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Sample records for monomorphic bacteria dna

  1. Major histocompatibility complex monomorphism and low levels of DNA fingerprinting variability in a reintroduced and rapidly expanding population of beavers.

    PubMed Central

    Ellegren, H; Hartman, G; Johansson, M; Andersson, L

    1993-01-01

    Loss of genetic variation due to population bottlenecks may be a severe threat for the survival of endangered species. Assessment and maintenance of genetic variability are thus crucial for conservation programs related to endangered populations. Scandinavian beavers went through an extensive bottleneck during the last century due to overhunting. In Sweden the species became extirpated but in Norway extinction was avoided by legal protection. Following reintroductions of small numbers of remaining Norwegian animals in 1922-1939, the Swedish population has increased tremendously, now harboring 100,000 animals. We show here that this viable population of beavers possesses extremely low levels of genetic variability at DNA fingerprinting loci and monomorphism at major histocompatibility complex (MHC) class I and class II loci. A similar pattern was also evident among Norwegian beavers but low levels of genetic variability were not a characteristic of the species since Russian conspecifics displayed substantial DNA fingerprinting polymorphism. However, the Russian animals were monomorphic at MHC loci, indicating that the European beaver is exceptional in its low level of MHC variability. The results demonstrate that a conservation program can be successful despite low levels of genetic variation in the founder population. Images Fig. 2 PMID:8367476

  2. Phylogenetic Analysis of Geographically Diverse Radopholus similis via rDNA Sequence Reveals a Monomorphic Motif.

    PubMed

    Kaplan, D T; Thomas, W K; Frisse, L M; Sarah, J L; Stanton, J M; Speijer, P R; Marin, D H; Opperman, C H

    2000-06-01

    The nucleic acid sequences of rDNA ITS1 and the rDNA D2/D3 expansion segment were compared for 57 burrowing nematode isolates collected from Australia, Cameroon, Central America, Cuba, Dominican Republic, Florida, Guadeloupe, Hawaii, Nigeria, Honduras, Indonesia, Ivory Coast, Puerto Rico, South Africa, and Uganda. Of the 57 isolates, 55 were morphologically similar to Radopholus similis and seven were citrus-parasitic. The nucleic acid sequences for PCR-amplified ITS1 and for the D2/D3 expansion segment of the 28S rDNA gene were each identical for all putative R. similis. Sequence divergence for both the ITS1 and the D2/D3 was concordant with morphological differences that distinguish R. similis from other burrowing nematode species. This result substantiates previous observations that the R. similis genome is highly conserved across geographic regions. Autapomorphies that would delimit phylogenetic lineages of non-citrus-parasitic R. similis from those that parasitize citrus were not observed. The data presented herein support the concept that R. similis is comprised of two pathotypes-one that parasitizes citrus and one that does not.

  3. Monomorphic subtelomeric DNA in the filamentous fungus, Metarhizium anisopliae,contains a RecQ helicase-like gene.

    PubMed

    Inglis, Peter W; Rigden, Daniel J; Mello, Luciane V; Louis, Edward J; Valadares-Inglis, M Cléria

    2005-08-01

    In most filamentous fungi, telomere-associated sequences (TASs) are polymorphic, and the presence of restriction fragment length polymorphisms (RFLPs) may permit the number of chromosome ends to be estimated from the number of telomeric bands obtained by restriction digestion. Here, we describe strains of Metarhizium, Gliocladium and Paecilomyces species in which only one or a few telomeric bands of unequal intensity are detectable by Southern hybridization, indicating that interchromosomal TAS exchange occurs. We also studied an anomalous strain of Metarhizium anisopliae, which produces polymorphic telomeric bands larger than 8 kb upon digestion of genomic DNA with XhoI. In this case, the first XhoI site in from the chromosome end must lie beyond the presumed monomorphic region. Cloned telomeres from this strain comprise 18-26 TTAGGG repeats, followed at the internal end of the telomere tract by five repeats of the telomere-like sequence TAAACGCTGG. An 8.1-kb TAS clone also contains a gene for a RecQ-like helicase, designated TAH1, suggesting that this TAS is analogous to the Y' elements in yeast and the subtelomeric helicase ORFs of Ustilago maydis (UTASRecQ) and Magnaporthe grisea (TLH1). The TAS in the anomalous strain of M. anisopliae, however, appears distinct from these in that it is found at most telomeres and its predicted protein product possesses a significantly longer N-terminal region in comparison to the M. grisea and U. maydis helicases. Hybridization analyses showed that TAH1 homologues are present in all other anomalous M. anisopliae strains studied, as well as in some other polymorphic strains, where the recQ-like gene also appears to be telomere-associated.

  4. [Repetitive monomorphic ventricular tachycardia].

    PubMed

    Maia, I G; Cruz Filho, F; Costa, A M; Boghossian, S H; Fagundes, M; Ribeiro, J C; Sá, R; Alves, P A

    1994-01-01

    To evaluate retrospectively clinical features of repetitive monomorphic ventricular tachycardia (RMVT). Files of 11 patients with RMVT were analyzed (9 females, mean-age 37 +/- 17 years). All patients were submitted to clinical evaluation, ECG, Holter monitoring stress test, high-resolution ECG and echocardiogram; they were treated with antiarrhythmic drugs. Patients were in NYHA class I or II, 9 asymptomatics and 2 with palpitations. The ECG was normal in all of them. Cardiac memory was observed in 3. A left bundle branch block with inferior axis deviation in the frontal plane was present during RMVT in all patients (right ventricular outflow tract focus). Holter monitoring revealed mean of 12031 +/- 8345 isolated PVC/24h; 2892 +/- 234 ventricular couplets/24h and 1367 +/- 890 VTs/24h (mainly nonsustained). In 6 patients RMVT was suppressed during maximal exercise treadmill. High-resolution ECG was negative in all group. Five patients had a normal echocardiogram while 5 showed mitral valve prolapse. One patient developed tachycardiomyopathy. The arrhythmia was controlled with 320mg of oral sotalol in 3 of 4 that used this drug and with 120mg oral propranolol in one of 6 that used this drug. Drug resistance was present in the others. The mean follow-up period was 38 +/- 16 months. The results demonstrate that RMVT is a benign form of VT with no detectable anatomic substract by the currently used methods. It is probably induced by nonreentrant mechanism and frequently drug resistance is observed. Among the antiarrhythmic drugs commonly used, sotalol showed to be the most effective.

  5. Preparation of genomic DNA from bacteria.

    PubMed

    Andreou, Lefkothea-Vasiliki

    2013-01-01

    The purpose of this protocol is the isolation of bulk cellular DNA from bacteria (alternatively see Preparation of genomic DNA from Saccharomyces cerevisiae or Isolation of Genomic DNA from Mammalian Cells protocols). Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Phosphorothioate DNA as an antioxidant in bacteria.

    PubMed

    Xie, Xinqiang; Liang, Jingdan; Pu, Tianning; Xu, Fei; Yao, Fen; Yang, Yan; Zhao, Yi-Lei; You, Delin; Zhou, Xiufen; Deng, Zixin; Wang, Zhijun

    2012-10-01

    Diverse bacteria contain DNA with sulfur incorporated stereo-specifically into their DNA backbone at specific sequences (phosphorothioation). We found that in vitro oxidation of phosphorothioate (PT) DNA by hydrogen peroxide (H(2)O(2)) or peracetic acid has two possible outcomes: DNA backbone cleavage or sulfur removal resulting in restoration of normal DNA backbone. The physiological relevance of this redox reaction was investigated by challenging PT DNA hosting Salmonella enterica cells using H(2)O(2). DNA phosphorothioation was found to correlate with increasing resistance to the growth inhibition by H(2)O(2). Resistance to H(2)O(2) was abolished when each of the three dnd genes, required for phosphorothioation, was inactivated. In vivo, PT DNA is more resistant to the double-strand break damage caused by H(2)O(2) than PT-free DNA. Furthermore, sulfur on the modified DNA was consumed and the DNA was converted to PT-free state when the bacteria were incubated with H(2)O(2). These findings are consistent with a hypothesis that phosphorothioation modification endows DNA with reducing chemical property, which protects the hosting bacteria against peroxide, explaining why this modification is maintained by diverse bacteria.

  7. Phosphorothioate DNA as an antioxidant in bacteria

    PubMed Central

    Xie, Xinqiang; Liang, Jingdan; Pu, Tianning; Xu, Fei; Yao, Fen; Yang, Yan; Zhao, Yi-Lei; You, Delin; Zhou, Xiufen; Deng, Zixin; Wang, Zhijun

    2012-01-01

    Diverse bacteria contain DNA with sulfur incorporated stereo-specifically into their DNA backbone at specific sequences (phosphorothioation). We found that in vitro oxidation of phosphorothioate (PT) DNA by hydrogen peroxide (H2O2) or peracetic acid has two possible outcomes: DNA backbone cleavage or sulfur removal resulting in restoration of normal DNA backbone. The physiological relevance of this redox reaction was investigated by challenging PT DNA hosting Salmonella enterica cells using H2O2. DNA phosphorothioation was found to correlate with increasing resistance to the growth inhibition by H2O2. Resistance to H2O2 was abolished when each of the three dnd genes, required for phosphorothioation, was inactivated. In vivo, PT DNA is more resistant to the double-strand break damage caused by H2O2 than PT-free DNA. Furthermore, sulfur on the modified DNA was consumed and the DNA was converted to PT-free state when the bacteria were incubated with H2O2. These findings are consistent with a hypothesis that phosphorothioation modification endows DNA with reducing chemical property, which protects the hosting bacteria against peroxide, explaining why this modification is maintained by diverse bacteria. PMID:22772986

  8. Isolating DNA from Gram-Negative Bacteria.

    PubMed

    Green, Michael R; Sambrook, Joseph

    2017-01-03

    The isolation of DNA from bacteria, described in this protocol, relies upon the use of sodium dodecyl sulfate and proteinase K to lyse the cells. High-molecular-weight DNA is then sheared (to reduce its viscosity and make it more manageable), extracted with phenol:chloroform, and precipitated with isopropanol. DNA isolated according to this procedure ranges from 30 to 80 kb in length.

  9. DNA Uptake by Transformable Bacteria

    SciTech Connect

    Lacks, Sanford A.

    1999-03-31

    The various processes of DNA uptake by cells can be categorized as: viral DNA entry, conjugation, or transformation. Within each category, a variety of mechanisms have been found. However, considerable similarities occur among the different mechanisms of conjugation and, especially, transformation. All of these natural mechanisms of DNA transfer are quite elaborate and involve multiple protein components, as the case may be, of the virus, the donor cell, and the recipient cell. The mechanisms of viral infection and conjugation will be discussed mainly with respect to their relevance to transformation.

  10. DNA UPTAKE BY TRANSFORMABLE BACTERIA

    SciTech Connect

    LACKS,S.A.

    1999-09-07

    The various processes of DNA uptake by cells can be categorized as: viral DNA entry, conjugation, or transformation. Within each category, a variety of mechanisms have been found. However, considerable similarities occur among the different mechanisms of conjugation and, especially, transformation. All of these natural mechanisms of DNA transfer are quite elaborate and involve multiple protein components, as the case may be, of the virus, the donor cell, and the recipient cell. The mechanisms of viral infection and conjugation will be discussed mainly with respect to their relevance to transformation.

  11. Ancient bacteria show evidence of DNA repair

    PubMed Central

    Johnson, Sarah Stewart; Hebsgaard, Martin B.; Christensen, Torben R.; Mastepanov, Mikhail; Nielsen, Rasmus; Munch, Kasper; Brand, Tina; Gilbert, M. Thomas P.; Zuber, Maria T.; Bunce, Michael; Rønn, Regin; Gilichinsky, David; Froese, Duane; Willerslev, Eske

    2007-01-01

    Recent claims of cultivable ancient bacteria within sealed environments highlight our limited understanding of the mechanisms behind long-term cell survival. It remains unclear how dormancy, a favored explanation for extended cellular persistence, can cope with spontaneous genomic decay over geological timescales. There has been no direct evidence in ancient microbes for the most likely mechanism, active DNA repair, or for the metabolic activity necessary to sustain it. In this paper, we couple PCR and enzymatic treatment of DNA with direct respiration measurements to investigate long-term survival of bacteria sealed in frozen conditions for up to one million years. Our results show evidence of bacterial survival in samples up to half a million years in age, making this the oldest independently authenticated DNA to date obtained from viable cells. Additionally, we find strong evidence that this long-term survival is closely tied to cellular metabolic activity and DNA repair that over time proves to be superior to dormancy as a mechanism in sustaining bacteria viability. PMID:17728401

  12. Transfer of DNA from Bacteria to Eukaryotes

    PubMed Central

    2016-01-01

    ABSTRACT Historically, the members of the Agrobacterium genus have been considered the only bacterial species naturally able to transfer and integrate DNA into the genomes of their eukaryotic hosts. Yet, increasing evidence suggests that this ability to genetically transform eukaryotic host cells might be more widespread in the bacterial world. Indeed, analyses of accumulating genomic data reveal cases of horizontal gene transfer from bacteria to eukaryotes and suggest that it represents a significant force in adaptive evolution of eukaryotic species. Specifically, recent reports indicate that bacteria other than Agrobacterium, such as Bartonella henselae (a zoonotic pathogen), Rhizobium etli (a plant-symbiotic bacterium related to Agrobacterium), or even Escherichia coli, have the ability to genetically transform their host cells under laboratory conditions. This DNA transfer relies on type IV secretion systems (T4SSs), the molecular machines that transport macromolecules during conjugative plasmid transfer and also during transport of proteins and/or DNA to the eukaryotic recipient cells. In this review article, we explore the extent of possible transfer of genetic information from bacteria to eukaryotic cells as well as the evolutionary implications and potential applications of this transfer. PMID:27406565

  13. Electro-microchip DNA-biosensor for bacteria detection.

    PubMed

    Yeh, Chia Hsien; Chang, Yu Huai; Chang, Tsung Chain; Lin, Hong Ping; Lin, Yu Cheng

    2010-10-01

    This paper presents a bacteria biosensor based on DNA hybridization detection with an electro-microchip transducer. Acinetobacter baumannii was chosen as DNA sample source, because the occurrence of bacteremia caused by Acinetobacter baumannii is high in hospitals worldwide. Our strategy is based on DNA hybridization of PCR amplified bacteria DNA with biotin labelled primers and detection enhancement using gold-streptavidin nanoparticles and Ag(+)-hydroquinone solution. Gold nanoparticles catalyze silver ions reduction by hydroquinone. The gradually precipitated silver metal between the two electrodes of the electro-microchip allows electrons to pass. The detection limit for Acinetobacter baumannii genomic DNA sample is 0.825 ng mL(-1) (1.2 fM). Probe specificity was investigated by screening various species of bacteria, various strains of a single species and various species of a single genus. The proposed DNA hybridization method is easy, convenient, and rapid. Moreover, it has potential applications in detection of bacteria causing infections and clinical diagnosis.

  14. Factors Behind Junk DNA in Bacteria

    PubMed Central

    Gil, Rosario; Latorre, Amparo

    2012-01-01

    Although bacterial genomes have been traditionally viewed as being very compact, with relatively low amounts of repetitive and non-coding DNA, this view has dramatically changed in recent years. The increase of available complete bacterial genomes has revealed that many species present abundant repetitive DNA (i.e., insertion sequences, prophages or paralogous genes) and that many of these sequences are not functional but can have evolutionary consequences as concerns the adaptation to specialized host-related ecological niches. Comparative genomics analyses with close relatives that live in non-specialized environments reveal the nature and fate of this bacterial junk DNA. In addition, the number of insertion sequences and pseudogenes, as well as the size of the intergenic regions, can be used as markers of the evolutionary stage of a genome. PMID:24705080

  15. Liquid-Crystalline Mesophases of Plasmid DNA in Bacteria

    NASA Astrophysics Data System (ADS)

    Reich, Ziv; Wachtel, Ellen J.; Minsky, Abraham

    1994-06-01

    Bacterial plasmids may often reach a copy number larger than 1000 per cell, corresponding to a total amount of DNA that may exceed the amount of DNA within the bacterial chromosome. This observation highlights the problem of cellular accommodation of large amounts of closed-circular nucleic acids, whose interwound conformation offers negligible DNA compaction. As determined by x-ray scattering experiments conducted on intact bacteria, supercoiled plasmids segregate within the cells into dense clusters characterized by a long-range order. In vitro studies performed at physiological DNA concentrations indicated that interwound DNA spontaneously forms liquid crystalline phases whose macroscopic structural properties are determined by the features of the molecular supercoiling. Because these features respond to cellular factors, DNA supercoiling may provide a sensitive regulatory link between cellular parameters and the packaging modes of interwound DNA in vivo.

  16. DNA Repair Is Associated with Information Content in Bacteria, Archaea, and DNA Viruses.

    PubMed

    Acosta, Sharlene; Carela, Miguelina; Garcia-Gonzalez, Aurian; Gines, Mariela; Vicens, Luis; Cruet, Ricardo; Massey, Steven E

    2015-01-01

    The concept of a "proteomic constraint" proposes that DNA repair capacity is positively correlated with the information content of a genome, which can be approximated to the size of the proteome (P). This in turn implies that DNA repair genes are more likely to be present in genomes with larger values of P. This stands in contrast to the common assumption that informational genes have a core function and so are evenly distributed across organisms. We examined the presence/absence of 18 DNA repair genes in bacterial genomes. A positive relationship between gene presence and P was observed for 17 genes in the total dataset, and 16 genes when only nonintracellular bacteria were examined. A marked reduction of DNA repair genes was observed in intracellular bacteria, consistent with their reduced value of P. We also examined archaeal and DNA virus genomes, and show that the presence of DNA repair genes is likewise related to a larger value of P. In addition, the products of the bacterial genes mutY, vsr, and ndk, involved in the correction of GC/AT mutations, are strongly associated with reduced genome GC content. We therefore propose that a reduction in information content leads to a loss of DNA repair genes and indirectly to a reduction in genome GC content in bacteria by exposure to the underlying AT mutation bias. The reduction in P may also indirectly lead to the increase in substitution rates observed in intracellular bacteria via loss of DNA repair genes.

  17. Immunochemical Detection and Isolation of DNA from Metabolically Active Bacteria

    PubMed Central

    Urbach, Ena; Vergin, Kevin L.; Giovannoni, Stephen J.

    1999-01-01

    Most techniques used to assay the growth of microbes in natural communities provide no information on the relationship between microbial productivity and community structure. To identify actively growing bacteria, we adapted a technique from immunocytochemistry to detect and selectively isolate DNA from bacteria incorporating bromodeoxyuridine (BrdU), a thymidine analog. In addition, we developed an immunocytochemical protocol to visualize BrdU-labeled microbial cells. Cultured bacteria and natural populations of aquatic bacterioplankton were pulse-labeled with exogenously supplied BrdU. Incorporation of BrdU into microbial DNA was demonstrated in DNA dot blots probed with anti-BrdU monoclonal antibodies and either peroxidase- or Texas red-conjugated secondary antibodies. BrdU-containing DNA was physically separated from unlabeled DNA by using antibody-coated paramagnetic beads, and the identities of bacteria contributing to both purified, BrdU-containing fractions and unfractionated, starting-material DNAs were determined by length heterogeneity PCR (LH-PCR) analysis. BrdU-containing DNA purified from a mixture of DNAs from labeled and unlabeled cultures showed >90-fold enrichment for the labeled bacterial taxon. The LH-PCR profile for BrdU-containing DNA from a labeled, natural microbial community differed from the profile for the community as a whole, demonstrating that BrdU was incorporated by a taxonomic subset of the community. Immunocytochemical detection of cells with BrdU-labeled DNA was accomplished by in situ probing with anti-BrdU monoclonal antibodies and Texas red-labeled secondary antibodies. Using this suite of techniques, microbial cells incorporating BrdU into their newly synthesized DNA can be quantified and the identities of these actively growing cells can be compared to the composition of the microbial community as a whole. Since not all strains tested could incorporate BrdU, these methods may be most useful when used to gain an understanding

  18. Characterizing self-similarity in bacteria DNA sequences

    NASA Astrophysics Data System (ADS)

    Lu, Xin; Sun, Zhirong; Chen, Huimin; Li, Yanda

    1998-09-01

    In this paper some parametric methods are introduced to characterize the self-similarity of DNA sequences. Compared with Fourier analysis, these methods perform statistically more stably and yield more reliable results. Using these methods, eight whole genomes of bacteria provided by NCBI are analyzed. Long-range correlation properties in the nucleotide density distribution along these DNA sequences are explored. Estimation results show that the long-range correlation structure prevails through the entire molecule of DNA. Higher order statistics through coarse graining reveal that rather than multifractal, there are only monofractal phenomena presented in the sequences. Hence, the nucleotide density distribution can be modeled asymptotically as fractional Gaussian noise. This result points to a new direction for analyzing and understanding the intrinsic structures of DNA sequences.

  19. DNA Integrity and Shock Wave Transformation Efficiency of Bacteria and Fungi

    NASA Astrophysics Data System (ADS)

    Loske, Achim M.; Campos-Guillén, Juan; Fernández, Francisco; Pastrana, Xóchitl; Magaña-Ortíz, Denis; Coconi-Linares, Nancy; Ortíz-Vázquez, Elizabeth; Gómez-Lim, Miguel

    Delivery of DNA into bacteria and fungi is essential in medicine and biotechnology to produce metabolites, enzymes, antibiotics and proteins. So far, protocols to genetically transform bacteria and fungi are inefficient and have low reproducibility.

  20. Social amoebae trap and kill bacteria by casting DNA nets

    PubMed Central

    Zhang, Xuezhi; Zhuchenko, Olga; Kuspa, Adam; Soldati, Thierry

    2016-01-01

    Extracellular traps (ETs) from neutrophils are reticulated nets of DNA decorated with anti-microbial granules, and are capable of trapping and killing extracellular pathogens. Various phagocytes of mammals and invertebrates produce ETs, however, the evolutionary history of this DNA-based host defence strategy is unclear. Here we report that Sentinel (S) cells of the multicellular slug stage of the social amoeba Dictyostelium discoideum produce ETs upon stimulation with bacteria or lipopolysaccharide in a reactive oxygen species-dependent manner. The production of ETs by S cells requires a Toll/Interleukin-1 receptor domain-containing protein TirA and reactive oxygen species-generating NADPH oxidases. Disruption of these genes results in decreased clearance of bacterial infections. Our results demonstrate that D. discoideum is a powerful model organism to study the evolution and conservation of mechanisms of cell-intrinsic immunity, and suggest that the origin of DNA-based ETs as an innate immune defence predates the emergence of metazoans. PMID:26927887

  1. Social amoebae trap and kill bacteria by casting DNA nets.

    PubMed

    Zhang, Xuezhi; Zhuchenko, Olga; Kuspa, Adam; Soldati, Thierry

    2016-03-01

    Extracellular traps (ETs) from neutrophils are reticulated nets of DNA decorated with anti-microbial granules, and are capable of trapping and killing extracellular pathogens. Various phagocytes of mammals and invertebrates produce ETs, however, the evolutionary history of this DNA-based host defence strategy is unclear. Here we report that Sentinel (S) cells of the multicellular slug stage of the social amoeba Dictyostelium discoideum produce ETs upon stimulation with bacteria or lipopolysaccharide in a reactive oxygen species-dependent manner. The production of ETs by S cells requires a Toll/Interleukin-1 receptor domain-containing protein TirA and reactive oxygen species-generating NADPH oxidases. Disruption of these genes results in decreased clearance of bacterial infections. Our results demonstrate that D. discoideum is a powerful model organism to study the evolution and conservation of mechanisms of cell-intrinsic immunity, and suggest that the origin of DNA-based ETs as an innate immune defence predates the emergence of metazoans.

  2. From Rising Bubble to RNA/DNA and Bacteria

    NASA Astrophysics Data System (ADS)

    Marks, Roman; Cieszyńska, Agata; Wereszka, Marzena; Borkowski, Wojciech

    2017-04-01

    In this study we have focused on the movement of rising bubbles in a salty water body. Experiments reviled that free buoyancy movement of bubbles forces displacement of ions, located on the outer side of the bubble wall curvatures. During the short moment of bubble passage, all ions in the vicinity of rising bubble, are separated into anions that are gathered on the bubble upper half sphere and cations that slip along the bottom concave half-sphere of a bubble and develop a sub-bubble vortex. The principle of ions separation bases on the differences in displacement resistance. In this way, relatively heavier and larger, thus more resistant to displacement anions are gathered on the rising bubble upper half sphere, while smaller and lighter cations are assembled on the bottom half sphere and within the sub-bubble vortex. The acceleration of motion generates antiparallel rotary of bi-ionic domains, what implies that anions rotate in clockwise (CW) and cationic in counter-clockwise (CCW) direction. Then, both rotational systems may undergo splicing and extreme condensing by bi-pirouette narrowing of rotary. It is suggested that such double helix motion of bi-ionic domains creates RNA/DNA molecules. Finally, when the bubble reaches the water surface it burst and the preprocessed RNA/DNA matter is ejected into the droplets. Since that stage, droplet is suspended in positively charged troposphere, thus the cationic domain is located in the droplet center, whilst negative ions are attracted to configure the outer areola. According to above, the present study implies that the rising bubbles in salty waters may incept synergistic processing of matter resulting in its rotational/spherical organization that led to assembly of RNA/DNA molecules and bacteria cells.

  3. Salivary gland monomorphic adenoma. Ultrastructural, immunoperoxidase, and histogenetic aspects.

    PubMed Central

    Dardick, I.; Kahn, H. J.; Van Nostrand, A. W.; Baumal, R.

    1984-01-01

    Monomorphic adenoma of basal cell type is a salivary gland tumor believed to result from a proliferation of a single type of cell. However, ultrastructural and immunocytochemical investigations of 6 monomorphic adenomas (5 from parotid and 1 from intraoral minor salivary gland) indicate that there are two classes of these lesions, one composed of two types of tumor cells and the other wholly or predominantly made up of one type of cell (isomorphic). In the former group, the organization of the tumor cells closely mimicked that of normal and hyperplastic salivary gland intercalated ducts. Aggregates of tumor cells were arranged as an inner layer of luminal epithelial cells which were surrounded by an outer layer of cells that, in some cases, had ultrastructural and immunohistochemical features indicating myoepithelial cell differentiation. In some adenomas formed by two types of tumor cells, basal-lamina-lined extracellular spaces were identified ultrastructurally in relation to modified myoepithelial cells; such spaces had the same fine-structural features as those reported in pleomorphic adenoma and adenoid cystic carcinoma. Predominantly isomorphic adenomas were composed exclusively of luminal epithelial cells. These results indicate that despite the varied histologic patterns in the numerous subtypes of monomorphic adenoma, there is a central theme of differentiation and organization in this type of neoplasm which recapitulates the ductoacinar unit of normal salivary gland parenchyma. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 PMID:6375388

  4. The same but different: monomorphic microsatellite markers as a new tool for genetic analysis.

    PubMed

    Nazareno, Alison G; dos Reis, Mauricio S

    2011-10-01

    The nucleotide variation at a microsatellite locus lacking length polymorphisms among its alleles was assessed to generate an informative tool for genetic analysis. From a set of microsatellite markers, a monomorphic microsatellite locus developed for the palm species Butia eriospatha was used to elucidate whether there are polymorphic sites in its flanking regions. DNA sequences ≈133 bp long were obtained. Aligned sequences show variation at 17 polymorphic sites with both insertions and nucleotide substitutions. Fourteen distinct sequences (alleles) among 22 individuals were identified. The percent sequence difference varied from 0.0 to 5%, indicating that there is significant variation among sequences. Due to significant levels of information and sequence diversity on a simple sequence repeat (SSR) locus of identical size, our study highlights that this molecular marker class can be a useful tool for population genetics and evolutionary studies for many plant species.

  5. Working with DNA & Bacteria in Precollege Science Classrooms.

    ERIC Educational Resources Information Center

    Horn, Toby Mogollon; Frame, Kathy, Ed.

    This document describes ways to work with DNA and host organisms in precollege classrooms. The guidelines are intended to assist the teacher who already has training in working with microbes, DNA, and associated chemicals. The contents of the guidelines include: (1) Permitted DNA molecules, vectors, and recommended host organisms for constructing…

  6. Working with DNA & Bacteria in Precollege Science Classrooms.

    ERIC Educational Resources Information Center

    Horn, Toby Mogollon; Frame, Kathy, Ed.

    This document describes ways to work with DNA and host organisms in precollege classrooms. The guidelines are intended to assist the teacher who already has training in working with microbes, DNA, and associated chemicals. The contents of the guidelines include: (1) Permitted DNA molecules, vectors, and recommended host organisms for constructing…

  7. DNA-DNA relatedness and phylogenetic positions of Slackia exigua, Slackia heliotrinireducens, Eggerthella lenta, and other related bacteria.

    PubMed

    Nakazawa, F; Hoshino, E

    2004-10-01

    Recently, two asaccharolytic Eubacterium species, Eubacterium exiguum and Eubacterium lentum, and Peptostreptococcus heliotrinreducens have been reclassified as Slackia exigua, Eggerthella lenta and Slackia heliotrinireducens in the novel genera on the basis of 16S rDNA sequence analysis. But DNA-DNA relatedness among these species and other related bacteria have not been reported yet. DNA-DNA relatedness is the standard arbiter and the recommended method for the designation and evaluation of new species, particularly closely related ones. In the present study, DNA-DNA hybridization studies were performed on S. exigua, S. heliotrinireducens and E. lenta together with the other bacterial species in the related genera. The phylogenetic relationships of these species were also investigated by comparison analysis of 16S rDNA sequence data. In the DNA-DNA hybridization studies, S. exigua showed a DNA homology level of 33% to S. heliotrinireducens and 11% to E. lenta. DNA-DNA homology between S. heliotrinireducens and E. lenta was 10%. But these three species showed very low homology (less than 5%) to the related asaccharolytic species such as Eubacterium and Mogibacterium. In conclusion, the DNA-DNA relatedness data together with the evolutionary data in the present paper further support the reclassification of Eubacterium exiguum, Peptostreptococcus heliotrinreducens and Eubacterium lentum as Slackia exigua, Slackia heliotrinireducens and Eggerthella lenta, respectively.

  8. [The efficacy of verapamil in sustained monomorphic ventricular tachycardia].

    PubMed

    Rumoroso, J R; Bodegas, A; Subinas, J; Montes, P M; Sanz, R; Rodrigo, D; Barrenetxea, J I

    1994-09-01

    We present a 36-year-old male without overt cardiac disease who suffered, since he was 15 years old, from sustained monomorphic ventricular tachycardia of left bundle branch block with a right axis, lasting for several hours; sometimes, syncope was a clinical form of manifestation. Electrophysiologic study, twenty-four hours Holter recording, cardiac catheterization and blood analysis were not useful in order to find its etiology. Efficacy of different drugs, like Mexiletil, Amiodarone, Atenolol and Verapamil (at a dose of 240 mg/day) were tested through multistaged graded-treadmill stress-testing using the Bruce protocol. Ventricular tachycardia was suppressed by administration of Lidocaine. Oral verapamil given at a dose of 360 mg/day prevented the induction of the arrhythmia, the efficacy was tested with maximal treadmill exercise and twenty-four hours Holter recording.

  9. Isolation and characterization of yeast monomorphic mutants of Candida albicans.

    PubMed Central

    Elorza, M V; Sentandreu, R; Ruiz-Herrera, J

    1994-01-01

    A method was devised for the isolation of yeast monomorphic (LEV) mutants of Candida albicans. By this procedure, about 20 stable yeast-like mutants were isolated after mutagenesis with ethyl methane sulfonate. The growth rate of the mutants in different carbon sources, both fermentable and not, was indistinguishable from that of the parental strain, but they were unable to grow as mycelial forms after application of any of the common effective inducers, i.e., heat shock, pH alterations, proline addition, or use of GlcNAc as the carbon source. Studies performed with one selected strain demonstrated that it had severe alterations in the chemical composition of the cell wall, mainly in the levels of chitin and glucans, and in specific mannoproteins, some of them recognizable by specific polyclonal and monoclonal antibodies. It is suggested that these structural alterations hinder the construction of a normal hyphal wall. Images PMID:8157600

  10. How-to-Do-It: A Simple DNA Isolation Technique Using Halophilic Bacteria.

    ERIC Educational Resources Information Center

    Guilfoile, Patrick

    1989-01-01

    Described is a simple technique for isolating DNA from halophilic bacteria. Materials, procedure, and additional experiments are outlined. It is stated that the DNA obtained will be somewhat contaminated with cellular proteins and RNA. Offers a procedure for greater purification. (RT)

  11. How-to-Do-It: A Simple DNA Isolation Technique Using Halophilic Bacteria.

    ERIC Educational Resources Information Center

    Guilfoile, Patrick

    1989-01-01

    Described is a simple technique for isolating DNA from halophilic bacteria. Materials, procedure, and additional experiments are outlined. It is stated that the DNA obtained will be somewhat contaminated with cellular proteins and RNA. Offers a procedure for greater purification. (RT)

  12. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.” (a... killing or growth inhibition of repair deficient bacteria in a set of repair proficient and deficient...

  13. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.” (a... killing or growth inhibition of repair deficient bacteria in a set of repair proficient and deficient...

  14. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.” (a... killing or growth inhibition of repair deficient bacteria in a set of repair proficient and deficient...

  15. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.” (a... killing or growth inhibition of repair deficient bacteria in a set of repair proficient and deficient...

  16. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.” (a... killing or growth inhibition of repair deficient bacteria in a set of repair proficient and deficient...

  17. Determination of DNA Content of Aquatic Bacteria by Flow Cytometry

    PubMed Central

    Button, D. K.; Robertson, Betsy R.

    2001-01-01

    The distribution of DNA among bacterioplankton and bacterial isolates was determined by flow cytometry of DAPI (4′,6′-diamidino-2-phenylindole)-stained organisms. Conditions were optimized to minimize error from nonspecific staining, AT bias, DNA packing, changes in ionic strength, and differences in cell permeability. The sensitivity was sufficient to characterize the small 1- to 2-Mb-genome organisms in freshwater and seawater, as well as low-DNA cells (“dims”). The dims could be formed from laboratory cultivars; their apparent DNA content was 0.1 Mb and similar to that of many particles in seawater. Preservation with formaldehyde stabilized samples until analysis. Further permeabilization with Triton X-100 facilitated the penetration of stain into stain-resistant lithotrophs. The amount of DNA per cell determined by flow cytometry agreed with mean values obtained from spectrophotometric analyses of cultures. Correction for the DNA AT bias of the stain was made for bacterial isolates with known G+C contents. The number of chromosome copies per cell was determined with pure cultures, which allowed growth rate analyses based on cell cycle theory. The chromosome ratio was empirically related to the rate of growth, and the rate of growth was related to nutrient concentration through specific affinity theory to obtain a probe for nutrient kinetics. The chromosome size of a Marinobacter arcticus isolate was determined to be 3.0 Mb by this method. In a typical seawater sample the distribution of bacterial DNA revealed two major populations based on DNA content that were not necessarily similar to populations determined by using other stains or protocols. A mean value of 2.5 fg of DNA cell−1 was obtained for a typical seawater sample, and 90% of the population contained more than 1.1 fg of DNA cell−1. PMID:11282616

  18. Sex-specific foraging behaviour in a monomorphic seabird.

    PubMed Central

    Lewis, S; Benvenuti, S; Dall'Antonia, L; Griffiths, R; Money, L; Sherratt, T N; Wanless, S; Hamer, K C

    2002-01-01

    Sexual differences in the foraging behaviour of parents have been observed in a number of sexually sizedimorphic birds, particularly seabirds, and the usual inference has been that these sex-specific differences are mediated primarily by differences in body size. To test this explanation, we compared the foraging behaviour of parents in a monomorphic seabird species, the northern gannet Morus bassanus. Using specially designed instruments and radio telemetry we found that individuals of both sexes were consistent in the directions and durations of their foraging trips. However, there were significant differences in the foraging behaviour of males and females. Female gannets were not only more selective than males in the areas where they foraged, but they also made longer, deeper dives and spent more time on the sea surface than males. As the sexes are morphologically similar in this species, then these differences are unlikely to have been mediated by body size. Our work highlights the need to investigate sexual differences in the foraging behaviour of seabirds and other species more closely, in order to test alternative theories that do not rely on differences in body size. PMID:12204129

  19. Polymorphism, monomorphism, and sequences in conserved microsatellites in primate species.

    PubMed

    Blanquer-Maumont, A; Crouau-Roy, B

    1995-10-01

    Dimeric short tandem repeats are a source of highly polymorphic markers in the mammalian genome. Genetic variation at these hypervariable loci is extensively used for linkage analysis, for the identification of individuals, and may be useful for interpopulation and interspecies studies. In this paper, we analyze the variability and the sequences of a segment including three microsatellites, first described in man, in several species of primates (chimpanzee, orangutan, gibbon, and macaque) using the heterologous primers (man primers). This region is located on the human chromosome 6p, near the tumor necrosis factor genes, in the major histocompatibility complex. The fact that these primers work in all species studied indicates that they are conserved throughout the different lineages of the two superfamilies, the Hominoidea and the Cercopithecidea, represented by the macaques. However, the intervening sequence displays intraspecific and interspecific variability. The sites of base substitutions and the insertion/deletion events are not evenly distributed within this region. The data suggest that it is necessary to have a minimal number of repeats to increase the rate of mutation sufficiently to allow the development of polymorphism. In some species, the microsatellites present single base variations which reduce the number of contiguous repeats, thus apparently slowing the rate of additional slippage events. Species with such variations or a low number of repeats are monomorphic. These microsatellite sequences are informative in the comparison of closely related species and reflect the phylogeny of the Old World monkeys, apes, and man.

  20. Molecular detection of bacteria in plant tissues, using universal 16S ribosomal DNA degenerated primers

    PubMed Central

    Tsoktouridis, Georgios; Tsiamis, George; Koutinas, Nikolaos; Mantell, Sinclair

    2014-01-01

    Highly specific, sensitive and rapid tests are required for the detection and identification of covert bacterial contaminations in plant tissue cultures. Current methods available for this purpose are tedious, time consuming, highly error prone, expensive, require advanced technical expertise and are sometimes ineffective. We report here the development of a sensitive polymerase chain reaction (PCR) based method for the rapid detection and identification of bacteria occurring in plant tissue cultures. A total of 121 16S ribosomal DNA (rDNA) coding regions from 14 different groups of bacteria, algae and plants, available in the Gene Bank/European Molecular Biology Laboratory databases, were aligned and several conserved DNA sequences of bacterial origin were identified. From those, five degenerated primers were designed in order to amplify only the bacterial DNA present in mixed plant/bacteria genomic DNA extracts. A known amount of bacterial suspension of either covert Pseudomonas or covert Bacillus were added to in vitro plant leaves and total plant/bacterial DNA extracted using three different methods to determine the lowest number of bacteria required to be present in order to allow their detection. The highest sensitivity of the bacterial cell detection was 2.5 × 106 cells of both Bacillus and Pseudomonas inoculums, using template DNA prepared by the MiniPrep method. Generation of PCR amplification fragments was achieved only for the 16S rDNA bacterial gene by using four combinations of degenerated primers. Successive sequence analysis of these amplified fragments led to the rapid detection and molecular identification of bacteria covertly associated with plants. PMID:26019546

  1. DNA extraction protocol for rapid PCR detection of pathogenic bacteria.

    PubMed

    Brewster, Jeffrey D; Paoli, George C

    2013-11-01

    Twelve reagents were evaluated to develop a direct DNA extraction method suitable for PCR detection of foodborne bacterial pathogens. Many reagents exhibited strong PCR inhibition, requiring significant dilution of the extract with a corresponding reduction in sensitivity. Most reagents also exhibited much lower recovery of DNA from the gram-positive test organism (Listeria monocytogenes) than from the gram-negative organism (Escherichia coli O157:H7), preventing unbiased detection and quantitation of both organisms. The 5× HotSHOT+Tween reagent exhibited minimal inhibition and high extraction efficiency for both test organisms, providing a 15-min single-tube DNA-extraction protocol suitable for highly sensitive quantitative PCR assays. Published by Elsevier Inc.

  2. Monomorphic genotypes within a generalist lineage of Campylobacter jejuni show signs of global dispersion

    PubMed Central

    Zhang, Ji; Vehkala, Minna; Välimäki, Niko; Hakkinen, Marjaana; Hänninen, Marja-Liisa; Roasto, Mati; Mäesaar, Mihkel; Taboada, Eduardo; Barker, Dillon; Garofolo, Giuliano; Cammà, Cesare; Di Giannatale, Elisabetta; Corander, Jukka; Rossi, Mirko

    2016-01-01

    The decreased costs of genome sequencing have increased the capability to apply whole-genome sequencing to epidemiological surveillance of zoonotic Campylobacter jejuni. However, knowledge of the genetic diversity of this bacteria is vital for inferring relatedness between epidemiologically linked isolates and a necessary prerequisite for correct application of this methodology. To address this issue in C. jejuni we investigated the spatial and temporal signals in the genomes of a major clonal complex and generalist lineage, ST-45 CC, by analysing the population structure and genealogy as well as applying genome-wide association analysis of 340 isolates from across Europe collected over a wide time range. The occurrence and strength of the geographical signal varied between sublineages and followed the clonal frame when present, while no evidence of a temporal signal was found. Certain sublineages of ST-45 formed discrete and genetically isolated clades containing isolates with extremely similar genomes regardless of time and location of sampling. Based on a separate data set, these monomorphic genotypes represent successful C. jejuni clones, possibly spread around the globe by rapid animal (migrating birds), food or human movement. In addition, we observed an incongruence between the genealogy of the strains and multilocus sequence typing (MLST), challenging the existing clonal complex definition and the use of whole-genome gene-by-gene hierarchical nomenclature schemes for C. jejuni. PMID:28348829

  3. Utility of Filter Paper for Preserving Insects, Bacteria, and Host Reservoir DNA for Molecular Testing

    PubMed Central

    Karimian, F; Sedaghat, MM; Oshaghi, MA; Mohtarami, F; Dehkordi, A Sanei; Koosha, M; Akbari, S; Hashemi-Aghdam, SS

    2011-01-01

    Background: Appropriate methodology for storage biological materials, extraction of DNA, and proper DNA preservation is vital for studies involving genetic analysis of insects, bacteria, and reservoir hosts as well as for molecular diagnostics of pathogens carried by vectors and reservoirs. Here we tried to evaluate the utility of a simple filter paper-based for storage of insects, bacteria, rodent, and human DNAs using PCR assays. Methods: Total body or haemolymph of individual mosquitoes, sand flies or cockroaches squashed or placed on the paper respectively. Extracted DNA of five different bacteria species as well as blood specimens of human and great gerbil Rhombomys opimus was pipetted directly onto filter paper. The papers were stored in room temperature up to 12 months during 2009 until 2011. At monthly intervals, PCR was conducted using a 1-mm disk from the DNA impregnated filter paper as target DNA. PCR amplification was performed against different target genes of the organisms including the ITS2-rDNA of mosquitoes, mtDNA-COI of the sand flies and cockroaches, 16SrRNA gene of the bacteria, and the mtDNA-CytB of the vertebrates. Results: Successful PCR amplification was observed for all of the specimens regardless of the loci, taxon, or time of storage. The PCR amplification were ranged from 462 to 1500 bp and worked well for the specified target gene/s. Time of storage did not affect the amplification up to one year. Conclusion: The filter paper method is a simple and economical way to store, to preserve, and to distribute DNA samples for PCR analysis. PMID:22808417

  4. Cultivation-independent detection of autotrophic hydrogen-oxidizing bacteria by DNA stable-isotope probing.

    PubMed

    Pumphrey, Graham M; Ranchou-Peyruse, Anthony; Spain, Jim C

    2011-07-01

    Knallgas bacteria are a physiologically defined group that is primarily studied using cultivation-dependent techniques. Given that current cultivation techniques fail to grow most bacteria, cultivation-independent techniques that selectively detect and identify knallgas bacteria will improve our ability to study their diversity and distribution. We used stable-isotope probing (SIP) to identify knallgas bacteria in rhizosphere soil of legumes and in a microbial mat from Obsidian Pool in Yellowstone National Park. When samples were incubated in the dark, incorporation of (13)CO(2) was H(2) dependent. SIP enabled the detection of knallgas bacteria that were not detected by cultivation, and the majority of bacteria identified in the rhizosphere soils were betaproteobacteria predominantly related to genera previously known to oxidize hydrogen. Bacteria in soil grew on hydrogen at concentrations as low as 100 ppm. A hydB homolog encoding a putative high-affinity NiFe hydrogenase was amplified from (13)C-labeled DNA from both vetch and clover rhizosphere soil. The results indicate that knallgas bacteria can be detected by SIP and populations that respond to different H(2) concentrations can be distinguished. The methods described here should be applicable to a variety of ecosystems and will enable the discovery of additional knallgas bacteria that are resistant to cultivation.

  5. Atomic Resolution Structure of Monomorphic Aβ42 Amyloid Fibrils.

    PubMed

    Colvin, Michael T; Silvers, Robert; Ni, Qing Zhe; Can, Thach V; Sergeyev, Ivan; Rosay, Melanie; Donovan, Kevin J; Michael, Brian; Wall, Joseph; Linse, Sara; Griffin, Robert G

    2016-08-03

    Amyloid-β (Aβ) is a 39-42 residue protein produced by the cleavage of the amyloid precursor protein (APP), which subsequently aggregates to form cross-β amyloid fibrils that are a hallmark of Alzheimer's disease (AD). The most prominent forms of Aβ are Aβ1-40 and Aβ1-42, which differ by two amino acids (I and A) at the C-terminus. However, Aβ42 is more neurotoxic and essential to the etiology of AD. Here, we present an atomic resolution structure of a monomorphic form of AβM01-42 amyloid fibrils derived from over 500 (13)C-(13)C, (13)C-(15)N distance and backbone angle structural constraints obtained from high field magic angle spinning NMR spectra. The structure (PDB ID: 5KK3 ) shows that the fibril core consists of a dimer of Aβ42 molecules, each containing four β-strands in a S-shaped amyloid fold, and arranged in a manner that generates two hydrophobic cores that are capped at the end of the chain by a salt bridge. The outer surface of the monomers presents hydrophilic side chains to the solvent. The interface between the monomers of the dimer shows clear contacts between M35 of one molecule and L17 and Q15 of the second. Intermolecular (13)C-(15)N constraints demonstrate that the amyloid fibrils are parallel in register. The RMSD of the backbone structure (Q15-A42) is 0.71 ± 0.12 Å and of all heavy atoms is 1.07 ± 0.08 Å. The structure provides a point of departure for the design of drugs that bind to the fibril surface and therefore interfere with secondary nucleation and for other therapeutic approaches to mitigate Aβ42 aggregation.

  6. Sexually Monomorphic Maps and Dimorphic Responses in Rat Genital Cortex.

    PubMed

    Lenschow, Constanze; Copley, Sean; Gardiner, Jayne M; Talbot, Zoe N; Vitenzon, Ariel; Brecht, Michael

    2016-01-11

    Mammalian external genitals show sexual dimorphism [1, 2] and can change size and shape upon sexual arousal. Genitals feature prominently in the oldest pieces of figural art [3] and phallic depictions of penises informed psychoanalytic thought about sexuality [4, 5]. Despite this longstanding interest, the neural representations of genitals are still poorly understood [6]. In somatosensory cortex specifically, many studies did not detect any cortical representation of genitals [7-9]. Studies in humans debate whether genitals are represented displaced below the foot of the cortical body map [10-12] or whether they are represented somatotopically [13-15]. We wondered what a high-resolution mapping of genital representations might tell us about the sexual differentiation of the mammalian brain. We identified genital responses in rat somatosensory cortex in a region previously assigned as arm/leg cortex. Genital responses were more common in males than in females. Despite such response dimorphism, we observed a stunning anatomical monomorphism of cortical penis and clitoris input maps revealed by cytochrome-oxidase-staining of cortical layer 4. Genital representations were somatotopic and bilaterally symmetric, and their relative size increased markedly during puberty. Size, shape, and erect posture give the cortical penis representation a phallic appearance pointing to a role in sexually aroused states. Cortical genital neurons showed unusual multi-body-part responses and sexually dimorphic receptive fields. Specifically, genital neurons were co-activated by distant body regions, which are touched during mounting in the respective sex. Genital maps indicate a deep homology of penis and clitoris representations in line with a fundamentally bi-sexual layout [16] of the vertebrate brain.

  7. Purkinje-related arrhythmias part I: monomorphic ventricular tachycardias.

    PubMed

    Nogami, Akihiko

    2011-05-01

    Purkinje-related monomorphic ventricular tachycardias (VTs) can be classified into four distinct groups: (1) verapamil-sensitive left fascicular VT, (2) Purkinje fiber-mediated VT post infarction, (3) bundle branch reentry (BBR) and interfascicular reentry VTs, and (4) focal Purkinje VT. There are three subtypes of fascicular VTs: (1) left posterior fascicular VT with a right bundle branch block (RBBB) configuration and superior axis; (2) left anterior fascicular VT with an RBBB configuration and right-axis deviation; and (3) upper septal fascicular VT with a narrow QRS configuration. The mechanism of the fascicular VT is macroreentry. While the antegrade limb of the circuit is a midseptal abnormal Purkinje fiber in the anterior and posterior fascicular VTs, the antegrade limb of the upper septal fascicular VT is both the anterior and posterior fascicles, and the retrograde limb is a midseptal abnormal Purkinje fiber. Purkinje fiber-mediated VT post infarction also exhibits verapamil sensitivity, and the surviving muscle bundles within the myocardium and Purkinje system are components of the reentry circuit. BBR-VT and interfascicular reentry VT are amenable to being cured by the creation of bundle or fascicular block. The mechanism of focal Purkinje VT is abnormal automaticity from the distal Purkinje system, and the ablation target is the earliest Purkinje activation during the VT. It is difficult to distinguish verapamil-sensitive fascicular VT from focal Purkinje VT by the 12-lead electrocardiogram; however, focal Purkinje VT is not responsive to verapamil . The recognition of the heterogeneity of these VTs and their unique characteristics should facilitate an appropriate diagnosis and therapy.

  8. Identification of dairy lactic acid bacteria by tRNAAla-23S rDNA-RFLP.

    PubMed

    Mancini, Andrea; Lazzi, Camilla; Bernini, Valentina; Neviani, Erasmo; Gatti, Monica

    2012-12-01

    The aim of this study was to evaluate the potential of target tRNA(Ala)-23S ribosomal DNA for identification of lactic acid bacteria strains associated with dairy ecosystem. For this purpose tRNA(Ala)-23S ribosomal DNA Restriction Fragment Length Polymorphism (tRNA(Ala)-23S rDNA-RFLP) was compared with two widely used DNA fingerprinting methods - P1 Random Amplified Polymorphic DNA (RAPD), (GTG)5 repetitive extragenic palindromic PCR (rep-PCR) - for their ability to identify different species on a set of 10 type and 34 reference strains. Moreover, 75 unknown isolates collected during different stages of Grana Padano cheese production and ripening were identified using tRNA(Ala)-23S rDNA-RFLP and compared to the RFLP profiles of the strains in the reference database. This study demonstrated that the target tRNA(Ala)-23S rDNA has high potential in bacterial identification and tRNA(Ala)-23S rDNA-RFLP is a promising method for reliable species-level identification of lactic acid bacteria (LAB) in dairy products.

  9. Incorporation of DNA and protein precursors into macromolecules by bacteria at -15 degrees C.

    PubMed

    Christner, Brent C

    2002-12-01

    DNA and protein precursors were incorporated into trichloroacetic acid-precipitated material by bacterial cell suspensions during incubation for 50 to 100 days at -15 degrees C. Incorporation did not occur at -70 degrees C and was inhibited by antibiotics. The results demonstrate that bacteria can perform macromolecular synthesis under conditions that mimic entrapment in glacial ice.

  10. DNA from oral bacteria by sodium hydroxide-paper method suitable for polymerase chain reaction.

    PubMed

    Lefimil, Claudia; Lozano, Carla; Morales-Bozo, Irene; Plaza, Anita; Maturana, Cristian; Urzúa, Blanca

    2013-02-15

    In the oral cavity, we can find a complex mixture of microorganisms, commensals, and pathogens. The studies of normal oral microbiota, as well as the studies of much oral pathology (e.g., caries, periodontitis), involve the isolation and cultivation of these microorganisms and their molecular analysis. The aim of this study was to validate a quick, easy, efficient, and inexpensive DNA extraction method for the recovery of genomic DNA from gram-positive and gram-negative oral bacteria to be used in polymerase chain reaction amplification. This method worked great with all samples analyzed, providing an approach to extract DNA for different microorganisms.

  11. Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays.

    PubMed

    Polen, Tino; Wendisch, Volker F

    2004-01-01

    DNA microarray technology has become an important research tool for biotechnology and microbiology. It is now possible to characterize genetic diversity and gene expression in a genomewide manner. DNA microarrays have been applied extensively to study the biology of many bacteria including Escherichia coli, but only recently have they been developed for the Gram-positive Corynebacterium glutamicum. Both bacteria are widely used for biotechnological amino acid production. In this article, in addition to the design and generation of microarrays as well as their use in hybridization experiments and subsequent data analysis, we describe recent applications of DNA microarray technology regarding amino acid production in C. glutamicum and E. coli. We also discuss the impact of functional genomics studies on fundamental as well as applied aspects of amino acid production with C. glutamicum and E. coli.

  12. Identification of DNA Methyltransferase Genes in Human Pathogenic Bacteria by Comparative Genomics.

    PubMed

    Brambila-Tapia, Aniel Jessica Leticia; Poot-Hernández, Augusto Cesar; Perez-Rueda, Ernesto; Rodríguez-Vázquez, Katya

    2016-06-01

    DNA methylation plays an important role in gene expression and virulence in some pathogenic bacteria. In this report, we describe DNA methyltransferases (MTases) present in human pathogenic bacteria and compared them with related species, which are not pathogenic or less pathogenic, based in comparative genomics. We performed a search in the KEGG database of the KEGG database orthology groups associated with adenine and cytosine DNA MTase activities (EC: 2.1.1.37, EC: 2.1.1.113 and EC: 2.1.1.72) in 37 human pathogenic species and 18 non/less pathogenic relatives and performed comparisons of the number of these MTases sequences according to their genome size, the DNA MTase type and with their non-less pathogenic relatives. We observed that Helicobacter pylori and Neisseria spp. presented the highest number of MTases while ten different species did not present a predicted DNA MTase. We also detected a significant increase of adenine MTases over cytosine MTases (2.19 vs. 1.06, respectively, p < 0.001). Adenine MTases were the only MTases associated with restriction modification systems and DNA MTases associated with type I restriction modification systems were more numerous than those associated with type III restriction modification systems (0.84 vs. 0.17, p < 0.001); additionally, there was no correlation with the genome size and the total number of DNA MTases, indicating that the number of DNA MTases is related to the particular evolution and lifestyle of specific species, regulating the expression of virulence genes in some pathogenic bacteria.

  13. Principles and Concepts of DNA Replication in Bacteria, Archaea, and Eukarya

    PubMed Central

    O’Donnell, Michael; Langston, Lance; Stillman, Bruce

    2013-01-01

    The accurate copying of genetic information in the double helix of DNA is essential for inheritance of traits that define the phenotype of cells and the organism. The core machineries that copy DNA are conserved in all three domains of life: bacteria, archaea, and eukaryotes. This article outlines the general nature of the DNA replication machinery, but also points out important and key differences. The most complex organisms, eukaryotes, have to coordinate the initiation of DNA replication from many origins in each genome and impose regulation that maintains genomic integrity, not only for the sake of each cell, but for the organism as a whole. In addition, DNA replication in eukaryotes needs to be coordinated with inheritance of chromatin, developmental patterning of tissues, and cell division to ensure that the genome replicates once per cell division cycle. PMID:23818497

  14. Overexpression of a pea DNA helicase 45 in bacteria confers salinity stress tolerance.

    PubMed

    Tajrishi, Marjan M; Vaid, Neha; Tuteja, Renu; Tuteja, Narendra

    2011-09-01

    Salinity stress is one of the major factors negatively affecting growth and productivity in living organisms including plants and bacteria resulting in significant losses worldwide. Therefore, it would be fruitful to develop salinity stress tolerant useful species and also to understand the mechanism of stress tolerance. The pea DNA helicase 45 (PDH45) is a DNA and RNA helicase, homologous to eukaryotic translation initiation factor 4A (eIF-4A) and is involved in various processes including protein synthesis, maintaining the basic activities of the cell, upregulation of topoisomerase I activity and salinity stress tolerance in plant, but its role in salinity stress tolerance in bacteria has not heretofore been studied. This study provides an evidence for a novel function of the PDH45 gene in high salinity (NaCl) stress tolerance in bacteria (Eschericia coli, BL21 cells) also. Furthermore, it has been shown that the functionally active PDH45 gene is required to show the stress tolerance in bacteria because the single mutants (E183G or R363Q) and the double mutant (E183G + R363Q) of the gene could not confer the same function. The response was specific to Na+ ions as the bacteria could not grow in presence of LiCl. This study suggests that the cellular response to high salinity stress across prokaryotes and plant kingdom is conserved and also helps in our better understanding of mechanism of stress tolerance in bacteria and plants. It could also be very useful in developing high salinity stress tolerant useful bacteria of agronomic importance. Overall, this study provides an evidence for a novel function of the PDH45 gene in high salinity stress tolerance in bacteria.

  15. Horizontal DNA transfer from bacteria to eukaryotes and a lesson from experimental transfers.

    PubMed

    Suzuki, Katsunori; Moriguchi, Kazuki; Yamamoto, Shinji

    2015-12-01

    Horizontal gene transfer (HGT) is widespread among bacteria and plays a key role in genome dynamics. HGT is much less common in eukaryotes, but is being reported with increasing frequency in eukaryotes. The mechanism as to how eukaryotes acquired genes from distantly related organisms remains obscure yet. This paper cites examples of bacteria-derived genes found in eukaryotic organisms, and then describes experimental DNA transports to eukaryotes by bacterial type 4 secretion systems in optimized conditions. The mechanisms of the latter are efficient, quite reproducible in vitro and predictable, and thereby would provide insight into natural HGT and to the development of new research tools.

  16. Magnetic Scanometric DNA Microarray Detection of Methyl Tertiary Butyl Ether Degrading Bacteria for Environmental Monitoring

    PubMed Central

    Chan, Mei-Lin; Jaramillo, Gerardo; Hristova, Krassimira R.; Horsley, David A.

