foodborne Bacillus spp.

  17. Microbial diversity in uranium mining-impacted soils as revealed by high-density 16S microarray and clone library.

    PubMed

    Rastogi, Gurdeep; Osman, Shariff; Vaishampayan, Parag A; Andersen, Gary L; Stetler, Larry D; Sani, Rajesh K

    2010-01-01

    Microbial diversity was characterized in mining-impacted soils collected from two abandoned uranium mine sites, the Edgemont and the North Cave Hills, South Dakota, using a high-density 16S microarray (PhyloChip) and clone libraries. Characterization of the elemental compositions of soils by X-ray fluorescence spectroscopy revealed higher metal contamination including uranium at the Edgemont than at the North Cave Hills mine site. Microarray data demonstrated extensive phylogenetic diversity in soils and confirmed nearly all clone-detected taxonomic levels. Additionally, the microarray exhibited greater diversity than clone libraries at each taxonomic level at both the mine sites. Interestingly, the PhyloChip detected the largest number of taxa in Proteobacteria phylum for both the mine sites. However, clone libraries detected Acidobacteria and Bacteroidetes as the most numerically abundant phyla in the Edgemont and North Cave Hills mine sites, respectively. Several 16S rDNA signatures found in both the microarrays and clone libraries displayed sequence similarities with yet-uncultured bacteria representing a hitherto unidentified diversity. Results from this study demonstrated that highly diverse microbial populations were present in these uranium mine sites. Diversity indices indicated that microbial communities at the North Cave Hills mine site were much more diverse than those at the Edgemont mine site.

  18. Identification and phylogeny of Arabian snakes: Comparison of venom chromatographic profiles versus 16S rRNA gene sequences

    PubMed Central

    Al Asmari, Abdulrahman; Manthiri, Rajamohammed Abbas; Khan, Haseeb Ahmad

    2014-01-01

    Identification of snake species is important for various reasons including the emergency treatment of snake bite victims. We present a simple method for identification of six snake species using the gel filtration chromatographic profiles of their venoms. The venoms of Echis coloratus, Echis pyramidum, Cerastes gasperettii, Bitis arietans, Naja arabica, and Walterinnesia aegyptia were milked, lyophilized, diluted and centrifuged to separate the mucus from the venom. The clear supernatants were filtered and chromatographed on fast protein liquid chromatography (FPLC). We obtained the 16S rRNA gene sequences of the above species and performed phylogenetic analysis using the neighbor-joining method. The chromatograms of venoms from different snake species showed peculiar patterns based on the number and location of peaks. The dendrograms generated from similarity matrix based on the presence/absence of particular chromatographic peaks clearly differentiated Elapids from Viperids. Molecular cladistics using 16S rRNA gene sequences resulted in jumping clades while separating the members of these two families. These findings suggest that chromatographic profiles of snake venoms may provide a simple and reproducible chemical fingerprinting method for quick identification of snake species. However, the validation of this methodology requires further studies on large number of specimens from within and across species. PMID:25313278

  19. High or low correlation between co-occuring gene clusters and 16S rRNA gene phylogeny.

    PubMed

    Rudi, Knut; Sekelja, Monika

    2013-02-01

    Ribosomal RNA (rRNA) genes are universal for all living organisms. Yet, the correspondence between genome composition and rRNA phylogeny remains poorly known. The aim of this study was to use the information from genome sequence databases to address the correlation between rRNA gene phylogeny and total gene composition in bacteria. This was done by analysing 327 genomes with TIGRFAM functional gene annotations. Our approach consisted of two steps. First, we searched for discriminatory clusters of co-occurring genes. Using a multivariate statistical approach, we identified 11 such clusters which contain genes that were co-occurring only in a subset of genomes and contributed to explain the gene content differences between genome subsets. Second, we mapped the discovered clusters to 16S rRNA-based phylogeny and calculated the correlation between co-occuring genes and phylogeny. Six of the 11 clusters exhibited significant correlation with 16S rRNA gene phylogeny. The most distinct phylogenetic finding was a high correlation between iron-sulfur oxidoreductases in combination with carbon nitrogen ligases and Chlorobium. The other correlations identified covered relatively large phylogroups: Actinobacteria were positively associated with kinases, while Gammaproteobacteria were positively associated with methylases and acyltransferases. The suggested functional differences between higher phylogroups, however, need experimental verification.

  20. Identification and phylogeny of Arabian snakes: Comparison of venom chromatographic profiles versus 16S rRNA gene sequences.

    PubMed

    Al Asmari, Abdulrahman; Manthiri, Rajamohammed Abbas; Khan, Haseeb Ahmad

    2014-11-01

    Identification of snake species is important for various reasons including the emergency treatment of snake bite victims. We present a simple method for identification of six snake species using the gel filtration chromatographic profiles of their venoms. The venoms of Echis coloratus, Echis pyramidum, Cerastes gasperettii, Bitis arietans, Naja arabica, and Walterinnesia aegyptia were milked, lyophilized, diluted and centrifuged to separate the mucus from the venom. The clear supernatants were filtered and chromatographed on fast protein liquid chromatography (FPLC). We obtained the 16S rRNA gene sequences of the above species and performed phylogenetic analysis using the neighbor-joining method. The chromatograms of venoms from different snake species showed peculiar patterns based on the number and location of peaks. The dendrograms generated from similarity matrix based on the presence/absence of particular chromatographic peaks clearly differentiated Elapids from Viperids. Molecular cladistics using 16S rRNA gene sequences resulted in jumping clades while separating the members of these two families. These findings suggest that chromatographic profiles of snake venoms may provide a simple and reproducible chemical fingerprinting method for quick identification of snake species. However, the validation of this methodology requires further studies on large number of specimens from within and across species.

  1. The use of 16S and 16S-23S rDNA to easily detect and differentiate common Gram-negative orchard epiphytes.

    PubMed

    Jeng, R S; Svircev, A M; Myers, A L; Beliaeva, L; Hunter, D M; Hubbes, M

    2001-02-01

    The identification of Gram-negative pathogenic and non-pathogenic bacteria commonly isolated from an orchard phylloplane may result in a time consuming and tedious process for the plant pathologist. The paper provides a simple "one-step" protocol that uses the polymerase chain reaction (PCR) to amplify intergenic spacer regions between 16S and 23S genes and a portion of 16S gene in the prokaryotic rRNA genetic loci. Amplified 16S rDNA, and restriction fragment length polymorphisms (RFLP) following EcoRI digestion produced band patterns that readily distinguished between the plant pathogen Erwinia amylovora (causal agent of fire blight in pear and apple) and the orchard epiphyte Pantoea agglomerans (formerly E. herbicola). The amplified DNA patterns of 16S-23S spacer regions may be used to differentiate E. amylovora at the intraspecies level. Isolates of E. amylovora obtained from raspberries exhibited two major fragments while those obtained from apples showed three distinct amplified DNA bands. In addition, the size of the 16S-23S spacer region differs between Pseudomonas syringae and Pseudomonas fluorescens. The RFLP pattern generated by HaeIII digestion may be used to provide a rapid and accurate identification of these two common orchard epiphytes.

  2. Microbiological and 16S rRNA analysis of sulphite-reducing clostridia from river sediments in central Italy

    PubMed Central

    Marcheggiani, Stefania; Iaconelli, Marcello; D'angelo, Annamaria; Pierdominici, Elio; La Rosa, Giuseppina; Muscillo, Michele; Equestre, Michele; Mancini, Laura

    2008-01-01

    Background Microbiological indicators are commonly used in the assessment of public health risks associated with fecal contamination of freshwater ecosystems. Sediments are a reservoir of microorganisms, and can thus provide information on past pollution events, not obtainable through the testing of surface water. Moreover, pathogens present in sediment may represent future threats to human health. Clostridium perfringens, a typical colonizer of sediments, has been suggested as an alternative indicator of fecal pollution. In order to be suitable for such purpose, the microorganism should be widely distributed in contaminated environments. The objective of this study was thus to determine the composition of the anaerobic community in sediment samples of the lower Tiber basin, in central Italy, through a combined approach involving granulometric analysis of sediment samples, as well as a microbiological and molecular (16S rRNA) analysis of strains. Results Granulometry showed a similar, clayey sediment composition, in most sampling sites. The microbiological method, employing, an adaptation of the standard method, proved to be effective in isolating anaerobic bacteria from the environmental matrix for the purpose of genetic analysis. Eighty-three strains of bacteria were isolated and the partial 16S rRNA gene sequenced. While biochemical analysis detected only C. perfringens strains, phylogenetic analysis indicated the presence of three clusters: C. perfringens, C. bifermentans and B. cereus, comprising eight taxa. C. perfringens, the commonest in almost all sediment sampling sites, was present in all sites, and in both seasons (seasonal sampling was carried out only along the Tiber and Aniene rivers). None of the described genetic profiles showed complete similarity with GenBank sequences. Conclusion The study underlines the value of C. perfringens as an alternative microbial indicator of fecal contamination in river sediments. This is supported by the bacterium

  3. Bacteroides isolated from four mammalian hosts lack host-specific 16S rRNA gene phylogeny and carbon and nitrogen utilization patterns*

    PubMed Central

    Atherly, Todd; Ziemer, Cherie J

    2014-01-01

    One-hundred-and-three isolates of Bacteroides ovatus,B. thetaiotaomicron, and B. xylanisolvens were recovered from cow, goat, human, and pig fecal enrichments with cellulose or xylan/pectin. Isolates were compared using 16S rRNA gene sequencing, repetitive sequence-based polymerase chain reaction (rep-PCR), and phenotypic microarrays. Analysis of 16S rRNA gene sequences revealed high sequence identity in these Bacteroides; with distinct phylogenetic groupings by bacterial species but not host origin. Phenotypic microarray analysis demonstrated these Bacteroides shared the ability to utilize many of the same carbon substrates, without differences due to species or host origin, indicative of their broad carbohydrate fermentation abilities. Limited nitrogen substrates were utilized; in addition to ammonia, guanine, and xanthine, purine derivatives were utilized by most isolates followed by a few amino sugars. Only rep-PCR analysis demonstrated host-specific patterns, indicating that genomic changes due to coevolution with host did not occur by mutation in the 16S rRNA gene or by a gain or loss of carbohydrate utilization genes within these Bacteroides. This is the first report to indicate that host-associated genomic differences are outside of 16S rRNA gene and carbohydrate utilization genes and suggest conservation of specific bacterial species with the same functionality across mammalian hosts for this Bacteroidetes clade. PMID:24532571

  4. Validation of the 16S rDNA and COI DNA barcoding technique for rapid molecular identification of stored product psocids (Insecta: Psocodea: Liposcelididae).

    PubMed

    Yang, Qianqian; Zhao, Shuo; Kucerová, Zuzana; Stejskal, Václav; Opit, George; Qin, Meng; Cao, Yang; Li, Fujun; Li, Zhihong

    2013-02-01

    Psocids are serious storage pests, and their control is hampered by the fact that different species respond differently to insecticides used for the control of stored-product insect pests. Additionally, psocids of genus Liposcelis that are commonly associated with stored-products are difficult to identify using morphological characteristics. The goal of this study was to validate molecular identification of stored-product psocids of genus Liposcelis based on 16S rDNA and cytochrome oxidase I (COI) DNA barcoding. Unidentified liposcelids (Liposcelis DK) imported from Denmark to China were compared with 14 population samples of seven common species (L. bostrychophila, L. brunnea, L. corrodens, L. decolor, L. entomophila, L. mendax, and L. paeta). The explored species (DK) liposcelids shared >98% sequence similarity for both the 16S rDNA and COI genes with the reference L. corrodens samples (98.32 and 98.94% for 16S rDNA and COI, respectively). A neighbor-joining tree revealed that the explored DK sample and the reference L. corrodens samples belong to the same clade. These molecular results were verified by morphological identification of DK specimens, facilitated by SEM microphotography. The DNA barcoding method and the neighbor-joining phylogenetic analyses indicated that both the 16S rDNA and COI genes were suitable for Liposcelis species identification. DNA barcoding has great potential for use in fast and accurate liposcelid identification.

  5. Structure and organization of the rrnD operon of 'Brevibacterium lactofermentum': analysis of the 16S rRNA gene.

    PubMed

    Amador, E; Castro, J M; Correia, A; Martín, J F

    1999-04-01

    Five rRNA operons (rrn) were found by hybridization in the genome of 'Brevibacterium lactofermentum' ATCC 13869 and Corynebacterium glutamicum ATCC 13032. 'B. lactofermentum' DSM 20412 differed from the other corynebacteria tested in showing six hybridizing BamHI bands. Two of the rrn operons (rrnD and rrnE) were located in a single cosmid. Sequencing of the rrnD operon showed that it contains a complete 16S rRNA-23S RNA-5S rRNA gene cluster. Phylogenetic studies using the complete 16S rRNA sequence showed that 'B. lactofermentum' is closely related to several species of the genus Corynebacterium but only distantly related to the type species Brevibacterium linens and the authors suggest that it should be reclassified as Corynebacterium lactofermentum. The 5' end of mature 16S rRNA was identified by primer extension. Sequence elements similar to those of mycobacteria implicated in transcription antitermination (Boxes A, B, C) and in processing of the pre-rRNA to 16S rRNA were identified. An open reading frame encoding an rpoD-like sigma factor (named SigC) different from the previously reported SigA and SigB proteins was found upstream of rrnD in the opposite orientation. Both rpoD and sigC seem to be expressed from a bidirectional promoter region.

  6. Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin

    PubMed Central

    Chahine, Sarah; Okafor, Darius; Ong, Ana C.; Maybank, Rosslyn; Kwak, Yoon I.; Wilson, Kerry; Zapor, Michael; Lesho, Emil; Hinkle, Mary

    2015-01-01

    16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test. PMID:26537447

  7. Monitoring Precursor 16S rRNAs of Acinetobacter spp. in Activated Sludge Wastewater Treatment Systems

    PubMed Central

    Oerther, Daniel B.; Pernthaler, Jakob; Schramm, Andreas; Amann, Rudolf; Raskin, Lutgarde

    2000-01-01

    Recently, Cangelosi and Brabant used oligonucleotide probes targeting the precursor 16S rRNA of Escherichia coli to demonstrate that the levels of precursor rRNA were more sensitive to changes in growth phase than the levels of total rRNA (G. A. Cangelosi and W. H. Brabant, J. Bacteriol. 179:4457–4463, 1997). In order to measure changes in the levels of precursor rRNA in activated sludge systems, we designed oligonucleotide probes targeting the 3′ region of the precursor 16S rRNA of Acinetobacter spp. We used these probes to monitor changes in the level of precursor 16S rRNA during batch growth of Acinetobacter spp. in Luria-Bertani (LB) medium, filtered wastewater, and in lab- and full-scale wastewater treatment systems. Consistent with the previous reports for E. coli, results obtained with membrane hybridizations and fluorescence in situ hybridizations with Acinetobacter calcoaceticus grown in LB medium showed a more substantial and faster increase in precursor 16S rRNA levels compared to the increase in total 16S rRNA levels during exponential growth. Diluting an overnight culture of A. calcoaceticus grown in LB medium with filtered wastewater resulted in a pattern of precursor 16S rRNA levels that appeared to follow diauxic growth. In addition, fluorescence in situ hybridizations with oligonucleotide probes targeting total 16S rRNA and precursor 16S rRNA showed that individual cells of A. calcoaceticus expressed highly variable levels of precursor 16S rRNA when adapting from LB medium to filtered sewage. Precursor 16S rRNA levels of Acinetobacter spp. transiently increased when activated sludge was mixed with influent wastewater in lab- and full-scale wastewater treatment systems. These results suggest that Acinetobacter spp. experience a change in growth activity within wastewater treatment systems. PMID:10788395

  8. Phylogeny of Japanese stag beetles (Coleoptera: Lucanidae) inferred from 16S mtrRNA gene sequences, with reference to the evolution of sexual dimorphism of mandibles.

    PubMed

    Hosoya, Tadatsugu; Araya, Kunio

    2005-12-01

    As a first step in reconstructing the phylogeny of world stag beetles (Coleoptera: Lucanidae), phylogenetic relationships among the major members of Japanese stag beetles were explored by analyzing a sequence of 1030 nucleotides from the mitochondrial 16S ribosomal RNA (16S rRNA) gene. A total of 20 species and three additional subspecies representing 13 genera were examined to provide basic information on the phylogeny of world Lucanidae. The resultant phylogenetic tree indicates that the family Lucanidae is monophyletic, and contains two major lineages: one consists of the genera Platycerus, Aesalus, Ceruchus, and Nicagus, and the other includes Dorcus, Rhaetulus, Prosopocoilus, Aegus, Neolucanus, Prismognathus, Lucanus, Figulus, and Nigidius. Generic members of the latter lineage are further divided into the following four sublineages: i) Figulus and Nigidius; ii) Prismognathus and Lucanus; iii) Aegus and Neolucanus; and iv) Dorcus, Rhaetulus, and Prosopocoilus. These molecular phylogenetic relationships are used as a basis for a preliminary exploration of the evolution of sexual dimorphism in the shape of the mandible. The results of this investigation suggest that strong sexual dimorphism with well-developed mandibles in males evolved independently at least twice, once in the genus Aegus and once in the ancestor of the Lucanus-Prismognathus and Dorcus-Rhaetulus-Prosopocoilus clades. Alternatively, it is possible that sexual dimorphism of mandibles has undergone secondary loss in the genera Figulus and Nigidius.

  9. Comparison of plastid 16S rRNA (rrn16) genes from Helicosporidium spp.: evidence supporting the reclassification of Helicosporidia as green algae (Chlorophyta).

    PubMed

    Tartar, Aurélien; Boucias, Drion G; Becnel, James J; Adams, Byron J

    2003-11-01

    The Helicosporidia are invertebrate pathogens that have recently been identified as non-photosynthetic green algae (Chlorophyta). In order to confirm the algal nature of the genus Helicosporidium, the presence of a retained chloroplast genome in Helicosporidia cells was investigated. Fragments homologous to plastid 16S rRNA (rrn16) genes were amplified successfully from cellular DNA extracted from two different Helicosporidium isolates. The fragment sequences are 1269 and 1266 bp long, are very AT-rich (60.7 %) and are similar to homologous genes sequenced from non-photosynthetic green algae. Maximum-parsimony, maximum-likelihood and neighbour-joining methods were used to infer phylogenetic trees from an rrn16 sequence alignment. All trees depicted the Helicosporidia as sister taxa to the non-photosynthetic, pathogenic alga Prototheca zopfii. Moreover, the trees identified Helicosporidium spp. as members of a clade that included the heterotrophic species Prototheca spp. and the mesotrophic species Chlorella protothecoides. The clade is always strongly supported by bootstrap values, suggesting that all these organisms share a most recent common ancestor. Phylogenetic analyses inferred from plastid 16S rRNA genes confirmed that the Helicosporidia are non-photosynthetic green algae, close relatives of the genus Prototheca (Chlorophyta, Trebouxiophyceae). Such phylogenetic affinities suggest that Helicosporidium spp. are likely to possess Prototheca-like organelles and organelle genomes.

  10. A Comparison between Transcriptome Sequencing and 16S Metagenomics for Detection of Bacterial Pathogens in Wildlife

    PubMed Central

    Razzauti, Maria; Galan, Maxime; Bernard, Maria; Maman, Sarah; Klopp, Christophe; Charbonnel, Nathalie; Vayssier-Taussat, Muriel; Eloit, Marc; Cosson, Jean-François

    2015-01-01

    Background Rodents are major reservoirs of pathogens responsible for numerous zoonotic diseases in humans and livestock. Assessing their microbial diversity at both the individual and population level is crucial for monitoring endemic infections and revealing microbial association patterns within reservoirs. Recently, NGS approaches have been employed to characterize microbial communities of different ecosystems. Yet, their relative efficacy has not been assessed. Here, we compared two NGS approaches, RNA-Sequencing (RNA-Seq) and 16S-metagenomics, assessing their ability to survey neglected zoonotic bacteria in rodent populations. Methodology/Principal Findings We first extracted nucleic acids from the spleens of 190 voles collected in France. RNA extracts were pooled, randomly retro-transcribed, then RNA-Seq was performed using HiSeq. Assembled bacterial sequences were assigned to the closest taxon registered in GenBank. DNA extracts were analyzed via a 16S-metagenomics approach using two sequencers: the 454 GS-FLX and the MiSeq. The V4 region of the gene coding for 16S rRNA was amplified for each sample using barcoded universal primers. Amplicons were multiplexed and processed on the distinct sequencers. The resulting datasets were de-multiplexed, and each read was processed through a pipeline to be taxonomically classified using the Ribosomal Database Project. Altogether, 45 pathogenic bacterial genera were detected. The bacteria identified by RNA-Seq were comparable to those detected by 16S-metagenomics approach processed with MiSeq (16S-MiSeq). In contrast, 21 of these pathogens went unnoticed when the 16S-metagenomics approach was processed via 454-pyrosequencing (16S-454). In addition, the 16S-metagenomics approaches revealed a high level of coinfection in bank voles. Conclusions/Significance We concluded that RNA-Seq and 16S-MiSeq are equally sensitive in detecting bacteria. Although only the 16S-MiSeq method enabled identification of bacteria in each

  11. Use of 16S Ribosomal RNA Sequences to Infer Relationships among Archaebacteria.

    DTIC Science & Technology

    1987-04-16

    FIELD GROUP SUB-GROUP Archaebacteria; Eubacteria ; Eukaryotes; 16S Ribosomal RNA; 08 I Phylogeny; rRNA; RNA Sequencing; Molecular Clock; Urkingdoms; r...16S rRNA data were used to infer the relat onships among the archaebacteria, and of the archaebacteria to the eubacteria and eukaryotes. ur programs for...been published (1, 2, 16, 18). The analyses render untenable the suggestions of Lake and colleagues (Lake et al., 1985) that the eubacteria derive from

  12. Bacterial communities associated with host-adapted populations of pea aphids revealed by deep sequencing of 16S ribosomal DNA.

    PubMed

    Gauthier, Jean-Pierre; Outreman, Yannick; Mieuzet, Lucie; Simon, Jean-Christophe

    2015-01-01

    Associations between microbes and animals are ubiquitous and hosts may benefit from harbouring microbial communities through improved resource exploitation or resistance to environmental stress. The pea aphid, Acyrthosiphon pisum, is the host of heritable bacterial symbionts, including the obligate endosymbiont Buchnera aphidicola and several facultative symbionts. While obligate symbionts supply aphids with key nutrients, facultative symbionts influence their hosts in many ways such as protection against natural enemies, heat tolerance, color change and reproduction alteration. The pea aphid also encompasses multiple plant-specialized biotypes, each adapted to one or a few legume species. Facultative symbiont communities differ strongly between biotypes, although bacterial involvement in plant specialization is uncertain. Here, we analyse the diversity of bacterial communities associated with nine biotypes of the pea aphid complex using amplicon pyrosequencing of 16S rRNA genes. Combined clustering and phylogenetic analyses of 16S sequences allowed identifying 21 bacterial OTUs (Operational Taxonomic Unit). More than 98% of the sequencing reads were assigned to known pea aphid symbionts. The presence of Wolbachia was confirmed in A. pisum while Erwinia and Pantoea, two gut associates, were detected in multiple samples. The diversity of bacterial communities harboured by pea aphid biotypes was very low, ranging from 3 to 11 OTUs across samples. Bacterial communities differed more between than within biotypes but this difference did not correlate with the genetic divergence between biotypes. Altogether, these results confirm that the aphid microbiota is dominated by a few heritable symbionts and that plant specialization is an important structuring factor of bacterial communities associated with the pea aphid complex. However, since we examined the microbiota of aphid samples kept a few generations in controlled conditions, it may be that bacterial diversity was

  13. Bacterial Communities Associated with Host-Adapted Populations of Pea Aphids Revealed by Deep Sequencing of 16S Ribosomal DNA

    PubMed Central

    Gauthier, Jean-Pierre; Outreman, Yannick; Mieuzet, Lucie; Simon, Jean-Christophe

    2015-01-01

    Associations between microbes and animals are ubiquitous and hosts may benefit from harbouring microbial communities through improved resource exploitation or resistance to environmental stress. The pea aphid, Acyrthosiphon pisum, is the host of heritable bacterial symbionts, including the obligate endosymbiont Buchnera aphidicola and several facultative symbionts. While obligate symbionts supply aphids with key nutrients, facultative symbionts influence their hosts in many ways such as protection against natural enemies, heat tolerance, color change and reproduction alteration. The pea aphid also encompasses multiple plant-specialized biotypes, each adapted to one or a few legume species. Facultative symbiont communities differ strongly between biotypes, although bacterial involvement in plant specialization is uncertain. Here, we analyse the diversity of bacterial communities associated with nine biotypes of the pea aphid complex using amplicon pyrosequencing of 16S rRNA genes. Combined clustering and phylogenetic analyses of 16S sequences allowed identifying 21 bacterial OTUs (Operational Taxonomic Unit). More than 98% of the sequencing reads were assigned to known pea aphid symbionts. The presence of Wolbachia was confirmed in A. pisum while Erwinia and Pantoea, two gut associates, were detected in multiple samples. The diversity of bacterial communities harboured by pea aphid biotypes was very low, ranging from 3 to 11 OTUs across samples. Bacterial communities differed more between than within biotypes but this difference did not correlate with the genetic divergence between biotypes. Altogether, these results confirm that the aphid microbiota is dominated by a few heritable symbionts and that plant specialization is an important structuring factor of bacterial communities associated with the pea aphid complex. However, since we examined the microbiota of aphid samples kept a few generations in controlled conditions, it may be that bacterial diversity was

  14. Novel PCR primers for the archaeal phylum Thaumarchaeota designed based on the comparative analysis of 16S rRNA gene sequences.

    PubMed

    Hong, Jin-Kyung; Kim, Hye-Jin; Cho, Jae-Chang

    2014-01-01

    Based on comparative phylogenetic analysis of 16S rRNA gene sequences deposited in an RDP database, we constructed a local database of thaumarchaeotal 16S rRNA gene sequences and developed a novel PCR primer specific for the archaeal phylum Thaumarchaeota. Among 9,727 quality-filtered (chimeral-checked, size >1.2 kb) archaeal sequences downloaded from the RDP database, 1,549 thaumarchaeotal sequences were identified and included in our local database. In our study, Thaumarchaeota included archaeal groups MG-I, SAGMCG-I, SCG, FSCG, RC, and HWCG-III, forming a monophyletic group in the phylogenetic tree. Cluster analysis revealed 114 phylotypes for Thaumarchaeota. The majority of the phylotypes (66.7%) belonged to the MG-I and SCG, which together contained most (93.9%) of the thaumarchaeotal sequences in our local database. A phylum-directed primer was designed from a consensus sequence of the phylotype sequences, and the primer's specificity was evaluated for coverage and tolerance both in silico and empirically. The phylum-directed primer, designated THAUM-494, showed >90% coverage for Thaumarchaeota and <1% tolerance to non-target taxa, indicating high specificity. To validate this result experimentally, PCRs were performed with THAUM-494 in combination with a universal archaeal primer (ARC917R or 1017FAR) and DNAs from five environmental samples to construct clone libraries. THAUM-494 showed a satisfactory specificity in empirical studies, as expected from the in silico results. Phylogenetic analysis of 859 cloned sequences obtained from 10 clone libraries revealed that >95% of the amplified sequences belonged to Thaumarchaeota. The most frequently sampled thaumarchaeotal subgroups in our samples were SCG, MG-I, and SAGMCG-I. To our knowledge, THAUM-494 is the first phylum-level primer for Thaumarchaeota. Furthermore, the high coverage and low tolerance of THAUM-494 will make it a potentially valuable tool in understanding the phylogenetic diversity and

  15. Novel PCR Primers for the Archaeal Phylum Thaumarchaeota Designed Based on the Comparative Analysis of 16S rRNA Gene Sequences

    PubMed Central

    Hong, Jin-Kyung; Kim, Hye-Jin; Cho, Jae-Chang

    2014-01-01

    Based on comparative phylogenetic analysis of 16S rRNA gene sequences deposited in an RDP database, we constructed a local database of thaumarchaeotal 16S rRNA gene sequences and developed a novel PCR primer specific for the archaeal phylum Thaumarchaeota. Among 9,727 quality-filtered (chimeral-checked, size >1.2 kb) archaeal sequences downloaded from the RDP database, 1,549 thaumarchaeotal sequences were identified and included in our local database. In our study, Thaumarchaeota included archaeal groups MG-I, SAGMCG-I, SCG, FSCG, RC, and HWCG-III, forming a monophyletic group in the phylogenetic tree. Cluster analysis revealed 114 phylotypes for Thaumarchaeota. The majority of the phylotypes (66.7%) belonged to the MG-I and SCG, which together contained most (93.9%) of the thaumarchaeotal sequences in our local database. A phylum-directed primer was designed from a consensus sequence of the phylotype sequences, and the primer’s specificity was evaluated for coverage and tolerance both in silico and empirically. The phylum-directed primer, designated THAUM-494, showed >90% coverage for Thaumarchaeota and <1% tolerance to non-target taxa, indicating high specificity. To validate this result experimentally, PCRs were performed with THAUM-494 in combination with a universal archaeal primer (ARC917R or 1017FAR) and DNAs from five environmental samples to construct clone libraries. THAUM-494 showed a satisfactory specificity in empirical studies, as expected from the in silico results. Phylogenetic analysis of 859 cloned sequences obtained from 10 clone libraries revealed that >95% of the amplified sequences belonged to Thaumarchaeota. The most frequently sampled thaumarchaeotal subgroups in our samples were SCG, MG-I, and SAGMCG-I. To our knowledge, THAUM-494 is the first phylum-level primer for Thaumarchaeota. Furthermore, the high coverage and low tolerance of THAUM-494 will make it a potentially valuable tool in understanding the phylogenetic diversity and

  16. Comparative analyses of phenotypic methods and 16S rRNA, khe, rpoB genes sequencing for identification of clinical isolates of Klebsiella pneumoniae.

    PubMed

    He, Yanxia; Guo, Xianguang; Xiang, Shifei; Li, Jiao; Li, Xiaoqin; Xiang, Hui; He, Jinlei; Chen, Dali; Chen, Jianping

    2016-07-01

    The present work aimed to evaluate 16S rRNA, khe and rpoB gene sequencing for the identification of Klebsiella pneumoniae in comparison with phenotypic methods. Fifteen clinical isolates were examined, which were initially identified as K. pneumoniae subsp. pneumoniae using the automated VITEK 32 system in two hospitals in Enshi City, China. Their identity was further supported by conventional phenotypic methods on the basis of morphological and biochemical characteristics. Using Bayesian phylogenetic analyses and haplotypes network reconstruction, 13 isolates were identified as K. pneumoniae, whereas the other two isolates (K19, K24) were classified as Shigella sp. and Enterobacter sp., respectively. Of the three genes, 16S rRNA and khe gene could discriminate the clinical isolates at the genus level, whereas rpoB could discriminate Klebsiella at the species and even subspecies level. Overall, the gene tree based on rpoB is more compatible with the currently accepted classification of Klebsiella than those based on 16S rRNA and khe genes, showing that rpoB can be a powerful tool for identification of K. pneumoniae isolates. Above all, our study challenges the utility of khe as a species-specific marker for identification of K. pneumoniae.

  17. Intragenomic diversity of the V1 regions of 16S rRNA genes in high-alkaline protease-producing Bacillus clausii spp.

    PubMed

    Kageyama, Yasushi; Takaki, Yoshihiro; Shimamura, Shigeru; Nishi, Shinro; Nogi, Yuichi; Uchimura, Kohsuke; Kobayashi, Tohru; Hitomi, Jun; Ozaki, Katsuya; Kawai, Shuji; Ito, Susumu; Horikoshi, Koki

    2007-07-01

    Alkaliphilic Bacillus sp. strain KSM-K16, which produces high-alkaline M-protease, was characterized phenotypically, biochemically and genetically. This strain was identified as Bacillus clausii based on the results of taxonomic studies, including sequencing of the 16S rRNA gene and DNA-DNA hybridization. Seven rRNA operons in the genome were identified by pulsed-field gel electrophoresis. Sequencing of cloned 16S rRNA genes revealed two distinct types of variable region V1. Moreover, some cloned 16S rRNA genes in some of the reference strains of B. clausii had a V1 region of yet another type. The B. clausii strains could clearly be divided into at least two subgroups based on the frequencies of the types of cloned V1 sequence. Bacillus sp. strain KSM-K16 was found to be in a different phylogenetic position from other high-alkaline protease-producing strains of B. clausii.

  18. Bacterial Diversity Analysis of Huanglongbing Pathogen-Infected Citrus, Using PhyloChip Arrays and 16S rRNA Gene Clone Library Sequencing▿ †

    PubMed Central

    Sagaram, Uma Shankar; DeAngelis, Kristen M.; Trivedi, Pankaj; Andersen, Gary L.; Lu, Shi-En; Wang, Nian

    2009-01-01

    The bacterial diversity associated with citrus leaf midribs was characterized for citrus groves that contained the Huanglongbing (HLB) pathogen, which has yet to be cultivated in vitro. We employed a combination of high-density phylogenetic 16S rRNA gene microarrays and 16S rRNA gene clone library sequencing to determine the microbial community composition for symptomatic and asymptomatic citrus midribs. Our results revealed that citrus leaf midribs can support a diversity of microbes. PhyloChip analysis indicated that 47 orders of bacteria in 15 phyla were present in the citrus leaf midribs, while 20 orders in 8 phyla were observed with the cloning and sequencing method. PhyloChip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs than in asymptomatic midribs. “Candidatus Liberibacter asiaticus” was detected at a very low level in asymptomatic plants but was over 200 times more abundant in symptomatic plants. The PhyloChip analysis results were further verified by sequencing 16S rRNA gene clone libraries, which indicated the dominance of “Candidatus Liberibacter asiaticus” in symptomatic leaves. These data implicate “Candidatus Liberibacter asiaticus” as the pathogen responsible for HLB disease. PMID:19151177

  19. A phylogenetic analysis of the phylum Fibrobacteres.

    PubMed

    Jewell, Kelsea A; Scott, Jarrod J; Adams, Sandra M; Suen, Garret

    2013-09-01

    Members of the phylum Fibrobacteres are highly efficient cellulolytic bacteria, best known for their role in rumen function and as potential sources of novel enzymes for bioenergy applications. Despite being key members of ruminants and other digestive microbial communities, our knowledge of this phylum remains incomplete, as much of our understanding is focused on two recognized species, Fibrobacter succinogenes and F. intestinalis. As a result, we lack insights regarding the environmental niche, host range, and phylogenetic organization of this phylum. Here, we analyzed over 1000 16S rRNA Fibrobacteres sequences available from public databases to establish a phylogenetic framework for this phylum. We identify both species- and genus-level clades that are suggestive of previously unknown taxonomic relationships between Fibrobacteres in addition to their putative lifestyles as host-associated or free-living. Our results shed light on this poorly understood phylum and will be useful for elucidating the function, distribution, and diversity of these bacteria in their niches.

  20. Mitochondrial 16S ribosomal RNA gene for forensic identification of crocodile species.

    PubMed

    Naga Jogayya, K; Meganathan, P R; Dubey, Bhawna; Haque, I

    2013-05-01

    All crocodilians are under various threats due to over exploitation and these species have been listed in Appendix I or II of CITES. Lack of molecular techniques for the forensic identification of confiscated samples makes it difficult to enforce the law. Therefore, we herein present a molecular method developed on the basis on 16S rRNA gene of mitochondrial DNA for identification of crocodile species. We have developed a set of 16S rRNA primers for PCR based identification of crocodilian species. These novel primers amplify partial 16S rRNA sequences of six crocodile species which can be later combined to obtain a larger region (1290 bp) of 16S rRNA gene. This 16S rRNA gene could be used as an effective tool for forensic authentication of crocodiles. The described primers hold great promise in forensic identification of crocodile species, which can aid in the effective enforcement of law and conservation of these species.

  1. Accurate taxonomy assignments from 16S rRNA sequences produced by highly parallel pyrosequencers

    PubMed Central

    Liu, Zongzhi; DeSantis, Todd Z.; Andersen, Gary L.; Knight, Rob

    2008-01-01

    The recent introduction of massively parallel pyrosequencers allows rapid, inexpensive analysis of microbial community composition using 16S ribosomal RNA (rRNA) sequences. However, a major challenge is to design a workflow so that taxonomic information can be accurately and rapidly assigned to each read, so that the composition of each community can be linked back to likely ecological roles played by members of each species, genus, family or phylum. Here, we use three large 16S rRNA datasets to test whether taxonomic information based on the full-length sequences can be recaptured by short reads that simulate the pyrosequencer outputs. We find that different taxonomic assignment methods vary radically in their ability to recapture the taxonomic information in full-length 16S rRNA sequences: most methods are sensitive to the region of the 16S rRNA gene that is targeted for sequencing, but many combinations of methods and rRNA regions produce consistent and accurate results. To process large datasets of partial 16S rRNA sequences obtained from surveys of various microbial communities, including those from human body habitats, we recommend the use of Greengenes or RDP classifier with fragments of at least 250 bases, starting from one of the primers R357, R534, R798, F343 or F517. PMID:18723574

  2. A backward view from 16S rRNA to archaea to the universal tree of life to progenotes: reminiscences of Carl Woese.

    PubMed

    Garrett, Roger A

    2014-01-01

    I first became aware of Carl Woese in the mid-1970s when he and George Fox criticized a few of the 16S rRNA oligonucleotide sequences emerging from Strasbourg in the 10-12 y RNA sequencing project of the first 16S rRNA from Escherichia coli, some of which we were using for assembling RNA binding sites of ribosomal proteins. When I realized that they were attempting to sequence 16S rRNAs from a range of bacteria to classify them phylogenetically, I seriously questioned their sanity. Not because of the goal, which was admirable, but because of the sheer technical difficulty, and slowness, of sequencing large RNA molecules using the original Sanger RNA sequencing method, not to mention the health hazards of regularly preparing rRNA using 20-30 mCi [ (32)P]. My view changed radically, however, with their subsequent prediction of 5S rRNA secondary structures using a phylogenetic approach. Previously, the molecular biology community had been competing to generate the maximum numbers of base pairs in the model RNA molecule E. coli 5S RNA when Fox and Woese introduced the concept of compensatory base changes based on phylogeny for defining secondary structure and applied it to 5S RNA, they found evidence for only about 50% base pairing. This approach had previously been used for tRNA secondary structure predictions but its more general significance had never been acknowledged. Carl subsequently persuaded Harry Noller to apply the same method to predicting secondary structures of the large rRNAs.

  3. 16S ribosomal DNA clone libraries to reveal bacterial diversity in anaerobic reactor-degraded tetrabromobisphenol A.

    PubMed

    Peng, Xingxing; Zhang, Zaili; Zhao, Ziling; Jia, Xiaoshan

    2012-05-01

    Microorganisms able to rapidly degrade tetrabromobisphenol A (TBBPA) were domesticated in an anaerobic reactor and added to gradually increased concentrations of TBBPA. After 240 days of domestication, the degradation rate reached 96.0% in cultivated batch experiments lasting 20 days. The optimum cultivating temperature and pH were 30°C and 7.0. The bacterial community's composition and diversity in the reactor was studied by comparative analysis with 16S ribosomal DNA clone libraries. Amplified rDNA restriction analysis of 200 clones from the library indicate that the rDNA richness was high (Coverage C 99.5%) and that evenness was not high (Shannon-Weaver index 2.42). Phylogenetic analysis of 63 bacterial sequences from the reactor libraries demonstrated the presence of Betaproteobacteria (33.1%), Gammaproteobacteria (18.7%), Bacteroidetes (13.9%), Firmicutes (11.4%), Chloroflexi (3.6%), Actinobacteria (0.6%), the candidate division TM7 (4.2%) and other unknown, uncultured bacterial groups (14.5%). Comamonas, Achromobacter, Pseudomonas and Flavobacterium were the dominant types.

  4. Diversity and phylogeny of bacteria on Zimbabwe tobacco leaves estimated by 16S rRNA sequence analysis.

    PubMed

    Su, Can; Gu, Wen; Zhe, Wei; Zhang, Ke-Qin; Duan, Yanqing; Yang, Jinkui

    2011-12-01

    Microorganisms play important roles in the tobacco aging process. However, microbial communities on flue-cured tobacco leaves (FCTL) remain largely unknown. In this study, the total microbial genomic DNA of unaged and aging FCTL from Zimbabwe were isolated using a culture-independent method, and the bacterial communities were investigated through analyzing two 16S rRNA gene libraries. Eighty-four and 65 operational taxonomic units were obtained from the libraries of the unaged and aging FCTL, respectively. The following genera were represented more than 4% in both libraries (aging and unaged library): Sphingomonas (4.84%, 4.18%), Stenotrophomonas (4.84%, 5.23%), Erwinia (5.81%, 4.88%), Pantoea (19.35%, 18.47%), and Pseudomonas (21.29%, 24.04%). The dominant species varied between the two libraries. Specifically, several dominant species in unaged FCTL including Pseudomonas fulva, Pseudomonas sp. (AM909658), Klebsiella sp. (HM584796), and Pantoea sp. (AY501386) were not identified in aging FCTL, while several dominant species in aging FCTL such as Pantoea sp. (GU566350), Pseudomonas sp. (EF157292), and Buttiauxella izardii were not found in unaged FCTL. The phylogenetic analysis showed that bacteria from unaged and aging FCTL were divided into two clades, and two unique subclades were identified in aging FCTL. Our results revealed for the first time the bacterial diversities on Zimbabwe tobacco, and provided a basis for clarifying the roles of bacteria in aging process of FCTL.

  5. Comparative Evaluation of Four Bacteria-Specific Primer Pairs for 16S rRNA Gene Surveys

    PubMed Central

    Thijs, Sofie; Op De Beeck, Michiel; Beckers, Bram; Truyens, Sascha; Stevens, Vincent; Van Hamme, Jonathan D.; Weyens, Nele; Vangronsveld, Jaco

    2017-01-01

    Bacterial taxonomic community analyses using PCR-amplification of the 16S rRNA gene and high-throughput sequencing has become a cornerstone in microbiology research. To reliably detect the members, or operational taxonomic units (OTUs), that make up bacterial communities, taxonomic surveys rely on the use of the most informative PCR primers to amplify the broad range of phylotypes present in up-to-date reference databases. However, primers specific for the domain Bacteria were often developed some time ago against database versions that are now out of date. Here we evaluated the performance of four bacterial primers for characterizing complex microbial communities in explosives contaminated and non-contaminated forest soil and by in silico evaluation against the current SILVA123 database. Primer pair 341f/785r produced the highest number of bacterial OTUs, phylogenetic richness, Shannon diversity, low non-specificity and most reproducible results, followed by 967f/1391r and 799f/1193r. Primer pair 68f/518r showed overall low coverage and a bias toward Alphaproteobacteria. In silico, primer pair 341f/785r showed the highest coverage of the domain Bacteria (96.1%) with no obvious bias toward the majority of bacterial species. This suggests the high utility of primer pair 341f/785r for soil and plant-associated bacterial microbiome studies.

  6. Research Techniques Made Simple: Bacterial 16S Ribosomal RNA Gene Sequencing in Cutaneous Research.

    PubMed

    Jo, Jay-Hyun; Kennedy, Elizabeth A; Kong, Heidi H

    2016-03-01

    Skin serves as a protective barrier and also harbors numerous microorganisms collectively comprising the skin microbiome. As a result of recent advances in sequencing (next-generation sequencing), our understanding of microbial communities on skin has advanced substantially. In particular, the 16S ribosomal RNA gene sequencing technique has played an important role in efforts to identify the global communities of bacteria in healthy individuals and patients with various disorders in multiple topographical regions over the skin surface. Here, we describe basic principles, study design, and a workflow of 16S ribosomal RNA gene sequencing methodology, primarily for investigators who are not familiar with this approach. This article will also discuss some applications and challenges of 16S ribosomal RNA sequencing as well as directions for future development.

  7. Routine Molecular Identification of Enterococci by Gene-Specific PCR and 16S Ribosomal DNA Sequencing

    PubMed Central

    Angeletti, Silvia; Lorino, Giulia; Gherardi, Giovanni; Battistoni, Fabrizio; De Cesaris, Marina; Dicuonzo, Giordano

    2001-01-01

    For 279 clinically isolated specimens identified by commercial kits as enterococci, genotypic identification was performed by two multiplex PCRs, one with ddlE. faecalis and ddlE. faecium primers and another with vanC-1 and vanC-2/3 primers, and by 16S ribosomal DNA (rDNA) sequencing. For 253 strains, phenotypic and genotypic results were the same. Multiplex PCR allowed for the identification of 13 discordant results. Six strains were not enterococci and were identified by 16S rDNA sequencing. For 5 discordant and 10 concordant enterococcal strains, 16S rDNA sequencing was needed. Because many supplementary tests are frequently necessary for phenotypic identification, the molecular approach is a good alternative. PMID:11158155

  8. Sequence of the 16S ribosomal RNA from Halobacterium volcanii, an archaebacterium

    NASA Technical Reports Server (NTRS)

    Gupta, R.; Lanter, J. M.; Woese, C. R.

    1983-01-01

    The sequence of the 16S ribosomal RNA (rRNA) from the archaebacterium Halobacterium volcanii has been determined by DNA sequencing methods. The archaebacterial rRNA is similar to its eubacterial counterpart in secondary structure. Although it is closer in sequence to the eubacterial 16S rRNA than to the eukaryotic 16S-like rRNA, the H. volcanii sequence also shows certain points of specific similarity to its eukaryotic counterpart. Since the H. volcanii sequence is closer to both the eubacterial and the eukaryotic sequences than these two are to one another, it follows that the archaebacterial sequence resembles their common ancestral sequence more closely than does either of the other two versions.

  9. Processing pathway of Escherichia coli 16S precursor rRNA.

    PubMed Central

    Srivastava, A K; Schlessinger, D

    1989-01-01

    Immediate precursors of 16S rRNA are processed by endonucleolytic cleavage at both 5' and 3' mature termini, with the concomitant release of precursor fragments which are further metabolized by both exo- and endonucleases. In wild-type cells rapid cleavages by RNase III in precursor-specific sequences precede the subsequent formation of the mature ends; mature termini can, however, be formed directly from pre-16S rRNA with no intermediate species. The direct maturation is most evident in a strain deficient in RNase III, and the results in whole cells are consistent with results from maturation reactions in vitro. Thus, maturation does not require cleavages within the double-stranded stems that enclose mature rRNA sequences in the pre-16S rRNA. Images PMID:2646597

  10. Enumeration of bacteria from the Clostridium leptum subgroup in human faecal microbiota using Clep1156 16S rRNA probe in combination with helper and competitor oligonucleotides.

    PubMed

    Saunier, Katiana; Rougé, Carole; Lay, Christophe; Rigottier-Gois, Lionel; Doré, Joël

    2005-07-01

    Target site inaccessibility represents a significant problem for fluorescent in situ hybridisation (FISH) of 16S rRNA oligonucleotide probes. For this reason, the Clep1156 probe targeting 16S rRNA of the Clostridium leptum phylogenetic subgroup used for dot blot experiments could not be used until now for FISH. Considering that bacteria from the C. leptum subgroup are very abundant in the human faecal microbiota and may play a significant role in host health, we have used unlabelled helper and competitor oligonucleotides to improve the 16S rRNA in situ accessibility and specificity of the Clep1156 probe and applied this approach to enumerate C. leptum bacteria in this ecosystem. Nine C. leptum target strains and five non-target strains were selected to develop and validate the helper-competitor strategy. Depending on the target strains, the use of helpers enhanced the fluorescence intensity signal of Clep1156 from 0.4-fold to 8.4-fold with a mean value of 3.6-fold, switching this probe from the brightness class V-VI (masked sites) to III-IV (accessible sites). The simultaneous use of helper and competitor oligonucleotides with Clep1156 probe allowed the expected specificity without disturbing in situ accessibility. Quantified by FISH combined with flow cytometry, C. leptum bacteria in human faecal samples (n=22) represented 19 +/- 7% of bacteria on average [4.9-37.5]. We conclude that helper oligonucleotides are very useful to circumvent the problem of target site in situ accessibility, especially when probe design is limited to only one 16S rRNA area and that helpers and competitors may be efficiently combined.

  11. Molecular characterization and in situ localization of endosymbiotic 16S ribosomal RNA and RuBisCO genes in the pogonophoran tissue.

    PubMed

    Kimura, Hiroyuki; Sato, Makoto; Sasayama, Yuichi; Naganuma, Takeshi

    2003-01-01

    Gutless pogonophorans are generally thought to live in symbiosis with methane-oxidizing bacteria (methanotrophs). We identified a 16S ribosomal RNA gene (rDNA) and a ribulose-1,5-bisphosphate carboxlase/oxygenase (RuBisCO, E.C.4.1.1.39) gene that encode the form I large subunit ( cbbL) from symbiont-bearing tissue of the pogonophoran Oligobrachia mashikoi. Phylogenetic analysis of the 16S rDNA sequence suggested that the pogonophoran endosymbiont belonged to the gamma-subdivision of Proteobacteria. The endosymbiont was most closely related to an uncultured bacterium from a hydrocarbon seep, forming a unique clade adjacent to the known methanotrophic 16S rDNA cluster. The RuBisCO gene from the pogonophoran tissue was closely related to those of the chemoautotrophic genera Thiobacillus and Hydrogenovibrio. Presence of the RuBisCO gene suggested a methanotrophic symbiosis because some methanotrophic bacteria are known to be capable of autotrophy via the Calvin cycle. In contrast, particulate and soluble methane monooxygenase genes ( pmoA and mmoX) and the methanol dehydrogenase gene ( mxaF), which are indicators for methanotrophs or methylotrophs, were not detected by repeated trial of polymerase chain reaction. For 16S rRNA and RuBisCO genes, endosymbiotic localizations were confirmed by in situ hybridization. These results support the possibilities that the pogonophoran host has a novel endosymbiont which belongs to the gamma-subdivision of Proteobacteria, and that the endosymbiont has the gene of the autotrophic enzyme RuBisCO.

  12. Phylogenetic analysis and delineation of phytoplasmas based on the secY gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The secY gene, located in the operator-distal part of the spc ribosomal protein operon, codes for a protein translocase subunit secY. The secY gene sequence is more variable than that of the 16S rRNA gene. Comparative phylogenetic analyses with 16S rRNA and secY gene sequences from 80 and 83 phytop...

  13. Molecular diversity of soil and marine 16S rRNA gene sequences related to beta-subgroup ammonia-oxidizing bacteria.

    PubMed

    Stephen, J R; McCaig, A E; Smith, Z; Prosser, J I; Embley, T M

    1996-11-01

    We have conducted a preliminary phylogenetic survey of ammonia-oxidizing beta-proteobacteria, using 16S rRNA gene libraries prepared by selective PCR and DNA from acid and neutral soils and polluted and nonpolluted marine sediments. Enrichment cultures were established from samples and analyzed by PCR. Analysis of 111 partial sequences of c. 300 bases revealed that the environmental sequences formed seven clusters, four of which are novel, within the phylogenetic radiation defined by cultured autotrophic ammonia oxidizers. Longer sequences from 13 cluster representatives support their phylogenetic positions relative to cultured taxa. These data suggest that known taxa may not be representative of the ammonia-oxidizing beta-proteobacteria in our samples. Our data provide further evidence that molecular and culture-based enrichment methods can select for different community members. Most enrichments contained novel Nitrosomonas-like sequences whereas novel Nitrosospira-like sequences were more common from gene libraries of soils and marine sediments. This is the first evidence for the occurrence of Nitrosospira-like strains in marine samples. Clear differences between the sequences of soil and marine sediment libraries were detected. Comparison of 16S rRNA sequences from polluted and nonpolluted sediments provided no strong evidence that the community composition was determined by the degree of pollution. Soil clone sequences fell into four clusters, each containing sequences from acid and neutral soils in varying proportions. Our data suggest that some related strains may be present in both samples, but further work is needed to resolve whether there is selection due to pH for particular sequence types.

  14. Effect of gemini (alkanediyl-α,ω-bis(dimethylcetylammonium bromide)) (16-s-16, s=4, 5, 6) surfactants on the interaction of ninhydrin with chromium-glycylphenylalanine.

    PubMed

    Kumar, Dileep; Rub, Malik Abdul; Akram, Mohd; Kabir-ud-Din

    2014-11-11

    The effect of gemini (alkanediyl-α,ω-bis(dimethylcetylammonium bromide)) (16-s-16, s=4, 5, 6) surfactants on the interaction of ninhydrin with chromium(III) complex of glycylphenylalanine ([Cr(III)-Gly-Phe]2+) has been investigated using UV-visible spectrophotometer at different temperatures. The order of reaction with respect to [Cr(III)-Gly-Phe]2+ is unity while it is fractional with respect to ninhydrin. Whereas, the values of rate constant (kψ) increase and leveling-off regions, like conventional single chain cetyltrimethylammonium bromide (CTAB) surfactant, were observed with geminis, later produces a third region of increasing kψ at higher gemini surfactant concentrations. This unusual third-region effect of the gemini micelles is assigned to changes in their micellar morphologies. The results obtained in micellar media were treated in terms of pseudo-phase model. The values of thermodynamic parameters (Ea, ΔH# and ΔS#) and binding constants (KA and KNin) have been evaluated.

  15. Phylogenetic relationships among Ehrlichia ruminantium isolates.

    PubMed

    Allsopp, M T E P; Van Heerden, H; Steyn, H C; Allsopp, B A

    2003-06-01

    Ehrlichia ruminantium, the causative agent of heartwater, is a tick-borne pathogen infecting ruminants throughout sub-Saharan Africa and on some Caribbean islands. The most reliable test for E. ruminantium is PCR-based, but this gives positive results in some areas free of clinical heartwater and of the known Amblyomma spp. tick vectors. To investigate the molecular basis for this finding we have sequenced and carried out phylogenetic analysis of a range of genes from a number of E. ruminantium isolates. The genes include ribonuclease III and cytochrome c oxidase assembly protein genes (the pCS20 region), groESL, citrate synthase (gltA), and 16S ribosomal RNA. Relationships among major antigenic protein (map1) genes have been exhaustively investigated in a previous study that showed that the genes are variable in length, have non-synonymous mutations, and show no geographical specificity among isolates. The 16S sequences are highly conserved, except in the V1 loop region. The pCS20, groESL, and gltA genes show only single nucleotide polymorphisms (SNPs) dispersed throughout the sequenced regions. Phylogenetic analysis using pCS20 data differentiates the western African isolates into a single clade, which also includes a southern African isolate. All other southern African isolates and a Caribbean isolate fall into a further clade, which is subdivided into two groups. Sequence variation within this clade is greater than that within the western African clade, suggesting that E. ruminantium originated in southern Africa.

  16. Genetic relatedness and phylogenetics of five Indian pufferfishes.

    PubMed

    Lakra, Wazir S; Goswami, Mukunda; Singh, Akhilesh

    2013-10-01

    The taxonomy and phylogeny of the pufferfishes belonging to the family Tetraodontidae found in India are poorly understood. We investigated five species of freshwater and marine pufferfishes using partial sequences of 16S rRNA and cytochrome c oxidase subunit I (COI) of mitochondrial genes. The sequence alignment of 16S rRNA yielded 573 bp, whereas COI gene sequence alignment yielded 614 bp. The sequence analysis of the genes revealed two distinct groups of freshwater and marine origin, which are genetically distinct from each other and exhibit identical phylogenetic resolution. The partial sequences of both the genes provided sufficient phylogenetic resolution to distinguish all the five species of pufferfishes. The COI sequences could be used as DNA barcodes for identification of the pufferfishes.

  17. Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays

    PubMed Central

    Small, Jack; Call, Douglas R.; Brockman, Fred J.; Straub, Timothy M.; Chandler, Darrell P.

    2001-01-01

    We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 μg of total RNA, representing approximately 7.5 × 106 Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR. PMID:11571176

  18. Ovine pedomics: the first study of the ovine foot 16S rRNA-based microbiome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report the first study of the bacterial microbiome of ovine interdigital skin based on 16S rRNA by pyrosequencing and conventional cloning with Sanger-sequencing. Ovine foot rot is an infectious, contagious disease of sheep that causes severe lameness and economic loss from decreased flock produc...

  19. Molecular Diagnosis of Actinomadura madurae Infection by 16S rRNA Deep Sequencing

    PubMed Central

    SenGupta, Dhruba J.; Hoogestraat, Daniel R.; Cummings, Lisa A.; Bryant, Bronwyn H.; Natividad, Catherine; Thielges, Stephanie; Monsaas, Peter W.; Chau, Mimosa; Barbee, Lindley A.; Rosenthal, Christopher; Cookson, Brad T.; Hoffman, Noah G.

    2013-01-01

    Next-generation DNA sequencing can be used to catalog individual organisms within complex, polymicrobial specimens. Here, we utilized deep sequencing of 16S rRNA to implicate Actinomadura madurae as the cause of mycetoma in a diabetic patient when culture and conventional molecular methods were overwhelmed by overgrowth of other organisms. PMID:24108607

  20. Molecular diagnosis of Actinomadura madurae infection by 16S rRNA deep sequencing.

    PubMed

    Salipante, Stephen J; Sengupta, Dhruba J; Hoogestraat, Daniel R; Cummings, Lisa A; Bryant, Bronwyn H; Natividad, Catherine; Thielges, Stephanie; Monsaas, Peter W; Chau, Mimosa; Barbee, Lindley A; Rosenthal, Christopher; Cookson, Brad T; Hoffman, Noah G

    2013-12-01

    Next-generation DNA sequencing can be used to catalog individual organisms within complex, polymicrobial specimens. Here, we utilized deep sequencing of 16S rRNA to implicate Actinomadura madurae as the cause of mycetoma in a diabetic patient when culture and conventional molecular methods were overwhelmed by overgrowth of other organisms.

  1. 16S rRNA region based PCR protocol for identification and subtyping of Parvimonas micra.

    PubMed

    Ota-Tsuzuki, C; Brunheira, A T P; Mayer, M P A

    2008-10-01

    The present study established a PCR protocol in order to identify Parvimonas micra and to evaluate the intra-species diversity by PCR-RFLP of 16S rRNA partial sequence. The data indicated that the protocol was able to identify this species which could be clustered in five genotypes.

  2. Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects

    PubMed Central

    Elbrecht, Vasco; Taberlet, Pierre; Dejean, Tony; Valentini, Alice; Usseglio-Polatera, Philippe; Beisel, Jean-Nicolas; Coissac, Eric; Boyer, Frederic

    2016-01-01

    Cytochrome c oxidase I (COI) is a powerful marker for DNA barcoding of animals, with good taxonomic resolution and a large reference database. However, when used for DNA metabarcoding, estimation of taxa abundances and species detection are limited due to primer bias caused by highly variable primer binding sites across the COI gene. Therefore, we explored the ability of the 16S ribosomal DNA gene as an alternative metabarcoding marker for species level assessments. Ten bulk samples, each containing equal amounts of tissue from 52 freshwater invertebrate taxa, were sequenced with the Illumina NextSeq 500 system. The 16S primers amplified three more insect species than the Folmer COI primers and amplified more equally, probably due to decreased primer bias. Estimation of biomass might be less biased with 16S than with COI, although variation in read abundances of two orders of magnitudes is still observed. According to these results, the marker choice depends on the scientific question. If the goal is to obtain a taxonomic identification at the species level, then COI is more appropriate due to established reference databases and known taxonomic resolution of this marker, knowing that a greater proportion of insects will be missed using COI Folmer primers. If the goal is to obtain a more comprehensive survey the 16S marker, which requires building a local reference database, or optimised degenerated COI primers could be more appropriate. PMID:27114891

  3. 16S rRNA region based PCR protocol for identification and subtyping of Parvimonas micra

    PubMed Central

    Ota-Tsuzuki, C.; Brunheira, A.T.P.; Mayer, M.P.A.

    2008-01-01

    The present study established a PCR protocol in order to identify Parvimonas micra and to evaluate the intra-species diversity by PCR-RFLP of 16S rRNA partial sequence. The data indicated that the protocol was able to identify this species which could be clustered in five genotypes. PMID:24031274

  4. Problem-Based Test: Functional Analysis of Mutant 16S rRNAs

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2010-01-01

    Terms to be familiar with before you start to solve the test: ribosome, ribosomal subunits, antibiotics, point mutation, 16S, 5S, and 23S rRNA, Shine-Dalgarno sequence, mRNA, tRNA, palindrome, hairpin, restriction endonuclease, fMet-tRNA, peptidyl transferase, initiation, elongation, termination of translation, expression plasmid, transformation,…

  5. Phylogenetic Relationships and Coaggregation Ability of Freshwater Biofilm Bacteria

    PubMed Central

    Rickard, Alex H.; Leach, Stephen A.; Hall, Laurence S.; Buswell, Clive M.; High, Nicola J.; Handley, Pauline S.

    2002-01-01

    Nineteen numerically dominant heterotrophic bacteria from a freshwater biofilm were identified by 16S ribosomal DNA gene sequencing, and their coaggregation partnerships were determined. Phylogenetic trees showed that both distantly related and closely related strains coaggregated at intergeneric, intrageneric, and intraspecies levels. One strain, Blastomonas natatoria 2.1, coaggregated with all 18 other strains and may function as a bridging organism in biofilm development. PMID:12089055

  6. Inter- and intraspecific genomic variability of the 16S-23S intergenic spacer regions (ISR) in representatives of Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans.

    PubMed

    Ni, Yong-Qing; Yang, Yuan; Bao, Jing-Ting; He, Kai-Yu; Li, Hong-Yu

    2007-05-01

    The complete sequences of 32 intergenic spacer regions (ISR) from Acidithiobacillus strains, including 29 field strains isolated from coal, copper, molybdenum mine wastes or sediment of different geoclimatic regions in China, reference strain ATCC19859 and the type strains of the two species were determined. These data, together with other sequences available in the GenBank database, were used to carry out the first detailed assessment of the inter- and intraspecific genomic variability of the ISR sequences and to infer phylogenetic relationships within the genus. The total length of the 16S-23S rRNA intergenic spacer regions of the Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans strains ranged from 451 to 490 bp, and from 434 to 456 bp, respectively. The degree of intrageneric ISR sequence similarity was higher than the degree of intergeneric similarity, and the overall similarity values of the ISRs varied from 60.49% to 84.71% between representatives of different species of the genus Acidithiobacillus. Sequences from the spacer of the A. thiooxidans and A. ferrooxidans strains ranged from 86.71% to 99.56% and 92.36% to 100% similarity, respectively. All Acidithiobacillus strains were separated into three phylogenetic major clusters and seven phylogenetic groups. ISR may be a potential target for the development of in situ hybridization probe aimed at accurately detecting acidithiobacilli in the various acidic environments.

  7. Subcuticular bacteria associated with two common New Zealand echinoderms: Characterization using 16S rRNA sequence analysis and fluorescence in situ hybridization.

    PubMed

    Lawrence, Scott A; O'Toole, Ronan; Taylor, Michael W; Davy, Simon K

    2010-02-01

    Many echinoderms contain subcuticular bacteria (SCB), symbionts which reside in the lumen between the host's epidermal cells and outer cuticle. This relationship is common, existing in about 60% of echinoderms studied so far, yet the function of SCB remains largely unknown. In this study, phylogenetic analysis was carried out on 16S rRNA sequences obtained from echinoderm-associated bacteria, resulting in the identification of four species of putative SCB. All four bacteria were identified from the holothurian Stichopus mollis, and two of the four were also found in the asteroid Patiriella sp. Two of these bacteria belong to the Alphaproteobacteria, and two to the Gammaproteobacteria. In addition to phylogenetic analysis, fluorescence in situ hybridization (FISH) assays were carried out on Patiriella sp., S. mollis, and the asteroid Astrostole scabra. Results showed that Patiriella sp. and S. mollis contain SCB, in agreement with the phylogenetic analysis, while SCB were not detected in A. scabra. Of the bacteria detected using FISH, more than 80% were recognized as belonging to the Alphaproteobacteria in both host species. However, in S. mollis about 20% of the detected SCB successfully hybridized with the Gammaproteobacteria-specific probe, whereas bacteria belonging to this class were never observed in Patiriella sp. This is only the second study to characterize SCB by molecular means, and is the first to identify SCB in situ using FISH.

  8. 'Candidatus Phytoplasma pruni', a novel taxon associated with X-disease of stone fruits, Prunus spp.: multilocus characterization based on 16S rRNA, secY, and ribosomal protein genes.

    PubMed

    Davis, Robert E; Zhao, Yan; Dally, Ellen L; Lee, Ing-Ming; Jomantiene, Rasa; Douglas, Sharon M

    2013-02-01

    X-disease is one of the most serious diseases known in peach (Prunus persica). Based on RFLP analysis of 16S rRNA gene sequences, peach X-disease phytoplasma strains from eastern and western United States and eastern Canada were classified in 16S rRNA gene RFLP group 16SrIII, subgroup A. Phylogenetic analyses of 16S rRNA gene sequences revealed that the X-disease phytoplasma strains formed a distinct subclade within the phytoplasma clade, supporting the hypothesis that they represented a lineage distinct from those of previously described 'Candidatus Phytoplasma' species. Nucleotide sequence alignments revealed that all studied X-disease phytoplasma strains shared less than 97.5 % 16S rRNA gene sequence similarity with previously described 'Candidatus Phytoplasma' species. Based on unique properties of the DNA, we propose recognition of X-disease phytoplasma strain PX11CT1(R) as representative of a novel taxon, 'Candidatus Phytoplasma pruni'. Results from nucleotide and phylogenetic analyses of secY and ribosomal protein (rp) gene sequences provided additional molecular markers of the 'Ca. Phytoplasma pruni' lineage. We propose that the term 'Ca. Phytoplasma pruni' be applied to phytoplasma strains whose 16S rRNA gene sequences contain the oligonucleotide sequences of unique regions that are designated in the formally published description of the taxon. Such strains include X-disease phytoplasma and--within the tolerance of a single base difference in one unique sequence--peach rosette, peach red suture, and little peach phytoplasmas. Although not employed for taxon delineation in this work, we further propose that secY, rp, and other genetic loci from the reference strain of a taxon, and where possible oligonucleotide sequences of unique regions of those genes that distinguish taxa within a given 16Sr group, be incorporated in emended descriptions and as part of future descriptions of 'Candidatus Phytoplasma' taxa.

  9. 16S rRNA beacons for bacterial monitoring during human space missions.

    PubMed

    Larios-Sanz, Maia; Kourentzi, Katerina D; Warmflash, David; Jones, Jeffrey; Pierson, Duane L; Willson, Richard C; Fox, George E

    2007-04-01

    Microorganisms are unavoidable in space environments and their presence has, at times, been a source of problems. Concerns about disease during human space missions are particularly important considering the significant changes the immune system incurs during spaceflight and the history of microbial contamination aboard the Mir space station. Additionally, these contaminants may have adverse effects on instrumentation and life-support systems. A sensitive, highly specific system to detect, characterize, and monitor these microbial populations is essential. Herein we describe a monitoring approach that uses 16S rRNA targeted molecular beacons to successfully detect several specific bacterial groupings. This methodology will greatly simplify in-flight monitoring by minimizing sample handling and processing. We also address and provide solutions to target accessibility problems encountered in hybridizations that target 16S rRNA.

  10. An intron within the 16S ribosomal RNA gene of the archaeon Pyrobaculum aerophilum

    NASA Technical Reports Server (NTRS)

    Burggraf, S.; Larsen, N.; Woese, C. R.; Stetter, K. O.

    1993-01-01

    The 16S rRNA genes of Pyrobaculum aerophilum and Pyrobaculum islandicum were amplified by the polymerase chain reaction, and the resulting products were sequenced directly. The two organisms are closely related by this measure (over 98% similar). However, they differ in that the (lone) 16S rRNA gene of Pyrobaculum aerophilum contains a 713-bp intron not seen in the corresponding gene of Pyrobaculum islandicum. To our knowledge, this is the only intron so far reported in the small subunit rRNA gene of a prokaryote. Upon excision the intron is circularized. A secondary structure model of the intron-containing rRNA suggests a splicing mechanism of the same type as that invoked for the tRNA introns of the Archaea and Eucarya and 23S rRNAs of the Archaea. The intron contains an open reading frame whose protein translation shows no certain homology with any known protein sequence.

  11. A renaissance for the pioneering 16S rRNA gene

    SciTech Connect

    Tringe, Susannah; Hugenholtz, Philip

    2008-09-07

    Culture-independent molecular surveys using the 16S rRNA gene have become a mainstay for characterizing microbial community structure over the last quarter century. More recently this approach has been overshadowed by metagenomics, which provides a global overview of a community's functional potential rather than just an inventory of its inhabitants. However, the pioneering 16S rRNA gene is making a comeback in its own right thanks to a number of methodological advancements including higher resolution (more sequences), analysis of multiple related samples (e.g. spatial and temporal series) and improved metadata and use of metadata. The standard conclusion that microbial ecosystems are remarkably complex and diverse is now being replaced by detailed insights into microbial ecology and evolution based only on this one historically important marker gene.

  12. An update of the structure and 16S rRNA gene sequence-based definition of higher ranks of the class Actinobacteria, with the proposal of two new suborders and four new families and emended descriptions of the existing higher taxa.

    PubMed

    Zhi, Xiao-Yang; Li, Wen-Jun; Stackebrandt, Erko

    2009-03-01

    The higher ranks of the class Actinobacteria were proposed and described in 1997. At each rank, the taxa were delineated from each other solely on the basis of 16S rRNA gene sequence phylogenetic clustering and taxon-specific 16S rRNA signature nucleotides. In the past 10 years, many novel members have been assigned to this class while, at the same time, some members have been reclassified. The new 16S rRNA gene sequence information and the changes in phylogenetic positions of some taxa influence decisions about which 16S rRNA nucleotides to define as taxon-specific. As a consequence, the phylogenetic relationships of Actinobacteria at higher levels may need to be reconstructed. Here, we present new 16S rRNA signature nucleotide patterns of taxa above the family level and indicate the affiliation of genera to families. These sets replace the signatures published in 1997. In addition, Actinopolysporineae subord. nov. and Actinopolysporaceae fam. nov. are proposed to accommodate the genus Actinopolyspora, Kineosporiineae subord. nov. and Kineosporiaceae fam. nov. are proposed to accommodate the genera Kineococcus, Kineosporia and Quadrisphaera, Beutenbergiaceae fam. nov. is proposed to accommodate the genera Beutenbergia, Georgenia and Salana and Cryptosporangiaceae fam. nov. is proposed to accommodate the genus Cryptosporangium. The families Nocardiaceae and Gordoniaceae are proposed to be combined in an emended family Nocardiaceae. Emended descriptions are also proposed for most of the other higher taxa.

  13. [Characterization of Black and Dichothrix Cyanobacteria Based on the 16S Ribosomal RNA Gene Sequence

    NASA Technical Reports Server (NTRS)

    Ortega, Maya

    2010-01-01

    My project focuses on characterizing different cyanobacteria in thrombolitic mats found on the island of Highborn Cay, Bahamas. Thrombolites are interesting ecosystems because of the ability of bacteria in these mats to remove carbon dioxide from the atmosphere and mineralize it as calcium carbonate. In the future they may be used as models to develop carbon sequestration technologies, which could be used as part of regenerative life systems in space. These thrombolitic communities are also significant because of their similarities to early communities of life on Earth. I targeted two cyanobacteria in my research, Dichothrix spp. and whatever black is, since they are believed to be important to carbon sequestration in these thrombolitic mats. The goal of my summer research project was to molecularly identify these two cyanobacteria. DNA was isolated from each organism through mat dissections and DNA extractions. I ran Polymerase Chain Reactions (PCR) to amplify the 16S ribosomal RNA (rRNA) gene in each cyanobacteria. This specific gene is found in almost all bacteria and is highly conserved, meaning any changes in the sequence are most likely due to evolution. As a result, the 16S rRNA gene can be used for bacterial identification of different species based on the sequence of their 16S rRNA gene. Since the exact sequence of the Dichothrix gene was unknown, I designed different primers that flanked the gene based on the known sequences from other taxonomically similar cyanobacteria. Once the 16S rRNA gene was amplified, I cloned the gene into specialized Escherichia coli cells and sent the gene products for sequencing. Once the sequence is obtained, it will be added to a genetic database for future reference to and classification of other Dichothrix sp.

  14. Greengenes: Chimera-checked 16S rRNA gene database and workbenchcompatible in ARB

    SciTech Connect

    DeSantis, T.Z.; Hugenholtz, P.; Larsen, N.; Rojas, M.; Brodie,E.L; Keller, K.; Huber, T.; Dalevi, D.; Hu, P.; Andersen, G.L.

    2006-02-01

    A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera-screening, standard alignments and taxonomic classification using multiple published taxonomies. It was revealed that incongruent taxonomic nomenclature exists among curators even at the phylum-level. Putative chimeras were identified in 3% of environmental sequences and 0.2% of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages within the Archaea and Bacteria.

  15. Molecular characterization of nocardioform actinomycetes in activated sludge by 16S rRNA analysis.

    PubMed

    Schuppler, M; Mertens, F; Schön, G; Göbel, U B

    1995-02-01

    The analysis of complex microbiota present in activated sludge is important for the understanding and possible control of severe separation problems in sewage treatment such as sludge bulking or sludge foaming. Previous studies have shown that nocardioform actinomycetes are responsible for these conditions, which not only affect the efficiency of sewage treatment but also represent a threat to public health due to spread of pathogens. However, isolation and identification of these filamentous, nocardioform actinomycetes is hampered by their fastidious nature. Most species are still uncultivable and their taxonomy is unresolved. To study the ecology of these micro-organisms at the molecular level, we have established a clone library of 16S rRNA gene fragments amplified from bulk sludge DNA. A rough indication of the predominant flora in the sludge was given by sequencing randomly chosen clones, which revealed a great diversity of bacteria from different taxa. Colony hybridization with oligonucleotide probe MNP1 detected 27 clones with 16S rDNA inserts from nocardioform actinomycetes and mycobacteria. The sequence data from these clones together with those from randomly chosen clones were used for comparative 16S rRNA analysis and construction of dendrograms. All sequences differed from those of previously sequenced species in the databases. Phenotypic characterization of isolates of nocardioform actinomycetes and mycobacteria cultivated in parallel from the same activated-sludge sample revealed a large discrepancy between the two approaches. Only one 16S rDNA sequence of a cultured isolate was represented in the clone library, indicating that culture conditions could select species which represent only a small fraction of the organisms in the activated sludge.

  16. Phenotypic characterisation and 16S rRNA sequence analysis of veterinary isolates of Streptococcus pluranimalium.

    PubMed

    Twomey, D F; Carson, T; Foster, G; Koylass, M S; Whatmore, A M

    2012-05-01

    Forty-two isolates of Streptococcus pluranimalium were identified from cattle (n=38), sheep (n=2), an alpaca (n=1) and a pheasant (n=1) in the United Kingdom. The isolates were confirmed as S. pluranimalium by 16S rRNA sequence analysis but could not be differentiated reliably from Streptococcus acidominimus by phenotypic characterisation using commercial kits routinely used in veterinary laboratories. The alanyl-phenylalanyl-proline arylamidase reaction could be used to differentiate S. pluranimalium (positive) from Aerococcus urinae (negative).

  17. How conserved are the conserved 16S-rRNA regions?

    PubMed Central

    Ortiz Suarez, Luis Enrique

    2017-01-01

    The 16S rRNA gene has been used as master key for studying prokaryotic diversity in almost every environment. Despite the claim of several researchers to have the best universal primers, the reality is that no primer has been demonstrated to be truly universal. This suggests that conserved regions of the gene may not be as conserved as expected. The aim of this study was to evaluate the conservation degree of the so-called conserved regions flanking the hypervariable regions of the 16S rRNA gene. Data contained in SILVA database (release 123) were used for the study. Primers reported as matches of each conserved region were assembled to form contigs; sequences sizing 12 nucleotides (12-mers) were extracted from these contigs and searched into the entire set of SILVA sequences. Frequency analysis shown that extreme regions, 1 and 10, registered the lowest frequencies. 12-mer frequencies revealed segments of contigs that were not as conserved as expected (≤90%). Fragments corresponding to the primer contigs 3, 4, 5b and 6a were recovered from all sequences in SILVA database. Nucleotide frequency analysis in each consensus demonstrated that only a small fraction of these so-called conserved regions is truly conserved in non-redundant sequences. It could be concluded that conserved regions of the 16S rRNA gene exhibit considerable variation that has to be considered when using this gene as biomarker. PMID:28265511

  18. Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing.

    PubMed

    Ranjan, Ravi; Rani, Asha; Metwally, Ahmed; McGee, Halvor S; Perkins, David L

    2016-01-22

    The human microbiome has emerged as a major player in regulating human health and disease. Translational studies of the microbiome have the potential to indicate clinical applications such as fecal transplants and probiotics. However, one major issue is accurate identification of microbes constituting the microbiota. Studies of the microbiome have frequently utilized sequencing of the conserved 16S ribosomal RNA (rRNA) gene. We present a comparative study of an alternative approach using whole genome shotgun sequencing (WGS). In the present study, we analyzed the human fecal microbiome compiling a total of 194.1 × 10(6) reads from a single sample using multiple sequencing methods and platforms. Specifically, after establishing the reproducibility of our methods with extensive multiplexing, we compared: 1) The 16S rRNA amplicon versus the WGS method, 2) the Illumina HiSeq versus MiSeq platforms, 3) the analysis of reads versus de novo assembled contigs, and 4) the effect of shorter versus longer reads. Our study demonstrates that whole genome shotgun sequencing has multiple advantages compared with the 16S amplicon method including enhanced detection of bacterial species, increased detection of diversity and increased prediction of genes. In addition, increased length, either due to longer reads or the assembly of contigs, improved the accuracy of species detection.

  19. Detection of bacterial 16S ribosomal RNA genes for forensic identification of vaginal fluid.

    PubMed

    Akutsu, Tomoko; Motani, Hisako; Watanabe, Ken; Iwase, Hirotaro; Sakurada, Koichi

    2012-05-01

    To preliminarily evaluate the applicability of bacterial DNA as a marker for the forensic identification of vaginal fluid, we developed and performed PCR-based detection of 16S ribosomal RNA genes of Lactobacillus spp. dominating the vagina and of bacterial vaginosis-related bacteria from DNA extracted from body fluids and stains. As a result, 16S ribosomal RNA genes of Lactobacillus crispatus, Lactobacillus jensenii and Atopobium vaginae were specifically detected in vaginal fluid and female urine samples. Bacterial genes detected in female urine might have originated from contaminated vaginal fluid. In addition, those of Lactobacillus iners, Lactobacillus gasseri and Gardnerella vaginalis were also detected in non-vaginal body fluids such as semen. Because bacterial genes were successfully amplified in DNA samples extracted by using the general procedure for animal tissues without any optional treatments, DNA samples prepared for the identification of vaginal fluid can also be used for personal identification. In conclusion, 16S ribosomal RNA genes of L. crispatus, L. jensenii and A. vaginae could be effective markers for forensic identification of vaginal fluid.

  20. The Role of 16S rRNA Gene Sequencing in Confirmation of Suspected Neonatal Sepsis

    PubMed Central

    El Gawhary, Somaia; El-Anany, Mervat; Ali, Doaa; El Gameel, El Qassem

    2016-01-01

    Different molecular assays for the detection of bacterial DNA in the peripheral blood represented a diagnostic tool for neonatal sepsis. We targeted to evaluate the role of 16S rRNA gene sequencing to screen for bacteremia to confirm suspected neonatal sepsis (NS) and compare with risk factors and septic screen testing. Sixty-two neonates with suspected NS were enrolled. White blood cells count, I/T ratio, C-reactive protein, blood culture and 16S rRNA sequencing were performed. Blood culture was positive in 26% of cases, and PCR was positive in 26% of cases. Evaluation of PCR for the diagnosis of NS showed sensitivity 62.5%, specificity 86.9%, PPV 62.5%, NPV 86.9% and accuracy of 79.7%. 16S rRNA PCR increased the sensitivity of detecting bacterial DNA in newborns with signs of sepsis from 26 to 35.4%, and its use can be limited to cases with the most significant risk factors and positive septic screen. PMID:26494728

  1. Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing

    PubMed Central

    Ranjan, Ravi; Rani, Asha; Metwally, Ahmed; McGee, Halvor S.; Perkins, David L.

    2016-01-01

    The human microbiome has emerged as a major player in regulating human health and disease. Translation studies of the microbiome have the potential to indicate clinical applications such as fecal transplants and probiotics. However, one major issue is accurate identification of microbes constituting the microbiota. Studies of the microbiome have frequently utilized sequencing of the conserved 16S ribosomal RNA (rRNA) gene. We present a comparative study of an alternative approach using shotgun whole genome sequencing (WGS). In the present study, we analyzed the human fecal microbiome compiling a total of 194.1×106 reads from a single sample using multiple sequencing methods and platforms. Specifically, after establishing the reproducibility of our methods with extensive multiplexing, we compared: 1) The 16S rRNA amplicon versus the WGS method, 2) the Illumina HiSeq versus MiSeq platforms, 3) the analysis of reads versus de novo assembled contigs, and 4) the effect of shorter versus longer reads. Our study demonstrates that shotgun whole genome sequencing has multiple advantages compared with the 16S amplicon method including enhanced detection of bacterial species, increased detection of diversity and increased prediction of genes. In addition, increased length, either due to longer reads or the assembly of contigs, improved the accuracy of species detection. PMID:26718401

  2. Simultaneous discrimination between 15 fish pathogens by using 16S ribosomal DNA PCR and DNA microarrays.

    PubMed

    Warsen, Adelaide E; Krug, Melissa J; LaFrentz, Stacey; Stanek, Danielle R; Loge, Frank J; Call, Douglas R

    2004-07-01

    We developed a DNA microarray suitable for simultaneous detection and discrimination between multiple bacterial species based on 16S ribosomal DNA (rDNA) polymorphisms using glass slides. Microarray probes (22- to 31-mer oligonucleotides) were spotted onto Teflon-masked, epoxy-silane-derivatized glass slides using a robotic arrayer. PCR products (ca. 199 bp) were generated using biotinylated, universal primer sequences, and these products were hybridized overnight (55 degrees C) to the microarray. Targets that annealed to microarray probes were detected using a combination of Tyramide Signal Amplification and Alexa Fluor 546. This methodology permitted 100% specificity for detection of 18 microbes, 15 of which were fish pathogens. With universal 16S rDNA PCR (limited to 28 cycles), detection sensitivity for purified control DNA was equivalent to <150 genomes (675 fg), and this sensitivity was not adversely impacted either by the presence of competing bacterial DNA (1.1 x 10(6) genomes; 5 ng) or by the addition of up to 500 ng of fish DNA. Consequently, coupling 16S rDNA PCR with a microarray detector appears suitable for diagnostic detection and surveillance for commercially important fish pathogens.

  3. The Role of 16S rRNA Gene Sequencing in Confirmation of Suspected Neonatal Sepsis.

    PubMed

    El Gawhary, Somaia; El-Anany, Mervat; Hassan, Reem; Ali, Doaa; El Gameel, El Qassem

    2016-02-01

    Different molecular assays for the detection of bacterial DNA in the peripheral blood represented a diagnostic tool for neonatal sepsis. We targeted to evaluate the role of 16S rRNA gene sequencing to screen for bacteremia to confirm suspected neonatal sepsis (NS) and compare with risk factors and septic screen testing. Sixty-two neonates with suspected NS were enrolled. White blood cells count, I/T ratio, C-reactive protein, blood culture and 16S rRNA sequencing were performed. Blood culture was positive in 26% of cases, and PCR was positive in 26% of cases. Evaluation of PCR for the diagnosis of NS showed sensitivity 62.5%, specificity 86.9%, PPV 62.5%, NPV 86.9% and accuracy of 79.7%. 16S rRNA PCR increased the sensitivity of detecting bacterial DNA in newborns with signs of sepsis from 26 to 35.4%, and its use can be limited to cases with the most significant risk factors and positive septic screen.

  4. Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification.

    PubMed

    Ziesemer, Kirsten A; Mann, Allison E; Sankaranarayanan, Krithivasan; Schroeder, Hannes; Ozga, Andrew T; Brandt, Bernd W; Zaura, Egija; Waters-Rist, Andrea; Hoogland, Menno; Salazar-García, Domingo C; Aldenderfer, Mark; Speller, Camilla; Hendy, Jessica; Weston, Darlene A; MacDonald, Sandy J; Thomas, Gavin H; Collins, Matthew J; Lewis, Cecil M; Hofman, Corinne; Warinner, Christina

    2015-11-13

    To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341-534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.

  5. Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification

    PubMed Central

    Ziesemer, Kirsten A.; Mann, Allison E.; Sankaranarayanan, Krithivasan; Schroeder, Hannes; Ozga, Andrew T.; Brandt, Bernd W.; Zaura, Egija; Waters-Rist, Andrea; Hoogland, Menno; Salazar-García, Domingo C.; Aldenderfer, Mark; Speller, Camilla; Hendy, Jessica; Weston, Darlene A.; MacDonald, Sandy J.; Thomas, Gavin H.; Collins, Matthew J.; Lewis, Cecil M.; Hofman, Corinne; Warinner, Christina

    2015-01-01

    To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341–534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions. PMID:26563586

  6. PCR Conditions for 16S Primers for Analysis of Microbes in the Colon of Rats

    PubMed Central

    Camacho, H.; Tuero, A. D.; Bacardí, D.; Palenzuela, D. O.; Aguilera, A.; Silva, J. A.; Estrada, R.; Gell, O.; Suárez, J.; Ancizar, J.; Brown, E.; Colarte, A. B.; Castro, J.; Novoa, L. I.

    2016-01-01

    The study of the composition of the intestinal flora is important to the health of the host, playing a key role in maintaining intestinal homeostasis and the evolution of the immune system. For these studies, various universal primers of the 16S rDNA gene are used in microbial taxonomy. Here, we report an evaluation of 5 universal primers to explore the presence of microbial DNA in colon biopsies preserved in RNAlater solution. The DNA extracted was used for the amplification of PCR products containing the variable (V) regions of the microbial 16S rDNA gene. The PCR products were studied by restriction fragment length polymorphism (RFLP) analysis and DNA sequence, whose percent of homology with microbial sequences reported in GenBank was verified using bioinformatics tools. The presence of microbes in the colon of rats was quantified by the quantitative PCR (qPCR) technique. We obtained microbial DNA from rat, useful for PCR analysis with the universal primers for the bacteria 16S rDNA. The sequences of PCR products obtained from a colon biopsy of the animal showed homology with the classes bacilli (Lactobacillus spp) and proteobacteria, normally represented in the colon of rats. The proposed methodology allowed the attainment of DNA of bacteria with the quality and integrity for use in qPCR, sequencing, and PCR-RFLP analysis. The selected universal primers provided knowledge of the abundance of microorganisms and the formation of a preliminary test of bacterial diversity in rat colon biopsies. PMID:27382362

  7. Processing of Escherichia coli 16S rRNA with bacteriophage lambda leader sequences.

    PubMed Central

    Krych, M; Sirdeshmukh, R; Gourse, R; Schlessinger, D

    1987-01-01

    To test whether any specific 5' precursor sequences are required for the processing of pre-16S rRNA, constructs were studied in which large parts of the 5' leader sequence were replaced by the coliphage lambda pL promoter and adjacent sequences. Unexpectedly, few full-length transcripts of the rRNA were detected after the pL promoter was induced, implying that either transcription was poor or most of the rRNA chains with lambda leader sequences were unstable. Nevertheless, sufficient transcription occurred to permit the detection of processing by S1 nuclease analysis. RNA transcripts in which 2/3 of the normal rRNA leader was deleted (from the promoter up to the normal RNase III cleavage site) were processed to form the normal 5' terminus. Thus, most of the double-stranded stem that forms from sequences bracketing wild-type 16S pre-rRNA is apparently not required for proper processing; the expression of such modified transcripts, however, must be increased before the efficiency of processing of the 16S rRNA formed can be assessed. Images PMID:2445728

  8. Interpreting 16S metagenomic data without clustering to achieve sub-OTU resolution

    PubMed Central

    Tikhonov, Mikhail; Leach, Robert W; Wingreen, Ned S

    2015-01-01

    The standard approach to analyzing 16S tag sequence data, which relies on clustering reads by sequence similarity into Operational Taxonomic Units (OTUs), underexploits the accuracy of modern sequencing technology. We present a clustering-free approach to multi-sample Illumina data sets that can identify independent bacterial subpopulations regardless of the similarity of their 16S tag sequences. Using published data from a longitudinal time-series study of human tongue microbiota, we are able to resolve within standard 97% similarity OTUs up to 20 distinct subpopulations, all ecologically distinct but with 16S tags differing by as little as one nucleotide (99.2% similarity). A comparative analysis of oral communities of two cohabiting individuals reveals that most such subpopulations are shared between the two communities at 100% sequence identity, and that dynamical similarity between subpopulations in one host is strongly predictive of dynamical similarity between the same subpopulations in the other host. Our method can also be applied to samples collected in cross-sectional studies and can be used with the 454 sequencing platform. We discuss how the sub-OTU resolution of our approach can provide new insight into factors shaping community assembly. PMID:25012900

  9. Strategy for microbiome analysis using 16S rRNA gene sequence analysis on the Illumina sequencing platform.

    PubMed

    Ram, Jeffrey L; Karim, Aos S; Sendler, Edward D; Kato, Ikuko

    2011-06-01

    Understanding the identity and changes of organisms in the urogenital and other microbiomes of the human body may be key to discovering causes and new treatments of many ailments, such as vaginosis. High-throughput sequencing technologies have recently enabled discovery of the great diversity of the human microbiome. The cost per base of many of these sequencing platforms remains high (thousands of dollars per sample); however, the Illumina Genome Analyzer (IGA) is estimated to have a cost per base less than one-fifth of its nearest competitor. The main disadvantage of the IGA for sequencing PCR-amplified 16S rRNA genes is that the maximum read-length of the IGA is only 100 bases; whereas, at least 300 bases are needed to obtain phylogenetically informative data down to the genus and species level. In this paper we describe and conduct a pilot test of a multiplex sequencing strategy suitable for achieving total reads of > 300 bases per extracted DNA molecule on the IGA. Results show that all proposed primers produce products of the expected size and that correct sequences can be obtained, with all proposed forward primers. Various bioinformatic optimization of the Illumina Bustard analysis pipeline proved necessary to extract the correct sequence from IGA image data, and these modifications of the data files indicate that further optimization of the analysis pipeline may improve the quality rankings of the data and enable more sequence to be correctly analyzed. The successful application of this method could result in an unprecedentedly deep description (800,000 taxonomic identifications per sample) of the urogenital and other microbiomes in a large number of samples at a reasonable cost per sample.

  10. Uncultured bacterial diversity in a seawater recirculating aquaculture system revealed by 16S rRNA gene amplicon sequencing.

    PubMed

    Lee, Da-Eun; Lee, Jinhwan; Kim, Young-Mog; Myeong, Jeong-In; Kim, Kyoung-Ho

    2016-04-01

    Bacterial diversity in a seawater recirculating aquaculture system (RAS) was investigated using 16S rRNA amplicon sequencing to understand the roles of bacterial communities in the system. The RAS was operated at nine different combinations of temperature (15°C, 20°C, and 25°C) and salinity (20‰, 25‰, and 32.5‰). Samples were collected from five or six RAS tanks (biofilters) for each condition. Fifty samples were analyzed. Proteobacteria and Bacteroidetes were most common (sum of both phyla: 67.2% to 99.4%) and were inversely proportional to each other. Bacteria that were present at an average of ≥ 1% included Actinobacteria (2.9%) Planctomycetes (2.0%), Nitrospirae (1.5%), and Acidobacteria (1.0%); they were preferentially present in packed bed biofilters, mesh biofilters, and maturation biofilters. The three biofilters showed higher diversity than other RAS tanks (aerated biofilters, floating bed biofilters, and fish tanks) from phylum to operational taxonomic unit (OTU) level. Samples were clustered into several groups based on the bacterial communities. Major taxonomic groups related to family Rhodobacteraceae and Flavobacteriaceae were distributed widely in the samples. Several taxonomic groups like [Saprospiraceae], Cytophagaceae, Octadecabacter, and Marivita showed a cluster-oriented distribution. Phaeobacter and Sediminicola-related reads were detected frequently and abundantly at low temperature. Nitrifying bacteria were detected frequently and abundantly in the three biofilters. Phylogenetic analysis of the nitrifying bacteria showed several similar OTUs were observed widely through the biofilters. The diverse bacterial communities and the minor taxonomic groups, except for Proteobacteria and Bacteroidetes, seemed to play important roles and seemed necessary for nitrifying activity in the RAS, especially in packed bed biofilters, mesh biofilters, and maturation biofilters.

  11. Profiling phylogenetic informativeness.

    PubMed

    Townsend, Jeffrey P

    2007-04-01

    The resolution of four controversial topics in phylogenetic experimental design hinges upon the informativeness of characters about the historical relationships among taxa. These controversies regard the power of different classes of phylogenetic character, the relative utility of increased taxonomic versus character sampling, the differentiation between lack of phylogenetic signal and a historical rapid radiation, and the design of taxonomically broad phylogenetic studies optimized by taxonomically sparse genome-scale data. Quantification of the informativeness of characters for resolution of phylogenetic hypotheses during specified historical epochs is key to the resolution of these controversies. Here, such a measure of phylogenetic informativeness is formulated. The optimal rate of evolution of a character to resolve a dated four-taxon polytomy is derived. By scaling the asymptotic informativeness of a character evolving at a nonoptimal rate by the derived asymptotic optimum, and by normalizing so that net phylogenetic informativeness is equivalent for all rates when integrated across all of history, an informativeness profile across history is derived. Calculation of the informativeness per base pair allows estimation of the cost-effectiveness of character sampling. Calculation of the informativeness per million years allows comparison across historical radiations of the utility of a gene for the inference of rapid adaptive radiation. The theory is applied to profile the phylogenetic informativeness of the genes BRCA1, RAG1, GHR, and c-myc from a muroid rodent sequence data set. Bounded integrations of the phylogenetic profile of these genes over four epochs comprising the diversifications of the muroid rodents, the mammals, the lobe-limbed vertebrates, and the early metazoans demonstrate the differential power of these genes to resolve the branching order among ancestral lineages. This measure of phylogenetic informativeness yields a new kind of information

  12. Cultivation-independent population analysis of bacterial endophytes in three potato varieties based on eubacterial and Actinomycetes-specific PCR of 16S rRNA genes.

    PubMed

    Sessitsch, Angela; Reiter, Birgit; Pfeifer, Ulrike; Wilhelm, Eva

    2002-01-01

    Abstract Endophytic bacteria are ubiquitous in most plants and colonise plants without exhibiting pathogenicity. Studies on the diversity of bacterial endophytes have been mainly approached by characterisation of isolates obtained from internal tissues. Despite the broad application of culture-independent techniques for the analysis of microbial communities in a wide range of natural habitats, little information is available on the species diversity of endophytes. In this study, microbial communities inhabiting stems, roots and tubers of three potato varieties were analysed by 16S rRNA-based techniques such as terminal restriction fragment length polymorphism analysis, denaturing gradient gel electrophoresis as well as 16S rDNA cloning and sequencing. Two individual plant experiments were conducted. In the first experiment plants suffered from light deficiency, whereas healthy and robust plants were obtained in the second experiment. Plants obtained from both experiments showed comparable endophytic populations, but healthy potato plants possessed a significantly higher diversity of endophytes than stressed plants. In addition, plant tissue and variety specific endophytes were detected. Sequence analysis of 16S rRNA genes indicated that a broad phylogenetic spectrum of bacteria is able to colonise plants internally including alpha-, beta-, and gamma-Proteobacteria, high-GC Gram-positives, microbes belonging to the Flexibacter/Cytophaga/Bacteroides group and Planctomycetales. Group-specific analysis of Actinomycetes indicated a higher abundance and diversity of Streptomyces scabiei-related species in the variety Mehlige Mühlviertler, which is known for its resistance against potato common scab caused by S. scabiei.

  13. Enterohemorrhagic Escherichia coli O157 in milk and dairy products from Libya: Isolation and molecular identification by partial sequencing of 16S rDNA

    PubMed Central

    Garbaj, Aboubaker M.; Awad, Enas M.; Azwai, Salah M.; Abolghait, Said K.; Naas, Hesham T.; Moawad, Ashraf A.; Gammoudi, Fatim T.; Barbieri, Ilaria; Eldaghayes, Ibrahim M.

    2016-01-01

    Aim: The aim of this work was to isolate and molecularly identify enterohemorrhagic Escherichia coli (EHEC) O157 in milk and dairy products in Libya, in addition; to clear the accuracy of cultural and biochemical identification as compared with molecular identification by partial sequencing of 16S rDNA for the existing isolates. Materials and Methods: A total of 108 samples of raw milk (cow, she-camel, and goat) and locally made dairy products (fermented cow’s milk, Maasora, Ricotta and ice cream) were collected from some regions (Janzour, Tripoli, Kremiya, Tajoura and Tobruk) in Libya. Samples were subjected to microbiological analysis for isolation of E. coli that was detected by conventional cultural and molecular method using polymerase chain reaction and partial sequencing of 16S rDNA. Results: Out of 108 samples, only 27 isolates were found to be EHEC O157 based on their cultural characteristics (Tellurite-Cefixime-Sorbitol MacConkey) that include 3 isolates from cow’s milk (11%), 3 isolates from she-camel’s milk (11%), two isolates from goat’s milk (7.4%) and 7 isolates from fermented raw milk samples (26%), isolates from fresh locally made soft cheeses (Maasora and Ricotta) were 9 (33%) and 3 (11%), respectively, while none of the ice cream samples revealed any growth. However, out of these 27 isolates, only 11 were confirmed to be E. coli by partial sequencing of 16S rDNA and E. coli O157 Latex agglutination test. Phylogenetic analysis revealed that majority of local E. coli isolates were related to E. coli O157:H7 FRIK944 strain. Conclusion: These results can be used for further studies on EHEC O157 as an emerging foodborne pathogen and its role in human infection in Libya. PMID:27956766

  14. Characterization of Enterococcus spp. from human and animal feces using 16S rRNA sequences, the esp gene, and PFGE for microbial source tracking in Korea.

    PubMed

    Kim, Sei-Yoon; Lee, Jung Eun; Lee, Sunghee; Lee, Hee Tae; Hur, Ho-Gil; Ko, Gwangpyo

    2010-05-01

    Contamination from human and animal fecal waste is a primary cause of water pollution. Microbial source tracking (MST) may be a useful tool for high-quality environmental management and for assessing human health risks associated with water pollution. The goal of this study was to evaluate Enterococcus spp. as a target organism for MST. Thirty-four fecal samples were collected from five different sources (human, chicken, pig, cow, and goose) in South Korea. In total, 237 Enterococcus spp. were isolated from feces using membrane- Enterococcus indoxyl-beta-d-glucoside agar. The 16S rRNA gene and the whole genome were analyzed using nucleic acid sequencing and pulsed-field gel electrophoresis (PFGE), respectively. Both phylogenetic analysis and principal coordinate analysis using UniFrac were performed on the nucleic acid sequences of the 16S rRNA gene. According to P-tests from UniFrac, significant differences existed between Enterococcus spp. isolated from human feces and those from animal feces. In addition, we evaluated whether the esp gene of Enterococcus faecium could be a specific target for Enterococcus spp. isolated from human feces. Of 58 E. faecium isolates tested, only three were esp-positive. The specificity of the esp gene of E. faecium isolated from human feces was 100%, but the sensitivity was <10%. These results suggest that Enterococcus spp. have different molecular characteristics according to their fecal source and that these characteristics can be further identified by analyzing the esp gene and 16S rRNA sequences, whereas PFGE provides limited information on the fecal sources of Enterococcus spp.

  15. Leptospira spp. strain identification by MALDI TOF MS is an equivalent tool to 16S rRNA gene sequencing and multi locus sequence typing (MLST)

    PubMed Central

    2012-01-01

    Background In this study mass spectrometry was used for evaluating extracted leptospiral protein samples and results were compared with molecular typing methods. For this, an extraction protocol for Leptospira spp. was independently established in two separate laboratories. Reference spectra were created with 28 leptospiral strains, including pathogenic, non-pathogenic and intermediate strains. This set of spectra was then evaluated on the basis of measurements with well-defined, cultured leptospiral strains and with 16 field isolates of veterinary or human origin. To verify discriminating peaks for the applied pathogenic strains, statistical analysis of the protein spectra was performed using the software tool ClinProTools. In addition, a dendrogram of the reference spectra was compared with phylogenetic trees of the 16S rRNA gene sequences and multi locus sequence typing (MLST) analysis. Results Defined and reproducible protein spectra using MALDI-TOF MS were obtained for all leptospiral strains. Evaluation of the newly-built reference spectra database allowed reproducible identification at the species level for the defined leptospiral strains and the field isolates. Statistical analysis of three pathogenic genomospecies revealed peak differences at the species level and for certain serovars analyzed in this study. Specific peak patterns were reproducibly detected for the serovars Tarassovi, Saxkoebing, Pomona, Copenhageni, Australis, Icterohaemorrhagiae and Grippotyphosa. Analysis of the dendrograms of the MLST data, the 16S rRNA sequencing, and the MALDI-TOF MS reference spectra showed comparable clustering. Conclusions MALDI-TOF MS analysis is a fast and reliable method for species identification, although Leptospira organisms need to be produced in a time-consuming culture process. All leptospiral strains were identified, at least at the species level, using our described extraction protocol. Statistical analysis of the three genomospecies L. borgpetersenii

  16. Microbial diversity in polluted harbor sediments I: Bacterial community assessment based on four clone libraries of 16S rDNA

    NASA Astrophysics Data System (ADS)

    Zhang, Wen; Ki, Jang-Seu; Qian, Pei-Yuan

    2008-02-01

    Bacteria, as the most abundant sediment organism, play a major role in the fate of pollutants. Therefore, many pollutant-related bacteria have been studied in harbor sediments, yet the entire bacterial profiles have not been reported. The bacterial diversity and community structures from sediments in Victoria Harbor (Hong Kong), including two polluted (VH and VHW) and two adjacent (open oceanic, TLC; estuary discharge affected, PC) sites, were characterized by analyses of four 16S rDNA clone libraries. Upon comparisons of RFLP patterns from 254 clones in the libraries, 178 unique phylotypes were retrieved. LIBSHUFF and Rarefaction analyses indicated that the sediment bacterial communities at the four sites showed high 16S rDNA richness and were significantly different from each other. Phylogenetic analysis of full-length 16S rDNA revealed 19 bacterial phyla in Victoria Harbor sediments. γ- and δ-proteobacteria, holophaga/acidobacteria, and planctomycetales were recorded in all the libraries. In addition, γ- and δ-proteobacteria were dominant at all sites (33.33-11.67%). Besides these two phyla, ɛ-proteobacteria, firmicutes, aminobacterium, holophaga/acidobacteria and bacteroidetes were judged to be major components of a given library since they constituted 10% or more of the total OTUs of the given library. The cyanobacteria, verrucomicrobia, β-proteobacteria, aminobacterium, chlorofiexi, and candidate division OP1, OP8 were detected in minor proportions in various libraries. A portion of the clones were only distantly related to sequences in the GenBank, suggesting bacteria in Victoria Harbor sediments were unique and diversified.

  17. Microdiversity of deep-sea Bacillales isolated from Tyrrhenian sea sediments as revealed by ARISA, 16S rRNA gene sequencing and BOX-PCR fingerprinting.

    PubMed

    Ettoumi, Besma; Guesmi, Amel; Brusetti, Lorenzo; Borin, Sara; Najjari, Afef; Boudabous, Abdellatif; Cherif, Ameur

    2013-01-01

    With respect to their terrestrial relatives, marine Bacillales have not been sufficiently investigated. In this report, the diversity of deep-sea Bacillales, isolated from seamount and non-seamount stations at 3,425 to 3,580 m depth in the Tyrrhenian Sea, was investigated using PCR fingerprinting and 16S rRNA sequence analysis. The isolate collection (n=120) was de-replicated by automated ribosomal intergenic spacer analysis (ARISA), and phylogenetic diversity was analyzed by 16S rRNA gene sequencing of representatives of each ARISA haplotype (n=37). Phylogenetic analysis of isolates showed their affiliation to six different genera of low G+C% content Gram-positive Bacillales: Bacillus, Staphylococcus, Exiguobacterium, Paenibacillus, Lysinibacillus and Terribacillus. Bacillus was the dominant genus represented by the species B. licheniformis, B. pumilus, B. subtilis, B. amyloliquefaciens and B. firmus, typically isolated from marine sediments. The most abundant species in the collection was B. licheniformis (n=85), which showed seven distinct ARISA haplotypes with haplotype H8 being the most dominant since it was identified by 63 isolates. The application of BOX-PCR fingerprinting to the B. licheniformis sub-collection allowed their separation into five distinct BOX genotypes, suggesting a high level of intraspecies diversity among marine B. licheniformis strains. This species also exhibited distinct strain distribution between seamount and non-seamount stations and was shown to be highly prevalent in non-seamount stations. This study revealed the great microdiversity of marine Bacillales and contributes to understanding the biogeographic distribution of marine bacteria in deep-sea sediments.

  18. Microdiversity of Deep-Sea Bacillales Isolated from Tyrrhenian Sea Sediments as Revealed by ARISA, 16S rRNA Gene Sequencing and BOX-PCR Fingerprinting

    PubMed Central

    Ettoumi, Besma; Guesmi, Amel; Brusetti, Lorenzo; Borin, Sara; Najjari, Afef; Boudabous, Abdellatif; Cherif, Ameur

    2013-01-01

    With respect to their terrestrial relatives, marine Bacillales have not been sufficiently investigated. In this report, the diversity of deep-sea Bacillales, isolated from seamount and non-seamount stations at 3,425 to 3,580 m depth in the Tyrrhenian Sea, was investigated using PCR fingerprinting and 16S rRNA sequence analysis. The isolate collection (n=120) was de-replicated by automated ribosomal intergenic spacer analysis (ARISA), and phylogenetic diversity was analyzed by 16S rRNA gene sequencing of representatives of each ARISA haplotype (n=37). Phylogenetic analysis of isolates showed their affiliation to six different genera of low G+C% content Gram-positive Bacillales: Bacillus, Staphylococcus, Exiguobacterium, Paenibacillus, Lysinibacillus and Terribacillus. Bacillus was the dominant genus represented by the species B. licheniformis, B. pumilus, B. subtilis, B. amyloliquefaciens and B. firmus, typically isolated from marine sediments. The most abundant species in the collection was B. licheniformis (n=85), which showed seven distinct ARISA haplotypes with haplotype H8 being the most dominant since it was identified by 63 isolates. The application of BOX-PCR fingerprinting to the B. licheniformis sub-collection allowed their separation into five distinct BOX genotypes, suggesting a high level of intraspecies diversity among marine B. licheniformis strains. This species also exhibited distinct strain distribution between seamount and non-seamount stations and was shown to be highly prevalent in non-seamount stations. This study revealed the great microdiversity of marine Bacillales and contributes to understanding the biogeographic distribution of marine bacteria in deep-sea sediments. PMID:24005887

  19. 16S rRNA gene sequences analysis of Ficus elastica rubber latex degrading thermophilic Bacillus strain ASU7 isolated from Egypt.

    PubMed

    Hesham, Abd El-Latif; Mohamed, Nadia H; Ismail, Mady A; Shoreit, Ahmed A M

    2012-09-01

    A thermophilic Bacillus strain ASU7 was isolated from soil sample collected from Assiut governorate in Upper Egypt on latex rubber-containing medium at 45 °C. Genetically, the 16S bacterial ribosomal RNA gene of the strain ASU7 was amplified by the polymerase chain reaction (PCR) and sequenced. The sequence of the PCR product was compared with known 16S rRNA gene sequences in the GenBank database. Based on phylogenetic analyses, strain ASU7 was identified as Bacillus amyloliquefaciens. The strain was able to utilize Ficus elastica rubber latex as a sole source for carbon and energy. The ability for degradation was determined by measuring the increase in protein content of bacterium (mg/g dry wt), reduction in molecular weight (g/mol), and inherent viscosity (dl/g) of the latex. Moreover, the degradation was also confirmed by observing the growth of bacterium and formation of aldehyde or keto group using scanning electron microscopy (SEM) and shiff's reagent, respectively.

  20. 16S rRNA Gene Sequencing, Multilocus Sequence Analysis, and Mass Spectrometry Identification of the Proposed New Species “Clostridium neonatale”

    PubMed Central

    Bouvet, Philippe; Ferraris, Laurent; Dauphin, Brunhilde; Popoff, Michel-Robert; Butel, Marie Jose

    2014-01-01

    In 2002, an outbreak of necrotizing enterocolitis in a Canadian neonatal intensive care unit was associated with a proposed novel species of Clostridium, “Clostridium neonatale.” To date, there are no data about the isolation, identification, or clinical significance of this species. Additionally, C. neonatale has not been formally classified as a new species, rendering its identification challenging. Indeed, the C. neonatale 16S rRNA gene sequence shows high similarity to another Clostridium species involved in neonatal necrotizing enterocolitis, Clostridium butyricum. By performing a polyphasic study combining phylogenetic analysis (16S rRNA gene sequencing and multilocus sequence analysis) and phenotypic characterization with mass spectrometry, we demonstrated that C. neonatale is a new species within the Clostridium genus sensu stricto, for which we propose the name Clostridium neonatale sp. nov. Now that the status of C. neonatale has been clarified, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) can be used for better differential identification of C. neonatale and C. butyricum clinical isolates. This is necessary to precisely define the role and clinical significance of C. neonatale, a species that may have been misidentified and underrepresented during previous neonatal necrotizing enterocolitis studies. PMID:25232167

  1. 16S rRNA gene sequencing, multilocus sequence analysis, and mass spectrometry identification of the proposed new species "Clostridium neonatale".