    2010-01-01

    A magnetoresistive biosensing platform based on a single magnetic tunnel junction (MTJ) scanning probe and DNA microarrays labeled with magnetic particles has been developed to provide an inexpensive, sensitive and reliable detection of DNA. The biosensing platform was demonstrated on a DNA microarray assay for quantifying bacteria capable of degrading methyl tertiary-butyl ether (MTBE), where concentrations as low as 10 pM were detectable. Synthetic probe bacterial DNA was immobilized on a microarray glass slide surface, hybridized with the 48 base pair long biotinylated target DNA and subsequently incubated with streptavidin-coated 2.8 μm diameter magnetic particles. The biosensing platform then makes use of a micron-sized MTJ sensor that was raster scanned across a 3 mm by 5 mm glass slide area to capture the stray magnetic field from the tagged DNA and extract two dimensional magnetic field images of the microarray. The magnetic field output is then averaged over each 100 μm diameter DNA array spot to extract the magnetic spot intensity, analogous to the fluorescence spot intensity used in conventional optical scanners. The magnetic scanning result is compared with results from a commercial laser scanner and particle coverage optical counting to demonstrate the dynamic range and linear sensitivity of the biosensing platform as a potentially inexpensive, sensitive and portable alternative for DNA microarray detection for field applications. PMID:20889328

  17. DNA/Ag Nanoparticles as Antibacterial Agents against Gram-Negative Bacteria

    PubMed Central

    Takeshima, Tomomi; Tada, Yuya; Sakaguchi, Norihito; Watari, Fumio; Fugetsu, Bunshi

    2015-01-01

    Silver (Ag) nanoparticles were produced using DNA extracted from salmon milt as templates. Particles spherical in shape with an average diameter smaller than 10 nm were obtained. The nanoparticles consisted of Ag as the core with an outermost thin layer of DNA. The DNA/Ag hybrid nanoparticles were immobilized over the surface of cotton based fabrics and their antibacterial efficiency was evaluated using E. coli as the typical Gram-negative bacteria. The antibacterial experiments were performed according to the Antibacterial Standard of Japanese Association for the Functional Evaluation of Textiles. The fabrics modified with DNA/Ag nanoparticles showed a high enough inhibitory and killing efficiency against E. coli at a concentration of Ag ≥ 10 ppm. PMID:28347012

  18. DNA/Ag Nanoparticles as Antibacterial Agents against Gram-Negative Bacteria.

    PubMed

    Takeshima, Tomomi; Tada, Yuya; Sakaguchi, Norihito; Watari, Fumio; Fugetsu, Bunshi

    2015-03-03

    Silver (Ag) nanoparticles were produced using DNA extracted from salmon milt as templates. Particles spherical in shape with an average diameter smaller than 10 nm were obtained. The nanoparticles consisted of Ag as the core with an outermost thin layer of DNA. The DNA/Ag hybrid nanoparticles were immobilized over the surface of cotton based fabrics and their antibacterial efficiency was evaluated using E. coli as the typical Gram-negative bacteria. The antibacterial experiments were performed according to the Antibacterial Standard of Japanese Association for the Functional Evaluation of Textiles. The fabrics modified with DNA/Ag nanoparticles showed a high enough inhibitory and killing efficiency against E. coli at a concentration of Ag ≥ 10 ppm.

  19. Microfluidic chip integrating high throughput continuous-flow PCR and DNA hybridization for bacteria analysis.

    PubMed

    Jiang, Xiran; Shao, Ning; Jing, Wenwen; Tao, Shengce; Liu, Sixiu; Sui, Guodong

    2014-05-01

    Rapid identification of clinical pathogens is the initial and essential step for antimicrobial therapy. Herein, we successfully developed a microfluidic device which combines high-throughput continuous-flow PCR and DNA hybridization for the detection of various bacterial pathogens. Universal primers were designed based on the conserved regions of bacterial 16S ribosomal DNA (16S rDNA), and specific probes were designed from a variable region of 16S rDNA within the amplicon sequences. In the chip operation, after the continuous flow PCR was achieved in the first microfluidic chip, the product was directly introduced into a hybridization chip integrated with microarray containing the immobilized DNA probes. The target-probe hybridization was completed within 1h at 55 °C, and fluorescence signals were obtained as the readout. The presented device is simple, versatile and with less sample consumption compared with traditional instruments. It can perform high-throughput bacteria detections continuously in a single assay, which makes it a promising platform for clinical bacteria identifications.

  20. Diversity of bacteria in ships ballast water as revealed by next generation DNA sequencing.

    PubMed

    Brinkmeyer, Robin

    2016-06-15

    The bacterial diversity in ballast water from five general cargo ships calling at the Port of Houston was determined with ion semiconductor DNA sequencing (Ion Torrent PGM) of PCR amplified 16S rRNA genes. Phylogenetic analysis revealed that the composition of bacteria in ballast water did not resemble that of typical marine habitats or even open ocean waters where BWEs occur. The predominant group of bacteria in ships conducting BWEs was the Roseobacter clade within the Alphaproteobacteria. In contrast, Gammaproteobacteria were predominant in the ship that did not conduct a BWE. All the ships contained human, fish, and terrestrial plant pathogens as well as bacteria indicative of fecal or activated sludge contamination. Most of the 60 pathogens had not been detected in ballast water previously. Among these were the human pathogens Corynebacterium diptheriae and several Legionella species and the fish pathogens Francisella piscicida and Piscirickettsia salmonis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Identification of active oxalotrophic bacteria by Bromodeoxyuridine DNA labeling in a microcosm soil experiments.

    PubMed

    Bravo, Daniel; Martin, Gaëtan; David, Maude M; Cailleau, Guillaume; Verrecchia, Eric; Junier, Pilar

    2013-11-01

    The oxalate-carbonate pathway (OCP) leads to a potential carbon sink in terrestrial environments. This process is linked to the activity of oxalotrophic bacteria. Although isolation and molecular characterizations are used to study oxalotrophic bacteria, these approaches do not give information on the active oxalotrophs present in soil undergoing the OCP. The aim of this study was to assess the diversity of active oxalotrophic bacteria in soil microcosms using the Bromodeoxyuridine (BrdU) DNA labeling technique. Soil was collected near an oxalogenic tree (Milicia excelsa). Different concentrations of calcium oxalate (0.5%, 1%, and 4% w/w) were added to the soil microcosms and compared with an untreated control. After 12 days of incubation, a maximal pH of 7.7 was measured for microcosms with oxalate (initial pH 6.4). At this time point, a DGGE profile of the frc gene was performed from BrdU-labeled soil DNA and unlabeled soil DNA. Actinobacteria (Streptomyces- and Kribbella-like sequences), Gammaproteobacteria and Betaproteobacteria were found as the main active oxalotrophic bacterial groups. This study highlights the relevance of Actinobacteria as members of the active bacterial community and the identification of novel uncultured oxalotrophic groups (i.e. Kribbella) active in soils.

  2. Monomorphic ventricular tachycardia in 'Brugada syndrome': clinical case and literature review.

    PubMed

    Allocca, Giuseppe; Proclemer, Alessandro; Nucifora, Gaetano; Dall'Armellina, Erica; Rebellato, Luca

    2008-08-01

    A 20-year-old white judoka was admitted for severe palpitations during exercise followed by syncope. The electrocardiogram on admission revealed a wide-complex monomorphic tachycardia at a rate of 260 beats/min, with right bundle brunch block morphology and right axis deviation. Following electrical cardioversion, the electrocardiogram showed sinus rhythm with type 1 pattern of Brugada syndrome. We describe in detail the clinical course, the results of electrophysiological study, and therapeutic management. We reviewed literature data concerning a few cases of 'atypical Brugada syndrome' characterized by monomorphic ventricular tachycardia as clinical arrhythmia.

  3. 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).

  4. 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).

  5. Relevance of GC content to the conservation of DNA polymerase III/mismatch repair system in Gram-positive bacteria

    PubMed Central

    Akashi, Motohiro; Yoshikawa, Hirofumi

    2013-01-01

    The mechanism of DNA replication is one of the driving forces of genome evolution. Bacterial DNA polymerase III, the primary complex of DNA replication, consists of PolC and DnaE. PolC is conserved in Gram-positive bacteria, especially in the Firmicutes with low GC content, whereas DnaE is widely conserved in most Gram-negative and Gram-positive bacteria. PolC contains two domains, the 3′-5′exonuclease domain and the polymerase domain, while DnaE only possesses the polymerase domain. Accordingly, DnaE does not have the proofreading function; in Escherichia coli, another enzyme DnaQ performs this function. In most bacteria, the fidelity of DNA replication is maintained by 3′-5′ exonuclease and a mismatch repair (MMR) system. However, we found that most Actinobacteria (a group of Gram-positive bacteria with high GC content) appear to have lost the MMR system and chromosomes may be replicated by DnaE-type DNA polymerase III with DnaQ-like 3′-5′ exonuclease. We tested the mutation bias of Bacillus subtilis, which belongs to the Firmicutes and found that the wild type strain is AT-biased while the mutS-deletant strain is remarkably GC-biased. If we presume that DnaE tends to make mistakes that increase GC content, these results can be explained by the mutS deletion (i.e., deletion of the MMR system). Thus, we propose that GC content is regulated by DNA polymerase and MMR system, and the absence of polC genes, which participate in the MMR system, may be the reason for the increase of GC content in Gram-positive bacteria such as Actinobacteria. PMID:24062730

  6. Biochemical and structural characterization of DNA ligases from bacteria and archaea

    PubMed Central

    Pergolizzi, Giulia; Wagner, Gerd K.; Bowater, Richard P.

    2016-01-01

    DNA ligases are enzymes that seal breaks in the backbones of DNA, leading to them being essential for the survival of all organisms. DNA ligases have been studied from many different types of cells and organisms and shown to have diverse sizes and sequences, with well conserved specific sequences that are required for enzymatic activity. A significant number of DNA ligases have been isolated or prepared in recombinant forms and, here, we review their biochemical and structural characterization. All DNA ligases contain an essential lysine that transfers an adenylate group from a co-factor to the 5′-phosphate of the DNA end that will ultimately be joined to the 3′-hydroxyl of the neighbouring DNA strand. The essential DNA ligases in bacteria use β-nicotinamide adenine dinucleotide (β-NAD+) as their co-factor whereas those that are essential in other cells use adenosine-5′-triphosphate (ATP) as their co-factor. This observation suggests that the essential bacterial enzyme could be targeted by novel antibiotics and the complex molecular structure of β-NAD+ affords multiple opportunities for chemical modification. Several recent studies have synthesized novel derivatives and their biological activity against a range of DNA ligases has been evaluated as inhibitors for drug discovery and/or non-natural substrates for biochemical applications. Here, we review the recent advances that herald new opportunities to alter the biochemical activities of these important enzymes. The recent development of modified derivatives of nucleotides highlights that the continued combination of structural, biochemical and biophysical techniques will be useful in targeting these essential cellular enzymes. PMID:27582505

  7. Reduction in the structural instability of cloned eukaryotic tandem-repeat DNA by low-temperature culturing of host bacteria.

    PubMed

    Thapana, Watcharaporn; Sujiwattanarat, Penporn; Srikulnath, Kornsorn; Hirai, Hirohisa; Koga, Akihiko

    2014-10-27

    Summary For accurate analyses of eukaryotic tandem-repeat DNA, it is often required to clone a genomic DNA fragment into a bacterial plasmid. It is, however, a serious problem that tandem-repeat DNA is frequently subjected to structural changes during maintenance or amplification in the host bacteria. Here, we show an example of a clear difference in the instability of tandem-repeat DNA between different culturing temperatures. A fragment of monkey centromeric DNA carried by pUC19 was considerably degraded by culturing bacteria at 37 °C, but the damage was reduced at 25 °C. Thus, culturing temperature is a significant factor for avoiding degradation, in addition to the genotype of the host bacteria.

  8. Algae–bacteria association inferred by 16S rDNA similarity in established microalgae cultures

    PubMed Central

    Schwenk, Dagmar; Nohynek, Liisa; Rischer, Heiko

    2014-01-01

    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. PMID:24799387

  9. 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.

  10. Ingestion of bacteria overproducing DnaK attenuates Vibrio infection of Artemia franciscana larvae

    PubMed Central

    Dhaene, Till; Defoirdt, Tom; Boon, Nico; MacRae, Thomas H.; Sorgeloos, Patrick; Bossier, Peter

    2009-01-01

    Feeding of bacterially encapsulated heat shock proteins (Hsps) to invertebrates is a novel way to limit Vibrio infection. As an example, ingestion of Escherichia coli overproducing prokaryotic Hsps significantly improves survival of gnotobiotically cultured Artemia larvae upon challenge with pathogenic Vibrio campbellii. The relationship between Hsp accumulation and enhanced resistance to infection may involve DnaK, the prokaryotic equivalent to Hsp70, a major molecular chaperone in eukaryotic cells. In support of this proposal, heat-stressed bacterial strains LVS 2 (Bacillus sp.), LVS 3 (Aeromonas hydrophila), LVS 8 (Vibrio sp.), GR 8 (Cytophaga sp.), and GR 10 (Roseobacter sp.) were shown in this work to be more effective than nonheated bacteria in protecting gnotobiotic Artemia larvae against V. campbellii challenge. Immunoprobing of Western blots and quantification by enzyme-linked immunosorbent assay revealed that the amount of DnaK in bacteria and their ability to enhance larval resistance to infection by V. campbellii are correlated. Although the function of DnaK is uncertain, it may improve tolerance to V. campbellii via immune stimulation, a possibility of significance from a fundamental perspective and also because it could be applied in aquaculture, a major method of food production. PMID:19373565

  11. Ingestion of bacteria overproducing DnaK attenuates Vibrio infection of Artemia franciscana larvae.

    PubMed

    Sung, Yeong Yik; Dhaene, Till; Defoirdt, Tom; Boon, Nico; MacRae, Thomas H; Sorgeloos, Patrick; Bossier, Peter

    2009-11-01

    Feeding of bacterially encapsulated heat shock proteins (Hsps) to invertebrates is a novel way to limit Vibrio infection. As an example, ingestion of Escherichia coli overproducing prokaryotic Hsps significantly improves survival of gnotobiotically cultured Artemia larvae upon challenge with pathogenic Vibrio campbellii. The relationship between Hsp accumulation and enhanced resistance to infection may involve DnaK, the prokaryotic equivalent to Hsp70, a major molecular chaperone in eukaryotic cells. In support of this proposal, heat-stressed bacterial strains LVS 2 (Bacillus sp.), LVS 3 (Aeromonas hydrophila), LVS 8 (Vibrio sp.), GR 8 (Cytophaga sp.), and GR 10 (Roseobacter sp.) were shown in this work to be more effective than nonheated bacteria in protecting gnotobiotic Artemia larvae against V. campbellii challenge. Immunoprobing of Western blots and quantification by enzyme-linked immunosorbent assay revealed that the amount of DnaK in bacteria and their ability to enhance larval resistance to infection by V. campbellii are correlated. Although the function of DnaK is uncertain, it may improve tolerance to V. campbellii via immune stimulation, a possibility of significance from a fundamental perspective and also because it could be applied in aquaculture, a major method of food production.

  12. Speciation is not necessarily easier in species with sexually monomorphic mating signals.

    PubMed

    Noh, S; Henry, C S

    2015-11-01

    Should we have different expectations regarding the likelihood and pace of speciation by sexual selection when considering species with sexually monomorphic mating signals? Two conditions that can facilitate rapid species divergence are Felsenstein's one-allele mechanism and a genetic architecture that includes a genetic association between signal and preference loci. In sexually monomorphic species, the former can manifest in the form of mate choice based on phenotype matching. The latter can be promoted by selection acting upon genetic loci for divergent signals and preferences expressed simultaneously in each individual, rather than acting separately on signal loci in males and preference loci in females. Both sexes in the Chrysoperla carnea group of green lacewings (Insecta, Neuroptera, Chrysopidae) produce sexually monomorphic species-specific mating signals. We hybridized the two species C. agilis and C. carnea to test for evidence of these speciation-facilitating conditions. Hybrid signals were more complex than the parents and we observed a dominant influence of C. carnea. We found a dominant influence of C. agilis on preferences in the form of hybrid discrimination against C. carnea. Preferences in hybrids followed patterns predicting preference loci that determine mate choice rather than a one-allele mechanism. The genetic association between signal and preference we detected in the segregating hybrid crosses indicates that speciation in these species with sexually monomorphic mating signals can have occurred rapidly. However, we need additional evidence to determine whether such genetic associations form more readily in sexually monomorphic species compared to dimorphic species and consequently facilitate speciation.

  13. Transferring whole genomes from bacteria to yeast spheroplasts using entire bacterial cells to reduce DNA shearing.

    PubMed

    Karas, Bogumil J; Jablanovic, Jelena; Irvine, Edward; Sun, Lijie; Ma, Li; Weyman, Philip D; Gibson, Daniel G; Glass, John I; Venter, J Craig; Hutchison, Clyde A; Smith, Hamilton O; Suzuki, Yo

    2014-04-01

    Direct cell-to-cell transfer of genomes from bacteria to yeast facilitates genome engineering for bacteria that are not amenable to genetic manipulation by allowing instead for the utilization of the powerful yeast genetic tools. Here we describe a protocol for transferring whole genomes from bacterial cells to yeast spheroplasts without any DNA purification process. The method is dependent on the treatment of the bacterial and yeast cellular mixture with PEG, which induces cell fusion, engulfment, aggregation or lysis. Over 80% of the bacterial genomes transferred in this way are complete, on the basis of structural and functional tests. Excluding the time required for preparing starting cultures and for incubating cells to form final colonies, the protocol can be completed in 3 h.

  14. DNA fingerprinting of thermophilic lactic acid bacteria using repetitive sequence-based polymerase chain reaction.

    PubMed

    De Urraza, P J; Gómez-Zavaglia, A; Lozano, M E; Romanowski, V; De Antoni, G L

    2000-08-01

    DNA fingerprints of lactic acid bacteria were generated by polymerase chain reaction using a primer based on the repetitive elements found in the genome of Streptococcus pneumoniae (BOX-PCR). The method made it possible to identify 37 isolates from raw milk. industrial starters and yogurt. Differentiation at species, subspecies and strain level was possible for Lactobacillus delbrueckii subsp. lactis, Lb. delbrueckii subsp bulgaricus and Str. thermophilus. BOX-PCR was also applied to studying the strain composition of a starter culture and the direct detection of strains in commercial fermented milk.

  15. Deoxynybomycins inhibit mutant DNA gyrase and rescue mice infected with fluoroquinolone-resistant bacteria

    PubMed Central

    Parkinson, Elizabeth I.; Bair, Joseph S.; Nakamura, Bradley A.; Lee, Hyang Y.; Kuttab, Hani I.; Southgate, Emma H.; Lezmi, Stéphane; Lau, Gee W.; Hergenrother, Paul J.

    2015-01-01

    Fluoroquinolones are one of the most commonly prescribed classes of antibiotics, but fluoroquinolone resistance (FQR) is widespread and increasing. Deoxynybomycin (DNM) is a natural-product antibiotic with an unusual mechanism of action, inhibiting the mutant DNA gyrase that confers FQR. Unfortunately, isolation of DNM is difficult and DNM is insoluble in aqueous solutions, making it a poor candidate for development. Here we describe a facile chemical route to produce DNM and its derivatives. These compounds possess excellent activity against FQR methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci clinical isolates and inhibit mutant DNA gyrase in-vitro. Bacteria that develop resistance to DNM are re-sensitized to fluoroquinolones, suggesting that resistance that emerges to DNM would be treatable. Using a DNM derivative, the first in-vivo efficacy of the nybomycin class is demonstrated in a mouse infection model. Overall, the data presented suggest the promise of DNM derivatives for the treatment of FQR infections. PMID:25907309

  16. Automated DNA-preparation system for bacteria out of air sampler liquids

    NASA Astrophysics Data System (ADS)

    Gransee, Rainer; Röser, Tina; Drese, Klaus Stefan; Düchs, Dominik; Disqué, Claudia; Zoll, Gudrun; Köhne, Stefan; Ritzi-Lehnert, Marion

    2012-06-01

    Preventing bacterial contaminations is a significant challenge in applications across a variety of industries, e.g. in food processing, the life sciences or biohazard detection. Here we present a fully automated lab-on-a-chip system wherein a disposable microfluidic chip moulded by polymeric injection is inserted into an operating device. Liquid samples, here obtained from an air sampler, can be processed to extract and lyse bacteria, and subsequently to purify their DNA using a silica matrix. After the washing and elution steps, the DNA solution is dispensed into a reaction vessel for further analysis in a conventional laboratory polymerase chain reaction (PCR) device. We demonstrate the workability and efficiency of our approach with results from a 9 ml liquid sample spiked with E. coli.

  17. Deoxynybomycins inhibit mutant DNA gyrase and rescue mice infected with fluoroquinolone-resistant bacteria.

    PubMed

    Parkinson, Elizabeth I; Bair, Joseph S; Nakamura, Bradley A; Lee, Hyang Y; Kuttab, Hani I; Southgate, Emma H; Lezmi, Stéphane; Lau, Gee W; Hergenrother, Paul J

    2015-04-24

    Fluoroquinolones are one of the most commonly prescribed classes of antibiotics, but fluoroquinolone resistance (FQR) is widespread and increasing. Deoxynybomycin (DNM) is a natural-product antibiotic with an unusual mechanism of action, inhibiting the mutant DNA gyrase that confers FQR. Unfortunately, isolation of DNM is difficult and DNM is insoluble in aqueous solutions, making it a poor candidate for development. Here we describe a facile chemical route to produce DNM and its derivatives. These compounds possess excellent activity against FQR methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci clinical isolates and inhibit mutant DNA gyrase in-vitro. Bacteria that develop resistance to DNM are re-sensitized to fluoroquinolones, suggesting that resistance that emerges to DNM would be treatable. Using a DNM derivative, the first in-vivo efficacy of the nybomycin class is demonstrated in a mouse infection model. Overall, the data presented suggest the promise of DNM derivatives for the treatment of FQR infections.

  18. Classification of Plant Associated Bacteria Using RIF, a Computationally Derived DNA Marker

    PubMed Central

    Schneider, Kevin L.; Marrero, Glorimar; Alvarez, Anne M.; Presting, Gernot G.

    2011-01-01

    A DNA marker that distinguishes plant associated bacteria at the species level and below was derived by comparing six sequenced genomes of Xanthomonas, a genus that contains many important phytopathogens. This DNA marker comprises a portion of the dnaA replication initiation factor (RIF). Unlike the rRNA genes, dnaA is a single copy gene in the vast majority of sequenced bacterial genomes, and amplification of RIF requires genus-specific primers. In silico analysis revealed that RIF has equal or greater ability to differentiate closely related species of Xanthomonas than the widely used ribosomal intergenic spacer region (ITS). Furthermore, in a set of 263 Xanthomonas, Ralstonia and Clavibacter strains, the RIF marker was directly sequenced in both directions with a success rate approximately 16% higher than that for ITS. RIF frameworks for Xanthomonas, Ralstonia and Clavibacter were constructed using 682 reference strains representing different species, subspecies, pathovars, races, hosts and geographic regions, and contain a total of 109 different RIF sequences. RIF sequences showed subspecific groupings but did not place strains of X. campestris or X. axonopodis into currently named pathovars nor R. solanacearum strains into their respective races, confirming previous conclusions that pathovar and race designations do not necessarily reflect genetic relationships. The RIF marker also was sequenced for 24 reference strains from three genera in the Enterobacteriaceae: Pectobacterium, Pantoea and Dickeya. RIF sequences of 70 previously uncharacterized strains of Ralstonia, Clavibacter, Pectobacterium and Dickeya matched, or were similar to, those of known reference strains, illustrating the utility of the frameworks to classify bacteria below the species level and rapidly match unknown isolates to reference strains. The RIF sequence frameworks are available at the online RIF database, RIFdb, and can be queried for diagnostic purposes with RIF sequences obtained

  19. Use of DNA probes to study tetracycline resistance determinants in gram-negative bacteria from swine

    SciTech Connect

    Lee, C.Y.

    1989-01-01

    Specific {sup 32}P-labeled DNA probes were prepared and used to evaluate the distribution of tetracycline resistance determinants carried by gram-negative enteric bacteria isolated from pigs in 3 swine herds with different histories of antibiotic exposure. Plasmid DNA, ranging in size from 2.1 to 186 Kb, was observed in over 84% of 114 isolates studied. Two of 78 tetracycline resistant strains did not harbor plasmids. The DNA probes were isolated from plasmids pSL18, pRT29/Tn10, pBR322 and pSL106, respectively, and they represented class A, B, C and D tetracycline resistance determinants. Hybridization conditions using 0.5X SSPE at 65{degrees}C minimize cross-hybridization between the different class of tetracycline resistance genes. Cross-hybridization between class A and class C determinants could be distinguished by simultaneous comparison of the intensity of their hybridization signals. Plasmids from over 44% of the tetracycline resistant isolates did not hybridize to DNA probes for the determinants tested. Class B determinant occurred more frequently than class A or C. None of the isolates hybridized with the class D probe.

  20. DNA repair and sequence context affect 1O2-induced mutagenesis in bacteria

    PubMed Central

    Agnez-Lima, L. F.; Napolitano, R. L.; Fuchs, R. P. P.; Mascio, P. Di; Muotri, A. R.; Menck, C. F. M.

    2001-01-01

    Electronic excited molecular oxygen (singlet oxygen, 1O2) is known to damage DNA, yielding mutations. In this work, the mutagenicity induced by 1O2 in a defined sequence of DNA was investigated after replication in Escherichia coli mutants deficient for nucleotide and base excision DNA repair pathways. For this purpose a plasmid containing a 1O2-damaged 14 base oligonucleotide was introduced into E.coli by transfection and mutations were screened by hybridization with an oligonucleotide with the original sequence. Mutagenesis was observed in all strains tested, but it was especially high in the BH20 (fpg), AYM57 (fpg mutY) and AYM84 (fpg mutY uvrC) strains. The frequency of mutants in the fpg mutY strain was higher than in the triple mutant fpg mutY uvrC, suggesting that activity of the UvrABC excinuclease can favor the mutagenesis of these lesions. Additionally, most of the mutations were G→T and G→C transversions, but this was dependent on the position of the guanine in the sequence and on repair deficiency in the host bacteria. Thus, the kind of repair and the mutagenesis associated with 1O2-induced DNA damage are linked to the context of the damaged sequence. PMID:11433036

  1. Horizontal Gene Transfer Regulation in Bacteria as a “Spandrel” of DNA Repair Mechanisms

    PubMed Central

    Fall, Saliou; Mercier, Anne; Bertolla, Franck; Calteau, Alexandra; Gueguen, Laurent; Perrière, Guy; Vogel, Timothy M.; Simonet, Pascal

    2007-01-01

    the bacteria to hijack DNA repair mechanisms in order to generate genetic diversity without losing too much genomic stability. PMID:17957239

  2. Comparison of DNA extraction kits and modification of DNA elution procedure for the quantitation of subdominant bacteria from piggery effluents with real-time PCR.

    PubMed

    Desneux, Jérémy; Pourcher, Anne-Marie

    2014-08-01

    Four commercial DNA extraction kits and a minor modification in the DNA elution procedure were evaluated for the quantitation of bacteria in pig manure samples. The PowerSoil(®), PowerFecal(®), NucleoSpin(®) Soil kits and QIAamp(®) DNA Stool Mini kit were tested on raw manure samples and on lagoon effluents for their ability to quantify total bacteria and a subdominant bacteria specific of pig manure contamination: Lactobacillus amylovorus. The NucleoSpin(®) Soil kit (NS kit), and to a lesser extent the PowerFecal(®) kit were the most efficient methods. Regardless of the kit utilized, the modified elution procedure increased DNA yield in the lagoon effluent by a factor of 1.4 to 1.8. When tested on 10 piggery effluent samples, compared to the QIAamp kit, the NS kit combined with the modified elution step, increased by a factor up to 1.7 log10 the values of the concentration of L. amylovorus. Regardless of the type of manure, the best DNA quality and the highest concentrations of bacteria were obtained using the NS kit combined with the modification of the elution procedure. The method recommended here significantly improved quantitation of subdominant bacteria in manure.

  3. Comparison of DNA extraction kits and modification of DNA elution procedure for the quantitation of subdominant bacteria from piggery effluents with real-time PCR

    PubMed Central

    Desneux, Jérémy; Pourcher, Anne-Marie

    2014-01-01

    Four commercial DNA extraction kits and a minor modification in the DNA elution procedure were evaluated for the quantitation of bacteria in pig manure samples. The PowerSoil®, PowerFecal®, NucleoSpin® Soil kits and QIAamp® DNA Stool Mini kit were tested on raw manure samples and on lagoon effluents for their ability to quantify total bacteria and a subdominant bacteria specific of pig manure contamination: Lactobacillus amylovorus. The NucleoSpin® Soil kit (NS kit), and to a lesser extent the PowerFecal® kit were the most efficient methods. Regardless of the kit utilized, the modified elution procedure increased DNA yield in the lagoon effluent by a factor of 1.4 to 1.8. When tested on 10 piggery effluent samples, compared to the QIAamp kit, the NS kit combined with the modified elution step, increased by a factor up to 1.7 log10 the values of the concentration of L. amylovorus. Regardless of the type of manure, the best DNA quality and the highest concentrations of bacteria were obtained using the NS kit combined with the modification of the elution procedure. The method recommended here significantly improved quantitation of subdominant bacteria in manure. PMID:24838631

  4. Relationships between 16S-23S rRNA gene internal transcribed spacer DNA and genomic DNA similarities in the taxonomy of phototrophic bacteria

    NASA Astrophysics Data System (ADS)

    Okamura, K.; Hisada, T.; Takata, K.; Hiraishi, A.

    2013-04-01

    Rapid and accurate identification of microbial species is essential task in microbiology and biotechnology. In prokaryotic systematics, genomic DNA-DNA hybridization is the ultimate tool to determine genetic relationships among bacterial strains at the species level. However, a practical problem in this assay is that the experimental procedure is laborious and time-consuming. In recent years, information on the 16S-23S rRNA gene internal transcribed spacer (ITS) region has been used to classify bacterial strains at the species and intraspecies levels. It is unclear how much information on the ITS region can reflect the genome that contain it. In this study, therefore, we evaluate the quantitative relationship between ITS DNA and entire genomic DNA similarities. For this, we determined ITS sequences of several species of anoxygenic phototrophic bacteria belonging to the order Rhizobiales, and compared with DNA-DNA relatedness among these species. There was a high correlation between the two genetic markers. Based on the regression analysis of this relationship, 70% DNA-DNA relatedness corresponded to 92% ITS sequence similarity. This suggests the usefulness of the ITS sequence similarity as a criterion for determining the genospecies of the phototrophic bacteria. To avoid the effects of polymorphism bias of ITS on similarities, PCR products from all loci of ITS were used directly as genetic probes for comparison. The results of ITS DNA-DNA hybridization coincided well with those of genomic DNA-DNA relatedness. These collective data indicate that the whole ITS DNA-DNA similarity can be used as an alternative to genomic DNA-DNA similarity.

  5. Basal DNA repair machinery is subject to positive selection in ionizing-radiation-resistant bacteria.

    PubMed

    Sghaier, Haïtham; Ghedira, Kaïs; Benkahla, Alia; Barkallah, Insaf

    2008-06-21

    Ionizing-radiation-resistant bacteria (IRRB) show a surprising capacity for adaptation to ionizing radiation and desiccation. Positive Darwinian selection is expected to play an important role in this trait, but no data are currently available regarding the role of positive adaptive selection in resistance to ionizing-radiation and tolerance of desiccation. We analyzed the four known genome sequences of IRRB (Deinococcus geothermalis, Deinococcus radiodurans, Kineococcus radiotolerans, and Rubrobacter xylanophilus) to determine the role of positive Darwinian selection in the evolution of resistance to ionizing radiation and tolerance of desiccation. We used the programs MultiParanoid and DnaSP to deduce the sets of orthologs that potentially evolved due to positive Darwinian selection in IRRB. We find that positive selection targets 689 ortholog sets of IRRB. Among these, 58 ortholog sets are absent in ionizing-radiation-sensitive bacteria (IRSB: Escherichia coli and Thermus thermophilus). The most striking finding is that all basal DNA repair genes in IRRB, unlike many of their orthologs in IRSB, are subject to positive selection. Our results provide the first in silico prediction of positively selected genes with potential roles in the molecular basis of resistance to gamma-radiation and tolerance of desiccation in IRRB. Identification of these genes provides a basis for future experimental work aimed at understanding the metabolic networks in which they participate.

  6. Assessment of viable bacteria and bacterial DNA in blood and bloodstain specimens stored under various conditions.

    PubMed

    Hosokawa-Muto, Junji; Fujinami, Yoshihito; Mizuno, Natsuko

    2013-11-01

    Microbial forensic specimens that are collected at biocrime and bioterrorism scenes include blood, tissue, cloths containing biological fluids, swabs, water, soil, and aerosols. It is preferable that pathogens in such specimens are alive and kept in a steady state. Specimens may be stored for a prolonged period before analysis; therefore, it is important to understand the effect of the storage conditions on the pathogens contained within the specimens. In this study, we prepared blood and bloodstain specimens containing Gram-negative or -positive bacteria, stored the samples for 482 days under various conditions, and measured viable bacterial counts and total bacterial contents in the samples. Viable bacteria were preserved well in the samples stored at -30 and -80 °C, but were diminished or undetectable in the samples stored at 4 °C and room temperature. The total bacterial content was maintained in the blood samples stored at -30 and -80 °C and in the bloodstain samples stored under all temperature conditions, but decreased in the blood samples stored at 4 °C and room temperature. This study showed that the storage conditions affected viable bacteria and bacterial DNA and that freezing and drying were significant for their long-term storage. We provide important information for the storage of microbial forensic specimens.

  7. A Mimicking-of-DNA-Methylation-Patterns Pipeline for Overcoming the Restriction Barrier of Bacteria

    PubMed Central

    Zhang, Guoqiang; Wang, Wenzhao; Deng, Aihua; Sun, Zhaopeng; Zhang, Yun; Liang, Yong; Che, Yongsheng; Wen, Tingyi

    2012-01-01

    Genetic transformation of bacteria harboring multiple Restriction-Modification (R-M) systems is often difficult using conventional methods. Here, we describe a mimicking-of-DNA-methylation-patterns (MoDMP) pipeline to address this problem in three difficult-to-transform bacterial strains. Twenty-four putative DNA methyltransferases (MTases) from these difficult-to-transform strains were cloned and expressed in an Escherichia coli strain lacking all of the known R-M systems and orphan MTases. Thirteen of these MTases exhibited DNA modification activity in Southwestern dot blot or Liquid Chromatography–Mass Spectrometry (LC–MS) assays. The active MTase genes were assembled into three operons using the Saccharomyces cerevisiae DNA assembler and were co-expressed in the E. coli strain lacking known R-M systems and orphan MTases. Thereafter, results from the dot blot and restriction enzyme digestion assays indicated that the DNA methylation patterns of the difficult-to-transform strains are mimicked in these E. coli hosts. The transformation of the Gram-positive Bacillus amyloliquefaciens TA208 and B. cereus ATCC 10987 strains with the shuttle plasmids prepared from MoDMP hosts showed increased efficiencies (up to four orders of magnitude) compared to those using the plasmids prepared from the E. coli strain lacking known R-M systems and orphan MTases or its parental strain. Additionally, the gene coding for uracil phosphoribosyltransferase (upp) was directly inactivated using non-replicative plasmids prepared from the MoDMP host in B. amyloliquefaciens TA208. Moreover, the Gram-negative chemoautotrophic Nitrobacter hamburgensis strain X14 was transformed and expressed Green Fluorescent Protein (GFP). Finally, the sequence specificities of active MTases were identified by restriction enzyme digestion, making the MoDMP system potentially useful for other strains. The effectiveness of the MoDMP pipeline in different bacterial groups suggests a universal potential

  8. Thioaromatic DNA monolayers for target-amplification-free electrochemical sensing of environmental pathogenic bacteria.

    PubMed

    Miranda-Castro, Rebeca; Sánchez-Salcedo, Raquel; Suárez-Álvarez, Beatriz; de-Los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo J; Jesús Lobo-Castañón, María

    2017-06-15

    Genosensing technology has mostly based on mixed self-assembled monolayers (SAMs) of thiol-modified oligonucleotides and alkanethiols on gold surfaces. However, the typical backfilling approach, which incorporates the alkanethiol in a second step, gives rise to a heterogeneous distribution of oligonucleotide probes on the surface, negatively affecting to both hybridization efficiency and surface stability. Despite aromatic thiols present a remarkably different behavior from alkanethiols, with higher rigidity and stronger intermolecular interactions, they have been scarcely explored for the fabrication of DNA sensing platforms. We have investigated different approaches involving SAMs of aromatic thiols, namely p-mercaptobenzoic acid (p-MBA) and p-aminothiophenol (p-ATP), to yield DNA sensing layers for sequence-specific detection of target oligonucleotides. The studied monolayers were evaluated by DNA surface coverage and further information was obtained by determining their functionality in a sandwich hybridization assay with enzymatic amplification of the electrochemical read-out. The insertion of thiol-oligonucleotides into p-ATP monolayers previously oxidized, and the covalent binding of amino-oligonucleotides to pure p-MBA monolayers give rise to increased storage stability and better analytical performance. The quantification of RNA from Legionella pneumophila cellular lysates was successfully performed, illustrating the usefulness of these sensing architectures for detecting pathogenic bacteria.

  9. DNA methylation in thermophilic bacteria: N4-methylcytosine, 5-methylcytosine, and N6-methyladenine.

    PubMed Central

    Ehrlich, M; Gama-Sosa, M A; Carreira, L H; Ljungdahl, L G; Kuo, K C; Gehrke, C W

    1985-01-01

    While determining the minor and major base composition of the DNA from 17 types of thermophilic bacteria by high performance liquid chromatography (HPLC) of enzymatic digests, we have discovered a novel base, N4-methylcytosine (m4C). Its structure was proven by comparison of the DNA-derived nucleoside to the analogous authentic compound by HPLC, UV spectroscopy, and mass spectroscopy. Eight of the bacterial DNAs contained m4C. Only two contained the common minor base, 5-methylcytosine (m5C), and neither of these was from an extreme thermophile. The other prevalent modified base of bacterial DNA, N6-methyladenine (m6A), was found in nine of the DNAs. Restriction analysis revealed that four of the DNAs had dam-type (Gm6ATC) methylation patterns. Due to the propensity of m5C residues to be deaminated by heat to thymine residues and to inefficient repair of the resulting mismatched base pairs, thermophiles with optimal growth temperatures of greater than or equal to 60 degrees C generally may avoid having m5C in their genomes. Instead, some of them have deamination-resistant m4C residues. PMID:4000939

  10. Cultivation-Independent Detection of Autotrophic Hydrogen-Oxidizing Bacteria by DNA Stable-Isotope Probing ▿

    PubMed Central

    Pumphrey, Graham M.; Ranchou-Peyruse, Anthony; Spain, Jim C.

    2011-01-01

    Knallgas bacteria are a physiologically defined group that is primarily studied using cultivation-dependent techniques. Given that current cultivation techniques fail to grow most bacteria, cultivation-independent techniques that selectively detect and identify knallgas bacteria will improve our ability to study their diversity and distribution. We used stable-isotope probing (SIP) to identify knallgas bacteria in rhizosphere soil of legumes and in a microbial mat from Obsidian Pool in Yellowstone National Park. When samples were incubated in the dark, incorporation of 13CO2 was H2 dependent. SIP enabled the detection of knallgas bacteria that were not detected by cultivation, and the majority of bacteria identified in the rhizosphere soils were betaproteobacteria predominantly related to genera previously known to oxidize hydrogen. Bacteria in soil grew on hydrogen at concentrations as low as 100 ppm. A hydB homolog encoding a putative high-affinity NiFe hydrogenase was amplified from 13C-labeled DNA from both vetch and clover rhizosphere soil. The results indicate that knallgas bacteria can be detected by SIP and populations that respond to different H2 concentrations can be distinguished. The methods described here should be applicable to a variety of ecosystems and will enable the discovery of additional knallgas bacteria that are resistant to cultivation. PMID:21622787

  11. Detection of Tn5-like sequences in kanamycin-resistant stream bacteria and environmental DNA

    SciTech Connect

    Leff, L.G.; McArthur, J.V. ); Dana, J.R.; Shimkets, L.J. )

    1993-02-01

    This study investigates the occurrence of kanamycin and neomycin resistance in the culturable portion of the bacterial assemblage of a South Carolina stream. The constitutively expressed nptII gene was used to determine resistance. Spartial differences in the relative abundances of nptII taken from different locations and habitats in the stream were investigated. Results suggest that multiple probes will probably be necessary to assess kanamycin resistance potential of stream bacteria. At the largest patial scale there are not significant difference among the sites in abundances of nptII genes, though there were some differences in habitats. The authors conclude DNA hybridization appears to be a useful technique for assessing the abundance of genes in mixtures of nonculturable organisms.

  12. Why it pays for bacteria to delete disused DNA and to maintain megaplasmids.

    PubMed

    Stouthamer, A H; Kooijman, S A

    1993-01-01

    Genetic information for disused metabolic systems is easily lost. This can be understood on the basis of a mathematic model for the description of the growth of individual microbial cells. The essential features of the model are that the uptake of nutrients is proportional to the surface area of the cell and maintenance costs are proportional to the volume. This explains why disused genes are easily lost. Growth of the individual cell continues until DNA replication of the genome is completed. The consequence is that cells with a large genome size are bigger at the moment of cell division. This results in a less favourable surface to volume ratio, which has a negative influence on the population growth rate. This means that cells with a smaller genome size will have a selective advantage. In cells in which the total DNA is divided over several units which replicate simultaneously (e.g. a cell with more chromosomes or a cell with one chromosome and one or more megaplasmids) a high surface to volume ratio can be maintained. The great metabolic differences between phylogenetically strongly related bacteria are due to loss of genetic information for complex metabolic characters in order to maintain a small genome size and a favourable surface to volume ratio.

  13. Role of Protein Phosphorylation in the Regulation of Cell Cycle and DNA-Related Processes in Bacteria

    PubMed Central

    Garcia-Garcia, Transito; Poncet, Sandrine; Derouiche, Abderahmane; Shi, Lei; Mijakovic, Ivan; Noirot-Gros, Marie-Françoise

    2016-01-01

    In all living organisms, the phosphorylation of proteins modulates various aspects of their functionalities. In eukaryotes, protein phosphorylation plays a key role in cell signaling, gene expression, and differentiation. Protein phosphorylation is also involved in the global control of DNA replication during the cell cycle, as well as in the mechanisms that cope with stress-induced replication blocks. Similar to eukaryotes, bacteria use Hanks-type kinases and phosphatases for signal transduction, and protein phosphorylation is involved in numerous cellular processes. However, it remains unclear whether protein phosphorylation in bacteria can also regulate the activity of proteins involved in DNA-mediated processes such as DNA replication or repair. Accumulating evidence supported by functional and biochemical studies suggests that phospho-regulatory mechanisms also take place during the bacterial cell cycle. Recent phosphoproteomics and interactomics studies identified numerous phosphoproteins involved in various aspect of DNA metabolism strongly supporting the existence of such level of regulation in bacteria. Similar to eukaryotes, bacterial scaffolding-like proteins emerged as platforms for kinase activation and signaling. This review reports the current knowledge on the phosphorylation of proteins involved in the maintenance of genome integrity and the regulation of cell cycle in bacteria that reveals surprising similarities to eukaryotes. PMID:26909079

  14. Role of Protein Phosphorylation in the Regulation of Cell Cycle and DNA-Related Processes in Bacteria.

    PubMed

    Garcia-Garcia, Transito; Poncet, Sandrine; Derouiche, Abderahmane; Shi, Lei; Mijakovic, Ivan; Noirot-Gros, Marie-Françoise

    2016-01-01

    In all living organisms, the phosphorylation of proteins modulates various aspects of their functionalities. In eukaryotes, protein phosphorylation plays a key role in cell signaling, gene expression, and differentiation. Protein phosphorylation is also involved in the global control of DNA replication during the cell cycle, as well as in the mechanisms that cope with stress-induced replication blocks. Similar to eukaryotes, bacteria use Hanks-type kinases and phosphatases for signal transduction, and protein phosphorylation is involved in numerous cellular processes. However, it remains unclear whether protein phosphorylation in bacteria can also regulate the activity of proteins involved in DNA-mediated processes such as DNA replication or repair. Accumulating evidence supported by functional and biochemical studies suggests that phospho-regulatory mechanisms also take place during the bacterial cell cycle. Recent phosphoproteomics and interactomics studies identified numerous phosphoproteins involved in various aspect of DNA metabolism strongly supporting the existence of such level of regulation in bacteria. Similar to eukaryotes, bacterial scaffolding-like proteins emerged as platforms for kinase activation and signaling. This review reports the current knowledge on the phosphorylation of proteins involved in the maintenance of genome integrity and the regulation of cell cycle in bacteria that reveals surprising similarities to eukaryotes.

  15. Ancient bacteria in permafrost soils fact or artefact? Considerations in recovering microbial DNA from geological ancient settings

    NASA Astrophysics Data System (ADS)

    Willerslev, E.

    2003-04-01

    Several recent reports claim that prokaryotic genetic sequences or viable cultures can survive for millions of years in geological settings. If substantiated, these findings could fundamentally alter views about bacterial physiology, ecology and evolution. However, both the culturing of microbes and the amplification of ancient DNA molecules from fossil remains are beset with difficulties. First, theoretical and empirical studies have shown that small DNA fragments (100 200 bp) do not survive in the geosphere for more than 104 years in temperate environments and 105 years in colder ones due to hydrolytic and oxidative damage. Therefore, the revivals of dormant bacteria with no active DNA repair from remains hundreds of thousands to millions of years old is, from a theoretical point, expected to be difficult, if not impossible. Second, the no specificity of the media used to culture micro organisms, as well as the great sensitivity of PCR, makes the risk of contamination with contemporary ubiquitous microbial cells and exogenous DNA molecules extremely high. Contamination poses risks at all stages of sample processing (e.g.) within the samples themselves, in the chemical reagents, on laboratory disposables or through the air. The high risk of contamination strongly suggests the need for standardized procedures within the field such as independent replication of results. This criterion of authenticity has not yet been full field in any of the studies claiming million year old microbial cultures or DNA. In order to tests the long-term survival of ancient bacteria DNA a study on permafrost was conducted using ancient DNA precautions, controls and criteria. Permafrost must be considered among the most promising environments for long term DNA survival due to its constant low temperatures (-10C to 12C Siberian or 20C Antarctica) and high cell numbers (107). We found that bacteria DNA could reproducibly be obtained from samples dated up to 300-400,000 years B.P. but not

  16. Seasonal Variation in Parental Care Drives Sex-Specific Foraging by a Monomorphic Seabird.

    PubMed

    Burke, Chantelle M; Montevecchi, William A; Regular, Paul M

    2015-01-01

    Evidence of sex-specific foraging in monomorphic seabirds is increasing though the underlying mechanisms remain poorly understood. We investigate differential parental care as a mechanism for sex-specific foraging in monomorphic Common Murres (Uria aalge), where the male parent alone provisions the chick after colony departure. Using a combination of geolocation-immersion loggers and stable isotopes, we assess two hypotheses: the reproductive role specialization hypothesis and the energetic constraint hypothesis. We compare the foraging behavior of females (n = 15) and males (n = 9) during bi-parental at the colony, post-fledging male-only parental care and winter when parental care is absent. As predicted by the reproductive role specialization hypothesis, we found evidence of sex-specific foraging during post-fledging only, the stage with the greatest divergence in parental care roles. Single-parenting males spent almost twice as much time diving per day and foraged at lower quality prey patches relative to independent females. This implies a potential energetic constraint for males during the estimated 62.8 ± 8.9 days of offspring dependence at sea. Contrary to the predictions of the energetic constraint hypothesis, we found no evidence of sex-specific foraging during biparental care, suggesting that male parents did not forage for their own benefit before colony departure in anticipation of post-fledging energy constraints. We hypothesize that unpredictable prey conditions at Newfoundland colonies in recent years may limit male parental ability to allocate additional time and energy to self-feeding during biparental care, without compromising chick survival. Our findings support differential parental care as a mechanism for sex-specific foraging in monomorphic murres, and highlight the need to consider ecological context in the interpretation of sex-specific foraging behavior.

  17. Seasonal Variation in Parental Care Drives Sex-Specific Foraging by a Monomorphic Seabird

    PubMed Central

    Burke, Chantelle M.; Montevecchi, William A.; Regular, Paul M.

    2015-01-01

    Evidence of sex-specific foraging in monomorphic seabirds is increasing though the underlying mechanisms remain poorly understood. We investigate differential parental care as a mechanism for sex-specific foraging in monomorphic Common Murres (Uria aalge), where the male parent alone provisions the chick after colony departure. Using a combination of geolocation-immersion loggers and stable isotopes, we assess two hypotheses: the reproductive role specialization hypothesis and the energetic constraint hypothesis. We compare the foraging behavior of females (n = 15) and males (n = 9) during bi-parental at the colony, post-fledging male-only parental care and winter when parental care is absent. As predicted by the reproductive role specialization hypothesis, we found evidence of sex-specific foraging during post-fledging only, the stage with the greatest divergence in parental care roles. Single-parenting males spent almost twice as much time diving per day and foraged at lower quality prey patches relative to independent females. This implies a potential energetic constraint for males during the estimated 62.8 ± 8.9 days of offspring dependence at sea. Contrary to the predictions of the energetic constraint hypothesis, we found no evidence of sex-specific foraging during biparental care, suggesting that male parents did not forage for their own benefit before colony departure in anticipation of post-fledging energy constraints. We hypothesize that unpredictable prey conditions at Newfoundland colonies in recent years may limit male parental ability to allocate additional time and energy to self-feeding during biparental care, without compromising chick survival. Our findings support differential parental care as a mechanism for sex-specific foraging in monomorphic murres, and highlight the need to consider ecological context in the interpretation of sex-specific foraging behavior. PMID:26575646

  18. A microfluidic chip integrating DNA extraction and real-time PCR for the detection of bacteria in saliva.

    PubMed

    Oblath, Emily A; Henley, W Hampton; Alarie, Jean Pierre; Ramsey, J Michael

    2013-04-07

    A microfluidic chip integrating DNA extraction, amplification, and detection for the identification of bacteria in saliva is described. The chip design integrated a monolithic aluminum oxide membrane (AOM) for DNA extraction with seven parallel reaction wells for real-time polymerase chain reaction (rtPCR) amplification of the extracted DNA. Samples were first heated to lyse target organisms and then added to the chip and filtered through the nanoporous AOM to extract the DNA. PCR reagents were added to each of the wells and the chip was thermocycled. Identification of Streptococcus mutans in a saliva sample is demonstrated along with the detection of 300 fg (100-125 copies) of both methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) genomic DNA (gDNA) spiked into a saliva sample. Multiple target species and strains of bacteria can be simultaneously identified in the same sample by varying the primers and probes used in each of the seven reaction wells. In initial tests, as little as 30 fg (8-12 copies) of MSSA gDNA in buffer has been successfully amplified and detected with this device.

  19. Termination of idiopathic sustained monomorphic ventricular tachycardia by intravenous adenosine in a pregnant woman.

    PubMed

    Hasdemir, Can; Musayev, Oktay; Alkan, Mustafa B; Can, Levent H; Kultursay, Hakan

    2009-11-01

    A 34-year-old pregnant woman presented to the emergency department with the complaints of palpitations at 32 weeks gestation. The diagnosis of right ventricular outflow tract ventricular tachycardia (VT) was made. Intravenous 5 mg of metoprolol and 25 mg of diltiazem did not terminate the VT. Ten milligrams of adenosine were administered. Within 10 s of adenosine administration, sustained VT converted to repetitive monomorphic VT and within 30 s to normal sinus rhythm. The mother and the foetus tolerated the medications well. Non-stress test for the assessment of the foetal well-being was normal.

  20. Conversion to Purkinje-Related Monomorphic Ventricular Tachycardia After Ablation of Ventricular Fibrillation in Ischemic Heart Disease.

    PubMed

    Masuda, Keita; Nogami, Akihiko; Kuroki, Kenji; Igarashi, Miyako; Sekiguchi, Yukio; Komatsu, Yuki; Kowase, Shinya; Kurosaki, Kenji; Nishihara, Shuzo; Niwa, Koichiro; Tsuchiya, Taketsugu; Igawa, Masayuki; Aonuma, Kazutaka

    2016-09-01

    Catheter ablation is an effective therapy for ventricular fibrillation (VF) arising from the Purkinje system in ischemic heart disease. However, some patients experience newly emergent monomorphic ventricular tachycardia (VT) after the ablation of VF. We evaluated the prevalence and mechanism of monomorphic VT after VF ablation. Twenty-one consecutive patients with primary VF because of ischemic heart disease who underwent catheter ablation were retrospectively analyzed. Twenty of 21 patients were in electrical storm. Ventricular premature contractions triggering VF arose from the left Purkinje system and were targeted for ablation. Before the ablation, 14 of 21 patients had only VF, and the other 7 had VF and concomitant monomorphic VT. Four of the 14 patients with only VF (29%) exhibited newly emergent monomorphic VT after VF ablation. Three of these patients had Purkinje-related VTs, which were successfully eliminated by the ablation of a Purkinje network located in the same low-voltage area as the site of prior successful VF ablation. During a median follow-up of 28 months (interquartile range, 16-68 months), VF recurred in 6 of 21 patients (29%); however, there were neither electrical storms nor monomorphic VT, and all recurring arrhythmias were controlled by medical therapy alone. Over one fifth of patients with primary ischemic VF experienced newly emergent Purkinje-related monomorphic VT after VF ablation. The circuit of the monomorphic VT associated with the Purkinje network was located in the same low-voltage area as the Purkinje tissue that triggered VF and could be suppressed by additional ablation. © 2016 American Heart Association, Inc.

  1. Rapid identification of dairy mesophilic and thermophilic sporeforming bacteria using DNA high resolution melt analysis of variable 16S rDNA regions.

    PubMed

    Chauhan, Kanika; Dhakal, Rajat; Seale, R Brent; Deeth, Hilton C; Pillidge, Christopher J; Powell, Ian B; Craven, Heather; Turner, Mark S

    2013-07-15

    Due to their ubiquity in the environment and ability to survive heating processes, sporeforming bacteria are commonly found in foods. This can lead to product spoilage if spores are present in sufficient numbers and where storage conditions favour spore germination and growth. A rapid method to identify the major aerobic sporeforming groups in dairy products, including Bacillus licheniformis group, Bacillus subtilis group, Bacillus pumilus group, Bacillus megaterium, Bacillus cereus group, Geobacillus species and Anoxybacillus flavithermus was devised. This method involves real-time PCR and high resolution melt analysis (HRMA) of V3 (~70 bp) and V6 (~100 bp) variable regions in the 16S rDNA. Comparisons of HRMA curves from 194 isolates of the above listed sporeforming bacteria obtained from dairy products which were identified using partial 16S rDNA sequencing, allowed the establishment of criteria for differentiating them from each other and several non-sporeforming bacteria found in samples. A blinded validation trial on 28 bacterial isolates demonstrated complete accuracy in unambiguous identification of the 7 different aerobic sporeformers. The reliability of HRMA method was also verified using boiled extractions of crude DNA, thereby shortening the time needed for identification. The HRMA method described in this study provides a new and rapid approach to identify the dominant mesophilic and thermophilic aerobic sporeforming bacteria found in a wide variety of dairy products.

  2. Reverse Sample Genome Probing, a New Technique for Identification of Bacteria in Environmental Samples by DNA Hybridization, and Its Application to the Identification of Sulfate-Reducing Bacteria in Oil Field Samples

    PubMed Central

    Voordouw, Gerrit; Voordouw, Johanna K.; Karkhoff-Schweizer, Roxann R.; Fedorak, Phillip M.; Westlake, Donald W. S.

    1991-01-01

    A novel method for the identification of bacteria in environmental samples by DNA hybridization is presented. It is based on the fact that, even within a genus, the genomes of different bacteria may have little overall sequence homology. This allows the use of the labeled genomic DNA of a given bacterium (referred to as a “standard”) to probe for its presence and that of bacteria with highly homologous genomes in total DNA obtained from an environmental sample. Alternatively, total DNA extracted from the sample can be labeled and used to probe filters on which denatured chromosomal DNA from relevant bacterial standards has been spotted. The latter technique is referred to as reverse sample genome probing, since it is the reverse of the usual practice of deriving probes from reference bacteria for analyzing a DNA sample. Reverse sample genome probing allows identification of bacteria in a sample in a single step once a master filter with suitable standards has been developed. Application of reverse sample genome probing to the identification of sulfate-reducing bacteria in 31 samples obtained primarily from oil fields in the province of Alberta has indicated that there are at least 20 genotypically different sulfate-reducing bacteria in these samples. Images PMID:16348574

  3. 16S rDNA sequence analysis of culturable marine biofilm forming bacteria from a ship's hull.

    PubMed

    Inbakandan, D; Murthy, P Sriyutha; Venkatesan, R; Khan, S Ajmal

    2010-11-01

    Marine bacteria from the hull of a ship in the form of biofilms or microfouling were isolated, cultured, and identified by phylogenetic analysis using 16S rDNA sequences. With an average length of 946 bp, all the 16 sequences were classified using the Ribosomal database project (RDP) and were submitted to the National Center for Biotechnology Information. Phylogenetic analysis using 16S rDNA sequences indicated that the 16 strains belonged to the Firmicutes (IK-MB6 Exiguobacterium aurantiacum, IK-MB7 Exiguobacterium arabatum, IK-MB8 Exiguobacterium arabatum, IK-MB9 Jeotgalibacillus alimentarius, IK-MB10 Bacillus megaterium, IK-MB11 Bacillus pumilus, IK-MB12 Bacillus pumilus, IK-MB13 Bacillus pumilus, IK-MB14 Bacillus megaterium), High GC, Gram-positive bacteria (IK-MB2 Micrococcus luteus, IK-MB5 Micrococcus luteus, IK-MB16 Arthrobacter mysorens), G-Proteobacteria (IK-MB3 Halomonas aquamarina, IK-MB15 Halotalea alkalilenta), CFB group bacteria (IK-MB1 Myroides odoratimimus), and Enterobacteria (IK-MB4 Proteus mirabilis). Among the 16 strains, representatives of the Firmicutes were dominant (56.25%) compared to the high GC, Gram-positive bacteria (18.75%), G-Proteobacteria (12.5%), CFB group bacteria (6.25%), and Enterobacteria (6.25%). Analysis revealed that majority of marine species found in marine biofilm are of anthropogenic origin.

  4. Comparative analysis of conserved genetic markers and adjacent DNA regions identified in beer-spoilage lactic acid bacteria.

    PubMed

    Suzuki, K; Ozaki, K; Yamashita, H

    2004-01-01

    To conduct an inter-species comparative study on the nucleotide sequences of the conserved DNA regions surrounding ORF5, a genetic marker for differentiating beer-spoilage lactic acid bacteria. The conserved DNA regions surrounding ORF5 were examined by PCR analysis, using three beer-spoilage strains, Lactobacillus brevis ABBC45C, L. paracollinoides LA2T and Pediococcus damnosus ABBC478. As a result, the DNA regions containing ORF1-7, originally found in ABBC45C, appeared to be conserved among the three strains, while the downstream region was not found in L. paracollinoides LA2T and P. damnosus ABBC478. The sequencing analysis of the conserved DNA regions of LA2T and ABBC478 revealed ca 99% nucleotide sequence identities with that of ABBC45C. The nucleotide sequences of the ca 8.2 kb DNA regions containing ORF1-7 were virtually identical among the three strains belonging to different species. The internal organizations of the ORFs were found to be remarkably similar. The level of nucleotide sequence identities suggests the DNA regions surrounding ORF5 were horizontally acquired by these beer-spoilage strains belonging to the three different species of lactic acid bacteria.

  5. Tracking heterotrophic and autotrophic carbon cycling by magnetotactic bacteria in freshwater sediments using DNA stable isotope probing

    NASA Astrophysics Data System (ADS)

    Kürşat Coşkun, Ömer; Roud, Sophie; He, Kuang; Petersen, Nikolai; Gilder, Stuart; Orsi, William D.

    2017-04-01

    Magnetotactic bacteria (MTB) are diverse, widespread, motile prokaryotes which biomineralize nanosize magnetic minerals, either magnetite or gregite, under highly conserved genetic control and have magnetotaxis to align their position in aquatic environment according to Earth's magnetic field. They play important roles on some geobiological cycle of important minerals such as iron, sulphur, nitrogen and carbon. Yet, to date, their importance in carbon cycle and carbon source in their natural environment have not been previously studied. In this study, we focused on freshwater benthic carbon cycling of MTB and total bacteria using DNA stable isotope probing (DNA-SIP) technique coupled with quantitative PCR (qPCR). Pond sediments from Unterlippach (Germany) were amended with 13C-labelled sodium bicarbonate and 13C-labelled organic matter, and incubated in the dark over a two week time period. Applying separate qPCR assays specific for total bacteria and MTB, respectively, allowed us to estimate the contribution of MTB to total heterotrophic and autotrophic carbon cycling via DNA-SIP. After one week, there was a slight degree of autotrophic activity which increased markedly after two weeks. Comparing total DNA to the qPCR data revealed that changes in the buoyant density of DNA was due mainly to autotrophic bacterial production. DNA-SIP also identified heterotrophic utilization of 13C-labelled organic matter by MTB after 1 week. The qPCR data also allowed us to estimate uptake rates based on the incubation times for heterotrophic and autotrophic MTB. High-throughput DNA sequencing of 16S rRNA genes showed that most of the MTB involved in carbon cycling were related to the Magnetococcus genus. This study sheds light on the carbon sources for MTB in a natural environment and helps unravel their ecological role in the carbon cycle.

  6. Bacteria and bacterial DNA in atherosclerotic plaque and aneurysmal wall biopsies from patients with and without periodontitis

    PubMed Central

    Armingohar, Zahra; Jørgensen, Jørgen J.; Kristoffersen, Anne Karin; Abesha-Belay, Emnet; Olsen, Ingar

    2014-01-01

    Background Several studies have reported an association between chronic periodontitis (CP) and cardiovascular diseases. Detection of periodontopathogens, including red complex bacteria (RCB), in vascular lesions has suggested these bacteria to be involved in the pathogenesis of atherosclerosis and abdominal aortic aneurysms. Objective In this study, we investigate bacteria and their DNA in vascular biopsies from patients with vascular diseases (VD; i.e. abdominal aortic aneurysms, atherosclerotic carotid, and common femoral arteries), with and without CP. Methods DNA was extracted from vascular biopsies selected from 40 VD patients: 30 with CP and 10 without CP. The V3-V5 region of the 16S rDNA (V3-V5) was polymerase chain reaction (PCR)-amplified, and the amplicons were cloned into Escherichia coli, sequenced, and classified (GenBank and the Human Oral Microbiome database). Species-specific primers were used for the detection of Porphyromonas gingivalis. In addition, 10 randomly selected vascular biopsies from the CP group were subjected to scanning electron microscopy (SEM) for visualization of bacteria. Checkerboard DNA–DNA hybridization was performed to assess the presence of RCB in 10 randomly selected subgingival plaque samples from CP patients. Results A higher load and mean diversity of bacteria were detected in vascular biopsies from VD patients with CP compared to those without CP. Enterobacteriaceae were frequently detected in vascular biopsies together with cultivable, commensal oral, and not-yet-cultured bacterial species. While 70% of the subgingival plaque samples from CP patients showed presence of RCB, only P. gingivalis was detected in one vascular biopsy. Bacterial cells were seen in all 10 vascular biopsies examined by SEM. Conclusions A higher bacterial load and more diverse colonization were detected in VD lesions of CP patients as compared to patients without CP. This indicated that a multitude of bacterial species both from the gut and the

  7. Small queens and big-headed workers in a monomorphic ponerine ant

    NASA Astrophysics Data System (ADS)

    Kikuchi, Tomonori; Miyazaki, Satoshi; Ohnishi, Hitoshi; Takahashi, Junichi; Nakajima, Yumiko; Tsuji, Kazuki

    2008-10-01

    Evolution of caste is a central issue in the biology of social insects. Comparative studies on their morphology so far suggest the following three patterns: (1) a positive correlation between queen worker size dimorphism and the divergence in reproductive ability between castes, (2) a negative correlation among workers between morphological diversity and reproductive ability, and (3) a positive correlation between queen worker body shape difference and the diversity in worker morphology. We conducted morphological comparisons between castes in Pachycondyla luteipes, workers of which are monomorphic and lack their reproductive ability. Although the size distribution broadly overlapped, mean head width, head length, and scape length were significantly different between queens and workers. Conversely, in eye length, petiole width, and Weber’s length, the size differences were reversed. The allometries (head length/head width, scape length/head width, and Weber’s length/head width) were also significantly different between queens and workers. Morphological examinations showed that the body shape was different between queens and workers, and the head part of workers was disproportionately larger than that of queens. This pattern of queen worker dimorphism is novel in ants with monomorphic workers and a clear exception to the last pattern. This study suggests that it is possible that the loss of individual-level selection, the lack of reproductive ability, influences morphological modification in ants.

  8. Development of a DNA macroarray for simultaneous detection of multiple foodborne pathogenic bacteria in fresh chicken meat.

    PubMed

    Kupradit, Chanida; Rodtong, Sureelak; Ketudat-Cairns, Mariena

    2013-12-01

    A DNA macroarray was developed to provide the ability to detect multiple foodborne pathogens in fresh chicken meat. Probes targeted to the 16S rRNA and genus- and species-specific genes, including fimY, ipaH, prfA, and uspA, were selected for the specific detection of Salmonella spp., Shigella spp., Listeria monocytogenes, and Escherichia coli, respectively. The combination of target gene amplification by PCR and a DNA macroarray in our system was able to distinguish all target bacteria from pure cultures with a detection sensitivity of 10⁵ c.f.u. ml⁻¹. The DNA macroarray was also applied to 10 fresh chicken meat samples. The assay validation demonstrated that by combining the enrichment steps for the target bacteria and the DNA macroarray, all 4 target bacteria could be detected simultaneously from the fresh chicken samples. The sensitivity of L. monocytogenes and Shigella boydii detection in the fresh chicken samples was at least 10 and 3 c.f.u. of the initial contamination in 25 g samples, respectively. The advantages of our developed protocol are high accuracy and time reduction when compared to conventional culture. The macroarray developed in our investigation was cost effective compared to modern oligonucleotide microarray techniques because there was no expensive equipment required for the detection of multiple foodborne pathogens.

  9. Multiple DNA Extractions Coupled with Stable-Isotope Probing of Anthracene-Degrading Bacteria in Contaminated Soil▿†

    PubMed Central

    Jones, Maiysha D.; Singleton, David R.; Sun, Wei; Aitken, Michael D.

    2011-01-01

    In many of the DNA-based stable-isotope probing (SIP) studies published to date in which soil communities were investigated, a single DNA extraction was performed on the soil sample, usually using a commercial DNA extraction kit, prior to recovering the 13C-labeled (heavy) DNA by density-gradient ultracentrifugation. Recent evidence suggests, however, that a single extraction of a soil sample may not lead to representative recovery of DNA from all of the organisms in the sample. To determine whether multiple DNA extractions would affect the DNA yield, the eubacterial 16S rRNA gene copy number, or the identification of anthracene-degrading bacteria, we performed seven successive DNA extractions on the same aliquot of contaminated soil either untreated or enriched with [U-13C]anthracene. Multiple extractions were necessary to maximize the DNA yield and 16S rRNA gene copy number from both untreated and anthracene-enriched soil samples. Sequences within the order Sphingomonadales, but unrelated to any previously described genus, dominated the 16S rRNA gene clone libraries derived from 13C-enriched DNA and were designated “anthracene group 1.” Sequences clustering with Variovorax spp., which were also highly represented, and sequences related to the genus Pigmentiphaga were newly associated with anthracene degradation. The bacterial groups collectively identified across all seven extracts were all recovered in the first extract, although quantitative PCR analysis of SIP-identified groups revealed quantitative differences in extraction patterns. These results suggest that performing multiple DNA extractions on soil samples improves the extractable DNA yield and the number of quantifiable eubacterial 16S rRNA gene copies but have little qualitative effect on the identification of the bacterial groups associated with the degradation of a given carbon source by SIP. PMID:21398486

  10. Multiple DNA extractions coupled with stable-isotope probing of anthracene-degrading bacteria in contaminated soil.

    PubMed

    Jones, Maiysha D; Singleton, David R; Sun, Wei; Aitken, Michael D

    2011-05-01

    In many of the DNA-based stable-isotope probing (SIP) studies published to date in which soil communities were investigated, a single DNA extraction was performed on the soil sample, usually using a commercial DNA extraction kit, prior to recovering the (13)C-labeled (heavy) DNA by density-gradient ultracentrifugation. Recent evidence suggests, however, that a single extraction of a soil sample may not lead to representative recovery of DNA from all of the organisms in the sample. To determine whether multiple DNA extractions would affect the DNA yield, the eubacterial 16S rRNA gene copy number, or the identification of anthracene-degrading bacteria, we performed seven successive DNA extractions on the same aliquot of contaminated soil either untreated or enriched with [U-(13)C]anthracene. Multiple extractions were necessary to maximize the DNA yield and 16S rRNA gene copy number from both untreated and anthracene-enriched soil samples. Sequences within the order Sphingomonadales, but unrelated to any previously described genus, dominated the 16S rRNA gene clone libraries derived from (13)C-enriched DNA and were designated "anthracene group 1." Sequences clustering with Variovorax spp., which were also highly represented, and sequences related to the genus Pigmentiphaga were newly associated with anthracene degradation. The bacterial groups collectively identified across all seven extracts were all recovered in the first extract, although quantitative PCR analysis of SIP-identified groups revealed quantitative differences in extraction patterns. These results suggest that performing multiple DNA extractions on soil samples improves the extractable DNA yield and the number of quantifiable eubacterial 16S rRNA gene copies but have little qualitative effect on the identification of the bacterial groups associated with the degradation of a given carbon source by SIP.

  11. Detection of bacteria by hybridization of rRNA with DNA-latex and immunodetection of hybrids.

    PubMed Central

    Miller, C A; Patterson, W L; Johnson, P K; Swartzell, C T; Wogoman, F; Albarella, J P; Carrico, R J

    1988-01-01

    A novel nucleic acid hybridization assay with a DNA probe immobilized on 1.25-micron-diameter latex particles was developed. Hybridization of the immobilized probe DNA with sample rRNA was complete in 10 to 15 min. Alkaline phosphatase-labeled anti-DNA-RNA was allowed to bind to the DNA-RNA hybrids on the latex particles. Then the latex was collected on a small glass fiber filter pad, and bound alkaline phosphatase was quantitated by reflectance rate measurement. The method detected a broad range of bacterial species and had a detection limit of 500 cells per assay. The assay was used to screen urine samples for bacteriuria and had a sensitivity of 96.2% compared with conventional culture at a decision level of greater than or equal to 10(4) CFU/ml. The hybridization method could have broad application to the detection of bacteria and viruses. PMID:2457597

  12. Amplifiable DNA from Gram-negative and Gram-positive bacteria by a low strength pulsed electric field method

    PubMed Central

    Vitzthum, Frank; Geiger, Georg; Bisswanger, Hans; Elkine, Bentsian; Brunner, Herwig; Bernhagen, Jürgen

    2000-01-01

    An efficient electric field-based procedure for cell disruption and DNA isolation is described. Isoosmotic suspensions of Gram-negative and Gram-positive bacteria were treated with pulsed electric fields of <60 V/cm. Pulses had an exponential decay waveform with a time constant of 3.4 µs. DNA yield was linearly dependent on time or pulse number, with several thousand pulses needed. Electrochemical side-effects and electrophoresis were minimal. The lysates contained non-fragmented DNA which was readily amplifiable by PCR. As the method was not limited to samples of high specific resistance, it should be applicable to physiological fluids and be useful for genomic and DNA diagnostic applications. PMID:10734214

  13. Myocardial scar predicts monomorphic ventricular tachycardia but not polymorphic ventricular tachycardia or ventricular fibrillation in nonischemic dilated cardiomyopathy.

    PubMed

    Piers, Sebastiaan R D; Everaerts, Kimberly; van der Geest, Rob J; Hazebroek, Mark R; Siebelink, Hans-Marc; Pison, Laurent A F G; Schalij, Martin J; Bekkers, Sebastiaan C A M; Heymans, Stephane; Zeppenfeld, Katja

    2015-10-01

    The relation between myocardial scar and different types of ventricular arrhythmias in patients with nonischemic dilated cardiomyopathy (NIDCM) is unknown. The purpose of this study was to analyze the effect of myocardial scar, assessed by late gadolinium enhancement cardiac magnetic resonance imaging (LGE-CMR), on the occurrence and type of ventricular arrhythmia in patients with NIDCM. Consecutive patients with NIDCM who underwent LGE-CMR and implantable cardioverter-defibrillator (ICD) implantation at either of 2 centers were included. LGE was defined by signal intensity ≥35% of maximal signal intensity, subdivided into core and border zones (≥50% and 35%-50% of maximal signal intensity, respectively), and categorized according to location (basal or nonbasal) and transmurality. ICD recordings and electrocardiograms were reviewed to determine the occurrence and type of ventricular arrhythmia during follow-up. Of 87 patients (age 56 ± 13 y, 62% male, left ventricular ejection fraction 29% ± 12%), 55 (63%) had LGE (median 6.3 g, interquartile range 0.0-13.8 g). During a median follow-up of 45 months, monomorphic ventricular tachycardia (VT) occurred in 18 patients (21%) and polymorphic VT/ventricular fibrillation (VF) in 10 (11%). LGE predicted monomorphic VT (log-rank, P < .001), but not polymorphic VT/VF (log-rank, P = .40). The optimal cutoff value for the extent of LGE to predict monomorphic VT was 7.2 g (area under curve 0.84). Features associated with monomorphic VT were core extent, basal location, and area with 51%-75% LGE transmurality. Myocardial scar assessed by LGE-CMR predicts monomorphic VT, but not polymorphic VT/VF, in NIDCM. The risk for monomorphic VT is particularly high when LGE shows a basal transmural distribution and a mass ≥7.2 g. Importantly, patients without LGE on CMR remain at risk for potentially fatal polymorphic VT/VF. Copyright © 2015 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

  14. Acridine dyes and other DNA-intercalating agents induce the luminescence system of luminous bacteria and their dark variants.

    PubMed Central

    Ulitzur, S; Weiser, I

    1981-01-01

    Acridine dyes and other DNA-intercalating agents such as ethidium bromide, theophylline, and caffeine induce luminescence in dark variants (K variants) different luminous species of bacteria, as well as in their wild-type luminous cells, prior to induction. The increase in luminescence appears 10-20 min after addition of these agents and is inhibited by chloramphenicol or rifampicin. Addition of these agents affects the synthesis of both luciferase and aldehyde-synthesizing enzymes. It is hypothesized that these agents, through their intercalation into DNA, cause configurational changes resulting in derepressed transcription of the luminescence operon. PMID:6943543

  15. An efficient strategy for broad-range detection of low abundance bacteria without DNA decontamination of PCR reagents.

    PubMed

    Chang, Shy-Shin; Hsu, Hsung-Ling; Cheng, Ju-Chien; Tseng, Ching-Ping

    2011-01-01

    Bacterial DNA contamination in PCR reagents has been a long standing problem that hampers the adoption of broad-range PCR in clinical and applied microbiology, particularly in detection of low abundance bacteria. Although several DNA decontamination protocols have been reported, they all suffer from compromised PCR efficiency or detection limits. To date, no satisfactory solution has been found. We herein describe a method that solves this long standing problem by employing a broad-range primer extension-PCR (PE-PCR) strategy that obviates the need for DNA decontamination. In this method, we first devise a fusion probe having a 3'-end complementary to the template bacterial sequence and a 5'-end non-bacterial tag sequence. We then hybridize the probes to template DNA, carry out primer extension and remove the excess probes using an optimized enzyme mix of Klenow DNA polymerase and exonuclease I. This strategy allows the templates to be distinguished from the PCR reagent contaminants and selectively amplified by PCR. To prove the concept, we spiked the PCR reagents with Staphylococcus aureus genomic DNA and applied PE-PCR to amplify template bacterial DNA. The spiking DNA neither interfered with template DNA amplification nor caused false positive of the reaction. Broad-range PE-PCR amplification of the 16S rRNA gene was also validated and minute quantities of template DNA (10-100 fg) were detectable without false positives. When adapting to real-time and high-resolution melting (HRM) analytical platforms, the unique melting profiles for the PE-PCR product can be used as the molecular fingerprints to further identify individual bacterial species. Broad-range PE-PCR is simple, efficient, and completely obviates the need to decontaminate PCR reagents. When coupling with real-time and HRM analyses, it offers a new avenue for bacterial species identification with a limited source of bacterial DNA, making it suitable for use in clinical and applied microbiology

  16. 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.

  17. DNA isolation protocols affect the detection limit of PCR approaches of bacteria in samples from the human gastrointestinal tract.

    PubMed

    Zoetendal, E G; Ben-Amor, K; Akkermans, A D; Abee, T; de Vos, W M

    2001-11-01

    A major concern in molecular ecological studies is the lysis efficiency of different bacteria in a complex ecosystem. We used a PCR-based 16S rDNA approach to determine the effect of two DNA isolation protocols (i.e. the bead beating and Triton-X100 method) on the detection limit of seven feces-associated bacterial species of different genera. Glycogen was used in these protocols to improve the precipitation of small concentrations of DNA in ethanol without affecting the sequential procedures. The PCR detection limit of 16S rDNA amplicons on agarose gel from the seven strains tested varied between 8.0 (+/- 1.3) x 10(4) and 4.3 (+/- 1.6) x 10(6) cells for the bead beating method, and between 8.0 (+/- 1.3) x 10(4) and 5.4 (+/- 0.7) x 10(8) cells for the Triton X-100 method. These large differences are most like due to the difference in cell lysis efficiency, since a competitive PCR experiment did not indicate any preference for gram negative, low G+C gram positive or high G+C gram positive bacteria. Denaturing gradient gel electrophoresis (DGGE) analysis was performed to investigate the effect of both DNA isolation protocols on the lysis efficiency of bacteria in fecal samples. A higher diversity in fecal samples was observed with the bead beating method than with the Triton-X100 method. Bands in the bead beating method-derived DGGE profiles corresponding to bands of cloned sequences of the Clostridium coccoides-Eubacterium rectale group and uncultured Fusobacterium prausnitzii were absent or had low intensity in the Triton X-100 method-derived profiles. The applicability of the bead beating method was further investigated by analyzing biopsy samples from the human colon which contain approximately 10(6) cells.

  18. [Standardization of ICO-1 monoclonal antibodies against monomorphic Ia-like (Dr) antigens].

    PubMed

    Baryshnikov, A Iu; Blokhina, N G; Kadagidze, Z G; Tupitsyn, N N; Sokolov, A V

    1987-01-01

    ICO-1 Mab were obtained following BALB/c mouse immunization with 24-week human fetal thymocytes. Cloning for two times by the method of limited dilutions led to a hybridoma with a stable production of G3 isotype Mab. ICO-1 Mab have immunoprecipitated an antigen consisting of two polypeptide chains with molecular weight 29 and 34 kDal. The antigen expression was enhanced following PHA or alloantigen activation of blood mononuclear cells. ICO-1 Mab inhibited the alloantigenic response of blood mononuclear cells. Mab detected 29% of antigen-positive cells in the peripheral blood of healthy adults. When the reaction of ICO-1 Mab was compared with that of Mab against monomorphic Ia-like antigens OKLa, anti-Ia-BRL, BMA-021 and HLA-Dr on blood cells from healthy donors and patients with leukemia proved to be identical.

  19. Clinical and morphological features of undifferentiated monomorphous GH/TSH-secreting pituitary adenoma.

    PubMed

    Skorić, T; Korsić, M; Zarković, K; Plavsić, V; Besenski, N; Breskovac, L; Giljević, Z; Paladino, J

    1999-06-01

    A 41-year-old male presented with progressive visual defects, acromegaly and hyperthyroidism. After clinical evaluation a giant GH/TSH-secreting pituitary adenoma was diagnosed. Administration of the somatostatin analog octreotide at doses of 150 microg s.c. per day inhibited the secretion of both GH and TSH. A three-week treatment with octreotide prior to surgery led to slight visual improvement and CT scan showed some new necrotic areas within the tumor mass. Transcranial surgery was performed. By immunohistochemical analyses of the adenoma tissue GH, prolactin and beta-chorionic gonadotropin were detected; TSH was negative. Electron microscopy revealed an undifferentiated, monomorphous adenoma with morphological features of an acidophil stem cell adenoma such as the presence of misplaced exocytoses, fibrous bodies and mitochondrial gigantism. However, the tumor cells contained small secretory granules (up to 250 nm) accumulated along the cell membrane characteristic of thyrotrope cells. Furthermore, some adenoma cells were fusiform with long cytoplasmic processes resembling thyrotropes. Two months after the operation CT scan revealed a large residual tumor. Serum GH and TSH levels had increased again and the TSH level was even higher than before the treatment. The patient died suddenly, most probably of lethal arrhythmia. Specimens of the adenoma tissue obtained at autopsy confirmed the previous findings with the exception of positive immunostaining for TSH which was found in less than 1% of the adenoma cells. This undifferentiated, monomorphous GH/TSH-secreting pituitary adenoma represents an entity that is unusual both in its ultrastructural features and clinical manifestations suggesting a cytogenesis from an early, undifferentiated stem cell.

  20. [Repetitive monomorphic ventricular tachycardia (Gallavardin type): clinical and electrophysiological characteristics in 20 patients].

    PubMed

    Hoffmann, E; Reithmann, C; Neuser, H; Nimmermann, P; Remp, T; Steinbeck, G

    1998-05-01

    Repetitive monomorphic ventricular tachycardia (RMVT) is defined by the presence of numerous monomorphic isolated, premature ventricular complexes, couplets, and runs of unsustained ventricular tachycardia having the same morphology in patients without structural heart disease. Patients with RMVT mostly demonstrate the typical left bundle branch block morphology with normal or rightward axis during tachycardia. At our institution, 20 patients with RMVT have been systematically studied: a syncope had occurred in 35% of our patients, in three cases a syncope was the first manifestation of the RMVT. Of our RMVT patients, 25% developed sustained episodes (> 3 min) of ventricular tachycardia as documented by Holter ECG. The salvos of ventricular tachycardia are generally short in RMVT. This behavior and the typical exercise dependence differentiates RMVT from paroxysmal sustained idiopathic ventricular tachycardia. Exercise testing is mandatory for correct diagnosis of RMVT. In our institution, 85-90% of RMVT patients demonstrated runs of ventricular tachycardia or sustained ventricular tachycardia while on a treadmill (exercise test) or during isoproterenol infusion. RMVT was inducible by programmed electrical right ventricular stimulation in only 13% of our patients. Therefore, in patients with suspected RMVT programmed electrophysiological stimulation is only useful to differentiate a ventricular tachycardia from a supraventricular tachycardia with bundle brunch block or in patients with unexplained syncope. The prognosis is considered generally good; in our patients no life threatening ventricular tachyarrhythmias were observed during a follow-up of up to 4 years. Verapamil and beta-adrenoceptor antagonists generally offer symptomatic improvement. In some cases treatment with a class III antiarrhythmic agent is necessary. While drug-refractory paroxysmal sustained idiopathic ventricular tachycardia can be abladed with both immediate and long-term success, catheter

  1. Catheter ablation of arrhythmic storm triggered by monomorphic ectopic beats in patients with coronary artery disease.

    PubMed

    Peichl, Petr; Cihák, Robert; Kozeluhová, Markéta; Wichterle, Dan; Vancura, Vlastimil; Kautzner, Josef

    2010-01-01

    Frequent episodes of polymorphic ventricular tachycardias/ventricular fibrillation (VT/VF) in patients with coronary artery disease can be triggered by monomorphic ventricular premature beats (VPBs) and thus, amenable to catheter ablation. The goal of this study was to review single-center experience in catheter ablation of electrical storm caused by focally triggered polymorphic VT/VF. Catheter ablation of electrical storm due to focally triggered polymorphic VT/VF was performed in nine patients (mean age, 62+/-7 years; two females). All patients had previous myocardial infarction (interval of 3 days to 171 months). Mean left ventricular ejection fraction was 27+/-6 percent. All patients presented with repeated runs of polymorphic VT/VF triggered by monomorphic VPBs. Based on mapping data, the ectopic beats originated from scar border zone on interventricular septum (n=5), inferior wall (n=3), and lateral wall (n=1). Catheter ablation was performed to abolish the triggering ectopy and to modify the arrhythmogenic substrate by linear lesions within the infarct border zone. The ablation procedure was acutely successful in eight out of nine patients. During the follow-up of 13+/-7 months, two patients died due to progressive heart failure. One patient had late recurrence of electrical storm due to ectopic beats of different morphology and was successfully reablated. Electrical storm due to focally triggered polymorphic VT/VF may occur either in subacute phase of myocardial infarction or substantially later after index event. Catheter ablation of ectopic beats triggering these arrhythmias can successfully abolish electrical storm and become a life-saving procedure.

  2. An improved method for extracting bacteria from soil for high molecular weight DNA recovery and BAC library construction.

    PubMed

    Liu, Juan; Li, Jingquan; Feng, Li; Cao, Hui; Cui, Zhongli

    2010-12-01

    Separation of bacterial cells from soil is a key step in the construction of metagenomic BAC libraries with large DNA inserts. Our results showed that when combined with sodium pyro-phosphate and homogenization for soil dispersion, sucrose density gradient centrifugation (SDGC) was more effective at separating bacteria from soil than was low speed centrifugation (LSC). More than 70% of the cells, along with some soil colloids, were recovered with one round of centrifugation. A solution of 0.8% NaCl was used to resuspend these cell and soil pellets for purification with nycodenz density gradient centrifugation (NDGC). After purification, more than 30% of the bacterial cells in the primary soil were extracted. This procedure effectively removed soil contamination and yielded sufficient cells for high molecular weight (HMW) DNA isolation. Ribosomal intergenic spacer analysis (RISA) showed that the microbial community structure of the extracted cells was similar to that of the primary soil, suggesting that this extraction procedure did not significantly change the the soil bacteria community structure. HMW DNA was isolated from bacterial cells extracted from red soil for metagenomic BAC library construction. This library contained DNA inserts of more than 200 Mb with an average size of 75 kb.

  3. How long can culturable bacteria and total DNA persist in environmental waters? The role of sunlight and solid particles.

    PubMed

    Gutiérrez-Cacciabue, Dolores; Cid, Alicia G; Rajal, Verónica B

    2016-01-01

    In this work, sunlight inactivation of two indicator bacteria in freshwater, with and without solid particles, was studied and the persistence of culturable cells and total DNA was compared. Environmental water was used to prepare two matrices, with and without solid particles, which were spiked with Escherichia coli and Enterococcus faecalis. These matrices were used to prepare microcosm bags that were placed in two containers: one exposed to sunlight and the other in the dark. During one month, samples were removed from each container and detection was done by membrane filter technique and real-time PCR. Kinetic parameters were calculated to assess sunlight effect. Indicator bacteria without solid particles exposed to sunlight suffered an immediate decay (<4h) compared with the ones which were shielded from them. In addition, the survival of both bacteria with solid particles varied depending on the situation analyzed (T99 from 3 up to 60days), being always culturable E. coli more persistent than E. faecalis. On the other side, E. faecalis DNA persisted much longer than culturable cells (T99>40h in the dark with particles). In this case active cells were more prone to sunlight than total DNA and the protective effect of solid particles was also observed. Results highlight that the effects caused by the parameters which describe the behavior of culturable microorganisms and total DNA in water are different and must be included in simulation models but without forgetting that these parameters will also depend on bacterial properties, sensitizers, composition, type, and uses of the aquatic environment under assessment.

  4. Bacteria used for the production of yogurt inactivate carcinogens and prevent DNA damage in the colon of rats.

    PubMed

    Wollowski, I; Ji, S T; Bakalinsky, A T; Neudecker, C; Pool-Zobel, B L

    1999-01-01

    Lactic acid-producing bacteria prevent carcinogen-induced preneoplastic lesions and tumors in rat colon. Because the mechanisms responsible for these protective effects are unknown, two strains of lactic acid bacteria, Lactobacillus delbrueckii ssp. bulgaricus 191R and Streptococcus salivarius ssp. thermophilus CH3, that are used to produce yogurt, were investigated in vitro and in vivo to elucidate their potential to deactivate carcinogens. Using the "Comet assay" to detect genetic damage, we found that L. bulgaricus 191R applied orally to rats could prevent 1, 2-dimethylhydrazine-induced DNA breaks in the colon in vivo, whereas St. thermophilus CH3 were not effective. However, in vitro, both strains prevented DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in isolated primary rat colon cells. Extracts prepared from milk fermented with St. thermophilus CH3 were as efficient in deactivating MNNG as was L-cysteine. Isolated metabolites arising from bacteria during fermentation in the colon or in milk [L(+) lactate, D(-) lactate, palmitic acid and isopalmitic acid] were not effective. We postulate that thiol-containing breakdown products of proteins, via catalysis by bacterial proteases, could be one mechanism by which MNNG or other carcinogens are deactivated in the gut lumen resulting in reduced damage to colonic mucosal cells.

  5. Bacteria capture, lysate clearance, and plasmid DNA extraction using pH-sensitive multifunctional magnetic nanoparticles.

    PubMed

    Shan, Zhi; Wu, Qi; Wang, Xianxiang; Zhou, Zhongwu; Oakes, Ken D; Zhang, Xu; Huang, Qianming; Yang, Wanshen

    2010-03-01

    A multifunctional magnetic nanoparticle (MNP)-assisted bioseparation method was developed to isolate plasmid DNA (pDNA) from Escherichia coli culture. Using the pH-sensitive carboxyl-modified magnetic nanoparticles, both cell capture and the subsequent removal of genomic DNA/protein complex after lysis can be achieved simply by magnetic separation. Furthermore, the yield and purity of pDNA extracted by MNPs are comparable to those obtained using organic solvents or commercial kits. This time- and cost-effective protocol does not require centrifugation or precipitation steps and has the potential for automated DNA extraction, especially within miniaturized lab chip applications. Copyright 2009 Elsevier Inc. All rights reserved.

  6. Electrochemical sandwich assay for attomole analysis of DNA and RNA from beer spoilage bacteria Lactobacillus brevis.

    PubMed

    Shipovskov, Stepan; Saunders, Aaron M; Nielsen, Jesper S; Hansen, Majken H; Gothelf, Kurt V; Ferapontova, Elena E

    2012-01-01

    Attomole (10(-18)mol) levels of RNA and DNA isolated from beer spoilage bacterial cells Lactobacillus brevis have been detected by the electrochemical sandwich DNA hybridization assay exploiting enzymatic activity of lipase. DNA sequences specific exclusively to L. brevis DNA and RNA were selected and used for probe and target DNA design. The assay employs magnetic beads (MB) modified with a capture DNA sequence and a reporter DNA probe labeled with the enzyme, both made to be highly specific for L. brevis DNA. Lipase-labeled DNAs captured on MBs in the sandwich assay were collected on gold electrodes modified with a ferrocene (Fc)-terminated SAM formed by aliphatic esters. Lipase hydrolysis of the ester bond released a fraction of the Fc redox active groups from the electrode surface, decreasing the electrochemical signal from the surface-confined Fc. The assay, shown to be efficient for analysis of short synthetic DNA sequences, was ineffective with genomic double stranded bacterial DNA, but it allowed down to 16 amole detection of 1563 nts long RNA, isolated from bacterial ribosomes without the need for PCR amplification, and single DNA strands produced from ribosomal RNA. No interference from E. coli RNA was registered. The assay allowed analysis of 400 L. brevis cells isolated from 1L of beer, which fits the "alarm signal" range (from 1 to 100 cells per 100mL). Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Identification of Benzo[a]pyrene-Metabolizing Bacteria in Forest Soils by Using DNA-Based Stable-Isotope Probing

    PubMed Central

    Song, Mengke; Jiang, Longfei; Zhang, Dayi; Wang, Yujie; Zhang, Gan

    2015-01-01

    DNA-based stable-isotope probing (DNA-SIP) was used in this study to investigate the uncultivated bacteria with benzo[a]pyrene (BaP) metabolism capacities in two Chinese forest soils (Mt. Maoer in Heilongjiang Province and Mt. Baicaowa in Hubei Province). We characterized three different phylotypes with responsibility for BaP degradation, none of which were previously reported as BaP-degrading microorganisms by SIP. In Mt. Maoer soil microcosms, the putative BaP degraders were classified as belonging to the genus Terrimonas (family Chitinophagaceae, order Sphingobacteriales), whereas Burkholderia spp. were the key BaP degraders in Mt. Baicaowa soils. The addition of metabolic salicylate significantly increased BaP degradation efficiency in Mt. Maoer soils, and the BaP-metabolizing bacteria shifted to the microorganisms in the family Oxalobacteraceae (genus unclassified). Meanwhile, salicylate addition did not change either BaP degradation or putative BaP degraders in Mt. Baicaowa. Polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHD) genes were amplified, sequenced, and quantified in the DNA-SIP 13C heavy fraction to further confirm the BaP metabolism. By illuminating the microbial diversity and salicylate additive effects on BaP degradation across different soils, the results increased our understanding of BaP natural attenuation and provided a possible approach to enhance the bioremediation of BaP-contaminated soils. PMID:26253666

  8. High-throughput DNA microarray detection of pathogenic bacteria in shallow well groundwater in the Kathmandu Valley, Nepal.

    PubMed

    Inoue, Daisuke; Hinoura, Takuji; Suzuki, Noriko; Pang, Junqin; Malla, Rabin; Shrestha, Sadhana; Chapagain, Saroj Kumar; Matsuzawa, Hiroaki; Nakamura, Takashi; Tanaka, Yasuhiro; Ike, Michihiko; Nishida, Kei; Sei, Kazunari

    2015-01-01

    Because of heavy dependence on groundwater for drinking water and other domestic use, microbial contamination of groundwater is a serious problem in the Kathmandu Valley, Nepal. This study investigated comprehensively the occurrence of pathogenic bacteria in shallow well groundwater in the Kathmandu Valley by applying DNA microarray analysis targeting 941 pathogenic bacterial species/groups. Water quality measurements found significant coliform (fecal) contamination in 10 of the 11 investigated groundwater samples and significant nitrogen contamination in some samples. The results of DNA microarray analysis revealed the presence of 1-37 pathogen species/groups, including 1-27 biosafety level 2 ones, in 9 of the 11 groundwater samples. While the detected pathogens included several feces- and animal-related ones, those belonging to Legionella and Arthrobacter, which were considered not to be directly associated with feces, were detected prevalently. This study could provide a rough picture of overall pathogenic bacterial contamination in the Kathmandu Valley, and demonstrated the usefulness of DNA microarray analysis as a comprehensive screening tool of a wide variety of pathogenic bacteria.

  9. Characteristics and diversity of endophytic bacteria in moso bamboo (Phyllostachys edulis) based on 16S rDNA sequencing.

    PubMed

    Liu, Fang; Yuan, Zongsheng; Zhang, Xintao; Zhang, Guofang; Xie, Baogui

    2017-06-10

    To understand the diversity and distribution of endophytic bacteria in moso bamboo (Phyllostachys edulis), we used 16S rDNA sequencing to investigate the characteristics and diversity of endophytic bacteria in different moso bamboo tissues. After 454 pyrosequencing, we obtained 141,269 sequences from seven moso bamboo tissue samples. The taxonomic origins of unique sequences were identified using RDP classifier. The results showed that these sequences belonged to 26 bacterial orders, including the Actinomycetales, Rickettsiales, Burkholderiales, Enterobacteriales, and Rhizobiales. Among these, Enterobacteriales was widely found in all bamboo tissues. Endophytic bacterial communities differed between the moso bamboo shoot and pole. With continuous growth and development, the number of endophytic species in the moso bamboo pole increased gradually.

  10. An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents

    PubMed Central

    Chang, Shy-Shin; Hsu, Hsung-Ling; Cheng, Ju-Chien; Tseng, Ching-Ping

    2011-01-01

    Background Bacterial DNA contamination in PCR reagents has been a long standing problem that hampers the adoption of broad-range PCR in clinical and applied microbiology, particularly in detection of low abundance bacteria. Although several DNA decontamination protocols have been reported, they all suffer from compromised PCR efficiency or detection limits. To date, no satisfactory solution has been found. Methodology/Principal Findings We herein describe a method that solves this long standing problem by employing a broad-range primer extension-PCR (PE-PCR) strategy that obviates the need for DNA decontamination. In this method, we first devise a fusion probe having a 3′-end complementary to the template bacterial sequence and a 5′-end non-bacterial tag sequence. We then hybridize the probes to template DNA, carry out primer extension and remove the excess probes using an optimized enzyme mix of Klenow DNA polymerase and exonuclease I. This strategy allows the templates to be distinguished from the PCR reagent contaminants and selectively amplified by PCR. To prove the concept, we spiked the PCR reagents with Staphylococcus aureus genomic DNA and applied PE-PCR to amplify template bacterial DNA. The spiking DNA neither interfered with template DNA amplification nor caused false positive of the reaction. Broad-range PE-PCR amplification of the 16S rRNA gene was also validated and minute quantities of template DNA (10–100 fg) were detectable without false positives. When adapting to real-time and high-resolution melting (HRM) analytical platforms, the unique melting profiles for the PE-PCR product can be used as the molecular fingerprints to further identify individual bacterial species. Conclusions/Significance Broad-range PE-PCR is simple, efficient, and completely obviates the need to decontaminate PCR reagents. When coupling with real-time and HRM analyses, it offers a new avenue for bacterial species identification with a limited source of bacterial DNA

  11. Prognosis of patients with a first episode of sustained monomorphic ventricular tachycardia.

    PubMed

    Bodegas, A; Arana, J; Rumoroso, J R; Rodrigo, D; Barrenetxea, J I

    1998-07-01

    To evaluate the cardiac mortality in patients suffering from a first episode of sustained monomorphic ventricular tachycardia (SMVT). 100 patients less than 75 years old were evaluated during a 50-month follow-up period. Patients were classified into four groups: myocardial infarction, dilated cardiomyopathy, normal heart and miscellany. Seventeen patients underwent a cardioverter-defibrillator implantation, two heart transplant, three aneurysmectomy and 10 other types of cardiac surgical proceedings. Patients with a left ventricle ejection fraction (EF)> or =50% presented a cardiac mortality of 5% compared with 38% of those with EF<50%. Etiology of underlying cardiomyopathy with an EF> or =50% was associated with a cardiac mortality of 5% (normal heart), 5% (myocardial infarction) and 9% (miscellany) compared to those with EF<50%: 33% (dilated cardiomyopathy) and 40% (myocardial infarction). Patients who experienced syncope during the first episode of SMVT showed a cardiac mortality of 31% compared to those 14% (P < 0.05) who did not experience. Patients with syncope, myocardial infarction and EF<50% showed a cardiac mortality of 68%. The present study shows that survival after the first episode of SMVT is closely related to EF and the existence of syncope. Patients with myocardial infarction and EF<50% had a worse prognosis when the site was the inferior wall.

  12. Monomorphic ants undergo within-colony morphological changes along the metal-pollution gradient.

    PubMed

    Grześ, Irena M; Okrutniak, Mateusz; Woch, Marcin W

    2015-04-01

    In ants, intra and inter-colony variation in body size can be considerable, even in monomorphic species. It has been previously shown that size-related parameters can be environmentally sensitive. The shape of the body size distribution curve is, however, rarely investigated. In this study, we measured head widthes of the black garden ant Lasius niger workers using digital methods. The ants were sampled from 51 colonies originating from 19 sites located along a metal pollution gradient, established in a former mining area in Poland. Total zinc concentrations in random samples of small invertebrates were used as a measure of site pollution levels. We found that the skewness of head size distribution grows significantly in line with the pollution level of the site, ranging from values slightly below zero (about -0.5) in the least polluted site up to a positive value (about 1.5) in the most polluted site. This result indicates that the frequency of small ants grows as pollution levels increase. The coefficient of variation, as well as the measures of central tendency, was not related to the pollution level. Four hypotheses explaining the obtained results are proposed. The bias towards the higher frequency of small workers may result from energy limitation and/or metal toxicity, but may also have an adaptive function.

  13. DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats

    PubMed Central

    de Lange, Orlando; Wolf, Christina; Thiel, Philipp; Krüger, Jens; Kleusch, Christian; Kohlbacher, Oliver; Lahaye, Thomas

    2015-01-01

    Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats. PMID:26481363

  14. Genetic encoding of DNA nanostructures and their self-assembly in living bacteria

    PubMed Central

    Elbaz, Johann; Yin, Peng; Voigt, Christopher A.

    2016-01-01

    The field of DNA nanotechnology has harnessed the programmability of DNA base pairing to direct single-stranded DNAs (ssDNAs) to assemble into desired 3D structures. Here, we show the ability to express ssDNAs in Escherichia coli (32–205 nt), which can form structures in vivo or be purified for in vitro assembly. Each ssDNA is encoded by a gene that is transcribed into non-coding RNA containing a 3′-hairpin (HTBS). HTBS recruits HIV reverse transcriptase, which nucleates DNA synthesis and is aided in elongation by murine leukemia reverse transcriptase. Purified ssDNA that is produced in vivo is used to assemble large 1D wires (300 nm) and 2D sheets (5.8 μm2) in vitro. Intracellular assembly is demonstrated using a four-ssDNA crossover nanostructure that recruits split YFP when properly assembled. Genetically encoding DNA nanostructures provides a route for their production as well as applications in living cells. PMID:27091073

  15. Genetic encoding of DNA nanostructures and their self-assembly in living bacteria.

    PubMed

    Elbaz, Johann; Yin, Peng; Voigt, Christopher A

    2016-04-19

    The field of DNA nanotechnology has harnessed the programmability of DNA base pairing to direct single-stranded DNAs (ssDNAs) to assemble into desired 3D structures. Here, we show the ability to express ssDNAs in Escherichia coli (32-205 nt), which can form structures in vivo or be purified for in vitro assembly. Each ssDNA is encoded by a gene that is transcribed into non-coding RNA containing a 3'-hairpin (HTBS). HTBS recruits HIV reverse transcriptase, which nucleates DNA synthesis and is aided in elongation by murine leukemia reverse transcriptase. Purified ssDNA that is produced in vivo is used to assemble large 1D wires (300 nm) and 2D sheets (5.8 μm(2)) in vitro. Intracellular assembly is demonstrated using a four-ssDNA crossover nanostructure that recruits split YFP when properly assembled. Genetically encoding DNA nanostructures provides a route for their production as well as applications in living cells.

  16. A census of α-helical membrane proteins in double-stranded DNA viruses infecting bacteria and archaea.

    PubMed

    Kristensen, David M; Saeed, Usman; Frishman, Dmitrij; Koonin, Eugene V

    2015-11-10

    Viruses are the most abundant and genetically diverse biological entities on earth, yet the repertoire of viral proteins remains poorly explored. As the number of sequenced virus genomes grows into the thousands, and the number of viral proteins into the hundreds of thousands, we report a systematic computational analysis of the point of first-contact between viruses and their hosts, namely viral transmembrane (TM) proteins. The complement of α-helical TM proteins in double-stranded DNA viruses infecting bacteria and archaea reveals large-scale trends that differ from those of their hosts. Viruses typically encode a substantially lower fraction of TM proteins than archaea or bacteria, with the notable exception of viruses with virions containing a lipid component such as a lipid envelope, internal lipid core, or inner membrane vesicle. Compared to bacteriophages, archaeal viruses are substantially enriched in membrane proteins. However, this feature is not always stable throughout the evolution of a viral lineage; for example, TM proteins are not part of the common heritage shared between Lipothrixviridae and Rudiviridae. In contrast to bacteria and archaea, viruses almost completely lack proteins with complicated membrane topologies composed of more than 4 TM segments, with the few detected exceptions being obvious cases of relatively recent horizontal transfer from the host. The dramatic differences between the membrane proteomes of cells and viruses stem from the fact that viruses do not depend on essential membranes for energy transformation, ion homeostasis, nutrient transport and signaling.

  17. DNA analysis of fecal bacteria to augment an epikarst dye trace study at Crump's Cave, Kentucky

    USDA-ARS?s Scientific Manuscript database

    A rainfall simulation experiment was performed to investigate the transport behavior of fecal-derived bacteria through shallow karst soils and through the epikarst. The experiment was conducted at Cave Springs Cavern located just south of Mammoth Cave National Park on the Sinkhole Plain of South Cen...

  18. Monomorphic lymphomas arising in patients with Hodgkin's disease. Correlation of morphologic, immunophenotypic, and molecular genetic findings in 12 cases.

    PubMed Central

    Casey, T. T.; Cousar, J. B.; Mangum, M.; Williams, M. E.; Lee, J. T.; Greer, J. P.; Collins, R. D.

    1990-01-01

    Patients with Hodgkin's Disease (HD) occasionally develop monomorphic lymphomas in which mononuclear cells, usually large in size, grow in sheets, and in which there are few reacting cells or classic Reed-Sternberg (RS) cells. Twelve patients of this type were reviewed to determine the nature of the monomorphic growth. Paraffin-embedded tissue sections from the original diagnostic HD and the monomorphic growths were stained for Leu-M1 (CD15), leukocyte common antigen (LCA, CD45), pan B-cell markers LN1, LN2, and L26, and pan T-cell marker UCHL1 (CD45R) reactive in paraffin-embedded tissues. Cases were included only if the original diagnostic material had the classic histopathologic features of HD, if there was a separate monomorphic growth (in place or time), and if sufficient materials from both phases were available for study. Original diagnoses of HD included nodular sclerosing (NS; 8 cases); lymphocyte predominant (LP; 2 cases); mixed cellularity (MC; 1 case); and lymphocyte depleted (LD: 1 case) types. RS cells in the eight cases of NS HD and one case of MC HD were generally Leu-M1 and LN2 positive, and L26, LN1, UCHL1, and LCA negative. RS cells in one case of NS HD were LCA positive in addition to Leu-M1, LN1, and LN2. Two cases of NS HD showed L26 positive RS cells. Conversely, RS cells and lymphocytic-histiocytic (L and H) variants in the cases of LP HD were Leu-M1 and LN2 negative, and LCA and LN1 positive. The one case of LD HD possessed RS cells that were negative for Leu-M1, but positive for LCA, L26, LN1, and LN2. In seven cases (4 NS, 2 LP, 1 LD) the monomorphic growths possessed a B-cell phenotype (LCA, L26, and LN1 positive; Leu-M1 and UCHL1 negative). In the remaining cases (4 NS, 1 MC), the monomorphic growths were Leu-M1 positive, and displayed phenotypes similar to the RS cells of the original NS HD. Southern blot analysis was performed on the monomorphic components of five cases and showed some form of immunoglobulin gene rearrangement in each

  19. All-or-Nothing Character of DNA Degradation in Bacteria after Ionizing Radiation

    PubMed Central

    Pollard, Ernest C.; Kraus, Kathryn

    1973-01-01

    The response of single cells of Escherichia coli Bs-11 and 15JG151 to radiation-induced DNA degradation has been observed by autoradiography. For both cells it is concluded that the event which causes DNA degradation is of an all-or-nothing character. The unit which suffers degradation is not the whole cell, but each cell has between two and four such units. The results suggest that there is some resynthesis of degraded DNA. Evidence that this occurs is shown by examining the degradation of mass cultures of 15TAU/t3 and t7 below and above the permissive temperature for DNA synthesis. The results on the all-or-nothing character are in agreement with previous studies made by completely different techniques. PMID:4572361

  20. Assessment of methods to recover DNA from bacteria, fungi and archaea in complex environmental samples.

    PubMed

    Guillén-Navarro, Karina; Herrera-López, David; López-Chávez, Mariana Y; Cancino-Gómez, Máximo; Reyes-Reyes, Ana L

    2015-11-01

    DNA extraction from environmental samples is a critical step for metagenomic analysis to study microbial communities, including those considered uncultivable. Nevertheless, obtaining good quality DNA in sufficient quantities for downstream methodologies is not always possible, and it depends on the complexity and stability of each ecosystem, which could be more problematic for samples from tropical regions because those ecosystems are less stable and more complex. Three laboratory methods for the extraction of nucleic acids from samples representing unstable (decaying coffee pulp and mangrove sediments) and relatively stable (compost and soil) environments were tested. The results were compared with those obtained using two commercial DNA extraction kits. The quality of the extracted DNA was evaluated by PCR amplification to verify the recovery of bacterial, archaeal, and fungal genetic material. The laboratory method that gave the best results used a lysis procedure combining physical, chemical, and enzymatic steps.

  1. Bridgehead invasion of a monomorphic plant pathogenic bacterium: Xanthomonas citri pv. citri, an emerging citrus pathogen in Mali and Burkina Faso.

    PubMed

    Leduc, A; Traoré, Y N; Boyer, K; Magne, M; Grygiel, P; Juhasz, C C; Boyer, C; Guerin, F; Wonni, I; Ouedraogo, L; Vernière, C; Ravigné, V; Pruvost, O

    2015-11-01

    Molecular epidemiology studies further our understanding of migrations of phytopathogenic bacteria, the major determining factor in their emergence. Asiatic citrus canker, caused by Xanthomonas citri pv. citri, was recently reported in Mali and Burkina Faso, a region remote from other contaminated areas. To identify the origin and pathways of these emergences, we used two sets of markers, minisatellites and microsatellites, for investigating different evolutionary scales. Minisatellite typing suggested the introduction of two groups of strains in Mali (DAPC 1 and DAPC 2), consistent with microsatellite typing. DAPC 2 was restricted to Bamako district, whereas DAPC 1 strains were found much more invasive. The latter strains formed a major clonal complex based on microsatellite data with the primary and secondary founders detected in commercial citrus nurseries and orchards. This suggests that human activities played a major role in the spread of DAPC 1 strains via the movement of contaminated propagative material, further supported by the frequent lack of differentiation between populations from geographically distant nurseries and orchards. Approximate Bayesian Computation analyses supported the hypothesis that strains from Burkina Faso resulted from a bridgehead invasion from Mali. Multi-locus variable number of tandem repeat analysis and Approximate Bayesian Computation are useful for understanding invasion routes and pathways of monomorphic bacterial pathogens.

  2. Late Presentation of Recurrent Monomorphic Ventricular Tachycardia following Minimally Invasive Mitral Valve Repair due to Epicardial Injury.

    PubMed

    South, Harry L; Osoro, Moses; Overly, Tjuan

    2014-01-01

    We report a 73-year-old male with late onset monomorphic ventricular tachycardia following mitral valve repair (MVR). Typically, injury to epicardial arteries following mitral valve repair/replacement presents immediately as ventricular tachycardia/fibrillation, difficulty weaning from cardiopulmonary bypass, worsening ECG changes, increasing cardiac biomarkers, or new wall motion abnormalities. Our case illustrates a "late complication" of a distorted circumflex artery following mitral valve repair and the importance of early diagnostic angiography and percutaneous intervention.

  3. A matter of life or death: modeling DNA damage and repair in bacteria.

    PubMed

    Karschau, Jens; de Almeida, Camila; Richard, Morgiane C; Miller, Samantha; Booth, Ian R; Grebogi, Celso; de Moura, Alessandro P S

    2011-02-16

    DNA damage is a hazard all cells must face, and evolution has created a number of mechanisms to repair damaged bases in the chromosome. Paradoxically, many of these repair mechanisms can create double-strand breaks in the DNA molecule which are fatal to the cell. This indicates that the connection between DNA repair and death is far from straightforward, and suggests that the repair mechanisms can be a double-edged sword. In this report, we formulate a mathematical model of the dynamics of DNA damage and repair, and we obtain analytical expressions for the death rate. We predict a counterintuitive relationship between survival and repair. We can discriminate between two phases: below a critical threshold in the number of repair enzymes, the half-life decreases with the number of repair enzymes, but becomes independent of the number of repair enzymes above the threshold. We are able to predict quantitatively the dependence of the death rate on the damage rate and other relevant parameters. We verify our analytical results by simulating the stochastic dynamics of DNA damage and repair. Finally, we also perform an experiment with Escherichia coli cells to test one of the predictions of our model.

  4. Ada response - a strategy for repair of alkylated DNA in bacteria.

    PubMed

    Mielecki, Damian; Grzesiuk, Elżbieta

    2014-06-01

    Alkylating agents are widespread in the environment and also occur endogenously. They can be cytotoxic or mutagenic to the cells introducing alkylated bases to DNA or RNA. All organisms have evolved multiple DNA repair mechanisms to counteract the effects of DNA alkylation: the most cytotoxic lesion, N(3)-methyladenine (3meA), is excised by AlkA glycosylase initiating base excision repair (BER); toxic N(1)-methyladenine (1meA) and N(3)-methylcytosine (3meC), induced in DNA and RNA, are removed by AlkB dioxygenase; and mutagenic and cytotoxic O(6)-methylguanine (O(6) meG) is repaired by Ada methyltransferase. In Escherichia coli, Ada response involves the expression of four genes, ada, alkA, alkB, and aidB, encoding respective proteins Ada, AlkA, AlkB, and AidB. The Ada response is conserved among many bacterial species; however, it can be organized differently, with diverse substrate specificity of the particular proteins. Here, an overview of the organization of the Ada regulon and function of individual proteins is presented. We put special effort into the characterization of AlkB dioxygenases, their substrate specificity, and function in the repair of alkylation lesions in DNA/RNA.

  5. High-Voltage Electroporation of Bacteria: Genetic Transformation of Campylobacter jejuni with Plasmid DNA

    NASA Astrophysics Data System (ADS)

    Miller, Jeff F.; Dower, William J.; Tompkins, Lucy S.

    1988-02-01

    Electroporation permits the uptake of DNA by mammalian cells and plant protoplasts because it induces transient permeability of the cell membrane. We investigated the utility of high-voltage electroporation as a method for genetic transformation of intact bacterial cells by using the enteric pathogen Campylobacter jejuni as a model system. This report demonstrates that the application of high-voltage discharges to bacterial cells permits genetic transformation. Our method involves exposure of a Campylobacter cell suspension to a high-voltage exponential decay discharge (5-13 kV/cm) for a brief period of time (resistance-capacitance time constant = 2.4-26 msec) in the presence of plasmid DNA. Electrical transformation of C. jejuni results in frequencies as high as 1.2 × 106 transformants per μ g of DNA. We have investigated the effects of pulse amplitude and duration, cell growth conditions, divalent cations, and DNA concentration on the efficiency of transformation. Transformants of C. jejuni obtained by electroporation contained structurally intact plasmid molecules. In addition, evidence is presented that indicates that C. jejuni possesses DNA restriction and modification systems. The use of electroporation as a method for transforming other bacterial species and guidelines for its implementation are also discussed.

  6. Endocardial substrate mapping for monomorphic ventricular tachycardia ablation in ischemic and non-ischemic cardiomyopathy.

    PubMed

    Fukuzawa, Koji; Yoshida, Akihiro; Kubo, Shinya; Takano, Takatsugu; Kiuchi, Kunihiko; Kanda, Gaku; Takami, Kaoru; Kumagai, Hiroyuki; Torii, Satoko; Takami, Mitsuru; Yokoyama, Mitsuhiro; Hirata, Ken-ichi

    2008-07-18

    We investigated the differences in the endocardial substrates between ischemic cardiomyopathy (ICM) and non-ICM (NICM) by using electro-anatomical mapping and pace-mapping. We studied 18 patients (ICM and NICM, 9 each) with monomorphic ventricular tachycardia (VT) documented by 12-leads ECG. Low voltage area was defined by signal amplitude <1.5 mV. A pace-map QRS morphology that matched VT in >10 of the 12-leads ECG was regarded as a pace-map match. And conduction delay during pace-mapping was defined as the stimulus to QRS interval >or=40 ms. Low voltage area was 53.8 +/- 21.5 and 20.8 +/- 16.7 cm2 in ICM and NICM patients, respectively (P = 0.002). Pace-mapping was assessed in 6 ICM and 9 NICM. Pace-map match with conduction delay were obtained in all the 6 ICM patients. But in NICM patients, pace-map match with conduction delay was obtained in 3 patients. Pace-map match sites where conduction delay was not observed were obtained in 5 patients. Pace-map match could not be obtained in 1 patient. We attempted ablation in 6 ICM and 7 NICM patients. Subsequently, VT recurrence was not observed in ICM but it was observed in 6 of 7 NICM patients (log-rank P = 0.0016). In NICM patients, the arrhythmogenic substrate that represented the abnormal electrogram and conduction delay was observed less within the endocardial surface when compared with that observed in ICM. VT recurrence rate subsequent to endocardial ablation was higher in NICM than in ICM patients.

  7. Local adaptation and divergence in colour signal conspicuousness between monomorphic and polymorphic lineages in a lizard.

    PubMed

    McLean, C A; Moussalli, A; Stuart-Fox, D

    2014-12-01

    Population differences in visual environment can lead to divergence in multiple components of animal coloration including signalling traits and colour patterns important for camouflage. Divergence may reflect selection imposed by different receivers (conspecifics, predators), which depends in turn on the location of the colour patch. We tested for local adaptation of two genetically and phenotypically divergent lineages of a rock-inhabiting lizard, Ctenophorus decresii, by comparing the visual contrast of colour patches to different receivers in native and non-native environments. The lineages differ most notably in male throat coloration, which is polymorphic in the northern lineage and monomorphic in the southern lineage, but also differ in dorsal and lateral coloration, which is visible to both conspecifics and potential predators. Using models of animal colour vision, we assessed whether lineage-specific throat, dorsal and lateral coloration enhanced conspicuousness to conspecifics, increased crypsis to birds or both, respectively, when viewed against the predominant backgrounds from each lineage. Throat colours were no more conspicuous against native than non-native rock but contrasted more strongly with native lichen, which occurs patchily on rocks inhabited by C. decresii. Conversely, neck coloration (lateral) more closely matched native lichen. Furthermore, although dorsal coloration of southern males was consistently more conspicuous to birds than that of northern males, both lineages had similar absolute conspicuousness against their native backgrounds. Combined, our results are consistent with local adaptation of multiple colour traits in relation to multiple receivers, suggesting that geographic variation in background colour has influenced the evolution of lineage-specific coloration in C. decresii.

  8. Fatty acid and DNA analyses of Permian bacteria isolated from ancient salt crystals reveal differences with their modern relatives.

    PubMed

    Vreeland, Russell H; Rosenzweig, William D; Lowenstein, Tim; Satterfield, Cindy; Ventosa, Antonio

    2006-02-01

    The isolation of living microorganisms from primary 250-million-year-old (MYA) salt crystals has been questioned by several researchers. The most intense discussion has arisen from questions about the texture and age of the crystals used, the ability of organisms to survive 250 million years when exposed to environmental factors such as radiation and the close similarity between 16S rRNA sequences in the Permian and modern microbes. The data in this manuscript are not meant to provide support for the antiquity of the isolated bacterial strains. Rather, the data presents several comparisons between the Permian microbes and other isolates to which they appear related. The analyses include whole cell fatty acid profiling, DNA-DNA hybridizations, ribotyping, and random amplified polymorphic DNA amplification (RAPD). These data show that the Permian strains, studied here, differ significantly from their more modern relatives. These differences are accumulating in both phenotypic and molecular areas of the cells. At the fatty acid level the differences are approaching but have not reached separate species status. At the molecular level the variation appears to be distributed across the genome and within the gene regions flanking the highly conserved 16S rRNA itself. The data show that these bacteria are not identical and help to rule out questions of contamination by putatively modern strains.