    PubMed

    Bouvet, Philippe; Ferraris, Laurent; Dauphin, Brunhilde; Popoff, Michel-Robert; Butel, Marie Jose; Aires, Julio

    2014-12-01

    In 2002, an outbreak of necrotizing enterocolitis in a Canadian neonatal intensive care unit was associated with a proposed novel species of Clostridium, "Clostridium neonatale." To date, there are no data about the isolation, identification, or clinical significance of this species. Additionally, C. neonatale has not been formally classified as a new species, rendering its identification challenging. Indeed, the C. neonatale 16S rRNA gene sequence shows high similarity to another Clostridium species involved in neonatal necrotizing enterocolitis, Clostridium butyricum. By performing a polyphasic study combining phylogenetic analysis (16S rRNA gene sequencing and multilocus sequence analysis) and phenotypic characterization with mass spectrometry, we demonstrated that C. neonatale is a new species within the Clostridium genus sensu stricto, for which we propose the name Clostridium neonatale sp. nov. Now that the status of C. neonatale has been clarified, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can be used for better differential identification of C. neonatale and C. butyricum clinical isolates. This is necessary to precisely define the role and clinical significance of C. neonatale, a species that may have been misidentified and underrepresented during previous neonatal necrotizing enterocolitis studies.

  2. Analysis of 16S rRNA and mxaF genes revealing insights into Methylobacterium niche-specific plant association

    PubMed Central

    Dourado, Manuella Nóbrega; Andreote, Fernando Dini; Dini-Andreote, Francisco; Conti, Raphael; Araújo, Janete Magali; Araújo, Welington Luiz

    2012-01-01

    The genus Methylobacterium comprises pink-pigmented facultative methylotrophic (PPFM) bacteria, known to be an important plant-associated bacterial group. Species of this group, described as plant-nodulating, have the dual capacity of producing cytokinin and enzymes, such as pectinase and cellulase, involved in systemic resistance induction and nitrogen fixation under specific plant environmental conditions. The aim hereby was to evaluate the phylogenetic distribution of Methylobacterium spp. isolates from different host plants. Thus, a comparative analysis between sequences from structural (16S rRNA) and functional mxaF (which codifies for a subunit of the enzyme methanol dehydrogenase) ubiquitous genes, was undertaken. Notably, some Methylobacterium spp. isolates are generalists through colonizing more than one host plant, whereas others are exclusively found in certain specific plant-species. Congruency between phylogeny and specific host inhabitance was higher in the mxaF gene than in the 16S rRNA, a possible indication of function-based selection in this niche. Therefore, in a first stage, plant colonization by Methylobacterium spp. could represent generalist behavior, possibly related to microbial competition and adaptation to a plant environment. Otherwise, niche-specific colonization is apparently impelled by the host plant. PMID:22481887

  3. Fluorescent Whole-Cell Hybridization with 16S rRNA-Targeted Oligonucleotide Probes To Identify Brucella spp. by Flow Cytometry

    PubMed Central

    Fernández-Lago, Luis; Vallejo, F. Javier; Trujillano, Ignacio; Vizcaíno, Nieves

    2000-01-01

    A whole-cell hybridization assay with fluorescent oligonucleotide probes derived from the 16S rRNA sequence of Brucella abortus in combination with flow cytometry has been developed. With the three fluorescent probes selected, a positive signal was observed with all the representative strains of the species and biovars of Brucella and with a total of nine different Brucella clinical isolates. Using the B9 probe in the hybridization assay, it was possible to discriminate between Brucella suis biovars 2, 3, 4, and 5 and almost all the other Brucella spp. On the basis of differences in fluorescence intensities, no discrimination was established between Brucella spp. and other phylogenetically related microorganisms. No positive fluorescence signals were detected with any of the bacteria showing serological cross-reactions with Brucella spp. and with a total of 17 clinical isolates not belonging to the genus Brucella. These results suggest that the 16S rRNA whole-cell hybridization technique could be a valuable diagnostic tool for the detection and identification of Brucella spp. PMID:10878084

  4. Identification of forensically important sarcophagid flies (Diptera: Sarcophagidae) in China, based on COI and 16S rDNA gene sequences.

    PubMed

    Guo, Yadong; Cai, Jifeng; Chang, Yunfeng; Li, Xiang; Liu, Qinlai; Wang, Xinghua; Wang, Xiang; Zhong, Ming; Wen, Jifang; Wang, Jiangfeng

    2011-11-01

    Insects attracted to cadavers may provide important indications of the postmortem interval (PMI). However, use of the flesh flies (Diptera: Sarcophagidae) for PMI estimation is limited as the species are often not morphologically distinct, especially as immatures. In this study, 23 forensically important flesh flies were collected from 13 locations in 10 Chinese provinces. Then, a 278-bp segment of the cytochrome oxidase subunits one (COI) gene and a 289-bp segment of the 16S rDNA gene of all specimens were successfully sequenced. Phylogenetic analysis of the sequenced segments showed that all sarcophagid specimens were properly assigned into four species (Boerttcherisca peregrina [Robineau-Desvoidy, 1830], Helicophagella melanura [Meigen, 1826], Parasarcophaga albiceps [Meigen, 1826], and Parasarcophaga dux [Thompson, 1869]) with relatively strong supporting values, thus indicating that the COI and 16S rDNA regions are suitable for identification of sarcophagid species. The difference between intraspecific threshold and interspecific divergence confirmed the potential of the two regions for sarcophagid species identification.

  5. Detection and Diversity Assessment of Xylella fastidiosa in Field-Collected Plant and Insect Samples by Using 16S rRNA and gyrB Sequences

    PubMed Central

    Rodrigues, Jorge L. M.; Silva-Stenico, M. E.; Gomes, J. E.; Lopes, J. R. S.; Tsai, S. M.

    2003-01-01

    The causal agent of diseases in many economically important plants is attributed to the xylem-limited bacterium Xylella fastidiosa. The detection of this plant pathogen has been hampered due to its difficult isolation and slow growth on plates. Nearly complete nucleotide sequences of the 16S rRNA gene and partial sequences of the gyrB gene were determined for 18 strains of X. fastidiosa isolated from different plant hosts. A phylogenetic analysis, based on gyrB, grouped strains in three clusters; grape-isolated strains formed one cluster, citrus-coffee strains formed another cluster, and a third cluster resulted from all other strains. Primer pairs designed for the 16S rRNA and gyrB genes were extensively searched in databases to verify their in silico specificity. Primer pairs were certified with 30 target and 36 nontarget pure cultures of microorganisms, confirming 100% specificity. A multiplex PCR protocol was developed and its sensitivity tested. Sequencing of PCR products confirmed the validity of the multiplex PCR. Xylella fastidiosa was detected in field-collected plants, disease vector insects, and nonsymptomatic but infected plants. Specific detection of X. fastidiosa may facilitate the understanding of its ecological significance and prevention of spread of the disease. PMID:12839807

  6. Phylogenetic Trees From Sequences

    NASA Astrophysics Data System (ADS)

    Ryvkin, Paul; Wang, Li-San

    In this chapter, we review important concepts and approaches for phylogeny reconstruction from sequence data.We first cover some basic definitions and properties of phylogenetics, and briefly explain how scientists model sequence evolution and measure sequence divergence. We then discuss three major approaches for phylogenetic reconstruction: distance-based phylogenetic reconstruction, maximum parsimony, and maximum likelihood. In the third part of the chapter, we review how multiple phylogenies are compared by consensus methods and how to assess confidence using bootstrapping. At the end of the chapter are two sections that list popular software packages and additional reading.

  7. Phylogenetic ecology of the freshwater Actinobacteria acI lineage.

    PubMed

    Newton, Ryan J; Jones, Stuart E; Helmus, Matthew R; McMahon, Katherine D

    2007-11-01

    The acI lineage of freshwater Actinobacteria is a cosmopolitan and often numerically dominant member of lake bacterial communities. We conducted a survey of acI 16S rRNA genes and 16S-23S rRNA internal transcribed spacer regions from 18 Wisconsin lakes and used standard nonphylogenetic and phylogenetic statistical approaches to investigate the factors that determine acI community composition at the local scale (within lakes) and at the regional scale (across lakes). Phylogenetic reconstruction of 434 acI 16S rRNA genes revealed a well-defined and highly resolved phylogeny. Eleven previously unrecognized monophyletic clades, each with > or =97.9% within-clade 16S rRNA gene sequence identity, were identified. Clade community similarity positively correlated with lake environmental similarity but not with geographic distance, implying that the lakes represent a single biotic region containing environmental filters for communities that have similar compositions. Phylogenetically disparate clades within the acI lineage were most abundant at the regional scale, and local communities were comprised of more closely related clades. Lake pH was a strong predictor of the community composition, but only when lakes with a pH below 6 were included in the data set. In the remaining lakes (pH above 6) biogeographic patterns in the landscape were instead a predictor of the observed acI community structure. The nonrandom distribution of the newly defined acI clades suggests potential ecophysiological differences between the clades, with acI clades AI, BII, and BIII preferring acidic lakes and acI clades AII, AVI, and BI preferring more alkaline lakes.

  8. Combined Use of 16S Ribosomal DNA and 16S rRNA To Study the Bacterial Community of Polychlorinated Biphenyl-Polluted Soil

    PubMed Central

    Nogales, Balbina; Moore, Edward R. B.; Llobet-Brossa, Enrique; Rossello-Mora, Ramon; Amann, Rudolf; Timmis, Kenneth N.

    2001-01-01

    The bacterial diversity assessed from clone libraries prepared from rRNA (two libraries) and ribosomal DNA (rDNA) (one library) from polychlorinated biphenyl (PCB)-polluted soil has been analyzed. A good correspondence of the community composition found in the two types of library was observed. Nearly 29% of the cloned sequences in the rDNA library were identical to sequences in the rRNA libraries. More than 60% of the total cloned sequence types analyzed were grouped in phylogenetic groups (a clone group with sequence similarity higher than 97% [98% for Burkholderia and Pseudomonas-type clones]) represented in both types of libraries. Some of those phylogenetic groups, mostly represented by a single (or pair) of cloned sequence type(s), were observed in only one of the types of library. An important difference between the libraries was the lack of clones representative of the Actinobacteria in the rDNA library. The PCB-polluted soil exhibited a high bacterial diversity which included representatives of two novel lineages. The apparent abundance of bacteria affiliated to the beta-subclass of the Proteobacteria, and to the genus Burkholderia in particular, was confirmed by fluorescence in situ hybridization analysis. The possible influence on apparent diversity of low template concentrations was assessed by dilution of the RNA template prior to amplification by reverse transcription-PCR. Although differences in the composition of the two rRNA libraries obtained from high and low RNA concentrations were observed, the main components of the bacterial community were represented in both libraries, and therefore their detection was not compromised by the lower concentrations of template used in this study. PMID:11282645

  9. Phylogenetic lineages in Entomophthoromycota

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Entomophthoromycota Humber is one of five major phylogenetic lineages among the former phylum Zygomycota. These early terrestrial fungi share evolutionarily ancestral characters such as coenocytic mycelium and gametangiogamy as a sexual process resulting in zygospore formation. Previous molecular st...

  10. Universal bacterial identification by mass spectrometry of 16S ribosomal RNA cleavage products

    NASA Astrophysics Data System (ADS)

    Jackson, George W.; McNichols, Roger J.; Fox, George E.; Willson, Richard C.

    2007-03-01

    The public availability of over 180,000 bacterial 16S ribosomal RNA (rRNA) sequences has facilitated microbial identification and classification using nucleic acid hybridization and other molecular approaches. Species-specific PCR, microarrays, and in situ hybridization are based on the presence of unique subsequences in the target sequence and therefore require prior knowledge of what organisms are likely to be present in a sample. Mass spectrometry is not limited by a pre-synthesized inventory of probe/primer sequences. It has already been demonstrated that organism identification can be recovered from mass spectra using various methods including base-specific cleavage of nucleic acids. The feasibility of broad bacterial identification by comparing such mass spectral patterns to predictive databases derived from virtually all previously sequenced strains has yet to be demonstrated, however. Herein, we present universal bacterial identification by base-specific cleavage, mass spectrometry, and an efficient coincidence function for rapid spectral scoring against a large database of predicted "mass catalogs". Using this approach in conjunction with universal PCR of the 16S rDNA gene, four bacterial isolates and an uncultured clone were successfully identified against a database of predicted cleavage products derived 6rom over 47,000 16S rRNA sequences representing all major bacterial taxaE At present, the conventional DNA isolation and PCR steps require approximately 2 h, while subsequent transcription, enzymatic cleavage, mass spectrometric analysis, and database comparison require less than 45 min. All steps are amenable to high-throughput implementation.

  11. Analysis, Optimization and Verification of Illumina-Generated 16S rRNA Gene Amplicon Surveys

    PubMed Central

    Nelson, Michael C.; Morrison, Hilary G.; Benjamino, Jacquelynn; Grim, Sharon L.; Graf, Joerg

    2014-01-01

    The exploration of microbial communities by sequencing 16S rRNA genes has expanded with low-cost, high-throughput sequencing instruments. Illumina-based 16S rRNA gene sequencing has recently gained popularity over 454 pyrosequencing due to its lower costs, higher accuracy and greater throughput. Although recent reports suggest that Illumina and 454 pyrosequencing provide similar beta diversity measures, it remains to be demonstrated that pre-existing 454 pyrosequencing workflows can transfer directly from 454 to Illumina MiSeq sequencing by simply changing the sequencing adapters of the primers. In this study, we modified 454 pyrosequencing primers targeting the V4-V5 hyper-variable regions of the 16S rRNA gene to be compatible with Illumina sequencers. Microbial communities from cows, humans, leeches, mice, sewage, and termites and a mock community were analyzed by 454 and MiSeq sequencing of the V4-V5 region and MiSeq sequencing of the V4 region. Our analysis revealed that reference-based OTU clustering alone introduced biases compared to de novo clustering, preventing certain taxa from being observed in some samples. Based on this we devised and recommend an analysis pipeline that includes read merging, contaminant filtering, and reference-based clustering followed by de novo OTU clustering, which produces diversity measures consistent with de novo OTU clustering analysis. Low levels of dataset contamination with Illumina sequencing were discovered that could affect analyses that require highly sensitive approaches. While moving to Illumina-based sequencing platforms promises to provide deeper insights into the breadth and function of microbial diversity, our results show that care must be taken to ensure that sequencing and processing artifacts do not obscure true microbial diversity. PMID:24722003

  12. Greengenes, a Chimera-checked 16S rRNA gene database and workbenchcompatible with ARB

    SciTech Connect

    DeSantis, Todd Z.; Hugenholtz, Philip; Larsen, Neils; Rojas,Mark; Brodie, Eoin L.; Keller, Keith; Huber, Thomas; Dalevi, Daniel; Hu,Ping; Andersen, Gary L.

    2006-04-10

    A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera-screening, standard alignments and taxonomic classification using multiple published taxonomies. It was revealed that in congruent taxonomic nomenclature exists among curators even at the phylum-level. Putative chimeras were identified in 3 percent of environmental sequences and 0.2 percent of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages within the Archaea and Bacteria.

  13. Circular code motifs in transfer and 16S ribosomal RNAs: a possible translation code in genes.

    PubMed

    Michel, Christian J

    2012-04-01

    In 1996, a common trinucleotide circular code, called X, is identified in genes of eukaryotes and prokaryotes (Arquès and Michel, 1996). This circular code X is a set of 20 trinucleotides allowing the reading frames in genes to be retrieved locally, i.e. anywhere in genes and in particular without start codons. This reading frame retrieval needs a window length l of 12 nucleotides (l ≥ 12). With a window length strictly less than 12 nucleotides (l < 12), some words of X, called ambiguous words, are found in the shifted frames (the reading frame shifted by one or two nucleotides) preventing the reading frame in genes to be retrieved. Since 1996, these ambiguous words of X were never studied. In the first part of this paper, we identify all the ambiguous words of the common trinucleotide circular code X. With a length l varying from 1 to 11 nucleotides, the type and the occurrence number (multiplicity) of ambiguous words of X are given in each shifted frame. Maximal ambiguous words of X, words which are not factors of another ambiguous words, are also determined. Two probability definitions based on these results show that the common trinucleotide circular code X retrieves the reading frame in genes with a probability of about 90% with a window length of 6 nucleotides, and a probability of 99.9% with a window length of 9 nucleotides (100% with a window length of 12 nucleotides, by definition of a circular code). In the second part of this paper, we identify X circular code motifs (shortly X motifs) in transfer RNA and 16S ribosomal RNA: a tRNA X motif of 26 nucleotides including the anticodon stem-loop and seven 16S rRNA X motifs of length greater or equal to 15 nucleotides. Window lengths of reading frame retrieval with each trinucleotide of these X motifs are also determined. Thanks to the crystal structure 3I8G (Jenner et al., 2010), a 3D visualization of X motifs in the ribosome shows several spatial configurations involving mRNA X motifs, A-tRNA and E-tRNA X

  14. Abiotrophia defectiva bleb-associated endophthalmitis confirmed with 16s ribosomal RNA sequencing.

    PubMed

    Hugo Lee, Ming-Han; Lawlor, Mitchell; Lee, Anne J

    2015-01-01

    One recognized complication of trabeculectomy with visually devastating potential is blebitis. We present a case of a 74-year-old woman with a culture and polymerase chain reaction-positive Abiotrophia defectiva bleb-associated endophthalmitis. Abiotrophia defectiva is a rare but possible cause of endophthalmitis secondary to blebitis and should be considered in culture-negative cases. Prompt identification, hence directed eradication, of the causative organism in such visually threatening cases may be facilitated by requesting polymerase chain reaction and 16S ribosomal RNA sequencing.

  15. Phylogenetic congruence and ecological coherence in terrestrial Thaumarchaeota.

    PubMed

    Oton, Eduard Vico; Quince, Christopher; Nicol, Graeme W; Prosser, James I; Gubry-Rangin, Cécile

    2016-01-01

    Thaumarchaeota form a ubiquitously distributed archaeal phylum, comprising both the ammonia-oxidising archaea (AOA) and other archaeal groups in which ammonia oxidation has not been demonstrated (including Group 1.1c and Group 1.3). The ecology of AOA in terrestrial environments has been extensively studied using either a functional gene, encoding ammonia monooxygenase subunit A (amoA) or 16S ribosomal RNA (rRNA) genes, which show phylogenetic coherence with respect to soil pH. To test phylogenetic congruence between these two markers and to determine ecological coherence in all Thaumarchaeota, we performed high-throughput sequencing of 16S rRNA and amoA genes in 46 UK soils presenting 29 available contextual soil characteristics. Adaptation to pH and organic matter content reflected strong ecological coherence at various levels of taxonomic resolution for Thaumarchaeota (AOA and non-AOA), whereas nitrogen, total mineralisable nitrogen and zinc concentration were also important factors associated with AOA thaumarchaeotal community distribution. Other significant associations with environmental factors were also detected for amoA and 16S rRNA genes, reflecting different diversity characteristics between these two markers. Nonetheless, there was significant statistical congruence between the markers at fine phylogenetic resolution, supporting the hypothesis of low horizontal gene transfer between Thaumarchaeota. Group 1.1c Thaumarchaeota were also widely distributed, with two clusters predominating, particularly in environments with higher moisture content and organic matter, whereas a similar ecological pattern was observed for Group 1.3 Thaumarchaeota. The ecological and phylogenetic congruence identified is fundamental to understand better the life strategies, evolutionary history and ecosystem function of the Thaumarchaeota.

  16. Phylogeny of coral-inhabiting barnacles (Cirripedia; Thoracica; Pyrgomatidae) based on 12S, 16S and 18S rDNA analysis.

    PubMed

    Simon-Blecher, N; Huchon, D; Achituv, Y

    2007-09-01

    The traditional phylogeny of the coral-inhabiting barnacles, the Pyrgomatidae, is based on morphological characteristics, mainly of the hard parts. It has been difficult to establish the phylogenetic relationships among Pyrgomatidae because of the apparent convergence of morphological characteristics, and due to the use of non-cladistic systematics, which emphasize ancestor-descendant relationships rather than sister-clade relationships. We used partial sequences of two mithochondrial genes, 12S rDNA and 16S rDNA, and a nuclear gene, 18S rDNA, to infer the molecular phylogeny of the pyrgomatids. Our phylogenetic results allowed us to reject previous classifications of Pyrgomatidae based on morphological characteristics. Our results also suggested the possibility of paraphyly of the Pyrgomatidae. The hydrocoral barnacle Wanella is not found on the same clade as the other pyrgomatids, but rather, with the free-living balanids. The basal position of Megatrema and Ceratoconcha is supported. The archeaobalanid Armatobalanus is grouped with Cantellius at the base of the Indo-Pacific pyrgomatines. Fusion of the shell plate and modification of the opercular valves are homoplasious features that occurred more than three times on different clades. The monophyly of the "Savignium" group, comprising four nominal genera, is also not supported, and the different taxa are placed on different clades.

  17. Molecular phylogeny of the genus Dicronocephalus (Coleoptera, Scarabaeidae, Cetoniinae) based on mtCOI and 16S rRNA genes

    PubMed Central

    Lee, Ga-Eun; Han, Taeman; Jeong, Jongchel; Kim, Seong-Hyun; Park, In Gyun; Park, Haechul

    2015-01-01

    Abstract The seven species belonging to the genus Dicronocephalus are a very interesting group with a unique appearance and distinct sexual dimorphism. Only one species among them, Dicronocephalus adamsi, has been known in the Korean fauna. This species is recognized as having a wide distribution from Tibet to Korean Peninsula and is currently represented by two subspecies that have separated geographical ranges. The phylogenetic relationships of Dicronocephalus adamsi were still unclear. The phylogeny of Dicronocephalus is reconstructed with a phylogenetic study of five species including four subspecies based on a molecular approach using mitochondrial COI and 16S rRNA genes. Our results are compared with the results obtained by previous authors based on morphological characters. They show that the tested taxa are divided into two major clades. Clade A consists of two species (Dicronocephalus adamsi + Dicranocephalus yui) and Clade B includes the others (Dicronocephalus dabryi + Dicranocephalus uenoi + Dicranocephalus wallichii). This result generally supports Kurosawa’s proposal except that Dicronocephalus dabryi and Dicranocephalus uenoi are newly recognized as members of a monophyletic group. We propose that Dicronocephalus adamsi drumonti is a junior subjective synonym of Dicronocephalus adamsi adamsi. These results show that three members of the Dicranocephalus wallichii group should be treated as species rather than subspecies. However, further research including analyses of different genetic markers is needed to reconfirm our results. PMID:25987879

  18. Phylogenetic relationships within the Phyllidiidae (Opisthobranchia, Nudibranchia)

    PubMed Central

    Stoffels, Bart E.M.W.; van der Meij, Sancia E.T.; Hoeksema, Bert W.; van Alphen, Joris; van Alen, Theo; Meyers-Muñoz, Maria Angelica; de Voogd, Nicole J.; Tuti, Yosephine; van der Velde, Gerard

    2016-01-01

    Abstract The Phyllidiidae (Gastropoda, Heterobranchia, Nudibranchia) is a family of colourful nudibranchs found on Indo-Pacific coral reefs. Despite the abundant and widespread occurrence of many species, their phylogenetic relationships are not well known. The present study is the first contribution to fill the gap in our knowledge on their phylogeny by combining morphological and molecular data. For that purpose 99 specimens belonging to 16 species were collected at two localities in Indonesia. They were photographed and used to make a phylogeny reconstruction based on newly obtained cytochrome oxidase subunit (COI) sequences as well as sequence data from GenBank. All mitochondrial 16S sequence data available from GenBank were used in a separate phylogeny reconstruction to obtain information for species we did not collect. COI data allowed the distinction of the genera and species, whereas the 16S data gave a mixed result with respect to the genera Phyllidia and Phyllidiella. Specimens which could be ascribed to species level based on their external morphology and colour patterns showed low variation in COI sequences, but there were two exceptions: three specimens identified as Phyllidia cf. babai represent two to three different species, while Phyllidiella pustulosa showed highly supported subclades. The barcoding marker COI also confirms that the species boundaries in morphologically highly variable species such as Phyllidia elegans, Phyllidia varicosa, and Phyllidiopsis krempfi, are correct as presently understood. In the COI as well as the 16S cladogram Phyllidiopsis cardinalis was located separately from all other Phyllidiidae, whereas Phyllidiopsis fissuratus was positioned alone from the Phyllidiella species by COI data only. Future studies on phyllidiid systematics should continue to combine morphological information with DNA sequences to obtain a clearer insight in their phylogeny. PMID:27551210

  19. Relationships between parasitoid wasps (Hymenoptera: Braconidae: Opiinae), fruit flies (Diptera: Tephritidae) and their host plants based on 16S rRNA, 12S rRNA, and ND1 gene sequences

    NASA Astrophysics Data System (ADS)

    Ibrahim, N. J.; Md-Zain, B. M.; Yaakop, S.

    2013-11-01

    Opiinae is among the l0 largest subfamilies under the family Braconidae. Opiines species have great potential as natural enemies against fruit fly pests. Before using them as a biological control agent, construction of the phylogenetic trees could facilitate in the molecular identification of individual species and their relationships among members of the Opiines, as well as between Opiines and their host plants. Larval specimens of tephritids were collected from four crop species at five localities throughout the Peninsular Malaysia. A total of 44 specimens of opiines had successfully emerged from the hosts, fruit fly larvae. The DNA sequences of 12S and 16S rRNA were obtained for the braconids while the mitochondrial ND1 sequences were obtained for the tephritids species through polymerase chain reaction. Maximum Parsimony and Bayesian trees were constructed by using PAUP 4.0b10 and MrBayes 3.1.2 to identify the relationships among the taxa. This study illustrates the phylogenetic relationships among parasitoid opiines collected and reared from parasitized fruit flies. The phylogenetic trees constructed based on the mitochondrial 12S and 16S rRNA sequences exhibited similar topology and sequence divergence. The opiines were divided into several clades and subclades according to the genus and species. Each clade also was supported by the similar host plants with high support values. However, their pests were not specific, except for Bactrocera cucurbitae. This study has reconfirmed the associations between Opiinae, tephritids, and host plants based on molecular data.

  20. An unusual case of Streptococcus anginosus group pyomyositis diagnosed using direct 16S ribosomal DNA sequencing.

    PubMed

    Walkty, Andrew; Embil, John M; Nichol, Kim; Karlowsky, James

    2014-01-01

    Bacteria belonging to the Streptococcus anginosus group (Streptococcus intermedius, Streptococcus constellatus and Streptococcus anginosus) are capable of causing serious pyogenic infections, with a tendency for abscess formation. The present article reports a case of S anginosus group pyomyositis in a 47-year-old man. The pathogen was recovered from one of two blood cultures obtained from the patient, but speciation was initially not performed because the organism was considered to be a contaminant (viridans streptococci group). The diagnosis was ultimately confirmed using 16S ribosomal DNA sequencing of purulent fluid obtained from a muscle abscess aspirate. The present case serves to emphasize that finding even a single positive blood culture of an organism belonging to the S anginosus group should prompt careful evaluation of the patient for a pyogenic focus of infection. It also highlights the potential utility of 16S ribosomal DNA amplification and sequencing in direct pathogen detection from aspirated fluid in cases of pyomyositis in which antimicrobial therapy was initiated before specimen collection.

  1. Novel haloarchaeal 16S rRNA gene sequences from Alpine Permo-Triassic rock salt.

    PubMed

    Radax, C; Gruber, C; Stan-Lotter, H

    2001-08-01

    Prokaryotic diversity in Alpine salt sediments was investigated by polymerase chain reaction (PCR) amplification of 16S rRNA genes, sequencing of cloned products, and comparisons with culturable strains. DNA was extracted from the residue following filtration of dissolved Permo-Triassic rock salt. Fifty-four haloarchaeal sequences were obtained, which could be grouped into at least five distinct clusters. Similarity values of three clusters to known 16S rRNA genes were less than 90%-95%, suggesting the presence of uncultured novel taxa; two clusters were 98% and 99% similar to isolates from Permo-Triassic or Miocene salt from England and Poland, and to Halobacterium salinarum, respectively. Some rock salt samples, including drilling cores, yielded no amplifiable DNA and no cells or only a few culturable cells. This result suggested a variable distribution of haloarchaea within different strata, probably consistent with the known geologic heterogeneity of Alpine salt deposits. We recently reported identical culturable Halococcus salifodinae strains in Permo-Triassic salt sediments from England, Germany, and Austria; together with the data presented here, those results suggest one plausible scenario to be an ancient continuous hypersaline ocean (Zechstein sea) populated by haloarchaea, whose descendants are found today in the salt sediments. The novelty of the sequences also suggested avoidance of haloarchaeal contaminants during our isolation of strains, preparation of DNA, and PCR reactions.

  2. Identification of Clinical Isolates of Actinomyces Species by Amplified 16S Ribosomal DNA Restriction Analysis

    PubMed Central

    Hall, Val; Talbot, P. R.; Stubbs, S. L.; Duerden, B. I.

    2001-01-01

    Amplified 16S ribosomal DNA (rDNA) restriction analysis (ARDRA), using enzymes HaeIII and HpaII, was applied to 176 fresh and 299 stored clinical isolates of putative Actinomyces spp. referred to the Anaerobe Reference Unit of the Public Health Laboratory Service for confirmation of identity. Results were compared with ARDRA results obtained previously for reference strains and with conventional phenotypic reactions. Identities of some strains were confirmed by analysis of partial 16S rDNA sequences. Of the 475 isolates, 331 (70%) were clearly assigned to recognized Actinomyces species, including 94 isolates assigned to six recently described species. A further 52 isolates in 12 ARDRA profiles were designated as apparently resembling recognized species, and 44 isolates, in 18 novel profiles, were confirmed as members of genera other than Actinomyces. The identities of 48 isolates in nine profiles remain uncertain, and they may represent novel species of Actinomyces. For the majority of species, phenotypic results, published reactions for the species, and ARDRA profiles concurred. However, of 113 stored isolates originally identified as A. meyeri or resembling A. meyeri by phenotypic tests, only 21 were confirmed as A. meyeri by ARDRA; 63 were reassigned as A. turicensis, 7 as other recognized species, and 22 as unidentified actinomycetes. Analyses of incidence and clinical associations of Actinomyces spp. add to the currently sparse knowledge of some recently described species. PMID:11574572

  3. Rapid identification of marine bioluminescent bacteria by amplified 16S ribosomal RNA gene restriction analysis.

    PubMed

    Kita-Tsukamoto, Kumiko; Wada, Minoru; Yao, Katomi; Kamiya, Akiko; Yoshizawa, Susumu; Uchiyama, Nami; Kogure, Kazuhiro

    2006-03-01

    To rapidly identify natural isolates of marine bioluminescent bacteria, we developed amplified ribosomal DNA restriction analysis (ARDRA) methods. ARDRA, which is based on the restriction patterns of 16S rRNA gene digested with five enzymes (EcoRI, DdeI, HhaI, HinfI, RsaI), clearly distinguished the 14 species of marine bioluminescent bacteria currently known, which belong to the genera Vibrio, Photobacterium, and Shewanella. When we applied ARDRA to 129 natural isolates from two cruises in Sagami Bay, Japan, 127 were grouped into six ARDRA types with distinctive restriction patterns; these isolates represented the bioluminescent species, P. angustum, P. leiognathi, P. phosphoreum, S. woodyi, V. fischeri, and V. harveyi. The other two isolates showing unexpected ARDRA patterns turned out to have 16S rRNA gene sequences similar to P. leiognathi and P. phosphoreum. Nevertheless, ARDRA provides a simple and fairly robust means for rapid identification of the natural isolates of marine bioluminescent bacteria, and is therefore useful in studying their diversity.

  4. Mitochondrial swinger replication: DNA replication systematically exchanging nucleotides and short 16S ribosomal DNA swinger inserts.

    PubMed

    Seligmann, Hervé

    2014-11-01

    Assuming systematic exchanges between nucleotides (swinger RNAs) resolves genomic 'parenthood' of some orphan mitochondrial transcripts. Twenty-three different systematic nucleotide exchanges (bijective transformations) exist. Similarities between transcription and replication suggest occurrence of swinger DNA. GenBank searches for swinger DNA matching the 23 swinger versions of human and mouse mitogenomes detect only vertebrate mitochondrial swinger DNA for swinger type AT+CG (from five different studies, 149 sequences) matching three human and mouse mitochondrial genes: 12S and 16S ribosomal RNAs, and cytochrome oxidase subunit I. Exchange A<->T+C<->G conserves self-hybridization properties, putatively explaining swinger biases for rDNA, against protein coding genes. Twenty percent of the regular human mitochondrial 16S rDNA consists of short swinger repeats (from 13 exchanges). Swinger repeats could originate from recombinations between regular and swinger DNA: duplicated mitochondrial genes of the parthenogenetic gecko Heteronotia binoei include fewer short A<->T+C<->G swinger repeats than non-duplicated mitochondrial genomes of that species. Presumably, rare recombinations between female and male mitochondrial genes (and in parthenogenetic situations between duplicated genes), favors reverse-mutations of swinger repeat insertions, probably because most inserts affect negatively ribosomal function. Results show that swinger DNA exists, and indicate that swinger polymerization contributes to the genesis of genetic material and polymorphism.

  5. Two Distinct Mechanisms Cause Heterogeneity of 16S rRNA

    PubMed Central

    Ueda, Kumiko; Seki, Tatsuji; Kudo, Takuji; Yoshida, Toshiomi; Kataoka, Masakazu

    1999-01-01

    To investigate the frequency of heterogeneity among the multiple 16S rRNA genes within a single microorganism, we determined directly the 120-bp nucleotide sequences containing the hypervariable α region of the 16S rRNA gene from 475 Streptomyces strains. Display of the direct sequencing patterns revealed the existence of 136 heterogeneous loci among a total of 33 strains. The heterogeneous loci were detected only in the stem region designated helix 10. All of the substitutions conserved the relevant secondary structure. The 33 strains were divided into two groups: one group, including 22 strains, had less than two heterogeneous bases; the other group, including 11 strains, had five or more heterogeneous bases. The two groups were different in their combinations of heterogeneous bases. The former mainly contained transitional substitutions, and the latter was mainly composed of transversional substitutions, suggesting that at least two mechanisms, possibly misincorporation during DNA replication and horizontal gene transfer, cause rRNA heterogeneity. PMID:9864315

  6. Greengenes: 16S rRNA Database and Workbench Compatible with ARB

    DOE Data Explorer

    DeSantis, T. Z.; Hugenholtz, P.; Larsen, N.; Rojas, M.; Brodie, E. L.; Keller, K.; Huber, T.; Dalevi, D. Hu, P. Andersen, G. L.

    Greengenes was developed, as the abstract of an AEM reprint states, to "addresse limitations of public repositories by providing chimera screening, standard alignment, and taxonomic classification using multiple published taxonomies. It was found that there is incongruent taxonomic nomenclature among curators even at the phylum level. Putative chimeras were identified in 3% of environmental sequences and in 0.2% of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages in the Archaea and Bacteria....Greengenes is also a functional workbench to assist in analysis of user-generated 16S rRNA gene sequences. Batches of sequencing reads can be uploaded for quality-based trimming and creation of multiple-sequence alignments (9). Three types of non-MSA similarity searches are also available, seed extension by BLAST (1), similarity based on shared 7-mers by a tool called Simrank, and a direct degenerative pattern match for probe/primer evaluation. Results are displayed using user-preferred taxonomic nomenclature and can be saved between sessions. [Taken from DeSantis, T. Z., P. Hugenholtz, N. Larsen, M. Rojas, E. L. Brodie, K. Keller, T. Huber, D. Dalevi, P. Hu, and G. L. Andersen. 2006. Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB. Appl Environ Microbiol 72:5069-72, pages 1 and 3] (Specialized Interface)

  7. Characterization of the Gut Microbiome Using 16S or Shotgun Metagenomics

    PubMed Central

    Jovel, Juan; Patterson, Jordan; Wang, Weiwei; Hotte, Naomi; O'Keefe, Sandra; Mitchel, Troy; Perry, Troy; Kao, Dina; Mason, Andrew L.; Madsen, Karen L.; Wong, Gane K.-S.

    2016-01-01

    The advent of next generation sequencing (NGS) has enabled investigations of the gut microbiome with unprecedented resolution and throughput. This has stimulated the development of sophisticated bioinformatics tools to analyze the massive amounts of data generated. Researchers therefore need a clear understanding of the key concepts required for the design, execution and interpretation of NGS experiments on microbiomes. We conducted a literature review and used our own data to determine which approaches work best. The two main approaches for analyzing the microbiome, 16S ribosomal RNA (rRNA) gene amplicons and shotgun metagenomics, are illustrated with analyses of libraries designed to highlight their strengths and weaknesses. Several methods for taxonomic classification of bacterial sequences are discussed. We present simulations to assess the number of sequences that are required to perform reliable appraisals of bacterial community structure. To the extent that fluctuations in the diversity of gut bacterial populations correlate with health and disease, we emphasize various techniques for the analysis of bacterial communities within samples (α-diversity) and between samples (β-diversity). Finally, we demonstrate techniques to infer the metabolic capabilities of a bacteria community from these 16S and shotgun data. PMID:27148170

  8. Kanamycin-resistant alfalfa has a point mutation in the 16S plastid rRNA.

    PubMed

    Rosellini, D; LaFayette, P R; Barone, P; Veronesi, F; Parrott, W A

    2004-05-01

    Genes conferring resistance to kanamycin are frequently used to obtain transgenic plants as spontaneous resistance to kanamycin is not known to exist in higher plants. Nevertheless, mutations conferring kanamycin resistance have been identified in Chlamydomonas reinhardtii, raising the question as to why kanamycin-resistant mutants have not been found in higher plants. While attempting plastid transformation of alfalfa, we obtained non-transgenic but kanamycin-resistant somatic embryos following 2 months of culture in the presence of 50 mg l(-1) kanamycin. Sequencing of the plastid DNA region corresponding to the decoding site of the 16S rRNA in ten independent resistant events revealed an A to C transversion at position 1357 of the 16S plastid rDNA, the same site at which an A to G conversion confers kanamycin resistance to C. reinhardtii by reducing the ability of the antibiotic to bind to its target site. All plants derived from the resistant embryos through additional cycles of somatic embryogenesis in the absence of kanamycin retained the mutant phenotype, suggesting that the mutation was homoplastomic. Resistant plants produced 85% less biomass than controls; their leaves were chlorotic during early development and over time slowly turned green. The absence of kanamycin- resistant mutants in higher plants might be explained by the requirement for a regeneration system capable of resulting in homoplastomic individuals, or it may be the result of the detrimental effect of the mutation on the phenotype.

  9. Changes in 16s RNA Gene Microbial Community Profiling by Concentration of Prokaryotic DNA.

    PubMed

    Glassing, Angela; Dowd, Scot E; Galandiuk, Susan; Davis, Brian; Jorden, Jeffrey R; Chiodini, Rodrick J

    2015-12-01

    Microbial metagenomics are hindered in clinical tissue samples as a result of the large relative amount of human DNA in relation to microbial DNA acting as competitive inhibitors of downstream applications. We evaluated the LOOXSTER® Enrichment Kit to separate eukaryotic and prokaryotic DNA in submucosal intestinal tissue samples having a low microbial biomass and to determine the effects of enrichment on 16s rRNA microbiota sequencing. The enrichment kit reduced the amount of human DNA in the samples 40-70% resulting in a 3.5-fold increase in the number of 16s bacterial gene sequences detected on the Illumina MiSeq platform. This increase was accompanied by the detection of 41 additional bacterial genera and 94 tentative species. The additional bacterial taxa detected accounted for as much as 25% of the total bacterial population that significantly altered the relative prevalence and composition of the intestinal microbiota. The ability to reduce the competitive inhibition created by human DNA and the concentration of bacterial DNA may allow metagenomics to be performed on complex tissues containing a low bacterial biomass.

  10. Phylogenetically resolving epidemiologic linkage

    PubMed Central

    Romero-Severson, Ethan O.; Bulla, Ingo; Leitner, Thomas

    2016-01-01

    Although the use of phylogenetic trees in epidemiological investigations has become commonplace, their epidemiological interpretation has not been systematically evaluated. Here, we use an HIV-1 within-host coalescent model to probabilistically evaluate transmission histories of two epidemiologically linked hosts. Previous critique of phylogenetic reconstruction has claimed that direction of transmission is difficult to infer, and that the existence of unsampled intermediary links or common sources can never be excluded. The phylogenetic relationship between the HIV populations of epidemiologically linked hosts can be classified into six types of trees, based on cladistic relationships and whether the reconstruction is consistent with the true transmission history or not. We show that the direction of transmission and whether unsampled intermediary links or common sources existed make very different predictions about expected phylogenetic relationships: (i) Direction of transmission can often be established when paraphyly exists, (ii) intermediary links can be excluded when multiple lineages were transmitted, and (iii) when the sampled individuals’ HIV populations both are monophyletic a common source was likely the origin. Inconsistent results, suggesting the wrong transmission direction, were generally rare. In addition, the expected tree topology also depends on the number of transmitted lineages, the sample size, the time of the sample relative to transmission, and how fast the diversity increases after infection. Typically, 20 or more sequences per subject give robust results. We confirm our theoretical evaluations with analyses of real transmission histories and discuss how our findings should aid in interpreting phylogenetic results. PMID:26903617

  11. The phylogenetic likelihood library.

    PubMed

    Flouri, T; Izquierdo-Carrasco, F; Darriba, D; Aberer, A J; Nguyen, L-T; Minh, B Q; Von Haeseler, A; Stamatakis, A

    2015-03-01

    We introduce the Phylogenetic Likelihood Library (PLL), a highly optimized application programming interface for developing likelihood-based phylogenetic inference and postanalysis software. The PLL implements appropriate data structures and functions that allow users to quickly implement common, error-prone, and labor-intensive tasks, such as likelihood calculations, model parameter as well as branch length optimization, and tree space exploration. The highly optimized and parallelized implementation of the phylogenetic likelihood function and a thorough documentation provide a framework for rapid development of scalable parallel phylogenetic software. By example of two likelihood-based phylogenetic codes we show that the PLL improves the sequential performance of current software by a factor of 2-10 while requiring only 1 month of programming time for integration. We show that, when numerical scaling for preventing floating point underflow is enabled, the double precision likelihood calculations in the PLL are up to 1.9 times faster than those in BEAGLE. On an empirical DNA dataset with 2000 taxa the AVX version of PLL is 4 times faster than BEAGLE (scaling enabled and required). The PLL is available at http://www.libpll.org under the GNU General Public License (GPL).

  12. Phylogenetically resolving epidemiologic linkage

    SciTech Connect

    Romero-Severson, Ethan O.; Bulla, Ingo; Leitner, Thomas

    2016-02-22

    The use of phylogenetic trees in epidemiological investigations has become commonplace, but their epidemiological interpretation has not been systematically evaluated. Here, we use an HIV-1 within-host coalescent model to probabilistically evaluate transmission histories of two epidemiologically linked hosts. Previous critique of phylogenetic reconstruction has claimed that direction of transmission is difficult to infer, and that the existence of unsampled intermediary links or common sources can never be excluded. The phylogenetic relationship between the HIV populations of epidemiologically linked hosts can be classified into six types of trees, based on cladistic relationships and whether the reconstruction is consistent with the true transmission history or not. We show that the direction of transmission and whether unsampled intermediary links or common sources existed make very different predictions about expected phylogenetic relationships: (i) Direction of transmission can often be established when paraphyly exists, (ii) intermediary links can be excluded when multiple lineages were transmitted, and (iii) when the sampled individuals’ HIV populations both are monophyletic a common source was likely the origin. Inconsistent results, suggesting the wrong transmission direction, were generally rare. In addition, the expected tree topology also depends on the number of transmitted lineages, the sample size, the time of the sample relative to transmission, and how fast the diversity increases after infection. Typically, 20 or more sequences per subject give robust results. Moreover, we confirm our theoretical evaluations with analyses of real transmission histories and discuss how our findings should aid in interpreting phylogenetic results.

  13. Phylogenetically resolving epidemiologic linkage

    DOE PAGES

    Romero-Severson, Ethan O.; Bulla, Ingo; Leitner, Thomas

    2016-02-22

    The use of phylogenetic trees in epidemiological investigations has become commonplace, but their epidemiological interpretation has not been systematically evaluated. Here, we use an HIV-1 within-host coalescent model to probabilistically evaluate transmission histories of two epidemiologically linked hosts. Previous critique of phylogenetic reconstruction has claimed that direction of transmission is difficult to infer, and that the existence of unsampled intermediary links or common sources can never be excluded. The phylogenetic relationship between the HIV populations of epidemiologically linked hosts can be classified into six types of trees, based on cladistic relationships and whether the reconstruction is consistent with the truemore » transmission history or not. We show that the direction of transmission and whether unsampled intermediary links or common sources existed make very different predictions about expected phylogenetic relationships: (i) Direction of transmission can often be established when paraphyly exists, (ii) intermediary links can be excluded when multiple lineages were transmitted, and (iii) when the sampled individuals’ HIV populations both are monophyletic a common source was likely the origin. Inconsistent results, suggesting the wrong transmission direction, were generally rare. In addition, the expected tree topology also depends on the number of transmitted lineages, the sample size, the time of the sample relative to transmission, and how fast the diversity increases after infection. Typically, 20 or more sequences per subject give robust results. Moreover, we confirm our theoretical evaluations with analyses of real transmission histories and discuss how our findings should aid in interpreting phylogenetic results.« less

  14. Review of 16S and ITS Direct Sequencing Results for Clinical Specimens Submitted to a Reference Laboratory

    PubMed Central

    Payne, Michael; Azana, Robert; Hoang, Linda M. N.

    2016-01-01

    We evaluated the performance of 16S and internal transcribed spacer (ITS) region amplification and sequencing of rDNA from clinical specimens, for the respective detection and identification of bacterial and fungal pathogens. Direct rDNA amplification of 16S and ITS targets from clinical samples was performed over a 4-year period and reviewed. All specimens were from sterile sites and submitted to a reference laboratory for evaluation. Results of 16S and ITS were compared to histopathology, Gram and/or calcofluor stain microscopy results. A total of 277 16S tests were performed, with 64 (23%) positive for the presence of bacterial DNA. Identification of an organism was more likely in microscopy positive 16S samples 14/21 (67%), compared to 35/175 (20%) of microscopy negative samples. A total of 110 ITS tests were performed, with 14 (13%) positive. The yield of microscopy positive ITS samples, 9/44 (21%), was higher than microscopy negative samples 3/50 (6%). Given these findings, 16S and ITS are valuable options for culture negative specimens from sterile sites, particularly in the setting of positive microscopy findings. Where microscopy results are negative, the limited sensitivity of 16S and ITS in detecting and identifying an infectious agent needs to be considered. PMID:27366168

  15. Reconstruction of phylogenetic trees of prokaryotes using maximal common intervals.

    PubMed

    Heydari, Mahdi; Marashi, Sayed-Amir; Tusserkani, Ruzbeh; Sadeghi, Mehdi

    2014-10-01

    One of the fundamental problems in bioinformatics is phylogenetic tree reconstruction, which can be used for classifying living organisms into different taxonomic clades. The classical approach to this problem is based on a marker such as 16S ribosomal RNA. Since evolutionary events like genomic rearrangements are not included in reconstructions of phylogenetic trees based on single genes, much effort has been made to find other characteristics for phylogenetic reconstruction in recent years. With the increasing availability of completely sequenced genomes, gene order can be considered as a new solution for this problem. In the present work, we applied maximal common intervals (MCIs) in two or more genomes to infer their distance and to reconstruct their evolutionary relationship. Additionally, measures based on uncommon segments (UCS's), i.e., those genomic segments which are not detected as part of any of the MCIs, are also used for phylogenetic tree reconstruction. We applied these two types of measures for reconstructing the phylogenetic tree of 63 prokaryotes with known COG (clusters of orthologous groups) families. Similarity between the MCI-based (resp. UCS-based) reconstructed phylogenetic trees and the phylogenetic tree obtained from NCBI taxonomy browser is as high as 93.1% (resp. 94.9%). We show that in the case of this diverse dataset of prokaryotes, tree reconstruction based on MCI and UCS outperforms most of the currently available methods based on gene orders, including breakpoint distance and DCJ. We additionally tested our new measures on a dataset of 13 closely-related bacteria from the genus Prochlorococcus. In this case, distances like rearrangement distance, breakpoint distance and DCJ proved to be useful, while our new measures are still appropriate for phylogenetic reconstruction.

  16. Phylogenetic relationship among horseshoe crab species: effect of substitution models on phylogenetic analyses.

    PubMed

    Xia, X

    2000-03-01

    The horseshoe crabs, known as living fossils, have maintained their morphology almost unchanged for the past 150 million years. The little morphological differentiation among horseshoe crab lineages has resulted in substantial controversy concerning the phylogenetic relationship among the extant species of horseshoe crabs, especially among the three species in the Indo-Pacific region. Previous studies suggest that the three species constitute a phylogenetically unresolvable trichotomy, the result of a cladogenetic process leading to the formation of all three Indo-Pacific species in a short geological time. Data from two mitochondrial genes (for 16S ribosomal rRNA and cytochrome oxidase subunit I) and one nuclear gene (for coagulogen) in the four species of horseshoe crabs and outgroup species were used in a phylogenetic analysis with various substitution models. All three genes yield the same tree topology, with Tachypleus-gigas and Carcinoscorpius-rotundicauda grouped together as a monophyletic taxon. This topology is significantly better than all the alternatives when evaluated with the RELL (resampling estimated log-likelihood) method.

  17. [PCR rDNA 16S used for the etiological diagnosis of blood culture negative endocarditis].

    PubMed

    Baty, G; Lanotte, P; Hocqueloux, L; Prazuck, T; Bret, L; Romano, M; Mereghetti, L

    2010-06-01

    We report the case of a 55 year-old man presenting with a double aortic and mitral endocarditis for which resected valve culture was repeatedly negative. Specific PCR made on valves because of highly positive blood tests for Bartonella henselae remained negative. A molecular approach was made with 16S rDNA PCR, followed by sequencing. Bartonella quintana was identified as the etiology of endocarditis. B. quintana, "fastidious" bacteria, even if hard to identify in a laboratory, is often reported as a blood culture negative endocarditis (BCNE) agent. Molecular biology methods have strongly improved the diagnosis of BCNE. We propose a review of the literature focusing on the interest of broad-spectrum PCR on valve for the etiological diagnosis of BCNE.

  18. Isolation of bacteria and 16S rDNAs from Lake Vostok accretion ice.

    PubMed

    Christner, B C; Mosley-Thompson, E; Thompson, L G; Reeve, J N

    2001-09-01

    Lake Vostok, the largest subglacial lake in Antarctica, is separated from the surface by approximately 4 km of glacial ice. It has been isolated from direct surface input for at least 420 000 years, and the possibility of a novel environment and ecosystem therefore exists. Lake Vostok water has not been sampled, but an ice core has been recovered that extends into the ice accreted below glacial ice by freezing of Lake Vostok water. Here, we report the recovery of bacterial isolates belonging to the Brachybacteria, Methylobacterium, Paenibacillus and Sphingomonas lineages from a sample of melt water from this accretion ice that originated 3593 m below the surface. We have also amplified small-subunit ribosomal RNA-encoding DNA molecules (16S rDNAs) directly from this melt water that originated from alpha- and beta-proteobacteria, low- and high-G+C Gram-positive bacteria and a member of the Cytophaga/Flavobacterium/Bacteroides lineage.

  19. An unusual case of seronegative, 16S PCR positive Brucella infection

    PubMed Central

    Backhouse, Lucy; Rawat, David; Naik, Sandhia; Millar, Michael

    2016-01-01

    Introduction: Brucella is a zoonotic infection commonly diagnosed by isolation of the organism from blood culture or positive serological testing. It is an uncommon cause of a pyrexia of unknown origin in the United Kingdom. Case presentation: We describe the case of a 14-year-old girl with no history of travel who presented with pyrexia, weight loss, arthralgia, multiple splenic abscesses and a subsequent pleural effusion, the latter of which isolated a Brucella species on 16S rRNA PCR. The patient responded well to initiation of treatment for brucellosis and on repeat imaging, after 3 months, the splenic abscesses had resolved. Conclusion: This unique case demonstrates uncommon complications of brucellosis and the challenges of diagnosing the organism, the latter of which can be alleviated by the utilization of molecularbased technologies. This patient had a negative serology result for brucellosis, which highlights the need to interpret serology results with caution in non-endemic regions for brucellosis. PMID:28348782

  20. Towards a phylogeny of the genus Vibrio based on 16S rRNA sequences.

    PubMed

    Dorsch, M; Lane, D; Stackebrandt, E

    1992-01-01

    The inter- and intrageneric relationships of the genus Vibrio were investigated by performing a comparative analysis of the 16S rRNAs of 10 species, including four pathogenic representatives. The results of immunological and 5S rRNA studies were confirmed in that the genus is a neighboring taxon of the family Enterobacteriaceae. With regard to the intrageneric structure, Vibrio alginolyticus, Vibrio campbellii, Vibrio natriegens, Vibrio harveyi, Vibrio proteolyticus, Vibrio parahaemolyticus, and Vibrio vulnificus form the core of the genus, while Vibrio (Listonella) anguillarum, Vibrio diazotrophicus, and Vibrio hollisae are placed on the outskirts of the genus. Variable regions around positions 80, 180, and 450 could be used as target sites for genus- and species-specific oligonucleotide probes and polymerase chain reaction primers to be used in molecular identification.

  1. Fully stereocontrolled total syntheses of the prostacyclin analogues 16S-iloprost and 16S-3-oxa-iloprost by a common route, using alkenylcopper-azoalkene conjugate addition, asymmetric olefination, and allylic alkylation.

    PubMed

    Kramp, Guido J; Kim, Mikhail; Gais, Hans-Joachim; Vermeeren, Cornelia

    2005-12-21

    In this article we describe fully stereocontrolled total syntheses of 16S-iloprost (16S-2), the most active component of the drugs Ilomedin and Ventavis, and of 16S-3-oxa-iloprost (16S-3), a close analogue of 16S-2 having the potential for a high oral activity, by a new and common route. The key steps of this route are (1) the establishment of the complete C13-C20 omega side chain of the target molecules through a stereoselective conjugate addition of the alkenylcopper derivative 9 to the bicyclic C6-C12 azoalkene 10 with formation of hydrazone 8, (2) the diastereoselective olefination of ketone 7 with the chiral phosphoryl acetate 39, and (3) the regio- and stereoselective alkylation of the allylic acetate 43 with cuprate 42. These measures allowed the 5E,15S,16S-stereoselective synthesis of 16S-2 and 16S-3, a goal which had previously not been achieved. Azoalkene 10 was obtained from the achiral bicyclic C6-C12 ketone 11 as previously described by using as key step an enantioselective deprotonation. The configuration at C16 of omega-side chain building block 9 has been installed with high stereoselectivity by the oxazolidinone method and that at C15 by a diastereoselective oxazaborolidine-catalyzed reduction of the C13-C20 ketone 23 with catecholborane. Surprisingly, a high diastereoselectivity in the reduction of 23 was only obtained by using 2 equiv of oxazaborolidine 24. Application of substoichiometric amounts of 24 resulted in irreproducible diastereoselectivities ranging from very high to nil.

  2. Multi-site-specific 16S rRNA Methyltransferase RsmF from Thermus thermophilus

    SciTech Connect

    Demirci, H.; Larsen, L; Hansen, T; Rasmussen, A; Cadambi, A; Gregory, S; Kirpekar, F; Jogl, G

    2010-01-01

    Cells devote a significant effort toward the production of multiple modified nucleotides in rRNAs, which fine tune the ribosome function. Here, we report that two methyltransferases, RsmB and RsmF, are responsible for all four 5-methylcytidine (m{sup 5}C) modifications in 16S rRNA of Thermus thermophilus. Like Escherichia coli RsmB, T. thermophilus RsmB produces m{sup 5}C967. In contrast to E. coli RsmF, which introduces a single m{sup 5}C1407 modification, T. thermophilus RsmF modifies three positions, generating m{sup 5}C1400 and m{sup 5}C1404 in addition to m{sup 5}C1407. These three residues are clustered near the decoding site of the ribosome, but are situated in distinct structural contexts, suggesting a requirement for flexibility in the RsmF active site that is absent from the E. coli enzyme. Two of these residues, C1400 and C1404, are sufficiently buried in the mature ribosome structure so as to require extensive unfolding of the rRNA to be accessible to RsmF. In vitro, T. thermophilus RsmF methylates C1400, C1404, and C1407 in a 30S subunit substrate, but only C1400 and C1404 when naked 16S rRNA is the substrate. The multispecificity of T. thermophilus RsmF is potentially explained by three crystal structures of the enzyme in a complex with cofactor S-adenosyl-methionine at up to 1.3 {angstrom} resolution. In addition to confirming the overall structural similarity to E. coli RsmF, these structures also reveal that key segments in the active site are likely to be dynamic in solution, thereby expanding substrate recognition by T. thermophilus RsmF.

  3. Hosts, distribution and genetic divergence (16S rDNA) of Amblyomma dubitatum (Acari: Ixodidae).

    PubMed

    Nava, Santiago; Venzal, José M; Labruna, Marcelo B; Mastropaolo, Mariano; González, Enrique M; Mangold, Atilio J; Guglielmone, Alberto A

    2010-08-01

    We supply information about hosts and distribution of Amblyomma dubitatum. In addition, we carry out an analysis of genetic divergence among specimens of A. dubitatum from different localities and with respect to other Neotropical Amblyomma species, using sequences of 16S rDNA gene. Although specimens of A. dubitatum were collected on several mammal species as cattle horse, Tapirus terrestris, Mazama gouazoubira, Tayassu pecari, Sus scrofa, Cerdocyon thous, Myocastor coypus, Allouata caraya, Glossophaga soricina and man, most records of immature and adult stages of A. dubitatum were made on Hydrochoerus hydrochaeris, making this rodent the principal host for all parasitic stages of this ticks. Cricetidae rodents (Lundomys molitor, Scapteromys tumidus), opossums (Didelphis albiventris) and vizcacha (Lagostomus maximus) also were recorded as hosts for immature stages. All findings of A. dubitatum correspond to localities of Argentina, Brazil, Paraguay and Uruguay, and they were concentrated in the Biogeographical provinces of Pampa, Chaco, Cerrado, Brazilian Atlantic Forest, Parana Forest and Araucaria angustifolia Forest. The distribution of A. dubitatum is narrower than that of its principal host, therefore environmental variables rather than hosts determine the distributional ranges of this tick. The intraspecific genetic divergence among 16S rDNA sequences of A. dubitatum ticks collected in different localities from Argentina, Brazil and Uruguay was in all cases lower than 0.8%, whereas the differences with the remaining Amblyomma species included in the analysis were always bigger than 6.8%. Thus, the taxonomic status of A. dubitatum along its distribution appears to be certain at the specific level.

  4. 16S ribosomal RNA gene-based phylogenetic analysis of abundant bacteria in river, canal and potable water in Bangkok, Thailand.

    PubMed

    Yamaguchi, Nobuyasu; Nishiguchi, Takahiro; Utrarachkij, Fuangfa; Suthienkul, Orasa; Nasu, Masao

    2013-01-01

    In Southeast Asian countries, industrialization and urbanization is occurring rapidly, and water pollution in rivers and canals poses serious problems in some areas, especially in cities. Excess inflow of domestic, agricultural, and industrial wastewater to freshwater environments disturbs the aquatic microbial ecosystem, which can further pollute water by inhibiting biodegradation of pollutants. Therefore, monitoring of microbes in freshwater environment is important to identify changes in indigenous microbial populations and to estimate the influence of wastewater inflows on them. Polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis is suitable for monitoring changes in microbial communities caused by human activities, but this method can be difficult in eutrophic freshwater samples that contain PCR inhibitors. In this study, we optimized DNA extraction procedures and PCR conditions for DGGE analysis of bacterial populations in freshwater samples (canal, river, and tap water) collected in Bangkok, Thailand. A simple freeze-thaw procedure was effective for extracting DNA from bacterial cells in the samples, and LA Taq with added bovine serum albumin provided the best PCR amplification. The PCR-DGGE approach revealed that the most common bacteria in freshwater samples belonged to Gammaproteobacteria, while a Gram-positive bacterium was present at Bangkok Noi Canal. Temporally and spatially continuous analyses of bacterial populations in Bangkok canals and rivers by PCR-DGGE approach should be useful to recognize disturbances of microbial ecosystems caused by excess inflows of wastewater.

  5. A Universal Phylogenetic Tree.

    ERIC Educational Resources Information Center

    Offner, Susan

    2001-01-01

    Presents a universal phylogenetic tree suitable for use in high school and college-level biology classrooms. Illustrates the antiquity of life and that all life is related, even if it dates back 3.5 billion years. Reflects important evolutionary relationships and provides an exciting way to learn about the history of life. (SAH)

  6. Phylogenetic analysis and development of probes for differentiating methylotrophic bacteria.

    PubMed Central

    Brusseau, G A; Bulygina, E S; Hanson, R S

    1994-01-01

    Fifteen small-subunit rRNAs from methylotrophic bacteria have been sequenced. Comparisons of these sequences with 22 previously published sequences further defined the phylogenetic relationships among these bacteria and illustrated the agreement between phylogeny and physiological characteristics of the bacteria. Phylogenetic trees were constructed with 16S rRNA sequences from methylotrophic bacteria and representative organisms from subdivisions within the class Proteobacteria on the basis of sequence similarities by using a weighted least-mean-square difference method. The methylotrophs have been separated into coherent clusters in which bacteria shared physiological characteristics. The clusters distinguished bacteria which used either the ribulose monophosphate or serine pathway for carbon assimilation. In addition, methanotrophs and methylotrophs which do not utilize methane were found to form distinct clusters within these groups. Five new deoxyoligonucleotide probes were designed, synthesized, labelled with digoxigenin-11-ddUTP, and tested for the ability to hybridize to RNA extracted from the bacteria represented in the unique clusters and for the ability to detect RNAs purified from soils enriched for methanotrophs by exposure to a methane-air atmosphere for one month. The 16S rRNA purified from soil hybridized to the probe which was complementary to sequences present in 16S rRNA from serine pathway methanotrophs and hybridized to a lesser extent with a probe complementary to sequences in 16S rRNAs of ribulose monophosphate pathway methanotrophs. The nonradioactive detection system used performed reliably at amounts of RNA from pure cultures as small as 10 ng. Images PMID:7510941

  7. Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences.

    PubMed

    Yarza, Pablo; Yilmaz, Pelin; Pruesse, Elmar; Glöckner, Frank Oliver; Ludwig, Wolfgang; Schleifer, Karl-Heinz; Whitman, William B; Euzéby, Jean; Amann, Rudolf; Rosselló-Móra, Ramon

    2014-09-01

    Publicly available sequence databases of the small subunit ribosomal RNA gene, also known as 16S rRNA in bacteria and archaea, are growing rapidly, and the number of entries currently exceeds 4 million. However, a unified classification and nomenclature framework for all bacteria and archaea does not yet exist. In this Analysis article, we propose rational taxonomic boundaries for high taxa of bacteria and archaea on the basis of 16S rRNA gene sequence identities and suggest a rationale for the circumscription of uncultured taxa that is compatible with the taxonomy of cultured bacteria and archaea. Our analyses show that only nearly complete 16S rRNA sequences give accurate measures of taxonomic diversity. In addition, our analyses suggest that most of the 16S rRNA sequences of the high taxa will be discovered in environmental surveys by the end of the current decade.

  8. Opposing phylogenetic diversity gradients of plant and soil bacterial communities

    PubMed Central

    Goberna, Marta; Navarro-Cano, Jose A.; Verdú, Miguel

    2016-01-01

    Plants and soil microbes show parallel patterns of species-level diversity. Diverse plant communities release a wider range of organics that are consumed by more microbial species. We speculated, however, that diversity metrics accounting for the evolutionary distance across community members would reveal opposing patterns between plant and soil bacterial phylogenetic diversity. Plant phylogenetic diversity enhances plant productivity and thus expectedly soil fertility. This, in turn, might reduce bacterial phylogenetic diversity by favouring one (or a few) competitive bacterial clade. We collected topsoils in 15 semi-arid plant patches and adjacent low-cover areas configuring a plant phylodiversity gradient, pyrosequenced the 16S rRNA gene to identify bacterial taxa and analysed soil fertility parameters. Structural equation modelling showed positive effects of both plant richness and phylogenetic diversity on soil fertility. Fertility increased bacterial richness but reduced bacterial phylogenetic diversity. This might be attributed to the competitive dominance of a lineage based on its high relative fitness. This suggests biotic interactions as determinants of the soil bacterial community assembly, while emphasizing the need to use phylogeny-informed metrics to tease apart the processes underlying the patterns of diversity. PMID:26888037

  9. Opposing phylogenetic diversity gradients of plant and soil bacterial communities.

    PubMed

    Goberna, Marta; Navarro-Cano, Jose A; Verdú, Miguel

    2016-02-24

    Plants and soil microbes show parallel patterns of species-level diversity. Diverse plant communities release a wider range of organics that are consumed by more microbial species. We speculated, however, that diversity metrics accounting for the evolutionary distance across community members would reveal opposing patterns between plant and soil bacterial phylogenetic diversity. Plant phylogenetic diversity enhances plant productivity and thus expectedly soil fertility. This, in turn, might reduce bacterial phylogenetic diversity by favouring one (or a few) competitive bacterial clade. We collected topsoils in 15 semi-arid plant patches and adjacent low-cover areas configuring a plant phylodiversity gradient, pyrosequenced the 16S rRNA gene to identify bacterial taxa and analysed soil fertility parameters. Structural equation modelling showed positive effects of both plant richness and phylogenetic diversity on soil fertility. Fertility increased bacterial richness but reduced bacterial phylogenetic diversity. This might be attributed to the competitive dominance of a lineage based on its high relative fitness. This suggests biotic interactions as determinants of the soil bacterial community assembly, while emphasizing the need to use phylogeny-informed metrics to tease apart the processes underlying the patterns of diversity.

  10. Abiotrophia defectiva infection of a total hip arthroplasty diagnosed by 16S rRNA gene sequencing.

    PubMed

    Rozemeijer, Wouter; Jiya, Timothy U; Rijnsburger, Martine; Heddema, Edou; Savelkoul, Paul; Ang, Wim

    2011-05-01

    We describe a case of a total hip arthroplasty infection caused by Abiotrophia defectiva, identified by 16S rRNA gene sequencing. Removal of the prosthesis followed by antibiotic treatment resulted in a good clinical outcome. 16S rRNA gene sequencing can be a useful tool in diagnosing infection with this fastidious microorganism that can easily be misidentified using phenotypic identification methods.

  11. Diversity and Structure of the Methanogenic Community in Anoxic Rice Paddy Soil Microcosms as Examined by Cultivation and Direct 16S rRNA Gene Sequence Retrieval

    PubMed Central

    Großkopf, Regine; Janssen, Peter H.; Liesack, Werner

    1998-01-01

    A dual approach consisting of cultivation and molecular retrieval of partial archaeal 16S rRNA genes was carried out to characterize the diversity and structure of the methanogenic community inhabiting the anoxic bulk soil of flooded rice microcosms. The molecular approach identified four groups of known methanogens. Three environmental sequences clustered with Methanobacterium bryantii and Methanobacterium formicicum, six were closely related but not identical to those of strains of Methanosaeta concilii, two grouped with members of the genus Methanosarcina, and two were related to the methanogenic endosymbiont of Plagiopyla nasuta. The cultivation approach via most-probable-number counts with a subsample of the same soil as an inoculum yielded cell numbers of up to 107 per g of dry soil for the H2-CO2-utilizing methanogens and of up to 106 for the acetate-utilizing methanogens. Strain VeH52, isolated from the terminal positive dilution on H2-CO2, grouped within the phylogenetic radiation characterized by M. bryantii and M. formicicum and the environmental sequences of the Methanobacterium-like group. A consortium of two distinct methanogens grew in the terminal positive culture on acetate. These two organisms showed absolute 16S rRNA gene identities with environmental sequences of the novel Methanosaeta-like group and the Methanobacterium-like group. Methanosarcina spp. were identified only in the less-dilute levels of the same dilution series on acetate. These data correlate well with acetate concentrations of about 11 μM in the pore water of this rice paddy soil. These concentrations are too low for the growth of known Methanosarcina spp. but are at the acetate utilization threshold of Methanosaeta spp. Thus, our data indicated Methanosaeta spp. and Methanobacterium spp. to be the dominant methanogenic groups in the anoxic rice soil, whereas Methanosarcina spp. appeared to be less abundant. PMID:9501436

  12. 16S rRNA partial gene sequencing for the differentiation and molecular subtyping of Listeria species.

    PubMed

    Hellberg, Rosalee S; Martin, Keely G; Keys, Ashley L; Haney, Christopher J; Shen, Yuelian; Smiley, R Derike

    2013-12-01

    Use of 16S rRNA partial gene sequencing within the regulatory workflow could greatly reduce the time and labor needed for confirmation and subtyping of Listeria monocytogenes. The goal of this study was to build a 16S rRNA partial gene reference library for Listeria spp. and investigate the potential for 16S rRNA molecular subtyping. A total of 86 isolates of Listeria representing L. innocua, L. seeligeri, L. welshimeri, and L. monocytogenes were obtained for use in building the custom library. Seven non-Listeria species and three additional strains of Listeria were obtained for use in exclusivity and food spiking tests. Isolates were sequenced for the partial 16S rRNA gene using the MicroSeq ID 500 Bacterial Identification Kit (Applied Biosystems). High-quality sequences were obtained for 84 of the custom library isolates and 23 unique 16S sequence types were discovered for use in molecular subtyping. All of the exclusivity strains were negative for Listeria and the three Listeria strains used in food spiking were consistently recovered and correctly identified at the species level. The spiking results also allowed for differentiation beyond the species level, as 87% of replicates for one strain and 100% of replicates for the other two strains consistently matched the same 16S type.

  13. 16S rRNA Amplicon Sequencing for Epidemiological Surveys of Bacteria in Wildlife

    PubMed Central

    Razzauti, Maria; Bard, Emilie; Bernard, Maria; Brouat, Carine; Charbonnel, Nathalie; Dehne-Garcia, Alexandre; Loiseau, Anne; Tatard, Caroline; Tamisier, Lucie; Vayssier-Taussat, Muriel; Vignes, Helene

    2016-01-01

    ABSTRACT The human impact on natural habitats is increasing the complexity of human-wildlife interactions and leading to the emergence of infectious diseases worldwide. Highly successful synanthropic wildlife species, such as rodents, will undoubtedly play an increasingly important role in transmitting zoonotic diseases. We investigated the potential for recent developments in 16S rRNA amplicon sequencing to facilitate the multiplexing of the large numbers of samples needed to improve our understanding of the risk of zoonotic disease transmission posed by urban rodents in West Africa. In addition to listing pathogenic bacteria in wild populations, as in other high-throughput sequencing (HTS) studies, our approach can estimate essential parameters for studies of zoonotic risk, such as prevalence and patterns of coinfection within individual hosts. However, the estimation of these parameters requires cleaning of the raw data to mitigate the biases generated by HTS methods. We present here an extensive review of these biases and of their consequences, and we propose a comprehensive trimming strategy for managing these biases. We demonstrated the application of this strategy using 711 commensal rodents, including 208 Mus musculus domesticus, 189 Rattus rattus, 93 Mastomys natalensis, and 221 Mastomys erythroleucus, collected from 24 villages in Senegal. Seven major genera of pathogenic bacteria were detected in their spleens: Borrelia, Bartonella, Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia. Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia have never before been detected in West African rodents. Bacterial prevalence ranged from 0% to 90% of individuals per site, depending on the bacterial taxon, rodent species, and site considered, and 26% of rodents displayed coinfection. The 16S rRNA amplicon sequencing strategy presented here has the advantage over other molecular surveillance tools of dealing with a large spectrum of bacterial

  14. Novel Diagnostic Algorithm for Identification of Mycobacteria Using Genus-Specific Amplification of the 16S-23S rRNA Gene Spacer and Restriction Endonucleases

    PubMed Central

    Roth, Andreas; Reischl, Udo; Streubel, Anna; Naumann, Ludmila; Kroppenstedt, Reiner M.; Habicht, Marion; Fischer, Marga; Mauch, Harald

    2000-01-01

    A novel genus-specific PCR for mycobacteria with simple identification to the species level by restriction fragment length polymorphism (RFLP) was established using the 16S-23S ribosomal RNA gene (rDNA) spacer as a target. Panspecificity of primers was demonstrated on the genus level by testing 811 bacterial strains (122 species in 37 genera from 286 reference strains and 525 clinical isolates). All mycobacterial isolates (678 strains among 48 defined species and 5 indeterminate taxons) were amplified by the new primers. Among nonmycobacterial isolates, only Gordonia terrae was amplified. The RFLP scheme devised involves estimation of variable PCR product sizes together with HaeIII and CfoI restriction analysis. It yielded 58 HaeIII patterns, of which 49 (84%) were unique on the species level. Hence, HaeIII digestion together with CfoI results was sufficient for correct identification of 39 of 54 mycobacterial taxons and one of three or four of seven RFLP genotypes found in Mycobacterium intracellulare and Mycobacterium kansasii, respectively. Following a clearly laid out diagnostic algorithm, the remaining unidentified organisms fell into five clusters of closely related species (i.e., the Mycobacterium avium complex or Mycobacterium chelonae-Mycobacterium abscessus) that were successfully separated using additional enzymes (TaqI, MspI, DdeI, or AvaII). Thus, next to slowly growing mycobacteria, all rapidly growing species studied, including M. abscessus, M. chelonae, Mycobacterium farcinogenes, Mycobacterium fortuitum, Mycobacterium peregrinum, and Mycobacterium senegalense (with a very high 16S rDNA sequence similarity) were correctly identified. A high intraspecies sequence stability and the good discriminative power of patterns indicate that this method is very suitable for rapid and cost-effective identification of a wide variety of mycobacterial species without the need for sequencing. Phylogenetically, spacer sequence data stand in good agreement with 16S r

  15. Bacterial diversity analysis of Huanglongbing pathogen-infected citrus, using PhyloChip and 16S rRNA gene clone library sequencing

    SciTech Connect

    Shankar Sagaram, U.; DeAngelis, K.M.; Trivedi, P.; Andersen, G.L.; Lu, S.-E.; Wang, N.

    2009-03-01

    The bacterial diversity associated with citrus leaf midribs was characterized 1 from citrus groves that contained the Huanglongbing (HLB) pathogen, which has yet to be cultivated in vitro. We employed a combination of high-density phylogenetic 16S rDNA microarray and 16S rDNA clone library sequencing to determine the microbial community composition of symptomatic and asymptomatic citrus midribs. Our results revealed that citrus leaf midribs can support a diversity of microbes. PhyloChip analysis indicated that 47 orders of bacteria from 15 phyla were present in the citrus leaf midribs while 20 orders from phyla were observed with the cloning and sequencing method. PhyloChip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs compared to asymptomatic midribs. Candidatus Liberibacter asiaticus (Las) was detected at a very low level in asymptomatic plants, but was over 200 times more abundant in symptomatic plants. The PhyloChip analysis was further verified by sequencing 16S rDNA clone libraries, which indicated the dominance of Las in symptomatic leaves. These data implicate Las as the pathogen responsible for HLB disease. Citrus is the most important commercial fruit crop in Florida. In recent years, citrus Huanglongbing (HLB), also called citrus greening, has severely affected Florida's citrus production and hence has drawn an enormous amount of attention. HLB is one of the most devastating diseases of citrus (6,13), characterized by blotchy mottling with green islands on leaves, as well as stunting, fruit decline, and small, lopsided fruits with poor coloration. The disease tends to be associated with a phloem-limited fastidious {alpha}-proteobacterium given a provisional Candidatus status (Candidatus Liberobacter spp. later changed to Candidatus Liberibacter spp.) in nomenclature (18,25,34). Previous studies indicate that HLB infection causes disorder in the phloem and severely impairs the translocation of assimilates in host

  16. Nucleotide sequencing and analysis of 16S rDNA and 16S-23S rDNA internal spacer region (ISR) of Taylorella equigenitalis, as an important pathogen for contagious equine metritis (CEM).

    PubMed

    Kagawa, S; Nagano, Y; Tazumi, A; Murayama, O; Millar, B C; Moore, J E; Matsuda, M

    2006-05-01

    The primer set for 16S rDNA amplified an amplicon of about 1500 bp in length for three strains of Taylorella equigenitalis (NCTC11184(T), Kentucky188 and EQ59). Sequence differences of the 16S rDNA among the six sequences, including three reference sequences, occurred at only a few nucleotide positions and thus, an extremely high sequence similarity of the 16S rDNA was first demonstrated among the six sequences. In addition, the primer set for 16S-23S rDNA internal spacer region (ISR) amplified two amplicons about 1300 bp and 1200 bp in length for the three strains. The ISRs were estimated to be about 920 bp in length for large ISR-A and about 830 bp for small ISR-B. Sequence alignment of the ISR-A and ISR-B demonstrated about 10 base differences between NCTC11184(T) and EQ59 and between Kentucky188 and EQ59. However, only minor sequence differences were demonstrated between the ISR-A and ISR-B from NCTC11184(T) and Kentucky188, respectively. A typical order of the intercistronic tRNAs with the 29 nucleotide spacer of 5'-16S rDNA-tRNA(Ile)-tRNA(Ala)-23S rDNA-3' was demonstrated in the all ISRs. The ISRs may be useful for the discrimination amongst isolates of T. equigenitalis if sequencing is employed.

  17. Algae-bacteria association inferred by 16S rDNA similarity in established microalgae cultures.

    PubMed

    Schwenk, Dagmar; Nohynek, Liisa; Rischer, Heiko

    2014-06-01

    Forty cultivable, visually distinct bacterial cultures were isolated from four Baltic microalgal cultures Chlorella pyrenoidosa, Scenedesmus obliquus, Isochrysis sp., and Nitzschia microcephala, which have been maintained for several years in the laboratory. Bacterial isolates were characterized with respect to morphology, antibiotic susceptibility, and 16S ribosomal DNA sequence. A total of 17 unique bacterial strains, almost all belonging to one of three families, Rhodobacteraceae, Rhizobiaceae, and Erythrobacteraceae, were subsequently isolated. The majority of isolated bacteria belong to Rhodobacteraceae. Literature review revealed that close relatives of the bacteria isolated in this study are not only often found in marine environments associated with algae, but also in lakes, sediments, and soil. Some of them had been shown to interact with organisms in their surroundings. A Basic Local Alignment Search Tool study indicated that especially bacteria isolated from the Isochrysis sp. culture were highly similar to microalgae-associated bacteria. Two of those isolates, I1 and I6, belong to the Cytophaga-Flavobacterium-Bacteroides phylum, members of which are known to occur in close communities with microalgae. An UniFrac analysis revealed that the bacterial community of Isochrysis sp. significantly differs from the other three communities.

  18. Molecular identification of adulteration in mutton based on mitochondrial 16S rRNA gene.

    PubMed

    Xu, Jia; Zhao, Wei; Zhu, Mengru; Wen, Yuanju; Xie, Tao; He, Xiaoqian; Zhang, Yongfeng; Cao, Suizhong; Niu, Lili; Zhang, Hongping; Zhong, Tao

    2016-01-01

    The aim of this study is to set up a protocol for identification of the adulteration in mutton based on mitochondrial 16S rRNA gene. The multiplex polymerase chain reaction (multi-PCR) assay was carried out to trace the impure DNA in mutton. A universal primer pair yielded an approximate 610 bp fragment in mutton, pork, duck, chicken, horse and cat meats. The amplicons of multi-PCR assay represented the species-specific products, which could be discriminated by the size ranging from 106 bp to 532 bp. Subsequently, the authentication of each fragment was also confirmed by sequencing. Random analyses of adulterants with various meats yielded the identical results to their components, showing the suitability of the multi-PCR assay for tracing of adulterant meats with high-accuracy and precision. This assay was sensitive to detect the species-specific DNA in different proportional mixtures of mutton and duck/pork (9.1%-90.9%). In conclusion, this multi-PCR assay successfully discriminated the double-, triple-, quadruple-, and quintuple-mixtures containing variant counterparts. This method will be particularly useful in the detection of mutton adulteration in processed foods further.

  19. Primer and platform effects on 16S rRNA tag sequencing

    DOE PAGES

    Tremblay, Julien; Singh, Kanwar; Fern, Alison; ...

    2015-08-04

    Sequencing of 16S rRNA gene tags is a popular method for profiling and comparing microbial communities. The protocols and methods used, however, vary considerably with regard to amplification primers, sequencing primers, sequencing technologies; as well as quality filtering and clustering. How results are affected by these choices, and whether data produced with different protocols can be meaningfully compared, is often unknown. Here we compare results obtained using three different amplification primer sets (targeting V4, V6–V8, and V7–V8) and two sequencing technologies (454 pyrosequencing and Illumina MiSeq) using DNA from a mock community containing a known number of species as wellmore » as complex environmental samples whose PCR-independent profiles were estimated using shotgun sequencing. We find that paired-end MiSeq reads produce higher quality data and enabled the use of more aggressive quality control parameters over 454, resulting in a higher retention rate of high quality reads for downstream data analysis. While primer choice considerably influences quantitative abundance estimations, sequencing platform has relatively minor effects when matched primers are used. In conclusion, beta diversity metrics are surprisingly robust to both primer and sequencing platform biases.« less

  20. Functional Specialization of Domains Tandemly Duplicated Witin 16S rRNA Methyltransferase RsmC

    SciTech Connect

    Sunita,S.; Purta, E.; Durawa, M.; Tkaczuk, K.; Swaathi, J.; Bujnicki, J.; Sivaraman, J.

    2007-01-01

    RNA methyltransferases (MTases) are important players in the biogenesis and regulation of the ribosome, the cellular machine for protein synthesis. RsmC is a MTase that catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to G1207 of 16S rRNA. Mutations of G1207 have dominant lethal phenotypes in Escherichia coli, underscoring the significance of this modified nucleotide for ribosome function. Here we report the crystal structure of E. coli RsmC refined to 2.1 Angstroms resolution, which reveals two homologous domains tandemly duplicated within a single polypeptide. We characterized the function of the individual domains and identified key residues involved in binding of rRNA and SAM, and in catalysis. We also discovered that one of the domains is important for the folding of the other. Domain duplication and subfunctionalization by complementary degeneration of redundant functions (in particular substrate binding versus catalysis) has been reported for many enzymes, including those involved in RNA metabolism. Thus, RsmC can be regarded as a model system for functional streamlining of domains accompanied by the development of dependencies concerning folding and stability.

  1. Investigation of the koala (Phascolarctos cinereus) hindgut microbiome via 16S pyrosequencing.

    PubMed

    Barker, Christopher J; Gillett, Amber; Polkinghorne, Adam; Timms, Peter

    2013-12-27

    As a dietary source, the foliage of Eucalyptus spp. is low in available protein and carbohydrate while containing polyphenolic compounds that interfere with enzymatic digestion. To overcome this, the koala (Phascolarctos cinereus) has evolved a range of anatomical and physiological adaptations to assist with digestion and absorption of nutrients from this food source. Microbial fermentation of partially digested eucalyptus leaves is thought to be critical in this process, however, little is known about the composition and diversity of microorganisms that are associated with digestive health in this native species. In this study, we performed 16S rRNA gene pyrosequencing of caecum, colon and faecal pellet samples from two wild, free ranging, Queensland koalas. Our results reveal a highly complex and diverse ecosystem with considerable intra-individual variation. Although samples were dominated by sequences from the Bacteroidetes and Firmicutes phyla there was considerable variation at the genus level. This study is the first non-culture based microbiota analysis, using 454-amplicon pyrosequencing, and provides preliminary data to expand our understanding of the koala hindgut.

  2. Molecular Analysis of Methanogen Richness in Landfill and Marshland Targeting 16S rDNA Sequences

    PubMed Central

    Yadav, Shailendra; Kundu, Sharbadeb; Ghosh, Sankar K.; Maitra, S. S.

    2015-01-01

    Methanogens, a key contributor in global carbon cycling, methane emission, and alternative energy production, generate methane gas via anaerobic digestion of organic matter. The methane emission potential depends upon methanogenic diversity and activity. Since they are anaerobes and difficult to isolate and culture, their diversity present in the landfill sites of Delhi and marshlands of Southern Assam, India, was analyzed using molecular techniques like 16S rDNA sequencing, DGGE, and qPCR. The sequencing results indicated the presence of methanogens belonging to the seventh order and also the order Methanomicrobiales in the Ghazipur and Bhalsawa landfill sites of Delhi. Sequences, related to the phyla Crenarchaeota (thermophilic) and Thaumarchaeota (mesophilic), were detected from marshland sites of Southern Assam, India. Jaccard analysis of DGGE gel using Gel2K showed three main clusters depending on the number and similarity of band patterns. The copy number analysis of hydrogenotrophic methanogens using qPCR indicates higher abundance in landfill sites of Delhi as compared to the marshlands of Southern Assam. The knowledge about “methanogenic archaea composition” and “abundance” in the contrasting ecosystems like “landfill” and “marshland” may reorient our understanding of the Archaea inhabitants. This study could shed light on the relationship between methane-dynamics and the global warming process. PMID:26568700

  3. Primer and platform effects on 16S rRNA tag sequencing

    SciTech Connect

    Tremblay, Julien; Singh, Kanwar; Fern, Alison; Kirton, Edward S.; He, Shaomei; Woyke, Tanja; Lee, Janey; Chen, Feng; Dangl, Jeffery L.; Tringe, Susannah G.

    2015-08-04

    Sequencing of 16S rRNA gene tags is a popular method for profiling and comparing microbial communities. The protocols and methods used, however, vary considerably with regard to amplification primers, sequencing primers, sequencing technologies; as well as quality filtering and clustering. How results are affected by these choices, and whether data produced with different protocols can be meaningfully compared, is often unknown. Here we compare results obtained using three different amplification primer sets (targeting V4, V6–V8, and V7–V8) and two sequencing technologies (454 pyrosequencing and Illumina MiSeq) using DNA from a mock community containing a known number of species as well as complex environmental samples whose PCR-independent profiles were estimated using shotgun sequencing. We find that paired-end MiSeq reads produce higher quality data and enabled the use of more aggressive quality control parameters over 454, resulting in a higher retention rate of high quality reads for downstream data analysis. While primer choice considerably influences quantitative abundance estimations, sequencing platform has relatively minor effects when matched primers are used. In conclusion, beta diversity metrics are surprisingly robust to both primer and sequencing platform biases.

  4. 16S rRNA Gene Mutations Associated with Decreased Susceptibility to Tetracycline in Mycoplasma bovis

    PubMed Central

    Amram, E.; Mikula, I.; Schnee, C.; Ayling, R. D.; Nicholas, R. A. J.; Rosales, R. S.; Harrus, S.

    2014-01-01

    Mycoplasma bovis isolates with decreased susceptibilities to tetracyclines are increasingly reported worldwide. The acquired molecular mechanisms associated with this phenomenon were investigated in 70 clinical isolates of M. bovis. Sequence analysis of the two 16S rRNA-encoding genes (rrs3 and rrs4 alleles) containing the primary binding pocket for tetracycline (Tet-1 site) was performed on isolates with tetracycline hydrochloride MICs of 0.125 to 16 μg/ml. Mutations at positions A965T, A967T/C (Escherichia coli numbering) of helix 31, U1199C of helix 34, and G1058A/C were identified. Decreased susceptibilities to tetracycline (MICs, ≥2 μg/ml) were associated with mutations present at two (A965 and A967) or three positions (A965, A967, and G1058) of the two rrs alleles. No tet(M), tet(O), or tet(L) determinants were found in the genome of any of the 70 M. bovis isolates. The data presented correlate (P < 0.0001) the mutations identified in the Tet-1 site of clinical isolates of M. bovis with decreased susceptibility to tetracycline. PMID:25403668

  5. Concurrent Nucleation of 16S Folding and Induced Fit in 30S Ribosome Assembly

    SciTech Connect

    Adilakshmi, T.; Bellur, D; Woodson, S

    2008-01-01

    Rapidly growing cells produce thousands of new ribosomes each minute, in a tightly regulated process that is essential to cell growth. How the Escherichia coli 16S ribosomal RNA and the 20 proteins that make up the 30S ribosomal subunit can assemble correctly in a few minutes remains a challenging problem, partly because of the lack of real-time data on the earliest stages of assembly. By providing snapshots of individual RNA and protein interactions as they emerge in real time, here we show that 30S assembly nucleates concurrently from different points along the rRNA. Time-resolved hydroxyl radical footprinting3 was used to map changes in the structure of the rRNA within 20 milliseconds after the addition of total 30S proteins. Helical junctions in each domain fold within 100 ms. In contrast, interactions surrounding the decoding site and between the 5', the central and the 3' domains require 2-200 seconds to form. Unexpectedly, nucleotides contacted by the same protein are protected at different rates, indicating that initial RNA-protein encounter complexes refold during assembly. Although early steps in assembly are linked to intrinsically stable rRNA structure, later steps correspond to regions of induced fit between the proteins and the rRNA.

  6. Phylogenetic relationships between flies of the Tephritinae subfamily (Diptera, Tephritidae) and their symbiotic bacteria.

    PubMed

    Mazzon, Luca; Martinez-Sañudo, Isabel; Simonato, Mauro; Squartini, Andrea; Savio, Claudia; Girolami, Vincenzo

    2010-07-01

    The Tephritinae is considered the most specialized subfamily of fruit flies, predominantly infesting flowerheads of Asteraceae. Some species are known to host specific non-culturable symbiont bacteria ("Candidatus Stammerula spp.") in the midgut. In this work we (i) examined the phylogenetic relationships among the insect hosts, (ii) investigated the presence of bacteria in other hitherto unexamined species, and (iii) evaluated the phylogenetic congruence between insects and symbionts. A total of 33 Tephritinae species in 17 different genera were analyzed. Two regions of the mitochondrial DNA (16S rDNA and COI-tRNALeu-COII) were examined in the insect host, while the 16S was analyzed in the bacteria. From the phylogenetic trees, four of the five tribes considered were statistically supported by each of the clustering methods used. Species belonging to the tribe Noeetini never clustered at significant levels. The phylogenetic COI-tRNALeu-COII tree showed internal nodes more highly supported than the 16S phylogeny. The analysis of the distribution of symbiosis across the subfamily has highlighted the presence of bacteria only in the tribe Tephritini and in the genus Noeeta from the tribe Noeetini. A cophylogenetic analysis revealed a substantial congruence between hosts and symbionts. The interesting exceptions can be justified by events like losses, duplications and hosts switching opportunities, which are likely to arise during the biological cycle of the fly in consideration of the extracellular status of these symbionts.

  7. International interlaboratory study comparing single organism 16S rRNA gene sequencing data: Beyond consensus sequence comparisons

    PubMed Central

    Olson, Nathan D.; Lund, Steven P.; Zook, Justin M.; Rojas-Cornejo, Fabiola; Beck, Brian; Foy, Carole; Huggett, Jim; Whale, Alexandra S.; Sui, Zhiwei; Baoutina, Anna; Dobeson, Michael; Partis, Lina; Morrow, Jayne B.

    2015-01-01

    This study presents the results from an interlaboratory sequencing study for which we developed a novel high-resolution method for comparing data from different sequencing platforms for a multi-copy, paralogous gene. The combination of PCR amplification and 16S ribosomal RNA gene (16S rRNA) sequencing has revolutionized bacteriology by enabling rapid identification, frequently without the need for culture. To assess variability between laboratories in sequencing 16S rRNA, six laboratories sequenced the gene encoding the 16S rRNA from Escherichia coli O157:H7 strain EDL933 and Listeria monocytogenes serovar 4b strain NCTC11994. Participants performed sequencing methods and protocols available in their laboratories: Sanger sequencing, Roche 454 pyrosequencing®, or Ion Torrent PGM®. The sequencing data were evaluated on three levels: (1) identity of biologically conserved position, (2) ratio of 16S rRNA gene copies featuring identified variants, and (3) the collection of variant combinations in a set of 16S rRNA gene copies. The same set of biologically conserved positions was identified for each sequencing method. Analytical methods using Bayesian and maximum likelihood statistics were developed to estimate variant copy ratios, which describe the ratio of nucleotides at each identified biologically variable position, as well as the likely set of variant combinations present in 16S rRNA gene copies. Our results indicate that estimated variant copy ratios at biologically variable positions were only reproducible for high throughput sequencing methods. Furthermore, the likely variant combination set was only reproducible with increased sequencing depth and longer read lengths. We also demonstrate novel methods for evaluating variable positions when comparing multi-copy gene sequence data from multiple laboratories generated using multiple sequencing technologies. PMID:27077030

  8. Identification of oral bacteria by 16S rRNA gene analysis in elderly persons requiring nursing care.

    PubMed

    Kurabayashi, Hirotaka; Kaneko, Akihiro; Sekiya, Ryo; Karakida, Kazunari; Sasaki, Masashi; Nakatogawa, Noriko; Aoki, Takayuki; Ota, Yoshihide; Sakamoto, Haruo

    2011-02-01

    After incubation of saliva from 58 semi-bedridden elderly persons, the cultures were identified based on the 16S rRNA gene base sequence to compare the identification by the conventional culture method. As a result, the 16S rRNA gene base sequence of 198 strains identified by the culture method showed 98.5% or more homology in some of the Human Oral Microbiome database, and the identification of bacterial species and genus was possible. When an organism identified by the 16S rRNA gene sequencing method was compared with that by the culture method, the concordance rates were 54.5% at the genus level and 35.9% at the species level. Streptococcus mitis strains most frequently isolated from saliva that were identified by the culture method were identified as the same species by the 16S rRNA gene sequencing method (32/35), and all the 11 Streptococcus salivarius strains identified by the culture method were identified as the same species by the 16S rRNA gene sequencing method. All the strains identified as Streptococcus anginosus group by the culture method and 8 of the 9 strains identified as Prevotella species by the culture method were identified as the same group and genus by the 16S rRNA gene sequencing method. When an oral microbial flora test with saliva samples from elderly persons is performed, the 16S rRNA gene sequence identification enables us to identify major indigenous bacteria and pathogenic bacteria and is considered useful as a means of supplementing the conventional culture method.

  9. Refuting phylogenetic relationships

    PubMed Central

    Bucknam, James; Boucher, Yan; Bapteste, Eric

    2006-01-01

    Background Phylogenetic methods are philosophically grounded, and so can be philosophically biased in ways that limit explanatory power. This constitutes an important methodologic dimension not often taken into account. Here we address this dimension in the context of concatenation approaches to phylogeny. Results We discuss some of the limits of a methodology restricted to verificationism, the philosophy on which gene concatenation practices generally rely. As an alternative, we describe a software which identifies and focuses on impossible or refuted relationships, through a simple analysis of bootstrap bipartitions, followed by multivariate statistical analyses. We show how refuting phylogenetic relationships could in principle facilitate systematics. We also apply our method to the study of two complex phylogenies: the phylogeny of the archaea and the phylogeny of the core of genes shared by all life forms. While many groups are rejected, our results left open a possible proximity of N. equitans and the Methanopyrales, of the Archaea and the Cyanobacteria, and as well the possible grouping of the Methanobacteriales/Methanoccocales and Thermosplasmatales, of the Spirochaetes and the Actinobacteria and of the Proteobacteria and firmicutes. Conclusion It is sometimes easier (and preferable) to decide which species do not group together than which ones do. When possible topologies are limited, identifying local relationships that are rejected may be a useful alternative to classical concatenation approaches aiming to find a globally resolved tree on the basis of weak phylogenetic markers. Reviewers This article was reviewed by Mark Ragan, Eugene V Koonin and J Peter Gogarten. PMID:16956399

  10. Analysis of a genome fragment of a deep-sea uncultivated Group II euryarchaeote containing 16S rDNA, a spectinomycin-like operon and several energy metabolism genes.

    PubMed

    Moreira, David; Rodríguez-Valera, Francisco; López-García, Purificación

    2004-09-01

    We have sequenced and analysed a 39.5 kbp genome fragment of a marine Group II euryarchaeote identified in a metagenomic library of 500 m deep plankton at the Antarctic Polar Front. The clone contains a 16S rRNA gene that is separated from the 23S rRNA gene in the genome. This appears to be a trait shared by Thermoplasmatales and Group II euryarchaeota. This genome fragment exhibits a compact organization, including a few overlapping genes in the canonical spectinomycin-like (spc) operon for ribosomal proteins that is immediately upstream the 16S rDNA. Most open reading frames (ORFs) encoded proteins involved in housekeeping processes and, as expected, exhibited a phylogenetic distribution congruent with that of the 16S rRNA. A considerable number of proteins with predicted transmembrane helices was identified. Among those, two proteins encoded by genes likely forming an operon appear to be part of a membrane terminal electron transport chain. One of these proteins has an unusual domain arrangement including ferredoxin, flavodoxin and one succinate dehydrogenase/fumarate reductase subunit. These proteins probably constitute a new succinate dehydrogenase-like oxidoreductase involved in what could be a novel pathway for energy metabolism in Group II euryarchaeota.

  11. Molecular phylogeny of isolates of Ctenocephalides felis and related species based on analysis of ITS1, ITS2 and mitochondrial 16S rDNA sequences and random binding primers.

    PubMed

    Vobis, M; D'Haese, J; Mehlhorn, H; Mencke, N; Blagburn, B L; Bond, R; Denholm, I; Dryden, M W; Payne, P; Rust, M K; Schroeder, I; Vaughn, M B; Bledsoe, D

    2004-10-01

    The phylogenetic relationships among 31 different flea isolates representing seven different species were studied by nucleotide sequence comparison of the internal transcribed spacer 1 (ITS1), internal transcribed spacer 2 (ITS2) and/or mitochondrial 16S ribosomal RNA gene (mt16S-rDNA) to examine the patterns of variation. Results show that all regions are useful in discriminating among flea species. In Ctenocephalides felis and Tunga penetrans, some differences in these gene regions occurred among different isolates within the same species. In the latter case, the differences are in the mt16S-rDNA region, with one isolate showing 48% divergence in nucleotide sequence. The taxonomic implications of this result are unclear at present. The gene regions revealed differences between C. felis isolates only after DNA sequencing the PCR products. Further differentiation among C. felis isolates was obtained using four different random binding primers (decamers) and primers for mammalian aldolase to amplify narrow differences in the genome. Using these primers we were able to discriminate between different C. felis isolates and determine that some of the genetic variation coincided with minor differences in response to the control agent imidacloprid. However, overall findings do not support the existence of subspecies of C. felis.

  12. Genetic Diversity of the Biofilm Covering Montacuta ferruginosa (Mollusca, Bivalvia) as Evaluated by Denaturing Gradient Gel Electrophoresis Analysis and Cloning of PCR-Amplified Gene Fragments Coding for 16S rRNA†

    PubMed Central

    Gillan, David C.; Speksnijder, Arjen G. C. L.; Zwart, Gabriel; De Ridder, Chantal

    1998-01-01

    The shell of the bivalve Montacuta ferruginosa, a symbiont living in the burrow of an echinoid, is covered with a rust-colored biofilm. This biofilm includes different morphotypes of bacteria that are encrusted with a mineral rich in ferric ion and phosphate. The aim of this research was to determine the genetic diversity and phylogenetic affiliation of the biofilm bacteria. Also, the possible roles of the microorganisms in the processes of mineral deposition within the biofilm, as well as their impact on the biology of the bivalve, were assessed by phenotypic inference. The genetic diversity was determined by denaturing gradient gel electrophoresis (DGGE) analysis of short (193-bp) 16S ribosomal DNA PCR products obtained with primers specific for the domain Bacteria. This analysis revealed a diverse consortium; 11 to 25 sequence types were detected depending on the method of DNA extraction used. Individual biofilms analyzed by using the same DNA extraction protocol did not produce identical DGGE profiles. However, different biofilms shared common bands, suggesting that similar bacteria can be found in different biofilms. The phylogenetic affiliations of the sequence types were determined by cloning and sequencing the 16S rRNA genes. Close relatives of the genera Pseudoalteromonas, Colwellia, and Oceanospirillum (members of the γ-Proteobacteria lineage), as well as Flexibacter maritimus (a member of the Cytophaga-Flavobacter-Bacteroides lineage), were found in the biofilms. We inferred from the results that some of the biofilm bacteria could play a role in the mineral formation processes. PMID:9726898

  13. Group-specific PCR primers for the phylum Acidobacteria designed based on the comparative analysis of 16S rRNA gene sequences.

    PubMed

    Lee, Sang-Hoon; Cho, Jae-Chang

    2011-08-01

    We performed a comprehensive phylogenetic analysis of the phylum Acidobacteria and developed novel, group-specific PCR primers for Acidobacteria and its class-level subgroups. Acidobacterial 16S rRNA gene sequences deposited in the RDP database were used to construct a local database then subsequently analyzed. A total of 556 phylotypes were observed and the majority of the phylotypes belonged to five major subgroups (subgroups 1, 2, 3, 4, and 6), which comprised >80% of the acidobacterial sequences in the RDP database. Phylum-specific and subgroup-specific primers were designed from the consensus sequences of the phylotype sequences, and the specificities of the designed primers were evaluated both in silico and empirically for coverage and tolerance. The phylum-specific primer ACIDO, which was designed in this study, showed increased coverage for Acidobacteria, as compared to the previous phylum-specific primer 31F. However, the tolerance of the primer ACIDO for non-target sequences was slightly higher than that of the primer 31F. We also developed subgroup-specific PCR primers for the major subgroups of Acidobacteria, except for subgroup 4. Subgroup-specific primers S1, S2, and S3, which targeted subgroups 1, 2, and 3, respectively, showed high coverage for their target subgroups and low tolerance for non-target sequences. However, the primer S6 targeting subgroup 6 showed a lower specificity in its empirical evaluation than expected from the in silico results. The subgroup-specific primers, as well as the phylum-specific primer designed in this study, will be valuable tools in understanding the phylogenetic diversity and ecological niche of the phylum Acidobacteria and its subgroups.

  14. Isolation of new bacterial species from drinking water biofilms and proof of their in situ dominance with highly specific 16S rRNA probes.

    PubMed Central

    Kalmbach, S; Manz, W; Szewzyk, U

    1997-01-01

    A polyphasic approach involving cultivation, direct viable counts, rRNA-based phylogenetic classification, and in situ probing was applied for the characterization of the dominant microbial population in a municipal drinking water distribution system. A total of 234 bacterial strains cultivated on R2A medium were screened for bacteria affiliated with the in situ dominating beta subclass of Proteobacteria. The isolates were grouped according to common features of their cell and colony morphologies, and eight representative strains were used for 16S rRNA sequencing and the development of a suite of strain-specific oligonucleotide probes. Phylogenetic analysis indicated that all of the isolates were hitherto unknown bacteria. Three of them, strains B4, B6, and B8, formed a separate cluster of closely related organisms within the beta 1 subclass of Proteobacteria. In situ probing revealed that (i) 67 to 72% of total bacteria, corresponding to more than 80% of beta-subclass bacteria, could be encompassed with the strain-specific probes and (ii) the dominating bacterial species were culturable on R2A medium. Additionally, two-thirds of the autochthonous drinking water population could be shown to be in a viable but nonculturable (VBNC) state by using a direct viable count approach. The comparison of isolation frequencies with the in situ abundances of the eight investigated strains revealed differences in their culturability, indicating variable ratios of culturable to VBNC cells among the strains. The further characterization of biofilms throughout the distribution network demonstrated strains B6 and B8 to be dominant bacterial strains in groundwater and distribution system biofilms. The other strains could be found at various frequencies in the different parts of the distribution system; several strains appeared exclusively in drinking water biofilms obtained from a house installation system. PMID:9361400

  15. 16S Ribosomal DNA Sequence Analysis of a Large Collection of Environmental and Clinical Unidentifiable Bacterial Isolates

    PubMed Central

    Drancourt, Michel; Bollet, Claude; Carlioz, Antoine; Martelin, Rolland; Gayral, Jean-Pierre; Raoult, Didier

    2000-01-01

    Some bacteria are difficult to identify with phenotypic identification schemes commonly used outside reference laboratories. 16S ribosomal DNA (rDNA)-based identification of bacteria potentially offers a useful alternative when phenotypic characterization methods fail. However, as yet, the usefulness of 16S rDNA sequence analysis in the identification of conventionally unidentifiable isolates has not been evaluated with a large collection of isolates. In this study, we evaluated the utility of 16S rDNA sequencing as a means to identify a collection of 177 such isolates obtained from environmental, veterinary, and clinical sources. For 159 isolates (89.8%) there was at least one sequence in GenBank that yielded a similarity score of ≥97%, and for 139 isolates (78.5%) there was at least one sequence in GenBank that yielded a similarity score of ≥99%. These similarity score values were used to defined identification at the genus and species levels, respectively. For isolates identified to the species level, conventional identification failed to produce accurate results because of inappropriate biochemical profile determination in 76 isolates (58.7%), Gram staining in 16 isolates (11.6%), oxidase and catalase activity determination in 5 isolates (3.6%) and growth requirement determination in 2 isolates (1.5%). Eighteen isolates (10.2%) remained unidentifiable by 16S rDNA sequence analysis but were probably prototype isolates of new species. These isolates originated mainly from environmental sources (P = 0.07). The 16S rDNA approach failed to identify Enterobacter and Pantoea isolates to the species level (P = 0.04; odds ratio = 0.32 [95% confidence interval, 0.10 to 1.14]). Elsewhere, the usefulness of 16S rDNA sequencing was compromised by the presence of 16S rDNA sequences with >1% undetermined positions in the databases. Unlike phenotypic identification, which can be modified by the variability of expression of characters, 16S rDNA sequencing provides

  16. Development of a 16S rRNA gene-based prototype microarray for the detection of selected actinomycetes genera.

    PubMed

    Kyselková, Martina; Kopecký, Jan; Felföldi, Tamás; Cermák, Ladislav; Omelka, Marek; Grundmann, Geneviève L; Moënne-Loccoz, Yvan; Ságová-Marecková, Markéta

    2008-10-01

    Actinomycetes are known for their secondary metabolites, which have been successfully used as drugs in human and veterinary medicines. However, information on the distribution of this group of Gram-positive bacteria in diverse ecosystems and a comprehension of their activities in ecosystem processes are still scarce. We have developed a 16S rRNA-based taxonomic microarray that targets key actinomycetes at the genus level. In total, 113 actinomycete 16S rRNA probes, corresponding to 55 of the 202 described genera, were designed. The microarray accuracy was evaluated by comparing signal intensities with probe/target-weighted mismatch values and the Gibbs energy of the probe/target duplex formation by hybridizing 17 non-actinomycete and 29 actinomycete strains/clones with the probe set. The validation proved that the probe set was specific, with only 1.3% of false results. The incomplete coverage of actinomycetes by a genus-specific probe was caused by the limited number of 16S rRNA gene sequences in databases or insufficient 16S rRNA gene polymorphism. The microarray enabled discrimination between actinomycete communities from three forest soil samples collected at one site. Cloning and sequencing of 16S rRNA genes from one of the soil samples confirmed the microarray results. We propose that this newly constructed microarray will be a valuable tool for genus-level comparisons of actinomycete communities in various ecological conditions.

  17. RiboFR-Seq: a novel approach to linking 16S rRNA amplicon profiles to metagenomes

    PubMed Central

    Zhang, Yanming; Ji, Peifeng; Wang, Jinfeng; Zhao, Fangqing

    2016-01-01

    16S rRNA amplicon analysis and shotgun metagenome sequencing are two main culture-independent strategies to explore the genetic landscape of various microbial communities. Recently, numerous studies have employed these two approaches together, but downstream data analyses were performed separately, which always generated incongruent or conflict signals on both taxonomic and functional classifications. Here we propose a novel approach, RiboFR-Seq (Ribosomal RNA gene flanking region sequencing), for capturing both ribosomal RNA variable regions and their flanking protein-coding genes simultaneously. Through extensive testing on clonal bacterial strain, salivary microbiome and bacterial epibionts of marine kelp, we demonstrated that RiboFR-Seq could detect the vast majority of bacteria not only in well-studied microbiomes but also in novel communities with limited reference genomes. Combined with classical amplicon sequencing and shotgun metagenome sequencing, RiboFR-Seq can link the annotations of 16S rRNA and metagenomic contigs to make a consensus classification. By recognizing almost all 16S rRNA copies, the RiboFR-seq approach can effectively reduce the taxonomic abundance bias resulted from 16S rRNA copy number variation. We believe that RiboFR-Seq, which provides an integrated view of 16S rRNA profiles and metagenomes, will help us better understand diverse microbial communities. PMID:26984526

  18. Comparative analysis of Pseudomonas syringae pv. actinidiae and pv. phaseolicola based on phaseolotoxin-resistant ornithine carbamoyltransferase gene (argK) and 16S-23S rRNA intergenic spacer sequences.

    PubMed

    Sawada, H; Takeuchi, T; Matsuda, I

    1997-01-01

    Pseudomonas syringae pv. phaseolicola, which causes halo blight on various legumes, and pv. actinidiae, responsible for canker or leaf spot on actinidia plants, are known as phaseolotoxin producers, and the former possesses phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) which confers resistance to the toxin. We confirmed that the latter is also resistant to phaseolotoxin and possesses ROCT, and we compared the two pathovars by using sequence data of the ROCT gene and the intergenic spacer region located between the 16S and 23S rRNA genes (16S-23S spacer region) as an index. It was found that the identical ROCT gene (argK) is contained not only in bean isolates of P. syringae pv. phaseolicola in Mexico and the United States but also in bean isolates in Japan and Canada, and that it is also distributed in the kudzu (Pueraria lobata) isolates of P. syringae pv. phaseolicola. Moreover, the kiwifruit and tara vine isolates of P. syringae pv. actinidiae were also found to possess the identical argK. On the contrary, the 16S-23S spacer regions showed a significant level of sequence variation between P. syringae pv. actinidiae and pv. phaseolicola, suggesting that these two pathovars evolved differently from each other in the phylogenetic development. The fact that even synonymous substitution has not occurred in argK among these strains despite their extreme differences in phylogenetic evolution and geographical distribution suggests that it was only recently in evolutionary time that argK was transferred from its origin to P. syringae pv. actinidiae and/or pv. phaseolicola.

  19. Comparative analysis of Pseudomonas syringae pv. actinidiae and pv. phaseolicola based on phaseolotoxin-resistant ornithine carbamoyltransferase gene (argK) and 16S-23S rRNA intergenic spacer sequences.