  9. Analysis of several methods for the extraction of high quality DNA from acetic acid bacteria in wine and vinegar for characterization by PCR-based methods.

    PubMed

    Jara, C; Mateo, E; Guillamón, J M; Torija, M J; Mas, A

    2008-12-10

    Acetic acid bacteria (AAB) are fastidious microorganisms with poor recovery in culture. Culture-independent methods are currently under examination. Good DNA extraction is a strict requirement of these methods. We compared five methods for extracting the DNA of AAB directly from wine and vinegar samples. Four matrices (white wine, red wine, superficial vinegar and submerged vinegar) contaminated with two AAB strains belonging to Acetobacter pasteurianus and Gluconacetobacter hansenii were assayed. To improve the yield and quality of the extracted DNA, a sample treatment (washing with polyvinyl pyrrolidone or NaCl) was also tested. DNA quality was measured by amplification of the 16S rRNA gene with conventional PCR. DNA recovery rate was assessed by real-time PCR. DNA amplification was always successful with the Wizard method though DNA recovery was poor. A CTAB-based method and NucleoSpin protocol extracted the highest DNA recoveries from wine and vinegar samples. Both of these methods require treatment to recover suitable DNA for amplification with maximum recovery. Both may therefore be good solutions for DNA extraction in wine and vinegar samples. DNA extraction of Ga hansenii was more effective than that of A. pasteurianus. The fastest and cheapest method we evaluated (the Thermal shock protocol) produced the worst results both for DNA amplification and DNA recovery.

  10. Dissecting the protein architecture of DNA-binding transcription factors in bacteria and archaea.

    PubMed

    Rivera-Gómez, Nancy; Martínez-Núñez, Mario Alberto; Pastor, Nina; Rodriguez-Vazquez, Katya; Perez-Rueda, Ernesto

    2017-08-01

    Gene regulation at the transcriptional level is a central process in all organisms where DNA-binding transcription factors play a fundamental role. This class of proteins binds specifically at DNA sequences, activating or repressing gene expression as a function of the cell's metabolic status, operator context and ligand-binding status, among other factors, through the DNA-binding domain (DBD). In addition, TFs may contain partner domains (PaDos), which are involved in ligand binding and protein-protein interactions. In this work, we systematically evaluated the distribution, abundance and domain organization of DNA-binding TFs in 799 non-redundant bacterial and archaeal genomes. We found that the distributions of the DBDs and their corresponding PaDos correlated with the size of the genome. We also identified specific combinations between the DBDs and their corresponding PaDos. Within each class of DBDs there are differences in the actual angle formed at the dimerization interface, responding to the presence/absence of ligands and/or crystallization conditions, setting the orientation of the resulting helices and wings facing the DNA. Our results highlight the importance of PaDos as central elements that enhance the diversity of regulatory functions in all bacterial and archaeal organisms, and our results also demonstrate the role of PaDos in sensing diverse signal compounds. The highly specific interactions between DBDs and PaDos observed in this work, together with our structural analysis highlighting the difficulty in predicting both inter-domain geometry and quaternary structure, suggest that these systems appeared once and evolved with diverse duplication events in all the analysed organisms.

  11. Use of DNA Markers for Investigating Sources of Bacteria in Contaminated Ground Water: Wooster Township, Wayne County, Ohio

    USGS Publications Warehouse

    Dumouchelle, Denise H.

    2006-01-01

    In 2004, a public-health nuisance was declared by the Wayne County Board of Health in the Scenic Heights Drive-Batdorf Road area of Wooster Township, Wayne County, Ohio, because of concerns about the safety of water from local wells. Repeated sampling had detected the presence of fecal-indicator bacteria and elevated nitrate concentrations. In June 2006, the U.S. Geological Survey (USGS), in cooperation with the Ohio Environmental Protection Agency (Ohio EPA), collected and analyzed samples from some of the affected wells to help investigate the possibility of human-origin bacterial contamination. Water samples from 12 wells and 5 home sewage-treatment systems (HSTS) were collected. Bromide concentrations were determined in samples from the 12 wells. Samples from 5 of the 12 wells were analyzed for wastewater compounds. Total coliform, enterococci and Escherichia coli (E. coli) bacteria concentrations were determined for samples from 8 of the 12 wells. In addition, two microbial source-tracking tools that employ DNA markers were used on samples from several wells and a composite sample of water from five septic tanks. The DNA markers from the Enterococcus faecium species and the order Bacteroidales are associated with specific sources, either human or ruminant sources. Bromide concentrations ranged from 0.04 to 0.18 milligrams per liter (mg/L). No wastewater compounds were detected at concentrations above the reporting limits. Samples from the 12 wells also were collected by Ohio EPA and analyzed for chloride and nitrate. Chloride concentrations ranged from 12.6 to 61.6 mg/L and nitrate concentrations ranged from 2.34 to 11.9 mg/L (as N). Total coliforms and enterococci were detected in samples from 8 wells, at concentrations from 2 to 200 colony-forming units per 100 milliliters (CFU/100 mL) and 0.5 to 17 CFU/100 mL, respectively. E. coli were detected in samples from three of the eight wells, at concentrations of 1 or 2 CFU/100 mL. Tests for the human

  12. Detection of beer spoilage bacteria Pectinatus and Megasphaera with acridinium ester labelled DNA probes using a hybridisation protection assay.

    PubMed

    Paradh, A D; Hill, A E; Mitchell, W J

    2014-01-01

    DNA probes specific for rRNA of selected target species were utilised for the detection of beer spoilage bacteria of the genera Pectinatus and Megasphaera using a hybridisation protection assay (HPA). All the probes were modified during synthesis by addition of an amino linker arm at the 5' end or were internally modified by inserting an amine modified thymidine base. Synthesised probes then were labelled with acridinium ester (AE) and purified using reverse phase HPLC. The internally AE labelled probes were able to detect target RNA within the range of 0.016-0.0032pmol. All the designed probes showed high specificity towards target RNA and could detect bacterial contamination within the range of ca. 5×10(2)1×10(3) CFU using the HPA. The developed assay was also compatible with MRS, NBB and SMMP beer enrichment media, routinely used in brewing laboratories.

  13. Research in Undergraduate Instruction: A Biotech Lab Project for Recombinant DNA Protein Expression in Bacteria

    NASA Astrophysics Data System (ADS)

    Brockman, Mark; Ordman, Alfred B.; Campbell, A. Malcolm

    1996-06-01

    In the sophomore-level Molecular Biology and Biotechnology course at Beloit College, students learn basic methods in molecular biology in the context of pursuing a semester-long original research project. We are exploring how DNA sequence affects expression levels of proteins. A DNA fragment encoding all or part of the guanylate monokinase (gmk) sequence is cloned into pSP73 and expressed in E. coli. A monoclonal antibody is made to gmk. The expression level of gmk is determined by SDS gel elctrophoresis, a Western blot, and an ELISA assay. Over four years, an increase in enrollment in the course from 9 to 34 students, the 85% of majors pursuing advanced degrees, and course evaluations all support the conclusion that involving students in research during undergraduate courses encourages them to pursue careers in science.

  14. Identification of bacteria directly from positive blood culture samples by DNA pyrosequencing of the 16S rRNA gene.

    PubMed

    Motoshima, Maiko; Yanagihara, Katsunori; Morinaga, Yoshitomo; Matsuda, Junichi; Hasegawa, Hiroo; Kohno, Shigeru; Kamihira, Shimeru

    2012-11-01

    Rapid identification of the causative bacteria of sepsis in patients can contribute to the selection of appropriate antibiotics and improvement of patients' prognosis. Genotypic identification is an emerging technology that may provide an alternative method to, or complement, established phenotypic identification procedures. We evaluated a rapid protocol for bacterial identification based on PCR and pyrosequencing of the V1 and V3 regions of the 16S rRNA gene using DNA extracted directly from positive blood culture samples. One hundred and two positive blood culture bottles from 68 patients were randomly selected and the bacteria were identified by phenotyping and pyrosequencing. The results of pyrosequencing identification displayed 84.3 and 64.7 % concordance with the results of phenotypic identification at the genus and species levels, respectively. In the monomicrobial samples, the concordance between the results of pyrosequencing and phenotypic identification at the genus level was 87.0 %. Pyrosequencing identified one isolate in 60 % of polymicrobial samples, which were confirmed by culture analysis. Of the samples identified by pyrosequencing, 55.7 % showed consistent results in V1 and V3 targeted sequencing; other samples were identified based on the results of V1 (12.5 %) or V3 (31.8 %) sequencing alone. One isolate was erroneously identified by pyrosequencing due to high sequence similarity with another isolate. Pyrosequencing identified one isolate that was not detected by phenotypic identification. The process of pyrosequencing identification can be completed within ~4 h. The information provided by DNA-pyrosequencing for the identification of micro-organisms in positive blood culture bottles is accurate and could prove to be a rapid and useful tool in standard laboratory practice.

  15. DNA repair in microgravity: studies on bacteria and mammalian cells in the experiments REPAIR and KINETICS.

    PubMed

    Horneck, G; Rettberg, P; Baumstark-Khan, C; Rink, H; Kozubek, S; Schäfer, M; Schmitz, C

    1996-06-27

    The impact of microgravity on cellular repair processes was tested in the space experiments REPAIR and KINETICS, which were performed during the IML-2 mission in the Biorack of ESA: (a) survival of spores of Bacillus subtilis HA101 after UV-irradiation (up to 340 J m-2) in the experiment REPAIR; (b) in the experiment KINETICS the kinetics of DNA repair in three different test systems: rejoining of X-ray-induced DNA strand breaks (B1) in cells of Escherichia coli B/r (120 Gy) and (B2) in human fibroblasts (5 and 10 Gy) as well as (B3) induction of the SOS response after gamma-irradiation (300 Gy) of cells of Escherichia coli PQ37. Cells were irradiated prior to the space mission and were kept in a non-metabolic state (metabolically inactive spores of B. subtilis on membrane filters, frozen cells of E. coli and human fibroblasts) until incubation in orbit. Germination and growth of B. subtilis were initiated by humidification, E. coli and fibroblasts were thawed up and incubated at 37 degrees C for defined repair periods (up to 4.5 h), thereafter they were frozen again for laboratory analysis. Relevant controls were performed in-flight (1 x g reference centrifuge) and on ground (1 x g and 1.4 x g) The results show no significant differences between the microgravity samples and the corresponding controls neither in the survival curves nor in the kinetics of DNA strand break rejoining and induction of the SOS response (proven by Student's t-test, 2 P = 0.05). These observations provide evidence that in the microgravity environment cells are able to repair radiation-induced DNA damage close to normality. The results suggest that a disturbance of cellular repair processes in the microgravity environment might not be the explanation for the reported synergism of radiation and microgravity.

  16. Novel Phenanthrene-Degrading Bacteria Identified by DNA-Stable Isotope Probing

    PubMed Central

    Luo, Chunling; Zhang, Dayi; Zhang, Gan

    2015-01-01

    Microorganisms responsible for the degradation of phenanthrene in a clean forest soil sample were identified by DNA-based stable isotope probing (SIP). The soil was artificially amended with either 12C- or 13C-labeled phenanthrene, and soil DNA was extracted on days 3, 6 and 9. Terminal restriction fragment length polymorphism (TRFLP) results revealed that the fragments of 219- and 241-bp in HaeIII digests were distributed throughout the gradient profile at three different sampling time points, and both fragments were more dominant in the heavy fractions of the samples exposed to the 13C-labeled contaminant. 16S rRNA sequencing of the 13C-enriched fraction suggested that Acidobacterium spp. within the class Acidobacteria, and Collimonas spp. within the class Betaproteobacteria, were directly involved in the uptake and degradation of phenanthrene at different times. To our knowledge, this is the first report that the genus Collimonas has the ability to degrade PAHs. Two PAH-RHDα genes were identified in 13C-labeled DNA. However, isolation of pure cultures indicated that strains of Staphylococcus sp. PHE-3, Pseudomonas sp. PHE-1, and Pseudomonas sp. PHE-2 in the soil had high phenanthrene-degrading ability. This emphasizes the role of a culture-independent method in the functional understanding of microbial communities in situ. PMID:26098417

  17. Novel Phenanthrene-Degrading Bacteria Identified by DNA-Stable Isotope Probing.

    PubMed

    Jiang, Longfei; Song, Mengke; Luo, Chunling; Zhang, Dayi; Zhang, Gan

    2015-01-01

    Microorganisms responsible for the degradation of phenanthrene in a clean forest soil sample were identified by DNA-based stable isotope probing (SIP). The soil was artificially amended with either 12C- or 13C-labeled phenanthrene, and soil DNA was extracted on days 3, 6 and 9. Terminal restriction fragment length polymorphism (TRFLP) results revealed that the fragments of 219- and 241-bp in HaeIII digests were distributed throughout the gradient profile at three different sampling time points, and both fragments were more dominant in the heavy fractions of the samples exposed to the 13C-labeled contaminant. 16S rRNA sequencing of the 13C-enriched fraction suggested that Acidobacterium spp. within the class Acidobacteria, and Collimonas spp. within the class Betaproteobacteria, were directly involved in the uptake and degradation of phenanthrene at different times. To our knowledge, this is the first report that the genus Collimonas has the ability to degrade PAHs. Two PAH-RHDα genes were identified in 13C-labeled DNA. However, isolation of pure cultures indicated that strains of Staphylococcus sp. PHE-3, Pseudomonas sp. PHE-1, and Pseudomonas sp. PHE-2 in the soil had high phenanthrene-degrading ability. This emphasizes the role of a culture-independent method in the functional understanding of microbial communities in situ.

  18. [Cardiotropic DNA viruses and bacteria in the pathogenesis of dilated cardiomyopathy with or without inflammation].

    PubMed

    Pankuweit, S; Hufnagel, G; Eckhardt, H; Herrmann, H; Uttecht, S; Maisch, B

    1998-04-15

    In the report of the 1995 WHO/ISFC task force on the definition and classification of cardiomyopathies a new entity within the dilated cardiomyopathies was introduced as "inflammatory cardiomyopathy". It is defined as myocarditis associated with cardiac dysfunction. Idiopathic, autoimmune and infectious forms of inflammatory cardiomyopathy are now recognized through this definition. Dilated cardiomyopathy with inflammation (DCMi, chronic myocarditis) was also defined by a recent ISFC task force as > 14 lymphocytes/macrophages/mm3. Enteroviruses, adenoviruses and cytomegaloviruses are considered as main etiopathogenetic factors in the pathogenesis of inflammatory heart disease and have been demonstrated as important trigger for inflammatory cardiac disease. They may also cause dilated cardiomyopathy by viral persistence or secondary immunopathogenesis due to antigenic or molecular mimicry. For the detection of viral persistence the investigation of endomyocardial biopsies in patients with cardiomyopathy by the use of polymerase chain reaction and southern blot analysis is an important step for the standardization of diagnostic criteria on virally induced inflammatory cardiomyopathy. Present studies indicate an incidence of cytomegalovirus-DNA in patients with inflammatory cardiomyopathy in 10%, adenoviral-DNA in 17% and borreliosis only in rare cases (< 1%). In dilated cardiomyopathy without inflammation the respective incidences were for cytomegalovirus 12%, 15% for adenovirus and only 0.5% of cases for borreliosis. In addition the results of immunohistochemical analysis and molecular biological investigations of endomyocardial biopsies may have implications for future therapeutic studies. Depending on the etiology of the disease, immunosuppression may have benefit for patients with virus-negative cardiomyopathy with inflammation in contrast to patients with cytomegalo-, adenovirus-DNA or enteroviral persistence, in whom immunomodulation with hyperimmunoglobulins

  19. Breakthrough of ultraviolet light from various brands of fluorescent lamps: lethal effects on DNA repair-defective bacteria.

    PubMed

    Hartman, P E; Biggley, W H

    1996-01-01

    In a comparative study of 17 pairs of 15 W fluorescent lamps intended for use in homes and purchased in local stores, we detect over 10-fold differences in UVB + UVC emissions between various lamps. This breakthrough of ultraviolet (UV) light is in part correlated with ability of lamps to kill DNA repair-defective recA-uvrB- Salmonella. Relative proficiency of lamps in eliciting photoreactivation of UV-induced DNA lesions also plays a prominent role in the relative rates of bacterial inactivation by emissions from different lamps. Lamps made in Chile, such as Philips brand lamps and one type of General Electric lamp, produce far less UVB + UVC and fail to kill recA-uvrB- bacteria. In contrast, all tested lamps manufactured in the USA, Hungary, and Japan exhibit readily observed deleterious biological effects. When an E. coli recA-uvrB-phr- (photolyase-negative) triple mutant is used for assay, lethal radiations are detected from all lamps, and single-hit exponential inactivation rates rather closely correlate to amount of directly measured UVB + UVC output of each pair of lamps. Although all lamps tested may meet international and United States standards for radiation safety, optimal practices in lamp manufacture are clearly capable of decreasing human exposure to indoor UV light.

  20. Breakthrough of ultraviolet light from various brands of fluorescent lamps: Lethal effects on DNA repair-defective bacteria

    SciTech Connect

    Hartman, P.E.; Biggley, W.H.

    1996-12-31

    In a comparative study of 17 pairs of 15 W fluorescent lamps intended for use in homes and purchased in local stores, we detect over 10-fold differences in UVB + UVC emissions between various lamps. This breakthrough of ultraviolet (UV) light is in part correlated with ability of lamps to kill DNA repair-defective recA{sup {minus}}uvrB{sup {minus}} Salmonella. Relative proficiency of lamps in eliciting photoreactivation of UV-induced DNA lesions also plays a prominent role in the relative rates of bacterial inactivation by emissions from different lamps. Lamps made in Chile, such as Phillips brand lamps and one type of General Electric lamp, produce far less UVB + UVC and fail to kill recA{sup {minus}} uvrB{sup {minus}} bacteria. In contrast, all tested lamps manufactured in the USA, Hungary, and Japan exhibit readily observed deleterious biological effects. When an E. coli recA{sup {minus}} uvrB{sup {minus}} phr{sup {minus}} (photolyase-negative) triple mutant is used for assay, lethal radiations are detected from all lamps, and single-hit exponential inactivation rates rather closely correlate to amount of directly measured UVB + UVC output of each pair of lamps. Although all lamps tested may meet international and Unite States standards for radiation safely, optimal practices in lamp manufacture are clearly capable of decreasing human exposure to indoor UV light. 38 refs., 3 figs., 1 tab.

  1. RRNA and dnaK relationships of Bradyrhizobium sp. nodule bacteria from four papilionoid legume trees in Costa Rica.

    PubMed

    Parker, Matthew A

    2004-05-01

    Enzyme electrophoresis and sequencing of rRNA and dnaK genes revealed high genetic diversity among root nodule bacteria from the Costa Rican trees Andira inermis, Dalbergia retusa, Platymiscium pinnatum (Papilionoideae tribe Dalbergieae) and Lonchocarpus atropurpureus (Papilionoideae tribe Millettieae). A total of 21 distinct multilocus genotypes [ETs (electrophoretic types)] was found among the 36 isolates analyzed, and no ETs were shared in common by isolates from different legume hosts. However, three of the ETs from D. retusa were identical to Bradyrhizobium sp. isolates detected in prior studies of several other legume genera in both Costa Rica and Panama. Nearly full-length 16S rRNA sequences and partial 23S rRNA sequences confirmed that two isolates from D. retusa were highly similar or identical to Bradyrhizobium strains isolated from the legumes Erythrina and Clitoria (Papilionoideae tribe Phaseoleae) in Panama. rRNA sequences for five isolates from L. atropurpureus, P. pinnatum and A. inermis were not closely related to any currently known strains from Central America or elsewhere, but had affinities to the reference strains Bradyrhizobium japonicum USDA 110 (three isolates) or to B. elkanii USDA 76 (two isolates). A phylogenetic tree for 21 Bradyrhizobium strains based on 603 bp of the dnaK gene showed several significant conflicts with the rRNA tree, suggesting that genealogical relationships may have been altered by lateral gene transfer events.

  2. Horizontal DNA Transfer Mechanisms of Bacteria as Weapons of Intragenomic Conflict

    PubMed Central

    Croucher, Nicholas J.; Mostowy, Rafal; Wymant, Christopher; Turner, Paul; Bentley, Stephen D.; Fraser, Christophe

    2016-01-01

    Horizontal DNA transfer (HDT) is a pervasive mechanism of diversification in many microbial species, but its primary evolutionary role remains controversial. Much recent research has emphasised the adaptive benefit of acquiring novel DNA, but here we argue instead that intragenomic conflict provides a coherent framework for understanding the evolutionary origins of HDT. To test this hypothesis, we developed a mathematical model of a clonally descended bacterial population undergoing HDT through transmission of mobile genetic elements (MGEs) and genetic transformation. Including the known bias of transformation toward the acquisition of shorter alleles into the model suggested it could be an effective means of counteracting the spread of MGEs. Both constitutive and transient competence for transformation were found to provide an effective defence against parasitic MGEs; transient competence could also be effective at permitting the selective spread of MGEs conferring a benefit on their host bacterium. The coordination of transient competence with cell–cell killing, observed in multiple species, was found to result in synergistic blocking of MGE transmission through releasing genomic DNA for homologous recombination while simultaneously reducing horizontal MGE spread by lowering the local cell density. To evaluate the feasibility of the functions suggested by the modelling analysis, we analysed genomic data from longitudinal sampling of individuals carrying Streptococcus pneumoniae. This revealed the frequent within-host coexistence of clonally descended cells that differed in their MGE infection status, a necessary condition for the proposed mechanism to operate. Additionally, we found multiple examples of MGEs inhibiting transformation through integrative disruption of genes encoding the competence machinery across many species, providing evidence of an ongoing “arms race.” Reduced rates of transformation have also been observed in cells infected by MGEs that

  3. Isolation of bacterial DNA followed by sequencing and differing cytokine response in peritoneal dialysis effluent help in identifying bacteria in culture negative peritonitis.

    PubMed

    Prasad, Narayan; Singh, Kamini; Gupta, Amit; Prasad, Kashi Nath

    2016-11-16

    The treatment of peritoneal dialysis related culture negative peritonitis is empirical which increases the cost of therapy and moreover antibiotic resistance. We aimed the study to isolate bacterial DNA from PD effluent and indentify bacteria causing peritonitis in culture negative situations. We have also studied the cytokine response with different bacteria causing peritonitis. We have isolated bacterial DNA from PD effluent of culture negative and culture positive peritonitis patients. Bacterial DNA was subjected to polymerase chain reaction using universal bacteria specific primers and subsequently to Gram type specific primers for the differentiation of the etiologic agents into Gram positive and Gram negative. The amplified products were sequenced and subjected to blast search to identify agent at genus/ species level. Of the 30 molecular method positive samples, 16 (53.33%) samples were positive for Gram negative bacteria and 4 (13.33%) for Gram positive while remaining10 (33.33%) were positive for both Gram positive and Gram negative bacteria. We have found organisms which usually do not grow on normal culture methods. TNF- α was significantly associated with Gram positive peritonitis and regulatory cytokine IL-10 with Gram negative peritonitis. The molecular techniques are helpful in detecting and identifying organisms from culture negative PD effluent. This article is protected by copyright. All rights reserved.

  4. 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

  5. Immunological detection of UV induced cyclobutane pyrimidine dimers and (6-4) photoproducts in DNA from reference bacteria and natural aquatic populations.

    PubMed

    Kraft, Stephanie; Stephanie, Kraft; Obst, Ursula; Ursula, Obst; Schwartz, Thomas; Thomas, Schwartz

    2011-03-01

    UV light-caused DNA damage is a widespread field of study. As UV light has the biological effect of inactivating or killing bacteria, it is used for water disinfection. Due to this application, it is important to study the DNA damage efficiencies and regeneration capacities in bacteria after UV irradiation. Two monoclonal antibodies, anti-CPD and anti-(6-4) PP, were applied for an immunoassay of UV-induced DNA modifications. Cyclobutane pyrimidine dimer (CPD) and 6-4 photoproduct (6-4 PP) were detected in the reference bacteria Pseudomonas aeruginosa and Enterococcus faecium, and in natural water communities. The antibody-mediated detection signals increased with the UV doses from 100-400J/m(2). Here, the CPD-specific signals were stronger than the (6-4) PP-specific signals. These immunological results were in accordance with parallel cultivation experiments. All UV-irradiated bacteria showed a reduction of their growth rate depending on UV application by several orders of magnitudes. The immunoassay was also applied to three types of natural aquatic habitats with different cell densities. Besides artificial UV irradiation, it was possible to visualize natural sunlight effects on these natural bacterial communities. Light-dependent and dark repair processes were distinguished using the established immunological assays. The antibody-mediated analyses presented are fast methods to detect UV-induced DNA lesions and repair capacities in selected bacterial species as well as in entire natural mixed populations.

  6. On-chip concentration of bacteria using a 3D dielectrophoretic chip and subsequent laser-based DNA extraction in the same chip

    NASA Astrophysics Data System (ADS)

    Cho, Yoon-Kyoung; Kim, Tae-hyeong; Lee, Jeong-Gun

    2010-06-01

    We report the on-chip concentration of bacteria using a dielectrophoretic (DEP) chip with 3D electrodes and subsequent laser-based DNA extraction in the same chip. The DEP chip has a set of interdigitated Au post electrodes with 50 µm height to generate a network of non-uniform electric fields for the efficient trapping by DEP. The metal post array was fabricated by photolithography and subsequent Ni and Au electroplating. Three model bacteria samples (Escherichia coli, Staphylococcus epidermidis, Streptococcus mutans) were tested and over 80-fold concentrations were achieved within 2 min. Subsequently, on-chip DNA extraction from the concentrated bacteria in the 3D DEP chip was performed by laser irradiation using the laser-irradiated magnetic bead system (LIMBS) in the same chip. The extracted DNA was analyzed with silicon chip-based real-time polymerase chain reaction (PCR). The total process of on-chip bacteria concentration and the subsequent DNA extraction can be completed within 10 min including the manual operation time.

  7. 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.

  8. Fluorescent Lactic Acid Bacteria and Bifidobacteria as Vehicles of DNA Microbial Biosensors

    PubMed Central

    Arqués, Juan Luis

    2017-01-01

    Control and quantification of effector molecules such as heavy metals, toxins or other target molecules is of great biotechnological, social and economic interest. Microorganisms have regulatory proteins that recognize and modify the gene expression in the presence or absence of these compounds (effector molecules) by means of binding to gene sequences. The association of these recognizing gene sequences to reporter genes will allow the detection of effector molecules of interest with high sensitivity. Once investigators have these two elements—recognizing gene sequences and reporter genes that emit signals—we need a suitable vehicle to introduce both elements. Here, we suggest lactic acid bacteria (LAB) and bifidobacteria as promising carrier microorganisms for these molecular biosensors. The use of fluorescent proteins as well as food-grade vectors and clustered regularly interspaced short palindromic repeats (CRISPR) are indispensable tools for introducing biosensors into these microorganisms. The use of these LAB and bifidobacteria would be of special interest for studying the intestinal environment or other complex ecosystems. The great variety of species adapted to many environments, as well as the possibility of applying several protocols for their transformation with recognizing gene sequences and reporter genes are considerable advantages. Finally, an effort must be made to find recognizable gene sequences.

  9. Fatty acids, unusual glycophospholipids and DNA analyses of thermophilic bacteria isolated from hot springs.

    PubMed

    Siristova, Lucie; Melzoch, Karel; Rezanka, Tomas

    2009-01-01

    The composition of fatty acids in 12 strains of the genera Thermus, Meiothermus, Geobacillus and Alicyclobacillus was analyzed by gas chromatography-mass spectrometry. Major FAs found in the profiles included i-15:0, i-17:0, ai-15:0, i-16:0, 16:0, ai-17:0, together with some minor components. Branched FAs were predominant, forming more than 80% of all FAs measured. Fast atom bombardment-mass spectrometry was used for analysis of unusual glycophospholipids, i.e., acylglycosylcardiolipins from genera Geobacillus and Alicyclobacillus and 1-(hydroxy(2-(O-acylglycosyl-oxy)hexadecyloxy)phosphoryloxy) hexadecan-2-yl esters of C15-C17 acids from genera Thermus and Meiothermus. Cloning and preliminary sequence analysis of 16S rDNA showed that these isolates belong to the genera Thermus, Meiothermus, Geobacillus and Alicyclobacillus.

  10. Comprehensive Census of Bacteria in Clean Rooms by Using DNA Microarray and Cloning Methods▿ †

    PubMed Central

    La Duc, Myron T.; Osman, Shariff; Vaishampayan, Parag; Piceno, Yvette; Andersen, Gary; Spry, J. A.; Venkateswaran, Kasthuri

    2009-01-01

    A census of clean room surface-associated bacterial populations was derived from the results of both the cloning and sequencing of 16S rRNA genes and DNA microarray (PhyloChip) analyses. Samples from the Lockheed Martin Aeronautics Multiple Testing Facility (LMA-MTF), the Kennedy Space Center Payload Hazard and Servicing Facility (KSC-PHSF), and the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) clean rooms were collected during the various assembly phases of the Phoenix and Mars Science Laboratory (MSL) spacecraft. Clone library-derived analyses detected a larger bacterial diversity prior to the arrival of spacecraft hardware in these clean room facilities. PhyloChip results were in agreement with this trend but also unveiled the presence of anywhere from 9- to 70-fold more bacterial taxa than cloning approaches. Among the facilities sampled, the JPL-SAF (MSL mission) housed a significantly less diverse bacterial population than either the LMA-MTF or KSC-PHSF (Phoenix mission). Bacterial taxa known to thrive in arid conditions were frequently detected in MSL-associated JPL-SAF samples, whereas proteobacterial lineages dominated Phoenix-associated KSC-PHSF samples. Comprehensive bacterial censuses, such as that reported here, will help space-faring nations preemptively identify contaminant biomatter that may compromise extraterrestrial life detection experiments. The robust nature and high sensitivity of DNA microarray technologies should prove beneficial to a wide range of scientific, electronic, homeland security, medical, and pharmaceutical applications and to any other ventures with a vested interest in monitoring and controlling contamination in exceptionally clean environments. PMID:19700540

  11. Theoretical models for the regulation of DNA replication in fast-growing bacteria

    NASA Astrophysics Data System (ADS)

    Creutziger, Martin; Schmidt, Mischa; Lenz, Peter

    2012-09-01

    Growing in always changing environments, Escherichia coli cells are challenged by the task to coordinate growth and division. In particular, adaption of their growth program to the surrounding medium has to guarantee that the daughter cells obtain fully replicated chromosomes. Replication is therefore to be initiated at the right time, which is particularly challenging in media that support fast growth. Here, the mother cell initiates replication not only for the daughter but also for the granddaughter cells. This is possible only if replication occurs from several replication forks that all need to be correctly initiated. Despite considerable efforts during the last 40 years, regulation of this process is still unknown. Part of the difficulty arises from the fact that many details of the relevant molecular processes are not known. Here, we develop a novel theoretical strategy for dealing with this general problem: instead of analyzing a single model, we introduce a wide variety of 128 different models that make different assumptions about the unknown processes. By comparing the predictions of these models we are able to identify the key quantities that allow the experimental discrimination of the different models. Analysis of these quantities yields that out of the 128 models 94 are not consistent with available experimental data. From the remaining 34 models we are able to conclude that mass growth and DNA replication need either to be truly coupled, by coupling DNA replication initiation to the event of cell division, or to the amount of accumulated mass. Finally, we make suggestions for experiments to further reduce the number of possible regulation scenarios.

  12. Comprehensive census of bacteria in clean rooms by using DNA microarray and cloning methods.

    PubMed

    La Duc, Myron T; Osman, Shariff; Vaishampayan, Parag; Piceno, Yvette; Andersen, Gary; Spry, J A; Venkateswaran, Kasthuri

    2009-10-01

    A census of clean room surface-associated bacterial populations was derived from the results of both the cloning and sequencing of 16S rRNA genes and DNA microarray (PhyloChip) analyses. Samples from the Lockheed Martin Aeronautics Multiple Testing Facility (LMA-MTF), the Kennedy Space Center Payload Hazard and Servicing Facility (KSC-PHSF), and the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) clean rooms were collected during the various assembly phases of the Phoenix and Mars Science Laboratory (MSL) spacecraft. Clone library-derived analyses detected a larger bacterial diversity prior to the arrival of spacecraft hardware in these clean room facilities. PhyloChip results were in agreement with this trend but also unveiled the presence of anywhere from 9- to 70-fold more bacterial taxa than cloning approaches. Among the facilities sampled, the JPL-SAF (MSL mission) housed a significantly less diverse bacterial population than either the LMA-MTF or KSC-PHSF (Phoenix mission). Bacterial taxa known to thrive in arid conditions were frequently detected in MSL-associated JPL-SAF samples, whereas proteobacterial lineages dominated Phoenix-associated KSC-PHSF samples. Comprehensive bacterial censuses, such as that reported here, will help space-faring nations preemptively identify contaminant biomatter that may compromise extraterrestrial life detection experiments. The robust nature and high sensitivity of DNA microarray technologies should prove beneficial to a wide range of scientific, electronic, homeland security, medical, and pharmaceutical applications and to any other ventures with a vested interest in monitoring and controlling contamination in exceptionally clean environments.

  13. Purification of bacterial genomic DNA in less than 20 min using chelex-100 microwave: examples from strains of lactic acid bacteria isolated from soil samples.

    PubMed

    Reyes-Escogido, Lourdes; Balam-Chi, Mario; Rodríguez-Buenfil, Ingrid; Valdés, Jesús; Kameyama, Luis; Martínez-Pérez, Francisco

    2010-11-01

    We established a Chelex 100-Microwave method for the purification of bacterial genomic DNA (gDNA) in less than 20 min with high yield and good quality, useful for multiple purposes. It combines Chelex 100, proteinase K, RNase A and heating in a microwave oven. The resulting gDNA was used directly to identify bacterial species of the Order Lactobacillales by means of PCR amplification of their 16S rDNA gene, isolated from sediments on the Yucatan Peninsula, Mexico. This method produced gDNA free of phenolic and protein residual contaminants from 100 of these isolated bacteria. 16S rDNA amplification and sequencing showed Pediococcus acidilactici to prevail in inland lagoons, and Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus sp., and Lactobacillus fermentum to be most abundant in the soils of livestock farms. The combination of Chelex 100, enzymes and microwave heating used in the Chelex 100-Microwave method produced large amounts of highly pure gDNA from Gram-positive and Gram-negative bacteria, in less than 20 min.

  14. A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria

    PubMed Central

    Donovan, Catriona; Heyer, Antonia; Pfeifer, Eugen; Polen, Tino; Wittmann, Anja; Krämer, Reinhard; Frunzke, Julia; Bramkamp, Marc

    2015-01-01

    In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin. PMID:25916847

  15. A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria.

    PubMed

    Donovan, Catriona; Heyer, Antonia; Pfeifer, Eugen; Polen, Tino; Wittmann, Anja; Krämer, Reinhard; Frunzke, Julia; Bramkamp, Marc

    2015-05-26

    In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. Effect of DNA extraction procedure, repeated extraction and ethidium monoazide (EMA)/propidium monoazide (PMA) treatment on overall DNA yield and impact on microbial fingerprints for bacteria, fungi and archaea in a reference soil

    PubMed Central

    Wagner, Andreas O.; Praeg, Nadine; Reitschuler, Christoph; Illmer, Paul

    2015-01-01

    Different DNA extraction protocols were evaluated on a reference soil. A wide difference was found in the total extractable DNA as derived from different extraction protocols. Concerning the DNA yield phenol–chloroform–isomyl alcohol extraction resulted in high DNA yield but also in a remarkable co-extraction of contaminants making PCR from undiluted DNA extracts impossible. By comparison of two different extraction kits, the Macherey&Nagel SoilExtract II kit resulted in the highest DNA yields when buffer SL1 and the enhancer solution were applied. The enhancer solution not only significantly increased the DNA yield but also the amount of co-extracted contaminates, whereas additional disintegration strategies did not. Although a three times repeated DNA extraction increased the total amount of extracted DNA, microbial fingerprints were merely affected. However, with the 5th extraction this changed. A reduction of total DGGE band numbers was observed for archaea and fungi, whereas for bacteria the diversity increased. The application of ethidium monoazide (EMA) or propidium monoazide (PMA) treatment aiming on the selective removal of soil DNA derived from cells lacking cell wall integrity resulted in a significant reduction of total extracted DNA, however, the hypothesized effect on microbial fingerprints failed to appear indicating the need for further investigations. PMID:26339125

  17. Effect of DNA extraction procedure, repeated extraction and ethidium monoazide (EMA)/propidium monoazide (PMA) treatment on overall DNA yield and impact on microbial fingerprints for bacteria, fungi and archaea in a reference soil.

    PubMed

    Wagner, Andreas O; Praeg, Nadine; Reitschuler, Christoph; Illmer, Paul

    2015-09-01

    Different DNA extraction protocols were evaluated on a reference soil. A wide difference was found in the total extractable DNA as derived from different extraction protocols. Concerning the DNA yield phenol-chloroform-isomyl alcohol extraction resulted in high DNA yield but also in a remarkable co-extraction of contaminants making PCR from undiluted DNA extracts impossible. By comparison of two different extraction kits, the Macherey&Nagel SoilExtract II kit resulted in the highest DNA yields when buffer SL1 and the enhancer solution were applied. The enhancer solution not only significantly increased the DNA yield but also the amount of co-extracted contaminates, whereas additional disintegration strategies did not. Although a three times repeated DNA extraction increased the total amount of extracted DNA, microbial fingerprints were merely affected. However, with the 5th extraction this changed. A reduction of total DGGE band numbers was observed for archaea and fungi, whereas for bacteria the diversity increased. The application of ethidium monoazide (EMA) or propidium monoazide (PMA) treatment aiming on the selective removal of soil DNA derived from cells lacking cell wall integrity resulted in a significant reduction of total extracted DNA, however, the hypothesized effect on microbial fingerprints failed to appear indicating the need for further investigations.

  18. The 16S rDNA Phylogenetic Composition of Bacteria Implicated in Sulfur Redox Cycles and Associated Sulfur Isotope Fractionation

    NASA Astrophysics Data System (ADS)

    Bicknell, B. T.; Batts, J. E.; Krouse, H. R.

    2006-12-01

    The reduction of sulfate ion to sulfide species by sulfate reducing bacteria (SRB) is accompanied by sulfur isotope fractionation, measured in terms of changes in the δ^{34}S values for sulfate and sulfide ions relative to a defined standard. In open environments, the S-isotope compositions of sulfate and sulfide can be affected by loss from the system of sulfide species as gaseous H2S, insoluble metal sulfides such as FeS2, organic complexes or by re-oxidation. The S-isotope fractionation accompanying bacterial sulfate reduction in nature is often much larger than the maxima obtained in chemical and bacterial sulfate reduction experiments in the laboratory. One mechanism postulated for the large natural S-isotope selectivity depends on repetitive reduction-oxidation cycles. In turn, this would require a level of tolerance to oxygen by SRB in the sedimentary environment, contrary to laboratory experience with SRB strains. Bird Lake (The Coorong, South Australia) is a small calcareous, evaporative lake, where average Δ^{34}S (δ^{34}Ssulfate - δ^{34}Ssulfide) values for groundwater at 16 of the 27 sites sampled periodically since 1974, vary from 15.0 ‰ to 62.3 ‰ within the range -1.8 ‰ to 70.6 ‰. Wide fluctuations in δ34Ssulfide values at individual sites are the significant factor affecting the variability of Δ^{34}S values. Values for δ18Osulfate are elevated over that of the sulfate source to an unusual extent, reflecting re-oxidation of sulfur species and O- isotope exchange between some of these species and water. One aspect of investigations at Bird Lake was the evaluation of bacterial populations in subsurface sediments and their role in sulfur cycling. To achieve this, microcosms were established with subsurface sediment and incubated under a nitrogen atmosphere, for up to 119 days. These were sampled at various times to determine sulfur species concentrations and sulfur isotope fractionation and to generate 16S rDNA clone libraries. Results

  19. Detection of Sequence Polymorphism in Rubus Occidentalis L. Monomorphic Microsatellite Markers by High Resolution Melting

    USDA-ARS?s Scientific Manuscript database

    Microsatellite, or simple sequence repeat (SSR) markers, are valuable as co-dominant genetic markers with a variety of applications such as DNA fingerprinting, linkage mapping, and population structure analysis. Development of microsatellite primers through the identification of appropriate repeate...

  20. Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories.

    PubMed

    Woo, P C Y; Lau, S K P; Teng, J L L; Tse, H; Yuen, K-Y

    2008-10-01

    In the last decade, as a result of the widespread use of PCR and DNA sequencing, 16S rDNA sequencing has played a pivotal role in the accurate identification of bacterial isolates and the discovery of novel bacteria in clinical microbiology laboratories. For bacterial identification, 16S rDNA sequencing is particularly important in the case of bacteria with unusual phenotypic profiles, rare bacteria, slow-growing bacteria, uncultivable bacteria and culture-negative infections. Not only has it provided insights into aetiologies of infectious disease, but it also helps clinicians in choosing antibiotics and in determining the duration of treatment and infection control procedures. With the use of 16S rDNA sequencing, 215 novel bacterial species, 29 of which belong to novel genera, have been discovered from human specimens in the past 7 years of the 21st century (2001-2007). One hundred of the 215 novel species, 15 belonging to novel genera, have been found in four or more subjects. The largest number of novel species discovered were of the genera Mycobacterium (n = 12) and Nocardia (n = 6). The oral cavity/dental-related specimens (n = 19) and the gastrointestinal tract (n = 26) were the most important sites for discovery and/or reservoirs of novel species. Among the 100 novel species, Streptococcus sinensis, Laribacter hongkongensis, Clostridium hathewayi and Borrelia spielmanii have been most thoroughly characterized, with the reservoirs and routes of transmission documented, and S. sinensis, L. hongkongensis and C. hathewayi have been found globally. One of the greatest hurdles in putting 16S rDNA sequencing into routine use in clinical microbiology laboratories is automation of the technology. The only step that can be automated at the moment is input of the 16S rDNA sequence of the bacterial isolate for identification into one of the software packages that will generate the result of the identity of the isolate on the basis of its sequence database. However

  1. 16S rDNA-based metagenomic analysis of dental plaque and lung bacteria in patients with severe acute exacerbations of chronic obstructive pulmonary disease.

    PubMed

    Tan, L; Wang, H; Li, C; Pan, Y

    2014-12-01

    Acute exacerbations of chronic obstructive pulmonary disease (AE-COPD) are leading causes of mortality in hospital intensive care units. We sought to determine whether dental plaque biofilms might harbor pathogenic bacteria that can eventually cause lung infections in patients with severe AE-COPD. Paired samples of subgingival plaque biofilm and tracheal aspirate were collected from 53 patients with severe AE-COPD. Total bacterial DNA was extracted from each sample individually for polymerase chain reaction amplification and/or generation of bacterial 16S rDNA sequences and cDNA libraries. We used a metagenomic approach, based on bacterial 16S rDNA sequences, to compare the distribution of species present in dental plaque and lung. Analysis of 1060 sequences (20 clones per patient) revealed a wide range of aerobic, anaerobic, pathogenic, opportunistic, novel and uncultivable bacterial species. Species indistinguishable between the paired subgingival plaque and tracheal aspirate samples (97-100% similarity in 16S rDNA sequence) were dental plaque pathogens (Aggregatibacter actinomycetemcomitans, Capnocytophaga sputigena, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola) and lung pathogens (Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Streptococcus pneumoniae). Real-time polymerase chain reaction of 16S rDNA indicated lower levels of Pseudomonas aeruginosa and Porphyromonas gingivalis colonizing the dental plaques compared with the paired tracheal aspirate samples. These results support the hypothesis that dental bacteria may contribute to the pathology of severe AE-COPD. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. Lactic acid bacteria associated with vacuum-packed cooked meat product spoilage: population analysis by rDNA-based methods.

    PubMed

    Chenoll, E; Macián, M C; Elizaquível, P; Aznar, R

    2007-02-01

    To determine the lactic acid bacteria (LAB) implicated in bloating spoilage of vacuum-packed and refrigerated meat products. A total of 18 samples corresponding to four types of meat products, with and without spoilage symptoms, were studied. In all, 387 colonies growing on de Man, Rogosa and Sharpe, yeast glucose lactose peptone and trypticase soy yeast extract plates were identified by internal spacer region (ISR), ISR-restriction fragment length polymorphism and rapid amplified ribosomal DNA restriction analysis profiles as Lactobacillus (37%), Leuconostoc (43%), Carnobacterium (11%), Enterococcus (4%) and Lactococcus (2%). Leuconostoc mesenteroides dominated the microbial population of spoiled products and was always present at the moment bloating occurred. Lactobacillus sakei, Lactobacillus plantarum and Lactobacillus curvatus were found in decreasing order of abundance. The analysis of two meat products, 'morcilla' and 'fiambre de magro adobado' obtained from production lines revealed a common succession pattern in LAB populations in both products and showed that Leuc. mesenteroides became the main species during storage, despite being below the detection level of culture methods after packing. Our results pointed to Leuc. mesenteroides as the main species responsible for bloating spoilage in vacuum-packed meat products. Prevention of bloating spoilage in vacuum-packed cooked meat products requires the sensitive detection of Leuc. mesenteroides (i.e. by PCR).

  3. The art of strain improvement of industrial lactic acid bacteria without the use of recombinant DNA technology.

    PubMed

    Derkx, Patrick M F; Janzen, Thomas; Sørensen, Kim I; Christensen, Jeffrey E; Stuer-Lauridsen, Birgitte; Johansen, Eric

    2014-08-29

    The food industry is constantly striving to develop new products to fulfil the ever changing demands of consumers and the strict requirements of regulatory agencies. For foods based on microbial fermentation, this pushes the boundaries of microbial performance and requires the constant development of new starter cultures with novel properties. Since the use of ingredients in the food industry is tightly regulated and under close scrutiny by consumers, the use of recombinant DNA technology to improve microbial performance is currently not an option. As a result, the focus for improving strains for microbial fermentation is on classical strain improvement methods. Here we review the use of these techniques to improve the functionality of lactic acid bacteria starter cultures for application in industrial-scale food production. Methods will be described for improving the bacteriophage resistance of specific strains, improving their texture forming ability, increasing their tolerance to stress and modulating both the amount and identity of acids produced during fermentation. In addition, approaches to eliminating undesirable properties will be described. Techniques include random mutagenesis, directed evolution and dominant selection schemes.

  4. The art of strain improvement of industrial lactic acid bacteria without the use of recombinant DNA technology

    PubMed Central

    2014-01-01

    The food industry is constantly striving to develop new products to fulfil the ever changing demands of consumers and the strict requirements of regulatory agencies. For foods based on microbial fermentation, this pushes the boundaries of microbial performance and requires the constant development of new starter cultures with novel properties. Since the use of ingredients in the food industry is tightly regulated and under close scrutiny by consumers, the use of recombinant DNA technology to improve microbial performance is currently not an option. As a result, the focus for improving strains for microbial fermentation is on classical strain improvement methods. Here we review the use of these techniques to improve the functionality of lactic acid bacteria starter cultures for application in industrial-scale food production. Methods will be described for improving the bacteriophage resistance of specific strains, improving their texture forming ability, increasing their tolerance to stress and modulating both the amount and identity of acids produced during fermentation. In addition, approaches to eliminating undesirable properties will be described. Techniques include random mutagenesis, directed evolution and dominant selection schemes. PMID:25186244

  5. From bacteria to humans: lessons learned from a reductionist's view of ultraviolet light-induced DNA lesions.

    PubMed

    Trosko, J E

    2001-01-01

    What follows is a personal remembrance of how Dr. Richard Setlow influenced me as a young postdoctoral fellow at Oak Ridge National laboratory between 1963 and 1966. The narrative tries to place my "maturation" as a young, inexperienced scientist in the context of the cultural upheaval caused by the Vietnam war, of a Northerner facing a "culture-shock" living in the South and in a revolution in molecular and radiation biology taking place at Oak Ridge National Laboratory at that time. The unique historic juxtaposition of Dr. Setlow's contribution of the discovery of UV-induced pyrimidine dimers in bacterial DNA, being potentially the molecular lesion responsible for cell killing and mutagenesis, occurring as I was at Oak Ridge, and the wonderful working relationship I had with William Carrier, his technician, led to our discovery with James Regan that normal human cells repaired these lesion from their DNA. Amazingly, because of Dr. Setlow's challenge to me about my thoughts of the implications of his findings in bacteria, the chance visit to Oak Ridge National Laboratory by Dr. James Cleaver and my background as a human geneticist provided me the extraordinary opportunity to carry out a collaboration to test if human cancer prone syndromes might be deficient in the repair of these UV-induced DNA lesions. With our finding that the direct demonstration of a lack of repair of UV-induced pyrimidine dimers in cells from the skin cancer prone syndrome, xeroderma pigmentosum, opened up a new paradigm for the understanding of the molecular mechanism of carcinogenesis of both radiation and chemical carcinogenesis. From this investigator's vantage point in the history of the understanding of carcinogenesis, which has led us to the present point of "oncogenes" and "tumor suppressor genes", the old adage by Newton, "I only saw further because I stood on the shoulder of giants", is so applicable here. Dr. Setlow's shoulders were indeed among those of all of us that have made

  6. Clonal growth is enhanced in the absence of a mating morph: a comparative study of fertile stylar polymorphic and sterile monomorphic populations of Nymphoides montana (Menyanthaceae).

    PubMed

    Haddadchi, Azadeh; Fatemi, Mohammad; Gross, C L

    2014-02-01

    Many aquatic species with stylar polymorphisms have the capacity for clonal and sexual reproduction and are sensitive to the balance of the two reproductive modes when there are a limited number of mating morphs within a population. This study asked how the clonal and sexual reproductive modes perform in populations that contain only a single morph and where fitness gain through sexual reproduction is rare. In clonal aquatic Nymphoides montana, polymorphic populations normally contain two mating morphs in equal frequencies. Populations are sexually fertile and appear to be maintained by pollen transfer between the two partners. However, in a monomorphic population of N. montana where mating opportunities are unavailable, female and male function is impaired and clonality maintains the population. Here, the consequences of intraspecific variation in sexuality were explored between monomorphic and polymorphic N. montana populations in eastern Australia. Comparative measurements of male and female fertility, total dry mass and genotypic diversity using ISSR markers were made between populations with variable sexuality. Very few seeds were produced in the monomorphic population under natural and glasshouse conditions due to dysfunctional pollen and ovules. Stigma-anther separation was minimal in the monomorphic population, which may be a consequence of the relaxed selective pressures that regulate the maintenance of sexual function. However, clonal reproduction was favoured at the expense of sexual reproduction in the monomorphic population; this may facilitate the establishment of sterility throughout the population via resource reallocation or pleiotropic effects. The ISSR results showed that the monomorphic population was one large, single genotype, unlike the multi-genotypic fertile polymorphic populations. Evolutionary loss of sex in a clonal population in which a mating morph is absent was evident; under these conditions clonal growth may assure reproduction and

  7. In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA.

    PubMed

    Choi, Yun Sik; Kim, Yong Cheol; Baek, Keum Jin; Choi, Youngnim

    2015-05-21

    The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

  8. Recipient-derived EBV-positive Monomorphic Plasmacytoma Type Posttransplant Lymphoproliferative Disorder After Allogeneic Stem Cell Transplant for Severe Aplastic Anemia: A Case Report.

    PubMed

    Carden, Marcus A; Caltharp, Shelley; Yee, Marianne E; Haight, Ann E; Westblade, Lars F; Park, Sunita

    2016-11-01

    Monomorphic plasmacytoma-type posttransplant lymphoproliferative disorder (PTLD) has not been reported after pediatric hematopoietic stem cell transplantation. We present a child with hepatitis-associated severe aplastic anemia who underwent an unrelated allogeneic hematopoietic stem cell transplantation and subsequently developed graft failure and an Epstein-Barr virus-positive monomorphic plasmacytoma-type PTLD of recipient origin. Despite broad-spectrum antimicrobials, weaning immunosuppression, rituximab administration, and a stem cell boost she died from complications of PTLD and a fungal pulmonary infection on day +78.

  9. Image findings of monomorphic non-hogdkin lymphoproliferative disorder in a post renal transplant patient diagnosed with fluorine-18 fluorodeoxyglucose-positron emission tomography/computed tomography

    PubMed Central

    Kamaleshwaran, Koramadai Karuppusamy; Rajasekar, Thirugnanam; Shibu, Deepu; Radhakrishnan, Edathurthy Kalarikal; Shinto, Ajit Sugunan

    2014-01-01

    Post-transplant lymphoproliferative disorder (PTLD) is a heterogeneous group of lymphoid proliferations caused by immunosuppression after solid organ or bone marrow transplantation. PTLD is categorized by early lesion, polymorphic PTLD and monomorphic PTLD. Fluorine-18 fluorodeoxyglucose-positron emission tomography/computed tomography (F-18 FDG-PET/CT) scans have clinical significance in the evaluation of PTLD following renal transplantation. We report imaging findings of a monomorphic non-Hodgkin lymphoma, post renal transplant seen on FDG PET/CT in a 32-year-old lactating woman. Whole body FDG- ET/CT demonstrated uptake in right external iliac and inguinal lymph nodes. PMID:25210292

  10. Ribosomal PCR and DNA sequencing for detection and identification of bacteria: experience from 6 years of routine analyses of patient samples.

    PubMed

    Jensen, Kristine Helander; Dargis, Rimtas; Christensen, Jens Jørgen; Kemp, Michael

    2014-03-01

    The use of broad range PCR and DNA sequencing of bacterial 16S ribosomal RNA genes for routine diagnostics of bacterial infections was evaluated. Here, the results from more than 2600 analyses during a 6-year period (2003-2009) are presented. Almost half of the samples were from joints and bones, and the second most frequent origin of samples was from the central nervous system. Overall, 26% of all samples were positive for bacterial DNA and bacterial identification was obtained in 80% of the PCR-positive samples by subsequent DNA sequencing. Ambiguous species identification was noticed among non-haemolytic streptococci, especially within the mitis group. The data show that ribosomal PCR with subsequent DNA sequencing of the PCR product is a most valuable supplement to culture for identifying bacterial agents of both acute and prolonged infections. However, some bacteria, including non-haemolytic streptococci, may not be precisely identified.

  11. Inconsistent shock advisories for monomorphic VT and Torsade de Pointes--A prospective experimental study on AEDs and defibrillators.

    PubMed

    Fitzgerald, Abi; Johnson, Meshell; Hirsch, Jan; Rich, Mary-Ann; Fidler, Richard

    2015-07-01

    Cardiovascular disease and sudden cardiac arrest are the leading causes of death in the United States. Early defibrillation is key to successful resuscitation for patients who experience shockable rhythms during a cardiac arrest. It is therefore vital that the shock advisory of AEDs (automated external defibrillators) or defibrillators in AED mode be reliable and appropriate. The goal of this study was to better understand the performance of multiple lay-rescuer and hospital professional defibrillators in AED mode in their analysis of ventricular arrhythmias. The measurable objectives of this study sought to quantify: 1. No shock advisory for sinus rhythms at any rate. 2. Recognition and shock advisory for ventricular fibrillation (VF). 3. Recognition and shock advisory for monomorphic ventricular tachycardia (VT). 4. Recognition and shock advisory for Torsades de Pointes (TdP). This is a prospective evaluation of two AEDs and four semi-automatic, hospital professional defibrillators. This study represents post-marketing evaluation of FDA approved devices. Each defibrillator was connected to multiple rhythm simulators and presented with simulated ECG waveforms 20 consecutive times at various rates when possible. All four defibrillators and both AEDs tested consistently recognized normal sinus rhythm (NSR) from all rhythm sources, and did not recommend a shock for NSR at any rate (from 80 to 220 bpm). All four defibrillators and both AEDs recognized VF from all rhythm sources tested and recommended a shock 100% of the time. Variations were found in the shock advisory rates among defibrillators when testing simulated VT heart rates at or below 150 bpm. One AED tested did not consistently advise a shock for monomorphic VT or TdP at any tested rate. Lay-rescuer AEDs and professional hospital defibrillators tested in AED mode did not reliably recommend a shock for sustained monomorphic VT or TdP at certain rates, despite the fact that it is a critical component of the

  12. High resolution melting detects sequence polymorphism in rubus occidentalis L. monomorphic microsatellite markers

    USDA-ARS?s Scientific Manuscript database

    Microsatellite, or simple sequence repeat (SSR) markers, are valuable as co-dominant genetic markers with a variety of applications such as DNA fingerprinting, linkage mapping, and population structure analysis. However, primer pairs designed from the regions that flank SSRs often generate fragment...

  13. 16S ribosomal DNA sequence-based identification of bacteria in laboratory rodents: a practical approach in laboratory animal bacteriology diagnostics.

    PubMed

    Benga, Laurentiu; Benten, W Peter M; Engelhardt, Eva; Köhrer, Karl; Gougoula, Christina; Sager, Martin

    2014-10-01

    Correct identification of bacteria is crucial for the management of rodent colonies. Some bacteria are difficult to identify phenotypically outside reference laboratories. In this study, we evaluated the utility of 16S ribosomal DNA (rDNA) sequencing as a means of identifying a collection of 30 isolates of rodent origin which are conventionally difficult to identify. Sequence analysis of the first approximate 720 to 880 bp of the 5'- end of 16S rDNA identified 25 isolates (83.33%) with ≥ 99% similarity to a sequence of a type strain, whereas three isolates (10%) displayed a sequence similarity ≥ 97% but <99% to the type strain sequences. These similarity scores were used to define identification to species and genus levels, respectively. Two of the 30 isolates (6.67%) displayed a sequence similarity of ≥ 95 but <97% to the reference strains and were thus allocated to a family. This technique allowed us to document the association of mice with bacteria relevant for the colonies management such as Pasteurellaceae, Bordetella hinzii or Streptococcus danieliae. In addition, human potential pathogens such as Acinetobacter spp., Ochrobactrum anthropi and Paracoccus yeei or others not yet reported in mouse bacterial species such as Leucobacter chironomi, Neisseria perflava and Pantoea dispersa were observed. In conclusion, the sequence analysis of 16S rDNA proved to be a useful diagnostic tool, with higher performance characteristics than the classical phenotypic methods, for identification of laboratory animal bacteria. For the first time this method allowed us to document the association of certain bacterial species with the laboratory mouse.

  14. Acquired long QT syndrome and monomorphic ventricular tachycardia after alternative treatment with cesium chloride for brain cancer.

    PubMed

    Dalal, Anuj K; Harding, John D; Verdino, Ralph J

    2004-08-01

    Individuals searching for symptomatic relief or a potential cure are increasingly seeking and using nontraditional therapies for their various diseases. Little is known about the potential adverse effects that patients may encounter while undergoing these alternative treatments. Cesium chloride is an unregulated agent that has been reported to have antineoplastic properties. Cesium chloride is advertised as an alternative agent for many different types of cancers and can be purchased easily on the Internet. Recently, QT prolongation and polymorphic ventricular tachycardia were reported in several patients taking cesium chloride as alternative treatment for cancer. We report acquired QT prolongation and sustained monomorphic ventricular tachycardia in a patient who self-initiated and completed a course of cesium chloride as adjunctive treatment for brain cancer.

  15. Oligo-DNA Custom Macroarray for Monitoring Major Pathogenic and Non-Pathogenic Fungi and Bacteria in the Phyllosphere of Apple Trees

    PubMed Central

    He, Ying-Hong; Isono, Sayaka; Shibuya, Makoto; Tsuji, Masaharu; Adkar Purushothama, Charith-Raj; Tanaka, Kazuaki; Sano, Teruo

    2012-01-01

    Background To monitor the richness in microbial inhabitants in the phyllosphere of apple trees cultivated under various cultural and environmental conditions, we developed an oligo-DNA macroarray for major pathogenic and non-pathogenic fungi and bacteria inhabiting the phyllosphere of apple trees. Methods and Findings First, we isolated culturable fungi and bacteria from apple orchards by an agar-plate culture method, and detected 32 fungal and 34 bacterial species. Alternaria, Aureobasidium, Cladosporium, Rhodotorula, Cystofilobasidium, and Epicoccum genera were predominant among the fungi, and Bacillus, Pseudomonas, Sphingomonas, Methylobacterium, and Pantoea genera were predominant among the bacteria. Based on the data, we selected 29 major non-pathogenic and 12 phytopathogenic fungi and bacteria as the targets of macroarray. Forty-one species-specific 40-base pair long oligo-DNA sequences were selected from the nucleotide sequences of rDNA-internal transcribed spacer region for fungi and 16S rDNA for bacteria. The oligo-DNAs were fixed on nylon membrane and hybridized with digoxigenin-labeled cRNA probes prepared for each species. All arrays except those for Alternaria, Bacillus, and their related species, were specifically hybridized. The array was sensitive enough to detect 103 CFU for Aureobasidium pullulans and Bacillus cereus. Nucleotide sequencing of 100 each of independent fungal rDNA-ITS and bacterial 16S-rDNA sequences from apple tree was in agreement with the macroarray data obtained using the same sample. Finally, we analyzed the richness in the microbial inhabitants in the samples collected from apple trees in four orchards. Major apple pathogens that cause scab, Alternaria blotch, and Marssonina blotch were detected along with several non-phytopathogenic fungal and bacterial inhabitants. Conclusions The macroarray technique presented here is a strong tool to monitor the major microbial species and the community structures in the phyllosphere of

  16. Cell lysis and DNA extraction of gram-positive and gram-negative bacteria from whole blood in a disposable microfluidic chip.

    PubMed

    Mahalanabis, Madhumita; Al-Muayad, Hussam; Kulinski, M Dominika; Altman, Dave; Klapperich, Catherine M

    2009-10-07

    Sepsis caused by gram positive and gram negative bacteria is the leading cause of death in noncoronary ICUs and the tenth leading cause of death in the United States. We have developed a microfluidic sample preparation platform for rapid on-chip detection of infectious organisms for point-of-care diagnostics. The microfluidic chips are made of a robust thermoplastic and can be easily multiplexed for high throughput applications. Bacteria are lysed on-chip via hybrid chemical/mechanical method. Once lysed, the bacterial DNA is isolated using a microscale silica bead/polymer composite solid-phase-extraction (SPE) column. Lysis was confirmed using off-chip real time PCR. We isolated and detected both gram-negative (Escherichia coli) and gram-positive (Bacillussubtilis and Enterococcus faecalis) bacterial genomic DNA from microliter scale spiked whole human blood samples. The system performs better for gram-negative bacteria than it does for gram-positive bacteria, with limits of detection at 10(2) CFU/ml and 10(3)-10(4) CFU/ml, respectively. Total extraction times are less than one hour and can be further decreased by altering the channel geometry and pumping configuration.

  17. A broad-range method to detect genomic DNA of multiple pathogenic bacteria based on the aggregation strategy of gold nanorods.

    PubMed

    Wang, Xiaohui; Li, Yuan; Wang, Jidong; Wang, Quanli; Xu, Lijuan; Du, Juan; Yan, Shaoduo; Zhou, Yong; Fu, Qiuxia; Wang, Yingli; Zhan, Linsheng

    2012-09-21

    It remains challenging to detect unknown pathogenic bacteria in diagnostic, clinical and environmental fields. This work describes the approach to the development of a sensitive, broad-range genosensing assay targeting the conserved 16S rDNA region existing in most bacteria, by monitoring the aggregation level of gold nanorods (GNRs)-based nanoprobes through their localized surface plasmon resonance (LSPR) property. In the quantitative detection of artificial sequence, the limit of detection (LOD) of such a bioassay is demonstrated to reach the 5 pM level. This pair of universal GNRs-based nanoprobes can further identify at least 6 species of bacteria that were most prevalent in platelet concentrates (PCs) and have no cross-reaction with other pathogens. Moreover, it also exhibits higher sensitivity than other broad-range methods in analysing Serratia marcescens-spiked PCs. Therefore, the presented strategy not only provides a novel and effective DNA analysis method to detect multiple bacterial contaminations in PCs, but also opens up possibilities for its future use of detecting unknown bacteria in other systems, such as food and water, even at ultralow levels.

  18. Seasonal Sexual Segregation by Monomorphic Sooty Shearwaters Puffinus griseus Reflects Different Reproductive Roles during the Pre-Laying Period

    PubMed Central

    Hedd, April; Montevecchi, William A.; Phillips, Richard A.; Fifield, David A.

    2014-01-01

    Tracking technology has revolutionized knowledge of seabird movements; yet, few studies have examined sex differences in distribution and behavior of small to medium-sized, sexually-monomorphic seabirds. Application of bird-borne geolocation-immersion loggers revealed seasonal segregation in the sexually-monomorphic Sooty Shearwater Puffinus griseus, mainly in the pre-laying period, when there were clear differences in reproductive roles. Shearwaters first returned to the Falkland Islands on 27 Sept±8 d; males, on average, 8 d earlier than females. Prior to egg-laying, distribution at sea, colony attendance and behaviour depended on sex. Males foraged locally over the southern Patagonian Shelf and Burdwood Bank, spending mainly single days at sea and intervening nights in the burrow. Females, who flew for more of the day during this time, foraged in more distant areas of the northern Patagonian Shelf and Argentine Basin that were deeper, warmer and relatively more productive. Attendance of females at the colony was also more variable than that of males and, overall, males were present for significantly more of the pre-laying period (38 vs. 19% of time). Sex differences were reduced following egg-laying, with males and females using similar foraging areas and making trips of similar mean duration in incubation (7.6±2.7 d) and chick-rearing (1.4±1.3 d). Congruence continued into the non-breeding period, with both sexes showing similar patterns of activity and areas of occupancy in the NW Atlantic. Thus, seasonal changes in reproductive roles influenced patterns of sexual segregation; this occurred only early in the season, when male Sooty Shearwaters foraged locally, returning regularly to the colony to defend (or maintain) the burrow or the mate, while females concentrated on building resources for egg development in distant and relatively more productive waters. PMID:24416429

  19. Could sustained monomorphic ventricular tachycardia in the early phase of a prime acute myocardial infarction affect patient outcome?

    PubMed

    Hatzinikolaou-Kotsakou, E; Tziakas, D; Hotidis, A; Stakos, D; Floros, D; Mavridis, A; Papanas, N; Chalikias, G; Maltezos, E; Hatseras, D I

    2007-01-01

    Sustained monomorphic ventricular tachycardia (SMVT) in the course of a prime acute myocardial infarction is not a common arrhythmia and its prognostic significance has not been specifically elucidated. The aim of the study was to estimate the prognostic implications of the occurrence of sustained monomorphic ventricular tachycardia in the early phase (<72 h) of a prime acute myocardial infarction. We studied 690 consecutive patients admitted to the coronary care unit with a diagnosis of a prime myocardial infarction. SMVT was observed in 18 (2.6%) patients and we followed these patients for establishing the prognostic value of the arrhythmia according to the clinical characteristics. Patients with SMVT had a more extensive myocardial infarction based on the peak of the CK-MB isoenzyme activity (480+/-290 IU/L, vs 270+/-190 IU/L, P < .01), and higher mortality rate (40% vs 9%, P < .001). The independent predictors of SMVT were CK-MB (odds ratio [OR] 12.4), presence of complex ventricular arrhythmias (OR = 5.7), a wide QRS complex > or =130 milliseconds (OR = 4.8) and Killip class (OR = 4.8). The SMVT was itself an independent predictor of mortality (OR = 5.0). Compared with patients with ventricular fibrillation or polymorphic ventricular tachycardia, those with SMVT had a higher CK-MB activity, higher rate of wide QRS > or =130 milliseconds (33% vs 8%, P < .002), had a worse hemodynamic condition (Killip class >I:58% vs 23%, P < .04) and higher recurrence rate of ischemic events (68% vs 16%, P < .05). During the one year follow-up period, 4 patients (36.3%) of the 11 survivors from those with SMVT died of cardiac related causes. SMVT during the first 72 h of a prime myocardial infarction is an index of a larger healing myocardium with acute very complexed electrophysiological changes and it is an independent predictor of in-hospital mortality and a prognostic factor of a poor one year outcome.

  20. [Photoreactivating Activity of Bioluminescence: Repair of UV-damaged DNA of Escherichia coli Occurs with Assistance of lux-Genes of Marine Bacteria].

    PubMed

    Zavilgelsky, G B; Melkina, O E; Kotova, V Yu; Konopleva, M N; Manukhov, I V; Pustovoit, K Ss

    2015-01-01

    The UV resistance of luminescent bacteria Escherichia coli AB1886 uvrA6 (pLeo1) containing the plasmid with luxCDABE genes of marine bacteria Photobacterium leiognathi is approximately two times higher than the UV resistance of non-luminous bacteria E. coli AB1886 uvrA6. Introduction of phr::kan(r) mutations (a defect in the functional activity of photolyase) into the genome of E. coli AB1886 uvrA6 (pLeo1) completely removes the high UV resistance of the cells. Therefore, photoreactivation that involves bacterial photolyase contributes mainly to the bioluminescence-induced DNA repair. It is shown that photoreactivating activity of bioluminescence of P. leiognathi is about 2.5 times lower compared with that one induced by a light source with λ > 385 nm. It is also shown that an increase in the bioluminescence intensity, induced by UV radiation in E. coli bacterial cells with a plasmid containing the luxCD ABE genes under RecA-LexA-regulated promoters, occurs only 25-30 min later after UV irradiation of cells and does not contribute to DNA repair. A quorum sensing regulatory system is not involved in the DNA repair by photolyase.

  1. [Obtaining and characterization of DNA-containing micromummies of yeasts and gram-positive bacteria with enhanced cell wall permeability: application in PCR].

    PubMed

    Danilevich, V N; Duda, V I; Suzina, N E; Grishin, E V

    2007-01-01

    The procedure of obtaining DNA-containing cell envelopes ("micromummies") of bacteria, yeasts, and fungi using chaotropic salts has been developed previously and the possibility of their direct application in PCR has been demonstrated. The fine structure of micromummies has been studied by electron microscopic methods. This work has demonstrated that additional treatment of micromummies of yeasts and gram-positive bacteria with proteinase K results in hydrolytic degradation of cell proteins and drastic enhancement of cell wall permeability for macromolecules (DNA). Thus, the efficiency of PCR ex situ using resultant micromummies after washing off the products of protein hydrolysis and proteinase K can be increased. The results of electron microscopic study of ultrathin sections of yeasts (Pichia pastoris, Saccharomyces cerevisiae) and gram-positive bacteria (Micrococcus luteus, Arthrobacter globiformis, Bacillus subtilis) support the biochemical data that treatment with chaotropic salts and proteinase K results in the loosening of microbial cell walls and in a decrease in the intracellular protein content. At the same time, cell walls generally maintain their integrity (continuity) and initial spherical or rodlike shape. The optimal modes of treatment of the cells of different microbial species with chaotropic salts and proteinase K have been selected to obtain permeabilized cell envelopes containing denatured or native DNA.

  2. Hydrocarbon-degrading bacteria enriched by the Deepwater Horizon oil spill identified by cultivation and DNA-SIP

    PubMed Central

    Gutierrez, Tony; Singleton, David R; Berry, David; Yang, Tingting; Aitken, Michael D; Teske, Andreas

    2013-01-01

    The massive influx of crude oil into the Gulf of Mexico during the Deepwater Horizon (DWH) disaster triggered dramatic microbial community shifts in surface oil slick and deep plume waters. Previous work had shown several taxa, notably DWH Oceanospirillales, Cycloclasticus and Colwellia, were found to be enriched in these waters based on their dominance in conventional clone and pyrosequencing libraries and were thought to have had a significant role in the degradation of the oil. However, this type of community analysis data failed to provide direct evidence on the functional properties, such as hydrocarbon degradation of organisms. Using DNA-based stable-isotope probing with uniformly 13C-labelled hydrocarbons, we identified several aliphatic (Alcanivorax, Marinobacter)- and polycyclic aromatic hydrocarbon (Alteromonas, Cycloclasticus, Colwellia)-degrading bacteria. We also isolated several strains (Alcanivorax, Alteromonas, Cycloclasticus, Halomonas, Marinobacter and Pseudoalteromonas) with demonstrable hydrocarbon-degrading qualities from surface slick and plume water samples collected during the active phase of the spill. Some of these organisms accounted for the majority of sequence reads representing their respective taxa in a pyrosequencing data set constructed from the same and additional water column samples. Hitherto, Alcanivorax was not identified in any of the previous water column studies analysing the microbial response to the spill and we discuss its failure to respond to the oil. Collectively, our data provide unequivocal evidence on the hydrocarbon-degrading qualities for some of the dominant taxa enriched in surface and plume waters during the DWH oil spill, and a more complete understanding of their role in the fate of the oil. PMID:23788333

  3. Hydrocarbon-degrading bacteria enriched by the Deepwater Horizon oil spill identified by cultivation and DNA-SIP.

    PubMed

    Gutierrez, Tony; Singleton, David R; Berry, David; Yang, Tingting; Aitken, Michael D; Teske, Andreas

    2013-11-01

    The massive influx of crude oil into the Gulf of Mexico during the Deepwater Horizon (DWH) disaster triggered dramatic microbial community shifts in surface oil slick and deep plume waters. Previous work had shown several taxa, notably DWH Oceanospirillales, Cycloclasticus and Colwellia, were found to be enriched in these waters based on their dominance in conventional clone and pyrosequencing libraries and were thought to have had a significant role in the degradation of the oil. However, this type of community analysis data failed to provide direct evidence on the functional properties, such as hydrocarbon degradation of organisms. Using DNA-based stable-isotope probing with uniformly (13)C-labelled hydrocarbons, we identified several aliphatic (Alcanivorax, Marinobacter)- and polycyclic aromatic hydrocarbon (Alteromonas, Cycloclasticus, Colwellia)-degrading bacteria. We also isolated several strains (Alcanivorax, Alteromonas, Cycloclasticus, Halomonas, Marinobacter and Pseudoalteromonas) with demonstrable hydrocarbon-degrading qualities from surface slick and plume water samples collected during the active phase of the spill. Some of these organisms accounted for the majority of sequence reads representing their respective taxa in a pyrosequencing data set constructed from the same and additional water column samples. Hitherto, Alcanivorax was not identified in any of the previous water column studies analysing the microbial response to the spill and we discuss its failure to respond to the oil. Collectively, our data provide unequivocal evidence on the hydrocarbon-degrading qualities for some of the dominant taxa enriched in surface and plume waters during the DWH oil spill, and a more complete understanding of their role in the fate of the oil.

  4. 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. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Distribution of Human-Specific Bacteroidales and Fecal Indicator Bacteria in an Urban Watershed Impacted by Sewage Pollution, Determined Using RNA- and DNA-Based Quantitative PCR Assays

    PubMed Central

    Kapoor, Vikram; Pitkänen, Tarja; Ryu, Hodon; Elk, Michael

    2014-01-01

    The identification of fecal pollution sources is commonly carried out using DNA-based methods. However, there is evidence that DNA can be associated with dead cells or present as “naked DNA” in the environment. Furthermore, it has been shown that rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) assays can be more sensitive than rRNA gene-based qPCR assays since metabolically active cells usually contain higher numbers of ribosomes than quiescent cells. To this end, we compared the detection frequency of host-specific markers and fecal bacteria using RNA-based RT-qPCR and DNA-based qPCR methods for water samples collected in sites impacted by combined sewer overflows. As a group, fecal bacteria were more frequently detected in most sites using RNA-based methods. Specifically, 8, 87, and 85% of the samples positive for general enterococci, Enterococcus faecalis, and Enterococcus faecium markers, respectively, were detected using RT-qPCR, but not with the qPCR assay counterpart. On average, two human-specific Bacteroidales markers were not detected when using DNA in 12% of the samples, while they were positive for all samples when using RNA (cDNA) as the template. Moreover, signal intensity was up to three orders of magnitude higher in RT-qPCR assays than in qPCR assays. The human-specific Bacteroidales markers exhibited moderate correlation with conventional fecal indicators using RT-qPCR results, suggesting the persistence of nonhuman sources of fecal pollution or the presence of false-positive signals. In general, the results from this study suggest that RNA-based assays can increase the detection sensitivity of fecal bacteria in urban watersheds impacted with human fecal sources. PMID:25326295

  6. An increase in negative supercoiling in bacteria reveals topology-reacting gene clusters and a homeostatic response mediated by the DNA topoisomerase I gene.

    PubMed

    Ferrándiz, María-José; Martín-Galiano, Antonio J; Arnanz, Cristina; Camacho-Soguero, Isabel; Tirado-Vélez, José-Manuel; de la Campa, Adela G

    2016-09-06

    We studied the transcriptional response to an increase in DNA supercoiling in Streptococcus pneumoniae by using seconeolitsine, a new topoisomerase I inhibitor. A homeostatic response allowing recovery of supercoiling was observed in cells treated with subinhibitory seconeolitsine concentrations. Supercoiling increases of 40.7% (6 μM) and 72.9% (8 μM) were lowered to 8.5% and 44.1%, respectively. Likewise, drug removal facilitated the recovery of cell viability and DNA-supercoiling. Transcription of topoisomerase I depended on the supercoiling level. Also specific binding of topoisomerase I to the gyrase A gene promoter was detected by chromatin-immunoprecipitation. The transcriptomic response to 8 μM seconeolitsine had two stages. An early stage, associated to an increase in supercoiling, affected 10% of the genome. A late stage, manifested by supercoiling recovery, affected 2% of the genome. Nearly 25% of the early responsive genes formed 12 clusters with a coordinated transcription. Clusters were 6.7-31.4 kb in length and included 9-22 responsive genes. These clusters partially overlapped with those observed under DNA relaxation, suggesting that bacteria manage supercoiling stress using pathways with common components. This is the first report of a coordinated global transcriptomic response that is triggered by an increase in DNA supercoiling in bacteria. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. An increase in negative supercoiling in bacteria reveals topology-reacting gene clusters and a homeostatic response mediated by the DNA topoisomerase I gene

    PubMed Central

    Ferrándiz, María-José; Martín-Galiano, Antonio J.; Arnanz, Cristina; Camacho-Soguero, Isabel; Tirado-Vélez, José-Manuel; de la Campa, Adela G.

    2016-01-01

    We studied the transcriptional response to an increase in DNA supercoiling in Streptococcus pneumoniae by using seconeolitsine, a new topoisomerase I inhibitor. A homeostatic response allowing recovery of supercoiling was observed in cells treated with subinhibitory seconeolitsine concentrations. Supercoiling increases of 40.7% (6 μM) and 72.9% (8 μM) were lowered to 8.5% and 44.1%, respectively. Likewise, drug removal facilitated the recovery of cell viability and DNA-supercoiling. Transcription of topoisomerase I depended on the supercoiling level. Also specific binding of topoisomerase I to the gyrase A gene promoter was detected by chromatin-immunoprecipitation. The transcriptomic response to 8 μM seconeolitsine had two stages. An early stage, associated to an increase in supercoiling, affected 10% of the genome. A late stage, manifested by supercoiling recovery, affected 2% of the genome. Nearly 25% of the early responsive genes formed 12 clusters with a coordinated transcription. Clusters were 6.7–31.4 kb in length and included 9–22 responsive genes. These clusters partially overlapped with those observed under DNA relaxation, suggesting that bacteria manage supercoiling stress using pathways with common components. This is the first report of a coordinated global transcriptomic response that is triggered by an increase in DNA supercoiling in bacteria. PMID:27378778

  8. The High Prevalence and Diversity of Chlamydiales DNA within Ixodes ricinus Ticks Suggest a Role for Ticks as Reservoirs and Vectors of Chlamydia-Related Bacteria

    PubMed Central

    Pilloux, Ludovic; Aeby, Sébastien; Gaümann, Rahel; Burri, Caroline; Beuret, Christian

    2015-01-01

    The Chlamydiales order is composed of nine families of strictly intracellular bacteria. Among them, Chlamydia trachomatis, C. pneumoniae, and C. psittaci are established human pathogens, whereas Waddlia chondrophila and Parachlamydia acanthamoebae have emerged as new pathogens in humans. However, despite their medical importance, their biodiversity and ecology remain to be studied. Even if arthropods and, particularly, ticks are well known to be vectors of numerous infectious agents such as viruses and bacteria, virtually nothing is known about ticks and chlamydia. This study investigated the prevalence of Chlamydiae in ticks. Specifically, 62,889 Ixodes ricinus ticks, consolidated into 8,534 pools, were sampled in 172 collection sites throughout Switzerland and were investigated using pan-Chlamydiales quantitative PCR (qPCR) for the presence of Chlamydiales DNA. Among the pools, 543 (6.4%) gave positive results and the estimated prevalence in individual ticks was 0.89%. Among those pools with positive results, we obtained 16S rRNA sequences for 359 samples, allowing classification of Chlamydiales DNA at the family level. A high level of biodiversity was observed, since six of the nine families belonging to the Chlamydiales order were detected. Those most common were Parachlamydiaceae (33.1%) and Rhabdochlamydiaceae (29.2%). “Unclassified Chlamydiales” (31.8%) were also often detected. Thanks to the huge amount of Chlamydiales DNA recovered from ticks, this report opens up new perspectives on further work focusing on whole-genome sequencing to increase our knowledge about Chlamydiales biodiversity. This report of an epidemiological study also demonstrates the presence of Chlamydia-related bacteria within Ixodes ricinus ticks and suggests a role for ticks in the transmission of and as a reservoir for these emerging pathogenic Chlamydia-related bacteria. PMID:26386066

  9. PprA Protein Is Involved in Chromosome Segregation via Its Physical and Functional Interaction with DNA Gyrase in Irradiated Deinococcus radiodurans Bacteria

    PubMed Central

    Devigne, Alice; Guérin, Philippe; Lisboa, Johnny; Quevillon-Cheruel, Sophie; Armengaud, Jean; Sommer, Suzanne; Bouthier de la Tour, Claire

    2016-01-01

    ABSTRACT PprA, a radiation-induced Deinococcus-specific protein, was previously shown to be required for cell survival and accurate chromosome segregation after exposure to ionizing radiation. Here, we used an in vivo approach to determine, by shotgun proteomics, putative PprA partners coimmunoprecipitating with PprA when cells were exposed to gamma rays. Among them, we found the two subunits of DNA gyrase and, thus, chose to focus our work on characterizing the activities of the deinococcal DNA gyrase in the presence or absence of PprA. Loss of PprA rendered cells hypersensitive to novobiocin, an inhibitor of the B subunit of DNA gyrase. We showed that treatment of bacteria with novobiocin resulted in induction of the radiation desiccation response (RDR) regulon and in defects in chromosome segregation that were aggravated by the absence of PprA. In vitro, the deinococcal DNA gyrase, like other bacterial DNA gyrases, possesses DNA negative supercoiling and decatenation activities. These two activities are inhibited in vitro by novobiocin and nalidixic acid, whereas PprA specifically stimulates the decatenation activity of DNA gyrase. Together, these results suggest that PprA plays a major role in chromosome decatenation via its interaction with the deinococcal DNA gyrase when D. radiodurans cells are recovering from exposure to ionizing radiation. IMPORTANCE D. radiodurans is one of the most radiation-resistant organisms known. This bacterium is able to cope with high levels of DNA lesions generated by exposure to extreme doses of ionizing radiation and to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments. Here, we identified partners of PprA, a radiation-induced Deinococcus-specific protein, previously shown to be required for radioresistance. Our study leads to three main findings: (i) PprA interacts with DNA gyrase after irradiation, (ii) treatment of cells with novobiocin results in defects in chromosome segregation

  10. Eukaryote-Made Thermostable DNA Polymerase Enables Rapid PCR-Based Detection of Mycoplasma, Ureaplasma and Other Bacteria in the Amniotic Fluid of Preterm Labor Cases

    PubMed Central

    Yoneda, Noriko; Yoneda, Satoshi; Mori, Masashi; Tabata, Homare; Minami, Hiroshi; Saito, Shigeru; Kitajima, Isao

    2015-01-01

    Background Intra-amniotic infection has long been recognized as the leading cause of preterm delivery. Microbial culture is the gold standard for the detection of intra-amniotic infection, but several days are required, and many bacterial species in the amniotic fluid are difficult to cultivate. Methods We developed a novel nested-PCR-based assay for detecting Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples within three hours of sample collection. To detect prokaryotes, eukaryote-made thermostable DNA polymerase, which is free from bacterial DNA contamination, is used in combination with bacterial universal primers. In contrast, to detect eukaryotes, conventional bacterially-made thermostable DNA polymerase is used in combination with fungal universal primers. To assess the validity of the PCR assay, we compared the PCR and conventional culture results using 300 amniotic fluid samples. Results Based on the detection level (positive and negative), 93.3% (280/300) of Mycoplasma, 94.3% (283/300) of Ureaplasma, 89.3% (268/300) of other bacteria and 99.7% (299/300) of fungi matched the culture results. Meanwhile, concerning the detection of bacteria other than Mycoplasma and Ureaplasma, 228 samples were negative according to the PCR method, 98.2% (224/228) of which were also negative based on the culture method. Employing the devised primer sets, mixed amniotic fluid infections of Mycoplasma, Ureaplasma and/or other bacteria could be clearly distinguished. In addition, we also attempted to compare the relative abundance in 28 amniotic fluid samples with mixed infection, and judged dominance by comparing the Ct values of quantitative real-time PCR. Conclusions We developed a novel PCR assay for the rapid detection of Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples. This assay can also be applied to accurately diagnose the absence of bacteria in samples. We believe that this assay will positively contribute to the

  11. One step forwards for the routine use of high-throughput DNA sequencing in environmental monitoring. An efficient and standardizable method to maximize the detection of environmental bacteria.

    PubMed

    Bruno, Antonia; Sandionigi, Anna; Galimberti, Andrea; Siani, Eleonora; Labra, Massimo; Cocuzza, Clementina; Ferri, Emanuele; Casiraghi, Maurizio

    2017-02-01

    We propose an innovative, repeatable, and reliable experimental workflow to concentrate and detect environmental bacteria in drinking water using molecular techniques. We first concentrated bacteria in water samples using tangential flow filtration and then we evaluated two methods of environmental DNA extraction. We performed tests on both artificially contaminated water samples and real drinking water samples. The efficiency of the experimental workflow was measured through qPCR. The successful applicability of the high-throughput DNA sequencing (HTS) approach was demonstrated on drinking water samples. Our results demonstrate the feasibility of our approach in high-throughput-based studies, and we suggest incorporating it in monitoring strategies to have a better representation of the microbial community. In the recent years, HTS techniques have become key tools in the study of microbial communities. To make the leap from academic laboratories to the routine monitoring (e.g., water treatment plants laboratories), we here propose an experimental workflow suitable for the introduction of HTS as a standard method for detecting environmental bacteria. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  12. History of infection with different male-killing bacteria in the two-spot ladybird beetle Adalia bipunctata revealed through mitochondrial DNA sequence analysis.

    PubMed Central

    v d Schulenburg, J Hinrich G; Hurst, Gregory D D; Tetzlaff, Dagmar; Booth, Gwendolen E; Zakharov, Ilia A; Majerus, Michael E N

    2002-01-01

    The two-spot ladybird beetle Adalia bipunctata (Coleoptera: Coccinellidae) is host to four different intracellular maternally inherited bacteria that kill male hosts during embryogenesis: one each of the genus Rickettsia (alpha-Proteobacteria) and Spiroplasma (Mollicutes) and two distinct strains of Wolbachia (alpha-Proteobacteria). The history of infection with these male-killers was explored using host mitochondrial DNA, which is linked with the bacteria due to joint maternal inheritance. Two variable regions, 610 bp of cytochrome oxidase subunit I and 563 bp of NADH dehydrogenase subunit 5, were isolated from 52 A. bipunctata with known infection status and different geographic origin from across Eurasia. Two outgroup taxa were also considered. DNA sequence analysis revealed that the distribution of mitochondrial haplotypes is not associated with geography. Rather, it correlates with infection status, confirming linkage disequilibrium between mitochondria and bacteria. The data strongly suggest that the Rickettsia male-killer invaded the host earlier than the other taxa. Further, the male-killing Spiroplasma is indicated to have undergone a recent and extensive spread through host populations. In general, male-killing in A. bipunctata seems to represent a highly dynamic system, which should prove useful in future studies on the evolutionary dynamics of this peculiar type of symbiont-host association. PMID:11901123

  13. Bacteria capable of degrading anthracene, phenanthrene, and fluoranthene as revealed by DNA based stable-isotope probing in a forest soil.

    PubMed

    Song, Mengke; Jiang, Longfei; Zhang, Dayi; Luo, Chunling; Wang, Yan; Yu, Zhiqiang; Yin, Hua; Zhang, Gan

    2016-05-05

    Information on microorganisms possessing the ability to metabolize different polycyclic aromatic hydrocarbons (PAHs) in complex environments helps in understanding PAHs behavior in natural environment and developing bioremediation strategies. In the present study, stable-isotope probing (SIP) was applied to investigate degraders of PAHs in a forest soil with the addition of individually (13)C-labeled phenanthrene, anthracene, and fluoranthene. Three distinct phylotypes were identified as the active phenanthrene-, anthracene- and fluoranthene-degrading bacteria. The putative phenanthrene degraders were classified as belonging to the genus Sphingomona. For anthracene, bacteria of the genus Rhodanobacter were the putative degraders, and in the microcosm amended with fluoranthene, the putative degraders were identified as belonging to the phylum Acidobacteria. Our results from DNA-SIP are the first to directly link Rhodanobacter- and Acidobacteria-related bacteria with anthracene and fluoranthene degradation, respectively. The results also illustrate the specificity and diversity of three- and four-ring PAHs degraders in forest soil, contributes to our understanding on natural PAHs biodegradation processes, and also proves the feasibility and practicality of DNA-based SIP for linking functions with identity especially uncultured microorganisms in complex microbial biota.

  14. Time-Resolved DNA Stable Isotope Probing Links Desulfobacterales- and Coriobacteriaceae-Related Bacteria to Anaerobic Degradation of Benzene under Methanogenic Conditions

    PubMed Central

    Noguchi, Mana; Kurisu, Futoshi; Kasuga, Ikuro; Furumai, Hiroaki

    2014-01-01

    To identify the microorganisms involved in benzene degradation, DNA-stable isotope probing (SIP) with 13C-benzene was applied to a methanogenic benzene-degrading enrichment culture. Pyrosequencing of ribosomal RNA (rRNA) gene sequences revealed that the community structure was highly complex in spite of a 3-year incubation only with benzene. The culture degraded 98% of approximately 1 mM 13C-benzene and mineralized 72% of that within 63 d. The terminal restriction fragment length polymorphism (T-RFLP) profiles of the buoyant density fractions revealed the incorporation of 13C into two phylotypes after 64 d. These two phylotypes were determined to be Desulfobacterales- and Coriobacteriaceae-related bacteria by cloning and sequencing of the 16S rRNA gene in the 13C-labeled DNA abundant fraction. Comparative pyrosequencing analysis of the buoyant density fractions of 12C- and 13C-labeled samples indicated the incorporation of 13C into three bacterial and one archaeal OTUs related to Desulfobacterales, Coriobacteriales, Rhodocyclaceae, and Methanosarcinales. The first two OTUs included the bacteria detected by T-RFLP-cloning-sequencing analysis. Furthermore, time-resolved SIP analysis confirmed that the activity of all these microbes appeared at the earliest stage of degradation. In this methanogenic culture, Desulfobacterales- and Coriobacteriaceae-related bacteria were most likely to be the major benzene degraders. PMID:24909708

  15. Microbial culture and checkerboard DNA-DNA hybridization assessment of bacteria in root canals of primary teeth pre- and post-endodontic therapy with a calcium hydroxide/chlorhexidine paste.

    PubMed

    Ito, Izabel Yoko; Junior, Fumio Matoba; Paula-Silva, Francisco Wanderley Garcia; Da Silva, Léa Assed Bezerra; Leonardo, Mário Roberto; Nelson-Filho, Paulo

    2011-09-01

    To investigate the root canal microbiota of primary teeth with apical periodontitis and the in vivo antimicrobial effects of a calcium hydroxide/chlorhexidine paste used as root canal dressing. Baseline samples were collected from 30 root canals of primary teeth with apical periodontitis. Then, the root canals were filled with a calcium hydroxide paste containing 1% chlorhexidine for 14 days and the second bacteriologic samples were taken prior to root canal filling. Samples were submitted to microbiologic culture procedure to detect root canal bacteria and processed for checkerboard DNA-DNA hybridization. Baseline microbial culture revealed high prevalence and cfu number of anaerobic, black-pigmented bacteroides, Streptococcus, and aerobic microorganisms. Following root canal dressing, the overall number of cfu was dramatically diminished compared to initial contamination (P <0.05), although prevalence did not change (P > 0.05). Of 35 probes used for checkerboard DNA-DNA hybridization, 31 (88.57%) were present at baseline, and following root canal dressing, the number of positive probes reduced to 13 (37.14%). Similarly, the number of bacterial cells diminished folowing application of calcium hydroxide/chlorhexidine root canal dressing (P = 0.006). Apical periodontitis is caused by a polymicrobial infection, and a calcium hydroxide/chlorhexidine paste is effective in reducing the number of bacteria inside root canals when applied as a root canal dressing. 2011 The Authors. International Journal of Paediatric Dentistry © 2011 BSPD, IAPD and Blackwell Publishing Ltd.

  16. 16S rDNA-based phylogeny of non-symbiotic bacteria of Entorno-pathogenic nematodes from infected insect cadavers.

    PubMed

    Razia, M; Karthikraja, R; Padmanaban, K; Chellapandi, P; Sivaramakrishnan, S

    2011-06-01

    Using 16S rDNA gene sequencing technique, three different species of non-symbiotic bacteria of entomopatho-genic nematodes (EPNs) (Steinernema sp. and Heterorhabditis sp.) were isolated and identified from infected insect cadavers {Galleria mellonella larvae) after 48-hour post infections. Sequence similarity analysis revealed that the strains SRK3, SRK4 and SRK5 belong to Ochrobactrum cytisi, Schineria larvae and Ochrobactrum anthropi, respectively. The isolates O. anthropi and S. larvae were found to be associated with Heterorhabditis indica strains BDU-17 and Yer-136, respectively, whereas O. cytisi was associated with Steinernema siamkayai strain BDU-87. Phenotypically, temporal EPN bacteria were fairly related to symbiotic EPN bacteria (Photorhabdus and Xenorhabdus genera). The strains SRK3 and SRK5 were phylogeographically similar to several non-symbionts and contaminated EPN bacteria isolated in Germany (LMG3311T) and China (X-14), while the strain SRK4 was identical to the isolates of S. larvae (Ll/57, Ll/58, Ll/68 and L2/11) from Wohlfahrtia magnifica in Hungary. The result was further confirmed by RNA secondary structure and minimum energy calculations of aligned sequences. This study suggested that the non-symbionts of these nematodes are phylogeographically diverged in some extent due to phase variation. Therefore, these strains are not host-dependent, but environment-specific isolates. Copyright © 2011 Beijing Genomics Institute. Published by Elsevier Ltd. All rights reserved.

  17. Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water.

    PubMed

    Li, Ru; Tun, Hein Min; Jahan, Musarrat; Zhang, Zhengxiao; Kumar, Ayush; Fernando, Dilantha; Farenhorst, Annemieke; Khafipour, Ehsan

    2017-07-18

    The limitation of 16S rRNA gene sequencing (DNA-based) for microbial community analyses in water is the inability to differentiate live (dormant cells as well as growing or non-growing metabolically active cells) and dead cells, which can lead to false positive results in the absence of live microbes. Propidium-monoazide (PMA) has been used to selectively remove DNA from dead cells during downstream sequencing process. In comparison, 16S rRNA sequencing (RNA-based) can target live microbial cells in water as both dormant and metabolically active cells produce rRNA. The objective of this study was to compare the efficiency and sensitivity of DNA-based, PMA-based and RNA-based 16S rRNA Illumina sequencing methodologies for live bacteria detection in water samples experimentally spiked with different combination of bacteria (2 gram-negative and 2 gram-positive/acid fast species either all live, all dead, or combinations of live and dead species) or obtained from different sources (First Nation community drinking water; city of Winnipeg tap water; water from Red River, Manitoba, Canada). The RNA-based method, while was superior for detection of live bacterial cells still identified a number of 16S rRNA targets in samples spiked with dead cells. In environmental water samples, the DNA- and PMA-based approaches perhaps overestimated the richness of microbial community compared to RNA-based method. Our results suggest that the RNA-based sequencing was superior to DNA- and PMA-based methods in detecting live bacterial cells in water.

  18. Competitive binding-based optical DNA mapping for fast identification of bacteria--multi-ligand transfer matrix theory and experimental applications on Escherichia coli.

    PubMed

    Nilsson, Adam N; Emilsson, Gustav; Nyberg, Lena K; Noble, Charleston; Stadler, Liselott Svensson; Fritzsche, Joachim; Moore, Edward R B; Tegenfeldt, Jonas O; Ambjörnsson, Tobias; Westerlund, Fredrik

    2014-09-01

    We demonstrate a single DNA molecule optical mapping assay able to resolve a specific Escherichia coli strain from other strains. The assay is based on competitive binding of the fluorescent dye YOYO-1 and the AT-specific antibiotic netropsin. The optical map is visualized by stretching the DNA molecules in nanofluidic channels. We optimize the experimental conditions to obtain reproducible barcodes containing as much information as possible. We implement a multi-ligand transfer matrix method for calculating theoretical barcodes from known DNA sequences. Our method extends previous theoretical approaches for competitive binding of two types of ligands to many types of ligands and introduces a recursive approach that allows long barcodes to be calculated with standard computer floating point formats. The identification of a specific E. coli strain (CCUG 10979) is based on mapping of 50-160 kilobasepair experimental DNA fragments onto the theoretical genome using the developed theory. Our identification protocol introduces two theoretical constructs: a P-value for a best experiment-theory match and an information score threshold. The developed methods provide a novel optical mapping toolbox for identification of bacterial species and strains. The protocol does not require cultivation of bacteria or DNA amplification, which allows for ultra-fast identification of bacterial pathogens. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Competitive binding-based optical DNA mapping for fast identification of bacteria - multi-ligand transfer matrix theory and experimental applications on Escherichia coli

    PubMed Central

    Nilsson, Adam N.; Emilsson, Gustav; Nyberg, Lena K.; Noble, Charleston; Stadler, Liselott Svensson; Fritzsche, Joachim; Moore, Edward R. B.; Tegenfeldt, Jonas O.; Ambjörnsson, Tobias; Westerlund, Fredrik

    2014-01-01

    We demonstrate a single DNA molecule optical mapping assay able to resolve a specific Escherichia coli strain from other strains. The assay is based on competitive binding of the fluorescent dye YOYO-1 and the AT-specific antibiotic netropsin. The optical map is visualized by stretching the DNA molecules in nanofluidic channels. We optimize the experimental conditions to obtain reproducible barcodes containing as much information as possible. We implement a multi-ligand transfer matrix method for calculating theoretical barcodes from known DNA sequences. Our method extends previous theoretical approaches for competitive binding of two types of ligands to many types of ligands and introduces a recursive approach that allows long barcodes to be calculated with standard computer floating point formats. The identification of a specific E. coli strain (CCUG 10979) is based on mapping of 50–160 kilobasepair experimental DNA fragments onto the theoretical genome using the developed theory. Our identification protocol introduces two theoretical constructs: a P-value for a best experiment-theory match and an information score threshold. The developed methods provide a novel optical mapping toolbox for identification of bacterial species and strains. The protocol does not require cultivation of bacteria or DNA amplification, which allows for ultra-fast identification of bacterial pathogens. PMID:25013180

  20. A novel approach to propagate flavivirus infectious cDNA clones in bacteria by introducing tandem repeat sequences upstream of virus genome.

    PubMed

    Pu, Szu-Yuan; Wu, Ren-Huang; Tsai, Ming-Han; Yang, Chi-Chen; Chang, Chung-Ming; Yueh, Andrew

    2014-07-01

    Despite tremendous efforts to improve the methodology for constructing flavivirus infectious cDNAs, the manipulation of flavivirus cDNAs remains a difficult task in bacteria. Here, we successfully propagated DNA-launched type 2 dengue virus (DENV2) and Japanese encephalitis virus (JEV) infectious cDNAs by introducing seven repeats of the tetracycline-response element (7×TRE) and a minimal cytomegalovirus (CMVmin) promoter upstream of the viral genome. Insertion of the 7×TRE-CMVmin sequence upstream of the DENV2 or JEV genome decreased the cryptic E. coli promoter (ECP) activity of the viral genome in bacteria, as measured using fusion constructs containing DENV2 or JEV segments and the reporter gene Renilla luciferase in an empty vector. The growth kinetics of recombinant viruses derived from DNA-launched DENV2 and JEV infectious cDNAs were similar to those of parental viruses. Similarly, RNA-launched DENV2 infectious cDNAs were generated by inserting 7×TRE-CMVmin, five repeats of the GAL4 upstream activating sequence, or five repeats of BamHI linkers upstream of the DENV2 genome. All three tandem repeat sequences decreased the ECP activity of the DENV2 genome in bacteria. Notably, 7×TRE-CMVmin stabilized RNA-launched JEV infectious cDNAs and reduced the ECP activity of the JEV genome in bacteria. The growth kinetics of recombinant viruses derived from RNA-launched DENV2 and JEV infectious cDNAs displayed patterns similar to those of the parental viruses. These results support a novel methodology for constructing flavivirus infectious cDNAs, which will facilitate research in virology, viral pathogenesis and vaccine development of flaviviruses and other RNA viruses. © 2014 The Authors.

  1. Molecular cloning of the DNA gyrase genes from Methylovorus sp. strain SS1 and the mechanism of intrinsic quinolone resistance in methylotrophic bacteria.

    PubMed

    Kim, Kwang-Seo; Kim, Jeong Hoon; Kim, Do Yeob; Kim, Hyun Jong; Park, Sang Tae; Kim, Young Min

    2005-12-31

    The genes encoding the DNA gyrase A (GyrA) and B subunits (GyrB) of Methylovorus sp. strain SS1 were cloned and sequenced. gyrA and gyrB coded for proteins of 846 and 799 amino acids with calculated molecular weights of 94,328 and 88,714, respectively, and complemented Escherichia coli gyrA and gyrB temperature sensitive (ts) mutants. To analyze the role of type II topoisomerases in the intrinsic quinolone resistance of methylotrophic bacteria, the sequences of the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase and the C subunit (ParC) of topoisomerase IV (Topo IV) of Methylovorus sp. strain SS1, Methylobacterium extorquens AM1 NCIB 9133, Methylobacillus sp, strain SK1 DSM 8269, and Methylophilus methylotrophus NCIB 10515 were determined. The deduced amino acid sequences of the QRDRs of the ParCs in the four methylotrophic bacteria were identical to that of E. coli ParC. The sequences of the QRDR in GyrA were also identical to those in E. coli GyrA except for the amino acids at positions 83, 87, or 95. The Ser83 to Thr substitution in Methylovorus sp. strain SS1, and the Ser83 to Leu and Asp87 to Asn substitutions in the three other methylotrophs, agreed well with the minimal inhibitory concentrations of quinolones in the four bacteria, suggesting that these residues play a role in the intrinsic susceptibility of methylotrophic bacteria to quinolones.

  2. Diagnosis and quantification of glycerol assimilating denitrifying bacteria in an integrated fixed-film activated sludge reactor via 13C DNA stable-isotope probing.

    PubMed

    Lu, Huijie; Chandran, Kartik

    2010-12-01

    Glycerol, a byproduct of biodiesel and oleo-chemical manufacturing operations, represents an attractive alternate to methanol as a carbon and electron donor for enhanced denitrification. However, unlike methanol, little is known about the diversity and activity of glycerol assimilating bacteria in activated sludge. In this study, the microbial ecology of glycerol assimilating denitrifying bacteria in a sequencing batch integrated fixed film activated sludge (SB-IFAS) reactor was investigated using (13)C-DNA stable isotope probing (SIP). During steady state SB-IFAS reactor operation, near complete nitrate removal (92.7 ± 5.8%) was achieved. Based on (13)C DNA clone libraries obtained after 360 days of SB-IFAS reactor operation, bacteria related to Comamonas spp. and Diaphorobacter spp. dominated in the suspended phase communities. (13)C assimilating members in the biofilm community were phylogenetically more diverse and were related to Comamonas spp., Bradyrhizobium spp., and Tessaracoccus spp. Possibly owing to greater substrate availability in the suspended phase, the glycerol-assimilating denitrifying populations (quantified by real-time PCR) were more abundant therein than in the biofilm phase. The biomass in the suspended phase also had a higher specific denitrification rate than the biofilm phase (p = 4.33e-4), and contributed to 69.7 ± 4.5% of the overall N-removal on a mass basis. The kinetics of glycerol based denitrification by suspended phase biomass were approximately 3 times higher than with methanol. Previously identified methanol assimilating denitrifying bacteria were not associated with glycerol assimilation, thereby suggesting limited cross-utilization of these two substrates for denitrification in the system tested.

  3. A Novel High-Throughput Cell-Based Assay Aimed at Identifying Inhibitors of DNA Metabolism in Bacteria

    PubMed Central

    Fan, Jun; de Jonge, Boudewijn L. M.; MacCormack, Kathy; Sriram, Shubha; McLaughlin, Robert E.; Plant, Helen; Preston, Marian; Fleming, Paul R.; Albert, Robert; Foulk, Melinda

    2014-01-01

    Bacterial biosensor strains can be useful tools for the discovery and characterization of antibacterial compounds. A plasmid-based reporter vector containing a transcriptional fusion between the recA promoter and green fluorescence protein gene was introduced into an Escherichia coli ΔtolC strain to create a biosensor strain that selectively senses inhibitors of DNA metabolism via the SOS response. The strain was used to develop a high-throughput assay to identify new inhibitors of DNA metabolism. Screening of the AstraZeneca compound library with this strain identified known inhibitors of DNA metabolism, as well as novel chemotypes. The cellular target of one novel series was elucidated as DNA gyrase through genetic characterization of laboratory-generated resistant mutants followed by 50% inhibitory concentration measurements in a DNA gyrase activity assay. These studies validated the use of this antibiotic biosensor strain to identify novel selective inhibitors of DNA metabolism by high-throughput screening. PMID:25246396

  4. Development and applications of a DNA labeling method with magnetic nanoparticles to study the role of horizontal gene transfer events between bacteria in soil pollutant bioremediation processes.

    PubMed

    Pivetal, J; Frénéa-Robin, M; Haddour, N; Vézy, C; Zanini, L F; Ciuta, G; Dempsey, N M; Dumas-Bouchiat, F; Reyne, G; Bégin-Colin, S; Felder-Flesh, D; Ghobril, C; Pourroy, G; Simonet, P

    2015-12-01

    Horizontal gene transfers are critical mechanisms of bacterial evolution and adaptation that are involved to a significant level in the degradation of toxic molecules such as xenobiotic pesticides. However, understanding how these mechanisms are regulated in situ and how they could be used by man to increase the degradation potential of soil microbes is compromised by conceptual and technical limitations. This includes the physical and chemical complexity and heterogeneity in such environments leading to an extreme bacterial taxonomical diversity and a strong redundancy of genes and functions. In addition, more than 99 % of soil bacteria fail to develop colonies in vitro, and even new DNA-based investigation methods (metagenomics) are not specific and sensitive enough to consider lysis recalcitrant bacteria and those belonging to the rare biosphere. The objective of the ANR funded project “Emergent” was to develop a new culture independent approach to monitor gene transfer among soil bacteria by labeling plasmid DNA with magnetic nanoparticles in order to specifically capture and isolate recombinant cells using magnetic microfluidic devices. We showed the feasibility of the approach by using electrotransformation to transform a suspension of Escherichia coli cells with biotin-functionalized plasmid DNA molecules linked to streptavidin-coated superparamagnetic nanoparticles. Our results have demonstrated that magnetically labeled cells could be specifically retained on micromagnets integrated in a microfluidic channel and that an efficient selective separation can be achieved with the microfluidic device. Altogether, the project offers a promising alternative to traditional culture-based approaches for deciphering the extent of horizontal gene transfer events mediated by electro or natural genetic transformation mechanisms in complex environments such as soil.

  5. Occult recurrence of monomorphic epitheliotropic intestinal T-cell lymphoma and the role of MATK gene expression in diagnosis.

    PubMed

    Chen, Yunxin; Tan, Soo-Yong; Petersson, Bengt Fredrik; Khor, Yiu Ming; Gopalakrishnan, Sathish Kumar; Tan, Daryl

    2016-03-07

    Monomorphic epitheliotropic intestinal T-cell lymphomas (MEITL), formerly Type II enteropathy-associated T-cell lymphomas (EATL), are rare peripheral T-cell lymphomas. They are associated with poor survival outcomes, in part because of their late diagnosis. Although MEITLs may be reliably diagnosed based on histological and immunophenotypic findings, overlaps with other NK/T and T-cell lymphomas may confound the diagnosis. The distinctive high-level nuclear staining of the novel marker Megakaryocyte-associated tyrosine kinase (MATK) in MEITLs is an invaluable tool in distinguishing MEITL from classical EATL and other NK/T or T-cell lymphomas. 18-Fluorine-2-fluorodeoxyglucose positron emission tomography ((18) F-FDG PET) has been shown to be a useful tool in the staging and follow-up of aggressive lymphomas. Herein, we describe an unusual case of occult hepatic recurrence of MEITL that was non-avid on (18) F-FDG PET, in which diagnosis was confirmed based on the expression of MATK in tumour cells. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  6. The costs of risky male behaviour: sex differences in seasonal survival in a small sexually monomorphic primate

    PubMed Central

    Kraus, Cornelia; Eberle, Manfred; Kappeler, Peter M

    2008-01-01

    Male excess mortality is widespread among mammals and frequently interpreted as a cost of sexually selected traits that enhance male reproductive success. Sex differences in the propensity to engage in risky behaviours are often invoked to explain the sex gap in survival. Here, we aim to isolate and quantify the survival consequences of two potentially risky male behavioural strategies in a small sexually monomorphic primate, the grey mouse lemur Microcebus murinus: (i) most females hibernate during a large part of the austral winter, whereas most males remain active and (ii) during the brief annual mating season males roam widely in search of receptive females. Using a 10-year capture–mark–recapture dataset from a population of M. murinus in Kirindy Forest, western Madagascar, we statistically modelled sex-specific seasonal survival probabilities. Surprisingly, we did not find any evidence for direct survival benefits of hibernation—winter survival did not differ between males and females. By contrast, during the breeding season males survived less well than females (sex gap: 16%). Consistent with the ‘risky male behaviour’ hypothesis, the period for lowered male survival was restricted to the short mating season. Thus, sex differences in survival in a promiscuous mammal can be substantial even in the absence of sexual dimorphism. PMID:18426751

  7. Small and ugly? Phylogenetic analyses of the "selfing syndrome" reveal complex evolutionary fates of monomorphic primrose flowers.

    PubMed

    de Vos, Jurriaan M; Wüest, Rafael O; Conti, Elena

    2014-04-01

    One of the most common trends in plant evolution, loss of self-incompatibility and ensuing increases in selfing, is generally assumed to be associated with a suite of phenotypic changes, notably a reduction of floral size, termed the selfing syndrome. We investigate whether floral morphological traits indeed decrease in a deterministic fashion after losses of self-incompatibility, as traditionally expected, using a phylogeny of 124 primrose species containing nine independent transitions from heterostyly (heteromorphic incompatibility) to homostyly (monomorphic self-compatibility), a classic system for evolution of selfing. We find similar overall variability of homostylous and heterostylous species, except for diminished herkogamy in homostyles. Bayesian mixed models demonstrate differences between homostylous and heterostylous species in all traits, but net effects across species are small (except herkogamy) and directionality differs among traits. Strongly drift-like evolutionary trajectories of corolla tube length and corolla diameter inferred by Ornstein-Uhlenbeck models contrast with expected deterministic trajectories toward small floral size. Lineage-specific population genetic effects associated with evolution of selfing may explain that reductions of floral size represent one of several possible outcomes of floral evolution after loss of heterostyly in primroses. Contrary to the traditional paradigm, selfing syndromes may, but do not necessarily evolve in response to increased selfing. © 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.

  8. The Structure and Stability of the Monomorphic HLA-G Are Influenced by the Nature of the Bound Peptide

    SciTech Connect

    Walpole, Nicholas G.; Kjer-Nielsen, Lars; Kostenko, Lyudmila; McCluskey, James; Brooks, Andrew G.; Rossjohn, Jamie; Clements, Craig S.

    2010-03-26

    The highly polymorphic major histocompatibility complex class Ia (MHC-Ia) molecules present a broad array of peptides to the clonotypically diverse {alpha}{beta} T-cell receptors. In contrast, MHC-Ib molecules exhibit limited polymorphism and bind a more restricted peptide repertoire, in keeping with their major role in innate immunity. Nevertheless, some MHC-Ib molecules do play a role in adaptive immunity. While human leukocyte antigen E (HLA-E), the MHC-Ib molecule, binds a very restricted repertoire of peptides, the peptide binding preferences of HLA-G, the class Ib molecule, are less stringent, although the basis by which HLA-G can bind various peptides is unclear. To investigate how HLA-G can accommodate different peptides, we compared the structure of HLA-G bound to three naturally abundant self-peptides (RIIPRHLQL, KGPPAALTL and KLPQAFYIL) and their thermal stabilities. The conformation of HLA-G{sup KGPPAALTL} was very similar to that of the HLA-G{sup RIIPRHLQL} structure. However, the structure of HLA-G{sup KLPQAFYIL} not only differed in the conformation of the bound peptide but also caused a small shift in the {alpha}2 helix of HLA-G. Furthermore, the relative stability of HLA-G was observed to be dependent on the nature of the bound peptide. These peptide-dependent effects on the substructure of the monomorphic HLA-G are likely to impact on its recognition by receptors of both innate and adaptive immune systems.

  9. Kinetics of killing Listeria monocytogenes by macrophages: correlation of /sup 3/H-DNA release from labeled bacteria and changes in numbers of viable organisms by mathematical model

    SciTech Connect

    Davies, W.A.

    1982-12-01

    Conventional methods of assessing antibacterial activities of macrophages by viable counting are limited by the precision of the statistics and are difficult to interpret quantitatively because of unrestrained extracellular growth of bacteria. An alternative technique based on the release of radioactive DNA from labeled bacteria has been offered as overcoming these drawbacks. To assess it for use with macrophages I have made a correlation with the conventional viable counting method using a mathematical model. Opsonized Listeria monocytogenes labeled with /sup 3/H-thymidine were exposed to rat macrophages for periods up to 4 hr. Numbers of viable bacteria determined after sonication increased exponentially in the absence of live cells and this growth rate was progressively inhibited by increasing numbers of macrophages. After a lag period of 30-60 min soluble /sup 3/H appeared in the supernatant, the amount increasing with time and numbers of macrophages. To correlate these data I developed a mathematical model that considered that changes in numbers of viable organisms were due to the difference between rates of 1) growth of extracellular bacteria and 2) killing within the macrophage. On the basis of this model curves of best fit to the viable counts data were used to predict the release of radioactivity, assuming that death of a bacterium led to the total release of its label. These predictions and the experimental data agreed well, the lag period of 30-60 min between death of the bacterium and release of radioactivity being consistent with intracellular digestion. Release of soluble radioactivity appears to be an accurate reflection of the number of bacteria killed within the macrophage.

  10. A non-invasive method for studying viral DNA delivery to bacteria reveals key requirements for phage SPP1 DNA entry in Bacillus subtilis cells.

    PubMed

    Fernandes, Sofia; Labarde, Audrey; Baptista, Catarina; Jakutytè, Lina; Tavares, Paulo; São-José, Carlos

    2016-08-01

    Bacteriophages use most frequently a tail apparatus to create a channel across the entire bacterial cell envelope to transfer the viral genome to the host cell cytoplasm, initiating infection. Characterization of this critical step remains a major challenge due to the difficulty to monitor DNA entry in the bacterium and its requirements. In this work we developed a new method to study phage DNA entry that has the potential to be extended to many tailed phages. Its application to study genome delivery of bacteriophage SPP1 into Bacillus subtilis disclosed a key role of the host cell membrane potential in the DNA entry process. An energized B. subtilis membrane and a millimolar concentration of calcium ions are shown to be major requirements for SPP1 DNA entry following the irreversible binding of phage particles to the receptor YueB. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Common mechanisms of DNA translocation motors in bacteria and viruses using one-way revolution mechanism without rotation.

    PubMed

    Guo, Peixuan; Zhao, Zhengyi; Haak, Jeannie; Wang, Shaoying; Wu, Dong; Meng, Bing; Weitao, Tao

    2014-01-01

    Biomotors were once described into two categories: linear motor and rotation motor. Recently, a third type of biomotor with revolution mechanism without rotation has been discovered. By analogy, rotation resembles the Earth rotating on its axis in a complete cycle every 24h, while revolution resembles the Earth revolving around the Sun one circle per 365 days (see animations http://nanobio.uky.edu/movie.html). The action of revolution that enables a motor free of coiling and torque has solved many puzzles and debates that have occurred throughout the history of viral DNA packaging motor studies. It also settles the discrepancies concerning the structure, stoichiometry, and functioning of DNA translocation motors. This review uses bacteriophages Phi29, HK97, SPP1, P22, T4, and T7 as well as bacterial DNA translocase FtsK and SpoIIIE or the large eukaryotic dsDNA viruses such as mimivirus and vaccinia virus as examples to elucidate the puzzles. These motors use ATPase, some of which have been confirmed to be a hexamer, to revolve around the dsDNA sequentially. ATP binding induces conformational change and possibly an entropy alteration in ATPase to a high affinity toward dsDNA; but ATP hydrolysis triggers another entropic and conformational change in ATPase to a low affinity for DNA, by which dsDNA is pushed toward an adjacent ATPase subunit. The rotation and revolution mechanisms can be distinguished by the size of channel: the channels of rotation motors are equal to or smaller than 2 nm, that is the size of dsDNA, whereas channels of revolution motors are larger than 3 nm. Rotation motors use parallel threads to operate with a right-handed channel, while revolution motors use a left-handed channel to drive the right-handed DNA in an anti-chiral arrangement. Coordination of several vector factors in the same direction makes viral DNA-packaging motors unusually powerful and effective. Revolution mechanism that avoids DNA coiling in translocating the lengthy genomic

  12. Spatial Dependence of DNA Damage in Bacteria due to Low-Temperature Plasma Application as Assessed at the Single Cell Level

    NASA Astrophysics Data System (ADS)

    Privat-Maldonado, Angela; O'Connell, Deborah; Welch, Emma; Vann, Roddy; van der Woude, Marjan W.