    PubMed Central

    Sawada, H; Takeuchi, T; Matsuda, I

    1997-01-01

    Pseudomonas syringae pv. phaseolicola, which causes halo blight on various legumes, and pv. actinidiae, responsible for canker or leaf spot on actinidia plants, are known as phaseolotoxin producers, and the former possesses phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) which confers resistance to the toxin. We confirmed that the latter is also resistant to phaseolotoxin and possesses ROCT, and we compared the two pathovars by using sequence data of the ROCT gene and the intergenic spacer region located between the 16S and 23S rRNA genes (16S-23S spacer region) as an index. It was found that the identical ROCT gene (argK) is contained not only in bean isolates of P. syringae pv. phaseolicola in Mexico and the United States but also in bean isolates in Japan and Canada, and that it is also distributed in the kudzu (Pueraria lobata) isolates of P. syringae pv. phaseolicola. Moreover, the kiwifruit and tara vine isolates of P. syringae pv. actinidiae were also found to possess the identical argK. On the contrary, the 16S-23S spacer regions showed a significant level of sequence variation between P. syringae pv. actinidiae and pv. phaseolicola, suggesting that these two pathovars evolved differently from each other in the phylogenetic development. The fact that even synonymous substitution has not occurred in argK among these strains despite their extreme differences in phylogenetic evolution and geographical distribution suggests that it was only recently in evolutionary time that argK was transferred from its origin to P. syringae pv. actinidiae and/or pv. phaseolicola. PMID:8979356

  20. Systematics of Plant-Pathogenic and Related Streptomyces Species Based on Phylogenetic Analyses of Multiple Gene Loci

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 10 species of Streptomyces implicated as the etiological agents in scab disease of potatoes or soft rot disease of sweet potatoes are distributed among 7 different phylogenetic clades in analyses based on 16S rRNA gene sequences, but high sequence similarity of this gene among Streptomyces speci...

  1. Comprehensive Molecular Characterization of Bacterial Communities in Feces of Pet Birds Using 16S Marker Sequencing.

    PubMed

    Garcia-Mazcorro, Jose F; Castillo-Carranza, Stephany A; Guard, Blake; Gomez-Vazquez, Jose P; Dowd, Scot E; Brigthsmith, Donald J

    2017-01-01

    Birds and other animals live and evolve in close contact with millions of microorganisms (microbiota). While the avian microbiota has been well characterized in domestic poultry, the microbiota of other bird species has been less investigated. The aim of this study was to describe the fecal bacterial communities of pet birds. Pooled fecal samples from 22 flocks representing over 150 individual birds of three different species (Melopsittacus undulatus or budgerigars, Nymphicus hollandicus or cockatiels, and Serinus canaria or domestic canaries) were used for analysis using the 16S rRNA gene sequencing in the MiSeq platform (Illumina). Firmicutes was the most abundant phylum (median 88.4 %; range 12.9-98.4 %) followed by other low-abundant phyla such as Proteobacteria (median 2.3 %; 0.1-85.3 %) and Actinobacteria (median 1.7 %; 0-18.3 %). Lactobacillaceae (mostly Lactobacillus spp.) was the most abundant family (median 78.1 %; 1.4-97.5 %), especially in budgerigars and canaries, and it deserves attention because of the ascribed beneficial properties of lactic acid bacteria. Importantly, feces from birds contain intestinal, urinary, and reproductive-associated microbiota thus posing a serious problem to study one anatomical region at a time. Other groups of interest include the family Clostridiaceae that showed very low abundance (overall median <0.1 %) with the exception of two samples from cockatiels (14 and 45.9 %) and one sample from budgerigars (19.9 %). Analysis of UniFrac metrics showed that overall, the microbial communities from the 22 flocks tended to cluster together for each bird species, meaning each species shed distinctive bacterial communities in feces. This descriptive analysis provides insight into the fecal microbiota of pet birds.

  2. 16S rRNA Gene Survey of Microbial Communities in Winogradsky Columns

    PubMed Central

    Rundell, Ethan A.; Banta, Lois M.; Ward, Doyle V.; Watts, Corey D.; Birren, Bruce; Esteban, David J.

    2014-01-01

    A Winogradsky column is a clear glass or plastic column filled with enriched sediment. Over time, microbial communities in the sediment grow in a stratified ecosystem with an oxic top layer and anoxic sub-surface layers. Winogradsky columns have been used extensively to demonstrate microbial nutrient cycling and metabolic diversity in undergraduate microbiology labs. In this study, we used high-throughput 16s rRNA gene sequencing to investigate the microbial diversity of Winogradsky columns. Specifically, we tested the impact of sediment source, supplemental cellulose source, and depth within the column, on microbial community structure. We found that the Winogradsky columns were highly diverse communities but are dominated by three phyla: Proteobacteria, Bacteroidetes, and Firmicutes. The community is structured by a founding population dependent on the source of sediment used to prepare the columns and is differentiated by depth within the column. Numerous biomarkers were identified distinguishing sample depth, including Cyanobacteria, Alphaproteobacteria, and Betaproteobacteria as biomarkers of the soil-water interface, and Clostridia as a biomarker of the deepest depth. Supplemental cellulose source impacted community structure but less strongly than depth and sediment source. In columns dominated by Firmicutes, the family Peptococcaceae was the most abundant sulfate reducer, while in columns abundant in Proteobacteria, several Deltaproteobacteria families, including Desulfobacteraceae, were found, showing that different taxonomic groups carry out sulfur cycling in different columns. This study brings this historical method for enrichment culture of chemolithotrophs and other soil bacteria into the modern era of microbiology and demonstrates the potential of the Winogradsky column as a model system for investigating the effect of environmental variables on soil microbial communities. PMID:25101630

  3. Beyond 16S rRNA Community Profiling: Intra-Species Diversity in the Gut Microbiota.

    PubMed

    Ellegaard, Kirsten M; Engel, Philipp

    2016-01-01

    Interactions with microbes affect many aspects of animal biology, including immune system development, nutrition and health. In vertebrates, the gut microbiota is dominated by a small subset of phyla, but the species composition within these phyla is typically not conserved. Moreover, several recent studies have shown that bacterial species in the gut are composed of a multitude of strains, which frequently co-exist in their host, and may be host-specific. However, since the study of intra-species diversity is challenging, particularly in the setting of complex, host-associated microbial communities, our current understanding of the distribution, evolution and functional relevance of intra-species diversity in the gut is scarce. In order to unravel how genomic diversity translates into phenotypic diversity, community analyses going beyond 16S rRNA profiling, in combination with experimental approaches, are needed. Recently, the honeybee has emerged as a promising model for studying gut bacterial communities, particularly in terms of strain-level diversity. Unlike most other invertebrates, the honeybee gut is colonized by a remarkably consistent and specific core microbiota, which is dominated by only eight bacterial species. As for the vertebrate gut microbiota, these species are composed of highly diverse strains suggesting that similar evolutionary forces shape gut community structures in vertebrates and social insects. In this review, we outline current knowledge on the evolution and functional relevance of strain diversity within the gut microbiota, including recent insights gained from mammals and other animals such as the honeybee. We discuss methodological approaches and propose possible future avenues for studying strain diversity in complex bacterial communities.

  4. Beyond 16S rRNA Community Profiling: Intra-Species Diversity in the Gut Microbiota

    PubMed Central

    Ellegaard, Kirsten M.; Engel, Philipp

    2016-01-01

    Interactions with microbes affect many aspects of animal biology, including immune system development, nutrition and health. In vertebrates, the gut microbiota is dominated by a small subset of phyla, but the species composition within these phyla is typically not conserved. Moreover, several recent studies have shown that bacterial species in the gut are composed of a multitude of strains, which frequently co-exist in their host, and may be host-specific. However, since the study of intra-species diversity is challenging, particularly in the setting of complex, host-associated microbial communities, our current understanding of the distribution, evolution and functional relevance of intra-species diversity in the gut is scarce. In order to unravel how genomic diversity translates into phenotypic diversity, community analyses going beyond 16S rRNA profiling, in combination with experimental approaches, are needed. Recently, the honeybee has emerged as a promising model for studying gut bacterial communities, particularly in terms of strain-level diversity. Unlike most other invertebrates, the honeybee gut is colonized by a remarkably consistent and specific core microbiota, which is dominated by only eight bacterial species. As for the vertebrate gut microbiota, these species are composed of highly diverse strains suggesting that similar evolutionary forces shape gut community structures in vertebrates and social insects. In this review, we outline current knowledge on the evolution and functional relevance of strain diversity within the gut microbiota, including recent insights gained from mammals and other animals such as the honeybee. We discuss methodological approaches and propose possible future avenues for studying strain diversity in complex bacterial communities. PMID:27708630

  5. Identification of Candidate Periodontal Pathogens and Beneficial Species by Quantitative 16S Clonal Analysis†

    PubMed Central

    Kumar, Purnima S.; Griffen, Ann L.; Moeschberger, Melvin L.; Leys, Eugene J.

    2005-01-01

    Most studies of the bacterial etiology of periodontitis have used either culture-based or targeted DNA approaches, and so it is likely that pathogens remain undiscovered. The purpose of this study was to use culture-independent, quantitative analysis of biofilms associated with chronic periodontitis and periodontal health to identify pathogens and beneficial species. Samples from subjects with periodontitis and controls were analyzed using ribosomal 16S cloning and sequencing. Several genera, many of them uncultivated, were associated with periodontitis, the most numerous of which were gram positive, including Peptostreptococcus and Filifactor. The genera Megasphaera and Desulfobulbus were elevated in periodontitis, and the levels of several species or phylotypes of Campylobacter, Selenomonas, Deferribacteres, Dialister, Catonella, Tannerella, Streptococcus, Atopobium, Eubacterium, and Treponema were elevated in disease. Streptococcus and Veillonella spp. were found in high numbers in all samples and accounted for a significantly greater fraction of the microbial community in healthy subjects than in those with periodontitis. The microbial profile of periodontal health also included the less-abundant genera Campylobacter, Abiotrophia, Gemella, Capnocytophaga, and Neisseria. These newly identified candidates outnumbered Porphyromonas gingivalis and other species previously implicated as periodontopathogens, and it is not clear if newly identified and more numerous species may play a more important role in pathogenesis. Finally, more differences were found in the bacterial profile between subjects with periodontitis and healthy subjects than between deep and shallow sites within the same subject. This suggests that chronic periodontitis is the result of a global perturbation of the oral bacterial ecology rather than a disease-site specific microbial shift. PMID:16081935

  6. Sources for sedimentary bacteriohopanepolyols as revealed by 16S rDNA stratigraphy.

    PubMed

    Coolen, Marco J L; Talbot, Helen M; Abbas, Ben A; Ward, Christopher; Schouten, Stefan; Volkman, John K; Damsté, Jaap S Sinninghe

    2008-07-01

    Bacteriohopanoids are widespread lipid biomarkers in the sedimentary record. Many aerobic and anaerobic bacteria are potential sources of these lipids which sometimes complicates the use of these biomarkers as proxies for ecological and environmental changes. Therefore, we applied preserved 16S ribosomal RNA genes to identify likely Holocene biological sources of bacteriohopanepolyols (BHPs) in the sulfidic sediments of the permanently stratified postglacial Ace Lake, Antarctica. A suite of intact BHPs were identified, which revealed a variety of structural forms whose composition differed through the sediment core reflecting changes in bacterial populations induced by large changes in lake salinity. Stable isotopic compositions of the hopanols formed from periodic acid-cleaved BHPs, showed that some were substantially depleted in (13)C, indicative of their methanotrophic origin. Using sensitive molecular tools, we found that Type I and II methanotrophic bacteria (respectively Methylomonas and Methylocystis) were unique to the oldest lacustrine sediments (> 9400 years BP), but quantification of fossil DNA revealed that the Type I methanotrophs, including methanotrophs related to methanotrophic gill symbionts of deep-sea cold-seep mussels, were the main precursors of the 35-amino BHPs (i.e. aminopentol, -tetrol and -triols). After isolation of the lake approximately 3000 years ago, one Type I methanotroph of the 'methanotrophic gill symbionts cluster' remained the most obvious source of aminotetrol and -triol. We, furthermore, identified a Synechococcus phylotype related to pelagic freshwater strains in the oldest lacustrine sediments as a putative source of 2-methylbacteriohopanetetrol (2-Me BHT). This combined application of advanced geochemical and paleogenomical tools further refined our knowledge about Holocene biogeochemical processes in Ace Lake.

  7. Vikodak - A Modular Framework for Inferring Functional Potential of Microbial Communities from 16S Metagenomic Datasets

    PubMed Central

    Nagpal, Sunil; Haque, Mohammed Monzoorul; Mande, Sharmila S.

    2016-01-01

    Background The overall metabolic/functional potential of any given environmental niche is a function of the sum total of genes/proteins/enzymes that are encoded and expressed by various interacting microbes residing in that niche. Consequently, prior (collated) information pertaining to genes, enzymes encoded by the resident microbes can aid in indirectly (re)constructing/ inferring the metabolic/ functional potential of a given microbial community (given its taxonomic abundance profile). In this study, we present Vikodak—a multi-modular package that is based on the above assumption and automates inferring and/ or comparing the functional characteristics of an environment using taxonomic abundance generated from one or more environmental sample datasets. With the underlying assumptions of co-metabolism and independent contributions of different microbes in a community, a concerted effort has been made to accommodate microbial co-existence patterns in various modules incorporated in Vikodak. Results Validation experiments on over 1400 metagenomic samples have confirmed the utility of Vikodak in (a) deciphering enzyme abundance profiles of any KEGG metabolic pathway, (b) functional resolution of distinct metagenomic environments, (c) inferring patterns of functional interaction between resident microbes, and (d) automating statistical comparison of functional features of studied microbiomes. Novel features incorporated in Vikodak also facilitate automatic removal of false positives and spurious functional predictions. Conclusions With novel provisions for comprehensive functional analysis, inclusion of microbial co-existence pattern based algorithms, automated inter-environment comparisons; in-depth analysis of individual metabolic pathways and greater flexibilities at the user end, Vikodak is expected to be an important value addition to the family of existing tools for 16S based function prediction. Availability and Implementation A web implementation of Vikodak

  8. Bacterial fauna associating with chironomid larvae from lakes of Bengaluru city, India - A 16s rRNA gene based identification.

    PubMed

    Kuncham, Ramprasad; Sivaprakasam, Thiyagarajan; Puneeth Kumar, R; Sreenath, P; Nayak, Ravi; Thayumanavan, Tha; Subba Reddy, Gopireddy V

    2017-06-01

    Chironomid larvae that inhabit in aquatic sediments play an important role as vector for bacterial pathogens. Its life cycle consists of four stages i.e. eggs, larvae, pupae and adult. In the present study we identified bacterial species associated with whole larvae of chironomids from 11 lake sediments of Bangalore region using 16s rRNA gene Sanger sequencing. We found that larvae from all lake sediments associated with bacterial species which include key pathogens. Totally we identified 65 bacterial isolates and obtained GenBank accession numbers (KX980423 - KX980487). Phylogenetic tree constructed using MEGA 7 software and tree analysis highlight the predominant bacterial community associated with larvae which include Enterobacteriaceae (43.08%; 28 isolates) and Aeromonas (24.62%; 16 isolates), Shewanella, Delftia, Bacillus (6.15%; 4 isolates each), Pseudomonas (4.62%; 3 isolates) and Exiguobacterium (3.08%; 2 isolates). Current findings state that among bacterial population Aeromonas, Enterobacter and Escherichia with serotypes are commonly associated with larvae in maximum lake points. In other hand Vibrio, Pseudomonas, Klebsiella, Shigella, Bacillus, and other bacterial species were identified moderately in all lakes. Interestingly, we identified first time Shigella Gram negative, rod shaped pathogenic organism of Enterobacteriaceae and Rheinheimera Gram negative, rod shaped organism associating chironomid larvae.

  9. Culturable bacteria present in the fluid of the hooded-pitcher plant Sarracenia minor based on 16S rDNA gene sequence data.

    PubMed

    Siragusa, Alex J; Swenson, Janice E; Casamatta, Dale A

    2007-08-01

    The culturable microbial community within the pitcher fluid of 93 Sarracenia minor carnivorous plants was examined over a 2-year study. Many aspects of the plant/bacterial/insect interaction within the pitcher fluid are minimally understood because the bacterial taxa present in these pitchers have not been identified. Thirteen isolates were characterized by 16S rDNA sequencing and subsequent phylogenetic analysis. The Proteobacteria were the most abundant taxa and included representatives from Serratia, Achromobacter, and Pantoea. The Actinobacteria Micrococcus was also abundant while Bacillus, Lactococcus, Chryseobacterium, and Rhodococcus were infrequently encountered. Several isolates conformed to species identifiers (>98% rDNA gene sequence similarity) including Serratia marcescens (isolates found in 27.5% of pitchers), Achromobacter xylosoxidans (37.6%), Micrococcus luteus (40.9%), Bacillus cereus (isolates found in 10.2%), Bacillus thuringiensis (5.4%), Lactococcus lactis (17.2%), and Rhodococcus equi (2.2%). Species-area curves suggest that sampling efforts were sufficient to recover a representative culturable bacterial community. The bacteria present represent a diverse community probably as a result of introduction by insect vectors, but the ecological significance remains under explored.

  10. Mitochondrial COI and 16sRNA evidence for a single species hypothesis of E. vitis, J. formosana and E. onukii in East Asia.

    PubMed

    Fu, Jian-Yu; Han, Bao-Yu; Xiao, Qiang

    2014-01-01

    Tea green leafhopper is one of the most damaging tea pests in main tea production regions of East Asia. For lack of recognized morphological characters, the dominant species of tea green leafhoppers in Mainland China, Taiwan and Japan have always been named as Empoasca vitis Göthe, Jacobiasca formosana Paoli and Empoasca onukii MATSUDA, respectively. Furthermore, nothing is known about the genetic relationships among them. In this study, we collected six populations from Mainland China, four populations from Japan and one population from Taiwan, and examined the genetic distances in the COI and 16sRNA regions of mtDNA among them. The results showed that the genetic distances based on single gene or the combined sequences among eleven leafhopper populations were 0.3-1.2%, which were all less than the species boundary of 2%. Moreover, there were at least two haplotypes shared by two distinct populations from different regions. The phylogenetic analysis based on single gene or combined sets also supported that tea green leafhoppers from Mainland China, Taiwan and Japan were closely related to each other, and there were at least two specimens from different regions clustered ahead of those from the same region. Therefore, we propose that the view of recognizing the dominant species of tea green leafhoppers in three adjacent tea production regions of East Asia as different species is unreliable or questionable and suggest that they are a single species.

  11. Use of 16S rRNA gene based clone libraries to assess microbial communities potentially involved in anaerobic methane oxidation in a Mediterranean cold seep.

    PubMed

    Heijs, Sander K; Haese, Ralf R; van der Wielen, Paul W J J; Forney, Larry J; van Elsas, Jan Dirk

    2007-04-01

    This study provides data on the diversities of bacterial and archaeal communities in an active methane seep at the Kazan mud volcano in the deep Eastern Mediterranean sea. Layers of varying depths in the Kazan sediments were investigated in terms of (1) chemical parameters and (2) DNA-based microbial population structures. The latter was accomplished by analyzing the sequences of directly amplified 16S rRNA genes, resulting in the phylogenetic analysis of the prokaryotic communities. Sequences of organisms potentially associated with processes such as anaerobic methane oxidation and sulfate reduction were thus identified. Overall, the sediment layers revealed the presence of sequences of quite diverse bacterial and archaeal communities, which varied considerably with depth. Dominant types revealed in these communities are known as key organisms involved in the following processes: (1) anaerobic methane oxidation and sulfate reduction, (2) sulfide oxidation, and (3) a range of (aerobic) heterotrophic processes. In the communities in the lowest sediment layer sampled (22-34 cm), sulfate-reducing bacteria and archaea of the ANME-2 cluster (likely involved in anaerobic methane oxidation) were prevalent, whereas heterotrophic organisms abounded in the top sediment layer (0-6 cm). Communities in the middle layer (6-22 cm) contained organisms that could be linked to either of the aforementioned processes. We discuss how these phylogeny (sequence)-based findings can support the ongoing molecular work aimed at unraveling both the functioning and the functional diversities of the communities under study.

  12. High-density 16S microarray and clone library-based microbial community composition of the Phoenix spacecraft assembly clean room.

    PubMed

    Vaishampayan, Parag; Osman, Shariff; Andersen, Gary; Venkateswaran, Kasthuri

    2010-06-01

    The bacterial diversity and comparative community structure of a clean room used for assembling the Phoenix spacecraft was characterized throughout the spacecraft assembly process by using 16S rRNA gene cloning/sequencing and DNA microarray (PhyloChip) technologies. Samples were collected from several locations of the clean room at three time points: before Phoenix's arrival (PHX-B), during hardware assembly (PHX-D), and after the spacecraft was removed for launch (PHX-A). Bacterial diversity comprised of all major bacterial phyla of PHX-B was found to be statistically different from PHX-D and PHX-A samples. Due to stringent cleaning and decontamination protocols during assembly, PHX-D bacterial diversity was dramatically reduced when compared to PHX-B and PHX-A samples. Comparative community analysis based on PhyloChip results revealed similar overall trends as were seen in clone libraries, but the high-density phylogenetic microarray detected larger diversity in all sampling events. The decrease in community complexity in PHX-D compared to PHX-B, and the subsequent recurrence of these organisms in PHX-A, speaks to the effectiveness of NASA cleaning protocols. However, the persistence of a subset of bacterial signatures throughout all spacecraft assembly phases underscores the need for continued refinement of sterilization technologies and the implementation of safeguards that monitor and inventory microbial contaminants.

  13. Dynamic changes in the composition of photosynthetic picoeukaryotes in the northwestern Pacific Ocean revealed by high-throughput tag sequencing of plastid 16S rRNA genes.

    PubMed

    Choi, Dong H; An, Sung M; Chun, Sungjun; Yang, Eun C; Selph, Karen E; Lee, Charity M; Noh, Jae H

    2016-02-01

    Photosynthetic picoeukaryotes (PPEs) are major oceanic primary producers. However, the diversity of such communities remains poorly understood, especially in the northwestern (NW) Pacific. We investigated the abundance and diversity of PPEs, and recorded environmental variables, along a transect from the coast to the open Pacific Ocean. High-throughput tag sequencing (using the MiSeq system) revealed the diversity of plastid 16S rRNA genes. The dominant PPEs changed at the class level along the transect. Prymnesiophyceae were the only dominant PPEs in the warm pool of the NW Pacific, but Mamiellophyceae dominated in coastal waters of the East China Sea. Phylogenetically, most Prymnesiophyceae sequences could not be resolved at lower taxonomic levels because no close relatives have been cultured. Within the Mamiellophyceae, the genera Micromonas and Ostreococcus dominated in marginal coastal areas affected by open water, whereas Bathycoccus dominated in the lower euphotic depths of oligotrophic open waters. Cryptophyceae and Phaeocystis (of the Prymnesiophyceae) dominated in areas affected principally by coastal water. We also defined the biogeographical distributions of Chrysophyceae, prasinophytes, Bacillariophyceaea and Pelagophyceae. These distributions were influenced by temperature, salinity and chlorophyll a and nutrient concentrations.

  14. Mitochondrial COI and 16sRNA Evidence for a Single Species Hypothesis of E. vitis, J. formosana and E. onukii in East Asia

    PubMed Central

    Fu, Jian-Yu; Han, Bao-Yu; Xiao, Qiang

    2014-01-01

    Tea green leafhopper is one of the most damaging tea pests in main tea production regions of East Asia. For lack of recognized morphological characters, the dominant species of tea green leafhoppers in Mainland China, Taiwan and Japan have always been named as Empoasca vitis Göthe, Jacobiasca formosana Paoli and Empoasca onukii MATSUDA, respectively. Furthermore, nothing is known about the genetic relationships among them. In this study, we collected six populations from Mainland China, four populations from Japan and one population from Taiwan, and examined the genetic distances in the COI and 16sRNA regions of mtDNA among them. The results showed that the genetic distances based on single gene or the combined sequences among eleven leafhopper populations were 0.3–1.2%, which were all less than the species boundary of 2%. Moreover, there were at least two haplotypes shared by two distinct populations from different regions. The phylogenetic analysis based on single gene or combined sets also supported that tea green leafhoppers from Mainland China, Taiwan and Japan were closely related to each other, and there were at least two specimens from different regions clustered ahead of those from the same region. Therefore, we propose that the view of recognizing the dominant species of tea green leafhoppers in three adjacent tea production regions of East Asia as different species is unreliable or questionable and suggest that they are a single species. PMID:25506929

  15. Comprehensive Meta-analysis of Ontology Annotated 16S rRNA Profiles Identifies Beta Diversity Clusters of Environmental Bacterial Communities

    PubMed Central

    Henschel, Andreas; Anwar, Muhammad Zohaib; Manohar, Vimitha

    2015-01-01

    Comprehensive mapping of environmental microbiomes in terms of their compositional features remains a great challenge in understanding the microbial biosphere of the Earth. It bears promise to identify the driving forces behind the observed community patterns and whether community assembly happens deterministically. Advances in Next Generation Sequencing allow large community profiling studies, exceeding sequencing data output of conventional methods in scale by orders of magnitude. However, appropriate collection systems are still in a nascent state. We here present a database of 20,427 diverse environmental 16S rRNA profiles from 2,426 independent studies, which forms the foundation of our meta-analysis. We conducted a sample size adaptive all-against-all beta diversity comparison while also respecting phylogenetic relationships of Operational Taxonomic Units(OTUs). After conventional hierarchical clustering we systematically test for enrichment of Environmental Ontology terms and their abstractions in all possible clusters. This post-hoc algorithm provides a novel formalism that quantifies to what extend compositional and semantic similarity of microbial community samples coincide. We automatically visualize significantly enriched subclusters on a comprehensive dendrogram of microbial communities. As a result we obtain the hitherto most differentiated and comprehensive view on global patterns of microbial community diversity. We observe strong clusterability of microbial communities in ecosystems such as human/mammal-associated, geothermal, fresh water, plant-associated, soils and rhizosphere microbiomes, whereas hypersaline and anthropogenic samples are less homogeneous. Moreover, saline samples appear less cohesive in terms of compositional properties than previously reported. PMID:26458130

  16. Rapid qualitative characterization of bacterial community in eutrophicated wastewater stabilization plant by T-RFLP method based on 16S rRNA genes.

    PubMed

    Belila, Abdelaziz; Snoussi, Mejdi; Hassan, Abdennaceur

    2012-01-01

    Waste stabilization ponds are a simple, low-cost extensive process for treating wastewater, and well adapted to low socio-economic conditions in developing countries where the microbial populations in these systems are not well characterized. The phylogenetic bacterial community structure within a Tunisian wastewater stabilization plant treating domestic wastewater was assessed by Terminal Restriction Fragment Length Polymorphism method targeting 16S rRNA genes and by the APLAUS+ software of the Microbial Community Analysis (MiCA) web based tool. The dimeric enzymatic digestion with HaeIII and HinfI restriction enzymes revealed high bacterial diversity within the plant where 11 bacterial phyla were identified. The total bacterial community structure includes bacteria catalysing nitrogen and phosphorus removal and bacteria involved in the sulfur cycle. The bacterial community was characterized by the dominance of Proteobacteria which was the most populous phylum (60%) followed by the Actinobacteria (20%), the Firmicutes (10.3%), the Bacteroidetes (2.3%), the Nitrospira (2.2%). Minor bacterial phyla groups occupied smaller fractions such as Chloroflexi, Deferribacteres and Verrumicrobia. T-RFLP analysis revealed also that The Proteobacteria phylum was characterized by the dominance of bacteria of The Gammaproteobacteria class.

  17. How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity

    NASA Technical Reports Server (NTRS)

    Fox, G. E.; Wisotzkey, J. D.; Jurtshuk, P. Jr

    1992-01-01

    16S rRNA (genes coding for rRNA) sequence comparisons were conducted with the following three psychrophilic strains: Bacillus globisporus W25T (T = type strain) and Bacillus psychrophilus W16AT, and W5. These strains exhibited more than 99.5% sequence identity and within experimental uncertainty could be regarded as identical. Their close taxonomic relationship was further documented by phenotypic similarities. In contrast, previously published DNA-DNA hybridization results have convincingly established that these strains do not belong to the same species if current standards are used. These results emphasize the important point that effective identity of 16S rRNA sequences is not necessarily a sufficient criterion to guarantee species identity. Thus, although 16S rRNA sequences can be used routinely to distinguish and establish relationships between genera and well-resolved species, very recently diverged species may not be recognizable.

  18. PCR Primer Design for 16S rRNAs for Experimental Horizontal Gene Transfer Test in Escherichia coli

    PubMed Central

    Miyazaki, Kentaro; Sato, Mitsuharu; Tsukuda, Miyuki

    2017-01-01

    We recently demonstrated that the Escherichia coli ribosome is robust enough to accommodate foreign 16S rRNAs from diverse gamma- and betaproteobacteria bacteria (Kitahara et al., 2012). Therein, we used the common universal primers Bac8f and UN1541r to obtain a nearly full-length gene. However, we noticed that these primers overlap variable sites at 19[A/C] and 1527[U/C] in Bac8f and UN1541r, respectively, and thus, the amplicon could contain mutations. This is problematic, particularly for the former site, because the 19th nucleotide pairs with the 916th nucleotide, which is a part of the “central pseudoknot” and is critical for function. Therefore, we mutationally investigated the role of the base pair using several 16S rRNAs from gamma- and betaproteobacteria. We found that both the native base pairs (gammaproteobacterial 19A–916U and betaproteobacterial 19C–916G) and the non-native 19A–916G pair retained function, whereas the non-native 19C–916U was defective 16S rRNAs. We next designed a new primer set, Bac1f and UN1542r, so that they do not overlap the potential mismatch sites. 16S rRNA amplicons obtained from the environmental metagenome using the new primer set were dominated by proteobacterial species (~85%). Subsequent functional screening identified various 16S rRNAs from proteobacteria, all of which contained native 19A–916U or 19C–916G base pairs. The primers developed in this study are thus advantageous for functional characterization of foreign 16S rRNA in E. coli with no artifacts. PMID:28293553

  19. PCR Primer Design for 16S rRNAs for Experimental Horizontal Gene Transfer Test in Escherichia coli.

    PubMed

    Miyazaki, Kentaro; Sato, Mitsuharu; Tsukuda, Miyuki

    2017-01-01

    We recently demonstrated that the Escherichia coli ribosome is robust enough to accommodate foreign 16S rRNAs from diverse gamma- and betaproteobacteria bacteria (Kitahara et al., 2012). Therein, we used the common universal primers Bac8f and UN1541r to obtain a nearly full-length gene. However, we noticed that these primers overlap variable sites at 19[A/C] and 1527[U/C] in Bac8f and UN1541r, respectively, and thus, the amplicon could contain mutations. This is problematic, particularly for the former site, because the 19th nucleotide pairs with the 916th nucleotide, which is a part of the "central pseudoknot" and is critical for function. Therefore, we mutationally investigated the role of the base pair using several 16S rRNAs from gamma- and betaproteobacteria. We found that both the native base pairs (gammaproteobacterial 19A-916U and betaproteobacterial 19C-916G) and the non-native 19A-916G pair retained function, whereas the non-native 19C-916U was defective 16S rRNAs. We next designed a new primer set, Bac1f and UN1542r, so that they do not overlap the potential mismatch sites. 16S rRNA amplicons obtained from the environmental metagenome using the new primer set were dominated by proteobacterial species (~85%). Subsequent functional screening identified various 16S rRNAs from proteobacteria, all of which contained native 19A-916U or 19C-916G base pairs. The primers developed in this study are thus advantageous for functional characterization of foreign 16S rRNA in E. coli with no artifacts.

  20. Cockroaches that lack Blattabacterium endosymbionts: the phylogenetically divergent genus Nocticola.

    PubMed

    Lo, Nathan; Beninati, Tiziana; Stone, Fred; Walker, James; Sacchi, Luciano

    2007-06-22

    Phylogenetic relationships among termites, mantids and the five traditionally recognized cockroach families have been the subject of several studies during the last half-century. One cockroach lineage that has remained notably absent from such studies is the Nocticolidae. This group of small, elusive surface- and cave-dwelling species from the Old World Tropics has been proposed to represent an additional family. Using molecular sequences, we performed an initial phylogenetic examination of Nocticola spp. The hypothesis that they are phylogenetically divergent was confirmed from the analyses of three genes and a combined dataset. To supplement our phylogenetic analyses, we attempted to amplify 16S rRNA from the obligate mutualistic endosymbiont Blattabacterium cuenoti, present in all cockroaches studied to date. Unexpectedly, amplification was unsuccessful in all Nocticola spp. examined. This result was confirmed by microscopic examinations of fat body tissue. These Nocticola spp. are the first cockroaches found to be uninfected by B. cuenoti, which raise questions about when the bacterium first infected cockroaches.

  1. Phylogenetic Comparative Assembly

    NASA Astrophysics Data System (ADS)

    Husemann, Peter; Stoye, Jens

    Recent high throughput sequencing technologies are capable of generating a huge amount of data for bacterial genome sequencing projects. Although current sequence assemblers successfully merge the overlapping reads, often several contigs remain which cannot be assembled any further. It is still costly and time consuming to close all the gaps in order to acquire the whole genomic sequence. Here we propose an algorithm that takes several related genomes and their phylogenetic relationships into account to create a contig adjacency graph. From this a layout graph can be computed which indicates putative adjacencies of the contigs in order to aid biologists in finishing the complete genomic sequence.

  2. PCR amplification of 16S rDNA from lyophilized cell cultures facilitates studies in molecular systematics

    NASA Technical Reports Server (NTRS)

    Wisotzkey, J. D.; Jurtshuk, P. Jr; Fox, G. E.

    1990-01-01

    The sequence of the major portion of a Bacillus cycloheptanicus strain SCH(T) 16S rRNA gene is reported. This sequence suggests that B. cycloheptanicus is genetically quite distinct from traditional Bacillus strains (e.g., B. subtilis) and may be properly regarded as belonging to a different genus. The sequence was determined from DNA that was produced by direct amplification of ribosomal DNA from a lyophilized cell pellet with straightforward polymerase chain reaction (PCR) procedures. By obviating the need to revive cell cultures from the lyophile pellet, this approach facilitates rapid 16S rDNA sequencing and thereby advances studies in molecular systematics.

  3. Phylogenetic relationships of Nembrothinae (Mollusca: Doridacea: Polyceridae) inferred from morphology and mitochondrial DNA.

    PubMed

    Pola, Marta; Cervera, J Lucas; Gosliner, Terrence M

    2007-06-01

    Within the Polyceridae, Nembrothinae includes some of the most striking and conspicuous sea slugs known, although several features of their biology and phylogenetic relationships remain unknown. This paper reports a phylogenetic analysis based on partial sequences of two mitochondrial genes (cytochrome c oxidase subunit I and 16S rRNA) and morphology for most species included in Nembrothinae. Our phylogenetic reconstructions using both molecular and combined morphological and molecular data support the taxonomic splitting of Nembrothinae into several taxa. Excluding one species (Tambja tentaculata), the monophyly of Roboastra was supported by all the phylogenetic analyses of the combined molecular data. Nembrotha was monophyletic both in the morphological and molecular analyses, always with high support. However, Tambja was recovered as para- or polyphyletic, depending on the analysis performed. Our study also rejects the monophyly of "phanerobranch" dorids based on molecular data.

  4. Targeted recovery of novel phylogenetic diversity from next-generation sequence data.

    PubMed

    Lynch, Michael D J; Bartram, Andrea K; Neufeld, Josh D

    2012-11-01

    Next-generation sequencing technologies have led to recognition of a so-called 'rare biosphere'. These microbial operational taxonomic units (OTUs) are defined by low relative abundance and may be specifically adapted to maintaining low population sizes. We hypothesized that mining of low-abundance next-generation 16S ribosomal RNA (rRNA) gene data would lead to the discovery of novel phylogenetic diversity, reflecting microorganisms not yet discovered by previous sampling efforts. Here, we test this hypothesis by combining molecular and bioinformatic approaches for targeted retrieval of phylogenetic novelty within rare biosphere OTUs. We combined BLASTN network analysis, phylogenetics and targeted primer design to amplify 16S rRNA gene sequences from unique potential bacterial lineages, comprising part of the rare biosphere from a multi-million sequence data set from an Arctic tundra soil sample. Demonstrating the feasibility of the protocol developed here, three of seven recovered phylogenetic lineages represented extremely divergent taxonomic entities. These divergent target sequences correspond to (a) a previously unknown lineage within the BRC1 candidate phylum, (b) a sister group to the early diverging and currently recognized monospecific Cyanobacteria Gloeobacter, a genus containing multiple plesiomorphic traits and (c) a highly divergent lineage phylogenetically resolved within mitochondria. A comparison to twelve next-generation data sets from additional soils suggested persistent low-abundance distributions of these novel 16S rRNA genes. The results demonstrate this sequence analysis and retrieval pipeline as applicable for exploring underrepresented phylogenetic novelty and recovering taxa that may represent significant steps in bacterial evolution.

  5. Entanglement, Invariants, and Phylogenetics

    NASA Astrophysics Data System (ADS)

    Sumner, J. G.

    2007-10-01

    This thesis develops and expands upon known techniques of mathematical physics relevant to the analysis of the popular Markov model of phylogenetic trees required in biology to reconstruct the evolutionary relationships of taxonomic units from biomolecular sequence data. The techniques of mathematical physics are plethora and have been developed for some time. The Markov model of phylogenetics and its analysis is a relatively new technique where most progress to date has been achieved by using discrete mathematics. This thesis takes a group theoretical approach to the problem by beginning with a remarkable mathematical parallel to the process of scattering in particle physics. This is shown to equate to branching events in the evolutionary history of molecular units. The major technical result of this thesis is the derivation of existence proofs and computational techniques for calculating polynomial group invariant functions on a multi-linear space where the group action is that relevant to a Markovian time evolution. The practical results of this thesis are an extended analysis of the use of invariant functions in distance based methods and the presentation of a new reconstruction technique for quartet trees which is consistent with the most general Markov model of sequence evolution.

  6. Phylogenetic trees in bioinformatics

    SciTech Connect

    Burr, Tom L

    2008-01-01

    Genetic data is often used to infer evolutionary relationships among a collection of viruses, bacteria, animal or plant species, or other operational taxonomic units (OTU). A phylogenetic tree depicts such relationships and provides a visual representation of the estimated branching order of the OTUs. Tree estimation is unique for several reasons, including: the types of data used to represent each OTU; the use ofprobabilistic nucleotide substitution models; the inference goals involving both tree topology and branch length, and the huge number of possible trees for a given sample of a very modest number of OTUs, which implies that fmding the best tree(s) to describe the genetic data for each OTU is computationally demanding. Bioinformatics is too large a field to review here. We focus on that aspect of bioinformatics that includes study of similarities in genetic data from multiple OTUs. Although research questions are diverse, a common underlying challenge is to estimate the evolutionary history of the OTUs. Therefore, this paper reviews the role of phylogenetic tree estimation in bioinformatics, available methods and software, and identifies areas for additional research and development.

  7. Comparative evaluation of prokaryotic 16S rDNA clone libraries and SSCP in groundwater samples.

    PubMed

    Larentis, Michael; Alfreider, Albin

    2011-06-01

    A comparison of ribosomal RNA sequence analysis methods based on clone libraries and single-strand conformational polymorphism technique (SSCP) was performed with groundwater samples obtained between 523-555 meters below surface. The coverage of analyzed clones by phylotype-richness estimates was between 88-100%, confirming that the clone libraries were adequately examined. Analysis of individual bands retrieved from SSCP gels identified 1-6 different taxonomic units per band, suggesting that a single SSCP band does often represent more than one single prokaryotic species. The prokaryotic diversity obtained by both methods showed an overall difference of 42-80%. In comparison to SSCP, clone libraries underestimated the phylogenetic diversity and only 36-66% of the phylotypes observed with SSCP were also detected with the clone libraries. An exception was a sample where the SSCP analysis of Archaea identified only half of the phylotypes retrieved by the clone library. Overall, this study suggests that the clone library and the SSCP approach do not provide an identical picture of the prokaryotic diversity in groundwater samples. The results clearly show that the SSCP method, although this approach is prone to generate methodological artifacts, was able to detect significantly more phylotypes than microbial community analysis based on clone libraries.

  8. Characterization of Bacterial Communities in Selected Smokeless Tobacco Products Using 16S rDNA Analysis.

    PubMed

    Tyx, Robert E; Stanfill, Stephen B; Keong, Lisa M; Rivera, Angel J; Satten, Glen A; Watson, Clifford H

    2016-01-01

    The bacterial communities present in smokeless tobacco (ST) products have not previously reported. In this study, we used Next Generation Sequencing to study the bacteria present in U.S.-made dry snuff, moist snuff and Sudanese toombak. Sample diversity and taxonomic abundances were investigated in these products. A total of 33 bacterial families from four phyla, Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes, were identified. U.S.-produced dry snuff products contained a diverse distribution of all four phyla. Moist snuff products were dominated by Firmicutes. Toombak samples contained mainly Actinobacteria and Firmicutes (Aerococcaceae, Enterococcaceae, and Staphylococcaceae). The program PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was used to impute the prevalence of genes encoding selected bacterial toxins, antibiotic resistance genes and other pro-inflammatory molecules. PICRUSt also predicted the presence of specific nitrate reductase genes, whose products can contribute to the formation of carcinogenic nitrosamines. Characterization of microbial community abundances and their associated genomes gives us an indication of the presence or absence of pathways of interest and can be used as a foundation for further investigation into the unique microbiological and chemical environments of smokeless tobacco products.

  9. Characterization of Bacterial Communities in Selected Smokeless Tobacco Products Using 16S rDNA Analysis

    PubMed Central

    Tyx, Robert E.; Stanfill, Stephen B.; Keong, Lisa M.; Rivera, Angel J.; Satten, Glen A.; Watson, Clifford H.

    2016-01-01

    The bacterial communities present in smokeless tobacco (ST) products have not previously reported. In this study, we used Next Generation Sequencing to study the bacteria present in U.S.-made dry snuff, moist snuff and Sudanese toombak. Sample diversity and taxonomic abundances were investigated in these products. A total of 33 bacterial families from four phyla, Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes, were identified. U.S.-produced dry snuff products contained a diverse distribution of all four phyla. Moist snuff products were dominated by Firmicutes. Toombak samples contained mainly Actinobacteria and Firmicutes (Aerococcaceae, Enterococcaceae, and Staphylococcaceae). The program PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was used to impute the prevalence of genes encoding selected bacterial toxins, antibiotic resistance genes and other pro-inflammatory molecules. PICRUSt also predicted the presence of specific nitrate reductase genes, whose products can contribute to the formation of carcinogenic nitrosamines. Characterization of microbial community abundances and their associated genomes gives us an indication of the presence or absence of pathways of interest and can be used as a foundation for further investigation into the unique microbiological and chemical environments of smokeless tobacco products. PMID:26784944

  10. Phylogenetic analysis of dissimilatory Fe(III)-reducing bacteria

    USGS Publications Warehouse

    Lonergan, D.J.; Jenter, H.L.; Coates, J.D.; Phillips, E.J.P.; Schmidt, T.M.; Lovley, D.R.

    1996-01-01

    Evolutionary relationships among strictly anaerobic dissimilatory Fe(III)- reducing bacteria obtained from a diversity of sedimentary environments were examined by phylogenetic analysis of 16S rRNA gene sequences. Members of the genera Geobacter, Desulfuromonas, Pelobacter, and Desulfuromusa formed a monophyletic group within the delta subdivision of the class Proteobacteria. On the basis of their common ancestry and the shared ability to reduce Fe(III) and/or S0, we propose that this group be considered a single family, Geobacteraceae. Bootstrap analysis, characteristic nucleotides, and higher- order secondary structures support the division of Geobacteraceae into two subgroups, designated the Geobacter and Desulfuromonas clusters. The genus Desulfuromusa and Pelobacter acidigallici make up a distinct branch with the Desulfuromonas cluster. Several members of the family Geobacteraceae, none of which reduce sulfate, were found to contain the target sequences of probes that have been previously used to define the distribution of sulfate-reducing bacteria and sulfate-reducing bacterium-like microorganisms. The recent isolations of Fe(III)-reducing microorganisms distributed throughout the domain Bacteria suggest that development of 16S rRNA probes that would specifically target all Fe(III) reducers may not be feasible. However, all of the evidence suggests that if a 16S rRNA sequence falls within the family Geobacteraceae, then the organism has the capacity for Fe(III) reduction. The suggestion, based on geological evidence, that Fe(III) reduction was the first globally significant process for oxidizing organic matter back to carbon dioxide is consistent with the finding that acetate-oxidizing Fe(III) reducers are phylogenetically diverse.

  11. Phenotypic and Phylogenetic Characterization of Ruminal Tannin-Tolerant Bacteria

    PubMed Central

    Nelson, Karen E.; Thonney, Michael L.; Woolston, Tina K.; Zinder, Stephen H.; Pell, Alice N.

    1998-01-01

    The 16S rRNA sequences and selected phenotypic characteristics were determined for six recently isolated bacteria that can tolerate high levels of hydrolyzable and condensed tannins. Bacteria were isolated from the ruminal contents of animals in different geographic locations, including Sardinian sheep (Ovis aries), Honduran and Colombian goats (Capra hircus), white-tail deer (Odocoileus virginianus) from upstate New York, and Rocky Mountain elk (Cervus elaphus nelsoni) from Oregon. Nearly complete sequences of the small-subunit rRNA genes, which were obtained by PCR amplification, cloning, and sequencing, were used for phylogenetic characterization. Comparisons of the 16S rRNA of the six isolates showed that four of the isolates were members of the genus Streptococcus and were most closely related to ruminal strains of Streptococcus bovis and the recently described organism Streptococcus gallolyticus. One of the other isolates, a gram-positive rod, clustered with the clostridia in the low-G+C-content group of gram-positive bacteria. The sixth isolate, a gram-negative rod, was a member of the family Enterobacteriaceae in the gamma subdivision of the class Proteobacteria. None of the 16S rRNA sequences of the tannin-tolerant bacteria examined was identical to the sequence of any previously described microorganism or to the sequence of any of the other organisms examined in this study. Three phylogenetically distinct groups of ruminal bacteria were isolated from four species of ruminants in Europe, North America, and South America. The presence of tannin-tolerant bacteria is not restricted by climate, geography, or host animal, although attempts to isolate tannin-tolerant bacteria from cows on low-tannin diets failed. PMID:9758806

  12. Physiological and phylogenetic study of microbes from geochemically and hydrogeologically diverse subsurface environments

    SciTech Connect

    Balkwill, D.L.; Reeves, R.H.

    1991-01-01

    The present document is an interim technical report in which we describe the research which has been completed during the seven-month period since the start of the grant. Progress is summarized in two main areas. The first is microbiological characterization of subsurface materials from the Hanford reservation and the Idaho National Engineering Laboratory, and the second is phylogenetic characterization of these microorganisms. The major tools used for phylogenetic characterization are RFLP analysis of PCR derived material and 16S rRNA sequencing. A description of manuscripts ready for publication is also provided. 4 refs. (MHB)

  13. Phylogenetic relationship of phototrophic purple sulfur bacteria according to pufL and pufM genes.

    PubMed

    Tank, Marcus; Thiel, Vera; Imhoff, Johannes F

    2009-09-01

    The phylogenetic relationship of purple sulfur bacteria (PSB), of the order Chromatiales (class Gammaproteobacteria), was analyzed based on photosynthetic gene sequences of the pufL and pufM genes, and the results compared to phylogenetic trees and groupings of the 16S rRNA gene. Primers for pufL and pufM genes were constructed and successfully used to amplify the pufLM genes of members of 16 genera of Chromatiales. In total, pufLM and 16S rRNA gene sequences of 66 PSB strains were analyzed, including 29 type strains and 28 new isolates. The inferred phylogenetic trees of the pufLM and 16S rRNA genes reflected a largely similar phylogenetic development suggesting coevolution of these essential genes within the PSB. It is concluded that horizontal gene transfer of pufLM genes within the PSB is highly unlikely, in contrast to the situation in other groups of anoxygenic phototrophic bacteria belonging to Alpha- and Betaproteobacteria. The phylogeny of pufLM is therefore in good agreement with the current taxonomic classification of PSB. A phylogenetic classification of PSB to the genus level is possible based on their pufL or pufM sequences, and in many cases even to the species level. In addition, our data support a correlation between Puf protein structure and the type of internal photosynthetic membranes (vesicular, lamellar, or tubular).

  14. A Mutation in the 16S rRNA Decoding Region Attenuates the Virulence of Mycobacterium tuberculosis

    PubMed Central

    Watanabe, Shinya; Matsumura, Kazunori; Iwai, Hiroki; Funatogawa, Keiji; Haishima, Yuji; Fukui, Chie; Okumura, Kayo; Kato-Miyazawa, Masako; Hashimoto, Masahito; Teramoto, Kanae; Kirikae, Fumiko; Miyoshi-Akiyama, Tohru

    2016-01-01

    Mycobacterium tuberculosis contains a single rRNA operon that encodes targets for antituberculosis agents, including kanamycin. To date, only four mutations in the kanamycin binding sites of 16S rRNA have been reported in kanamycin-resistant clinical isolates. We hypothesized that another mutation(s) in the region may dramatically decrease M. tuberculosis viability and virulence. Here, we describe an rRNA mutation, U1406A, which was generated in vitro and confers resistance to kanamycin while highly attenuating M. tuberculosis virulence. The mutant showed decreased expression of 20% (n = 361) of mycobacterial proteins, including central metabolic enzymes, mycolic acid biosynthesis enzymes, and virulence factors such as antigen 85 complexes and ESAT-6. The mutation also induced three proteins, including KsgA (Rv1010; 16S rRNA adenine dimethyltransferase), which closely bind to the U1406A mutation site on the ribosome; these proteins were associated with ribosome maturation and translation initiation processes. The mutant showed an increase in 17S rRNA (precursor 16S rRNA) and a decrease in the ratio of 30S subunits to the 70S ribosomes, suggesting that the U1406A mutation in 16S rRNA attenuated M. tuberculosis virulence by affecting these processes. PMID:27245411

  15. Molecular Diagnosis of Kingella kingae Pericarditis by Amplification and Sequencing of the 16S rRNA Gene▿

    PubMed Central

    Matta, Matta; Wermert, Delphine; Podglajen, Isabelle; Sanchez, Olivier; Buu-Hoï, Annie; Gutmann, Laurent; Meyer, Guy; Mainardi, Jean-Luc

    2007-01-01

    Kingella kingae is a fastidious gram-negative bacillus that is considered an emerging pathogen in pediatric settings but remains less common in adults. Here we describe a case of pericarditis in an immunocompetent adult host. The microorganism was identified directly from the clinical sample by molecular techniques, i.e., 16S rRNA gene amplification and sequencing. PMID:17634294

  16. Molecular diagnosis of Kingella kingae pericarditis by amplification and sequencing of the 16S rRNA gene.

    PubMed

    Matta, Matta; Wermert, Delphine; Podglajen, Isabelle; Sanchez, Olivier; Buu-Hoï, Annie; Gutmann, Laurent; Meyer, Guy; Mainardi, Jean-Luc

    2007-09-01

    Kingella kingae is a fastidious gram-negative bacillus that is considered an emerging pathogen in pediatric settings but remains less common in adults. Here we describe a case of pericarditis in an immunocompetent adult host. The microorganism was identified directly from the clinical sample by molecular techniques, i.e., 16S rRNA gene amplification and sequencing.

  17. 16S rRNA methyltransferase KsgA contributes to oxidative stress resistance and virulence in Staphylococcus aureus.

    PubMed

    Kyuma, Tatsuhiko; Kizaki, Hayato; Ryuno, Hiroki; Sekimizu, Kazuhisa; Kaito, Chikara

    2015-12-01

    We previously reported that the rRNA methyltransferases RsmI and RsmH, which are responsible for cytidine dimethylation at position 1402 of 16S rRNA in the decoding center of the ribosome, contribute to Staphylococcus aureus virulence. Here we evaluated other 16S rRNA methyltransferases, including KsgA (RsmA), RsmB/F, RsmC, RsmD, RsmE, and RsmG. Knockout of KsgA, which methylates two adjacent adenosines at positions 1518 and 1519 of 16S rRNA in the intersubunit bridge of the ribosome, attenuated the S. aureus killing ability against silkworms. The ksgA knockout strain was sensitive to oxidative stress and had a lower survival rate in murine macrophages than the parent strain. The ksgA knockout strain exhibited decreased translational fidelity in oxidative stress conditions. Administration of N-acetyl-l-cysteine, a free-radical scavenger, restored the killing ability of the ksgA knockout strain against silkworms. These findings suggest that the methyl-modifications of 16S rRNA by KsgA contribute to maintain ribosome function under oxidative conditions and thus to S. aureus virulence.

  18. Species identification and profiling of complex microbial communities using shotgun Illumina sequencing of 16S rRNA amplicon sequences.

    PubMed

    Ong, Swee Hoe; Kukkillaya, Vinutha Uppoor; Wilm, Andreas; Lay, Christophe; Ho, Eliza Xin Pei; Low, Louie; Hibberd, Martin Lloyd; Nagarajan, Niranjan

    2013-01-01

    The high throughput and cost-effectiveness afforded by short-read sequencing technologies, in principle, enable researchers to perform 16S rRNA profiling of complex microbial communities at unprecedented depth and resolution. Existing Illumina sequencing protocols are, however, limited by the fraction of the 16S rRNA gene that is interrogated and therefore limit the resolution and quality of the profiling. To address this, we present the design of a novel protocol for shotgun Illumina sequencing of the bacterial 16S rRNA gene, optimized to amplify more than 90% of sequences in the Greengenes database and with the ability to distinguish nearly twice as many species-level OTUs compared to existing protocols. Using several in silico and experimental datasets, we demonstrate that despite the presence of multiple variable and conserved regions, the resulting shotgun sequences can be used to accurately quantify the constituents of complex microbial communities. The reconstruction of a significant fraction of the 16S rRNA gene also enabled high precision (>90%) in species-level identification thereby opening up potential application of this approach for clinical microbial characterization.

  19. Evolutionary and Diagnostic Implications of Intragenomic Heterogeneity in the 16S rRNA Gene in Aeromonas Strains

    PubMed Central

    Morandi, Alessia; Zhaxybayeva, Olga; Gogarten, J. Peter; Graf, Joerg

    2005-01-01

    Sequencing 16S rRNA genes (SSU) cloned from Aeromonas strains revealed that strains contained up to six copies differing by ≤1.5%. The SSU copies from Aeromonas veronii LMG13695 clustered with sequences from four Aeromonas species. These results demonstrate intragenomic heterogeneity of SSU and suggest caution when using SSU to identify aeromonads. PMID:16159790

  20. Comparison of gull-specific assays targeting 16S rRNA gene of Catellicoccus marimammalium and Streptococcus spp.

    EPA Science Inventory

    Gulls have been implicated as a source of fecal contamination in inland and coastal waters. Only one gull-specific assay is currently available (i.e., gull2 qPCR assay). This assay is based on the 16S rRNA gene of Catellicocclls marimammalium and has showed a high level of host-s...

  1. A Mutation in the 16S rRNA Decoding Region Attenuates the Virulence of Mycobacterium tuberculosis.

    PubMed

    Watanabe, Shinya; Matsumura, Kazunori; Iwai, Hiroki; Funatogawa, Keiji; Haishima, Yuji; Fukui, Chie; Okumura, Kayo; Kato-Miyazawa, Masako; Hashimoto, Masahito; Teramoto, Kanae; Kirikae, Fumiko; Miyoshi-Akiyama, Tohru; Kirikae, Teruo

    2016-08-01

    Mycobacterium tuberculosis contains a single rRNA operon that encodes targets for antituberculosis agents, including kanamycin. To date, only four mutations in the kanamycin binding sites of 16S rRNA have been reported in kanamycin-resistant clinical isolates. We hypothesized that another mutation(s) in the region may dramatically decrease M. tuberculosis viability and virulence. Here, we describe an rRNA mutation, U1406A, which was generated in vitro and confers resistance to kanamycin while highly attenuating M. tuberculosis virulence. The mutant showed decreased expression of 20% (n = 361) of mycobacterial proteins, including central metabolic enzymes, mycolic acid biosynthesis enzymes, and virulence factors such as antigen 85 complexes and ESAT-6. The mutation also induced three proteins, including KsgA (Rv1010; 16S rRNA adenine dimethyltransferase), which closely bind to the U1406A mutation site on the ribosome; these proteins were associated with ribosome maturation and translation initiation processes. The mutant showed an increase in 17S rRNA (precursor 16S rRNA) and a decrease in the ratio of 30S subunits to the 70S ribosomes, suggesting that the U1406A mutation in 16S rRNA attenuated M. tuberculosis virulence by affecting these processes.

  2. ASSESSMENT OF FECAL POLLUTION SOURCES IN PLUM CREEK WATERSHED USING BACTEROIDETES 16S RDNA-BASED ASSAYS

    EPA Science Inventory

    Recently, 16S rDNA Bacteroidetes-targeted PCR assays were developed to discriminate between ruminant and human fecal pollution. These assays are rapid and relatively inexpensive but have been used in a limited number of studies. In this study, we evaluated the efficacy o...

  3. Minimization of chloroplast contamination in 16S rRNA gene pyrosequencing of insect herbivore bacterial communities

    PubMed Central

    Hanshew, Alissa S.; Mason, Charles J.; Raffa, Kenneth F.; Currie, Cameron R.

    2014-01-01

    Chloroplast sequence contamination in 16S ribosomal RNA gene (16S) analyses can be particularly problematic when sampling microbial communities in plants and folivorous arthropods. We previously encountered high levels of plastid contamination in herbivorous insect samples when we used the predominant 454 pyrosequencing 16S methodologies described in the literature. 799F, a primer previously found to exclude chloroplast sequences, was modified to enhance its efficacy, and we describe, in detail, our methodology throughout amplicon pyrosequencing. Thirteen versions of 799F were assessed for the exclusion of chloroplast sequences from our samples. We found that a shift in the mismatch between 799F and chloroplast 16S resulted in significant reduction of chloroplast reads. Our results also indicate that amplifying sequences from environmental samples in a two-step PCR process, with the addition of the multiplex identifiers and 454 adapters in a second round of PCR, further improved primer specificity. Primers that included 3′ phosphorothioate bonds, which were designed to block primer degradation, did not amplify consistently across samples. The different forward primers do not appear to bias the bacterial communities detected. We provide a methodological framework for reducing chloroplast reads in high-throughput sequencing data sets that can be applied to a number of environmental samples and sequencing techniques. PMID:23968645

  4. Species Identification and Profiling of Complex Microbial Communities Using Shotgun Illumina Sequencing of 16S rRNA Amplicon Sequences

    PubMed Central

    Lay, Christophe; Ho, Eliza Xin Pei; Low, Louie; Hibberd, Martin Lloyd; Nagarajan, Niranjan

    2013-01-01

    The high throughput and cost-effectiveness afforded by short-read sequencing technologies, in principle, enable researchers to perform 16S rRNA profiling of complex microbial communities at unprecedented depth and resolution. Existing Illumina sequencing protocols are, however, limited by the fraction of the 16S rRNA gene that is interrogated and therefore limit the resolution and quality of the profiling. To address this, we present the design of a novel protocol for shotgun Illumina sequencing of the bacterial 16S rRNA gene, optimized to amplify more than 90% of sequences in the Greengenes database and with the ability to distinguish nearly twice as many species-level OTUs compared to existing protocols. Using several in silico and experimental datasets, we demonstrate that despite the presence of multiple variable and conserved regions, the resulting shotgun sequences can be used to accurately quantify the constituents of complex microbial communities. The reconstruction of a significant fraction of the 16S rRNA gene also enabled high precision (>90%) in species-level identification thereby opening up potential application of this approach for clinical microbial characterization. PMID:23579286

  5. Campylobacter jejuni, an uncommon cause of splenic abscess diagnosed by 16S rRNA gene sequencing.

    PubMed

    Seng, Piseth; Quenard, Fanny; Menard, Amélie; Heyries, Laurent; Stein, Andreas

    2014-12-01

    Splenic abscess is a rare disease that primarily occurs in patients with splenic trauma, endocarditis, sickle cell anemia, or other diseases that compromise the immune system. This report describes a culture-negative splenic abscess in an immunocompetent patient caused by Campylobacter jejuni, as determined by 16S rRNA gene sequencing.

  6. A Web-Hosted R Workflow to Simplify and Automate the Analysis of 16S NGS Data

    EPA Science Inventory

    Next-Generation Sequencing (NGS) produces large data sets that include tens-of-thousands of sequence reads per sample. For analysis of bacterial diversity, 16S NGS sequences are typically analyzed in a workflow that containing best-of-breed bioinformatics packages that may levera...

  7. MULTIPLE ENZYME RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS FOR HIGH RESOLUTION DISTINCTION OF PSEUDOMONAS (SENSU STRICTO) 16S RRNA GENES

    EPA Science Inventory

    Pseudomonas specific 16S rDNA PCR amplification and multiple enzyme restriction fragment length polymorphism (MERFLP) analysis using a single digestion mixture of Alu I, Hinf I, Rsa I, and Tru 9I distinguished 150 published sequences and reference strains of authentic Pseudomonas...

  8. COI is better than 16S rRNA for DNA barcoding Asiatic salamanders (Amphibia: Caudata: Hynobiidae).

    PubMed

    Xia, Yun; Gu, Hai-Feng; Peng, Rui; Chen, Qin; Zheng, Yu-Chi; Murphy, Robert W; Zeng, Xiao-Mao

    2012-01-01

    The 5' region of the mitochondrial DNA (mtDNA) gene cytochrome c oxidase I (COI) is the standard marker for DNA barcoding. However, because COI tends to be highly variable in amphibians, sequencing is often challenging. Consequently, another mtDNA gene, 16S rRNA gene, is often advocated for amphibian barcoding. Herein, we directly compare the usefulness of COI and 16S in discriminating species of hynobiid salamanders using 130 individuals. Species identification and classification of these animals, which are endemic to Asia, are often based on morphology only. Analysis of Kimura 2-parameter genetic distances (K2P) documents the mean intraspecific variation for COI and 16S rRNA genes to be 1.4% and 0.3%, respectively. Whereas COI can always identify species, sometimes 16S cannot. Intra- and interspecific genetic divergences occasionally overlap in both markers, thus reducing the value of a barcoding gap to identify genera. Regardless, COI is the better DNA barcoding marker for hynobiids. In addition to the comparison of two potential markers, high levels of intraspecific divergence in COI (>5%) suggest that both Onychodactylus fischeri and Salamandrella keyserlingii might be composites of cryptic species.

  9. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and standard protocol for genomovar assignment.

    PubMed

    LaFrentz, B R; Waldbieser, G C; Welch, T J; Shoemaker, C A

    2014-07-01

    Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare, and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing of this important fish pathogen. Interpretation of restriction patterns can be difficult due to the lack of a formal description of the expected number and sizes of DNA fragments generated for each of the described genomovars. In this study, partial 16S rRNA gene sequences (ca. 1250-bp fragment) from isolates representing each described genomovar and isolates generating unique restriction patterns were cloned and sequenced. The results demonstrated that some isolates contained up to three different 16S rRNA genes whose sequences generate different RFLP patterns due to intragenomic heterogeneity within HaeIII restriction sites. The occurrence of HaeIII restriction sites within the portion of the 16S rRNA gene used for typing the F. columnare isolates and intragenomic heterogeneity within these sites explained the restriction patterns observed following RFLP analyses. This research provides a standard protocol for typing isolates of F. columnare by RFLP and a formal description of the expected restriction patterns for the previously described genomovars I, II, II-B and III. Additionally, we describe a new genomovar, I/II.

  10. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and standard protocol for genomovar assignment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing this important fish pathogen. Interpretation of restriction patterns can be difficult due to th...

  11. The phylogenetic diversity of metagenomes.

    PubMed

    Kembel, Steven W; Eisen, Jonathan A; Pollard, Katherine S; Green, Jessica L

    2011-01-01

    Phylogenetic diversity--patterns of phylogenetic relatedness among organisms in ecological communities--provides important insights into the mechanisms underlying community assembly. Studies that measure phylogenetic diversity in microbial communities have primarily been limited to a single marker gene approach, using the small subunit of the rRNA gene (SSU-rRNA) to quantify phylogenetic relationships among microbial taxa. In this study, we present an approach for inferring phylogenetic relationships among microorganisms based on the random metagenomic sequencing of DNA fragments. To overcome challenges caused by the fragmentary nature of metagenomic data, we leveraged fully sequenced bacterial genomes as a scaffold to enable inference of phylogenetic relationships among metagenomic sequences from multiple phylogenetic marker gene families. The resulting metagenomic phylogeny can be used to quantify the phylogenetic diversity of microbial communities based on metagenomic data sets. We applied this method to understand patterns of microbial phylogenetic diversity and community assembly along an oceanic depth gradient, and compared our findings to previous studies of this gradient using SSU-rRNA gene and metagenomic analyses. Bacterial phylogenetic diversity was highest at intermediate depths beneath the ocean surface, whereas taxonomic diversity (diversity measured by binning sequences into taxonomically similar groups) showed no relationship with depth. Phylogenetic diversity estimates based on the SSU-rRNA gene and the multi-gene metagenomic phylogeny were broadly concordant, suggesting that our approach will be applicable to other metagenomic data sets for which corresponding SSU-rRNA gene sequences are unavailable. Our approach opens up the possibility of using metagenomic data to study microbial diversity in a phylogenetic context.

  12. Relating Phylogenetic and Functional Diversity among Denitrifiers and Quantifying their Capacity to Predict Community Functioning.

    PubMed

    Salles, Joana Falcão; Le Roux, Xavier; Poly, Franck

    2012-01-01

    Genetic diversity of phylogenetic or functional markers is widely used as a proxy of microbial diversity. However, it remains unclear to what extent functional diversity (FD), gene sequence diversity and community functioning are linked. For a range of denitrifying bacteria, we analyzed the relationships between (i) the similarity of functional traits evaluated from metabolic profiles (BIOLOG plates) or from N(2)O accumulation patterns on different carbon sources and (ii) the similarity of phylogenetic (16S rRNA gene) or functional (nir gene) markers. We also calculated different proxies for the diversity of denitrifier community based on taxa richness, phylogenetic (16S rRNA gene) or functional similarities (based either on metabolic profiles or N(2)O accumulation patterns), and evaluated their performance in inferring the functioning of assembled denitrifying communities. For individual strains, the variation in the 16S rRNA gene sequence was weakly correlated with the variation in metabolic patterns (ρ = 0.35) and was not related to N(2)O accumulation. The latter was correlated with the similarity of nitrite reductase residues. When nir genes were analyzed separately, the similarity in amino acids coded by the nirS genes was highly correlated with the observed patterns of N(2)O accumulation (ρ = 0.62), whereas nirK amino acid residues were unrelated to N(2)O accumulation. For bacterial assemblages, phylogenetic diversity (average similarity among species in a community) and mean community dissimilarity (average distance between species) calculated using 16S rRNA gene sequences, and FD measures associated with metabolic profiles, poorly predicted the variation in the functioning of assembled communities (≤15%). In contrast, the proxies of FD based on N(2)O accumulation patterns performed better and explained from 23 to 42% of the variation in denitrification. Amongst those, community niche was the best metric, indicating the importance of

  13. Phylogenetic Diversity of Bacteria Associated with the Marine Sponge Rhopaloeides odorabile†

    PubMed Central

    Webster, Nicole S.; Wilson, Kate J.; Blackall, Linda L.; Hill, Russell T.

    2001-01-01

    Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of the Actinobacteria, low-G+C gram-positive bacteria, the β- and γ-subdivisions of the Proteobacteria, Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R. odorabile. High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge. PMID:11133476

  14. Species identification and phylogenetic analysis of genus Nassarius (Nassariidae) based on mitochondrial genes

    NASA Astrophysics Data System (ADS)

    Li, Haitao; Lin, Duan; Fang, Hongda; Zhu, Aijia; Gao, Yang

    2010-05-01

    Genus Nassarius contains many subgenera, such as Zeuxis, Telasco, Niotha, Varicinassa, Plicarcularia, Nassarius s. str. and Reticunassa. On the basis of morphological characteristics of the shell and radula and sequences of mitochondrial cytochrome oxidase subunit I (COI) and 16S rRNA genes, Nassarius specimens collected from the South China Sea were identified and phylogenetically analyzed. Although Nassarius sp. and Nassarius ( Varicinassa) variciferus were morphologically different in their shells, few variations were found among their radular teeth and sequences of mtCOI and mt16S RNA genes. Therefore, Nassarius sp. should be classified as N. (Varicinassa) variciferus. Nassarius (Zeuxis) sp. has only a subtle difference from Nassarius (Zeuxis) algidus on the shell, but it shows obvious differences in radular teeth and DNA sequence, indicating that they are two distinct species. Sequence divergence of mtCOI and mt16S RNA genes within Nassarius species was much lower than that between species, suggesting that these two genes are suitable for Nassarius species identification. Phylogenetic analysis (neighbor-joining and maximum parsimony) based on mtCOI and mt16S rRNA genes revealed the presence of two groups in genus Nassarius and a closest relationship between subgenera Zeuxis and Telasco. Species of subgenus Plicarcularia did not form a single clade. The molecular phylogeny was not congruent with the previous morphological phylogeny. The subgeneric divisions of genus Nassarius appear to be uncertain and unreliable.

  15. A preliminary report of phylogenetic diversity of bacterial strains isolated from marine creatures.

    PubMed

    Kurahashi, Midori; Yokota, Akira

    2002-10-01

    Bacterial diversity among marine creatures, especially molluscs, as a source for searching out novel lineages of bacteria, was studied. Marine creatures were collected at the coasts of the Kanto area in Japan. A total of 116 strains of bacteria were isolated from the intestines of 19 species of marine creatures includings molluscs, pisces and protochordata. Partial sequencing of 16S rDNA revealed that most of the isolates belonged to the gamma subclass of the Proteobacteria and Cytophaga-Flavobacterium-Bacteroides group. The BLAST searches revealed that the complete 16S rDNA sequence of 17 strains out of 116 isolates showed less than 94% similarity with 16S rDNA sequences deposited in the database. Four strains out of the 17 isolates belonged to the Rhodobacter group, 8 strains to the Alteromonas group, and the remaining 5 strains to the Cytophaga-Flavobacterium-Bacteroides group. Phylogenetic positions of 6 strains belonging to the Alteromonas group, which were isolated from different marine creatures, were close to each other, and represented a novel 16S rDNA lineage within the gamma subclass of Proteobacteria. Therefore, it may be inferred that these 6 strains belong to a new genus of Proteobacteria. Phylogenetic positions of the other strains are also independent from neighboring taxa, and they were suggested to respectively form a novel lineage. From these results, it is clear that the biodiversity of bacteria in marine creatures is much wider than was previously thought, and unknown microbiological resources are buried in these organisms.

  16. Incompletely resolved phylogenetic trees inflate estimates of phylogenetic conservatism.

    PubMed

    Davies, T Jonathan; Kraft, Nathan J B; Salamin, Nicolas; Wolkovich, Elizabeth M

    2012-02-01

    The tendency for more closely related species to share similar traits and ecological strategies can be explained by their longer shared evolutionary histories and represents phylogenetic conservatism. How strongly species traits co-vary with phylogeny can significantly impact how we analyze cross-species data and can influence our interpretation of assembly rules in the rapidly expanding field of community phylogenetics. Phylogenetic conservatism is typically quantified by analyzing the distribution of species values on the phylogenetic tree that connects them. Many phylogenetic approaches, however, assume a completely sampled phylogeny: while we have good estimates of deeper phylogenetic relationships for many species-rich groups, such as birds and flowering plants, we often lack information on more recent interspecific relationships (i.e., within a genus). A common solution has been to represent these relationships as polytomies on trees using taxonomy as a guide. Here we show that such trees can dramatically inflate estimates of phylogenetic conservatism quantified using S. P. Blomberg et al.'s K statistic. Using simulations, we show that even randomly generated traits can appear to be phylogenetically conserved on poorly resolved trees. We provide a simple rarefaction-based solution that can reliably retrieve unbiased estimates of K, and we illustrate our method using data on first flowering times from Thoreau's woods (Concord, Massachusetts, USA).

  17. Phylogenetic comparisons of bacterial communities from serpentine and nonserpentine soils.

    PubMed

    Oline, David K

    2006-11-01

    I present the results of a culture-independent survey of soil bacterial communities from serpentine soils and adjacent nonserpentine comparator soils using a variety of newly developed phylogenetically based statistical tools. The study design included site-based replication of the serpentine-to-nonserpentine community comparison over a regional scale ( approximately 100 km) in Northern California and Southern Oregon by producing 16S rRNA clone libraries from pairs of samples taken on either side of the serepentine-nonserpentine edaphic boundary at three geographical sites. At the division level, the serpentine and nonserpentine communities were similar to each other and to previous data from forest soils. Comparisons of both richness and Shannon diversity produced no significant differences between any of the libraries, but the vast majority of phylogenetically based tests were significant, even with only 50 sequences per library. These results suggest that most samples were distinct, consisting of a collection of lineages generally not found in other samples. The pattern of results showed that serpentine communities tended to be more similar to each other than they were to nonserpentine communities, and these differences were at a lower taxonomic scale. Comparisons of two nonserpentine communities generally showed differences, and some results suggest that the geographical site may control community composition as well. These results show the power of phylogenetic tests to discern differences between 16S rRNA libraries compared to tests that discard DNA data to bin sequences into operational taxonomic units, and they stress the importance of replication at larger scales for inferences regarding microbial biogeography.

  18. Phylogenetic Comparisons of Bacterial Communities from Serpentine and Nonserpentine Soils▿

    PubMed Central

    Oline, David K.

    2006-01-01

    I present the results of a culture-independent survey of soil bacterial communities from serpentine soils and adjacent nonserpentine comparator soils using a variety of newly developed phylogenetically based statistical tools. The study design included site-based replication of the serpentine-to-nonserpentine community comparison over a regional scale (∼100 km) in Northern California and Southern Oregon by producing 16S rRNA clone libraries from pairs of samples taken on either side of the serepentine-nonserpentine edaphic boundary at three geographical sites. At the division level, the serpentine and nonserpentine communities were similar to each other and to previous data from forest soils. Comparisons of both richness and Shannon diversity produced no significant differences between any of the libraries, but the vast majority of phylogenetically based tests were significant, even with only 50 sequences per library. These results suggest that most samples were distinct, consisting of a collection of lineages generally not found in other samples. The pattern of results showed that serpentine communities tended to be more similar to each other than they were to nonserpentine communities, and these differences were at a lower taxonomic scale. Comparisons of two nonserpentine communities generally showed differences, and some results suggest that the geographical site may control community composition as well. These results show the power of phylogenetic tests to discern differences between 16S rRNA libraries compared to tests that discard DNA data to bin sequences into operational taxonomic units, and they stress the importance of replication at larger scales for inferences regarding microbial biogeography. PMID:16950906

  19. Phylogenetic analysis suggests that habitat filtering is structuring marine bacterial communities across the globe.

    PubMed

    Pontarp, Mikael; Canbäck, Björn; Tunlid, Anders; Lundberg, Per

    2012-07-01

    The phylogenetic structure and community composition were analysed in an existing data set of marine bacterioplankton communities to elucidate the evolutionary and ecological processes dictating the assembly. The communities were sampled from coastal waters at nine locations distributed worldwide and were examined through the use of comprehensive clone libraries of 16S ribosomal RNA genes. The analyses show that the local communities are phylogenetically different from each other and that a majority of them are phylogenetically clustered, i.e. the species (operational taxonomic units) were more related to each other than expected by chance. Accordingly, the local communities were assembled non-randomly from the global pool of available bacterioplankton. Further, the phylogenetic structures of the communities were related to the water temperature at the locations. In agreement with similar studies, including both macroorganisms and bacteria, these results suggest that marine bacterial communities are structured by “habitat filtering”, i.e. through non-random colonization and invasion determined by environmental characteristics. Different bacterial types seem to have different ecological niches that dictate their survival in different habitats. Other eco-evolutionary processes that may contribute to the observed phylogenetic patterns are discussed. The results also imply a mapping between phenotype and phylogenetic relatedness which facilitates the use of community phylogenetic structure analysis to infer ecological and evolutionary assembly processes.

  20. Phylogenetic constraints on ecosystem functioning.

    PubMed

    Gravel, Dominique; Bell, Thomas; Barbera, Claire; Combe, Marine; Pommier, Thomas; Mouquet, Nicolas

    2012-01-01

    There is consensus that biodiversity losses will result in declining ecosystem functioning if species have different functional traits. Phylogenetic diversity has recently been suggested as a predictor of ecosystem functioning because it could approximate the functional complementarity among species. Here we describe an experiment that takes advantage of the rapid evolutionary response of bacteria to disentangle the role of phylogenetic and species diversity. We impose a strong selection regime on marine bacterial lineages and assemble the ancestral and evolved lines in microcosms of varying lineage and phylogenetic diversity. We find that the relationship between phylogenetic diversity and productivity is strong for the ancestral lineages but brakes down for the evolved lineages. Our results not only emphasize the potential of using phylogeny to evaluate ecosystem functioning, but also they warn against using phylogenetics as a proxy for functional diversity without good information on species evolutionary history.

  1. Phylogenetic analysis of Salmonella, Shigella, and Escherichia coli strains on the basis of the gyrB gene sequence.

    PubMed

    Fukushima, Masao; Kakinuma, Kenichi; Kawaguchi, Ryuji

    2002-08-01

    Phylogenetic analysis of about 200 strains of Salmonella, Shigella, and Escherichia coli was carried out using the nucleotide sequence of the gene for DNA gyrase B (gyrB), which was determined by directly sequencing PCR fragments. The results establish a new phylogenetic tree for the classification of Salmonella, Shigella, and Escherichia coli in which Salmonella forms a cluster separate from but closely related to Shigella and E. coli. In comparison with 16S rRNA analysis, the gyrB sequences indicated a greater evolutionary divergence for the bacteria. Thus, in screening for the presence of bacteria, the gyrB gene might be a useful tool for differentiating between closely related species of bacteria such as Shigella spp. and E. coli. At present, 16S rRNA sequence analysis is an accurate and rapid method for identifying most unknown bacteria to the genus level because the highly conserved 16S rRNA region is easy to amplify; however, analysis of the more variable gyrB sequence region can identify unknown bacteria to the species level. In summary, we have shown that gyrB sequence analysis is a useful alternative to 16S rRNA analysis for constructing the phylogenetic relationships of bacteria, in particular for the classification of closely related bacterial species.

  2. Isolation and 16S DNA characterization of soil microorganisms from tropical soils capable of utilizing the herbicides hexazinone and tebuthiuron.

    PubMed

    Mostafa, Fadwa I Y; Helling, Charles S

    2003-11-01

    Six non-fermentative bacteria were isolated from Colombian (South America) and Hawaiian (USA) soils after enrichment with minimal medium supplemented with two herbicides, hexazinone (Hex) and tebuthiuron (Teb). Microscopic examination and physiological tests were followed by partial 16S DNA sequence analysis, using the first 527 bp of the 16S rRNA gene for bacterial identification. The isolated microorganisms (and in brackets, the herbicide that each degraded) were identified as: from Colombia. Methylobacterium organophilum [Teb], Paenibacillus pabuli [Teb], and Micrmbacterium foliorum [Hex]; and from Hawaii, Methylobacterium radiotolerans [Teb], Paenibacillus illinoisensis [Hex], and Rhodococcus equi [Hex]. The findings further explain how these herbicides, which have potential for illicit coca (Erythroxylum sp.) control, dissipate following their application to tropical soils.

  3. Phylogenetic molecular function annotation

    NASA Astrophysics Data System (ADS)

    Engelhardt, Barbara E.; Jordan, Michael I.; Repo, Susanna T.; Brenner, Steven E.

    2009-07-01

    It is now easier to discover thousands of protein sequences in a new microbial genome than it is to biochemically characterize the specific activity of a single protein of unknown function. The molecular functions of protein sequences have typically been predicted using homology-based computational methods, which rely on the principle that homologous proteins share a similar function. However, some protein families include groups of proteins with different molecular functions. A phylogenetic approach for predicting molecular function (sometimes called "phylogenomics") is an effective means to predict protein molecular function. These methods incorporate functional evidence from all members of a family that have functional characterizations using the evolutionary history of the protein family to make robust predictions for the uncharacterized proteins. However, they are often difficult to apply on a genome-wide scale because of the time-consuming step of reconstructing the phylogenies of each protein to be annotated. Our automated approach for function annotation using phylogeny, the SIFTER (Statistical Inference of Function Through Evolutionary Relationships) methodology, uses a statistical graphical model to compute the probabilities of molecular functions for unannotated proteins. Our benchmark tests showed that SIFTER provides accurate functional predictions on various protein families, outperforming other available methods.

  4. Phylogenetic molecular function annotation

    PubMed Central

    Engelhardt, Barbara E; Jordan, Michael I; Repo, Susanna T; Brenner, Steven E

    2010-01-01

    It is now easier to discover thousands of protein sequences in a new microbial genome than it is to biochemically characterize the specific activity of a single protein of unknown function. The molecular functions of protein sequences have typically been predicted using homology-based computational methods, which rely on the principle that homologous proteins share a similar function. However, some protein families include groups of proteins with different molecular functions. A phylogenetic approach for predicting molecular function (sometimes called “phylogenomics”) is an effective means to predict protein molecular function. These methods incorporate functional evidence from all members of a family that have functional characterizations using the evolutionary history of the protein family to make robust predictions for the uncharacterized proteins. However, they are often difficult to apply on a genome-wide scale because of the time-consuming step of reconstructing the phylogenies of each protein to be annotated. Our automated approach for function annotation using phylogeny, the SIFTER (Statistical Inference of Function Through Evolutionary Relationships) methodology, uses a statistical graphical model to compute the probabilities of molecular functions for unannotated proteins. Our benchmark tests showed that SIFTER provides accurate functional predictions on various protein families, outperforming other available methods. PMID:20664722

  5. Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens.

    PubMed

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; MacKichan, Joanna; Kato-Maeda, Midori; Miller, Steve; Nadarajan, Rohan; Brodie, Eoin L; Lynch, Susan V

    2015-01-01

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n = 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci.

  6. RmtC and RmtF 16S rRNA Methyltransferase in NDM-1-Producing Pseudomonas aeruginosa.

    PubMed

    Rahman, Mohibur; Prasad, Kashi Nath; Pathak, Ashutosh; Pati, Binod Kumar; Singh, Avinash; Ovejero, Cristina M; Ahmad, Saheem; Gonzalez-Zorn, Bruno

    2015-11-01

    We investigated 16S rRNA methyltransferases in 38 blaNDM-1-positive Pseudomonas aeruginosa isolates and found RmtC in 3 isolates, 1 of which also harbored RmtF. The isolates were clonally unrelated; rmtC and rmtF genes were located on a chromosome with the blaNDM-1 gene. Strategies are needed to limit the spread of such isolates.

  7. Use of 16S rRNA Sequencing for Identification of Actinobacillus ureae Isolated from a Cerebrospinal Fluid Sample

    PubMed Central

    Whitelaw, A. C.; Shankland, I. M.; Elisha, B. G.

    2002-01-01

    Actinobacillus ureae, previously Pasteurella ureae, has on rare occasions been described as a cause of human infection. Owing to its rarity, it may not be easily identified in clinical microbiology laboratories by standard tests. This report describes a patient with acute bacterial meningitis due to A. ureae. The identity of the isolate was determined by means of DNA sequence analysis of a portion of the 16S rRNA gene. PMID:11825992

  8. Usefulness of 16S rDNA sequencing for the diagnosis of infective endocarditis caused by Corynebacterium diphtheriae.

    PubMed

    Pathipati, Padmaja; Menon, Thangam; Kumar, Naveen; Francis, Thara; Sekar, Prem; Cherian, Kotturathu Mammen

    2012-08-01

    We report a rare case of infective endocarditis caused by Corynebacterium diphtheriae in an 8-year-old boy, 2 years after a right ventricular outflow tract reconstruction with a bovine Contegra valved conduit. The patient recovered well after an RV-PA conduit enblock explantation and replacement with an aortic homograft with antibiotic treatment. All bacteriological cultures of excised tissue and blood were negative. The aetiological agent was identified as C. diphtheriae subsp. gravis by 16s rDNA sequencing.

  9. Use of 16S rRNA Gene for Identification of a Broad Range of Clinically Relevant Bacterial Pathogens

    PubMed Central

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; MacKichan, Joanna; Kato-Maeda, Midori; Miller, Steve; Nadarajan, Rohan; Brodie, Eoin L.; Lynch, Susan V.

    2015-01-01

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n = 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci. PMID:25658760

  10. Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens

    DOE PAGES

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; ...

    2015-02-06

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n =more » 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci.« less

  11. Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens

    SciTech Connect

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; MacKichan, Joanna; Kato-Maeda, Midori; Miller, Steve; Nadarajan, Rohan; Brodie, Eoin L.; Lynch, Susan V.; Heimesaat, Markus M.

    2015-02-06

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n = 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci.

  12. Short-read assembly of full-length 16S amplicons reveals bacterial diversity in subsurface sediments.

    PubMed

    Miller, Christopher S; Handley, Kim M; Wrighton, Kelly C; Frischkorn, Kyle R; Thomas, Brian C; Banfield, Jillian F

    2013-01-01

    In microbial ecology, a fundamental question relates to how community diversity and composition change in response to perturbation. Most studies have had limited ability to deeply sample community structure (e.g. Sanger-sequenced 16S rRNA libraries), or have had limited taxonomic resolution (e.g. studies based on 16S rRNA hypervariable region sequencing). Here, we combine the higher taxonomic resolution of near-full-length 16S rRNA gene amplicons with the economics and sensitivity of short-read sequencing to assay the abundance and identity of organisms that represent as little as 0.01% of sediment bacterial communities. We used a new version of EMIRGE optimized for large data size to reconstruct near-full-length 16S rRNA genes from amplicons sheared and sequenced with Illumina technology. The approach allowed us to differentiate the community composition among samples acquired before perturbation, after acetate amendment shifted the predominant metabolism to iron reduction, and once sulfate reduction began. Results were highly reproducible across technical replicates, and identified specific taxa that responded to the perturbation. All samples contain very high alpha diversity and abundant organisms from phyla without cultivated representatives. Surprisingly, at the time points measured, there was no strong loss of evenness, despite the selective pressure of acetate amendment and change in the terminal electron accepting process. However, community membership was altered significantly. The method allows for sensitive, accurate profiling of the "long tail" of low abundance organisms that exist in many microbial communities, and can resolve population dynamics in response to environmental change.

  13. Phylogenetic congruence of mealybugs and their primary endosymbionts.

    PubMed

    Downie, D A; Gullan, P J

    2005-03-01

    Tight interactions between unrelated organisms such as is seen in plant-insect, host-parasite, or host-symbiont associations may lead to speciation of the smaller partners when their hosts speciate. Totally congruent phylogenies of interacting taxa have not been observed often but a number of studies have provided evidence that various hemipteran insect taxa and their primary bacterial endosymbionts share phylogenetic histories. Like other hemipterans, mealybugs (Pseudococcidae) harbour multiple intracellular bacterial symbionts, which are thought to be strictly vertically inherited, implying codivergence of hosts and symbionts. Here, robust estimates of phylogeny were generated from four fragments of three nuclear genes for mealybugs of the subfamily Pseudococcinae, and a substantial fragment of the 16S-23S rDNA of their P-endosymbionts. Phylogenetic congruence was highly significant, with 75% of nodes on the two trees identical, and significant correlation of branch lengths indicated coincident timing of cladogenesis. It is suggested that the low level of observed incongruence was influenced by uncertainty in phylogenetic estimation, but evolutionary outcomes other than congruence, including host shifts, could not be rejected.

  14. Mesoamerican tree squirrels evolution (Rodentia: Sciuridae): a molecular phylogenetic analysis.

    PubMed

    Villalobos, Federico; Gutierrez-Espeleta, Gustavo

    2014-06-01

    The tribe Sciurini comprehends the genera Sciurus, Syntheosiurus, Microsciurus, Tamiasciurus and Rheinthrosciurus. The phylogenetic relationships within Sciurus have been only partially done, and the relationship between Mesoamerican species remains unsolved. The phylogenetic relationships of the Mesoamerican tree squirrels were examined using molecular data. Sequence data publicly available (12S, 16S, CYTB mitochondrial genes and IRBP nuclear gene) and cytochrome B gene sequences of four previously not sampled Mesoamerican Sciurus species were analyzed under a Bayesian multispecies coalescence model. Phylogenetic analysis of the multilocus data set showed the neotropical tree squirrels as a monophyletic clade. The genus Sciurus was paraphyletic due to the inclusion of Microsciurus species (M. alfari and M. flaviventer). The South American species S. aestuans and S. stramineus showed a sister taxa relationship. Single locus analysis based on the most compact and complete data set (i.e. CYTB gene sequences), supported the monophyly of the South American species and recovered a Mesoamerican clade including S. aureogaster, S. granatensis and S. variegatoides. These results corroborated previous findings based on cladistic analysis of cranial and post-cranial characters. Our data support a close relationship between Mesoamerican Sciurus species and a sister relationship with South American species, and corroborates previous findings in relation to the polyphyly of Microsciurus and Syntheosciurus paraphyly.

  15. IMNGS: A comprehensive open resource of processed 16S rRNA microbial profiles for ecology and diversity studies

    PubMed Central

    Lagkouvardos, Ilias; Joseph, Divya; Kapfhammer, Martin; Giritli, Sabahattin; Horn, Matthias; Haller, Dirk; Clavel, Thomas

    2016-01-01

    The SRA (Sequence Read Archive) serves as primary depository for massive amounts of Next Generation Sequencing data, and currently host over 100,000 16S rRNA gene amplicon-based microbial profiles from various host habitats and environments. This number is increasing rapidly and there is a dire need for approaches to utilize this pool of knowledge. Here we created IMNGS (Integrated Microbial Next Generation Sequencing), an innovative platform that uniformly and systematically screens for and processes all prokaryotic 16S rRNA gene amplicon datasets available in SRA and uses them to build sample-specific sequence databases and OTU-based profiles. Via a web interface, this integrative sequence resource can easily be queried by users. We show examples of how the approach allows testing the ecological importance of specific microorganisms in different hosts or ecosystems, and performing targeted diversity studies for selected taxonomic groups. The platform also offers a complete workflow for de novo analysis of users’ own raw 16S rRNA gene amplicon datasets for the sake of comparison with existing data. IMNGS can be accessed at www.imngs.org. PMID:27659943

  16. IMNGS: A comprehensive open resource of processed 16S rRNA microbial profiles for ecology and diversity studies.

    PubMed

    Lagkouvardos, Ilias; Joseph, Divya; Kapfhammer, Martin; Giritli, Sabahattin; Horn, Matthias; Haller, Dirk; Clavel, Thomas

    2016-09-23

    The SRA (Sequence Read Archive) serves as primary depository for massive amounts of Next Generation Sequencing data, and currently host over 100,000 16S rRNA gene amplicon-based microbial profiles from various host habitats and environments. This number is increasing rapidly and there is a dire need for approaches to utilize this pool of knowledge. Here we created IMNGS (Integrated Microbial Next Generation Sequencing), an innovative platform that uniformly and systematically screens for and processes all prokaryotic 16S rRNA gene amplicon datasets available in SRA and uses them to build sample-specific sequence databases and OTU-based profiles. Via a web interface, this integrative sequence resource can easily be queried by users. We show examples of how the approach allows testing the ecological importance of specific microorganisms in different hosts or ecosystems, and performing targeted diversity studies for selected taxonomic groups. The platform also offers a complete workflow for de novo analysis of users' own raw 16S rRNA gene amplicon datasets for the sake of comparison with existing data. IMNGS can be accessed at www.imngs.org.

  17. Quantifying Microbial Diversity: Morphotypes, 16S rRNA Genes, and Carotenoids of Oxygenic Phototrophs in Microbial Mats

    PubMed Central

    Nübel, Ulrich; Garcia-Pichel, Ferran; Kühl, Michael; Muyzer, Gerard

    1999-01-01

    We quantified the diversity of oxygenic phototrophic microorganisms present in eight hypersaline microbial mats on the basis of three cultivation-independent approaches. Morphological diversity was studied by microscopy. The diversity of carotenoids was examined by extraction from mat samples and high-pressure liquid chromatography analysis. The diversity of 16S rRNA genes from oxygenic phototrophic microorganisms was investigated by extraction of total DNA from mat samples, amplification of 16S rRNA gene segments from cyanobacteria and plastids of eukaryotic algae by phylum-specific PCR, and sequence-dependent separation of amplification products by denaturing-gradient gel electrophoresis. A numerical approach was introduced to correct for crowding the results of chromatographic and electrophoretic analyses. Diversity estimates typically varied up to twofold among mats. The congruence of richness estimates and Shannon-Weaver indices based on numbers and proportional abundances of unique morphotypes, 16S rRNA genes, and carotenoids unveiled the underlying diversity of oxygenic phototrophic microorganisms in the eight mat communities studied. PMID:9925563

  18. Multiplex 16S rRNA‐derived geno‐biochip for detection of 16 bacterial pathogens from contaminated foods

    PubMed Central

    Shin, Hwa Hui; Hwang, Byeong Hee

    2016-01-01

    Abstract Foodborne diseases caused by various pathogenic bacteria occur worldwide. To prevent foodborne diseases and minimize their impacts, it is important to inspect contaminated foods and specifically detect many types of pathogenic bacteria. Several DNA oligonucleotide biochips based on 16S rRNA have been investigated to detect bacteria; however, a mode of detection that can be used to detect diverse pathogenic strains and to examine the safety of food matrixes is still needed. In the present work, a 16S rRNA gene‐derived geno‐biochip detection system was developed after screening DNA oligonucleotide specific capture probes, and it was validated for multiple detection of 16 pathogenic strains that frequently occur with a signature pattern. rRNAs were also used as detection targets directly obtained from cell lysates without any purification and amplification steps in the bacterial cells separated from 8 food matrixes by simple pretreatments. Thus, the developed 16S rRNA‐derived geno‐biochip can be successfully used for the rapid and multiple detection of the 16 pathogenic bacteria frequently isolated from contaminated foods that are important for food safety. PMID:27492058

  19. Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing

    PubMed Central

    Shin, Jongoh; Lee, Sooin; Go, Min-Jeong; Lee, Sang Yup; Kim, Sun Chang; Lee, Chul-Ho; Cho, Byung-Kwan

    2016-01-01

    Demands for faster and more accurate methods to analyze microbial communities from natural and clinical samples have been increasing in the medical and healthcare industry. Recent advances in next-generation sequencing technologies have facilitated the elucidation of the microbial community composition with higher accuracy and greater throughput than was previously achievable; however, the short sequencing reads often limit the microbial composition analysis at the species level due to the high similarity of 16S rRNA amplicon sequences. To overcome this limitation, we used the nanopore sequencing platform to sequence full-length 16S rRNA amplicon libraries prepared from the mouse gut microbiota. A comparison of the nanopore and short-read sequencing data showed that there were no significant differences in major taxonomic units (89%) except one phylotype and three taxonomic units. Moreover, both sequencing data were highly similar at all taxonomic resolutions except the species level. At the species level, nanopore sequencing allowed identification of more species than short-read sequencing, facilitating the accurate classification of the bacterial community composition. Therefore, this method of full-length 16S rRNA amplicon sequencing will be useful for rapid, accurate and efficient detection of microbial diversity in various biological and clinical samples. PMID:27411898

  20. Designation of Streptomycete 16S and 23S rRNA-based target regions for oligonucleotide probes.

    PubMed

    Stackebrandt, E; Witt, D; Kemmerling, C; Kroppenstedt, R; Liesack, W

    1991-05-01

    The 16S and 23S rRNA of various Streptomyces species were partially sequenced and screened for the presence of stretches that could define all members of the genus, groups of species, or individual species. Nucleotide 929 (Streptomyces ambofaciens nomenclature [J.L. Pernodet, M.T. Alegre, F. Boccard, and M. Guerineau, Gene 79:33-46, 1989]) is a nucleotide highly unique to Streptomyces species which, in combination with flanking regions, allowed the designation of a genus-specific probe. Regions 158 through 203 of the 16S rRNA and 1518 through 1645 of the 23S rRNA (helix 54 [Pernodet et al., Gene 79:33-46, 1989]) have a high potential to define species, whereas the degree of variation in regions 982 through 998 and 1102 through 1122 of the 16S rRNA is less pronounced but characteristic for at least certain species. Alone or in combination with each other, these regions may serve as target sites for synthetic oligonucleotide probes and primers to be used in the determination of pure cultures and in the characterization of community structures. The specificity of several probes is demonstrated by dot blot hybridization.

  1. Escherichia coli 16S rRNA 3'-end formation requires a distal transfer RNA sequence at a proper distance.

    PubMed Central

    Srivastava, A K; Schlessinger, D

    1989-01-01

    The 16S rRNA species in bacterial precursor rRNAs is followed by two evolutionarily conserved features: (i) a double-stranded stem formed by complementary sequences adjacent to the 5' and 3' ends of the 16S rRNA; and (ii) a 3'-transfer RNA sequence. To assess the possible role of these features, plasmid constructs with precursor-specific features deleted were tested for their capacity to form mature rRNA. Stem-forming sequences were dispensable for both 5' and 3' terminus formation; whereas an intact spacer tRNA positioned greater than 24 nucleotides downstream of the 16S RNA sequence was required for correct 3'-end maturation. These results suggest that spacer tRNA at an appropriate location helps form a conformation obligate for pre-rRNA processing, perhaps by binding to a nascent binding site in preribosomes. Thus, spacer tRNAs may be an obligate participant in ribosome formation. Images PMID:2684637

  2. Structure of ERA in complex with the 3′ end of 16S rRNA: Implications for ribosome biogenesis

    SciTech Connect

    Tu, Chao; Zhou, Xiaomei; Tropea, Joseph E.; Austin, Brian P.; Waugh, David S.; Court, Donald L.; Ji, Xinhua

    2009-10-09

    ERA, composed of an N-terminal GTPase domain followed by an RNA-binding KH domain, is essential for bacterial cell viability. It binds to 16S rRNA and the 30S ribosomal subunit. However, its RNA-binding site, the functional relationship between the two domains, and its role in ribosome biogenesis remain unclear. We have determined two crystal structures of ERA, a binary complex with GDP and a ternary complex with a GTP-analog and the {sub 1531}AUCACCUCCUUA{sub 1542} sequence at the 3' end of 16S rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein. We show that GTP binding is a prerequisite for RNA recognition by ERA and that RNA recognition stimulates its GTP-hydrolyzing activity. Based on these and other data, we propose a functional cycle of ERA, suggesting that the protein serves as a chaperone for processing and maturation of 16S rRNA and a checkpoint for assembly of the 30S ribosomal subunit. The AUCA sequence is highly conserved among bacteria, archaea, and eukaryotes, whereas the CCUCC, known as the anti-Shine-Dalgarno sequence, is conserved in noneukaryotes only. Therefore, these data suggest a common mechanism for a highly conserved ERA function in all three kingdoms of life by recognizing the AUCA, with a 'twist' for noneukaryotic ERA proteins by also recognizing the CCUCC.

  3. Structure of ERA in Complex with the 3 End of 16s rRNBA Implications for Ribosome Biogenesis

    SciTech Connect

    Tu, C.; Zhou, X; Tropea, J; Austin, B; Waugh, D; Court, D; Ji, X

    2009-01-01

    ERA, composed of an N-terminal GTPase domain followed by an RNA-binding KH domain, is essential for bacterial cell viability. It binds to 16S rRNA and the 30S ribosomal subunit. However, its RNA-binding site, the functional relationship between the two domains, and its role in ribosome biogenesis remain unclear. We have determined two crystal structures of ERA, a binary complex with GDP and a ternary complex with a GTP-analog and the 1531AUCACCUCCUUA1542 sequence at the 3? end of 16S rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein. We show that GTP binding is a prerequisite for RNA recognition by ERA and that RNA recognition stimulates its GTP-hydrolyzing activity. Based on these and other data, we propose a functional cycle of ERA, suggesting that the protein serves as a chaperone for processing and maturation of 16S rRNA and a checkpoint for assembly of the 30S ribosomal subunit. The AUCA sequence is highly conserved among bacteria, archaea, and eukaryotes, whereas the CCUCC, known as the anti-Shine-Dalgarno sequence, is conserved in noneukaryotes only. Therefore, these data suggest a common mechanism for a highly conserved ERA function in all three kingdoms of life by recognizing the AUCA, with a 'twist' for noneukaryotic ERA proteins by also recognizing the CCUCC.

  4. Assignment of fatty acid-beta-oxidizing syntrophic bacteria to Syntrophomonadaceae fam. nov. on the basis of 16S rRNA sequence analyses

    NASA Technical Reports Server (NTRS)

    Zhao, H.; Yang, D.; Woese, C. R.; Bryant, M. P.

    1993-01-01

    After enrichment from Chinese rural anaerobic digestor sludge, anaerobic, sporing and nonsporing, saturated fatty acid-beta-oxidizing syntrophic bacteria were isolated as cocultures with H2- and formate-utilizing Methanospirillum hungatei or Desulfovibrio sp. strain G-11. The syntrophs degraded C4 to C8 saturated fatty acids, including isobutyrate and 2-methylbutyrate. They were adapted to grow on crotonate and were isolated as pure cultures. The crotonate-grown pure cultures alone did not grow on butyrate in either the presence or the absence of some common electron acceptors. However, when they were reconstituted with M. hungatei, growth on butyrate again occurred. In contrast, crotonate-grown Clostridium kluyveri and Clostridium sticklandii, as well as Clostridium sporogenes, failed to grow on butyrate when these organisms were cocultured with M. hungatei. The crotonate-grown pure subcultures of the syntrophs described above were subjected to 16S rRNA sequence analysis. Several previously documented fatty acid-beta-oxidizing syntrophs grown in pure cultures with crotonate were also subjected to comparative sequence analyses. The sequence analyses revealed that the new sporing and nonsporing isolates and other syntrophs that we sequenced, which had either gram-negative or gram-positive cell wall ultrastructure, all belonged to the phylogenetically gram-positive phylum. They were not closely related to any of the previously known subdivisions in the gram-positive phylum with which they were compared, but were closely related to each other, forming a new subdivision in the phylum. We recommend that this group be designated Syntrophomonadaceae fam. nov.; a description is given.

  5. 16S rDNA analysis of archaea indicates dominance of Methanobacterium and high abundance of Methanomassiliicoccaceae in rumen of Nili-Ravi buffalo.

    PubMed

    Paul, S S; Deb, S M; Dey, A; Somvanshi, S P S; Singh, D; Rathore, R; Stiverson, J

    2015-10-01

    The molecular diversity of rumen methanogens was investigated using 16S rDNA gene library prepared from the rumen contents of Nili-Ravi buffaloes. Microbial genomic DNA was isolated from four adult male fistulated buffaloes and PCR conditions were set up using specific primers. Amplified product was cloned into a suitable vector, and the inserts of positive clones were sequenced. A total of 142 clones were examined, and the analysis revealed 46 species level (0.01 distance) operational taxonomic units (OTUs). Twenty six OTUs comprising 89 clones (63% of the total clones) were taxonomically assigned to Methanobacterium genus and the majority of them had highest percent identity with Methanobacterium flexile among cultured methanogens. Five OTUs comprising 27 clones (19% of total clones) were taxonomically assigned to Methanomicrobium genus and these clones showed highest sequence identity with Methanomicrobium mobile. Only two OTUs comprising 6 clones (4% of total clones) were assigned to Methanobrevibacter genus. A total of 17 clones belonging to 10 species level OTUs showed highest percent identity (ranging from 85 to 95%) with Methanomassilicoccus luminyensis and were taxonomically classified as Methanomassiliicocaceae. Out of the 142 rDNA clones, 112 clones, which constitute 79% of the total clones representing 42 OTUs, had less than 98.5% sequence identity with any of the cultured strains of methanogens and represent novel species of methanogens. This study has revealed the largest assortment of hydrogenotrophic methanogen phylotypes ever identified from the rumen of Nili-Ravi buffaloes. The study indicates that Methanobacterium is the most dominant methanogen in the rumen of Nili-Ravi buffalo. This is also the first report on the presence of methanogens phylogenetically close to M. luminyensis, an H2 dependent methylotrophic methanogen, in the rumen of buffaloes at such a high level of abundance.

  6. Phylogenetic metrics of community similarity.

    PubMed

    Ives, Anthony R; Helmus, Matthew R

    2010-11-01

    We derive a new metric of community similarity that takes into account the phylogenetic relatedness among species. This metric, phylogenetic community dissimilarity (PCD), can be partitioned into two components, a nonphylogenetic component that reflects shared species between communities (analogous to Sørensen' s similarity metric) and a phylogenetic component that reflects the evolutionary relationships among nonshared species. Therefore, even if a species is not shared between two communities, it will increase the similarity of the two communities if it is phylogenetically related to species in the other community. We illustrate PCD with data on fish and aquatic macrophyte communities from 59 temperate lakes. Dissimilarity between fish communities associated with environmental differences between lakes often has a phylogenetic component, whereas this is not the case for macrophyte communities. With simulations, we then compare PCD with two other metrics of phylogenetic community similarity, II(ST) and UniFrac. Of the three metrics, PCD was best at identifying environmental drivers of community dissimilarity, showing lower variability and greater statistical power. Thus, PCD is a statistically powerful metric that separates the effects of environmental drivers on compositional versus phylogenetic components of community structure.

  7. Molecular phylogeny of the butterfly tribe Satyrini (Nymphalidae: Satyrinae) with emphasis on the utility of ribosomal mitochondrial genes 16s rDNA and nuclear 28s rDNA.

    PubMed

    Yang, Mingsheng; Zhang, Yalin

    2015-07-09

    The tribe Satyrini is one of the most diverse groups of butterflies, but no robust phylogenetic hypothesis for this group has been achieved. Two rarely used 16s and 28s ribosomal and another seven protein-coding genes were used to reconstruct the phylogeny of the Satyrini, with further aim to evaluate the informativeness of the ribosomal genes. Our maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) analyses consistently recovered three well-supported clades for the eleven sampled subtribes of Satyrini: clade I includes Eritina and Coenonymphina, being sister to the clade II + clade III; clade II contains Parargina, Mycalesina and Lethina, and the other six subtribes constitute clade III. The placements of the taxonomically unstable Davidina Oberthür and geographically restricted Paroeneis Moore in Satyrina are confirmed for the first time based on molecular evidence. The close relationships of Callerebia Butler, Loxerebia Watkins and Argestina Riley are well-supported. We suggest that Rhaphicera Butler belongs to Lethina. The partitioned Bremer support (PBS) values of MP analysis show that the 16s rDNA contributes well to the nodes representing all the taxa from subtribe to species levels, and the 28s rDNA is informative at the subtribe level. Furthermore, our ML analyses show that the ribosomal genes 16s rDNA and 28s rDNA are informative, because most node support values are lower in the ML tree after the removal of them than that in ML tree constructed based on the full nine-gene dataset. This indicates that some other ribosomal genes should be tentatively used through combining with traditionally used protein-coding genes in further analysis on phylogeny of Satyrini, providing that proper representatives are sampled.

  8. Microbial diversity and activity in the Nematostella vectensis holobiont: insights from 16S rRNA gene sequencing, isolate genomes, and a pilot-scale survey of gene expression

    PubMed Central

    Har, Jia Y.; Helbig, Tim; Lim, Ju H.; Fernando, Samodha C.; Reitzel, Adam M.; Penn, Kevin; Thompson, Janelle R.

    2015-01-01

    We have characterized the molecular and genomic diversity of the microbiota of the starlet sea anemone Nematostella vectensis, a cnidarian model for comparative developmental and functional biology and a year-round inhabitant of temperate salt marshes. Molecular phylogenetic analysis of 16S rRNA gene clone libraries revealed four ribotypes associated with N. vectensis at multiple locations and times. These associates include two novel ribotypes within the ε-Proteobacterial order Campylobacterales and the Spirochetes, respectively, each sharing <85% identity with cultivated strains, and two γ-Proteobacterial ribotypes sharing >99% 16S rRNA identity with Endozoicomonas elysicola and Pseudomonas oleovorans, respectively. Species-specific PCR revealed that these populations persisted in N. vectensis asexually propagated under laboratory conditions. cDNA indicated expression of the Campylobacterales and Endozoicomonas 16S rRNA in anemones from Sippewissett Marsh, MA. A collection of bacteria from laboratory raised N. vectensis was dominated by isolates from P. oleovorans and Rhizobium radiobacter. Isolates from field-collected anemones revealed an association with Limnobacter and Stappia isolates. Genomic DNA sequencing was carried out on 10 cultured bacterial isolates representing field- and laboratory-associates, i.e., Limnobacter spp., Stappia spp., P. oleovorans and R. radiobacter. Genomes contained multiple genes identified as virulence (host-association) factors while S. stellulata and L. thiooxidans genomes revealed pathways for mixotrophic sulfur oxidation. A pilot metatranscriptome of laboratory-raised N. vectensis was compared to the isolate genomes and indicated expression of ORFs from L. thiooxidans with predicted functions of motility, nutrient scavenging (Fe and P), polyhydroxyalkanoate synthesis for carbon storage, and selective permeability (porins). We hypothesize that such activities may mediate acclimation and persistence of bacteria in a N

  9. Molecular phylogenetics before sequences: oligonucleotide catalogs as k-mer spectra.

    PubMed

    Ragan, Mark A; Bernard, Guillaume; Chan, Cheong Xin

    2014-01-01

    From 1971 to 1985, Carl Woese and colleagues generated oligonucleotide catalogs of 16S/18S rRNAs from more than 400 organisms. Using these incomplete and imperfect data, Carl and his colleagues developed unprecedented insights into the structure, function, and evolution of the large RNA components of the translational apparatus. They recognized a third domain of life, revealed the phylogenetic backbone of bacteria (and its limitations), delineated taxa, and explored the tempo and mode of microbial evolution. For these discoveries to have stood the test of time, oligonucleotide catalogs must carry significant phylogenetic signal; they thus bear re-examination in view of the current interest in alignment-free phylogenetics based on k-mers. Here we consider the aims, successes, and limitations of this early phase of molecular phylogenetics. We computationally generate oligonucleotide sets (e-catalogs) from 16S/18S rRNA sequences, calculate pairwise distances between them based on D 2 statistics, compute distance trees, and compare their performance against alignment-based and k-mer trees. Although the catalogs themselves were superseded by full-length sequences, this stage in the development of computational molecular biology remains instructive for us today.

  10. Phylogenetic perspective and the search for life on earth and elsewhere

    NASA Technical Reports Server (NTRS)

    Pace, Norman R.