    2016-10-01

    Low temperature plasmas (LTPs) generate a cocktail of reactive nitrogen and oxygen species (RNOS) with bactericidal activity. The RNOS however are spatially unevenly distributed in the plasma. Here we test the hypothesis that this distribution will affect the mechanisms underpinning plasma bactericidal activity focussing on the level of DNA damage in situ. For the first time, a quantitative, single cell approach was applied to assess the level of DNA damage in bacteria as a function of the radial distance from the centre of the plasma jet. Salmonella enterica on a solid, dry surface was treated with two types of LTP: an atmospheric-pressure dielectric barrier discharge plasma jet (charged and neutral species) and a radio-frequency atmospheric-pressure plasma jet (neutral species). In both cases, there was an inverse correlation between the degree of DNA damage and the radial distance from the centre of the plasma, with the highest DNA damage occurring directly under the plasma. This trend was also observed with Staphylococcus aureus. LTP-generated UV radiation was eliminated as a contributing factor. Thus valuable mechanistic information can be obtained from assays on biological material, which can inform the development of LTP as a complementary or alternative therapy for (topical) bacterial infections.

  13. Spatial Dependence of DNA Damage in Bacteria due to Low-Temperature Plasma Application as Assessed at the Single Cell Level

    PubMed Central

    Privat-Maldonado, Angela; O’Connell, Deborah; Welch, Emma; Vann, Roddy; van der Woude, Marjan W.

    2016-01-01

    Low temperature plasmas (LTPs) generate a cocktail of reactive nitrogen and oxygen species (RNOS) with bactericidal activity. The RNOS however are spatially unevenly distributed in the plasma. Here we test the hypothesis that this distribution will affect the mechanisms underpinning plasma bactericidal activity focussing on the level of DNA damage in situ. For the first time, a quantitative, single cell approach was applied to assess the level of DNA damage in bacteria as a function of the radial distance from the centre of the plasma jet. Salmonella enterica on a solid, dry surface was treated with two types of LTP: an atmospheric-pressure dielectric barrier discharge plasma jet (charged and neutral species) and a radio-frequency atmospheric-pressure plasma jet (neutral species). In both cases, there was an inverse correlation between the degree of DNA damage and the radial distance from the centre of the plasma, with the highest DNA damage occurring directly under the plasma. This trend was also observed with Staphylococcus aureus. LTP-generated UV radiation was eliminated as a contributing factor. Thus valuable mechanistic information can be obtained from assays on biological material, which can inform the development of LTP as a complementary or alternative therapy for (topical) bacterial infections. PMID:27759098

  14. Common Mechanisms of DNA translocation motors in Bacteria and Viruses Using One-way Revolution Mechanism without Rotation

    PubMed Central

    Guo, Peixuan; Zhao, Zhengyi; Haak, Jeannie; Wang, Shaoying; Weitao, Tao

    2014-01-01

    Biomotors were once classified into two categories: linear motor and rotation motor. For decades, the viral DNA-packaging motor has been popularly believed to be a five-fold rotation motor. Recently, a third type of biomotor with revolution mechanism without rotation has been discovered. By analogy, rotation resembles the Earth rotating on its axis in a complete cycle every 24 hours, while revolution resembles the Earth revolving around the Sun one circle per 365 days (see animations http://nanobio.uky.edu/movie.html). The action of revolution that enables a motor free of coiling and torque has solved many puzzles and debates that have occurred throughout the history of viral DNA packaging motor studies. It also settles the discrepancies concerning the structure, stoichiometry, and functioning of DNA translocation motors. This review uses bacteriophages Phi29, HK97, SPP1, P22, T4, T7 as well as bacterial DNA translocase FtsK and SpoIIIE as examples to elucidate the puzzles. These motors use a ATPase, some of which have been confirmed to be a hexamer, to revolve around the dsDNA sequentially. ATP binding induces conformational change and possibly an entropy alteration in ATPase to a high affinity toward dsDNA; but ATP hydrolysis triggers another entropic and conformational change in ATPase to a low affinity for DNA, by which dsDNA is pushed toward an adjacent ATPase subunit. The rotation and revolution mechanisms can be distinguished by the size of channel: the channels of rotation motors are equal to or smaller than 2 nm, whereas channels of revolution motors are larger than 3 nm. Rotation motors use parallel threads to operate with a right-handed channel, while revolution motors use a left-handed channel to drive the right-handed DNA in an anti-parallel arrangement. Coordination of several vector factors in the same direction makes viral DNA-packaging motors unusually powerful and effective. Revolution mechanism avoids DNA coiling in translocating the lengthy

  15. Analysis of the distribution and evolution of the ATP-dependent DNA ligases of bacteria delineates a distinct phylogenetic group 'Lig E'.

    PubMed

    Williamson, Adele; Hjerde, Erik; Kahlke, Tim

    2016-01-01

    Prior to the discovery of a minimal ATP-dependent DNA ligase in Haemophilus influenzae, bacteria were thought to only possess a NAD-dependent ligase, which was involved in sealing of Okazaki fragments. We now know that a diverse range of bacterial species possess up to six of these accessory bacterial ATP-dependent DNA ligases (b-ADLs), which vary in size and enzymatic domain associations. Here we compare the domain structure of different types of b-ADLs and investigate their distribution among the bacterial domain to describe possible evolutionary trajectories that gave rise to the sequence and structural diversity of these enzymes. Previous biochemical and genetic analyses have delineated three main classes of these enzymes: Lig B, Lig C and Lig D, which appear to have descended from a common ancestor within the bacterial domain. In the present study, we delineate a fourth group of b-ADLs, Lig E, which possesses a number of unique features at the primary and tertiary structural levels. The biochemical characteristics, domain structure and inferred extracellular location sets this group apart from the other b-ADLs. The results presented here indicate that the Lig E type ligases were horizontally transferred into bacteria in a separate event from other b-ADLs possibly from a bacteriophage. © 2015 John Wiley & Sons Ltd.

  16. Biofouling of ultrafiltration membrane by dairy fluids: Characterization of pioneer colonizer bacteria using a DNA metabarcoding approach.

    PubMed

    Chamberland, Julien; Lessard, Marie-Hélène; Doyen, Alain; Labrie, Steve; Pouliot, Yves

    2017-02-01

    Biofouling of filtration membranes is a major quality and performance issue for the dairy industry. Because biofilms that survive cleaning cycles become resistant over time, prevention strategies limiting the adhesion of bacteria to membranes should be prioritized for sustainable control of biofouling. However, this cannot be achieved because the pioneer bacteria colonizing these membranes are still unknown. Consequently, the objective of this study was to characterize pioneer bacteria on the filtration membrane surface and to measure the effect of filtration operational parameters on their diversity. Thus, milk and cheese whey were filtered for 5 h in concentration mode at 10 and 40°C using a laboratory-scale crossflow filtration system equipped with flat-sheet ultrafiltration membranes. Pioneer colonizer bacteria found on membranes after a chlorinated alkaline cleaning cycle were identified using a metabarcoding approach targeting the 16S ribosomal RNA genes. Our results suggested that prevention strategies targeting biofouling should consider the nature of the filtered fluid and the feed temperature (36.15 and 5.09% of the variances observed on membranes, respectively), as well as the microbial environment of the dairy processing plant. In the future, it is hypothesized that cleaning prevention strategies will be specific to each dairy processor and their operational parameters. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  17. USE OF COMPETITIVE DNA HYBRIDIZATION TO IDENTIFY DIFFERENCES IN THE GENOMES OF TWO CLOSELY RELATED FECAL INDICATOR BACTERIA

    EPA Science Inventory

    Although recent technological advances in DNA sequencing and computational biology now allow scientists to compare entire microbial genomes, comparisons of closely related bacterial species and individual isolates by whole-genome sequencing approaches remains prohibitively expens...

  18. USE OF COMPETITIVE DNA HYBRIDIZATION TO IDENTIFY DIFFERENCES IN THE GENOMES OF TWO CLOSELY RELATED FECAL INDICATOR BACTERIA

    EPA Science Inventory

    Although recent technological advances in DNA sequencing and computational biology now allow scientists to compare entire microbial genomes, comparisons of closely related bacterial species and individual isolates by whole-genome sequencing approaches remains prohibitively expens...

  19. Seasonal succession leads to habitat-dependent differentiation in ribosomal RNA:DNA ratios among freshwater lake bacteria

    DOE PAGES

    Denef, Vincent J.; Fujimoto, Masanori; Berry, Michelle A.; ...

    2016-04-29

    Relative abundance profiles of bacterial populations measured by sequencing DNA or RNA of marker genes can widely differ. These differences, made apparent when calculating ribosomal RNA:DNA ratios, have been interpreted as variable activities of bacterial populations. However, inconsistent correlations between ribosomal RNA:DNA ratios and metabolic activity or growth rates have led to a more conservative interpretation of this metric as the cellular protein synthesis potential (PSP). Little is known, particularly in freshwater systems, about how PSP varies for specific taxa across temporal and spatial environmental gradients and how conserved PSP is across bacterial phylogeny. Here, we generated 16S rRNA genemore » sequencing data using simultaneously extracted DNA and RNA from fractionated (free-living and particulate) water samples taken seasonally along a eutrophic freshwater estuary to oligotrophic pelagic transect in Lake Michigan. In contrast to previous reports, we observed frequent clustering of DNA and RNA data from the same sample. Analysis of the overlap in taxa detected at the RNA and DNA level indicated that microbial dormancy may be more common in the estuary, the particulate fraction, and during the stratified period. Across spatiotemporal gradients, PSP was often conserved at the phylum and class levels. PSPs for specific taxa were more similar across habitats in spring than in summer and fall. This was most notable for PSPs of the same taxa when located in the free-living or particulate fractions, but also when contrasting surface to deep, and estuary to Lake Michigan communities. Our results show that community composition assessed by RNA and DNA measurements are more similar than previously assumed in freshwater systems. Furthermore, the similarity between RNA and DNA measurements and taxa-specific PSPs that drive community-level similarities are conditional on spatiotemporal factors.« less

  20. Seasonal succession leads to habitat-dependent differentiation in ribosomal RNA:DNA ratios among freshwater lake bacteria

    SciTech Connect

    Denef, Vincent J.; Fujimoto, Masanori; Berry, Michelle A.; Schmidt, Marian L.

    2016-04-29

    Relative abundance profiles of bacterial populations measured by sequencing DNA or RNA of marker genes can widely differ. These differences, made apparent when calculating ribosomal RNA:DNA ratios, have been interpreted as variable activities of bacterial populations. However, inconsistent correlations between ribosomal RNA:DNA ratios and metabolic activity or growth rates have led to a more conservative interpretation of this metric as the cellular protein synthesis potential (PSP). Little is known, particularly in freshwater systems, about how PSP varies for specific taxa across temporal and spatial environmental gradients and how conserved PSP is across bacterial phylogeny. Here, we generated 16S rRNA gene sequencing data using simultaneously extracted DNA and RNA from fractionated (free-living and particulate) water samples taken seasonally along a eutrophic freshwater estuary to oligotrophic pelagic transect in Lake Michigan. In contrast to previous reports, we observed frequent clustering of DNA and RNA data from the same sample. Analysis of the overlap in taxa detected at the RNA and DNA level indicated that microbial dormancy may be more common in the estuary, the particulate fraction, and during the stratified period. Across spatiotemporal gradients, PSP was often conserved at the phylum and class levels. PSPs for specific taxa were more similar across habitats in spring than in summer and fall. This was most notable for PSPs of the same taxa when located in the free-living or particulate fractions, but also when contrasting surface to deep, and estuary to Lake Michigan communities. Our results show that community composition assessed by RNA and DNA measurements are more similar than previously assumed in freshwater systems. Furthermore, the similarity between RNA and DNA measurements and taxa-specific PSPs that drive community-level similarities are conditional on spatiotemporal factors.

  1. The Plant Pathogen Pseudomonas syringae pv. tomato Is Genetically Monomorphic and under Strong Selection to Evade Tomato Immunity

    PubMed Central

    Yan, Shuangchun; Liu, Haijie; Clarke, Christopher R.; Campanile, Francesco; Almeida, Nalvo F.; Studholme, David J.; Lindeberg, Magdalen; Schneider, David; Zaccardelli, Massimo; Setubal, Joao C.; Morales-Lizcano, Nadia P.; Bernal, Adriana; Coaker, Gitta; Baker, Christy; Bender, Carol L.; Leman, Scotland; Vinatzer, Boris A.

    2011-01-01

    Recently, genome sequencing of many isolates of genetically monomorphic bacterial human pathogens has given new insights into pathogen microevolution and phylogeography. Here, we report a genome-based micro-evolutionary study of a bacterial plant pathogen, Pseudomonas syringae pv. tomato. Only 267 mutations were identified between five sequenced isolates in 3,543,009 nt of analyzed genome sequence, which suggests a recent evolutionary origin of this pathogen. Further analysis with genome-derived markers of 89 world-wide isolates showed that several genotypes exist in North America and in Europe indicating frequent pathogen movement between these world regions. Genome-derived markers and molecular analyses of key pathogen loci important for virulence and motility both suggest ongoing adaptation to the tomato host. A mutational hotspot was found in the type III-secreted effector gene hopM1. These mutations abolish the cell death triggering activity of the full-length protein indicating strong selection for loss of function of this effector, which was previously considered a virulence factor. Two non-synonymous mutations in the flagellin-encoding gene fliC allowed identifying a new microbe associated molecular pattern (MAMP) in a region distinct from the known MAMP flg22. Interestingly, the ancestral allele of this MAMP induces a stronger tomato immune response than the derived alleles. The ancestral allele has largely disappeared from today's Pto populations suggesting that flagellin-triggered immunity limits pathogen fitness even in highly virulent pathogens. An additional non-synonymous mutation was identified in flg22 in South American isolates. Therefore, MAMPs are more variable than expected differing even between otherwise almost identical isolates of the same pathogen strain. PMID:21901088

  2. The plant pathogen Pseudomonas syringae pv. tomato is genetically monomorphic and under strong selection to evade tomato immunity.

    PubMed

    Cai, Rongman; Lewis, James; Yan, Shuangchun; Liu, Haijie; Clarke, Christopher R; Campanile, Francesco; Almeida, Nalvo F; Studholme, David J; Lindeberg, Magdalen; Schneider, David; Zaccardelli, Massimo; Setubal, Joao C; Morales-Lizcano, Nadia P; Bernal, Adriana; Coaker, Gitta; Baker, Christy; Bender, Carol L; Leman, Scotland; Vinatzer, Boris A

    2011-08-01

    Recently, genome sequencing of many isolates of genetically monomorphic bacterial human pathogens has given new insights into pathogen microevolution and phylogeography. Here, we report a genome-based micro-evolutionary study of a bacterial plant pathogen, Pseudomonas syringae pv. tomato. Only 267 mutations were identified between five sequenced isolates in 3,543,009 nt of analyzed genome sequence, which suggests a recent evolutionary origin of this pathogen. Further analysis with genome-derived markers of 89 world-wide isolates showed that several genotypes exist in North America and in Europe indicating frequent pathogen movement between these world regions. Genome-derived markers and molecular analyses of key pathogen loci important for virulence and motility both suggest ongoing adaptation to the tomato host. A mutational hotspot was found in the type III-secreted effector gene hopM1. These mutations abolish the cell death triggering activity of the full-length protein indicating strong selection for loss of function of this effector, which was previously considered a virulence factor. Two non-synonymous mutations in the flagellin-encoding gene fliC allowed identifying a new microbe associated molecular pattern (MAMP) in a region distinct from the known MAMP flg22. Interestingly, the ancestral allele of this MAMP induces a stronger tomato immune response than the derived alleles. The ancestral allele has largely disappeared from today's Pto populations suggesting that flagellin-triggered immunity limits pathogen fitness even in highly virulent pathogens. An additional non-synonymous mutation was identified in flg22 in South American isolates. Therefore, MAMPs are more variable than expected differing even between otherwise almost identical isolates of the same pathogen strain.

  3. A novel universal DNA labeling and amplification system for rapid microarray-based detection of 117 antibiotic resistance genes in Gram-positive bacteria.

    PubMed

    Strauss, Christian; Endimiani, Andrea; Perreten, Vincent

    2015-01-01

    A rapid and simple DNA labeling system has been developed for disposable microarrays and has been validated for the detection of 117 antibiotic resistance genes abundant in Gram-positive bacteria. The DNA was fragmented and amplified using phi-29 polymerase and random primers with linkers. Labeling and further amplification were then performed by classic PCR amplification using biotinylated primers specific for the linkers. The microarray developed by Perreten et al. (Perreten, V., Vorlet-Fawer, L., Slickers, P., Ehricht, R., Kuhnert, P., Frey, J., 2005. Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria. J.Clin.Microbiol. 43, 2291-2302.) was improved by additional oligonucleotides. A total of 244 oligonucleotides (26 to 37 nucleotide length and with similar melting temperatures) were spotted on the microarray, including genes conferring resistance to clinically important antibiotic classes like β-lactams, macrolides, aminoglycosides, glycopeptides and tetracyclines. Each antibiotic resistance gene is represented by at least 2 oligonucleotides designed from consensus sequences of gene families. The specificity of the oligonucleotides and the quality of the amplification and labeling were verified by analysis of a collection of 65 strains belonging to 24 species. Association between genotype and phenotype was verified for 6 antibiotics using 77 Staphylococcus strains belonging to different species and revealed 95% test specificity and a 93% predictive value of a positive test. The DNA labeling and amplification is independent of the species and of the target genes and could be used for different types of microarrays. This system has also the advantage to detect several genes within one bacterium at once, like in Staphylococcus aureus strain BM3318, in which up to 15 genes were detected. This new microarray-based detection system offers a large potential for applications in clinical diagnostic, basic research, food safety and

  4. Design and Performance Testing of a DNA Extraction Assay for Sensitive and Reliable Quantification of Acetic Acid Bacteria Directly in Red Wine Using Real Time PCR

    PubMed Central

    Longin, Cédric; Guilloux-Benatier, Michèle; Alexandre, Hervé

    2016-01-01

    Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence, there is a real need for a rapid, specific, sensitive, and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR). Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP) at 1% (v/v) during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability, and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 to 10 mL. Thus, the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage. PMID:27313572

  5. Design and Performance Testing of a DNA Extraction Assay for Sensitive and Reliable Quantification of Acetic Acid Bacteria Directly in Red Wine Using Real Time PCR.

    PubMed

    Longin, Cédric; Guilloux-Benatier, Michèle; Alexandre, Hervé

    2016-01-01

    Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence, there is a real need for a rapid, specific, sensitive, and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR). Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP) at 1% (v/v) during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability, and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 to 10 mL. Thus, the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage.

  6. Design and testing of a functional group-specific DNA probe for the study of natural populations of acetogenic bacteria.

    PubMed Central

    Lovell, C R; Hui, Y

    1991-01-01

    The acetogens, although phylogenetically diverse, can be characterized by their possession of the acetyl coenzyme A (acetyl-CoA) pathway for autotrophic CO2 fixation. The gene encoding formyltetrahydrofolate synthetase, a key enzyme of the acetyl-CoA pathway, was previously cloned from the thermophilic acetogen Clostridium thermoaceticum and has now been tested as a group-specific probe for acetogens. Stable hybrids were formed between the probe and single DNA fragments from eight known acetogens representing six genera. A hybrid was also formed between the probe and a DNA fragment from one sulfate reducer known to be capable of both autotrophic CO2 fixation and acetate catabolism. No such hybrid was formed between the probe and DNA from a homoacetate fermenter not known to use the acetyl-CoA pathway, with two known formyltetrahydrofolate synthetase-producing purine fermenters, or with DNA from 27 other species representing 16 genera of organisms that do not use the acetyl-CoA pathway. DNA purified from cells extracted from horse manure was also screened with the acetogen probe. Six hybrids, indicating at least six detectable acetogen "strains," were observed. Images PMID:1768134

  7. Using a DNA microarray to investigate the distribution of insect virulence factors in strains of photorhabdus bacteria.

    PubMed

    Marokhazi, Judit; Waterfield, Nicholas; LeGoff, Gaelle; Feil, Edward; Stabler, Richard; Hinds, Jason; Fodor, Andras; ffrench-Constant, Richard H

    2003-08-01

    Photorhabdus is an insect-pathogenic bacterium in which oral toxicity to insects is found in two distinct taxonomic groups. Using a DNA microarray and comparative genomics, we show that oral toxicity is associated with toxin complex genes tcaABC and that this locus can be mobilized or deleted within different strains.

  8. Using a DNA Microarray To Investigate the Distribution of Insect Virulence Factors in Strains of Photorhabdus Bacteria

    PubMed Central

    Marokhazi, Judit; Waterfield, Nicholas; LeGoff, Gaelle; Feil, Edward; Stabler, Richard; Hinds, Jason; Fodor, Andras; ffrench-Constant, Richard H.

    2003-01-01

    Photorhabdus is an insect-pathogenic bacterium in which oral toxicity to insects is found in two distinct taxonomic groups. Using a DNA microarray and comparative genomics, we show that oral toxicity is associated with toxin complex genes tcaABC and that this locus can be mobilized or deleted within different strains.   PMID:12867479

  9. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  10. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  11. A New Double Digestion Ligation Mediated Suppression PCR Method for Simultaneous Bacteria DNA-Typing and Confirmation of Species: An Acinetobacter sp. Model

    PubMed Central

    Stojowska, Karolina; Krawczyk, Beata

    2014-01-01

    We have designed a new ddLMS PCR (double digestion Ligation Mediated Suppression PCR) method based on restriction site polymorphism upstream from the specific target sequence for the simultaneous identification and differentiation of bacterial strains. The ddLMS PCR combines a simple PCR used for species or genus identification and the LM PCR strategy for strain differentiation. The bacterial identification is confirmed in the form of the PCR product(s), while the length of the PCR product makes it possible to differentiate between bacterial strains. If there is a single copy of the target sequence within genomic DNA, one specific PCR product is created (simplex ddLMS PCR), whereas for multiple copies of the gene the fingerprinting patterns can be obtained (multiplex ddLMS PCR). The described ddLMS PCR method is designed for rapid and specific strain differentiation in medical and microbiological studies. In comparison to other LM PCR it has substantial advantages: enables specific species' DNA-typing without the need for pure bacterial culture selection, is not sensitive to contamination with other cells or genomic DNA, and gives univocal “band-based” results, which are easy to interpret. The utility of ddLMS PCR was shown for Acinetobacter calcoaceticus-baumannii (Acb) complex, the genetically closely related and phenotypically similar species and also important nosocomial pathogens, for which currently, there are no recommended methods for screening, typing and identification. In this article two models are proposed: 3′ recA-ddLMS PCR-MaeII/RsaI for Acb complex interspecific typing and 5′ rrn-ddLMS PCR-HindIII/ApaI for Acinetobacter baumannii intraspecific typing. ddLMS PCR allows not only for DNA-typing but also for confirmation of species in one reaction. Also, practical guidelines for designing a diagnostic test based on ddLMS PCR for genotyping different species of bacteria are provided. PMID:25522278

  12. 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.

  13. Molecular characterization by amplified ribosomal DNA restriction analysis and antimicrobial potential of endophytic fungi isolated from Luehea divaricata (Malvaceae) against plant pathogenic fungi and pathogenic bacteria.

    PubMed

    Bernardi-Wenzel, J; Garcia, A; Azevedo, J L; Pamphile, J A

    2013-10-29

    Luehea divaricata is an important plant in popular medicine; it is used for its depurative, anti-inflammatory, and other therapeutic activities. We evaluated the antimicrobial activity of endophytic fungi isolated from leaves of L. divaricata against phytopathogens and pathogenic bacteria, and characterized the isolates based on amplified ribosomal DNA restriction analysis (ARDRA). The in vitro antagonistic activity of these endophytes against the phytopathogen Alternaria alternata was assayed by dual culture technique. Based on this evaluation of antimicrobial activity, we extracted secondary metabolites from nine endophytic fungi by partitioning in ethyl acetate and methanol. These were tested against the phytopathogens A. alternata, Colletotrichum sp and Moniliophthora perniciosa, and against the human pathogenic bacteria Escherichia coli and Staphylococcus aureus. Molecular characterization by ARDRA technique was used for phylogenetic analysis, based on comparison with sequences in GenBank. The endophytes had varied effects on A. alternata. One isolate produced an inhibition halo against M. perniciosa and against E. coli. This antibiosis activity indicates a role in the protection of the plant against microbial pathogens in nature, with potential for pharmaceutical and agricultural applications. Based on ARDRA, the 13 isolates were grouped. We found three different haplotypes of Phomopsis sp, showing interspecific variability. It appears that examination of the microbial community associated with medicinal plants of tropical regions has potential as a useful strategy to look for species with biotechnological applications.

  14. Geographic Separation of Domestic and Wild Strains of Toxoplasma gondii in French Guiana Correlates with a Monomorphic Version of Chromosome1a

    PubMed Central

    Khan, Asis; Ajzenberg, Daniel; Mercier, Aurélien; Demar, Magalie; Simon, Stéphane; Dardé, Marie Laure; Wang, Qiuling; Verma, Shiv Kumar; Rosenthal, Benjamin M.; Dubey, Jitender P.; Sibley, L. David

    2014-01-01

    Background Previous studies have stressed the genetic divergence and high pathogenicity of strains of T. gondii from French Guiana. Although strains from coastal, human adapted environments (so called anthropized) resemble those found in other regions of the Caribbean, strains collected from inland jungle environment are genetically quite diverse. To better understand the composition of these distinct strain types, we undertook a more in depth analysis of T. gondii strains from French Guiana including profiling of chromosome 1a (Chr1a), which is often shared as a single monomorphic haplotype among lineages that are otherwise genetically distinct. Methodology/Principal Findings Comparison of intron sequences from selectively neutral genes indicated that anthropized strains were most closely related to clonal type III strains from North America, although wider RFLP analysis revealed that they are natural hybrids. In contrast, strains isolated from the jungle were genetically very diverse. Remarkably, nearly all anthropized strains contained the monomorphic version of Chr1a while wild stains were extremely divergent. The presence of the monomorphic Chr1a strongly correlated with greater transmission in domestic cats, although there were several exceptions, indicating that other factors also contribute. Anthropized strains also varied in their virulence in laboratory mice, and this pattern could not be explained by the simple combination of previously identified virulence factors, indicating that other genetic determinants influence pathogenicity. Conclusions/Significance Our studies underscore the marked genetic separation of anthropized and wild strains of T. gondii in French Guiana and provide additional evidence that the presence of Chr1a is associated with successful expansion of widely different lineages within diverse geographic areas. The predominance of Chr1a among strains in the anthropized environment suggests that it may confer an advantage for transmission

  15. Anaerobic bacteria

    MedlinePlus

    Anaerobic bacteria are bacteria that do not live or grow when oxygen is present. In humans, these bacteria ... Goldstein EJ. Diseases caused by non-spore forming anaerobic bacteria. In: Goldman L, Schafer AI, eds. Goldman's Cecil ...

  16. A monoclonal antibody (1B7) specific for polymorphic determinant on mouse I-A antigens recognizes monomorphic epitope shared by most of RT-1 haplotypes in rats.

    PubMed

    Iwabuchi, K; Ishikawa, N; Mizuno, K; Kojima, H; Natori, T; Ogasawara, K; Ogasawara, M; Fujita, M; Onoé, K

    1986-10-01

    A monoclonal antibody, 1B7, which was established by immunizing C57BL/10 mice with splenocytes from B10.BR, was investigated by serological, immunochemical and functional analyses in mouse, rat, guinea pig and human systems. 1B7 recognized a polymorphic determinant on class II antigens in the mouse system. In the rat system, however, this antibody appeared to recognize a monomorphic epitope shared by all RT-1 haplotypes. 1B7 showed no reactivity in the human and guinea pig strains tested.

  17. Identification of dominant bacteria in feces and colonic mucosa from healthy Spanish adults by culturing and by 16S rDNA sequence analysis.

    PubMed

    Delgado, Susana; Suárez, Adolfo; Mayo, Baltasar

    2006-04-01

    The aim of this work was to examine by culturing the changes in the total and indicator populations of the feces of two individuals over 1 year and to identify the dominant microbial components of a single sample of feces from each donor. Populations and dominant bacteria from a sample of colonic mucosa from a further individual were also assessed. The culture results were then compared to those obtained with the same samples by 16S rDNA cloning and sequencing. High interindividual variation in representative microbial populations of the gastrointestinal tract (GIT) was revealed by both the culture and the culture-independent techniques. Species belonging to Clostridium clusters (XIVa, IV, and XVIII) predominated in both the fecal and the mucosal samples (except in the mucose cultured isolates), members of Clostridium coccoides cluster XIVa being the most numerous microorganisms. Species of gamma-proteobacteria (Escherichia coli and Shigella spp.), bifidobacteria, and actinobacteria appeared in lower numbers than those of clostridia. From the mucosal cultured sample, only facultative anaerobes and bifidobacteria were recovered, suggesting destruction of the anaerobe population during processing. In accordance with this, the microbial diversity revealed by 16S rDNA sequence analysis was greater than that revealed by culturing. Despite large interindividual differences, distinct human communities may have group-associated GIT microbiota characteristics, such as the low number of Bacteroides seen in the subjects in this study.

  18. Easy DNA extraction method and optimisation of PCR-Temporal Temperature Gel Electrophoresis to identify the predominant high and low GC-content bacteria from dairy products.

    PubMed

    Parayre, Sandrine; Falentin, Hélène; Madec, Marie-Noëlle; Sivieri, Katia; Le Dizes, Anne-Sophie; Sohier, Danièle; Lortal, Sylvie

    2007-06-01

    Molecular fingerprinting of bacterial ecosystems has recently increased in food microbiology. The aim of this work was to develop a rapid and easy method to extract DNA from various cheeses, and to optimize the separation of low and high GC-content bacteria by PCR-Temporal Temperature Gel Electrophoresis (PCR-TTGE). Seventy six strains belonging to 50 of the most frequently encountered bacterial species in dairy products were used to construct a database. Specific PCR-TTGE ladders containing 17 species forming a regular scale were created. Amplicons of these species were sequenced and the GC-content plotted against the migration distance: the correlation coefficients obtained were r(2)=0.97 and r(2)=0.99, respectively for high and low GC-contents. The extraction method developed did not use any harmful solvent such as phenol/chloroform. The concentrations of DNA extracted from hard cooked and pressed cheeses, quantified by picogreen molecular probes, were between 0.7 and 6 microg/g for core samples and 8 to 30 microg/g for rind samples. Experimental as well as commercial dairy products were analysed using the developed method and the reproducibility of the profiles was 89%. The method appears to be particularly efficient in the characterization of the ecosystem of cheese rinds.

  19. Removal of Contaminant DNA by Combined UV-EMA Treatment Allows Low Copy Number Detection of Clinically Relevant Bacteria Using Pan-Bacterial Real-Time PCR

    PubMed Central

    Humphrey, Bruce; McLeod, Neil; Turner, Carrie; Sutton, J. Mark; Dark, Paul M.; Warhurst, Geoffrey

    2015-01-01

    Background More than two decades after its discovery, contaminant microbial DNA in PCR reagents continues to impact the sensitivity and integrity of broad-range PCR diagnostic techniques. This is particularly relevant to their use in the setting of human sepsis, where a successful diagnostic on blood samples needs to combine universal bacterial detection with sensitivity to 1-2 genome copies, because low levels of a broad range of bacteria are implicated. Results We investigated the efficacy of ethidium monoazide (EMA) and propidium monoazide (PMA) treatment as emerging methods for the decontamination of PCR reagents. Both treatments were able to inactivate contaminating microbial DNA but only at concentrations that considerably affected assay sensitivity. Increasing amplicon length improved EMA/PMA decontamination efficiency but at the cost of assay sensitivity. The same was true for UV exposure as an alternative decontamination strategy, likely due to damage sustained by oligonucleotide primers which were a significant source of contamination. However, a simple combination strategy with UV-treated PCR reagents paired with EMA-treated primers produced an assay capable of two genome copy detection and a <5% contamination rate. This decontamination strategy could have important utility in developing improved pan-bacterial assays for rapid diagnosis of low pathogen burden conditions such as in the blood of patients with suspected blood stream infection. PMID:26172943

  20. Removal of Contaminant DNA by Combined UV-EMA Treatment Allows Low Copy Number Detection of Clinically Relevant Bacteria Using Pan-Bacterial Real-Time PCR.

    PubMed

    Humphrey, Bruce; McLeod, Neil; Turner, Carrie; Sutton, J Mark; Dark, Paul M; Warhurst, Geoffrey

    2015-01-01

    More than two decades after its discovery, contaminant microbial DNA in PCR reagents continues to impact the sensitivity and integrity of broad-range PCR diagnostic techniques. This is particularly relevant to their use in the setting of human sepsis, where a successful diagnostic on blood samples needs to combine universal bacterial detection with sensitivity to 1-2 genome copies, because low levels of a broad range of bacteria are implicated. We investigated the efficacy of ethidium monoazide (EMA) and propidium monoazide (PMA) treatment as emerging methods for the decontamination of PCR reagents. Both treatments were able to inactivate contaminating microbial DNA but only at concentrations that considerably affected assay sensitivity. Increasing amplicon length improved EMA/PMA decontamination efficiency but at the cost of assay sensitivity. The same was true for UV exposure as an alternative decontamination strategy, likely due to damage sustained by oligonucleotide primers which were a significant source of contamination. However, a simple combination strategy with UV-treated PCR reagents paired with EMA-treated primers produced an assay capable of two genome copy detection and a <5% contamination rate. This decontamination strategy could have important utility in developing improved pan-bacterial assays for rapid diagnosis of low pathogen burden conditions such as in the blood of patients with suspected blood stream infection.

  1. 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.

  2. 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.

  3. Mechanisms of recombination and function of DNA in bacteria. Progress report, January 15, 1983-January 17, 1984

    SciTech Connect

    Guild, W.R.

    1984-01-01

    Studies on gene transfer in pneumococcus are described. Specifically transformation by chromosomal and plasmid DNAs, transfection by phage and plasmid DNA, and the novel kind of conjugative transfer of drug resistance elements among the chromosomes of streptococci are examined. Some of these elements appear to be transposons. There are numerous questions about their structure, functions, and relation to plasmids and other better characterized genetic elements. The experiments involve extensive use of transformation, cloning in both pneumococcal and E. coli systems, and analysis of DNAs by gel electrophoresis. For restriction mapping, we have concentrated on the insertion carrying genes for resistance to chloramphenicol and tetracycline.

  4. Repair of hydrolytic DNA deamination damage in thermophilic bacteria: cloning and characterization of a Vsr endonuclease homolog from Bacillus stearothermophilus

    PubMed Central

    Laging, Martin; Lindner, Eric; Fritz, Hans-Joachim; Kramer, Wilfried

    2003-01-01

    Hydrolytic deamination of 5-methyl cytosine in double stranded DNA results in formation of a T/G mismatch that—if left unrepaired—leads to a C→T transition mutation in half of the progeny. In addition to several mismatch-specific glycosylases that have been found in both pro- and eukaryotes to channel this lesion into base excision repair by removing the T from the mismatch, Vsr endonuclease from Escherichia coli has been described which initiates repair by an endonucleolytic strand incision 5′ to the mismatched T. We have isolated a gene coding for a homolog of E.coli Vsr endonuclease from the thermophilic bacterium Bacillus stearothermophilus H3 (Vsr.Bst) using a method that allows PCR amplification with degenerated primers of gene segments which code for only one highly conserved amino acid region. Vsr.Bst was produced heterologously in E.coli and purified to apparent homogeneity. Vsr.Bst specifically incises heteroduplex DNA with a preference for T/G mismatches. The selectivity of Vsr.Bst for the sequence context of the T/G mismatch appears less pronounced than for Vsr.Eco. PMID:12655008

  5. Repair of hydrolytic DNA deamination damage in thermophilic bacteria: cloning and characterization of a Vsr endonuclease homolog from Bacillus stearothermophilus.

    PubMed

    Laging, Martin; Lindner, Eric; Fritz, Hans-Joachim; Kramer, Wilfried

    2003-04-01

    Hydrolytic deamination of 5-methyl cytosine in double stranded DNA results in formation of a T/G mismatch that-if left unrepaired-leads to a C-->T transition mutation in half of the progeny. In addition to several mismatch-specific glycosylases that have been found in both pro- and eukaryotes to channel this lesion into base excision repair by removing the T from the mismatch, Vsr endonuclease from Escherichia coli has been described which initiates repair by an endonucleolytic strand incision 5' to the mismatched T. We have isolated a gene coding for a homolog of E.coli Vsr endonuclease from the thermophilic bacterium Bacillus stearothermophilus H3 (Vsr.Bst) using a method that allows PCR amplification with degenerated primers of gene segments which code for only one highly conserved amino acid region. Vsr.Bst was produced heterologously in E.coli and purified to apparent homogeneity. Vsr.Bst specifically incises heteroduplex DNA with a preference for T/G mismatches. The selectivity of Vsr.Bst for the sequence context of the T/G mismatch appears less pronounced than for Vsr.Eco.

  6. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  7. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  8. High-throughput DNA sequencing of the ruminal bacteria from moose (Alces alces) in Vermont, Alaska, and Norway.

    PubMed

    Ishaq, Suzanne L; Wright, André-Denis

    2014-08-01

    In the present study, the rumen bacteria of moose (Alces alces) from three distinct geographic locations were investigated. Moose are large, browsing ruminants in the deer family, which subsist on fibrous, woody browse, and aquatic plants. Subspecies exist which are distinguished by differing body and antler size, and these are somewhat geographically isolated. Seventeen rumen samples were collected from moose in Vermont, Alaska, and Norway, and bacterial 16S ribosomal RNA genes were sequenced using Roche 454 pyrosequencing with titanium chemistry. Overall, 109,643 sequences were generated from the 17 individual samples, revealing 33,622 unique sequences. Members of the phylum Bacteroidetes were dominant in samples from Alaska and Norway, but representatives of the phylum Firmicutes were dominant in samples from Vermont. Within the phylum Bacteroidetes, Prevotellaceae was the dominant family in all three sample locations, most of which belonged to the genus Prevotella. Within the phylum Firmicutes, the family Lachnospiraceae was the most prevalent in all three sample locations. The data set supporting the results of this article is available in the Sequence Read Archive (SRA), available through NCBI [study accession number SRP022590]. Samples clustered by geographic location and by weight and were heterogenous based on gender, location, and weight class (p < 0.05). Location was a stronger factor in determining the core microbiome than either age or weight, but gender did not appear to be a strong factor. There were no shared operational taxonomic units across all 17 samples, which indicates that these moose may have been isolated long enough to preclude a core microbiome among moose. Other potential factors discussed include differences in climate, food quality and availability, gender, and life cycle.

  9. High-throughput DNA sequence analysis reveals stable engraftment of gut microbiota following transplantation of previously frozen fecal bacteria.

    PubMed

    Hamilton, Matthew J; Weingarden, Alexa R; Unno, Tatsuya; Khoruts, Alexander; Sadowsky, Michael J

    2013-01-01

    Fecal microbiota transplantation (FMT) is becoming a more widely used technology for treatment of recurrent Clostridum difficile infection (CDI). While previous treatments used fresh fecal slurries as a source of microbiota for FMT, we recently reported the successful use of standardized, partially purified and frozen fecal microbiota to treat CDI. Here we report that high-throughput 16S rRNA gene sequencing showed stable engraftment of gut microbiota following FMT using frozen fecal bacteria from a healthy donor. Similar bacterial taxa were found in post-transplantation samples obtained from the recipients and donor samples, but the relative abundance varied considerably between patients and time points. Post FMT samples from patients showed an increase in the abundance of Firmicutes and Bacteroidetes, representing 75-80% of the total sequence reads. Proteobacteria and Actinobacteria were less abundant (< 5%) than that found in patients prior to FMT. Post FMT samples from two patients were very similar to donor samples, with the Bacteroidetes phylum represented by a great abundance of members of the families Bacteroidaceae, Rikenellaceae and Porphyromonadaceae, and were largely comprised of Bacteroides, Alistipes and Parabacteroides genera. Members of the phylum Firmicutes were represented by Ruminococcaceae, Lachnospiraceae, Verrucomicrobiaceae and unclassified Clostridiales and members of the Firmicutes. One patient subsequently received antibiotics for an unrelated infection, resulting in an increase in the number of intestinal Proteobacteria, primarily Enterobacteriaceae. Our results demonstrate that frozen fecal microbiota from a healthy donor can be used to effectively treat recurrent CDI resulting in restoration of the structure of gut microbiota and clearing of Clostridum difficile.

  10. A hot pepper cDNA encoding ascorbate peroxidase is induced during the incompatible interaction with virus and bacteria.

    PubMed

    Yoo, Tae Hyoung; Park, Chang-Jin; Lee, Gil-Je; Shin, Ryoung; Yun, Ji-Hyun; Kim, Ki-Jeong; Rhee, Ki-Hyeong; Paek, Kyung-Hee

    2002-08-31

    Capsicum annuum L. is infected by a number of viruses, including the tobacco mosaic virus (TMV). To study the defense-related genes that are induced by TMV in hot peppers, the pepper plant, which is susceptible to P1.2 but resistant to the P0 pathotype of TMV, was inoculated with TMV-P0. Differential screening isolated the genes that were specifically up- or down-regulated during the hypersensitive response (HR). The CaAPX1 cDNA clone that putatively encodes a polypeptide of cytosolic ascorbate peroxidase was selected as an up-regulated gene. It was isolated for further study. The full-length cDNA for CaAPX1, which is 972 bp long, contained the open-reading frame of 250-amino acid residues. A genomic Southern blot analysis showed that there were only limited copies of the CaAPX1 gene in the hot pepper genome. In hot pepper cv. Bugang, which is resistant to TMV-P0 and susceptible to TMV-P1.2, the CaAPX1 gene transcript was accumulated by TMV-P0, but not by TMV-P1.2 inoculation. CaAPX1 transcripts began to accumulate 24 h post-inoculation of TMV-P0, and increased gradually until 96 h. To investigate whether each transcript is induced by other stimuli, the plants were treated with various chemicals and wounding. A striking induction of the CaAPX1 transcript was observed at 2 h. It subsided 12 h after salicylic acid (SA), ethephon, and methyl jasmonate (MeJA) treatments. The response of the gene upon other pathogen infection was also examined by a bacterial pathogen (Xanthomonas campestris pv. vesicatoria race 3) inoculation. The CaAPX1 gene was induced in a hot pepper (C. annuum cv. ECW 20R) that was resistant to this bacterial pathogen, but not in a susceptible hot pepper (C. annuum cv. ECW). These results suggest the possible role(s) for the CaAPX1 gene in plant defense against viral and bacterial pathogen.

  11. Protective role of probiotic lactic acid bacteria against dietary fumonisin B1-induced toxicity and DNA-fragmentation in sprague-dawley rats.

    PubMed

    Khalil, Ashraf A; Abou-Gabal, Ashgan E; Abdellatef, Amira A; Khalid, Ahmed E

    2015-08-18

    The genus Fusarium, especially F. verticillioides and F. proliferatum, has been found in several agricultural products worldwide, especially in maize. Regardless the occurrence of symptoms, the presence of Fusarium in maize constitutes an imminent risk due to its ability to produce fumonisins, mycotoxins with proven carcinogenic effect on rats, swine, and equines and already classified as possible carcinogens to humans. The toxicity of incremental levels of fumonisin B1 (FB1), that is, 50, 100, and 200 mg FB1/kg diet, and the role of Lactobacillus delbrueckii subsp. lactis DSM 20076 (LL) and Pediococcus acidilactici NNRL B-5627 (PA) supplementation in counteracting the FB1 effects in intoxicated rats were monitored over a period of 4 weeks. Effects on the feed intake and body weight gain were noticed. A significant (p ≤ 0.05) increase in the level of liver and kidney functions markers and DNA fragmentation was also noticed in rat groups T100 and T200. The lactic acid bacteria (LAB) supplementation could bring back the normal serum biochemical parameters in rats fed on fumonisin B1-contaminated diets (T50 and T100) compared to FB1-treated groups. In rats of high-dosage dietary groups supplemented with LAB (T200-LL and T200-PA), the supplementation reduced the serum activity levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and creatinine by 11.3, 11.9, 32, and 20%, respectively. DNA fragmentations were observed in the rat group treated with 200 mg FB1 after 3 weeks, while fragmentation was noticed in treated groups with 100 and 200 mg FB1 after 4 weeks. No DNA fragmentation was apparent in FB1-treated rats co-administered the LL or PA strain. These results suggest that in male rats consuming diets containing FB1, there is a time- and dose-dependent increase in serum enzyme activities and DNA lesions. Moreover, Lb. delbrueckii subsp. lactis (LL) and P. acidilactici (PA) strains have a protective effect

  12. In situ DNA hybridized chain reaction (FISH-HCR) as a better method for quantification of bacteria and archaea within marine sediment

    NASA Astrophysics Data System (ADS)

    Buongiorno, J.; Lloyd, K. G.; Shumaker, A.; Schippers, A.; Webster, G.; Weightman, A.; Turner, S.

    2015-12-01

    Nearly 75% of the Earth's surface is covered by marine sediment that is home to an estimated 2.9 x 1029 microbial cells. A substantial impediment to understanding the abundance and distribution of cells within marine sediment is the lack of a consistent and reliable method for their taxon-specific quantification. Catalyzed reporter fluorescent in situ hybridization (CARD-FISH) provides taxon-specific enumeration, but this process requires passing a large enzyme through cell membranes, decreasing its precision relative to general cell counts using a small DNA stain. In 2015, Yamaguchi et al. developed FISH hybridization chain reaction (FISH-HCR) as an in situ whole cell detection method for environmental microorganisms. FISH-HCR amplifies the fluorescent signal, as does CARD-FISH, but it allows for milder cell permeation methods that might prevent yield loss. To compare FISH-HCR to CARD-FISH, we examined bacteria and archaea cell counts within two sediment cores, Lille Belt (~78 meters deep) and Landsort Deep (90 meters deep), which were retrieved from the Baltic Sea Basin during IODP Expedition 347. Preliminary analysis shows that CARD-FISH counts are below the quantification limit for most depths across both cores. By contrast, quantification of cells was possible with FISH-HCR in all examined depths. When quantification with CARD-FISH was above the limit of detection, counts with FISH-HCR were up to 11 fold higher for Bacteria and 3 fold higher for Archaea from the same sediment sample. Further, FISH-HCR counts follow the trends of on board counts nicely, indicating that FISH-HCR may better reflect the cellular abundance within marine sediment than other quantification methods, including qPCR. Using FISH-HCR, we found that archaeal cell counts were on average greater than bacterial cell counts, but within the same order of magnitude.

  13. Amplified ribosomal DNA restriction analysis of free-living bacteria present in the headbox of a Canadian paper machine.

    PubMed

    Prince, Véronique; Simao-Beaunoir, Anne-Marie; Beaulieu, Carole

    2009-07-01

    The headbox water is the main source of bacterial contamination of paper machines. Identification of these bacterial contaminants could be an asset in developing specific control methods. An amplified ribosomal DNA restriction analysis (ARDRA) was carried out to characterize the bacterial communities associated with the headbox water of a paper machine in a Canadian mill in February and July 2006. Eight bacterial genera were identified as the main colonizers present in the headbox water. The genus Meiothermus appeared to be the dominant bacterial group in the Canadian paper machine. Some variation was observed between the February and July clone libraries. Bacterial genera such as Chelatococcus and Hydrogenophilus were only detected in February or in July, respectively. Furthermore, the proportion of Tepidimonas clones in the libraries was higher in July than in February. The metabolic profile of the February and July communities, determined using Biolog EcoPlates, also suggested that temporal variation occurred within the bacterial populations that colonized the headbox of the paper machine.

  14. Interspersed DNA repeats bcr1-bcr18 of Bacillus cereus group bacteria form three distinct groups with different evolutionary and functional patterns.

    PubMed

    Kristoffersen, Simen M; Tourasse, Nicolas J; Kolstø, Anne-Brit; Økstad, Ole Andreas

    2011-02-01

    Many short (<400 bp) interspersed sequence repeats exist in bacteria, yet little is known about their origins, mode of generation, or possible function. Here, we present a comprehensive analysis of 18 different previously identified repeated DNA elements, bcr1-bcr18 (Økstad OA, Hegna I, Lindback T, Rishovd AL, Kolstø AB. 1999. Genome organization is not conserved between Bacillus cereus and Bacillus subtilis. Microbiology. 145:621-631.; Tourasse NJ, Helgason E, Økstad OA, Hegna IK, Kolstø AB. 2006. The Bacillus cereus group: novel aspects of population structure and genome dynamics. J Appl Microbiol. 101:579-593.), in 36 sequenced genomes from the Bacillus cereus group of bacteria. This group consists of genetically closely related species with variable pathogenic specificity toward different hosts and includes among others B. anthracis, B. cereus, and B. thuringiensis. The B. cereus group repeat elements could be classified into three categories with different properties: Group A elements (bcr1-bcr3) exhibited highly variable copy numbers ranging from 4 to 116 copies per strain, showed a nonconserved chromosomal distribution pattern between strains, and displayed several features characteristic of mobile elements. Group B repeats (bcr4-bcr6) were present in 0-10 copies per strain and were associated with strain-specific genes and disruptions of genome synteny, implying a possible contribution to genome rearrangements and/or horizontal gene transfer events. bcr5, in particular, was associated with large gene clusters showing resemblance to integrons. In agreement with their potentially mobile nature or involvement in horizontal transfers, the sequences of the repeats from Groups A and B (bcr1-bcr6) followed a phylogeny different from that of the host strains. Conversely, repeats from Group C (bcr7-bcr18) had a conserved chromosomal location and orthologous gene neighbors in the investigated B. cereus group genomes, and their phylogeny matched that of the host

  15. Prevalence of lysogeny among soil bacteria and presence of 16S rRNA and trzN genes in viral-community DNA.

    PubMed

    Ghosh, Dhritiman; Roy, Krishnakali; Williamson, Kurt E; White, David C; Wommack, K Eric; Sublette, Kerry L; Radosevich, Mark

    2008-01-01

    Bacteriophages are very abundant in the biosphere, and viral infection is believed to affect the activity and genetic diversity of bacterial communities in aquatic environments. Lysogenic conversion, for example, can improve host fitness and lead to phage-mediated horizontal gene transfer. However, little is known about lysogeny and transduction in the soil environment. In this study we employed atrazine-impregnated Bio-Sep beads (a cell immobilization matrix) to sample active microbiota from soils with prior pesticide exposure history. Once recovered from soil, the bead communities were induced with mitomycin C (MC), and viral and bacterial abundances were determined to evaluate the incidence of inducible prophage in soil bacteria. The inducible fraction calculated within bead communities was high (ca. 85%) relative to other studies in aquatic and sedimentary environments. Moreover, the bacterial genes encoding 16S rRNA and trzN, a chlorohydrolase gene responsible for dehalogenation of atrazine, were detected by PCR in the viral DNA fraction purified from MC-induced bead communities. A diverse collection of actinobacterial 16S rRNA gene sequences occurred within the viral DNA fraction of induced, water-equilibrated beads. Similar results were observed in induced atrazine-equilibrated beads, where 77% of the cloned sequences were derived from actinobacterial lineages. Heterogeneous 16S rRNA gene sequences consisting of fragments from two different taxa were detected in the clone libraries. The results suggest that lysogeny is a prevalent reproductive strategy among soil bacteriophages and that the potential for horizontal gene transfer via transduction is significant in soil microbial communities.

  16. Proteomics, DNA arrays and the analysis of still unknown regulons and unknown proteins of Bacillus subtilis and pathogenic gram-positive bacteria.

    PubMed

    Hecker, M; Engelmann, S

    2000-05-01

    The complete sequence of the bacterial genomes provides new perspectives for the study of gene expression and gene function. By the combination of the highly sensitive 2-dimensional (2D) protein gel electrophoresis with the identification of the protein spots by microsequencing or mass spectrometry we established a 2D protein index of Bacillus subtilis that currently comprises almost 400 protein entries. A computer-aided evaluation of the 2D gels loaded with radioactively-labelled proteins from growing or stressed/starved cells proved to be a powerful tool in the analysis of global regulation of the expression of the entire genome. For the general stress regulon it is demonstrated how the proteomics approach can be used to analyse the regulation, structure and function of still unknown regulons. The application of this approach is illustrated for the sigmaB dependent general stress regulon. For the comprehensive description of proteins/genes belonging to stimulons or regulons it is generally recommended to complement the proteome approach with DNA array techniques in order to identify and allocate still undiscovered members of individual regulons. This approach is also very attractive to uncover the function of still unknown global regulators and regulons and to dissect the entire genome into its basic modules of global regulation. The same strategy can be used to analyse the regulation, structure and function of regulons encoding virulence factors of pathogenic bacteria for a comprehensive understanding of the pathogenicity and for the identification of new antibacterial targets.

  17. Sensitive Visual Detection of AHPND Bacteria Using Loop-Mediated Isothermal Amplification Combined with DNA-Functionalized Gold Nanoparticles as Probes

    PubMed Central

    Arunrut, Narong; Kampeera, Jantana; Sirithammajak, Sarawut; Sanguanrut, Piyachat; Proespraiwong, Porranee; Suebsing, Rungkarn; Kiatpathomchai, Wansika

    2016-01-01

    Acute hepatopancreatic necrosis disease (AHPND) is a component cause of early mortality syndrome (EMS) of shrimp. In 2013, the causative agent was found to be unique isolates of Vibrio parahaemolyticus (VPAHPND) that contained a 69 kbp plasmid (pAP1) carrying binary Pir-like toxin genes PirvpA and PirvpB. In Thailand, AHPND was first recognized in 2012, prior to knowledge of the causative agent, and it subsequently led to a precipitous drop in shrimp production. After VPAHPND was characterized, a major focus of the AHPND control strategy was to monitor broodstock shrimp and post larvae for freedom from VPAHPND by nucleic acid amplification methods, most of which required use of expensive and sophisticated equipment not readily available in a shrimp farm setting. Here, we describe a simpler but equally sensitive approach for detection of VPAHPND based on loop-mediated isothermal amplification (LAMP) combined with unaided visual reading of positive amplification products using a DNA-functionalized, ssDNA-labled nanogold probe (AuNP). The target for the special set of six LAMP primers used was the VPAHPND PirvpA gene. The LAMP reaction was carried out at 65°C for 45 min followed by addition of the red AuNP solution and further incubation at 65°C for 5 min, allowing any PirvpA gene amplicons present to hybridize with the probe. Hybridization protected the AuNP against aggregation, so that the solution color remained red upon subsequent salt addition (positive test result) while unprotected AuNP aggregated and underwent a color change from red to blue and eventually precipitated (negative result). The total assay time was approximately 50 min. The detection limit (100 CFU) was comparable to that of other commonly-used methods for nested PCR detection of VPAHPND and 100-times more sensitive than 1-step PCR detection methods (104 CFU) that used amplicon detection by electrophoresis or spectrophotometry. There was no cross reaction with DNA templates derived from non

  18. Development of a real-time PCR method for the detection of fossil 16S rDNA fragments of phototrophic sulfur bacteria in the sediments of Lake Cadagno.

    PubMed

    Ravasi, D F; Peduzzi, S; Guidi, V; Peduzzi, R; Wirth, S B; Gilli, A; Tonolla, M

    2012-05-01

    Lake Cadagno is a crenogenic meromictic lake situated in the southern range of the Swiss Alps characterized by a compact chemocline that has been the object of many ecological studies. The population dynamics of phototrophic sulfur bacteria in the chemocline has been monitored since 1994 with molecular methods such as 16S rRNA gene clone library analysis. To reconstruct paleo-microbial community dynamics, we developed a quantitative real-time PCR methodology for specific detection of 16S rRNA gene sequences of purple and green sulfur bacteria populations from sediment samples. We detected fossil 16S rDNA of nine populations of phototrophic sulfur bacteria down to 9-m sediment depth, corresponding to about 9500 years of the lake's biogeological history. These results provide the first evidence for the presence of 16S rDNA of anoxygenic phototrophic bacteria in Holocene sediments of an alpine meromictic lake and indicate that the water column stratification and the bacterial plume were already present in Lake Cadagno thousands of years ago. The finding of Chlorobium clathratiforme remains in all the samples analyzed shows that this population, identified in the water column only in 2001, was already a part of the lake's biota in the past.

  19. DNase I and Proteinase K eliminate DNA from injured or dead bacteria but not from living bacteria in microbial reference systems and natural drinking water biofilms for subsequent molecular biology analyses.

    PubMed

    Villarreal, Jessica Varela; Jungfer, Christina; Obst, Ursula; Schwartz, Thomas

    2013-09-01

    Molecular techniques, such as polymerase chain reaction (PCR) and quantitative PCR (qPCR), are very sensitive, but may detect total DNA present in a sample, including extracellular DNA (eDNA) and DNA coming from live and dead cells. DNase I is an endonuclease that non-specifically cleaves single- and double-stranded DNA. This enzyme was tested in this study to analyze its capacity of digesting DNA coming from dead cells with damaged cell membranes, leaving DNA from living cells with intact cell membranes available for DNA-based methods. For this purpose, an optimized DNase I/Proteinase K (DNase/PK) protocol was developed. Intact Staphylococcus aureus cells, heat-killed Pseudomonas aeruginosa cells, free genomic DNA of Salmonella enterica, and a mixture of these targets were treated according to the developed DNase/PK protocol. In parallel, these samples were treated with propidium monoazide (PMA) as an already described assay for live-dead discrimination. Quantitative PCR and PCR-DGGE of the eubacterial 16S rDNA fragment were used to test the ability of the DNase/PK and PMA treatments to distinguish DNA coming from cells with intact cell membranes in the presence of DNA from dead cells and free genomic DNA. The methods were applied to three months old autochthonous drinking water biofilms from a pilot facility built at a German waterworks. Shifts in the DNA patterns observed after DGGE analysis demonstrated the applicability of DNase/PK as well as of the PMA treatment for natural biofilm investigation. However, the DNase/PK treatment demonstrated some practical advantages in comparison with the PMA treatment for live/dead discrimination of bacterial targets in drinking water systems.

  20. Cloning and identification of a novel NhaD-type Na+/H+ antiporter from metagenomic DNA of the halophilic bacteria in soil samples around Daban Salt Lake.

    PubMed

    Zhang, Hua; Wang, Zhenhui; Wang, Lei; Mu, Ren; Zou, Zhi; Yuan, Kun; Wang, Yuekun; Wu, Haiping; Jiang, Juquan; Yang, Lifu

    2014-01-01

    In this study, metagenomic DNA was screened for the Na(+)/H(+) antiporter gene from the halophilic bacteria in Daban Salt Lake by selection in Escherichia coli KNabc lacking three major Na(+)/H(+) antiporters. One gene designated as Hb_nhaD encoding a novel NhaD-type Na(+)/H(+) antiporter was finally cloned. The presence of Hb_NhaD conferred tolerance of E. coli KNabc to up to 0.5 M NaCl and 0.2 M LiCl, and an alkaline pH. Hb_NhaD has the highest identity (70.6%) with a putative NhaD-type Na(+)/H(+) antiporter from an uncharacterized Clostridiaceae species, and also has lower identity with known NhaD-type Na(+)/H(+) antiporters from Halomonas elongata (20.8%), Alkalimonas amylolytica (19.0%), Vibrio parahaemolyticus (18.9%) and Vibrio cholerae (18.7 %). pH-dependent Na(+)(Li(+))/H(+) antiport activity was detected from everted membrane vesicles prepared from E. coli KNabc carrying Hb_nhaD. Hb_NhaD exhibited very high Na(+)(Li(+))/H(+) antiport activity over a wide pH range from 6.5 to 9.0 with the highest activity at pH 7.0 which is significantly different from those of the above known NhaD-type Na(+)/H(+) antiporters. Also, the apparent K m values of Hb_NhaD for Na(+) and Li(+) at pH 7.0 were determined to be 1.31 and 2.16, respectively. Based on the above results, we proposed that Hb_NhaD should be categorized as a novel NhaD-type Na(+)/H(+) antiporter.

  1. Detection of fecal bacteria and source tracking identifiers in environmental waters using rRNA-based RT-qPCR and rDNA-based qPCR assays.

    PubMed

    Pitkänen, Tarja; Ryu, Hodon; Elk, Michael; Hokajärvi, Anna-Maria; Siponen, Sallamaari; Vepsäläinen, Asko; Räsänen, Pia; Santo Domingo, Jorge W

    2013-01-01

    In this study, we evaluated the use of RT-qPCR assays targeting rRNA gene sequences for the detection of fecal bacteria in water samples. We challenged the RT-qPCR assays against RNA extracted from sewage effluent (n = 14), surface water (n = 30), and treated source water (n = 15) samples. Additionally, we applied the same assays using DNA as the qPCR template. The targeted fecal bacteria were present in most of the samples tested, although in several cases, the detection frequency increased when RNA was used as the template. For example, the majority of samples that tested positive for E. coli and Campylobacter spp. in surface waters, and for human-specific Bacteroidales, E. coli, and Enterococcus spp. in treated source waters were only detected when rRNA was used as the original template. The difference in detection frequency using rRNA or rDNA (rRNA gene) was sample- and assay-dependent, suggesting that the abundance of active and nonactive populations differed between samples. Statistical analyses for each population exhibiting multiple quantifiable results showed that the rRNA copy numbers were significantly higher than the rDNA counterparts (p < 0.05). Moreover, the detection frequency of rRNA-based assays were in better agreement with the culture-based results of E. coli, intestinal enterococci, and thermotolerant Campylobacter spp. in surface waters than that of rDNA-based assays, suggesting that rRNA signals were associated to active bacterial populations. Our data show that using rRNA-based approaches significantly increases detection sensitivity for common fecal bacteria in environmental waters. These findings have important implications for microbial water quality monitoring and public health risk assessments.

  2. Diversity and Distribution of DNA Sequences with Affinity to Ammonia-Oxidizing Bacteria of the β Subdivision of the Class Proteobacteria in the Arctic Ocean

    PubMed Central

    Bano, Nasreen; Hollibaugh, James T.

    2000-01-01

    The spatial distribution and diversity of ammonia-oxidizing bacteria of the β subdivision of the class Proteobacteria (hereinafter referred to as ammonia oxidizers) in the Arctic Ocean were determined. The presence of ammonia oxidizers was detected by PCR amplification of 16S rRNA genes using a primer set specific for this group of organisms (nitA and nitB, which amplifies a 1.1-kb fragment between positions 137 and 1234, corresponding to Escherichia coli 16S rDNA numbering). We analyzed 246 samples collected from the upper water column (5 to 235 m) during March and April 1995, September and October 1996, and September 1997. Ammonia oxidizers were detected in 25% of the samples from 5 m, 80% of the samples from 55 m, 88% of the samples from 133 m, and 50% of the samples from 235 m. Analysis of nitA-nitB PCR product by nested PCR-denaturing gradient gel electrophoresis (DGGE) showed that all positive samples contained the same major band (band A), indicating the presence of a dominant, ubiquitous ammonia oxidizer in the Arctic Ocean basin. Twenty-two percent of the samples contained additional major bands. These samples were restricted to the Chukchi Sea shelf break, the Chukchi cap, and the Canada basin; areas likely influenced by Pacific inflow. The nucleotide sequence of the 1.1-kb nitA-nitB PCR product from a sample that contained only band A grouped with sequences designated group 1 marine Nitrosospira-like sequences. PCR-DGGE analysis of 122 clones from four libraries revealed that 67 to 71% of the inserts contained sequences with the same mobility as band A. Nucleotide sequences (1.1 kb) of another distinct group of clones, found only in 1995 samples (25%), fell into the group 5 marine Nitrosomonas-like sequences. Our results suggest that the Arctic Ocean β-proteobacterial ammonia oxidizers have low diversity and are dominated by marine Nitrosospira-like organisms. Diversity appears to be higher in Western Arctic Ocean regions influenced by inflow from the

  3. Simple & Safe Genomic DNA Isolation.

    ERIC Educational Resources Information Center

    Moss, Robert; Solomon, Sondra

    1991-01-01

    A procedure for purifying DNA using either bacteria or rat liver is presented. Directions for doing a qualitative DNA assay using diphenylamine and a quantitative DNA assay using spectroscopy are included. (KR)

  4. Simple & Safe Genomic DNA Isolation.

    ERIC Educational Resources Information Center

    Moss, Robert; Solomon, Sondra

    1991-01-01

    A procedure for purifying DNA using either bacteria or rat liver is presented. Directions for doing a qualitative DNA assay using diphenylamine and a quantitative DNA assay using spectroscopy are included. (KR)

  5. Isolation and Identification of Concrete Environment Bacteria

    NASA Astrophysics Data System (ADS)

    Irwan, J. M.; Anneza, L. H.; Othman, N.; Husnul, T.; Alshalif, A. F.

    2016-07-01

    This paper presents the isolation and molecular method for bacteria identification through PCR and DNA sequencing. Identification of the bacteria species is required in order to fully utilize the bacterium capability for precipitation of calcium carbonate in concrete. This process is to enable the addition of suitable catalyst according to the bacterium enzymatic pathway that is known through the bacteria species used. The objective of this study is to isolate, enriched and identify the bacteria species. The bacteria in this study was isolated from fresh urine and acid mine drainage water, Kota Tinggi, Johor. Enrichment of the isolated bacteria was conducted to ensure the bacteria survivability in concrete. The identification of bacteria species was done through polymerase chain reaction (PCR) and rRDNA sequencing. The isolation and enrichment of the bacteria was done successfully. Whereas, the results for bacteria identification showed that the isolated bacteria strains are Bacillus sp and Enterococus faecalis.

  6. Monomorphic Epithelial Proliferations of the Breast: A Possible Precursor Lesion Associated With Ipsilateral Breast Failure After Breast Conserving Therapy in Patients With Negative Lumpectomy Margins

    SciTech Connect

    Goldstein, Neal S.; Kestin, Larry L.; Vicini, Frank A.

    2011-03-01

    Background: It is generally believed that ipsilateral breast failures (IBFs) after breast-conserving therapy (BCT) develop from incompletely eradicated carcinoma. We previously suggested that monomorphic epithelial proliferations (MEPs) in the breast may be a pool of partially transformed clones from which breast carcinomas can arise and that radiation therapy (RT) may also reduce the risk of IBF by eradicating MEPs. We examined salvage mastectomy specimens in patients experiencing an IBF to define the relationship between MEPs and IBFs and an additional potential mechanism for IBF risk reduction by RT. Methods and Materials: The location, number, and distribution of radiation changes and MEPs relative to 51 IBFs were mapped in salvage mastectomy specimens from BCT patients with adequately excised, initial carcinomas (negative lumpectomy margins). Results: All 51 salvage mastectomies had diffuse, late radiation changes. None had active fibrocystic lesions. MEPs were predominantly located in the immediate vicinity of the IBFs. A mean of 39% of MEP cases were located within the IBF, 46% were located within 2 cm of the IBF, and 14% were 2-3 cm from the IBF. Conclusions: MEPs appear to be a pool of partially transformed precursor lesions that can give rise to ductal carcinoma in situ and invasive carcinomas (CAs). Many IBFs may arise from MEPs that reemerge after RT. Radiation may also reduce IBF risk after BCT (including in patients with negative margins) by primarily eradicating MEPs.

  7. Analyzing arthropods for the presence of bacteria.

    PubMed

    Andrews, Elizabeth S

    2013-02-01

    Bacteria within arthropods can be identified using culture-independent methods. This unit describes protocols for surface sterilization of arthropods, DNA extraction of whole bodies and tissues, touchdown PCR amplification using 16S rDNA general bacteria primers, and profiling the bacterial community using denaturing gradient gel electrophoresis. © 2013 by John Wiley & Sons, Inc.

  8. Genetic and functional characterization of a yet-unclassified rhizobial Dtr (DNA-transfer-and-replication) region from a ubiquitous plasmid conjugal system present in Sinorhizobium meliloti, in Sinorhizobium medicae, and in other nonrhizobial Gram-negative bacteria.

    PubMed

    Giusti, María de los Ángeles; Pistorio, Mariano; Lozano, Mauricio J; Tejerizo, Gonzalo A Torres; Salas, María Eugenia; Martini, María Carla; López, José Luis; Draghi, Walter O; Del Papa, María Florencia; Pérez-Mendoza, Daniel; Sanjuán, Juan; Lagares, Antonio

    2012-05-01

    Rhizobia are Gram-negative bacteria that live in soils and associate with leguminous plants to establish nitrogen-fixing symbioses. The ability of these bacteria to undergo horizontal gene transfer (HGT) is thought to be one of the main features to explain both the origin of their symbiotic life-style and the plasticity and dynamics of their genomes. In our laboratory we have previously characterized at the species level the non-pSym plasmid mobilome in Sinorhizobium meliloti, the symbiont of Medicago spp., and have found a high incidence of conjugal activity in many plasmids (Pistorio et al., 2008). In this work we characterized the Dtr (DNA-transfer-and-replication) region of one of those plasmids, pSmeLPU88b. This mobilization region was found to represent a previously unclassified Dtr type in rhizobia (hereafter type-IV), highly ubiquitous in S. meliloti and found in other genera of Gram-negative bacteria as well; including Agrobacterium, Ochrobactrum, and Chelativorans. The oriT of the type-IV Dtr described here could be located by function within a DNA fragment of 278 bp, between the divergent genes parA and mobC. The phylogenetic analysis of the cognate relaxase MobZ indicated that this protein groups close to the previously defined MOB(P3) and MOB(P4) type of enzymes, but is located in a separate and novel cluster that we have designated MOB(P0). Noteworthy, MOB(P0) and MOB(P4) relaxases were frequently associated with plasmids present in rhizospheric soil bacteria. A comparison of the nod-gene locations with the phylogenetic topology of the rhizobial relaxases revealed that the symbiotic genes are found on diverse plasmids bearing any of the four Dtr types, thus indicating that pSym plasmids are not specifically associated with any particular mobilization system. Finally, we demonstrated that the type-IV Dtr promoted the mobilization of plasmids from S. meliloti to Sinorhizobium medicae as well as from these rhizobia to other bacteria by means of their own

  9. Tachycardia-related channel in the scar tissue in patients with sustained monomorphic ventricular tachycardias: influence of the voltage scar definition.

    PubMed

    Arenal, Angel; del Castillo, Silvia; Gonzalez-Torrecilla, Esteban; Atienza, Felipe; Ortiz, Mercedes; Jimenez, Javier; Puchol, Alberto; García, Javier; Almendral, Jesús

    2004-10-26

    Endocardial mapping before sustained monomorphic ventricular tachycardia (SMVT) induction may reduce mapping time during tachycardia and facilitate the ablation of unmappable VT. Left ventricular electroanatomic voltage maps obtained during right ventricular apex pacing in 26 patients with chronic myocardial infarction referred for VT ablation were analyzed to identify conducting channels (CCs) inside the scar tissue. A CC was defined by the presence of a corridor of consecutive electrograms differentiated by higher voltage amplitude than the surrounding area. The effect of different levels of voltage scar definition, from 0.5 to 0.1 mV, was analyzed. Twenty-three channels were identified in 20 patients. The majority of CCs were identified when the voltage scar definition was < or =0.2 mV. Electrograms with > or =2 components were recorded more frequently at the inner than at the entrance of CCs (100% versus 75%, P< or =0.01). The activation time of the latest component was longer at the inner than at the entrance of CCs (200+/-40 versus 164+/-53 ms, P< or =0.001). Pacing from these CCs gave rise to a long-stimulus QRS interval (110+/-49 ms). Radiofrequency lesion applied to CCs suppressed the inducibility in 88% of CC-related tachycardias. During a follow-up of 17+/-11 months, 23% of the patients experienced a VT recurrence. CCs represent areas of slow conduction that can be identified in 75% of patients with SMVT. A tiered decreasing-voltage definition of the scar is critical for CC identification.

  10. A novel HURRAH protocol reveals high numbers of monomorphic MHC class II loci and two asymmetric multi-locus haplotypes in the Père David's deer.

    PubMed

    Wan, Qiu-Hong; Zhang, Pei; Ni, Xiao-Wei; Wu, Hai-Long; Chen, Yi-Yan; Kuang, Ye-Ye; Ge, Yun-Fa; Fang, Sheng-Guo

    2011-01-18

    The Père David's deer is a highly inbred, but recovered, species, making it interesting to consider their adaptive molecular evolution from an immunological perspective. Prior to this study, genomic sequencing was the only method for isolating all functional MHC genes within a certain species. Here, we report a novel protocol for isolating MHC class II loci from a species, and its use to investigate the adaptive evolution of this endangered deer at the level of multi-locus haplotypes. This protocol was designated "HURRAH" based on its various steps and used to estimate the total number of MHC class II loci. We confirmed the validity of this novel protocol in the giant panda and then used it to examine the Père David's deer. Our results revealed that the Père David's deer possesses nine MHC class II loci and therefore has more functional MHC class II loci than the eight genome-sequenced mammals for which full MHC data are currently available. This could potentially account at least in part for the strong survival ability of this species in the face of severe bottlenecking. The results from the HURRAH protocol also revealed that: (1) All of the identified MHC class II loci were monomorphic at their antigen-binding regions, although DRA was dimorphic at its cytoplasmic tail; and (2) these genes constituted two asymmetric functional MHC class II multi-locus haplotypes: DRA1*01 ∼ DRB1 ∼ DRB3 ∼ DQA1 ∼ DQB2 (H1) and DRA1*02 ∼ DRB2 ∼ DRB4 ∼ DQA2 ∼ DQB1 (H2). The latter finding indicates that the current members of the deer species have lost the powerful ancestral MHC class II haplotypes of nine or more loci, and have instead fixed two relatively weak haplotypes containing five genes. As a result, the Père David's deer are currently at risk for increased susceptibility to infectious pathogens.

  11. Leading ladies: leadership of group movements in a pair-living, co-dominant, monomorphic primate across reproductive stages and fruit availability seasons.

    PubMed

    Tecot, Stacey R; Romine, Natalie K

    2012-07-01

    For gregarious species, individuals must maintain cohesion while minimizing the costs of coordinated travel. Leaders of group movements potentially influence energy expenditure, energy intake, and predation risk for individuals in the group, which can have important fitness consequences. Models of pair-living species predict that energetic asymmetries lead to an emergent leader, with those in greater need leading. We investigated sex differences in leadership in pairs of red-bellied lemurs, Eulemur rubriventer, a monomorphic species with bisexual dispersal and no discernible hierarchy, to determine whether higher energetic requirements by adult females lead to female leadership. We collected leadership data in Ranomafana National Park, Madagascar on six groups of habituated E. rubriventer for 13 consecutive months between 2004-2005. To determine whether females led group movements more than males, we examined the difference in leadership frequencies of progressions in adult males and adult females within each group (n = 1,346 progressions). We further investigated the behavioral context (i.e. travel followed by feeding or not) and seasonal contexts (fruit availability, reproduction) of leadership. Group leadership was distributed, with different individuals leading the group at different times. However, females led significantly more than males, a pattern which was consistent in both feeding and non-feeding contexts and throughout all fruiting seasons and reproductive stages. While disparities in energetic status among the sexes may impact leadership in this species, leadership did not differ with changes in food availability or reproductive stage, and thus we were unable to determine whether female leadership might be related to changes in energetic status. Females may have higher energetic needs than males at all times, not merely seasonally, or female leadership may be unrelated to immediate energetic need. Rather, female leadership may be a legacy of female

  12. [Vector cardiographic evaluation of monomorphic ventricular tachycardia; its relation to the type of cardiopathy, vagal tonus and the prevalence of late potentials].

    PubMed

    Rosas Peralta, M; Casanova Garcés, J M; González Hermosillo, J A

    1994-01-01

    The most common cause of sudden death is malignant ventricular arrhytHmia. In order to identify the predictive value of the vectospatial evaluation in the surface electrocardiogram during a monomorphic ventricular tachycardia (MVT), and the equilibrium state of AutonomOus Nervous System (ANS), 89 patients of both sexes were studied with mean age of 47 +/- 16.2 years. They were grouped as follows: Group I included 43 patients (P), with a coronary heart disease; Group II (n = 24P) with a noncoronary myocardiopathy and Group III (n = 22P) with unknown origin MVT (Cryptogenic). Relationship between QRS configuration in the frontal plane (QRSf) during MVT episode with transverse plane, cardiac position in the chest X-rays, presence and duration of late potentials (LPs) in their two types of analysis (time domain and spectral mapping by high-resolution electrocardiogram), heart rate variability and ejection fraction by echocardiography were determined in all patients. The QRSf configuration with left bundle-branch block (LBBB) was the most common in group I, the sustained MVT (SMVT) + LBBB was associated with both prevalence and duration of late potentials (p = 0.005), low-rate heart variability and ejection fraction < 40%. SMVT + LBBB was the most common type in group III and if it has shown and inferior axis, an elevated rate of LPs (+) was seen. Situation that oriented to an arrhythmogenic right ventricular dysplasia. Low amplitude signals with short duration in the time domain were seen in group I with LBBB; and with SMVT + RBBB in group II. We suggest that vectospatial evaluation of QRSf during a MVT is a greater importance in the risk stratification for sudden death and it can guide to anatomic origin and the diagnosis-therapeutic approach.

  13. Magnetic Bacteria.

    ERIC Educational Resources Information Center

    Nelson, Jane Bray; Nelson, Jim

    1992-01-01

    Describes the history of Richard Blakemore's discovery of magnetotaxic organisms. Discusses possible reasons why the magnetic response in bacteria developed. Proposes research experiments integrating biology and physics in which students investigate problems using cultures of magnetotaxic organisms. (MDH)

  14. Design and Assembly of DNA Sequence Libraries for Chromosomal Insertion in Bacteria Based on a Set of Modified MoClo Vectors.

    PubMed

    Schindler, Daniel; Milbredt, Sarah; Sperlea, Theodor; Waldminghaus, Torsten

    2016-12-16

    Efficient assembly of large DNA constructs is a key technology in synthetic biology. One of the most popular assembly systems is the MoClo standard in which restriction and ligation of multiple fragments occurs in a one-pot reaction. The system is based on a smart vector design and type IIs restriction enzymes, which cut outside their recognition site. While the initial MoClo vectors had been developed for the assembly of multiple transcription units of plants, some derivatives of the vectors have been developed over the last years. Here we present a new set of MoClo vectors for the assembly of fragment libraries and insertion of constructs into bacterial chromosomes. The vectors are accompanied by a computer program that generates a degenerate synthetic DNA sequence that excludes "forbidden" DNA motifs. We demonstrate the usability of the new approach by construction of a stable fluorescence repressor operator system (FROS).

  15. Use of single-strand conformation polymorphism of amplified 16S rDNA for grouping of bacteria isolated from foods.

    PubMed

    Takahashi, Hajime; Kimura, Bon; Tanaka, Yuichiro; Mori, Mayumi; Yokoi, Asami; Fujii, Tateo

    2008-04-01

    The grouping method for isolated strains from foods using single-strand conformation polymorphism (SSCP) after PCR amplification of a portion of 16S rDNA was developed. This method was able to group the strains from various food samples based on 16S rDNA sequence. As 97.8% of the isolated strains from various foods were grouped correctly, use of the PCR-SSCP method enables the prompt and labor-saving analysis of microbial population of food-derived bacterial strains. Advantages in speed and accuracy of bacterial population identification by the PCR-SSCP method have practical application for food suppliers and testing laboratories.

  16. Interactive and individual effects of dietary non-digestible carbohydrates and oils on DNA damage, SCFA and bacteria in the large bowel of rats.

    PubMed

    Conlon, Michael A; Bird, Anthony R

    2009-04-01

    Dietary non-digestible carbohydrates (NDC) play an important role in large-bowel health and one form of NDC, resistant starch (RS), can promote low levels of DNA damage and other markers of colonic health. The objective of the present study was to determine whether the ability of dietary RS or other NDC to influence colonic health, particularly DNA damage, is dependent on the type of dietary oil. We compared the effects of diets containing 10 % of NDC from cellulose, wheat bran, high-amylose maize starch (HAS, a rich source of RS type 2) or a retrograded HAS (RHAS, a rich source of RS type 3) on DNA damage, SCFA production and bacterial changes in the large bowel of rats. Each carbohydrate source was combined with 10 % fish oil (FO) or Sunola oil (SO; rich in oleic acid). There was a significant interaction between NDC and oil treatments on single-strand DNA breaks in colonocytes isolated from the colon. The damage in rats consuming RHAS was greater for FO consumption than for SO consumption. There was a significant interaction between NDC and oils on caecum weights and treatment effects of NDC and oils were observed for the weights and lengths of other gut tissues. Significant differences were found in colonic SCFA pools and caecal numbers of lactobacilli, bifidobacteria, Escherichia coli and Bacteroides fragilis with the various NDC and oil treatments. The present results demonstrate that the effects of NDC and oils, particularly on colonic DNA damage, can depend on how they are combined within the diet.

  17. Methanotrophic bacteria.

    PubMed Central

    Hanson, R S; Hanson, T E

    1996-01-01

    Methane-utilizing bacteria (methanotrophs) are a diverse group of gram-negative bacteria that are related to other members of the Proteobacteria. These bacteria are classified into three groups based on the pathways used for assimilation of formaldehyde, the major source of cell carbon, and other physiological and morphological features. The type I and type X methanotrophs are found within the gamma subdivision of the Proteobacteria and employ the ribulose monophosphate pathway for formaldehyde assimilation, whereas type II methanotrophs, which employ the serine pathway for formaldehyde assimilation, form a coherent cluster within the beta subdivision of the Proteobacteria. Methanotrophic bacteria are ubiquitous. The growth of type II bacteria appears to be favored in environments that contain relatively high levels of methane, low levels of dissolved oxygen, and limiting concentrations of combined nitrogen and/or copper. Type I methanotrophs appear to be dominant in environments in which methane is limiting and combined nitrogen and copper levels are relatively high. These bacteria serve as biofilters for the oxidation of methane produced in anaerobic environments, and when oxygen is present in soils, atmospheric methane is oxidized. Their activities in nature are greatly influenced by agricultural practices and other human activities. Recent evidence indicates that naturally occurring, uncultured methanotrophs represent new genera. Methanotrophs that are capable of oxidizing methane at atmospheric levels exhibit methane oxidation kinetics different from those of methanotrophs available in pure cultures. A limited number of methanotrophs have the genetic capacity to synthesize a soluble methane monooxygenase which catalyzes the rapid oxidation of environmental pollutants including trichloroethylene. PMID:8801441

  18. Studies of the repair of O6-alkylguanine and O4-alkylthymine in DNA by alkyltransferases from mammalian cells and bacteria.

    PubMed Central

    Pegg, A E; Dolan, M E; Scicchitano, D; Morimoto, K

    1985-01-01

    O6-Methylguanine in DNA is repaired by the action of a protein termed O6-alkylguanine-DNA alkyltransferase (AT) which transfers the methyl group to a cysteine residue in its own sequence. Since the cysteine which is methylated is not regenerated rapidly, if at all, the capacity for repair of O6-methylguanine is limited by the number of molecules of the AT available within the cell. The level and inducibility of the AT differed greatly in different mammalian cell types and species with the highest levels in human tissues and in liver and the lowest levels in brain. Only a small induction occurred in rat liver in response to exposure to alkylating agents. In E. coli such exposure increased the activity more than 100-fold. The At was not specific for methyl groups but also removed ethyl, 2-hydroxyethyl, n-propyl, isopropyl and n-butyl groups from the O6-position in DNA. The protein isolated from E. coli removed methyl groups much more rapidly than the larger alkyl groups but the mammalian AT isolated from rat liver showed much less difference in rate with adducts of different size. Ethyl and n-propyl groups were removed by the rat liver AT only three to four times more slowly than methyl groups. Another important difference between the bacterial and mammalian ATs is that the bacterial protein was also able to remove methyl groups from the O4-position of thymine in methylated DNA or poly(dT) but the AT from rat liver or human fibroblasts did not repair O4-methylthymidine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3910413

  19. Precise Identification of Genome-Wide Transcription Start Sites in Bacteria by 5'-Rapid Amplification of cDNA Ends (5'-RACE).

    PubMed

    Matteau, Dominick; Rodrigue, Sébastien

    2015-01-01

    Transcription start sites are commonly used to locate promoter elements in bacterial genomes. TSS were previously studied one gene at a time, often through 5'-rapid amplification of cDNA ends (5'-RACE). This technique has now been adapted for high-throughput sequencing and can be used to precisely identify TSS in a genome-wide fashion for practically any bacterium, which greatly contributes to our understanding of gene regulatory networks in microorganisms.

  20. Studies on DNA binding behaviour of biologically active transition metal complexes of new tetradentate N2O2 donor Schiff bases: Inhibitory activity against bacteria

    NASA Astrophysics Data System (ADS)

    Sobha, S.; Mahalakshmi, R.; Raman, N.

    A series of Cu(II), Ni(II) and Zn(II) complexes of the type ML have been synthesized with Schiff bases derived from o-acetoacetotoluidide, 2-hydroxybenzaldehyde and o-phenylenediamine/1,4-diaminobutane. The complexes are insoluble in common organic solvents but soluble in DMF and DMSO. The measured molar conductance values in DMSO indicate that the complexes are non-electrolytic in nature. All the six metal complexes have been fully characterized with the help of elemental analyses, molecular weights, molar conductance values, magnetic moments and spectroscopic data. The analytical data helped to elucidate the structure of the metal complexes. The Schiff bases are found to act as tetradentate ligands using N2O2 donor set of atoms leading to a square-planar geometry for the complexes around all the metal ions. The binding properties of metal complexes with DNA were investigated by absorption spectra, viscosity measurements and cyclic voltammetry. Detailed analysis reveals that the metal complexes intercalate into the DNA base stack as intercalators. All the metal complexes cleave the pUC19 DNA in presence of H2O2. The Schiff bases and their complexes have been screened for their antibacterial activity against five bacterial strains (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, Klebsiella pneumoniae) by disk diffusion method. All the metal complexes have potent biocidal activity than the free ligands.

  1. The 3'-5' exonuclease site of DNA polymerase III from gram-positive bacteria: definition of a novel motif structure.

    PubMed

    Barnes, M H; Spacciapoli, P; Li, D H; Brown, N C

    1995-11-07

    The primary structure of the 3'-5' exonuclease (Exo) site of the Gram+ bacterial DNA polymerase III (Pol III) was examined by site-directed mutagenesis of Bacillus subtilis Pol III (BsPol III). It was found to differ significantly from the conventional three-motif substructure established for the Exo site of DNA polymerase I of Escherichia coli (EcPol I) and the majority of other DNA polymerase-exonucleases. Motifs I and II were conventionally organized and anchored functionally by the predicted carboxylate residues. However, the conventional downstream motif, motif III, was replaced by motif III epsilon, a novel 55-amino-acid (aa) segment incorporating three essential aa (His565, Asp533 and Asp570) which are strictly conserved in three Gram+ Pol III and in the Ec Exo epsilon (epsilon). Despite its unique substructure, the Gram+ Pol III-specific Exo site was conventionally independent of Pol, the site of 2'-deoxyribonucleoside 5-triphosphate (dNTP) binding and polymerization. The entire Exo site, including motif III epsilon, could be deleted without profoundly affecting the enzyme's capacity to polymerize dNTPs. Conversely, Pol and all other sequences downstream of the Exo site could be deleted with little apparent effect on Exo activity. Whether the three essential aa within the unique motif III epsilon substructure participate in the conventional two-metal-ion mechanism elucidated for the model Exo site of EcPol I, remains to be established.

  2. Studies on DNA binding behaviour of biologically active transition metal complexes of new tetradentate N2O2 donor Schiff bases: inhibitory activity against bacteria.

    PubMed

    Sobha, S; Mahalakshmi, R; Raman, N

    2012-06-15

    A series of Cu(II), Ni(II) and Zn(II) complexes of the type ML have been synthesized with Schiff bases derived from o-acetoacetotoluidide, 2-hydroxybenzaldehyde and o-phenylenediamine/1,4-diaminobutane. The complexes are insoluble in common organic solvents but soluble in DMF and DMSO. The measured molar conductance values in DMSO indicate that the complexes are non-electrolytic in nature. All the six metal complexes have been fully characterized with the help of elemental analyses, molecular weights, molar conductance values, magnetic moments and spectroscopic data. The analytical data helped to elucidate the structure of the metal complexes. The Schiff bases are found to act as tetradentate ligands using N(2)O(2) donor set of atoms leading to a square-planar geometry for the complexes around all the metal ions. The binding properties of metal complexes with DNA were investigated by absorption spectra, viscosity measurements and cyclic voltammetry. Detailed analysis reveals that the metal complexes intercalate into the DNA base stack as intercalators. All the metal complexes cleave the pUC19 DNA in presence of H(2)O(2.) The Schiff bases and their complexes have been screened for their antibacterial activity against five bacterial strains (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, Klebsiella pneumoniae) by disk diffusion method. All the metal complexes have potent biocidal activity than the free ligands.

  3. Genomics of oral bacteria.

    PubMed

    Duncan, Margaret J

    2003-01-01

    Advances in bacterial genetics came with the discovery of the genetic code, followed by the development of recombinant DNA technologies. Now the field is undergoing a new revolution because of investigators' ability to sequence and assemble complete bacterial genomes. Over 200 genome projects have been completed or are in progress, and the oral microbiology research community has benefited through projects for oral bacteria and their non-oral-pathogen relatives. This review describes features of several oral bacterial genomes, and emphasizes the themes of species relationships, comparative genomics, and lateral gene transfer. Genomics is having a broad impact on basic research in microbial pathogenesis, and will lead to new approaches in clinical research and therapeutics. The oral microbiota is a unique community especially suited for new challenges to sequence the metagenomes of microbial consortia, and the genomes of uncultivable bacteria.

  4. Phylogenetic analysis and possible function of bro-like genes, a multigene family widespread among large double-stranded DNA viruses of invertebrates and bacteria.

    PubMed

    Bideshi, Dennis K; Renault, Sylvaine; Stasiak, Karine; Federici, Brian A; Bigot, Yves

    2003-09-01

    Baculovirus repeated open reading frame (bro) genes and their relatives constitute a multigene family, typically with multiple copies per genome, known to occur among certain insect dsDNA viruses and bacteriophages. Little is known about the evolutionary history and function of the proteins encoded by these genes. Here we have shown that bro and bro-like (bro-l) genes occur among viruses of two additional invertebrate viral families, Ascoviridae and Iridoviridae, and in prokaryotic class II transposons. Analysis of over 100 sequences showed that the N-terminal region, consisting of two subdomains, is the most conserved region and contains a DNA-binding motif that has been characterized previously. Phylogenetic analysis indicated that these proteins are distributed among eight groups, Groups 1-7 consisting of invertebrate virus proteins and Group 8 of proteins in bacteriophages and bacterial transposons. No bro genes were identified in databases of invertebrate or vertebrate genomes, vertebrate viruses and transposons, nor in prokaryotic genomes, except in prophages or transposons of the latter. The phylogenetic relationship between bro genes suggests that they have resulted from recombination of viral genomes that allowed the duplication and loss of genes, but also the acquisition of genes by horizontal transfer over evolutionary time. In addition, the maintenance and diversity of bro-l genes in different types of invertebrate dsDNA viruses, but not in vertebrate viruses, suggests that these proteins play an important role in invertebrate virus biology. Experiments with the unique orf2 bro gene of Autographa californica multicapsid nucleopolyhedrovirus showed that it is not required for replication, but may enhance replication during the occlusion phase of reproduction.

  5. Molecular cloning and expression in photosynthetic bacteria of a soybean cDNA coding for phytoene desaturase, an enzyme of the carotenoid biosynthesis pathway.

    PubMed Central

    Bartley, G E; Viitanen, P V; Pecker, I; Chamovitz, D; Hirschberg, J; Scolnik, P A

    1991-01-01

    Carotenoids are orange, yellow, or red photo-protective pigments present in all plastids. The first carotenoid of the pathway is phytoene, a colorless compound that is converted into colored carotenoids through a series of desaturation reactions. Genes coding for carotenoid desaturases have been cloned from microbes but not from plants. We report the cloning of a cDNA for pds1, a soybean (Glycine max) gene that, based on a complementation assay using the photosynthetic bacterium Rhodobacter capsulatus, codes for an enzyme that catalyzes the two desaturation reactions that convert phytoene into zeta-carotene, a yellow carotenoid. The 2281-base-pair cDNA clone analyzed contains an open reading frame with the capacity to code for a 572-residue protein of predicted Mr 63,851. Alignment of the deduced Pds1 peptide sequence with the sequences of fungal and bacterial carotenoid desaturases revealed conservation of several amino acid residues, including a dinucleotide-binding motif that could mediate binding to FAD. The Pds1 protein is synthesized in vitro as a precursor that, upon import into isolated chloroplasts, is processed to a smaller mature form. Hybridization of the pds1 cDNA to genomic blots indicated that this gene is a member of a low-copy-number gene family. One of these loci was genetically mapped using restriction fragment length polymorphisms between Glycine max and Glycine soja. We conclude that pds1 is a nuclear gene encoding a phytoene desaturase enzyme that, as its microbial counterparts, contains sequence motifs characteristic of flavoproteins. Images PMID:1862081

  6. Selection of indicator bacteria based on screening of 16S rDNA metagenomic library from a two-stage anoxic-oxic bioreactor system degrading azo dyes.

    PubMed

    Dafale, Nishant; Agrawal, Leena; Kapley, Atya; Meshram, Sudhir; Purohit, Hemant; Wate, Satish

    2010-01-01

    Dye degradation has gained attention of late due to indiscriminate disposal from user industries. Enhancing efficiency of biological treatment provides a cheaper alternative vis-à-vis other advanced technologies. Dye molecules are metabolized biologically via anoxic and oxic treatments. In this study, bacterial community surviving on dye effluent working in anoxic-oxic bioreactor was analyzed using 16S rDNA approach. Azo-dye decolorizing and degrading bacterial community was enriched in lab-scale two-stage anoxic-oxic bioreactor. 16S rDNA metagenomic libraries of enriched population were constructed, screened and phylogenetically analyzed separately. Removal of approximately 35% COD with complete decolorization was observed in anoxic bioreactor. Process was carried out by uncultured gamma proteobacterium constituting 48% of the total population and 12% clones having homology to Klebsiella. Aromatic amines generated during partial treatment under anoxic bioreactor were treated by aerobic population having 72% unculturable unidentified bacterium and rest of the population consisting of Thauera sp., Pseudoxanthomonas sp., Desulfomicrobium sp., Ottowia sp., Acidovorax sp., and Bacteriodetes bacterium sp.

  7. A New Broad Range Plasmid for DNA Delivery in Eukaryotic Cells Using Lactic Acid Bacteria: In Vitro and In Vivo Assays.

    PubMed

    Mancha-Agresti, Pamela; Drumond, Mariana Martins; Carmo, Fillipe Luiz Rosa do; Santos, Monica Morais; Santos, Janete Soares Coelho Dos; Venanzi, Franco; Chatel, Jean-Marc; Leclercq, Sophie Yvette; Azevedo, Vasco

    2017-03-17

    Lactococcus lactis is well documented as a promising candidate for development of novel oral live vaccines. It has been broadly engineered for heterologous expression, as well as for plasmid expression vector delivery, directly inside eukaryotic cells, for DNA vaccine, or as therapeutic vehicle. This work describes the characteristics of a new plasmid, pExu (extra chromosomal unit), for DNA delivery using L. lactis and evaluates its functionality both by in vitro and in vivo assays. This plasmid exhibits the following features: (1) a theta origin of replication and (2) an expression cassette containing a multiple cloning site and a eukaryotic promoter, the cytomegalovirus (pCMV). The functionality of pExu:egfp was evaluated by fluorescence microscopy. The L. lactis MG1363 (pExu:egfp) strains were administered by gavage to Balb/C mice and the eGFP expression was monitored by fluorescence microscopy. The pExu vector has demonstrated an excellent stability either in L. lactis or in Escherichia coli. The eGFP expression at different times in in vitro assay showed that 15.8% of CHO cells were able to express the protein after transfection. The enterocytes of mice showed the expression of eGFP protein. Thus, L. lactis carrying the pExu is a good candidate to deliver genes into eukaryotic cells.

  8. Evaluation of direct 16S rDNA sequencing as a metagenomics-based approach to screening bacteria in bottled water.

    PubMed

    Hansen, Trine; Skånseng, Beate; Hoorfar, Jeffrey; Löfström, Charlotta

    2013-09-01

    Deliberate or accidental contamination of food, feed, and water supplies poses a threat to human health worldwide. A rapid and sensitive detection technique that could replace the current labor-intensive and time-consuming culture-based methods is highly desirable. In addition to species-specific assays, such as PCR, there is a need for generic methods to screen for unknown pathogenic microorganisms in samples. This work presents a metagenomics-based direct-sequencing approach for detecting unknown microorganisms, using Bacillus cereus (as a model organism for B. anthracis) in bottled water as an example. Total DNA extraction and 16S rDNA gene sequencing were used in combination with principle component analysis and multicurve resolution to study detection level and possibility for identification. Results showed a detection level of 10(5) to 10(6) CFU/L. Using this method, it was possible to separate 2 B. cereus strains by the principal component plot, despite the close sequence resemblance. A linear correlation between the artificial contamination level and the relative amount of the Bacillus artificial contaminant in the metagenome was observed, and a relative amount value above 0.5 confirmed the presence of Bacillus. The analysis also revealed that background flora in the bottled water varied between the different water types that were included in the study. This method has the potential to be adapted to other biological matrices and bacterial pathogens for fast screening of unknown bacterial threats in outbreak situations.

  9. Transformation of gram positive bacteria by sonoporation

    DOEpatents

    Yang, Yunfeng; Li, Yongchao

    2014-03-11

    The present invention provides a sonoporation-based method that can be universally applied for delivery of compounds into Gram positive bacteria. Gram positive bacteria which can be transformed by sonoporation include, for example, Bacillus, Streptococcus, Acetobacterium, and Clostridium. Compounds which can be delivered into Gram positive bacteria via sonoporation include nucleic acids (DNA or RNA), proteins, lipids, carbohydrates, viruses, small organic and inorganic molecules, and nano-particles.

  10. Bacteria Counter

    NASA Technical Reports Server (NTRS)

    1981-01-01

    Science Applications, Inc.'s ATP Photometer makes a rapid and accurate count of the bacteria in a body fluid sample. Instrument provides information on the presence and quantity of bacteria by measuring the amount of light emitted by the reaction between two substances. Substances are ATP adenosine triphosphate and luciferase. The reactants are applied to a human body sample and the ATP Photometer observes the intensity of the light emitted displaying its findings in a numerical output. Total time lapse is usually less than 10 minutes, which represents a significant time savings in comparison of other techniques. Other applications are measuring organisms in fresh and ocean waters, determining bacterial contamination of foodstuffs, biological process control in the beverage industry, and in assay of activated sewage sludge.

  11. Evolutionary relationships of lactate dehydrogenases (LDHs) from mammals, birds, an amphibian, fish, barley, and bacteria: LDH cDNA sequences from Xenopus, pig, and rat.

    PubMed

    Tsuji, S; Qureshi, M A; Hou, E W; Fitch, W M; Li, S S

    1994-09-27

    The nucleotide sequences of the cDNAs encoding LDH (EC 1.1.1.27) subunits LDH-A (muscle), LDH-B (liver), and LDH-C (oocyte) from Xenopus laevis, LDH-A (muscle) and LDH-B (heart) from pig, and LDH-B (heart) and LDH-C (testis) from rat were determined. These seven newly deduced amino acid sequences and 22 other published LDH sequences, and three unpublished fish LDH-A sequences kindly provided by G. N. Somero and D. A. Powers, were used to construct the most parsimonious phylogenetic tree of these 32 LDH subunits from mammals, birds, an amphibian, fish, barley, and bacteria. There have been at least six LDH gene duplications among the vertebrates. The Xenopus LDH-A, LDH-B, and LDH-C subunits are most closely related to each other and then are more closely related to vertebrate LDH-B than LDH-A. Three fish LDH-As, as well as a single LDH of lamprey, also seem to be more related to vertebrate LDH-B than to land vertebrate LDH-A. The mammalian LDH-C (testis) subunit appears to have diverged very early, prior to the divergence of vertebrate LDH-A and LDH-B subunits, as reported previously.

  12. Evidence of DNA double strand breaks formation in Escherichia coli bacteria exposed to alpha particles of different LET assessed by the SOS response.

    PubMed

    Serment-Guerrero, Jorge; Breña-Valle, Matilde; Aguilar-Moreno, Magdalena; Balcázar, Miguel

    2012-12-01

    Ionizing radiation produces a plethora of lesion upon DNA which sometimes is generated among a relatively small region due to clustered energy deposition events, the so called locally multiply damaged sites that could change to DSB. Such clustered damages are more likely to occur in high LET radiation exposures. The effect of alpha particles of different LET was evaluated on the bacterium Escherichia coli either by survival properties or the SOS response activity. Alpha radiation and LET distribution was controlled by means of Nuclear Track Detectors. The results suggest that alpha particles produce two types of lesion: lethal lesions and SOS inducing-mutagenic, a proportion that varies depending on the LET values. The SOS response as a sensitive parameter to assess RBE is mentioned.

  13. Identification of Nitrogen-Incorporating Bacteria in Petroleum-Contaminated Arctic Soils by Using [15N]DNA-Based Stable Isotope Probing and Pyrosequencing ▿ †

    PubMed Central

    Bell, Terrence H.; Yergeau, Etienne; Martineau, Christine; Juck, David; Whyte, Lyle G.; Greer, Charles W.

    2011-01-01

    Arctic soils are increasingly susceptible to petroleum hydrocarbon contamination, as exploration and exploitation of the Arctic increase. Bioremediation in these soils is challenging due to logistical constraints and because soil temperatures only rise above 0°C for ∼2 months each year. Nitrogen is often added to contaminated soil in situ to stimulate the existing microbial community, but little is known about how the added nutrients are used by these microorganisms. Microbes vary widely in their ability to metabolize petroleum hydrocarbons, so the question becomes: which hydrocarbon-degrading microorganisms most effectively use this added nitrogen for growth? Using [15N]DNA-based stable isotope probing, we determined which taxonomic groups most readily incorporated nitrogen from the monoammonium phosphate added to contaminated and uncontaminated soil in Canadian Forces Station-Alert, Nunavut, Canada. Fractions from each sample were amplified with bacterial 16S rRNA and alkane monooxygenase B (alkB) gene-specific primers and then sequenced using lage-scale parallel-pyrosequencing. Sequence data was combined with 16S rRNA and alkB gene C quantitative PCR data to measure the presence of various phylogenetic groups in fractions at different buoyant densities. Several families of Proteobacteria and Actinobacteria that are directly involved in petroleum degradation incorporated the added nitrogen in contaminated soils, but it was the DNA of Sphingomonadaceae that was most enriched in 15N. Bacterial growth in uncontaminated soils was not stimulated by nutrient amendment. Our results suggest that nitrogen uptake efficiency differs between bacterial groups in contaminated soils. A better understanding of how groups of hydrocarbon-degraders contribute to the catabolism of petroleum will facilitate the design of more targeted bioremediation treatments. PMID:21498745

  14. The detection of microbial DNA but not cultured bacteria is associated with increased mortality in patients with suspected sepsis-a prospective multi-centre European observational study.

    PubMed

    O'Dwyer, M J; Starczewska, M H; Schrenzel, J; Zacharowski, K; Ecker, D J; Sampath, R; Brealey, D; Singer, M; Libert, N; Wilks, M; Vincent, J-L

    2017-03-01

    Blood culture results inadequately stratify the mortality risk in critically ill patients with sepsis. We sought to establish the prognostic significance of the presence of microbial DNA in the bloodstream of patients hospitalized with suspected sepsis. We analysed the data collected during the Rapid Diagnosis of Infections in the Critically Ill (RADICAL) study, which compared a novel culture-independent PCR/electrospray ionization-mass spectrometry (ESI-MS) assay with standard microbiological testing. Patients were eligible for the study if they had suspected sepsis and were either hospitalized or were referred to one of nine intensive care units from six European countries. The blood specimen for PCR/ESI-MS assay was taken along with initial blood culture taken for clinical indications. Of the 616 patients recruited to the RADICAL study, 439 patients had data on outcome, results of the blood culture and PCR/ESI-MS assay available for analysis. Positive blood culture and PCR/ESI-MSI result was found in 13% (56/439) and 40% (177/439) of patients, respectively. Either a positive blood culture (p 0.01) or a positive PCR/ESI-MS (p 0.005) was associated with higher SOFA scores on enrolment to the study. There was no difference in 28-day mortality observed in patients who had either positive or negative blood cultures (35% versus 32%, p 0.74). However, in patients with a positive PCR/ESI-MS assay, mortality was significantly higher in comparison to those with a negative result (42% versus 26%, p 0.001). Presence of microbial DNA in patients with suspected sepsis might define a patient group at higher risk of death. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  15. Trans-kingdom horizontal DNA transfer from bacteria to yeast is highly plastic due to natural polymorphisms in auxiliary nonessential recipient genes.

    PubMed

    Moriguchi, Kazuki; Yamamoto, Shinji; Tanaka, Katsuyuki; Kurata, Nori; Suzuki, Katsunori

    2013-01-01

    With the rapid accumulation of genomic information from various eukaryotes in the last decade, genes proposed to have been derived from recent horizontal gene transfer (HGT) events have been reported even in non-phagotrophic unicellular and multicellular organisms, but the molecular pathways underlying HGT remain to be explained. The development of in vitro HGT detection systems, which permit the molecular and genetic analyses of donor and recipient organisms and quantify HGT, are helpful in order to gain insight into mechanisms that may contribute to contemporary HGT events or may have contributed to past HGT events. We applied a horizontal DNA transfer system model based on conjugal gene transfer called trans-kingdom conjugation (TKC) from the prokaryote Escherichia coli to the eukaryote Saccharomyces cerevisiae, and assessed whether and to what extent genetic variations in the eukaryotic recipient affect its receptivity to TKC. Strains from a collection of 4,823 knock-out mutants of S. cerevisiae MAT-α haploids were tested for their individual TKC receptivity. Two types of mutants, an ssd1 mutant and respiratory mutants, which are also found in experimental strains and in nature widely, were identified as highly receptive mutants. The TKC efficiency for spontaneously accrued petite (rho (-/0)) mutants of the functional allele (SSD1-V) strain showed increased receptivity. The TKC efficiency of the ssd1Δ mutant was 36% for bacterial conjugation, while that of the petite/ssd1Δ double mutants was even higher (220% in average) compared to bacterial conjugation. This increased TKC receptivity was also observed when other conjugal transfer systems were applied and the donor bacterium was changed to Agrobacterium tumefaciens. These results support the idea that the genomes of certain eukaryotes have been exposed to exogenous DNA more frequently and continuously than previously thought.

  16. Trans-Kingdom Horizontal DNA Transfer from Bacteria to Yeast Is Highly Plastic Due to Natural Polymorphisms in Auxiliary Nonessential Recipient Genes

    PubMed Central

    Moriguchi, Kazuki; Yamamoto, Shinji; Tanaka, Katsuyuki; Kurata, Nori; Suzuki, Katsunori

    2013-01-01

    With the rapid accumulation of genomic information from various eukaryotes in the last decade, genes proposed to have been derived from recent horizontal gene transfer (HGT) events have been reported even in non-phagotrophic unicellular and multicellular organisms, but the molecular pathways underlying HGT remain to be explained. The development of in vitro HGT detection systems, which permit the molecular and genetic analyses of donor and recipient organisms and quantify HGT, are helpful in order to gain insight into mechanisms that may contribute to contemporary HGT events or may have contributed to past HGT events. We applied a horizontal DNA transfer system model based on conjugal gene transfer called trans-kingdom conjugation (TKC) from the prokaryote Escherichia coli to the eukaryote Saccharomyces cerevisiae, and assessed whether and to what extent genetic variations in the eukaryotic recipient affect its receptivity to TKC. Strains from a collection of 4,823 knock-out mutants of S. cerevisiae MAT-α haploids were tested for their individual TKC receptivity. Two types of mutants, an ssd1 mutant and respiratory mutants, which are also found in experimental strains and in nature widely, were identified as highly receptive mutants. The TKC efficiency for spontaneously accrued petite (rho−/0) mutants of the functional allele (SSD1-V) strain showed increased receptivity. The TKC efficiency of the ssd1Δ mutant was 36% for bacterial conjugation, while that of the petite/ssd1Δ double mutants was even higher (220% in average) compared to bacterial conjugation. This increased TKC receptivity was also observed when other conjugal transfer systems were applied and the donor bacterium was changed to Agrobacterium tumefaciens. These results support the idea that the genomes of certain eukaryotes have been exposed to exogenous DNA more frequently and continuously than previously thought. PMID:24058593

  17. Identification of nitrogen-incorporating bacteria in petroleum-contaminated arctic soils by using [15N]DNA-based stable isotope probing and pyrosequencing.

    PubMed

    Bell, Terrence H; Yergeau, Etienne; Martineau, Christine; Juck, David; Whyte, Lyle G; Greer, Charles W

    2011-06-01

    Arctic soils are increasingly susceptible to petroleum hydrocarbon contamination, as exploration and exploitation of the Arctic increase. Bioremediation in these soils is challenging due to logistical constraints and because soil temperatures only rise above 0°C for ∼2 months each year. Nitrogen is often added to contaminated soil in situ to stimulate the existing microbial community, but little is known about how the added nutrients are used by these microorganisms. Microbes vary widely in their ability to metabolize petroleum hydrocarbons, so the question becomes: which hydrocarbon-degrading microorganisms most effectively use this added nitrogen for growth? Using [(15)N]DNA-based stable isotope probing, we determined which taxonomic groups most readily incorporated nitrogen from the monoammonium phosphate added to contaminated and uncontaminated soil in Canadian Forces Station-Alert, Nunavut, Canada. Fractions from each sample were amplified with bacterial 16S rRNA and alkane monooxygenase B (alkB) gene-specific primers and then sequenced using large-scale parallel-pyrosequencing. Sequence data was combined with 16S rRNA and alkB gene C quantitative PCR data to measure the presence of various phylogenetic groups in fractions at different buoyant densities. Several families of Proteobacteria and Actinobacteria that are directly involved in petroleum degradation incorporated the added nitrogen in contaminated soils, but it was the DNA of Sphingomonadaceae that was most enriched in (15)N. Bacterial growth in uncontaminated soils was not stimulated by nutrient amendment. Our results suggest that nitrogen uptake efficiency differs between bacterial groups in contaminated soils. A better understanding of how groups of hydrocarbon-degraders contribute to the catabolism of petroleum will facilitate the design of more targeted bioremediation treatments.

  18. Replication Restart in Bacteria.

    PubMed

    Michel, Bénédicte; Sandler, Steven J

    2017-07-01

    In bacteria, replication forks assembled at a replication origin travel to the terminus, often a few megabases away. They may encounter obstacles that trigger replisome disassembly, rendering replication restart from abandoned forks crucial for cell viability. During the past 25 years, the genes that encode replication restart proteins have been identified and genetically characterized. In parallel, the enzymes were purified and analyzed in vitro, where they can catalyze replication initiation in a sequence-independent manner from fork-like DNA structures. This work also revealed a close link between replication and homologous recombination, as replication restart from recombination intermediates is an essential step of DNA double-strand break repair in bacteria and, conversely, arrested replication forks can be acted upon by recombination proteins and converted into various recombination substrates. In this review, we summarize this intense period of research that led to the characterization of the ubiquitous replication restart protein PriA and its partners, to the definition of several replication restart pathways in vivo, and to the description of tight links between replication and homologous recombination, responsible for the importance of replication restart in the maintenance of genome stability. Copyright © 2017 American Society for Microbiology.

  19. Mechanism of copper surface toxicity in Escherichia coli O157:H7 and Salmonella involves immediate membrane depolarization followed by slower rate of DNA destruction which differs from that observed for Gram-positive bacteria.

    PubMed

    Warnes, S L; Caves, V; Keevil, C W

    2012-07-01

    We have reported previously that copper I and II ionic species, and superoxide but not Fenton reaction generated hydroxyl radicals, are important in the killing mechanism of pathogenic enterococci on copper surfaces. In this new work we determined if the mechanism was the same in non-pathogenic ancestral (K12) and laboratory (DH5α) strains, and a pathogenic strain (O157), of Escherichia coli. The pathogenic strain exhibited prolonged survival on stainless steel surfaces compared with the other E. coli strains but all died within 10 min on copper surfaces using a 'dry' inoculum protocol (with approximately 10(7)  cfu cm(-2) ) to mimic dry touch contamination. We observed immediate cytoplasmic membrane depolarization, not seen with enterococci or methicillin resistant Staphylococcus aureus, and loss of outer membrane integrity, inhibition of respiration and in situ generation of reactive oxygen species on copper and copper alloy surfaces that did not occur on stainless steel. Chelation of copper (I) and (II) ionic species still had the most significant impact on bacterial survival but protection by d-mannitol suggests hydroxyl radicals are involved in the killing mechanism. We also observed a much slower rate of DNA destruction on copper surfaces compared with previous results for enterococci. This may be due to protection of the nucleic acid by the periplasm and the extensive cell aggregation that we observed on copper surfaces. Similar results were obtained for Salmonella species but partial quenching by d-mannitol suggests radicals other than hydroxyl may be involved. The results indicate that copper biocidal surfaces are effective for Gram-positive and Gram-negative bacteria but bacterial morphology affects the mechanism of toxicity. These surfaces could not only help to prevent infection spread but also prevent horizontal gene transmission which is responsible for the evolution of virulent toxin producing and antibiotic resistant bacteria.

  20. Identification of biomass utilizing bacteria in a carbon-depleted glacier forefield soil by the use of 13C DNA stable isotope probing.

    PubMed

    Zumsteg, Anita; Schmutz, Stefan; Frey, Beat

    2013-06-01

    As Alpine glaciers are retreating rapidly, bare soils with low organic C and N contents are becoming exposed. Carbon availability is a key factor regulating microbial diversity and ecosystem functioning in these soils. The aim of this study was to investigate how bacterial activity, community structure and composition are influenced by organic carbon availability. Bare soils were supplied with (13)C-labelled fungal (Penicillium sp.) and green algal (Chlorella sp.) biomass and the CO2 evolution and its δ(13)C signature were monitored up to 60 days. These organisms have previously been isolated near the glacier terminus. DNA stable isotope probing followed by T-RFLP profiling and sequencing of 16S rRNA genes was employed to identify consumers able to assimilate carbon from these biomass amendments. Higher respiration and higher bacterial activity indicated a more efficient utilization of algal cells than fungal cells. Flavobacterium sp. predominantly incorporated fungal-derived C, whereas the algal-derived C was mainly incorporated by Acidobacteria and Proteobacteria. This study emphasizes the important role of both fungal and algal biomass in increasing the carbon pool in recently deglaciated bare soils, as only 20% of the added C was respired as CO2, and the rest, we presume, remained in the soil.

  1. Deterioration to extinction of wastewater bacteria by non-thermal atmospheric pressure air plasma as assessed by 16S rDNA-DGGE fingerprinting

    PubMed Central

    El-Sayed, Wael S.; Ouf, Salama A.; Mohamed, Abdel-Aleam H.

    2015-01-01

    The use of cold plasma jets for inactivation of a variety of microorganisms has recently been evaluated via culture-based methods. Accordingly, elucidation of the role of cold plasma in decontamination would be inaccurate because most microbial populations within a system remain unexplored owing to the high amount of yet uncultured bacteria. The impact of cold atmospheric plasma on the bacterial community structure of wastewater from two different industries was investigated by metagenomic-based polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) utilizing 16S rRNA genes. Three doses of atmospheric pressure dielectric barrier discharge plasma were applied to wastewater samples on different time scales. DGGE revealed that the bacterial community gradually changed and overall abundance decreased to extinction upon plasma treatment. The bacterial community in food processing wastewater contained 11 key operational taxonomic units that remained almost completely unchanged when exposed to plasma irradiation at 75.5 mA for 30 or 60 s. However, when exposure time was extended to 90 s, only Escherichia coli, Coliforms, Aeromonas sp., Vibrio sp., and Pseudomonas putida survived. Only E. coli, Aeromonas sp., Vibrio sp., and P. putida survived treatment at 81.94 mA for 90 s. Conversely, all bacterial groups were completely eliminated by treatment at 85.34 mA for either 60 or 90 s. Dominant bacterial groups in leather processing wastewater also changed greatly upon exposure to plasma at 75.5 mA for 30 or 60 s, with Enterobacter aerogenes, Klebsiella sp., Pseudomonas stutzeri, and Acidithiobacillus ferrooxidans being sensitive to and eliminated from the community. At 90 s of exposure, all groups were affected except for Pseudomonas sp. and Citrobacter freundii. The same trend was observed for treatment at 81.94 mA. The variability in bacterial community response to different plasma treatment protocols revealed that plasma had a selective impact on bacterial

  2. DNA Methylation

    PubMed Central

    Marinus, M.G.; Løbner-Olesen, A.

    2014-01-01

    The DNA of E. coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcm methyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during repair of replication errors, controlling the frequency of initiation of chromosome replication at oriC, and regulation of transcription initiation at promoters containing GATC sequences. In contrast, there is no known function for Dcm methylation although Dcm recognition sites constitute sequence motifs for Very Short Patch repair of T/G base mismatches. In certain bacteria (e.g., Vibrio cholerae, Caulobacter crescentus) adenine methylation is essential and in C. crescentus, it is important for temporal gene expression which, in turn, is required for coordinating chromosome initiation, replication and division. In practical terms, Dam and Dcm methylation can inhibit restriction enzyme cleavage; decrease transformation frequency in certain bacteria; decrease the stability of short direct repeats; are necessary for site-directed mutagenesis; and to probe eukaryotic structure and function. PMID:26442938

  3. Phages of dairy bacteria.

    PubMed

    Brussow, H

    2001-01-01

    Bacteriophages of lactic acid bacteria are a threat to industrial milk fermentation. Owing to their economical importance, dairy phages became the most thoroughly sequenced phage group in the database. Comparative genomics identified related cos-site and pac-site phages, respectively, in lactococci, lactic streptococci and lactobacilli. Each group was represented with closely related temperate and virulent phages. Over the structural genes their gene maps resembled that of lambdoid coliphages, suggesting distant evolutionary relationships. Despite a lack of sequence similarity, a number of biochemical characteristics of these dairy phages are lambda-like (genetic switch, DNA packaging, head and tail morphogenesis, and integration, but not excision). These dairy phages thus provide interesting variations to the phage lambda paradigm. The structural gene cluster of Lactococcus phage r1t resembled that of phages from mycobacteria. Virulent lactococcal phages with prolate heads (c2-like genus of Siphoviridae), in contrast, have no known counterparts in other bacterial genera.