    1989-01-01

    Any search for microbial life on Mars cannot rely upon cultivation of indigenous organisms. Only a minority of even terrestrial organisms that are observed in mixed, naturally-occurring microbial populations can be cultivated in the laboratory. Consequently, methods are being developed for analyzing the phylogenetic affiliations of the constituents of natural microbial populations without the need for their cultivation. This is more than an exercise in taxonomy, for the extent of phylogenetic relatedness between unknown and known organisms is some measure of the extent of their biochemical commonalities. In one approach, total DNA is isolated from natural microbial populations and 16S rRNA genes are shotgun cloned for rapid sequence determinations and phylogenetic analyses. A second approach employs oligodeoxynucleotide hybridization probes that bind to phylogenetic group-specific sequences in 16S rRNA. Since each actively growing cell contains about 104 ribosomes, the binding of the diagnostic probes to single cells can be visualized by radioactivity or fluorescence. The application of these methods and the use of in situ cultivation techniques is illustrated using submarine hydrothermal vent communities. Recommendations are made regarding planning toward future Mars missions.

  11. Development of an Analysis Pipeline Characterizing Multiple Hypervariable Regions of 16S rRNA Using Mock Samples

    PubMed Central

    Barb, Jennifer J.; Oler, Andrew J.; Kim, Hyung-Suk; Chalmers, Natalia; Wallen, Gwenyth R.; Cashion, Ann; Munson, Peter J.; Ames, Nancy J.

    2016-01-01

    Objectives There is much speculation on which hypervariable region provides the highest bacterial specificity in 16S rRNA sequencing. The optimum solution to prevent bias and to obtain a comprehensive view of complex bacterial communities would be to sequence the entire 16S rRNA gene; however, this is not possible with second generation standard library design and short-read next-generation sequencing technology. Methods This paper examines a new process using seven hypervariable or V regions of the 16S rRNA (six amplicons: V2, V3, V4, V6-7, V8, and V9) processed simultaneously on the Ion Torrent Personal Genome Machine (Life Technologies, Grand Island, NY). Four mock samples were amplified using the 16S Ion Metagenomics Kit™ (Life Technologies) and their sequencing data is subjected to a novel analytical pipeline. Results Results are presented at family and genus level. The Kullback-Leibler divergence (DKL), a measure of the departure of the computed from the nominal bacterial distribution in the mock samples, was used to infer which region performed best at the family and genus levels. Three different hypervariable regions, V2, V4, and V6-7, produced the lowest divergence compared to the known mock sample. The V9 region gave the highest (worst) average DKL while the V4 gave the lowest (best) average DKL. In addition to having a high DKL, the V9 region in both the forward and reverse directions performed the worst finding only 17% and 53% of the known family level and 12% and 47% of the genus level bacteria, while results from the forward and reverse V4 region identified all 17 family level bacteria. Conclusions The results of our analysis have shown that our sequencing methods using 6 hypervariable regions of the 16S rRNA and subsequent analysis is valid. This method also allowed for the assessment of how well each of the variable regions might perform simultaneously. Our findings will provide the basis for future work intended to assess microbial abundance at

  12. Paenibacillus larvae 16S-23S rDNA intergenic transcribed spacer (ITS) regions: DNA fingerprinting and characterization.

    PubMed

    Dingman, Douglas W

    2012-07-01

    Paenibacillus larvae is the causative agent of American foulbrood in honey bee (Apis mellifera) larvae. PCR amplification of the 16S-23S ribosomal DNA (rDNA) intergenic transcribed spacer (ITS) regions, and agarose gel electrophoresis of the amplified DNA, was performed using genomic DNA collected from 134 P. larvae strains isolated in Connecticut, six Northern Regional Research Laboratory stock strains, four strains isolated in Argentina, and one strain isolated in Chile. Following electrophoresis of amplified DNA, all isolates exhibited a common migratory profile (i.e., ITS-PCR fingerprint pattern) of six DNA bands. This profile represented a unique ITS-PCR DNA fingerprint that was useful as a fast, simple, and accurate procedure for identification of P. larvae. Digestion of ITS-PCR amplified DNA, using mung bean nuclease prior to electrophoresis, characterized only three of the six electrophoresis bands as homoduplex DNA and indicating three true ITS regions. These three ITS regions, DNA migratory band sizes of 915, 1010, and 1474 bp, signify a minimum of three types of rrn operons within P. larvae. DNA sequence analysis of ITS region DNA, using P. larvae NRRL B-3553, identified the 3' terminal nucleotides of the 16S rRNA gene, 5' terminal nucleotides of the 23S rRNA gene, and the complete DNA sequences of the 5S rRNA, tRNA(ala), and tRNA(ile) genes. Gene organization within the three rrn operon types was 16S-23S, 16S-tRNA(ala)-23S, and l6S-5S-tRNA(ile)-tRNA(ala)-23S and these operons were named rrnA, rrnF, and rrnG, respectively. The 23S rRNA gene was shown by I-CeuI digestion and pulsed-field gel electrophoresis of genomic DNA to be present as seven copies. This was suggestive of seven rrn operon copies within the P. larvae genome. Investigation of the 16S-23S rDNA regions of this bacterium has aided the development of a diagnostic procedure and has helped genomic mapping investigations via characterization of the ITS regions.

  13. Phylogenetics and the Human Microbiome

    PubMed Central

    Matsen, Frederick A.

    2015-01-01

    The human microbiome is the ensemble of genes in the microbes that live inside and on the surface of humans. Because microbial sequencing information is now much easier to come by than phenotypic information, there has been an explosion of sequencing and genetic analysis of microbiome samples. Much of the analytical work for these sequences involves phylogenetics, at least indirectly, but methodology has developed in a somewhat different direction than for other applications of phylogenetics. In this article, I review the field and its methods from the perspective of a phylogeneticist, as well as describing current challenges for phylogenetics coming from this type of work. PMID:25102857

  14. Phylogenetics and the human microbiome.

    PubMed

    Matsen, Frederick A

    2015-01-01

    The human microbiome is the ensemble of genes in the microbes that live inside and on the surface of humans. Because microbial sequencing information is now much easier to come by than phenotypic information, there has been an explosion of sequencing and genetic analysis of microbiome samples. Much of the analytical work for these sequences involves phylogenetics, at least indirectly, but methodology has developed in a somewhat different direction than for other applications of phylogenetics. In this article, I review the field and its methods from the perspective of a phylogeneticist, as well as describing current challenges for phylogenetics coming from this type of work.

  15. [Foundations of the new phylogenetics].

    PubMed

    Pavlinov, I Ia

    2004-01-01

    Evolutionary idea is the core of the modern biology. Due to this, phylogenetics dealing with historical reconstructions in biology takes a priority position among biological disciplines. The second half of the 20th century witnessed growth of a great interest to phylogenetic reconstructions at macrotaxonomic level which replaced microevolutionary studies dominating during the 30s-60s. This meant shift from population thinking to phylogenetic one but it was not revival of the classical phylogenetics; rather, a new approach emerged that was baptized The New Phylogenetics. It arose as a result of merging of three disciplines which were developing independently during 60s-70s, namely cladistics, numerical phyletics, and molecular phylogenetics (now basically genophyletics). Thus, the new phylogenetics could be defined as a branch of evolutionary biology aimed at elaboration of "parsimonious" cladistic hypotheses by means of numerical methods on the basis of mostly molecular data. Classical phylogenetics, as a historical predecessor of the new one, emerged on the basis of the naturphilosophical worldview which included a superorganismal idea of biota. Accordingly to that view, historical development (the phylogeny) was thought an analogy of individual one (the ontogeny) so its most basical features were progressive parallel developments of "parts" (taxa), supplemented with Darwinian concept of monophyly. Two predominating traditions were diverged within classical phylogenetics according to a particular interpretation of relation between these concepts. One of them (Cope, Severtzow) belittled monophyly and paid most attention to progressive parallel developments of morphological traits. Such an attitude turned this kind of phylogenetics to be rather the semogenetics dealing primarily with evolution of structures and not of taxa. Another tradition (Haeckel) considered both monophyletic and parallel origins of taxa jointly: in the middle of 20th century it was split into

  16. Phylogenetic characterization of the epibiotic bacteria associated with the hydrothermal vent polychaete Alvinella pompejana.

    PubMed Central

    Haddad, A; Camacho, F; Durand, P; Cary, S C

    1995-01-01

    Alvinella pompejana is a polychaetous annelid that inhabits active deep-sea hydrothermal vent sites along the East Pacific Rise, where it colonizes the walls of actively venting high-temperature chimneys. An abundant, morphologically diverse epibiotic microflora is associated with the worm's dorsal integument, with a highly integrated filamentous morphotype clearly dominating the microbial biomass. It has been suggested that this bacterial population participates in either the nutrition of the worm or in detoxification of the worm's immediate environment. The primary goal of this study was to phylogenetically characterize selected epibionts through the analysis of 16S rRNA gene sequences. Nucleic acids were extracted from bacteria collected from the dorsal surface of A. pompejana. 16S rRNA genes were amplified with universal bacterial primers by the PCR. These genes were subsequently cloned, and the resulting clone library was screened by restriction fragment length polymorphism analysis to identify distinct clone types. The restriction fragment length polymorphism analysis identified 32 different clone families in the library. Four of these families were clearly dominant, representing more than 65% of the library. Representatives from the four most abundant clone families were chosen for complete 16S rRNA gene sequencing and phylogenetic analysis. These gene sequences were analyzed by a variety of phylogenetic inference methods and found to be related to the newly established epsilon subdivision of the division Proteobacteria. Secondary structural model comparisons and comparisons of established signature base positions in the 16S rRNA confirmed the placement of the Alvinella clones in the epsilon subdivision of the Proteobacteria. PMID:7544093

  17. Phylogenetic Ecology of the Freshwater Actinobacteria acI Lineage▿ †

    PubMed Central

    Newton, Ryan J.; Jones, Stuart E.; Helmus, Matthew R.; McMahon, Katherine D.

    2007-01-01

    The acI lineage of freshwater Actinobacteria is a cosmopolitan and often numerically dominant member of lake bacterial communities. We conducted a survey of acI 16S rRNA genes and 16S-23S rRNA internal transcribed spacer regions from 18 Wisconsin lakes and used standard nonphylogenetic and phylogenetic statistical approaches to investigate the factors that determine acI community composition at the local scale (within lakes) and at the regional scale (across lakes). Phylogenetic reconstruction of 434 acI 16S rRNA genes revealed a well-defined and highly resolved phylogeny. Eleven previously unrecognized monophyletic clades, each with ≥97.9% within-clade 16S rRNA gene sequence identity, were identified. Clade community similarity positively correlated with lake environmental similarity but not with geographic distance, implying that the lakes represent a single biotic region containing environmental filters for communities that have similar compositions. Phylogenetically disparate clades within the acI lineage were most abundant at the regional scale, and local communities were comprised of more closely related clades. Lake pH was a strong predictor of the community composition, but only when lakes with a pH below 6 were included in the data set. In the remaining lakes (pH above 6) biogeographic patterns in the landscape were instead a predictor of the observed acI community structure. The nonrandom distribution of the newly defined acI clades suggests potential ecophysiological differences between the clades, with acI clades AI, BII, and BIII preferring acidic lakes and acI clades AII, AVI, and BI preferring more alkaline lakes. PMID:17827330

  18. Novel green sulfur bacteria phylotypes detected in saline environments: ecophysiological characters versus phylogenetic taxonomy.

    PubMed

    Triadó-Margarit, Xavier; Vila, Xavier; Abella, Charles A

    2010-05-01

    The taxonomic significance of salt tolerance or requirements in green sulfur bacteria has been analyzed with environmental populations and enrichment cultures from several saline systems (inland and coastal water bodies) with different salinities (salt composition and concentration). Novel phylotypes of green sulfur bacteria have been found in hypersaline and brackish environments and 16S rRNA gene sequence analysis affiliated them into phylogenetic groups in which neither halotolerant nor halophilic species have been known to date. Therefore, salt tolerance does not seem to be restricted to members of any specific subgroup but is widespread among all the different phylogenetic branches of the green sulfur bacteria group, and closely-related phylotypes can have dissimilar salt tolerance capacities. Thus the phenotypic characteristics and phylogenetic structure of the green sulfur bacteria present some incongruities. Phenotypic traits should be studied further in order to determine the ecophysiological features of green sulfur bacteria phylotypes.

  19. Touchdown Enzyme Time Release-PCR for Detection and Identification of Chlamydia trachomatis, C. pneumoniae, and C. psittaci Using the 16S and 16S-23S Spacer rRNA Genes

    PubMed Central

    Madico, Guillermo; Quinn, Thomas C.; Boman, Jens; Gaydos, Charlotte A.

    2000-01-01

    Three touchdown enzyme time release (TETR)-PCR assays were used to amplify different DNA sequences in the variable regions of the 16S and 16S-23S spacer rRNA genes specific for Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci as improved tests for sensitive diagnosis and rapid species differentiation. The TETR-PCR protocol used 60 cycles of amplification, which provided improved analytical sensitivity (0.004 to 0.063 inclusion-forming unit of Chlamydia species per PCR). The sensitivity of TETR-PCR with primer set CTR 70-CTR 71 was 96.7%, and the specificity was 99.6%, compared to those of the AMPLICOR PCR for the detection of C. trachomatis in vaginal swab samples. TETR-PCR for C. pneumoniae with primer set CPN 90-CPN 91 was 90% sensitive and 93.3% specific compared with a nested PCR with primer set CP1/2-CPC/D for clinical respiratory samples. TETR-PCR for C. psittaci with primer set CPS 100-CPS 101 showed substantial agreement with cell culturing (κ, 0.78) for animal tissue samples. Primer sets were then combined into a single multiplex TETR-PCR test. The respective 315-, 195-, and 111-bp DNA target products were precisely amplified when DNA from each of the respective Chlamydia species or combinations of them was used. Multiplex chlamydia TETR-PCR correctly identified one strain of each of the 15 serovars of C. trachomatis, 22 isolates of C. pneumoniae, and 20 isolates of C. psittaci. The primer sets were specific for each species. No target products were amplified when DNA from C. pecorum or a variety of other microorganisms was tested for specificity. TETR-PCR with primers selected for specific sequences in the 16S and 16S-23S spacer rRNA genes is a valuable test that could be used either with individual primers or in a multiplex assay for the identification and differentiation of Chlamydia species from culture isolates or for the detection of chlamydiae in clinical samples. PMID:10699002

  20. Phylogenetics: bats united, microbats divided.

    PubMed

    Springer, Mark S

    2013-11-18

    Phylogenetic analyses on four new bat genomes provide convincing support for the placement of bats relative to other placental mammals, suggest that microbats are an unnatural group, and have important implications for understanding the evolution of echolocation.

  1. Phylogenetic lineages in Pseudocercospora

    PubMed Central

    Crous, P.W.; Braun, U.; Hunter, G.C.; Wingfield, M.J.; Verkley, G.J.M.; Shin, H.-D.; Nakashima, C.; Groenewald, J.Z.

    2013-01-01

    Pseudocercospora is a large cosmopolitan genus of plant pathogenic fungi that are commonly associated with leaf and fruit spots as well as blights on a wide range of plant hosts. They occur in arid as well as wet environments and in a wide range of climates including cool temperate, sub-tropical and tropical regions. Pseudocercospora is now treated as a genus in its own right, although formerly recognised as either an anamorphic state of Mycosphaerella or having mycosphaerella-like teleomorphs. The aim of this study was to sequence the partial 28S nuclear ribosomal RNA gene of a selected set of isolates to resolve phylogenetic generic limits within the Pseudocercospora complex. From these data, 14 clades are recognised, six of which cluster in Mycosphaerellaceae. Pseudocercospora s. str. represents a distinct clade, sister to Passalora eucalypti, and a clade representing the genera Scolecostigmina, Trochophora and Pallidocercospora gen. nov., taxa formerly accommodated in the Mycosphaerella heimii complex and characterised by smooth, pale brown conidia, as well as the formation of red crystals in agar media. Other clades in Mycosphaerellaceae include Sonderhenia, Microcyclosporella, and Paracercospora. Pseudocercosporella resides in a large clade along with Phloeospora, Miuraea, Cercospora and Septoria. Additional clades represent Dissoconiaceae, Teratosphaeriaceae, Cladosporiaceae, and the genera Xenostigmina, Strelitziana, Cyphellophora and Thedgonia. The genus Phaeomycocentrospora is introduced to accommodate Mycocentrospora cantuariensis, primarily distinguished from Pseudocercospora based on its hyaline hyphae, broad conidiogenous loci and hila. Host specificity was considered for 146 species of Pseudocercospora occurring on 115 host genera from 33 countries. Partial nucleotide sequence data for three gene loci, ITS, EF-1α, and ACT suggest that the majority of these species are host specific. Species identified on the basis of host, symptomatology and general

  2. Visual exploration of parameter influence on phylogenetic trees.

    PubMed

    Hess, Martin; Bremm, Sebastian; Weissgraeber, Stephanie; Hamacher, Kay; Goesele, Michael; Wiemeyer, Josef; von Landesberger, Tatiana

    2014-01-01

    Evolutionary relationships between organisms are frequently derived as phylogenetic trees inferred from multiple sequence alignments (MSAs). The MSA parameter space is exponentially large, so tens of thousands of potential trees can emerge for each dataset. A proposed visual-analytics approach can reveal the parameters' impact on the trees. Given input trees created with different parameter settings, it hierarchically clusters the trees according to their structural similarity. The most important clusters of similar trees are shown together with their parameters. This view offers interactive parameter exploration and automatic identification of relevant parameters. Biologists applied this approach to real data of 16S ribosomal RNA and protein sequences of ion channels. It revealed which parameters affected the tree structures. This led to a more reliable selection of the best trees.

  3. Phylogenetic comparison of two polycyclic aromatic hydrocarbon-degrading mycobacteria.

    PubMed Central

    Govindaswami, M; Feldhake, D J; Kinkle, B K; Mindell, D P; Loper, J C

    1995-01-01

    Two mycobacterial strains previously isolated from fossil-fuel-contaminated environments and shown to degrade four- and/or five-ring polycyclic aromatic hydrocarbons were further characterized. The two strains, PYR-I and RJGII-135, had similar growth characteristics, colony morphologies, and scotochromogenic pigmentations. DNA amplification fingerprints obtained with total genomic DNA indicated some strain similarities but with several distinctly different bands. Moreover, phylogenetic analysis based upon essentially full-length 16S rRNA gene sequences separates the two strains as distinct species within the fast-growing group of mycobacteria. Although both strains are thermosensitive, strain PYR-I has the bulged U between positions 184 and 193 characteristic of thermotolerant mycobacteria. Both strains are of potential use for reintroduction into and bioremediation of polycyclic aromatic hydrocarbon-contaminated soils. PMID:7574631

  4. Phylogenetic comparison of two polycyclic aromatic hydrogen-degrading mycobacteria

    SciTech Connect

    Govindaswami, M.; Loper, J.C.; Feldhake, D.J.

    1995-09-01

    Two mycobacterial strains previously isolated from fossil-fuel-contaminated environments and shown to degrade four- and/or five-ring polycyclic aromatic hydrocarbons were further characterized. The two strains, PYR-I and RJGII-135, had similar growth characteristics, colony morphologies, and scotochromogenic pigmentations. DNA amplification fringerprints obtained with total genomic DNA indicated some strain similarities but with several distinctly different bands. Moreover, phylogenetic analysis based upon essentially full-length 16S rRNA gene sequences separates the two strains as distinct species within the fast-growing group of mycobacteria. Although both strains are thermosensitive, strain PYR-I has the bulged U between positions 184 and 193 characteristic of thermotolerant mycobacteria. Both strains are of potential use for reintroduction into and bioremediation of polycyclic aromatic hydrocarbon-contaminated soils. 26 refs., 4 figs., 1 tab.

  5. Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system

    PubMed Central

    Jenior, Matthew L.; Koumpouras, Charles C.; Westcott, Sarah L.; Highlander, Sarah K.

    2016-01-01

    Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina’s MiSeq, have allowed researchers to obtain millions of high quality but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3–V5, V1–V3, V1–V5, V1–V6, and V1–V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1–V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina’s MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting. PMID:27069806

  6. Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system.

    PubMed

    Schloss, Patrick D; Jenior, Matthew L; Koumpouras, Charles C; Westcott, Sarah L; Highlander, Sarah K

    2016-01-01

    Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina's MiSeq, have allowed researchers to obtain millions of high quality but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3-V5, V1-V3, V1-V5, V1-V6, and V1-V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1-V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina's MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.

  7. CLUSTOM: A Novel Method for Clustering 16S rRNA Next Generation Sequences by Overlap Minimization

    PubMed Central

    Kim, Byung Kwon; Yu, Dong Su; Hou, Bo Kyeng; Caetano-Anollés, Gustavo; Hong, Soon Gyu; Kim, Kyung Mo

    2013-01-01

    The recent nucleic acid sequencing revolution driven by shotgun and high-throughput technologies has led to a rapid increase in the number of sequences for microbial communities. The availability of 16S ribosomal RNA (rRNA) gene sequences from a multitude of natural environments now offers a unique opportunity to study microbial diversity and community structure. The large volume of sequencing data however makes it time consuming to assign individual sequences to phylotypes by searching them against public databases. Since ribosomal sequences have diverged across prokaryotic species, they can be grouped into clusters that represent operational taxonomic units. However, available clustering programs suffer from overlap of sequence spaces in adjacent clusters. In natural environments, gene sequences are homogenous within species but divergent between species. This evolutionary constraint results in an uneven distribution of genetic distances of genes in sequence space. To cluster 16S rRNA sequences more accurately, it is therefore essential to select core sequences that are located at the centers of the distributions represented by the genetic distance of sequences in taxonomic units. Based on this idea, we here describe a novel sequence clustering algorithm named CLUSTOM that minimizes the overlaps between adjacent clusters. The performance of this algorithm was evaluated in a comparative exercise with existing programs, using the reference sequences of the SILVA database as well as published pyrosequencing datasets. The test revealed that our algorithm achieves higher accuracy than ESPRIT-Tree and mothur, few of the best clustering algorithms. Results indicate that the concept of an uneven distribution of sequence distances can effectively and successfully cluster 16S rRNA gene sequences. The algorithm of CLUSTOM has been implemented both as a web and as a standalone command line application, which are available at http://clustom.kribb.re.kr. PMID:23650520

  8. Flow Cytometric and 16S Sequencing Methodologies for Monitoring the Physiological Status of the Microbiome in Powdered Infant Formula Production

    PubMed Central

    Anvarian, Amir H. P.; Cao, Yu; Srikumar, Shabarinath; Fanning, Séamus; Jordan, Kieran

    2016-01-01

    The aim of this study was to develop appropriate protocols for flow cytometric (FCM) and 16S rDNA sequencing investigation of the microbiome in a powdered infant formula (PIF) production facility. Twenty swabs were collected from each of the three care zones of a PIF production facility and used for preparing composite samples. For FCM studies, the swabs were washed in 200 mL phosphate buffer saline (PBS). The cells were harvested by three-step centrifugation followed by a single stage filtration. Cells were dispersed in fresh PBS and analyzed with a flow cytometer for membrane integrity, metabolic activity, respiratory activity and Gram characteristics of the microbiome using various fluorophores. The samples were also plated on agar plates to determine the number of culturable cells. For 16S rDNA sequencing studies, the cells were harvested by centrifugation only. Genomic DNA was extracted using a chloroform-based method and used for 16S rDNA sequencing studies. Compared to the dry low and high care zones, the wet medium care zone contained a greater number of viable, culturable, and metabolically active cells. Viable but non-culturable cells were also detected in dry-care zones. In total, 243 genera were detected in the facility of which 42 were found in all three care zones. The greatest diversity in the microbiome was observed in low care. The genera present in low, medium and high care were mostly associated with soil, water, and humans, respectively. The most prevalent genera in low, medium and high care were Pseudomonas, Acinetobacter, and Streptococcus, respectively. The integration of FCM and metagenomic data provided further information on the density of different species in the facility. PMID:27446009

  9. High-density universal 16S rRNA microarray analysis revealsbroader diversity than typical clone library when sampling theenvironment

    SciTech Connect

    DeSantis, Todd Z.; Brodie, Eoin L.; Moberg, Jordan P.; Zubieta,Ingrid X.; Piceno, Yvette M.; Andersen, Gary L.

    2006-06-15

    Molecular approaches aimed at detection of a broad-range ofprokaryotes in the environment routinely rely upon classifyingheterogeneous 16S rRNA genes amplified by PCR using primers with broadspecificity. The general method of sampling and categorizing DNA has beento clone then sequence the PCR products. However, the number of clonesrequired to adequately catalogue the majority of taxa in a sample isunwieldy. Alternatively, hybridizing target sequences to a universal 16SrRNA gene microarray may provide a more rapid and comprehensive view ofprokaryotic community composition. This study investigated the breadthand accuracy of a microarray in detecting diverse 16S rRNA gene sequencetypes compared to clone-and-sequencing using three environmental samples:urban aerosol, subsurface soil and subsurface water. PCR productsgenerated from universal 16S rRNA gene-targeted primers were classifiedusing either the clone-and-sequence method or by hybridization to a novelhigh-density microarray of 297,851 probes complementary to 842prokaryotic sub-families. The three clone libraries comprised 1,391high-quality sequences. Approximately 8 percent of the clones could notbe placed into a known sub-family and were considered novel. Themicroarray results confirmed the majority of clone-detected sub-familiesand additionally demonstrated greater amplicon diversity extending intophyla not observed by the cloning method. Sequences matching OTUs withinthe phyla Nitrospira, Planctomycetes, and TM7, which were uniquelydetected by the array, were verified with specific primers and subsequentamplicon sequencing. Sub-family richness detected by the arraycorresponded well with non-parametric richness predictions extrapolatedfrom clone libraries except in the water community where clone-basedrichness predictions were greatly exceeded. It was concluded thatalthough the microarray is unreliable inidentifying novel prokaryotictaxa, it reveals greater diversity in environmental samples thansequencing a

  10. Using DGGE and 16S rRNA Gene Sequence Analysis to Evaluate Changes in Oral Bacterial Composition

    PubMed Central

    CHEN, Zhou; TRIVEDI, Harsh M.; CHHUN, Nok; BARNES, Virginia M.; SAXENA, Deepak; XU, Tao; LI, Yihong

    2015-01-01

    Objective To investigate whether a standard dental prophylaxis followed by tooth brushing with an antibacterial dentifrice will affect the oral bacterial community, as determined by denaturing gradient gel electrophoresis (DGGE) combined with 16S rRNA gene sequence analysis. Methods Twenty-four healthy adults were instructed to brush their teeth using commercial dentifrice for 1 week during a washout period. An initial set of pooled supragingival plaque samples was collected from each participant at baseline (0 h) before prophylaxis treatment. The subjects were given a clinical examination and dental prophylaxis and asked to brush for 1 min with a dentifrice containing 0.3% triclosan/2.0% PVM/MA copolymer/0.243% sodium fluoride (Colgate Total). On the following day, a second set of pooled supragingival plaque samples (24 h) was collected. Total bacterial genomic DNA was isolated from the samples. Differences in the microbial composition before and after the prophylactic procedure and tooth brushing were assessed by comparing the DGGE profiles of PCR-amplified and 16S rRNA gene segments sequence analysis. Results Two distinct clusters of DGGE profiles were found, suggesting that a shift in the microbial composition had occurred 24 h after the prophylaxis and brushing. A detailed sequencing analysis of 16S rRNA gene segments further identified six phyla and 29 genera, including known and unknown bacterial species. Importantly, an increase in bacterial diversity was observed after 24 h, including members of the Streptococcaceae family, Prevotella, Corynebacterium, TM7 and other commensal bacteria. Conclusion The results suggest that the use of a standard prophylaxis followed by the use of the dentifrice containing 0.3% triclosan/2.0% PVM/MA copolymer/0.243% sodium fluoride may promote a healthier composition within the oral bacterial community. PMID:22319750

  11. Assessment of Three Mitochondrial Genes (16S, Cytb, CO1) for Identifying Species in the Praomyini Tribe (Rodentia: Muridae)

    PubMed Central

    Nicolas, Violaine; Schaeffer, Brigitte; Missoup, Alain Didier; Kennis, Jan; Colyn, Marc; Denys, Christiane; Tatard, Caroline; Cruaud, Corinne; Laredo, Catherine

    2012-01-01

    The Praomyini tribe is one of the most diverse and abundant groups of Old World rodents. Several species are known to be involved in crop damage and in the epidemiology of several human and cattle diseases. Due to the existence of sibling species their identification is often problematic. Thus an easy, fast and accurate species identification tool is needed for non-systematicians to correctly identify Praomyini species. In this study we compare the usefulness of three genes (16S, Cytb, CO1) for identifying species of this tribe. A total of 426 specimens representing 40 species (sampled across their geographical range) were sequenced for the three genes. Nearly all of the species included in our study are monophyletic in the neighbour joining trees. The degree of intra-specific variability tends to be lower than the divergence between species, but no barcoding gap is detected. The success rate of the statistical methods of species identification is excellent (up to 99% or 100% for statistical supervised classification methods as the k-Nearest Neighbour or Random Forest). The 16S gene is 2.5 less variable than the Cytb and CO1 genes. As a result its discriminatory power is smaller. To sum up, our results suggest that using DNA markers for identifying species in the Praomyini tribe is a largely valid approach, and that the CO1 and Cytb genes are better DNA markers than the 16S gene. Our results confirm the usefulness of statistical methods such as the Random Forest and the 1-NN methods to assign a sequence to a species, even when the number of species is relatively large. Based on our NJ trees and the distribution of all intraspecific and interspecific pairwise nucleotide distances, we highlight the presence of several potentially new species within the Praomyini tribe that should be subject to corroboration assessments. PMID:22574186

  12. Multicenter quality assessment of 16S ribosomal DNA-sequencing for microbiome analyses reveals high inter-center variability.

    PubMed

    Hiergeist, Andreas; Reischl, Udo; Gessner, Andrè

    2016-08-01

    The composition of human as well as animal microbiota has increasingly gained in interest since metabolites and structural components of endogenous microorganisms fundamentally influence all aspects of host physiology. Since many of the bacteria are still unculturable, molecular techniques such as high-throughput sequencing have dramatically increased our knowledge of microbial communities. The majority of microbiome studies published thus far are based on bacterial 16S ribosomal RNA (rRNA) gene sequencing, so that they can, at least in principle, be compared to determine the role of the microbiome composition for host metabolism and physiology, developmental processes, as well as different diseases. However, differences in DNA preparation and purification, 16S rDNA PCR amplification, sequencing procedures and platforms, as well as bioinformatic analysis and quality control measures may strongly affect the microbiome composition results obtained in different laboratories. To systematically evaluate the comparability of results and identify the most influential methodological factors affecting these differences, identical human stool sample replicates spiked with quantified marker bacteria, and their subsequent DNA sequences were analyzed by nine different centers in an external quality assessment (EQA). While high intra-center reproducibility was observed in repetitive tests, significant inter-center differences of reported microbiota composition were obtained. All steps of the complex analysis workflow significantly influenced microbiome profiles, but the magnitude of variation caused by PCR primers for 16S rDNA amplification was clearly the largest. In order to advance microbiome research to a more standardized and routine medical diagnostic procedure, it is essential to establish uniform standard operating procedures throughout laboratories and to initiate regular proficiency testing.

  13. Changes in the Composition of Drinking Water Bacterial Clone Libraries Introduced by Using Two Different 16S rRna Gene PCR Primers

    EPA Science Inventory

    Sequence analysis of 16S rRNA gene clone libraries is a popular tool used to describe the composition of natural microbial communities. Commonly, clone libraries are developed by direct cloning of 16S rRNA gene PCR products. Different primers are often employed in the initial amp...

  14. Changes in the Composition of Drinking Water Bacterial Clone Libraries Introduced by Using Two Different 16S rRNA Gene PCR Primers

    EPA Science Inventory

    Sequence analysis of 16S rRNA gene clone libraries is a popular tool used to describe the composition of natural microbial communities. Commonly, clone libraries are developed by direct cloning of 16S rRNA gene PCR products. Different primers are often employed in the initial amp...

  15. Identification of bacterial pathogens from formalin-fixed, paraffin-embedded tissues by using 16S sequencing: retrospective correlation of results to clinicians' responses.

    PubMed

    Racsa, Lori D; DeLeon-Carnes, Marlene; Hiskey, Matthew; Guarner, Jeannette

    2017-01-01

    16S sequencing on formalin-fixed, paraffin-embedded (FFPE) material has been used to identify bacteria when culture-based phenotyping techniques have not worked. The objective of this study was to determine how frequently 16S sequencing used in FFPE material was helpful to clinicians in the diagnosis and treatment of infectious diseases. Requests for testing occurred upon consultation between an infectious disease pathologist and a surgical pathologist or an infectious disease physician. A selected paraffin block from each case was referred for 16S sequencing. Retrospectively, we correlated clinical history and management decisions on 27 cases that were tested by paneubacterial 16S sequencing. Samples included 24 surgical specimens, 1 autopsy, and 2 cytology blocks. Seventeen (63%) of the 27 cases had a positive 16S sequencing. Acute inflammation was present in 10 of these cases, and organisms were observed using special stains in 3. In 11 (65%) of the 17 cases, clinicians considered the organism identified by 16S sequencing to be the cause or possible cause of the infectious process. Organisms included common (Citrobacter) and fastidious bacteria (Haemophilus, Fusobacterium). In 3 cases, clinicians changed antibiotic treatment based on the bacteria identified, whereas in 8 (including 2 where no organism was found), clinicians continued the antibiotic treatment. The use of 16S sequencing on FFPE identified specific bacteria even when organisms were not observed histopathologically. 16S results had an impact in infectious disease management decisions.

  16. 16S Ribosomal DNA Characterization of Nitrogen-Fixing Bacteria Isolated from Banana (Musa spp.) and Pineapple (Ananas comosus (L.) Merril)

    PubMed Central

    Magalhães Cruz, Leonardo; Maltempi de Souza, Emanuel; Weber, Olmar Baler; Baldani, José Ivo; Döbereiner, Johanna; de Oliveira Pedrosa, Fábio

    2001-01-01

    Nitrogen-fixing bacteria isolated from banana (Musa spp.) and pineapple (Ananas comosus (L.) Merril) were characterized by amplified 16S ribosomal DNA restriction analysis and 16S rRNA sequence analysis. Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, Burkholderia brasilensis, and Burkholderia tropicalis were identified. Eight other types were placed in close proximity to these genera and other alpha and beta Proteobacteria. PMID:11319127

  17. RmtC introduces G1405 methylation in 16S rRNA and confers high-level aminoglycoside resistance on Gram-positive microorganisms.

    PubMed

    Wachino, Jun-Ichi; Shibayama, Keigo; Kimura, Kouji; Yamane, Kunikazu; Suzuki, Satowa; Arakawa, Yoshichika

    2010-10-01

    Seven plasmid-mediated 16S rRNA methyltransferases (MTases), RmtA, RmtB, RmtC, RmtD, RmtE, ArmA, and NpmA, conferring aminoglycoside resistance have so far been found in Gram-negative pathogenic microorganisms. In the present study, by performing an RNase protection assay, primer extension, and HPLC, we confirmed that RmtC indeed methylates at the N7 position of nucleotide G1405 in 16S rRNA as found in ArmA and RmtB. RmtC has an MTase activity specific for the bacterial 30S ribosomal subunit consisting of 16S rRNA and several ribosomal proteins, but not for the naked 16S rRNA, as seen in ArmA, RmtB, and NpmA. All seven 16S rRNA MTases have been found exclusively in Gram-negative bacilli to date, and no plasmid-mediated 16S rRNA MTase has been reported in Gram-positive pathogenic microorganisms. Thus, we checked whether or not the RmtC could function in Gram-positive bacilli, and found that RmtC could indeed confer high-level resistance to gentamicin and kanamycin in Bacillus subtilis and Staphylococcus aureus. 16S rRNA MTases seemed to be functional to some extent in any bacterial species, regardless of the provenance of the 16S rRNA MTase gene responsible for aminoglycoside resistance.

  18. Development of a Multiplex PCR Method for Detection of the Genes Encoding 16S rRNA, Coagulase, Methicillin Resistance and Enterotoxins in Staphylococcus aureus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A multiplex PCR method was developed for simultaneous detection of the genes encoding methicillin resistance (mecA), staphylococcal enterotoxins A, B and C (sea, seb and sec), coagulase (coa) and 16S rRNA. The primers for amplification of the 16S rRNA gene were specific for Staphylococcus spp., and ...

  19. 16S ribosomal DNA characterization of nitrogen-fixing bacteria isolated from banana (Musa spp.) and pineapple (Ananas comosus (L.) Merril).

    PubMed

    Magalhães Cruz, L; de Souza, E M; Weber, O B; Baldani, J I; Döbereiner, J; Pedrosa, F de O

    2001-05-01

    Nitrogen-fixing bacteria isolated from banana (Musa spp.) and pineapple (Ananas comosus (L.) Merril) were characterized by amplified 16S ribosomal DNA restriction analysis and 16S rRNA sequence analysis. Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, Burkholderia brasilensis, and Burkholderia tropicalis were identified. Eight other types were placed in close proximity to these genera and other alpha and beta Proteobacteria.

  20. Recovery of partial 16S rDNA sequences suggests the presence of Crenarchaeota in the human digestive ecosystem.

    PubMed

    Rieu-Lesme, Françoise; Delbès, Céline; Sollelis, Lauriane

    2005-11-01

    Human feces collected from 10 healthy teenagers was analyzed for the presence of Crenarchaeota. After a first polymerase chain reaction (PCR) with Archaea-specific primers, a nested real-time PCR was performed using Crenarchaeota-specific primers. Real-time Crenarchaeotal PCR products detected from four subjects were cloned and the sequencing revealed that most of the partial 16S rRNA gene sequences were highly similar (> or = 97% homology) to sequences affiliated to the Sulfolobus group of the Crenarchaeota phylum. Our findings suggest for the first time that Crenarchaeota might be present in the microbiota of the human digestive ecosystem in which this phylum has never been found yet.

  1. 16S rRNA gene sequences from bacteria associated with adult Anopheles darlingi (Diptera: Culicidae) mosquitoes.

    PubMed

    Terenius, Olle; de Oliveira, Caroline Dantas; Pinheiro, Waleria Dasso; Tadei, Wanderli Pedro; James, Anthony Amade; Marinotti, Osvaldo

    2008-01-01

    The microbial flora associated with Anopheles darlingi Root (Diptera: Culicidae), a major Neotropical malaria vector, was investigated for the development of a paratransgenesis-based approach to control malaria transmission in Brazil. Female mosquitoes were collected using human land catches and captured insects provided a bloodmeal. The controlled blood feeding resulted in increased detection of mosquito bacterial population because it was possible to retrieve bacterial DNA from all blood-fed mosquitoes. The 16S sequences of bacteria recovered, include some closely related to those found in other vector mosquitoes, including Aeromonas, Pantoea and Pseudomonas species.

  2. [Phylogenetic analysis of bacteria of extreme ecosystems].

    PubMed

    Romanovskaia, V A; Parfenova, V V; Bel'kova, N L; Sukhanova, E V; Gladka, G V; Tashireva, A A

    2014-01-01

    Phylogenetic analysis of aerobic chemoorganotrophic bacteria of the two extreme regions (Dead Sea and West Antarctic) was performed on the basis of the nucleotide sequences of the 16S rRNA gene. Thermotolerant and halotolerant spore-forming bacteria 7t1 and 7t3 of terrestrial ecosystems Dead Sea identified as Bacillus licheniformis and B. subtilis subsp. subtilis, respectively. Taking into account remote location of thermotolerant strain 6t1 from closely related strains in the cluster Staphylococcus, 6t1 strain can be regarded as Staphylococcus sp. In terrestrial ecosystems, Galindez Island (Antarctic) detected taxonomically diverse psychrotolerant bacteria. From ornithogenic soil were isolated Micrococcus luteus O-1 and Microbacterium trichothecenolyticum O-3. Strains 4r5, 5r5 and 40r5, isolated from grass and lichens, can be referred to the genus Frondihabitans. These strains are taxonomically and ecologically isolated and on the tree diagram form the joint cluster with three isolates Frondihabitans sp., isolated from the lichen Austrian Alps, and psychrotolerant associated with plants F. cladoniiphilus CafT13(T). Isolates from black lichen in the different stationary observation points on the south side of a vertical cliff identified as: Rhodococcus fascians 181n3, Sporosarcina aquimarina O-7, Staphylococcus sp. 0-10. From orange biofilm of fouling on top of the vertical cliff isolated Arthrobacter sp. 28r5g1, from the moss-- Serratia sp. 6r1g. According to the results, Frondihabitans strains most frequently encountered among chemoorganotrophic aerobic bacteria in the Antarctic phytocenoses.

  3. Phylogenetic and metabolic diversity of bacteria associated with cystic fibrosis.

    PubMed

    Guss, Adam M; Roeselers, Guus; Newton, Irene L G; Young, C Robert; Klepac-Ceraj, Vanja; Lory, Stephen; Cavanaugh, Colleen M

    2011-01-01

    In patients afflicted with cystic fibrosis (CF), morbidity and mortality are primarily associated with the adverse consequences of chronic microbial bronchial infections, which are thought to be caused by a few opportunistic pathogens. However, recent evidence suggests the presence of other microorganisms, which may significantly affect the course and outcome of the infection. Using a combination of 16S rRNA gene clone libraries, bacterial culturing and pyrosequencing of barcoded 16S rRNA amplicons, the microbial communities present in CF patient sputum samples were examined. In addition to previously recognized CF pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus, >60 phylogenetically diverse bacterial genera that are not typically associated with CF pathogenesis were also detected. A surprisingly large number of fermenting facultative and obligate anaerobes from multiple bacterial phyla was present in each sample. Many of the bacteria and sequences found were normal residents of the oropharyngeal microflora and with many containing opportunistic pathogens. Our data suggest that these undersampled organisms within the CF lung are part of a much more complex microbial ecosystem than is normally presumed. Characterization of these communities is the first step in elucidating potential roles of diverse bacteria in disease progression and to ultimately facilitate advances in CF therapy.

  4. Phylogenetic diversity and position of the genus Campylobacter

    NASA Technical Reports Server (NTRS)

    Lau, P. P.; DeBrunner-Vossbrinck, B.; Dunn, B.; Miotto, K.; MacDonnell, M. T.; Rollins, D. M.; Pillidge, C. J.; Hespell, R. B.; Colwell, R. R.; Sogin, M. L.; Fox, G. E.

    1987-01-01

    RNA sequence analysis has been used to examine the phylogenetic position and structure of the genus Campylobacter. A complete 5S rRNA sequence was determined for two strains of Campylobacter jejuni and extensive partial sequences of the 16S rRNA were obtained for several strains of C. jejuni and Wolinella succinogenes. In addition limited partial sequence data were obtained from the 16S rRNAs of isolates of C. coli, C. laridis, C. fetus, C. fecalis, and C. pyloridis. It was found that W. succinogenes is specifically related to, but not included, in the genus Campylobacter as presently constituted. Within the genus significant diversity was noted. C. jejuni, C. coli and C. laridis are very closely related but the other species are distinctly different from one another. C. pyloridis is without question the most divergent of the Campylobacter isolates examined here and is sufficiently distinct to warrant inclusion in a separate genus. In terms of overall position in bacterial phylogeny, the Campylobacter/Wolinella cluster represents a deep branching most probably located within an expanded version of the Division containing the purple photosynthetic bacteria and their relatives. The Campylobacter/Wolinella cluster is not specifically includable in either the alpha, beta or gamma subdivisions of the purple bacteria.

  5. Phylogenetic relationships among subsurface microorganisms. Project technical progress report

    SciTech Connect

    Nierzwicki-Bauer, S.A.

    1993-08-01

    The development of group-specific, 16S ribosomal RNA-targeted oligonucleotide hybridization probes for the rapid detection of specific types of subsurface microorganisms is described. Because portions of the 16S RRNA molecule are unique to particular organisms or groups, these unique sequences can serve as targets for hybridization probes with varied specificity. Target sequences for selected microbial groups have been identified by analysis of the available RRNA sequence data for subsurface microbes. Hybridization probes for these target sequences were produced and their effectiveness and specificity tested with RNA cell blot and in situ hybridizations. Selected probes were used to study phylogenetic relationships among subsurface microbes and to classify these organisms into the specific groups that the probes are designed to detect. To date, this work has been performed on the P24 and C10 borehole isolates from the Savannah River Site. The probes will also be used, with in situ hybridizations, to detect and monitor selected microbial groups in freshly collected subsurface samples and laboratory microcosms in collaboration with other investigators. In situ hybridizations permit detection of selected microbial types without the necessity to isolate and culture them in the laboratory.

  6. Phylogenetic analysis and antimicrobial activities of Streptomyces isolates from mangrove sediment.

    PubMed

    Satheeja, Santhi V; Jebakumar, Solomon R D

    2011-02-01

    The phylogeny of members of Streptomyces bacteria isolated from mangrove sediments in the Manakudi estuary near the Arabian Sea, India, was analyzed in the present study. Among the 35 different isolates, five organisms, JS-9, JS-11, JS-12, JS-13 and JS-20, exhibited potent antimicrobial effects against methicillin-resistant Staphylococcus aureus (clinical isolate) and methicillin-susceptible S. aureus MTCC 3160 and Salmonella typhi MTCC 733; all other isolates displayed intermediate antimicrobial effects. RFLP analysis of HaeIII and BstUI double-digested 16S rRNA gene fragments of the isolates were distinguished into 20 distinct RFLP types, with the genetic similarity coefficient varying from 0.57 to 0.97. On average, 17 RFLP markers were observed from approximately 50 to 350 bp size and all the RFLP types showed significant genetic polymorphism by clustering into three major clusters. Phylogenetic analysis showed that the 20-member Streptomyces isolates were divided into three major clusters and they shared 97.2-99.8% sequence identity to the 16S rRNA gene sequences of the Streptomyces taxons of marine origin. The distribution of the isolates revealed that the distinct Streptomyces groups were clustered in the phylogenetic tree and there was a good correlation between the diversity of the antimicrobial phenotype and that of the 16S rRNA gene.

  7. Distribution and phylogenetic diversity of the subsurface microbial community in a Japanese epithermal gold mine.

    PubMed

    Inagaki, Fumio; Takai, Ken; Hirayama, Hisako; Yamato, Yu; Nealson, Kenneth H; Horikoshi, Koki

    2003-08-01

    Distribution and phylogenetic diversity of microbial communities in hot, deep underground environments in the Hishikari epithermal gold mine, southern part of Kyushu, Japan, were evaluated using molecular phylogenetic analyses. Samples included drilled cores such as andesitic volcanic rock (0.95-1.78 Ma) and the oceanic sedimentary basement rock of Shimanto-Supergroup (100 Ma), as well as geothermal hot aquifer waters directly collected from two different sites: AW-site (71.5 degrees C, pH 6.19) and XW-site (85.0 degrees C, pH 6.80) at a depth of 350 mbls (meters below land surface). Based on PCR-amplified 16S rRNA gene clone analysis, the microbial communities in the drilled cores and the hot aquifer water from the XW-site consisted largely of the 16S rRNA gene sequences, closely related to the sequences often found in marine environments, while the aquifer water from the AW-site contained 16S rRNA gene sequences representing members of Aquificales, thermophilic methanotrophs within the gamma-subdivision of the Proteobacteria and uncultivated strains within the beta-subdivision of Proteobacteria. The cultivable microbial community detected by enrichment cultivation analysis largely matched that detected by the culture-independent molecular analysis.

  8. RAP, the sole octotricopeptide repeat protein in Arabidopsis, is required for chloroplast 16S rRNA maturation.

    PubMed

    Kleinknecht, Laura; Wang, Fei; Stübe, Roland; Philippar, Katrin; Nickelsen, Jörg; Bohne, Alexandra-Viola

    2014-02-01

    The biogenesis and activity of chloroplasts in both vascular plants and algae depends on an intracellular network of nucleus-encoded, trans-acting factors that control almost all aspects of organellar gene expression. Most of these regulatory factors belong to the helical repeat protein superfamily, which includes tetratricopeptide repeat, pentatricopeptide repeat, and the recently identified octotricopeptide repeat (OPR) proteins. Whereas green algae express many different OPR proteins, only a single orthologous OPR protein is encoded in the genomes of most land plants. Here, we report the characterization of the only OPR protein in Arabidopsis thaliana, RAP, which has previously been implicated in plant pathogen defense. Loss of RAP led to a severe defect in processing of chloroplast 16S rRNA resulting in impaired chloroplast translation and photosynthesis. In vitro RNA binding and RNase protection assays revealed that RAP has an intrinsic and specific RNA binding capacity, and the RAP binding site was mapped to the 5' region of the 16S rRNA precursor. Nucleoid localization of RAP was shown by transient green fluorescent protein import assays, implicating the nucleoid as the site of chloroplast rRNA processing. Taken together, our data indicate that the single OPR protein in Arabidopsis is important for a basic process of chloroplast biogenesis.

  9. Arrested development of the myxozoan parasite, Myxobolus cerebralis, in certain populations of mitochondrial 16S lineage III Tubifex tubifex

    USGS Publications Warehouse

    Baxa, D.V.; Kelley, G.O.; Mukkatira, K.S.; Beauchamp, K.A.; Rasmussen, C.; Hedrick, R.P.

    2008-01-01

    Laboratory populations of Tubifex tubifex from mitochondrial (mt)16S ribosomal DNA (rDNA) lineage III were generated from single cocoons of adult worms releasing the triactinomyxon stages (TAMs) of the myxozoan parasite, Myxobolus cerebralis. Subsequent worm populations from these cocoons, referred to as clonal lines, were tested for susceptibility to infection with the myxospore stages of M. cerebralis. Development and release of TAMs occurred in five clonal lines, while four clonal lines showed immature parasitic forms that were not expelled from the worm (non-TAM producers). Oligochaetes from TAM- and non-TAM-producing clonal lines were confirmed as lineage III based on mt16S rDNA and internal transcribed spacer region 1 (ITS1) sequences, but these genes did not differentiate these phenotypes. In contrast, random amplified polymorphic DNA analyses of genomic DNA demonstrated unique banding patterns that distinguished the phenotypes. Cohabitation of parasite-exposed TAM- and non-TAM-producing phenotypes showed an overall decrease in expected TAM production compared to the same exposure dose of the TAM-producing phenotype without cohabitation. These studies suggest that differences in susceptibility to parasite infection can occur in genetically similar T. tubifex populations, and their coexistence may affect overall M. cerebralis production, a factor that may influence the severity of whirling disease in wild trout populations. ?? 2007 Springer-Verlag.

  10. Use of acetate for enrichment of electrochemically active microorganisms and their 16S rDNA analyses.

    PubMed

    Lee, Jiyoung; Phung, Nguyet Thu; Chang, In Seop; Kim, Byung Hong; Sung, Ha Chin

    2003-06-27

    A fuel cell-type electrochemical device has been used to enrich microbes oxidizing acetate with concomitant electricity generation without using an electron mediator from activated sludge. The device generated a stable current of around 5 mA with complete oxidation of 5 mM acetate at the hydraulic retention time of 2.5 h after 4 weeks of enrichment. Over 70% of electrons available from acetate oxidation was recovered as current. Carbon monoxide or hydrogen did not influence acetate oxidation or current generation from the microbial fuel cell (MFC). Denaturing gradient gel electrophoresis showed that DNA extracted from the acetate-enriched MFC had different 16S rDNA patterns from those of sludge or glucose+glutamate-enriched MFCs. Nearly complete 16S rDNA sequence analyses showed that diverse bacteria were enriched in the MFC fed with acetate. Electron microscopic observations showed biofilm developed on the electrode, but not microbial clumps observed in MFCs fed with complex fuel such as glucose and wastewater from a corn-processing factory.

  11. Relative Prevalence and Antimicrobial Susceptibility of Clinical Isolates of Elizabethkingia Species Based on 16S rRNA Gene Sequencing.

    PubMed

    Han, Mi-Soon; Kim, Hyunsoo; Lee, Yangsoon; Kim, Myungsook; Ku, Nam Su; Choi, Jun Yong; Yong, Dongeun; Jeong, Seok Hoon; Lee, Kyungwon; Chong, Yunsop

    2017-01-01

    Some of the previously reported clinical isolates of Elizabethkingia meningoseptica may be later named species of Elizabethkingia We determined the accuracy of species identification (with two matrix-assisted laser desorption ionization-time of flight mass spectrometry [MALDI-TOF MS] systems and the Vitek 2 GN card), relative prevalence of three Elizabethkingia spp. in clinical specimens, and antimicrobial susceptibility of the species identified by 16S rRNA gene sequencing. Specimens for culture were collected from patients in a university hospital in Seoul, South Korea, between 2009 and 2015. All 3 Elizabethkingia spp. were detected in patients; among the 86 isolates identified by 16S rRNA gene sequencing, 17 (19.8%) were E. meningoseptica, 18 (20.9%) were Elizabethkingia miricola, and 51 (59.3%) were Elizabethkingia anophelis Only the MALDI-TOF Vitek MS system with an amended database correctly identified all of the isolates. The majority (76.7%) of the isolates were from the lower respiratory tract, and 8 (9.3%) were from blood. Over 90% of E. meningoseptica and E. anophelis isolates were susceptible to piperacillin-tazobactam and rifampin. In contrast, all E. miricola isolates were susceptible to fluoroquinolones except ciprofloxacin. Further studies are urgently needed to determine the optimal antimicrobial agents for the treatment of infections due to each individual Elizabethkingia species.

  12. Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons.

    PubMed

    Lagkouvardos, Ilias; Fischer, Sandra; Kumar, Neeraj; Clavel, Thomas

    2017-01-01

    The importance of 16S rRNA gene amplicon profiles for understanding the influence of microbes in a variety of environments coupled with the steep reduction in sequencing costs led to a surge of microbial sequencing projects. The expanding crowd of scientists and clinicians wanting to make use of sequencing datasets can choose among a range of multipurpose software platforms, the use of which can be intimidating for non-expert users. Among available pipeline options for high-throughput 16S rRNA gene analysis, the R programming language and software environment for statistical computing stands out for its power and increased flexibility, and the possibility to adhere to most recent best practices and to adjust to individual project needs. Here we present the Rhea pipeline, a set of R scripts that encode a series of well-documented choices for the downstream analysis of Operational Taxonomic Units (OTUs) tables, including normalization steps, alpha- and beta-diversity analysis, taxonomic composition, statistical comparisons, and calculation of correlations. Rhea is primarily a straightforward starting point for beginners, but can also be a framework for advanced users who can modify and expand the tool. As the community standards evolve, Rhea will adapt to always represent the current state-of-the-art in microbial profiles analysis in the clear and comprehensive way allowed by the R language. Rhea scripts and documentation are freely available at https://lagkouvardos.github.io/Rhea.

  13. Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates

    PubMed Central

    Bar-Yaacov, Dan; Frumkin, Idan; Yashiro, Yuka; Schlesinger, Orr; Bieri, Philipp; Greber, Basil; Ban, Nenad; Zarivach, Raz; Alfonta, Lital; Pilpel, Yitzhak; Suzuki, Tsutomu; Mishmar, Dan

    2016-01-01

    The mitochondrial ribosome, which translates all mitochondrial DNA (mtDNA)-encoded proteins, should be tightly regulated pre- and post-transcriptionally. Recently, we found RNA-DNA differences (RDDs) at human mitochondrial 16S (large) rRNA position 947 that were indicative of post-transcriptional modification. Here, we show that these 16S rRNA RDDs result from a 1-methyladenosine (m1A) modification introduced by TRMT61B, thus being the first vertebrate methyltransferase that modifies both tRNA and rRNAs. m1A947 is conserved in humans and all vertebrates having adenine at the corresponding mtDNA position (90% of vertebrates). However, this mtDNA base is a thymine in 10% of the vertebrates and a guanine in the 23S rRNA of 95% of bacteria, suggesting alternative evolutionary solutions. m1A, uridine, or guanine may stabilize the local structure of mitochondrial and bacterial ribosomes. Experimental assessment of genome-edited Escherichia coli showed that unmodified adenine caused impaired protein synthesis and growth. Our findings revealed a conserved mechanism of rRNA modification that has been selected instead of DNA mutations to enable proper mitochondrial ribosome function. PMID:27631568

  14. Assessment of microbial dynamics in the Pearl River Estuary by 16S rRNA terminal restriction fragment analysis

    NASA Astrophysics Data System (ADS)

    Wu, Madeline; Song, Liansheng; Ren, Jianping; Kan, Jianjun; Qian, Pei-Yuan

    2004-10-01

    We have evaluated the feasibility of using the terminal restriction fragment length polymorphism (T-RFLP) pattern of polymerase chain reaction (PCR) amplified 16S rRNA sequences to track the changes of the free-living bacterial community for the Pearl River Estuary surface waters. The suitability of specific PCR primers, PCR bias induced by thermal cycles, and field-sampling volumes were critically evaluated in laboratory tests. We established a workable protocol and obtained TRF patterns that reflected the changes in the bacterial population. The temporal dynamics over a 24 h period were examined at one anchored station, as well as the spatial distribution pattern of the bacterial community at several stations, covering the transects along the river discharge direction and across the river plume. The TRF pattern revealed 9 dominant bacterial groups. Changes in their relative abundance reflecting the changes in the bacterial community composition were documented. Many culturable species were isolated from each field sample and a portion of the 16S rRNA gene for each species was sequenced. The species was identified based on sequence data comparison. In this region, the dominant species belong to the γ-subdivision of proteobacteria and the Bacillus/Clostridium group of Firmicutes. We also detected the wide spread distribution of Acinetobacter spp.; many of these species are known nosocomial pathogen for humans.

  15. Comparison of the gut microbial community between obese and lean peoples using 16S gene sequencing in a Japanese population.

    PubMed

    Andoh, Akira; Nishida, Atsushi; Takahashi, Kenichiro; Inatomi, Osamu; Imaeda, Hirotsugu; Bamba, Shigeki; Kito, Katsuyuki; Sugimoto, Mitsushige; Kobayashi, Toshio

    2016-07-01

    Altered gut microbial ecology contributes to the development of metabolic diseases including obesity. In this study, we performed 16S rRNA sequence analysis of the gut microbiota profiles of obese and lean Japanese populations. The V3-V4 hypervariable regions of 16S rRNA of fecal samples from 10 obese and 10 lean volunteers were sequenced using the Illumina MiSeq(TM)II system. The average body mass index of the obese and lean group were 38.1 and 16.6 kg/m(2), respectively (p<0.01). The Shannon diversity index was significantly higher in the lean group than in the obese group (p<0.01). The phyla Firmicutes and Fusobacteria were significantly more abundant in obese people than in lean people. The abundance of the phylum Bacteroidetes and the Bacteroidetes/Firmicutes ratio were not different between the obese and lean groups. The genera Alistipes, Anaerococcus, Corpococcus, Fusobacterium and Parvimonas increased significantly in obese people, and the genera Bacteroides, Desulfovibrio, Faecalibacterium, Lachnoanaerobaculum and Olsenella increased significantly in lean people. Bacteria species possessing anti-inflammatory properties, such as Faecalibacterium prausnitzii, increased significantly in lean people, but bacteria species possessing pro-inflammatory properties increased in obese people. Obesity-associated gut microbiota in the Japanese population was different from that in Western people.

  16. Native Valve Endocarditis due to Corynebacterium striatum confirmed by 16S Ribosomal RNA Sequencing: A Case Report and Literature Review

    PubMed Central

    2016-01-01

    Corynebacterium species are non-fermentous Gram-positive bacilli that are normal flora of human skin and mucous membranes and are commonly isolated in clinical specimens. Non-diphtheriae Corynebacterium are regarded as contaminants when found in blood culture. Currently, Corynebacterium striatum is considered one of the emerging nosocomial agents implicated in endocarditis and serious infections. We report a case of native-valve infective endocarditis caused by C. striatum, which was misidentified by automated identification system but identified accurately by 16S ribosomal RNA sequencing, in a 55-year-old male patient. The patient had two mobile vegetations on his mitral valve, both of which had high embolic risk. Through surgical valve replacement and an antibiotic regimen, the patient recovered completely. In unusual clinical scenarios, C. striatum should not be simply dismissed as a contaminant when isolated from clinical specimens. The possibility of C. striatum infection should be considered even in an immunocompetent patient, and we suggest a genotypic assay, such as 16S rRNA sequencing, to confirm species identity. PMID:27659439

  17. PCR detection of colonization by Helicobacter pylori in conventional, euthymic mice based on the 16S ribosomal gene sequence.

    PubMed Central

    Smith, J G; Kong, L; Abruzzo, G K; Gill, C J; Flattery, A M; Scott, P M; Bramhill, D; Cioffe, C; Thompson, C M; Bartizal, K

    1996-01-01

    Many animal models of Helicobacter infection have been described, including infection in rhesus monkeys, ferrets, gnotobiotic piglets, and mice. These animal models utilize a combination of detection methods, including culture, urease testing, and histopathology, all of which may be unreliable, insensitive, or labor-intensive. Development of new animal models of Helicobacter pylori requires new methods of detection with increased sensitivity and specificity. We have developed sensitive and specific PCR primers based on the 16S ribosomal gene sequence of H. pylori. The primers detected single-copy 16S DNA representing 0.2 cell of pure H. pylori (2 cells in the presence of mouse stomach mucosal DNA) and did not cross-react with closely related bacteria. We were able to detect colonization by H. pylori in conventional, euthymic, outbred mice up to 4 weeks postinoculation with a high percentage of isolates tested. One isolate of H. pylori was detected by PCR in 100% of the mice at 6 months and 60% of the mice 1 year after inoculation. Approximately 10(3) to 10(4) H. pylori cells per stomach were detected by utilizing this PCR methodology semiquantitatively. These primers and PCR methodology have facilitated detection of H. pylori colonization in conventional, euthymic mice, colonization which may not have been detectable by other methods. PMID:8770506

  18. Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons

    PubMed Central

    Fischer, Sandra; Kumar, Neeraj

    2017-01-01

    The importance of 16S rRNA gene amplicon profiles for understanding the influence of microbes in a variety of environments coupled with the steep reduction in sequencing costs led to a surge of microbial sequencing projects. The expanding crowd of scientists and clinicians wanting to make use of sequencing datasets can choose among a range of multipurpose software platforms, the use of which can be intimidating for non-expert users. Among available pipeline options for high-throughput 16S rRNA gene analysis, the R programming language and software environment for statistical computing stands out for its power and increased flexibility, and the possibility to adhere to most recent best practices and to adjust to individual project needs. Here we present the Rhea pipeline, a set of R scripts that encode a series of well-documented choices for the downstream analysis of Operational Taxonomic Units (OTUs) tables, including normalization steps, alpha- and beta-diversity analysis, taxonomic composition, statistical comparisons, and calculation of correlations. Rhea is primarily a straightforward starting point for beginners, but can also be a framework for advanced users who can modify and expand the tool. As the community standards evolve, Rhea will adapt to always represent the current state-of-the-art in microbial profiles analysis in the clear and comprehensive way allowed by the R language. Rhea scripts and documentation are freely available at https://lagkouvardos.github.io/Rhea. PMID:28097056

  19. Identification of bacteria associated with underground parts of Crocus sativus by 16S rRNA gene targeted metagenomic approach.

    PubMed

    Ambardar, Sheetal; Sangwan, Naseer; Manjula, A; Rajendhran, J; Gunasekaran, P; Lal, Rup; Vakhlu, Jyoti

    2014-10-01

    Saffron (Crocus sativus L), an autumn-flowering perennial sterile plant, reproduces vegetatively by underground corms. Saffron has biannual corm-root cycle that makes it an interesting candidate to study microbial dynamics in its rhizosphere and cormosphere (area under influence of corm). Culture independent 16S rRNA gene metagenomic study of rhizosphere and cormosphere of Saffron during flowering stage revealed presence of 22 genera but none of the genus was common in all the three samples. Bulk soil bacterial community was represented by 13 genera with Acidobacteria being dominant. In rhizosphere, out of eight different genera identified, Pseudomonas was the most dominant genus. Cormosphere bacteria comprised of six different genera, dominated by the genus Pantoea. This study revealed that the bacterial composition of all the three samples is significantly different (P < 0.05) from each other. This is the first report on the identification of bacteria associated with rhizosphere, cormosphere and bulk soil of Saffron, using cultivation independent 16S rRNA gene targeted metagenomic approach.

  20. 16S rRNA gene phylogenesis of culturable predominant bacteria from diseased Apostichopus japonicus (Holothuroidea, Echinodermata)

    NASA Astrophysics Data System (ADS)

    Ma, Haiyan; Jiang, Guoliang; Wu, Zhiqiang; Wang, Xin

    2009-06-01

    Cultured Apostichopus japonicus in China suffers from a kind of skin ulceration disease that has caused severe economic loss in recent years. The disease, pathogens of which are supposed to be bacteria by most researchers, is highly infectious and can often cause all individuals in the same culture pool to die in a very short time. The 16S rRNA gene phylogenesis of the culturable bacteria from the lesions of diseased individuals was conducted to study the biodiversity of the bacterial communities in the lesions and to identify probable pathogen(s) associated with this kind of disease. S. japonica samples were selected from a hatchery located in the eastern part of Qingdao, China. Bacterial universal primers GM5F and DS907R were used to amplify the 16S rRNA gene of bacteria colonies, and touchdown PCR was performed to amplify the target sequences. The results suggest that γ- proteobacteria (Alteromonadales and Vibrionales) of CFB group, many strains of which have been also determined as pathogens in other marine species, are the predominant bacterial genera of the diseased Apostichopus japonicus individuals.

  1. Collagenase production and hemolytic activity related to 16S rRNA variability among Parvimonas micra oral isolates.

    PubMed

    Ota-Tsuzuki, Claudia; Alves Mayer, Marcia Pinto

    2010-02-01

    Parvimonas micra are gram positive anaerobic cocci isolated from the oral cavity and frequently related to polymicrobial infections in humans. Despite reports about phenotypic differences, the genotypic variation of P. micra and its role in virulence are still not elucidated. The aim of this study was to determine the genotypic diversity of P. micra isolates obtained from the subgingival biofilm of subjects with different periodontal conditions and to correlate these findings with phenotypic traits. Three reference strains and 35 isolates of P. micra were genotyped by 16S rRNA PCR-RFLP and phenotypic traits such as collagenase production, elastolytic and hemolytic activities were evaluated. 16S rRNA PCR-RFLP showed that P. micra could be grouped into two main clusters: C1 and C2; cluster C1 harbored three genotypes (HG1259-like, HG1467-like and ICBMO583-like) while cluster C2 harbored two genotypes (ATCC33270-like and ICBMO36). A wide variability in collagenolytic activity intensities was observed among all isolates, while elastolytic activity was detected in only two isolates. There was an association between hemolytic activity in rabbit erythrocytes and cluster C2. There was an association between hemolytic activity in rabbit erythrocytes and cluster C1. Although these data suggest a possible association between P. micra genetic diversity and their pathogenic potential, further investigations are needed to confirm this hypothesis.

  2. Comparison of the gut microbial community between obese and lean peoples using 16S gene sequencing in a Japanese population

    PubMed Central

    Andoh, Akira; Nishida, Atsushi; Takahashi, Kenichiro; Inatomi, Osamu; Imaeda, Hirotsugu; Bamba, Shigeki; Kito, Katsuyuki; Sugimoto, Mitsushige; Kobayashi, Toshio

    2016-01-01

    Altered gut microbial ecology contributes to the development of metabolic diseases including obesity. In this study, we performed 16S rRNA sequence analysis of the gut microbiota profiles of obese and lean Japanese populations. The V3–V4 hypervariable regions of 16S rRNA of fecal samples from 10 obese and 10 lean volunteers were sequenced using the Illumina MiSeqTMII system. The average body mass index of the obese and lean group were 38.1 and 16.6 kg/m2, respectively (p<0.01). The Shannon diversity index was significantly higher in the lean group than in the obese group (p<0.01). The phyla Firmicutes and Fusobacteria were significantly more abundant in obese people than in lean people. The abundance of the phylum Bacteroidetes and the Bacteroidetes/Firmicutes ratio were not different between the obese and lean groups. The genera Alistipes, Anaerococcus, Corpococcus, Fusobacterium and Parvimonas increased significantly in obese people, and the genera Bacteroides, Desulfovibrio, Faecalibacterium, Lachnoanaerobaculum and Olsenella increased significantly in lean people. Bacteria species possessing anti-inflammatory properties, such as Faecalibacterium prausnitzii, increased significantly in lean people, but bacteria species possessing pro-inflammatory properties increased in obese people. Obesity-associated gut microbiota in the Japanese population was different from that in Western people. PMID:27499582

  3. 16S rDNA-based probes for two polycyclic aromatic hydrocarbon (PAH)-degrading soil Mycobacteria

    SciTech Connect

    Govindaswami, M.; Feldhake, D.J.; Loper, J.C.

    1994-12-31

    PAHs are a class of widespread pollutants, some of which have been shown to be genotoxic, hence the fate of these compounds in the environment is of considerable interest. Research on the biodegradation of 4 and 5 ring PAHs has been limited by the general lack of microbial isolates or consortia which can completely degrade these toxicants. Heitkamp and Cerniglia have described an oxidative soil Mycobacterium-strain PYR-1 that metabolizes pyrene and fluoranthene more rapidly than the 2 and 3 ring naphthalene and phenanthrene; although some metabolites of benzo-(a)-pyrene (BaP) were detected, no mineralization of BaP was observed. In 1991 Grosser et al. reported the isolation of a Mycobacterium sp. which mineralizes pyrene and also causing some mineralization of BaP. Their study describes a comparative analysis of these two strains, which show very similar colony morphology, growth rate and yellow-orange pigmentation. Genetic differences were shown by DNA amplification fingerprinting (DAF) using two arbitrary GC-rich octanucleotide primers, and by sequence comparison of PCR amplified 16S rDNA, although both strains show similarity closest to that of the genus Mycobacteria. These 16S rDNA sequences are in use for the construction of strain-specific DNA probes to monitor the presence, survival and growth of these isolates in PAH-contaminated soils in studies of biodegradation.

  4. 16S rRNA gene probe quantitates residual host cell DNA in pharmaceutical-grade plasmid DNA.

    PubMed

    Wang, Kai-Yu; Guo, Ying-Jun; Sun, Shu-Han; Shi, Ke; Zhang, Shu; Wang, Kai-Hui; Yi-Zhang; Chen, Zu-Huan

    2006-03-24

    The development and widespread use of DNA-based vaccination against infectious pathogens have been a great triumph of medical science. Quality control of DNA vaccines as biopharmaceutical productions is a problem to solve. Residual genomic DNA of engineering bacteria has been identified as a potential risk factor, so whose level must be controlled under the regulatory standards. We report a dot-blot hybridization method to detect residual host cell DNA in purified DNA vaccines. The assay utilizes PCR amplified and digoxigenin-labeled Escherichia coli 16S rRNA gene as probe. The sensitivity of the dot-blot hybridization assay with E. coli 16S rRNA gene probe was evaluated in comparison with single copy UidR gene probe. The optimized dot-blot hybridization assay had both low background and a suitable sensitivity, detecting 10 pg of residual E. coli DNA. The method is suitable in the routine use of measuring the levels of residual E. coli DNA in the pharmaceutical-grade DNA vaccine.

  5. Studying long 16S rDNA sequences with ultrafast-metagenomic sequence classification using exact alignments (Kraken).

    PubMed

    Valenzuela-González, Fabiola; Martínez-Porchas, Marcel; Villalpando-Canchola, Enrique; Vargas-Albores, Francisco

    2016-03-01

    Ultrafast-metagenomic sequence classification using exact alignments (Kraken) is a novel approach to classify 16S rDNA sequences. The classifier is based on mapping short sequences to the lowest ancestor and performing alignments to form subtrees with specific weights in each taxon node. This study aimed to evaluate the classification performance of Kraken with long 16S rDNA random environmental sequences produced by cloning and then Sanger sequenced. A total of 480 clones were isolated and expanded, and 264 of these clones formed contigs (1352 ± 153 bp). The same sequences were analyzed using the Ribosomal Database Project (RDP) classifier. Deeper classification performance was achieved by Kraken than by the RDP: 73% of the contigs were classified up to the species or variety levels, whereas 67% of these contigs were classified no further than the genus level by the RDP. The results also demonstrated that unassembled sequences analyzed by Kraken provide similar or inclusively deeper information. Moreover, sequences that did not form contigs, which are usually discarded by other programs, provided meaningful information when analyzed by Kraken. Finally, it appears that the assembly step for Sanger sequences can be eliminated when using Kraken. Kraken cumulates the information of both sequence senses, providing additional elements for the classification. In conclusion, the results demonstrate that Kraken is an excellent choice for use in the taxonomic assignment of sequences obtained by Sanger sequencing or based on third generation sequencing, of which the main goal is to generate larger sequences.

  6. Rapid identification and classification of bacteria by 16S rDNA restriction fragment melting curve analyses (RFMCA).

    PubMed

    Rudi, Knut; Kleiberg, Gro H; Heiberg, Ragnhild; Rosnes, Jan T

    2007-08-01

    The aim of this work was to evaluate restriction fragment melting curve analyses (RFMCA) as a novel approach for rapid classification of bacteria during food production. RFMCA was evaluated for bacteria isolated from sous vide food products, and raw materials used for sous vide production. We identified four major bacterial groups in the material analysed (cluster I-Streptococcus, cluster II-Carnobacterium/Bacillus, cluster III-Staphylococcus and cluster IV-Actinomycetales). The accuracy of RFMCA was evaluated by comparison with 16S rDNA sequencing. The strains satisfying the RFMCA quality filtering criteria (73%, n=57), with both 16S rDNA sequence information and RFMCA data (n=45) gave identical group assignments with the two methods. RFMCA enabled rapid and accurate classification of bacteria that is database compatible. Potential application of RFMCA in the food or pharmaceutical industry will include development of classification models for the bacteria expected in a given product, and then to build an RFMCA database as a part of the product quality control.

  7. Bases in 16S rRNA Important for Subunit Association, tRNA binding, and Translocation

    PubMed Central

    Shi, Xinying; Chiu, Katie; Ghosh, Srikanta; Joseph, Simpson

    2009-01-01

    Ribosomes are the cellular machinery responsible for protein synthesis. A well-orchestrated step in the elongation cycle of protein synthesis is the precise translocation of the tRNA-mRNA complex within the ribosome. Here we report the application of a new in vitro modification-interference method for the identification of bases in 16S rRNA that are essential for translocation. Our results suggest that conserved bases U56, U723, A1306, A1319, and A1468 in 16S rRNA are important for translocation. These five bases were deleted or mutated in order to study their role in translation. Depending on the type of mutation, we observed inhibition of growth rate, subunit association, tRNA binding and/or translocation. Interestingly, deletion of U56 or A1319 or mutation of A1319 to C showed a lethal phenotype and were defective in protein synthesis in vitro. Further analysis showed that deletion of U56 or A1319 caused defects in 30S subunit assembly, subunit association and tRNA binding. In contrast, A1319C mutation showed no defects in subunit association; however, the extent of tRNA binding and translocation was significantly reduced. These results show that conserved bases located as far away as 100 Å from the tRNA binding sites can be important for translation. PMID:19545171

  8. Analysis of conformational changes in 16 S rRNA during the course of 30 S subunit assembly.

    PubMed

    Holmes, Kristi L; Culver, Gloria M

    2005-11-25

    Ribosome biogenesis involves an integrated series of binding events coupled with conformational changes that ultimately result in the formation of a functional macromolecular complex. In vitro, Escherichia coli 30 S subunit assembly occurs in a cooperative manner with the ordered addition of 20 ribosomal proteins (r-proteins) with 16 S rRNA. The assembly pathway for 30 S subunits has been dissected in vitro into three steps, where specific r-proteins associate with 16 S rRNA early in 30 S subunit assembly, followed by a mid-assembly conformational rearrangement of the complex that then enables the remaining r-proteins to associate in the final step. Although the three steps of 30 S subunit assembly have been known for some time, few details have been elucidated about changes that occur as a result of these three specific stages. Here, we present a detailed analysis of the concerted early and late stages of small ribosomal subunit assembly. Conformational changes, roles for base-pairing and r-proteins at specific stages of assembly, and a polar nature to the assembly process have been revealed. This work has allowed a more comprehensive and global view of E.coli 30 S ribosomal subunit assembly to be obtained.

  9. apex: phylogenetics with multiple genes.

    PubMed

    Jombart, Thibaut; Archer, Frederick; Schliep, Klaus; Kamvar, Zhian; Harris, Rebecca; Paradis, Emmanuel; Goudet, Jérome; Lapp, Hilmar

    2017-01-01

    Genetic sequences of multiple genes are becoming increasingly common for a wide range of organisms including viruses, bacteria and eukaryotes. While such data may sometimes be treated as a single locus, in practice, a number of biological and statistical phenomena can lead to phylogenetic incongruence. In such cases, different loci should, at least as a preliminary step, be examined and analysed separately. The r software has become a popular platform for phylogenetics, with several packages implementing distance-based, parsimony and likelihood-based phylogenetic reconstruction, and an even greater number of packages implementing phylogenetic comparative methods. Unfortunately, basic data structures and tools for analysing multiple genes have so far been lacking, thereby limiting potential for investigating phylogenetic incongruence. In this study, we introduce the new r package apex to fill this gap. apex implements new object classes, which extend existing standards for storing DNA and amino acid sequences, and provides a number of convenient tools for handling, visualizing and analysing these data. In this study, we introduce the main features of the package and illustrate its functionalities through the analysis of a simple data set.

  10. Mitochondrial DNA sequence-based phylogenetic relationship of Trichiurus lepturus (Perciformes: Trichiuridae) from the Persian Gulf

    PubMed Central

    Tamadoni Jahromi, S.; Mohd Noor, S. A.; Pirian, K.; Dehghani, R.; Nazemi, M.; Khazaali, A.

    2016-01-01

    In this study, mitochondrial DNA analysis using 16S ribosomal DNA (rDNA) was performed to investigate the phylogeny relationship of Trichiurus lepturus in the Persian Gulf compared to the other investigated area. The amplification of 16S rDNA resulted in a product of 600 bp in all samples. The results showed that the isolated strain belongs to T. lepturus showing 42 divergence sites among the same reported partial sequences of 16S rRNA gene from the other area (West Atlantic and Indo-Pacific area). Phylogeny results showed that all 18 haplotypes of the species clustered into five clades with reasonably high bootstrap support of values (>64%). Overall, the tree topology for both phylogenetic and phenetic trees for 16S rDNA was similar. Both trees exposed two major clusters, one wholly containing the haplotypes of the T. lepturus species belonging to Indo-Pacific area with two major sister groups including Persian Gulf specimen and the other cleared the Western Atlantic and Japan individuals clustered in another distinct clade supporting the differentiation between the two areas. Phylogenic relationship observed between the Persian Gulf and the other Indo-Pacific Individuals suggested homogeneity between two mentioned areas. PMID:27822250

  11. Phylogeny of the Genus Nocardia Based on Reassessed 16S rRNA Gene Sequences Reveals Underspeciation and Division of Strains Classified as Nocardia asteroides into Three Established Species and Two Unnamed Taxons

    PubMed Central

    Roth, Andreas; Andrees, Sebastian; Kroppenstedt, Reiner M.; Harmsen, Dag; Mauch, Harald

    2003-01-01

    Conventional identification of Nocardia in the routine laboratory remains problematic due to a paucity of reliable phenotypic tests and due to the yet-unresolved taxonomy of strains classified as belonging to the species Nocardia asteroides, which comprises the type strain and isolates with drug pattern types II and VI. The 16S rRNA gene of 74 representative strains of the genus Nocardia, encompassing 25 established species, was sequenced in order to provide a molecular basis for accurate species identification and with the aim of reassessing the phylogeny of taxons assigned to the species N. asteroides. The result of this phylogenetic analysis confirms that the interspecies heterogeneity of closely related nocardial species can be considerably low (a sequence divergence of only 0.5% was found between N. paucivorans and N. brevicatena). We observed a sequence microheterogeneity (sequence divergence of fewer than five bases) in 8 of 11 species of which more than one strain in the species was studied. At least 10 taxons were found that merit description as new species. Strains previously classified as N. asteroides fell into five distinct phylogenetic groups: the type strain cluster (N. asteroides sensu strictu), N. abscessus, N. cyriacigeorgica, and two clusters closely related to N. carnea or N. flavorosea. The strains within the latter two groups probably represent new species, pending further genetic and phenotypic evaluation. Restricted phenotypic data revealed that N. abscessus, N. cyriacigeorgica, and the two Nocardia species taxons are equivalent to drug patterns I, VI, and II, respectively. In the future, these data will help in finding species-specific markers after adoption of a more precise nomenclature for isolates closely related to N. asteroides and unravel confusing phenotypic data obtained in the past for unresolved groups of strains that definitely belong to separate taxons from a phylogenetic point of view. PMID:12574299

  12. Phylogenetic Approaches Toward Crocodylian History

    NASA Astrophysics Data System (ADS)

    Brochu, Christopher A.

    A review of crocodylian phylogeny reveals a more complex history than might have been anticipated from a direct reading of the fossil record without consideration of phylogenetic relationships. The three main extant crocodylian lineagesGavialoidea, Alligatoroidea, Crocodyloideaare known from fossils in the Late Cretaceous, and the group is found nearly worldwide during the Cenozoic. Some groups have distributions that are best explained by the crossing of marine barriers during the Tertiary. Early Tertiary crocodylian faunas are phylogenetically composite, and clades tend to be morphologically uniform and geographically widespread. Later in the Tertiary, Old World crocodylian faunas are more endemic. Crocodylian phylogeneticists face numerous challenges, the most important being the phylogenetic relationships and time of divergence of the two living gharials (Gavialis gangeticus and Tomistoma schlegelii), the relationships among living true crocodiles (Crocodylus), and the relationships among caimans.

  13. Development and evaluation of two novel oligonucleotide probes based on 16S rRNA sequence for the identification of Salmonella in foods.

    PubMed

    Lin, C K; Tsen, H Y

    1995-05-01

    DNA sequence in the V3 to V6 region of the 16S rRNA gene of Salmonella enteritidis was determined. By comparison of this sequence with those of Escherichia coli and Proteus vulgaris obtained from GenBank/EMBL database, three oligonucleotides termed as 16S I, 16S II and 16S III were synthesized. Hybridization of these oligonucleotides with 325 Salmonella isolates and some non-Salmonella isolates including the Salmonella closely related species of the family of Enterobacteriaceae showed that 16S II could not be used as a Salmonella specific-probe. 16S I and 16S III hybridized with all the Salmonella isolates tested, the former also hybridizing with Citrobacter spp. and the latter hybridizing with Klebsiella pneumoniae as well as Serratia marcescens. Since enrichment of the target cells in food samples was usually required prior to the DNA hybridization assay, the interference from those non-Salmonella isolates could be prevented by enrichment by culturing in lactose-combined tetrathionate (CTET) broth followed by Gram-negative (GN) broth at 37 degrees C and/or 43 degrees C. Such a culture step could inhibit the growth of Klebsiella spp., Ser. marcescens and/or Citrobacter spp. and allowed the specific detection of Salmonella.

  14. Rapid in situ hybridization technique using 16S rRNA segments for detecting and differentiating the closely related gram-positive organisms Bacillus polymyxa and Bacillus macerans

    NASA Technical Reports Server (NTRS)

    Jurtshuk, R. J.; Blick, M.; Bresser, J.; Fox, G. E.; Jurtshuk, P. Jr

    1992-01-01

    A rapid, sensitive, inexpensive in situ hybridization technique, using 30-mer 16S rRNA probes, can specifically differentiate two closely related Bacillus spp., B. polymyxa and B. macerans. The 16S rRNA probes were labeled with a rhodamine derivative (Texas Red), and quantitative fluorescence measurements were made on individual bacterial cells. The microscopic fields analyzed were selected by phase-contrast microscopy, and the fluorescence imaging analyses were performed on 16 to 67 individual cells. The labeled 16S rRNA probe, POL, whose sequence was a 100% match with B. polymyxa 16S rRNA but only a 60% match with B. macerans 16S rRNA, gave quantitative fluorescence ratio measurements that were 34.8-fold higher for B. polymyxa cells than for B. macerans cells. Conversely, the labeled probe, MAC, which matched B. polymyxa 16S rRNA in 86.6% of its positions and B. macerans 16S rRNA in 100% of its positions, gave quantitative fluorescence measurements that were 59.3-fold higher in B. macerans cells than in B. polymyxa cells. Control probes, whose 16S rRNA sequence segment (P-M) was present in both B. polymyxa and B. macerans as well as a panprokaryotic probe (16S), having a 100% match with all known bacteria, hybridized equally well with both organisms. These latter hybridizations generated very high fluorescence signals, but their comparative fluorescence ratios (the differences between two organisms) were low. The control paneukaryotic probe (28S), which had less than 30% identity for both B. macerans and B. polymyxa, did not hybridize with either organism.

  15. Cyanobacterial endobionts within a major marine planktonic calcifier (Globigerina bulloides, Foraminifera) revealed by 16S rRNA metabarcoding

    NASA Astrophysics Data System (ADS)

    Bird, Clare; Darling, Kate F.; Russell, Ann D.; Davis, Catherine V.; Fehrenbacher, Jennifer; Free, Andrew; Wyman, Michael; Ngwenya, Bryne T.

    2017-02-01

    We investigated the possibility of bacterial symbiosis in Globigerina bulloides, a palaeoceanographically important, planktonic foraminifer. This marine protist is commonly used in micropalaeontological investigations of climatically sensitive subpolar and temperate water masses as well as wind-driven upwelling regions of the world's oceans. G. bulloides is unusual because it lacks the protist algal symbionts that are often found in other spinose species. In addition, it has a large offset in its stable carbon and oxygen isotopic compositions compared to other planktonic foraminifer species, and also that predicted from seawater equilibrium. This is suggestive of novel differences in ecology and life history of G. bulloides, making it a good candidate for investigating the potential for bacterial symbiosis as a contributory factor influencing shell calcification. Such information is essential to evaluate fully the potential response of G. bulloides to ocean acidification and climate change. To investigate possible ecological interactions between G. bulloides and marine bacteria, 18S rRNA gene sequencing, fluorescence microscopy, 16S rRNA gene metabarcoding and transmission electron microscopy (TEM) were performed on individual specimens of G. bulloides (type IId) collected from two locations in the California Current. Intracellular DNA extracted from five G. bulloides specimens was subjected to 16S rRNA gene metabarcoding and, remarkably, 37-87 % of all 16S rRNA gene sequences recovered were assigned to operational taxonomic units (OTUs) from the picocyanobacterium Synechococcus. This finding was supported by TEM observations of intact Synechococcus cells in both the cytoplasm and vacuoles of G. bulloides. Their concentrations were up to 4 orders of magnitude greater inside the foraminifera than those reported for the California Current water column and approximately 5 % of the intracellular Synechococcus cells observed were undergoing cell division. This suggests

  16. Composition and Dynamics of Bacterial Communities of a Drinking Water Supply System as Assessed by RNA- and DNA-Based 16S rRNA Gene Fingerprinting

    PubMed Central

    Eichler, Stefan; Christen, Richard; Höltje, Claudia; Westphal, Petra; Bötel, Julia; Brettar, Ingrid; Mehling, Arndt; Höfle, Manfred G.

    2006-01-01

    Bacterial community dynamics of a whole drinking water supply system (DWSS) were studied from source to tap. Raw water for this DWSS is provided by two reservoirs with different water characteristics in the Harz mountains of Northern Germany. Samples were taken after different steps of treatment of raw water (i.e., flocculation, sand filtration, and chlorination) and at different points along the supply system to the tap. RNA and DNA were extracted from the sampled water. The 16S rRNA or its genes were partially amplified by reverse transcription-PCR or PCR and analyzed by single-strand conformation polymorphism community fingerprints. The bacterial community structures of the raw water samples from the two reservoirs were very different, but no major changes of these structures occurred after flocculation and sand filtration. Chlorination of the processed raw water strongly affected bacterial community structure, as reflected by the RNA-based fingerprints. This effect was less pronounced for the DNA-based fingerprints. After chlorination, the bacterial community remained rather constant from the storage containers to the tap. Furthermore, the community structure of the tap water did not change substantially for several months. Community composition was assessed by sequencing of abundant bands and phylogenetic analysis of the sequences obtained. The taxonomic compositions of the bacterial communities from both reservoirs were very different at the species level due to their different limnologies. On the other hand, major taxonomic groups, well known to occur in freshwater, such as Alphaproteobacteria, Betaproteobacteria, and Bacteroidetes, were found in both reservoirs. Significant differences in the detection of the major groups were observed between DNA-based and RNA-based fingerprints irrespective of the reservoir. Chlorination of the drinking water seemed to promote growth of nitrifying bacteria. Detailed analysis of the community dynamics of the whole DWSS

  17. Shift in prokaryotic diversity in Arctic sediment along a continuum Glacier -River - Fjord using massive 16S rRNA gene tag sequencing

    NASA Astrophysics Data System (ADS)

    Laghdass, M.; Deloffre, J.; Lafite, R.; Hänni, C.; Gillet, B.; Cecillon, S.; Simonet, P.; Petit, F.

    2012-04-01

    In Arctic environment, one of indirect consequences of the global climate warming is the significant amplification of the amount of inland water during the spring thaw resulting from the snow cover and permafrost melting. These freshwater transfers to the coast cause sedimentary transfers. The Arctic fjords that represent deep glacial valleys of the sea are particularly vulnerable systems. Although the previous studies have highlighted potentially the high bacterial diversity in Arctic environment by the pyrosequencing, a new-generation sequencing and high throughput method, does not escape the same bias as the one of classical molecular biology techniques involved at different stages of the analysis. In this context, our objective was to characterize the prokaryotic diversity associated to the sediment transfer along a gradient from the head of the glacier to mud patch sediment in the Goule river streaming in Kongsfjorden (Svalbard) during an active thaw. The prokaryotic diversity in sediment was characterized by combining a massive of 16S rRNA gene tag sequencing with a specific and original approach in order to overcome the bias associated to the sampling and extraction. The sediment was extracted by three different methods. One method was done in duplicate. Negative controls performed at extraction and PCR stages were also sequenced. The phylogenetic analysis of the environmental samples below phylum level revealed significantly changes in the diversity and the function of the prokaryotic community along the gradient. The subglacial Goule river sediment is characterized by bacteria with specific functions methylotroph bacteria, aerobic chemoautolithotrophic bacteria (Alphaproteobacteria with Methylobacteriaceae) whereas the mouth of the river Goule and the freshwater part of the Goule River was dominated by sulphate-reducing-bacteria, anaerobic chemooorganotroph (Deltaprotobacteria with the Desulfobulbaceae and Desulfuromonadaceae) and by

  18. Phylogenetic diversity of rhizobial species and symbiovars nodulating Phaseolus vulgaris in Iran.

    PubMed

    Rouhrazi, Kiomars; Khodakaramian, Gholam; Velázquez, Encarna

    2016-03-01

    The phylogenetic diversity of 29 rhizobial strains nodulating Phaseolus vulgaris in Iran was analysed on the basis of their core and symbiotic genes. These strains displayed five 16S rRNA-RFLP patterns and belong to eight ERIC-PCR clusters. The phylogenetic analyses of 16S rRNA, recA and atpD core genes allowed the identification of several strains as Rhizobium sophoriradicis, R. leguminosarum, R. tropici and Pararhizobium giardinii, whereas other strains represented a new phylogenetic lineage related to R. vallis. These strains and those identified as R. sophoriradicis and R. leguminosarum belong to the symbiovar phaseoli carrying the γ nodC allele distributed in P. vulgaris endosymbionts in America, Europe, Africa and Asia. The strain identified as R. tropici belongs to the symbiovar tropici carried by strains of R. tropici, R. leucaenae, R. lusitanum and R. freirei nodulating P. vulgaris in America, Africa and Asia. The strain identified as P. giardinii belongs to the symbiovar giardinii together with the type strain of this species nodulating P. vulgaris in France. It is remarkable that the recently described species R. sophoriradicis is worldwide distributed in P. vulgaris nodules carrying the γ nodC allele of symbiovar phaseoli harboured by rhizobia isolated in the American distribution centers of this legume.

  19. The fur Gene as a New Phylogenetic Marker for Vibrionaceae Species Identification

    PubMed Central

    Gram, Lone

    2015-01-01

    Microbial taxonomy is essential in all areas of microbial science. The 16S rRNA gene sequence is one of the main phylogenetic species markers; however, it does not provide discrimination in the family Vibrionaceae, where other molecular techniques allow better interspecies resolution. Although multilocus sequence analysis (MLSA) has been used successfully in the identification of Vibrio species, the technique has several limitations. They include the fact that several locus amplifications and sequencing have to be performed, which still sometimes lead to doubtful identifications. Using an in silico approach based on genomes from 103 Vibrionaceae strains, we demonstrate here the high resolution of the fur gene in the identification of Vibrionaceae species and its usefulness as a phylogenetic marker. The fur gene showed within-species similarity higher than 95%, and the relationships inferred from its use were in agreement with those observed for 16S rRNA analysis and MLSA. Furthermore, we developed a fur PCR sequencing-based method that allowed identification of Vibrio species. The discovery of the phylogenetic power of the fur gene and the development of a PCR method that can be used in amplification and sequencing of the gene are of general interest whether for use alone or together with the previously suggested loci in an MLSA. PMID:25662978

  20. Usefulness of the MicroSeq 500 16S rDNA bacterial identification system for identification of anaerobic Gram positive bacilli isolated from blood cultures

    PubMed Central

    Lau, S K P; Ng, K H L; Woo, P C Y; Yip, K‐t; Fung, A M Y; Woo, G K S; Chan, K‐m; Que, T‐l

    2006-01-01

    Using full 16S ribosomal RNA (rRNA) gene sequencing as the gold standard, 20 non‐duplicating anaerobic Gram positive bacilli isolated from blood cultures were analysed by the MicroSeq 500 16S rDNA bacterial identification system. The MicroSeq system successfully identified 13 of the 20 isolates. Four and three isolates were misidentified at the genus and species level, respectively. Although the MicroSeq 500 16S rDNA bacterial identification system is better than three commercially available identification systems also evaluated, its database needs to be expanded for accurate identification of anaerobic Gram positive bacilli. PMID:16443743

  1. Phylogenetic analyses of phylum Actinobacteria based on whole genome sequences.

    PubMed

    Verma, Mansi; Lal, Devi; Kaur, Jaspreet; Saxena, Anjali; Kaur, Jasvinder; Anand, Shailly; Lal, Rup

    2013-09-01

    Actinobacteria constitute one of the largest and ancient taxonomic phylum within the domain bacteria and are well known for their secondary metabolites. Considerable variation in the metabolic properties, genome size and GC content of the members of this phylum has been observed. Therefore, the placement of new or existing species based on 16S rRNA gene sometimes becomes problematic due to the low congruence level. In the present study, phylogeny of ninety actinobacterial genomes was reconstructed using single gene and whole genome based data. Where alignment-free phylogenetic method was found to be more robust, the concatenation of 94 proteins improved the resolution which all single gene based phylogenies failed to resolve. The comprehensive analysis of 94 conserved proteins resulted in a total of 42,447 informative sites, which is so far the largest meta-alignment obtained for this phylum. But the ultimate resolved phylogeny was obtained by generating a consensus tree by combining the information from single gene and genome based phylogenies. The present investigation clearly revealed that the consensus approach is a useful tool for phylogenetic inference and the taxonomic affiliations must be based on this approach. The consensus approach suggested that there is a need for taxonomic amendments of the orders Frankiales and Micrococcales.

  2. [Phylogenetic diversity of airborne microbes in Qingdao downtown in autumn].

    PubMed

    Wang, Lin; Song, Zhi-wen; Xu, Ai-ling; Wu, Deng-deng; Xia, Yan

    2015-04-01

    To determine the community structure of airborne microbes in Qingdao downtown in autumn, the airborne bacteria and fungi were collected by the KC-6120 air sampler and analyzed using the 16S/18S rDNA gene clone library method. Phylogenetic analysis of airborne bacteria showed that they belonged to six major phylogenetic groups: Proteobacteria (78. 8%), Firmicutes (14.6%), Actinobacteria (4.0%), Planctomycetes (1.3%), Cyanobacteria (0.7%), and Deinococcus-Thermus (0.7%). The dominant genera of airborne bacteria included Acinetobacter (39.7%), Staphylococcus (11.3%), Sphingomonas (8.6%), Paracoccus (6.0%) and Massilia (5.3%). The main types of airborne fungi were Ascomycota (97.5%) and Basidiomycota (2.5%). Dominant genera of airborne fungi included Pyrenophora (76.5%), Xylaria (13.6%) and Exophiala (2.5%). The pathogens or conditioned pathogens, such as Acinetobacter, Staphylococcus, or Sphingomonas were detected in the airborne bacteria, whereas certain kinds of fungi, such as P. graminea, X. hypoxylon and Zasmidium angulare that could cause a variety of crop diseases were also detected.

  3. [Comparison of MALDI-TOF and 16S rRNA methods in identification of viridans group streptococci].

    PubMed

    Süzük Yıldız, Serap; Kaşkatepe, Banu; Altınok, Salih; Çetin, Mustafa; Karagöz, Alper; Savaş, Sümeyra

    2017-01-01

    Accurate identification of viridans group streptococci (VGS) frequently encountered as a causative agent of infective endocarditis is always a challenge for the clinical microbiology laboratory. Clinical microbiology laboratories generally use semi automatic/full automatic systems, molecular methods and also conventional methods for the identification of these bacteria. There are recent published studies that have used MALDI-TOF (Matrix Assisted Laser Ionization Mass Spectrometry-Time of Flight) systems in the identification of VGS. The aim of the study was to compare the performance of the conventional methods, semi automatic and MALDI-TOF MS system used in identification of VGS in oral microbiota of persons under the risk of infective endocarditis, with the gold standard method 16S rRNA sequence analysis and to create a diagnosis algorithm for the identification of VGS in clinical microbiology laboratories according to the obtained data.The study was conducted with 51 VGS strains isolated from oral microbiota of the patients with rheumatologic cardiac, valve and/or prosthetic valve diseases, under the risk of development of infective endocarditis, who have admitted to Ankara Numune Training and Research Hospital, Department of Cardiology, between February-June 2015. Standard microbiology procedures, optochin susceptibility and bile solubility tests were done for the isolation of bacteria. Bacteria were also identified with APISTREP (bioMérieux, France) and MALDI-TOF MS Bruker Microflex (Bruker Biotyper; Bruker Daltonics, Bremen, Germany) methods. BSF-8 (5´-AGAGTTTGATCCTGGCTCAG-3´) and BSR-534(5´-ATTACCGCGGCTGCTGGC-3´) primers were used in the 16S rRNA sequence analysis of bacteria. ABI PRISM 3100 Avan t Genetic Analyzer (Applied Biossytems, Foster City, CA, USA) were used for the sequence analysis. Electropherograms were analyzed in SeqScape Software (Applied Biosystems, Foster City, CA, USA) and compared with the reference sequences in GenBank with BLASTN

  4. Differentiation of Acinetobacter baumannii biotypes by amplification of 16S-23S rRNA intergenic spacer sequences.

    PubMed

    Garcia, A; Montoya, R; Bello, H; Gonzalez, G; Dominguez, M; Zemelman, R

    1996-01-01

    Isolates of Acinetobacter baumannii (32 strains) from blood samples obtained from patients in five Chilean hospitals were identified and biotyped according to their phenotypic properties. They were also submitted to random amplified polymorphic DNA (RAPD) using eight randomly designed 10-mers and the core sequence of M13 phage (15-mers) as well as amplification of the spacer regions between 16S and 23S genes in the prokaryotic rRNA genetic loci. With some primers, RAPD discriminated between biotypes, whereas with others each isolate showed a particular profile. When amplification of spacer regions was performed, a clear correlation between patterns and biotypes was found. This last technique allowed correct biotyping of clinical isolates. Both genetic methods might be used for the identification of A. baumannii biotypes.

  5. MSClust: A Multi-Seeds Based Clustering Algorithm for microbiome profiling using 16S rRNA Sequence

    PubMed Central

    Chen, Wei; Cheng, Yongmei; Zhang, Clarence; Zhang, Shaowu; Zhao, Hongyu

    2013-01-01

    Recent developments of next generation sequencing technologies have led to rapid accumulation of 16s rRNA sequences for microbiome profiling. One key step in data processing is to cluster short sequences into operational taxonomic units (OTUs). Although many methods have been proposed for OTU inferences, a major challenge is the balance between inference accuracy and computational efficiency, where inference accuracy is often sacrificed to accommodate the need to analyze large numbers of sequences. Inspired by the hierarchical clustering method and a modified greedy network clustering algorithm, we propose a novel multi-seeds based heuristic clustering method, named MSClust, for OTU inference. MSClust first adaptively selects multi-seeds instead of one seed for each candidate cluster, and the reads are then processed using a greedy clustering strategy. Through many numerical examples, we demonstrate that MSClust enjoys less memory usage, and better biological accuracy compared to existing heuristic clustering methods while preserving efficiency and scalability. PMID:23899776

  6. Surface water-borne multidrug and heavy metal-resistant Staphylococcus isolates characterized by 16S rDNA sequencing.

    PubMed

    Yilmaz, Fadime; Orman, Nazlı; Serim, Gamze; Kochan, Ceren; Ergene, Aysun; Icgen, Bulent

    2013-12-01

    Four Staphylococcus isolates having both multidrug- and multimetal-resistant ability were isolated from surface water. Further identification of the isolates was obtained through biochemical tests and 16S rDNA gene sequencing. One methicillin-resistant and two methicillin-sensitive isolates were determined as Staphylococcus aureus. The other isolate was identified as Staphylococcus warneri. The antibiotic and heavy metal resistance profiles of the Staphylococcus isolates were determined by using 26 antibiotics and 17 heavy metals. S. aureus isolates displayed resistance to most of the β-lactam antibiotics tested. All Staphylococcus isolates were resistant to heavy metals including silver, lithium, and barium. Due to a possible health risk of these pathogenic bacteria, a need exists for an accurate assessment of their acquired resistance to multiple drugs and metals.

  7. Salmonella detection using 16S ribosomal DNA/RNA probe-gold nanoparticles and lateral flow immunoassay.

    PubMed

    Liu, Cheng-Che; Yeung, Chun-Yan; Chen, Po-Hao; Yeh, Ming-Kung; Hou, Shao-Yi

    2013-12-01

    An ultrasensitive, simple, and fast lateral flow immunoassay for Salmonella detection using gold nanoparticles conjugated with a DNA probe, which is complementary to the 16S ribosomal RNA and DNA of Salmonella, has been developed. The detection limit is 5 fmol for the synthetic single-stranded DNA. For the Salmonella cultured samples, the nucleic acids from 10(7) bacteria were rapidly detected in 30 min. After silver enhancement, the detection limit was as low as 10(4) cells which is lower than 10(5) bacteria cells, the human infective dose of food-borne Salmonella. Furthermore, the probes used in this study are specific to Salmonella compared to several other Enterobacteriaceae. This approach would be a useful tool for microbial detection regarding food safety or clinical diagnosis. It is also suitable for large-scale screening in developing countries because it is low-cost, sensitive, specific and convenient.

  8. Identification of Atypical Rhodococcus-Like Clinical Isolates as Dietzia spp. by 16S rRNA Gene Sequencing▿

    PubMed Central

    Pilares, Lilian; Agüero, Jesús; Vázquez-Boland, José A.; Martínez-Martínez, Luis; Navas, Jesús

    2010-01-01

    Rhodococcus equi and Dietzia spp. are closely related actinomycetes that show similar phenotypic properties. In humans, R. equi is an opportunistic pathogen associated with severe immunodeficiency. Dietzia spp. are environmental bacteria that have been isolated recently from clinical material and are presumptively associated with human infections. During the last 5 years, 15 bacterial isolates from human clinical samples collected at the Hospital Marqués de Valdecilla, Santander, Spain, were identified as R. equi by the API Coryne test. 16S rRNA gene sequencing confirmed seven isolates to be true R. equi strains, whereas the other eight were identified as members of the genus Dietzia, including Dietzia maris (four isolates), Dietzia natronolimnaea (two isolates), and Dietzia timorensis and Dietzia sp. (one isolate each). The eight Dietzia isolates were highly sensitive to 12 antimicrobial compounds. PMID:20220156

  9. Next-Generation Sequencing of the Bacterial 16S rRNA Gene for Forensic Soil Comparison: A Feasibility Study.

    PubMed

    Jesmok, Ellen M; Hopkins, James M; Foran, David R

    2016-05-01

    Soil has the potential to be valuable forensic evidence linking a person or item to a crime scene; however, there is no established soil individualization technique. In this study, the utility of soil bacterial profiling via next-generation sequencing of the 16S rRNA gene was examined for associating soils with their place of origin. Soil samples were collected from ten diverse and nine similar habitats over time, and within three habitats at various horizontal and vertical distances. Bacterial profiles were analyzed using four methods: abundance charts and nonmetric multidimensional scaling provided simplification and visualization of the massive datasets, potentially aiding in expert testimony, while analysis of similarities and k-nearest neighbor offered objective statistical comparisons. The vast majority of soil bacterial profiles (95.4%) were classified to their location of origin, highlighting the potential of bacterial profiling via next-generation sequencing for the forensic analysis of soil samples.

  10. Isolation and characterization of a novel chlorpyrifos degrading flavobacterium species EMBS0145 by 16S rRNA gene sequencing.

    PubMed

    Amareshwari, P; Bhatia, Mayuri; Venkatesh, K; Roja Rani, A; Ravi, G V; Bhakt, Priyanka; Bandaru, Srinivas; Yadav, Mukesh; Nayarisseri, Anuraj; Nair, Achuthsankar S

    2015-03-01

    Indiscriminate application of pesticides like chlorpyrifos, diazinon, or malathion contaminate the soil in addition has being unsafe often it has raised severe health concerns. Conversely, microorganisms like Trichoderma, Aspergillus and Bacteria like Rhizobium Bacillus, Azotobacter, Flavobacterium etc have evolved that are endowed with degradation of pesticides aforementioned to non-toxic products. The current study pitches into identification of a novel species of Flavobacterium bacteria capable to degrade the Organophosphorous pesticides. The bacterium was isolated from agricultural soil collected from Guntur District, Andhra Pradesh, India. The samples were serially diluted and the aliquots were incubated for a suitable time following which the suspected colony was subjected to 16S rDNA sequencing. The sequence thus obtained was aligned pairwise against Flavobacterium species, which resulted in identification of novel specie of Flavobacterium later named as EMBS0145, the sequence of which was deposited in in GenBank with accession number JN794045.

  11. A novel mutation 3090 G>A of the mitochondrial 16S ribosomal RNA associated with myopathy.

    PubMed

    Coulbault, L; Deslandes, B; Herlicoviez, D; Read, M H; Leporrier, N; Schaeffer, S; Mouadil, A; Lombès, A; Chapon, F; Jauzac, P; Allouche, S

    2007-10-26

    We describe a young woman who presented with a progressive myopathy since the age of 9. Spectrophotometric analysis of the respiratory chain in muscle tissue revealed combined and profound complex I, III, II+III, and IV deficiency ranging from 60% to 95% associated with morphological and histochemical abnormalities of the muscle. An exhaustive screening of mitochondrial transfer and ribosomal RNAs showed a novel G>A substitution at nucleotide position 3090 which was detected only in urine sediment and muscle of the patient and was not found in her mother's blood cells and urine sample. We suggest that this novel de novo mutation in the 16S ribosomal RNA, a nucleotide which is highly conserved in different species, would impair mitochondrial protein synthesis and would cause a severe myopathy.

  12. Evolutionary Phylogenetic Networks: Models and Issues

    NASA Astrophysics Data System (ADS)

    Nakhleh, Luay

    Phylogenetic networks are special graphs that generalize phylogenetic trees to allow for modeling of non-treelike evolutionary histories. The ability to sequence multiple genetic markers from a set of organisms and the conflicting evolutionary signals that these markers provide in many cases, have propelled research and interest in phylogenetic networks to the forefront in computational phylogenetics. Nonetheless, the term 'phylogenetic network' has been generically used to refer to a class of models whose core shared property is tree generalization. Several excellent surveys of the different flavors of phylogenetic networks and methods for their reconstruction have been written recently. However, unlike these surveys, this chapte focuses specifically on one type of phylogenetic networks, namely evolutionary phylogenetic networks, which explicitly model reticulate evolutionary events. Further, this chapter focuses less on surveying existing tools, and addresses in more detail issues that are central to the accurate reconstruction of phylogenetic networks.

  13. Structural Insights into the Methylation of C1402 in 16S rRNA by Methyltransferase RsmI

    PubMed Central

    Liu, Guangfeng; Wang, Li; Wang, Jian; Gao, Zengqiang; Dong, Yuhui; Zhang, Linbo; Gong, Yong

    2016-01-01

    RsmI and RsmH are conserved S-Adenosylmethionine (AdoMet)-dependent methyltransferases (MTases) that are responsible for the 2′-O-methylation and N4-methylation of C1402 in bacterial 16S rRNA, respectively. Methylation of m4Cm1402 plays a role in fine-tuning the shape and functions of the P-site to increase the decoding fidelity, and was recently found to contribute to the virulence of Staphylococcus aureus in host animals. Here we report the 2.20-Å crystal structure of homodimeric RsmI from Escherichia coli in complex with the cofactor AdoMet. RsmI consists of an N-terminal putative RNA-binding domain (NTD) and a C-terminal catalytic domain (CTD) with a Rossmann-like fold, and belongs to the class III MTase family. AdoMet is specifically bound into a negatively charged deep pocket formed by both domains by making extensive contacts. Structure-based mutagenesis and isothermal titration calorimetry (ITC) assays revealed Asp100 and Ala124 are vital for AdoMet-binding. Although the overall fold of RsmI shows remarkable similarities to the characterized MTases involved in vitamin B12 biosynthesis, it exhibits a distinct charge distribution especially around the AdoMet-binding pocket because of different substrate specificity. The docking model of RsmI-AdoMet-RNA ternary complex suggested a possible base-flipping mechanism of the substrate RNA that has been observed in several known RNA MTases. Our structural and biochemical studies provide novel insights into the catalytic mechanism of C1402 methylation in 16S rRNA. PMID:27711192

  14. PCR-based method for targeting 16