  4. Back To Bacteria.

    ERIC Educational Resources Information Center

    Flannery, Maura C.

    1997-01-01

    Explores new research about bacteria. Discusses bacterial genomes, archaea, unusual environments, evolution, pathogens, bacterial movement, biofilms, bacteria in the body, and a bacterial obsession. Contains 29 references. (JRH)

  5. Back To Bacteria.

    ERIC Educational Resources Information Center

    Flannery, Maura C.

    1997-01-01

    Explores new research about bacteria. Discusses bacterial genomes, archaea, unusual environments, evolution, pathogens, bacterial movement, biofilms, bacteria in the body, and a bacterial obsession. Contains 29 references. (JRH)

  6. SatDNA Analyzer: a computing tool for satellite-DNA evolutionary analysis.

    PubMed

    Navajas-Pérez, Rafael; Rubio-Escudero, Cristina; Aznarte, José Luis; Rejón, Manuel Ruiz; Garrido-Ramos, Manuel A

    2007-03-15

    satDNA Analyzer is a program, implemented in C++, for the analysis of the patterns of variation at each nucleotide position considered independently amongst all units of a given satellite-DNA family when comparing it between a pair of species. The program classifies each site accordingly as monomorphic or polymorphic, discriminates shared from non-shared polymorphisms and classifies each non-shared polymorphism according to the model proposed by Strachan et al. in six different stages of transition during the spread of a variant repeat unit toward its fixation. Furthermore, this program implements several other utilities for satellite-DNA analysis evolution such as the design of the average consensus sequences, the average base pair contents, the distribution of variant sites, the transition to transversion ratio and different estimates of intra-specific variation and inter-specific variation. Aprioristic hypotheses on factors influencing the molecular drive process and the rates and biases of concerted evolution can be tested with this program. Additionally, satDNA Analyzer generates an output file containing a sequence alignment without shared polymorphisms to be used for further evolutionary analysis by using different phylogenetic softwares. satDNA Analyzer is freely available at http://satdna.sourceforge.net/. SatDNA Analyzer has been designed to operate on Windows, Linux and Mac OS X.

  7. Functional Encyclopedia of Bacteria and Archaea

    SciTech Connect

    Blow, M. J.; Deutschbauer, A. M.; Hoover, C. A.; Lamson, J.; Lamson, J.; Price, M. N.; Waters, J.; Wetmore, K. M.; Bristow, J.; Arkin, A. P.

    2013-03-20

    Bacteria and Archaea exhibit a huge diversity of metabolic capabilities with fundamental importance in the environment, and potential applications in biotechnology. However, the genetic bases of these capabilities remain unclear due largely to an absence of technologies that link DNA sequence to molecular function. To address this challenge, we are developing a pipeline for high throughput annotation of gene function using mutagenesis, growth assays and DNA sequencing. By applying this pipeline to annotate gene function in 50 diverse microbes we hope to discover thousands of new gene functions and produce a proof of principle `Functional Encyclopedia of Bacteria and Archaea?.

  8. Comparison of mitochondrial DNA control region sequence and microsatellite DNA analyses in estimating population structure and gene flow rates in Atlantic sturgeon Acipenser oxyrinchus

    USGS Publications Warehouse

    Wirgin, I.; Waldman, J.; Stabile, J.; Lubinski, B.; King, T.

    2002-01-01

    Atlantic sturgeon Acipenser oxyrinchus is large, long-lived, and anadromous with subspecies distributed along the Atlantic (A. oxyrinchus oxyrinchus) and Gulf of Mexico (A. o. desotoi) coasts of North America. Although it is not certain if extirpation of some population units has occurred, because of anthropogenic influences abundances of all populations are low compared with historical levels. Informed management of A. oxyrinchus demands a detailed knowledge of its population structure, levels of genetic diversity, and likelihood to home to natal rivers. We compared the use of mitochondrial DNA (mtDNA) control region sequence and microsatellite nuclear DNA (nDNA) analyses in identifying the stock structure and homing fidelity of Atlantic and Gulf coast populations of A. oxyrinchus. The approaches were concordant in that they revealed moderate to high levels of genetic diversity and suggested that populations of Atlantic sturgeon are highly structured. At least six genetically distinct management units were detected using the two approaches among the rivers surveyed. Mitochondrial DNA sequences revealed a significant cline in haplotype diversity along the Atlantic coast with monomorphism observed in Canadian populations. High levels of nDNA diversity were also observed among populations along the Atlantic coast, including the two Canadian populations, probably resulting from the more rapid rate of mutational and evolutionary change at microsatellite loci. Estimates of gene flow among populations were similar between both approaches with the exception that because of mtDNA monomorphism in Canadian populations, gene flow estimates between them were unobtainable. Analyses of both genomes provided high resolution and confidence in characterizing the population structure of Atlantic sturgeon. Microsatellite analysis was particularly informative in delineating population structure in rivers that were recently glaciated and may prove diagnostic in rivers that are

  9. Phenotypic switching in bacteria

    NASA Astrophysics Data System (ADS)

    Merrin, Jack

    Living matter is a non-equilibrium system in which many components work in parallel to perpetuate themselves through a fluctuating environment. Physiological states or functionalities revealed by a particular environment are called phenotypes. Transitions between phenotypes may occur either spontaneously or via interaction with the environment. Even in the same environment, genetically identical bacteria can exhibit different phenotypes of a continuous or discrete nature. In this thesis, we pursued three lines of investigation into discrete phenotypic heterogeneity in bacterial populations: the quantitative characterization of the so-called bacterial persistence, a theoretical model of phenotypic switching based on those measurements, and the design of artificial genetic networks which implement this model. Persistence is the phenotype of a subpopulation of bacteria with a reduced sensitivity to antibiotics. We developed a microfluidic apparatus, which allowed us to monitor the growth rates of individual cells while applying repeated cycles of antibiotic treatments. We were able to identify distinct phenotypes (normal and persistent) and characterize the stochastic transitions between them. We also found that phenotypic heterogeneity was present prior to any environmental cue such as antibiotic exposure. Motivated by the experiments with persisters, we formulated a theoretical model describing the dynamic behavior of several discrete phenotypes in a periodically varying environment. This theoretical framework allowed us to quantitatively predict the fitness of dynamic populations and to compare survival strategies according to environmental time-symmetries. These calculations suggested that persistence is a strategy used by bacterial populations to adapt to fluctuating environments. Knowledge of the phenotypic transition rates for persistence may provide statistical information about the typical environments of bacteria. We also describe a design of artificial

  10. Bacteria isolated from amoebae/bacteria consortium

    DOEpatents

    Tyndall, R.L.

    1995-05-30

    New protozoan derived microbial consortia and method for their isolation are provided. Consortia and bacteria isolated therefrom are useful for treating wastes such as trichloroethylene and trinitrotoluene. Consortia, bacteria isolated therefrom, and dispersants isolated therefrom are useful for dispersing hydrocarbons such as oil, creosote, wax, and grease.

  11. Bacteria isolated from amoebae/bacteria consortium

    DOEpatents

    Tyndall, Richard L.

    1995-01-01

    New protozoan derived microbial consortia and method for their isolation are provided. Consortia and bacteria isolated therefrom are useful for treating wastes such as trichloroethylene and trinitrotoluene. Consortia, bacteria isolated therefrom, and dispersants isolated therefrom are useful for dispersing hydrocarbons such as oil, creosote, wax, and grease.

  12. Oligonucleotide recombination in gram negative bacteria

    USDA-ARS?s Scientific Manuscript database

    This report describes several key aspects of a novel form of RecA-independent homologous recombination. We found that synthetic single stranded DNA oligonucleotides (oligos) introduced into bacteria by transformation can site-specifically recombine with bacterial chromosomes in the absence of any a...

  13. Connecting chromosome replication with cell growth in bacteria.

    PubMed

    Murray, Heath

    2016-12-01

    For bacteria to proliferate they must duplicate their genetic material so that it can be passed to their progeny. This requires that DNA replication is coordinated with cell growth and division. In the natural environment bacterial growth is dynamic and strongly influenced by changes in nutrient availability. Recent studies have found that bacteria utilize a range of regulatory systems, many of them species-specific, to coordinate DNA replication with cell growth. This variability likely reflects the diverse lifestyles of different bacterial types.

  14. Bacteria Inactivation During Lithotripsy

    NASA Astrophysics Data System (ADS)

    del Sol Quintero, María; Mora, Ulises; Gutiérrez, Jorge; Mues, Enrique; Castaño, Eduardo; Fernández, Francisco; Loske, Achim M.

    2006-09-01

    The influence of extracorporeal and intracorporeal lithotripsy on the viability of bacteria contained inside artificial kidney stones was investigated in vitro. Two different bacteria were exposed to the action of one extracorporeal shock wave generator and four intracorporeal lithotripters.

  15. Bleach vs. Bacteria

    MedlinePlus

    ... Articles | Inside Life Science Home Page Bleach vs. Bacteria By Sharon Reynolds Posted April 2, 2014 Your ... hypochlorous acid to help kill invading microbes, including bacteria. Researchers funded by the National Institutes of Health ...

  16. Characterization of 35 novel microsatellite DNA markers from the duck (Anas platyrhynchos) genome and cross-amplification in other birds

    PubMed Central

    Huang, Yinhua; Tu, Jianfeng; Cheng, Xuebo; Tang, Bo; Hu, Xiaoxiang; Liu, Zhaoliang; Feng, Jidong; Lou, Yankun; Lin, Li; Xu, Ke; Zhao, Yulong; Li, Ning

    2005-01-01

    In order to study duck microsatellites, we constructed a library enriched for (CA)n, (CAG)n, (GCC)n and (TTTC)n. A total of 35 pairs of primers from these microsatellites were developed and used to detect polymorphisms in 31 unrelated Peking ducks. Twenty-eight loci were polymorphic and seven loci were monomorphic. A total of 117 alleles were observed from these polymorphic microsatellite markers, which ranged from 2 to 14 with an average of 4.18 per locus. The frequencies of the 117 alleles ranged from 0.02 to 0.98. The highest heterozygosity (0.97) was observed at the CAUD019 microsatellite locus and the lowest heterozygosity (0.04) at the CAUD008 locus, and 11 loci had heterozygosities greater than 0.50 (46.43%). The polymorphism information content (PIC) of 28 loci ranged from 0.04 to 0.88 with an average of 0.42. All the above markers were used to screen the polymorphism in other bird species. Two markers produced specific monomorphic products with the chicken DNA. Fourteen markers generated specific fragments with the goose DNA: 5 were polymorphic and 9 were monomorphic. But no specific product was detected with the peacock DNA. Based on sequence comparisons of the flanking sequence and repeat, we conclude that 2 chicken loci and 14 goose loci were true homologous loci of the duck loci. The microsatellite markers identified and characterized in the present study will contribute to the genetic map, quantitative traits mapping, and phylogenetic analysis in the duck and goose. PMID:15943922

  17. Detection of fecal bacteria and source tracking identifiers in environmental waters using rRNA-based RT-qPCR and rDNA-based qPCR assays

    EPA Science Inventory

    The identification of fecal pollution sources is commonly performed using DNA-based methods. However, there is evidence that DNA can be associated with dead cells or present as “naked DNA” in the environment. To this end, we compared the detection frequency of host specific marke...

  18. Detection of fecal bacteria and source tracking identifiers in environmental waters using rRNA-based RT-qPCR and rDNA-based qPCR assays

    EPA Science Inventory

    The identification of fecal pollution sources is commonly performed using DNA-based methods. However, there is evidence that DNA can be associated with dead cells or present as “naked DNA” in the environment. To this end, we compared the detection frequency of host specific marke...

  19. ANTI-DNA ANTIBODIES IN HYPERIMMUNIZED RABBITS

    PubMed Central

    Christian, Charles L.; DeSimone, Arthur R.; Abruzzo, John L.

    1965-01-01

    Complement-fixing anti-DNA antibodies were detected in a minority of sera of rabbits hyperimmunized with killed Gram-negative bacteria. The C'-fixing property of DNA was lost after DNase treatment. Preferential reactivity with denatured DNA was observed. The antisera reacted with DNA preparations derived from rabbit bone marrow and thymus, calf thymus, pneumococci, salmon sperm, and Escherichia coli. E. coli DNA was less effective than preparations of mammalian and salmon sperm DNA in fixation of C'. Inhibition of DNA C' fixation by nucleotides and nucleosides was observed. The bulk of anti-DNA activity was associated with the low molecular weight antibody fraction. PMID:14264274

  20. DNA replication in thermophiles.

    PubMed

    Majerník, A I; Jenkinson, E R; Chong, J P J

    2004-04-01

    DNA replication enzymes in the thermophilic Archaea have previously attracted attention due to their obvious use in methods such as PCR. The proofreading ability of the Pyrococcus furiosus DNA polymerase has resulted in a commercially successful product (Pfu polymerase). One of the many notable features of the Archaea is the fact that their DNA processing enzymes appear on the whole to be more like those found in eukaryotes than bacteria. These proteins also appear to be simpler versions of those found in eukaryotes. For these reasons, archaeal organisms make potentially interesting model systems to explore the molecular mechanisms of processes such as DNA replication, repair and recombination. Why archaeal DNA-manipulation systems were adopted over bacterial systems by eukaryotic cells remains a most interesting question that we suggest may be linked to thermophily.

  1. The use of dimorphic Alu insertions in human DNA fingerprinting

    SciTech Connect

    Novick, G.E.; Gonzalez, T.; Garrison, J.; Novick, C.C.; Herrera, R.J.; Batzer, M.A.; Deininger, P.L.

    1992-12-04

    We have characterized certain Human Specific Alu Insertions as either dimorphic (TPA25, PV92, APO), sightly dimorphic (C2N4 and C4N4) or monomorphic (C3N1, C4N6, C4N2, C4N5, C4N8), based on studies of Caucasian, Asian, American Black and African Black populations. Our approach is based upon: (1) PCR amplification using primers directed to the sequences that flank the site of insertion of the different Alu elements studied; (2) gel electrophoresis and scoring according to the presence or absence of an Alu insertion in one or both homologous chromosomes; (3) allelic frequencies calculated and compared according to Hardy-Weinberg equilibrium. Our DNA fingerprinting procedure using PCR amplification of dimorphic Human Specific Alu insertions, is stable enough to be used not only as a tool for genetic mapping but also to characterize populations, study migrational patterns and track the inheritance of human genetic disorders.

  2. Diversity of Bacteria at Healthy Human Conjunctiva

    PubMed Central

    Dong, Qunfeng; Brulc, Jennifer M.; Iovieno, Alfonso; Bates, Brandon; Garoutte, Aaron; Miller, Darlene; Revanna, Kashi V.; Gao, Xiang; Antonopoulos, Dionysios A.; Slepak, Vladlen Z.

    2011-01-01

    Purpose. Ocular surface (OS) microbiota contributes to infectious and autoimmune diseases of the eye. Comprehensive analysis of microbial diversity at the OS has been impossible because of the limitations of conventional cultivation techniques. This pilot study aimed to explore true diversity of human OS microbiota using DNA sequencing-based detection and identification of bacteria. Methods. Composition of the bacterial community was characterized using deep sequencing of the 16S rRNA gene amplicon libraries generated from total conjunctival swab DNA. The DNA sequences were classified and the diversity parameters measured using bioinformatics software ESPRIT and MOTHUR and tools available through the Ribosomal Database Project-II (RDP-II). Results. Deep sequencing of conjunctival rDNA from four subjects yielded a total of 115,003 quality DNA reads, corresponding to 221 species-level phylotypes per subject. The combined bacterial community classified into 5 phyla and 59 distinct genera. However, 31% of all DNA reads belonged to unclassified or novel bacteria. The intersubject variability of individual OS microbiomes was very significant. Regardless, 12 genera—Pseudomonas, Propionibacterium, Bradyrhizobium, Corynebacterium, Acinetobacter, Brevundimonas, Staphylococci, Aquabacterium, Sphingomonas, Streptococcus, Streptophyta, and Methylobacterium—were ubiquitous among the analyzed cohort and represented the putative “core” of conjunctival microbiota. The other 47 genera accounted for <4% of the classified portion of this microbiome. Unexpectedly, healthy conjunctiva contained many genera that are commonly identified as ocular surface pathogens. Conclusions. The first DNA sequencing-based survey of bacterial population at the conjunctiva have revealed an unexpectedly diverse microbial community. All analyzed samples contained ubiquitous (core) genera that included commensal, environmental, and opportunistic pathogenic bacteria. PMID:21571682

  3. Diversity of bacteria at healthy human conjunctiva.

    PubMed

    Dong, Qunfeng; Brulc, Jennifer M; Iovieno, Alfonso; Bates, Brandon; Garoutte, Aaron; Miller, Darlene; Revanna, Kashi V; Gao, Xiang; Antonopoulos, Dionysios A; Slepak, Vladlen Z; Shestopalov, Valery I

    2011-07-20

    Ocular surface (OS) microbiota contributes to infectious and autoimmune diseases of the eye. Comprehensive analysis of microbial diversity at the OS has been impossible because of the limitations of conventional cultivation techniques. This pilot study aimed to explore true diversity of human OS microbiota using DNA sequencing-based detection and identification of bacteria. Composition of the bacterial community was characterized using deep sequencing of the 16S rRNA gene amplicon libraries generated from total conjunctival swab DNA. The DNA sequences were classified and the diversity parameters measured using bioinformatics software ESPRIT and MOTHUR and tools available through the Ribosomal Database Project-II (RDP-II). Deep sequencing of conjunctival rDNA from four subjects yielded a total of 115,003 quality DNA reads, corresponding to 221 species-level phylotypes per subject. The combined bacterial community classified into 5 phyla and 59 distinct genera. However, 31% of all DNA reads belonged to unclassified or novel bacteria. The intersubject variability of individual OS microbiomes was very significant. Regardless, 12 genera-Pseudomonas, Propionibacterium, Bradyrhizobium, Corynebacterium, Acinetobacter, Brevundimonas, Staphylococci, Aquabacterium, Sphingomonas, Streptococcus, Streptophyta, and Methylobacterium-were ubiquitous among the analyzed cohort and represented the putative "core" of conjunctival microbiota. The other 47 genera accounted for <4% of the classified portion of this microbiome. Unexpectedly, healthy conjunctiva contained many genera that are commonly identified as ocular surface pathogens. The first DNA sequencing-based survey of bacterial population at the conjunctiva have revealed an unexpectedly diverse microbial community. All analyzed samples contained ubiquitous (core) genera that included commensal, environmental, and opportunistic pathogenic bacteria.

  4. Bacteriophage biosensors for antibiotic-resistant bacteria.

    PubMed

    Sorokulova, Irina; Olsen, Eric; Vodyanoy, Vitaly

    2014-03-01

    An increasing number of disease-causing bacteria are resistant to one or more anti-bacterial drugs utilized for therapy. Early and speedy detection of these pathogens is therefore very important. Traditional pathogen detection techniques, that include microbiological and biochemical assays are long and labor-intensive, while antibody or DNA-based methods require substantial sample preparation and purification. Biosensors based on bacteriophages have demonstrated remarkable potential to surmount these restrictions and to offer rapid, efficient and sensitive detection technique for antibiotic-resistant bacteria.

  5. Genomics of Probiotic Bacteria

    NASA Astrophysics Data System (ADS)

    O'Flaherty, Sarah; Goh, Yong Jun; Klaenhammer, Todd R.

    Probiotic bacteria from the Lactobacillus and Bifidobacterium species belong to the Firmicutes and the Actinobacteria phylum, respectively. Lactobacilli are members of the lactic acid bacteria (LAB) group, a broadly defined family of microorganisms that ferment various hexoses into primarily lactic acid. Lactobacilli are typically low G + C gram-positive species which are phylogenetically diverse, with over 100 species documented to date. Bifidobacteria are heterofermentative, high G + C content bacteria with about 30 species of bifidobacteria described to date.

  6. Identification of bacteria in scuba divers' rinse tanks.

    PubMed

    Washburn, Brian K; Levin, Andrew E; Hennessy, Kristen; Miller, Michael R

    2010-01-01

    Scuba divers typically rinse equipment in communal tanks. Studies show these tanks are contaminated with bacteria, but the types of bacteria have not been studied. We sought to identify bacteria in rinse tanks at a dive facility at San Pedro, Belize, to determine the origin of the bacteria and determine whether the bacteria represented potential threats to human health. The identity of bacteria was investigated using reverse line blot (RLB) assays based on 28 different rDNA probes designed to detect known pathogens of sepsis, as well as by sequencing 23S rDNA from isolates and performing VITEK identification of several isolates. Based on the identities of bacteria in divers' rinse tanks, many likely originate from the ocean, and others likely originate from the divers themselves. None of the bacteria identified would be considered overt human pathogens. However, some of the bacteria found in the tanks are known to be associated with unsanitary conditions and can cause opportunistic infections, which may pose health problems to some individuals. Rinsing scuba equipment in communal tanks has the potential to transmit disease among some divers. Equipment, especially regulators and masks, should be rinsed/cleaned individually and not be placed in communal tanks.

  7. The association between bacteria and urinary stones

    PubMed Central

    Wolfe, Alan J.

    2017-01-01

    Urinary stone disease (USD) is an increasing clinical problem in both children and adults. One in ten individuals will experience a urinary stone, yet the mechanisms responsible for urinary stones remain largely unknown. Bacteria have long been recognized to contribute to struvite urinary stones; however, the role of bacteria in the development of the more common calcium oxalate (CaOx) and calcium phosphate (CaPhos) stones has not been extensively investigated. However, several findings do indicate a possible association between urinary stones and bacteria, including the high rate of urinary tract infections (UTI) in urinary stone patients and multiple case series of culture-positive urinary stones, including stones composed of CaOx or CaPhos. New technology, such as next generation sequencing, may be used to lend additional insight regarding the association between urinary stones and bacteria. In 2015, we published the initial bacterial sequencing results from five urinary stones, from which we sequenced multiple types of bacterial DNA. Whether these bacteria are causal, disease modifying or passively present remains to be determined. However, initial exploration of underlying mechanisms for this association indicate that bacteria aggregate selectively to crystals, that their presence is associated with increased clumping of crystals, and that they stimulate incorporation of proteins into the stone matrix. PMID:28217697

  8. Translesion DNA Synthesis and Mutagenesis in Eukaryotes

    PubMed Central

    Sale, Julian E.

    2013-01-01

    The structural features that enable replicative DNA polymerases to synthesize DNA rapidly and accurately also limit their ability to copy damaged DNA. Direct replication of DNA damage is termed translesion synthesis (TLS), a mechanism conserved from bacteria to mammals and executed by an array of specialized DNA polymerases. This chapter examines how these translesion polymerases replicate damaged DNA and how they are regulated to balance their ability to replicate DNA lesions with the risk of undesirable mutagenesis. It also discusses how TLS is co-opted to increase the diversity of the immunoglobulin gene hypermutation and the contribution it makes to the mutations that sculpt the genome of cancer cells. PMID:23457261

  9. Hessian fly-associated bacteria: transmission, essentiality, and composition.

    PubMed

    Bansal, Raman; Hulbert, Scot; Schemerhorn, Brandi; Reese, John C; Whitworth, R Jeff; Stuart, Jeffrey J; Chen, Ming-Shun

    2011-01-01

    Plant-feeding insects have been recently found to use microbes to manipulate host plant physiology and morphology. Gall midges are one of the largest groups of insects that manipulate host plants extensively. Hessian fly (HF, Mayetiola destructor) is an important pest of wheat and a model system for studying gall midges. To examine the role of bacteria in parasitism, a systematic analysis of bacteria associated with HF was performed for the first time. Diverse bacteria were found in different developmental HF stages. Fluorescent in situ hybridization detected a bacteriocyte-like structure in developing eggs. Bacterial DNA was also detected in eggs by PCR using primers targeted to different bacterial groups. These results indicated that HF hosted different types of bacteria that were maternally transmitted to the next generation. Eliminating bacteria from the insect with antibiotics resulted in high mortality of HF larvae, indicating that symbiotic bacteria are essential for the insect to survive on wheat seedlings. A preliminary survey identified various types of bacteria associated with different HF stages, including the genera Enterobacter, Pantoea, Stenotrophomonas, Pseudomonas, Bacillus, Ochrobactrum, Acinetobacter, Alcaligenes, Nitrosomonas, Arcanobacterium, Microbacterium, Paenibacillus, and Klebsiella. Similar bacteria were also found specifically in HF-infested susceptible wheat, suggesting that HF larvae had either transmitted bacteria into plant tissue or brought secondary infection of bacteria to the wheat host. The bacteria associated with wheat seedlings may play an essential role in the wheat-HF interaction.

  10. An Optimized Pentaplex PCR for Detecting DNA Mismatch Repair-Deficient Colorectal Cancers

    PubMed Central

    Hamelin, Richard; Boland, C. Richard

    2010-01-01

    Purpose Microsatellite instability (MSI) is used to screen colorectal cancers (CRC) for Lynch Syndrome, and to predict outcome and response to treatment. The current technique for measuring MSI requires DNA from normal and neoplastic tissues, and fails to identify tumors with specific DNA mismatch repair (MMR) defects. We tested a panel of five quasi-monomorphic mononucleotide repeat markers amplified in a single multiplex PCR reaction (pentaplex PCR) to detect MSI. Experimental Design We investigated a cohort of 213 CRC patients, comprised of 114 MMR-deficient and 99 MMR-proficient tumors. Immunohistochemical (IHC) analysis evaluated the expression of MLH1, MSH2, PMS2 and MSH6. MSI status was defined by differences in the quasi-monomorphic variation range (QMVR) from a pool of normal DNA samples, and measuring differences in allele lengths in tumor DNA. Results Amplification of 426 normal alleles allowed optimization of the QMVR at each marker, and eliminated the requirement for matched reference DNA to define MSI in each sample. Using ≥2/5 unstable markers as the criteria for MSI resulted in a sensitivity of 95.6% (95% CI = 90.1–98.1%) and a positive predictive value of 100% (95% CI = 96.6%–100%). Detection of MSH6-deficiency was limited using all techniques. Data analysis with a three-marker panel (BAT26, NR21 and NR27) was comparable in sensitivity (97.4%) and positive predictive value (96.5%) to the five marker panel. Both approaches were superior to the standard approach to measuring MSI. Conclusions An optimized pentaplex (or triplex) PCR offers a facile, robust, very inexpensive, highly sensitive, and specific assay for the identification of MSI in CRC. PMID:20195377

  11. Analysis of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments.

    PubMed Central

    Kowalchuk, G A; Stephen, J R; De Boer, W; Prosser, J I; Embley, T M; Woldendorp, J W

    1997-01-01

    Denaturing gradient gel electrophoresis (DGGE) is a powerful and convenient tool for analyzing the sequence diversity of complex natural microbial populations. DGGE was evaluated for the identification of ammonia oxidizers of the beta subdivision of the Proteobacteria based on the mobility of PCR-amplified 16S rDNA fragments and for the analysis of mixtures of PCR products from this group generated by selective PCR of DNA extracted from coastal sand dunes. Degenerate PCR primers, CTO189f-GC and CTO654r, incorporating a 5' GC clamp, were designed to amplify a 465-bp 16S rDNA region spanning the V-2 and V-3 variable domains. The primers were tested against a representative selection of clones and cultures encompassing the currently recognized beta-subdivision ammonia oxidizer 16S rDNA sequence diversity. Analysis of these products by DGGE revealed that while many of the sequences could be separated, some which were known to be different migrated similarly in the denaturant system used. The CTO primer pair was used to amplify 16S rDNA sequences from DNA extracted from soil sampled from Dutch coastal dune locations of differing in pH and distance from the beach. The derived DGGE patterns were reproducible across multiple DNA isolations and PCRs. Ammonia oxidizer-like sequences from different phylogenetic groupings isolated from gene libraries made from the same sand dune DNA samples but prepared with different primers gave DGGE bands which comigrated with most of the bands detected from the sand dune samples. Bands from the DGGE gels of environmental samples were excised, reamplified, and directly sequenced, revealing strong similarity or identity of the recovered products to the corresponding regions of library clones. Six of the seven sequenced clusters of beta-subdivision ammonia oxidizers were detected in the dune systems, and differences in community structure between some sample sites were demonstrated. The most seaward dune site contained sequences showing

  12. Bacteria turn tiny gears

    SciTech Connect

    2009-01-01

    Swarms of bacteria turn two 380-micron long gears, opening the possibility of building hybrid biological machines at the microscopic scale. Read more at Wired: http://www.wired.com/wiredscience/2009/12/bacterial-micro-machine/#more-15684 or Scientific American: http://www.scientificamerican.com/article.cfm?id=brownian-motion-bacteria

  13. Some bacteria are beneficial!

    USGS Publications Warehouse

    McMahon, Peter B.

    1995-01-01

    Most people would agree that bacteria usually spell trouble where the quality of drinking water is con cerned. However, recent studies conducted by the U.S. Geological Survey (USGS) under the National Water-Quality Assessment (NAWQA) program have shown that some bacteria can improve the quality of water.

  14. Some bacteria are beneficial!

    USGS Publications Warehouse

    McMahon, Peter B.

    1995-01-01

    Most people would agree that bacteria usually spell trouble where the quality of drinking water is con cerned. However, recent studies conducted by the U.S. Geological Survey (USGS) under the National Water-Quality Assessment (NAWQA) program have shown that some bacteria can improve the quality of water.

  15. Chromosome-Membrane Interactions in Bacteria.

    PubMed

    Roggiani, Manuela; Goulian, Mark

    2015-01-01

    Prokaryotes, by definition, do not segregate their genetic material from the cytoplasm. Thus, there is no barrier preventing direct interactions between chromosomal DNA and the plasma membrane. The possibility of such interactions in bacteria was proposed long ago and supported by early electron microscopy and cell fractionation studies. However, the identification and characterization of chromosome-membrane interactions have been slow in coming. Recently, this subject has seen more progress, driven by advances in imaging techniques and in the exploration of diverse cellular processes. A number of loci have been identified in specific bacteria that depend on interactions with the membrane for their function. In addition, there is growing support for a general mechanism of DNA-membrane contacts based on transertion-concurrent transcription, translation, and insertion of membrane proteins. This review summarizes the history and recent results of chromosome-membrane associations and discusses the known and theorized consequences of these interactions in the bacterial cell.

  16. Whole genome plasticity in pathogenic bacteria.

    PubMed

    Dobrindt, U; Hacker, J

    2001-10-01

    The exploitation of bacterial genome sequences has so far provided a wealth of new general information about the genetic diversity of bacteria, such as that of many pathogens. Comparative genomics uncovered many genome variations in closely related bacteria and revealed basic principles involved in bacterial diversification, improving our knowledge of the evolution of bacterial pathogens. A correlation between metabolic versatility and genome size has become evident. The degenerated life styles of obligate intracellular pathogens correlate with significantly reduced genome sizes, a phenomenon that has been termed "evolution by reduction". These mechanisms can permanently alter bacterial genotypes and result in adaptation to their environment by genome optimization. In this review, we summarize the recent results of genome-wide approaches to studying the genetic diversity of pathogenic bacteria that indicate that the acquisition of DNA and the loss of genetic information are two important mechanisms that contribute to strain-specific differences in genome content.

  17. DNA/genetic vaccination (minireview).

    PubMed

    Kucerova, L

    1998-01-01

    An important new approach to vaccination is plasmid DNA injection in vivo that can elicit an immune response against protein(s) encoded. Antigen that is expressed from the in vivo transfected cells induces both humoral and cellular immune response. DNA immunization is generally applicable for a wide range of proteins. It can provide an organism with immunity against viruses, bacteria, parasites, and tumors. DNA vaccines can overcome the disadvantages of vaccines presently used as well as provide various new vaccines that are currently not available. This minireview provides an overview of evaluated DNA vaccine candidates against infectious agents and certain cancers.

  18. In vitro antibacterial activity of AZD0914, a new spiropyrimidinetrione DNA gyrase/topoisomerase inhibitor with potent activity against Gram-positive, fastidious Gram-Negative, and atypical bacteria.

    PubMed

    Huband, Michael D; Bradford, Patricia A; Otterson, Linda G; Basarab, Gregory S; Kutschke, Amy C; Giacobbe, Robert A; Patey, Sara A; Alm, Richard A; Johnstone, Michele R; Potter, Marie E; Miller, Paul F; Mueller, John P

    2015-01-01

    AZD0914 is a new spiropyrimidinetrione bacterial DNA gyrase/topoisomerase inhibitor with potent in vitro antibacterial activity against key Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae), fastidious Gram-negative (Haemophilus influenzae and Neisseria gonorrhoeae), atypical (Legionella pneumophila), and anaerobic (Clostridium difficile) bacterial species, including isolates with known resistance to fluoroquinolones. AZD0914 works via inhibition of DNA biosynthesis and accumulation of double-strand cleavages; this mechanism of inhibition differs from those of other marketed antibacterial compounds. AZD0914 stabilizes and arrests the cleaved covalent complex of gyrase with double-strand broken DNA under permissive conditions and thus blocks religation of the double-strand cleaved DNA to form fused circular DNA. Whereas this mechanism is similar to that seen with fluoroquinolones, it is mechanistically distinct. AZD0914 exhibited low frequencies of spontaneous resistance in S. aureus, and if mutants were obtained, the mutations mapped to gyrB. Additionally, no cross-resistance was observed for AZD0914 against recent bacterial clinical isolates demonstrating resistance to fluoroquinolones or other drug classes, including macrolides, β-lactams, glycopeptides, and oxazolidinones. AZD0914 was bactericidal in both minimum bactericidal concentration and in vitro time-kill studies. In in vitro checkerboard/synergy testing with 17 comparator antibacterials, only additivity/indifference was observed. The potent in vitro antibacterial activity (including activity against fluoroquinolone-resistant isolates), low frequency of resistance, lack of cross-resistance, and bactericidal activity of AZD0914 support its continued development. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  19. In Vitro Antibacterial Activity of AZD0914, a New Spiropyrimidinetrione DNA Gyrase/Topoisomerase Inhibitor with Potent Activity against Gram-Positive, Fastidious Gram-Negative, and Atypical Bacteria

    PubMed Central

    Bradford, Patricia A.; Otterson, Linda G.; Basarab, Gregory S.; Kutschke, Amy C.; Giacobbe, Robert A.; Patey, Sara A.; Alm, Richard A.; Johnstone, Michele R.; Potter, Marie E.; Miller, Paul F.; Mueller, John P.

    2014-01-01

    AZD0914 is a new spiropyrimidinetrione bacterial DNA gyrase/topoisomerase inhibitor with potent in vitro antibacterial activity against key Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae), fastidious Gram-negative (Haemophilus influenzae and Neisseria gonorrhoeae), atypical (Legionella pneumophila), and anaerobic (Clostridium difficile) bacterial species, including isolates with known resistance to fluoroquinolones. AZD0914 works via inhibition of DNA biosynthesis and accumulation of double-strand cleavages; this mechanism of inhibition differs from those of other marketed antibacterial compounds. AZD0914 stabilizes and arrests the cleaved covalent complex of gyrase with double-strand broken DNA under permissive conditions and thus blocks religation of the double-strand cleaved DNA to form fused circular DNA. Whereas this mechanism is similar to that seen with fluoroquinolones, it is mechanistically distinct. AZD0914 exhibited low frequencies of spontaneous resistance in S. aureus, and if mutants were obtained, the mutations mapped to gyrB. Additionally, no cross-resistance was observed for AZD0914 against recent bacterial clinical isolates demonstrating resistance to fluoroquinolones or other drug classes, including macrolides, β-lactams, glycopeptides, and oxazolidinones. AZD0914 was bactericidal in both minimum bactericidal concentration and in vitro time-kill studies. In in vitro checkerboard/synergy testing with 17 comparator antibacterials, only additivity/indifference was observed. The potent in vitro antibacterial activity (including activity against fluoroquinolone-resistant isolates), low frequency of resistance, lack of cross-resistance, and bactericidal activity of AZD0914 support its continued development. PMID:25385112

  20. Cell Size Regulation in Bacteria

    NASA Astrophysics Data System (ADS)

    Amir, Ariel

    2014-05-01

    Various bacteria such as the canonical gram negative Escherichia coli or the well-studied gram positive Bacillus subtilis divide symmetrically after they approximately double their volume. Their size at division is not constant, but is typically distributed over a narrow range. Here, we propose an analytically tractable model for cell size control, and calculate the cell size and interdivision time distributions, as well as the correlations between these variables. We suggest ways of extracting the model parameters from experimental data, and show that existing data for E. coli supports partial size control, and a particular explanation: a cell attempts to add a constant volume from the time of initiation of DNA replication to the next initiation event. This hypothesis accounts for the experimentally observed correlations between mother and daughter cells as well as the exponential dependence of size on growth rate.

  1. Structural investigation into physiological DNA phosphorothioate modification

    PubMed Central

    Lan, Wenxian; Hu, Zhongpei; Shen, Jie; Wang, Chunxi; Jiang, Feng; Liu, Huili; Long, Dewu; Liu, Maili; Cao, Chunyang

    2016-01-01

    DNA phosphorothioate (PT) modification, with sulfur replacing a nonbridging phosphate oxygen in a sequence and stereo specific manner, is a novel physiological variation in bacteria. But what effects on DNA properties PT modification has is still unclear. To address this, we prepared three double-stranded (ds) DNA decamers, d(CGPXGCCGCCGA) with its complementary strand d(TCGGCGPXGCCG) (where X = O or S, i.e., PT-free dsDNA, [Sp, Sp]-PT dsDNA or [Rp, Rp]-PT dsDNA) located in gene of Streptomyces lividans. Their melting temperature (Tm) measurement indicates that [Rp, Rp]-PT dsDNA is most unstable. Their electron transfer potential detection presents an order of anti-oxidation properties: Sp-PT DNA > Rp-PT DNA > PT-free DNA. Their NMR structures demonstrate that PT modification doesn’t change their B-form conformation. The sulfur in [Rp, Rp]-PT dsDNA locates in the major groove, with steric effects on protons in the sugar close to modification sites, resulting in its unstability, and facilitating its selectively interactions with ScoMcrA. We thought that PT modification was dialectical to the bacteria. It protects the hosting bacteria by working as antioxidant against H2O2, and acts as a marker, directing restriction enzyme observed in other hosts, like ScoMcrA, to correctly cleave the PT modified DNA, so that bacteria cannot spread and survive. PMID:27169778

  2. Structural investigation into physiological DNA phosphorothioate modification.

    PubMed

    Lan, Wenxian; Hu, Zhongpei; Shen, Jie; Wang, Chunxi; Jiang, Feng; Liu, Huili; Long, Dewu; Liu, Maili; Cao, Chunyang

    2016-05-12

    DNA phosphorothioate (PT) modification, with sulfur replacing a nonbridging phosphate oxygen in a sequence and stereo specific manner, is a novel physiological variation in bacteria. But what effects on DNA properties PT modification has is still unclear. To address this, we prepared three double-stranded (ds) DNA decamers, d(CG(PX)GCCGCCGA) with its complementary strand d(TCGGCG(PX)GCCG) (where X = O or S, i.e., PT-free dsDNA, [Sp, Sp]-PT dsDNA or [Rp, Rp]-PT dsDNA) located in gene of Streptomyces lividans. Their melting temperature (Tm) measurement indicates that [Rp, Rp]-PT dsDNA is most unstable. Their electron transfer potential detection presents an order of anti-oxidation properties: Sp-PT DNA > Rp-PT DNA > PT-free DNA. Their NMR structures demonstrate that PT modification doesn't change their B-form conformation. The sulfur in [Rp, Rp]-PT dsDNA locates in the major groove, with steric effects on protons in the sugar close to modification sites, resulting in its unstability, and facilitating its selectively interactions with ScoMcrA. We thought that PT modification was dialectical to the bacteria. It protects the hosting bacteria by working as antioxidant against H2O2, and acts as a marker, directing restriction enzyme observed in other hosts, like ScoMcrA, to correctly cleave the PT modified DNA, so that bacteria cannot spread and survive.

  3. Precision genome engineering in lactic acid bacteria

    PubMed Central

    2014-01-01

    Innovative new genome engineering technologies for manipulating chromosomes have appeared in the last decade. One of these technologies, recombination mediated genetic engineering (recombineering) allows for precision DNA engineering of chromosomes and plasmids in Escherichia coli. Single-stranded DNA recombineering (SSDR) allows for the generation of subtle mutations without the need for selection and without leaving behind any foreign DNA. In this review we discuss the application of SSDR technology in lactic acid bacteria, with an emphasis on key factors that were critical to move this technology from E. coli into Lactobacillus reuteri and Lactococcus lactis. We also provide a blueprint for how to proceed if one is attempting to establish SSDR technology in a lactic acid bacterium. The emergence of CRISPR-Cas technology in genome engineering and its potential application to enhancing SSDR in lactic acid bacteria is discussed. The ability to perform precision genome engineering in medically and industrially important lactic acid bacteria will allow for the genetic improvement of strains without compromising safety. PMID:25185700

  4. Chromosome dynamics in multichromosome bacteria.

    PubMed

    Jha, Jyoti K; Baek, Jong Hwan; Venkova-Canova, Tatiana; Chattoraj, Dhruba K

    2012-07-01

    On the basis of limited information, bacteria were once assumed to have no more than one chromosome. In the era of genomics, it has become clear that some, like eukaryotes, have more than one chromosome. Multichromosome bacteria provide opportunities to investigate how split genomes emerged, whether the individual chromosomes communicate to coordinate their replication and segregation, and what selective advantages split genomes might provide. Our current knowledge of these topics comes mostly from studies in Vibrio cholerae, which has two chromosomes, chr1 and chr2. Chr1 carries out most of the house-keeping functions and is considered the main chromosome, whereas chr2 appears to have originated from a plasmid and has acquired genes of mostly unknown origin and function. Nevertheless, unlike plasmids, chr2 replicates once and only once per cell cycle, like a bona fide chromosome. The two chromosomes replicate and segregate using separate programs, unlike eukaryotic chromosomes. They terminate replication synchronously, suggesting that there might be communication between them. Replication of the chromosomes is affected by segregation genes but in a chromosome specific fashion, a new development in the field of DNA replication control. The split genome allows genome duplication to complete in less time and with fewer replication forks, which could be beneficial for genome maintenance during rapid growth, which is the norm for V. cholerae in broth cultures and in the human host. In the latter, the expression of chr2 genes increases preferentially. Studies of chromosome maintenance in multichromosomal bacteria, although in their infancy, are already broadening our view of chromosome biology. This article is part of a Special Issue entitled: Chromatin in time and space. Published by Elsevier B.V.

  5. Genetic manipulation of acidophilic bacteria

    SciTech Connect

    Ward, T.E.; Rowland, M.L.; Glenn, A.W.; Watkins, C.S.; Bruhn, D.F.; Bulmer, D.; Roberto, F.F.

    1989-01-01

    Thiobacillus ferrooxidans is important in leaching of metals from mineral ores and in the removal of pyritic sulfur from coal. It is also intimately involved in production of acid mine drainage. Other acidophilic bacteria, including members of the genus Acidiphilium, are usually present in the same environments as T. ferrooxidans, and there is evidence to suggest that these acidophilic heterotrophs may increase the rate of T. ferrooxidans' attack on inorganic sulfides. Our laboratory is studying the genetic characteristics of these acidophilic bacteria and developing techniques for introducing desirable genes into them. Several endogenous plasmids from Acidiphilium strains have been cloned into E. coli vectors. Some of the resulting plasmids are able to confer antibiotic resistance to Acidiphilium after transformation by electroporation. In addition, a broad-host range plasmid conferring resistance to tetracycline has been introduced into Acidiphilium strains by electroporation. This same plasmid, has also been transferred to Acidiphilium from E. coli directly by conjugation. A temperate bacteriophage which infects a number of Acidiphilium isolates has been discovered and partially characterized. It has a lambdoid morphology and a genome of approximately 97 kb, comprised of double-stranded DNA which is probably modified. 16 refs., 2 figs., 4 tabs.

  6. Quorum sensing in bacteria.

    PubMed

    Miller, M B; Bassler, B L

    2001-01-01

    Quorum sensing is the regulation of gene expression in response to fluctuations in cell-population density. Quorum sensing bacteria produce and release chemical signal molecules called autoinducers that increase in concentration as a function of cell density. The detection of a minimal threshold stimulatory concentration of an autoinducer leads to an alteration in gene expression. Gram-positive and Gram-negative bacteria use quorum sensing communication circuits to regulate a diverse array of physiological activities. These processes include symbiosis, virulence, competence, conjugation, antibiotic production, motility, sporulation, and biofilm formation. In general, Gram-negative bacteria use acylated homoserine lactones as autoinducers, and Gram-positive bacteria use processed oligo-peptides to communicate. Recent advances in the field indicate that cell-cell communication via autoinducers occurs both within and between bacterial species. Furthermore, there is mounting data suggesting that bacterial autoinducers elicit specific responses from host organisms. Although the nature of the chemical signals, the signal relay mechanisms, and the target genes controlled by bacterial quorum sensing systems differ, in every case the ability to communicate with one another allows bacteria to coordinate the gene expression, and therefore the behavior, of the entire community. Presumably, this process bestows upon bacteria some of the qualities of higher organisms. The evolution of quorum sensing systems in bacteria could, therefore, have been one of the early steps in the development of multicellularity.

  7. Inactivation of biofilm bacteria.

    PubMed Central

    LeChevallier, M W; Cawthon, C D; Lee, R G

    1988-01-01

    The current project was developed to examine inactivation of biofilm bacteria and to characterize the interaction of biocides with pipe surfaces. Unattached bacteria were quite susceptible to the variety of disinfectants tested. Viable bacterial counts were reduced 99% by exposure to 0.08 mg of hypochlorous acid (pH 7.0) per liter (1 to 2 degrees C) for 1 min. For monochloramine, 94 mg/liter was required to kill 99% of the bacteria within 1 min. These results were consistent with those found by other investigators. Biofilm bacteria grown on the surfaces of granular activated carbon particles, metal coupons, or glass microscope slides were 150 to more than 3,000 times more resistant to hypochlorous acid (free chlorine, pH 7.0) than were unattached cells. In contrast, resistance of biofilm bacteria to monochloramine disinfection ranged from 2- to 100-fold more than that of unattached cells. The results suggested that, relative to inactivation of unattached bacteria, monochloramine was better able to penetrate and kill biofilm bacteria than free chlorine. For free chlorine, the data indicated that transport of the disinfectant into the biofilm was a major rate-limiting factor. Because of this phenomenon, increasing the level of free chlorine did not increase disinfection efficiency. Experiments where equal weights of disinfectants were used suggested that the greater penetrating power of monochloramine compensated for its limited disinfection activity. These studies showed that monochloramine was as effective as free chlorine for inactivation of biofilm bacteria. The research provides important insights into strategies for control of biofilm bacteria. Images PMID:2849380

  8. Isolation of isoprene degrading bacteria from soils, development of isoA gene probes and identification of the active isoprene-degrading soil community using DNA-stable isotope probing.

    PubMed

    El Khawand, Myriam; Crombie, Andrew T; Johnston, Antonia; Vavlline, Dmitrii V; McAuliffe, Joseph C; Latone, Jacob A; Primak, Yuliya A; Lee, Sang-Kyu; Whited, Gregg M; McGenity, Terry J; Murrell, J Colin

    2016-09-01

    Emissions of biogenic volatile organic compounds (bVOCs), are an important element in the global carbon cycle, accounting for a significant proportion of fixed carbon. They contribute directly and indirectly to global warming and climate change and have a major effect on atmospheric chemistry. Plants emit isoprene to the atmosphere in similar quantities to emissions of methane from all sources and each accounts for approximately one third of total VOCs. Although methanotrophs, capable of growth on methane, have been intensively studied, we know little of isoprene biodegradation. Here, we report the isolation of two isoprene-degrading strains from the terrestrial environment and describe the design and testing of polymerase chain reaction (PCR) primers targeting isoA, the gene encoding the active-site component of the conserved isoprene monooxygenase, which are capable of retrieving isoA sequences from isoprene-enriched environmental samples. Stable isotope probing experiments, using biosynthesized (13) C-labelled isoprene, identified the active isoprene-degrading bacteria in soil. This study identifies novel isoprene-degrading strains using both culture-dependent and, for the first time, culture-independent methods and provides the tools and foundations for continued investigation of the biogeography and molecular ecology of isoprene-degrading bacteria. © 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

  9. Antibiotics from predatory bacteria

    PubMed Central

    Korp, Juliane; Vela Gurovic, María S

    2016-01-01

    Summary Bacteria, which prey on other microorganisms, are commonly found in the environment. While some of these organisms act as solitary hunters, others band together in large consortia before they attack their prey. Anecdotal reports suggest that bacteria practicing such a wolfpack strategy utilize antibiotics as predatory weapons. Consistent with this hypothesis, genome sequencing revealed that these micropredators possess impressive capacities for natural product biosynthesis. Here, we will present the results from recent chemical investigations of this bacterial group, compare the biosynthetic potential with that of non-predatory bacteria and discuss the link between predation and secondary metabolism. PMID:27340451

  10. Antibiotics from predatory bacteria.

    PubMed

    Korp, Juliane; Vela Gurovic, María S; Nett, Markus

    2016-01-01

    Bacteria, which prey on other microorganisms, are commonly found in the environment. While some of these organisms act as solitary hunters, others band together in large consortia before they attack their prey. Anecdotal reports suggest that bacteria practicing such a wolfpack strategy utilize antibiotics as predatory weapons. Consistent with this hypothesis, genome sequencing revealed that these micropredators possess impressive capacities for natural product biosynthesis. Here, we will present the results from recent chemical investigations of this bacterial group, compare the biosynthetic potential with that of non-predatory bacteria and discuss the link between predation and secondary metabolism.

  11. Indicator For Pseudomonas Bacteria

    NASA Technical Reports Server (NTRS)

    Margalit, Ruth

    1990-01-01

    Characteristic protein extracted and detected. Natural protein marker found in Pseudomonas bacteria. Azurin, protein containing copper readily extracted, purified, and used to prepare antibodies. Possible to develop simple, fast, and accurate test for marker carried out in doctor's office.

  12. [Darwin and bacteria].

    PubMed

    Ledermann D, Walter

    2009-02-01

    As in 2009 the scientific world celebrates two hundreds years from the birthday of Charles Darwin and one hundred and fifty from the publication of The Origin of Species, an analysis of his complete work is performed, looking for any mention of bacteria. But it seems that the great naturahst never took knowledge about its existence, something rather improbable in a time when the discovery of bacteria shook the medical world, or he deliberately ignored them, not finding a place for such microscopic beings into his theory of evolution. But the bacteria badly affected his familiar life, killing scarlet fever one of his children and worsening to death the evolution of tuberculosis of his favourite Annie. Darwin himself could suffer the sickness of Chagas, whose etiological agent has a similar level to bacteria in the scale of evolution.

  13. Indicator For Pseudomonas Bacteria

    NASA Technical Reports Server (NTRS)

    Margalit, Ruth

    1990-01-01

    Characteristic protein extracted and detected. Natural protein marker found in Pseudomonas bacteria. Azurin, protein containing copper readily extracted, purified, and used to prepare antibodies. Possible to develop simple, fast, and accurate test for marker carried out in doctor's office.

  14. [Geographical distribution of magnetotactic bacteria].

    PubMed

    Zhang, Wen-yan; Zhang, Sheng-da; Xiao, Tian; Pan, Yong-xin; Wu, Long-fei

    2010-02-01

    Magnetotactic bacteria (MTB) are widely distributed in aquatic environments. To assess the correlation between their evolutionary relatedness and geographic distribution, we analyzed 239 16S rDNA sequences available in the Genbank, and constructed phylogenetic trees based on the sequences. After elimination of redundant sequences by grouping those with identity > 97% into a single one, we analyzed in detail total 139 16S rDNA sequences, including 55 from marine MTB and 82 from freshwater sequences, and belonging to Proteobacteria and Nitrospirae. Phylogeny analysis based on those sequences suggests that the geographical distribution of MTB has certain regional distribution character: marine MTB is distinct from freshwater MTB, and off coast MTB are remotely related with ocean MTB. In contrast, the MTB from similar habitats, such as offshore in Brazil and the United States or freshwater lakes in Germany and China, are closely related. It is found that similar species have a large geographic distribution and tend to adopt the similar habitats, morphotypes of MTB and their living environment conditions have a significant relevance. This observation suggests that MTB may have multiple evolutionary origins. And also, it suggests the environmental conditions, as an important evolutionary pressure, play an important role in the long-term evolution of MTB.

  15. Gut bacteria and cancer

    PubMed Central

    Erdman, Susan E.; Poutahidis, Theofilos

    2015-01-01

    Microbiota on the mucosal surfaces of the gastrointestinal (GI) tract greatly outnumber the cells in the human body. Effects of antibiotics indicate that GI tract bacteria may be determining the fate of distal cancers. Recent data implicate dysregulated host responses to enteric bacteria leading to cancers in extra-intestinal sites. Together these findings point to novel anti-cancer strategies aimed at promoting GI tract homeostasis. PMID:26050963

  16. Lipopolysaccharides in diazotrophic bacteria.

    PubMed

    Serrato, Rodrigo V

    2014-01-01

    Biological nitrogen fixation (BNF) is a process in which the atmospheric nitrogen (N2) is transformed into ammonia (NH3) by a select group of nitrogen-fixing organisms, or diazotrophic bacteria. In order to furnish the biologically useful nitrogen to plants, these bacteria must be in constant molecular communication with their host plants. Some of these molecular plant-microbe interactions are very specific, resulting in a symbiotic relationship between the diazotroph and the host. Others are found between associative diazotrophs and plants, resulting in plant infection and colonization of internal tissues. Independent of the type of ecological interaction, glycans, and glycoconjugates produced by these bacteria play an important role in the molecular communication prior and during colonization. Even though exopolysaccharides (EPS) and lipochitooligosaccharides (LCO) produced by diazotrophic bacteria and released onto the environment have their importance in the microbe-plant interaction, it is the lipopolysaccharides (LPS), anchored on the external membrane of these bacteria, that mediates the direct contact of the diazotroph with the host cells. These molecules are extremely variable among the several species of nitrogen fixing-bacteria, and there are evidences of the mechanisms of infection being closely related to their structure.

  17. Aerobic Anoxygenic Phototrophic Bacteria

    PubMed Central

    Yurkov, Vladimir V.; Beatty, J. Thomas

    1998-01-01

    The aerobic anoxygenic phototrophic bacteria are a relatively recently discovered bacterial group. Although taxonomically and phylogenetically heterogeneous, these bacteria share the following distinguishing features: the presence of bacteriochlorophyll a incorporated into reaction center and light-harvesting complexes, low levels of the photosynthetic unit in cells, an abundance of carotenoids, a strong inhibition by light of bacteriochlorophyll synthesis, and the inability to grow photosynthetically under anaerobic conditions. Aerobic anoxygenic phototrophic bacteria are classified in two marine (Erythrobacter and Roseobacter) and six freshwater (Acidiphilium, Erythromicrobium, Erythromonas, Porphyrobacter, Roseococcus, and Sandaracinobacter) genera, which phylogenetically belong to the α-1, α-3, and α-4 subclasses of the class Proteobacteria. Despite this phylogenetic information, the evolution and ancestry of their photosynthetic properties are unclear. We discuss several current proposals for the evolutionary origin of aerobic phototrophic bacteria. The closest phylogenetic relatives of aerobic phototrophic bacteria include facultatively anaerobic purple nonsulfur phototrophic bacteria. Since these two bacterial groups share many properties, yet have significant differences, we compare and contrast their physiology, with an emphasis on morphology and photosynthetic and other metabolic processes. PMID:9729607

  18. Phylogenetic analysis on the bacteria producing non-volatile fungistatic substances.

    PubMed

    Li, ZhiFang; Zou, ChangSong; He, YueQiu; Mo, MingHe; Zhang, KeQin

    2008-06-01

    This study characterized the soil bacteria producing non-volatile fungistatic substances. Among the 2,100 colonies of soil bacteria randomly isolated from seven agricultural soil samples, 518 isolates (24.67% of total) showed fungistatic activity toward nematophagous fungi Paecilomyces lilacinus and Trichoderma viride by producing non-volatile substances. A phylogenetic analysis based on amplified ribosomal DNA restriction analysis (ARDRA) and 16S rDNA sequence placed the 518 bacteria in three groups of the domain Bacteria: Actinomycetales, Bacillales, and Gammaproteobacteria. Three genera, Arthrobacter, Bacillus, and Pseudomonas, were the most frequently encountered groups.

  19. Mismatch repair in heteroduplex DNA.

    PubMed Central

    Wildenberg, J; Meselson, M

    1975-01-01

    DNA with base pair mismatches was prepared by annealing mixtures of genetically marked DNA from bacteriophage lambda. This heteroduplex DNA was used to transfect bacteria under conditions minimizing recombination. Genetic analysis of the progeny phages indicates that: (i) Mismatch repair occurs, usually giving rise to a DNA molecule with one chain with the genotype arising from repair and one parental chain. (ii) The frequency of repair of a given mismatch to wild type depends on the marker, ranging from 3 to 20%. (iii) Excision tracts may extend several hundred nucleotides but are usually shorter than about 2000 nucleotides. (iv) In Rec-mediated bacteriophage crosses, recombination of markers closer than about 10-3 nucleotide pairs frequently occurs by mismatch repair within heteroduplex DNA. (V) The average amount of heteroduplex DNA formed in a Rec-mediated recombination event is a few thousand nucleotide pairs. PMID:1094458

  20. The fecal bacteria

    USGS Publications Warehouse

    Sadowsky, Michael J.; Whitman, Richard L.

    2011-01-01

    The Fecal Bacteria offers a balanced, integrated discussion of fecal bacteria and their presence and ecology in the intestinal tract of mammals, in the environment, and in the food supply. This volume covers their use in examining and assessing water quality in order to offer protection from illnesses related to swimming in or ingesting contaminated water, in addition to discussing their use in engineering considerations of water quality, modeling, monitoring, and regulations. Fecal bacteria are additionally used as indicators of contamination of ready-to-eat foods and fresh produce. The intestinal environment, the microbial community structure of the gut microbiota, and the physiology and genomics of this broad group of microorganisms are explored in the book. With contributions from an internationally recognized group of experts, the book integrates medicine, public health, environmental, and microbiological topics in order to provide a unique, holistic understanding of fecal bacteria. Moreover, it shows how the latest basic science and applied research findings are helping to solve problems and develop effective management strategies. For example, readers will discover how the latest tools and molecular approaches have led to our current understanding of fecal bacteria and enabled us to improve human health and water quality. The Fecal Bacteria is recommended for microbiologists, clinicians, animal scientists, engineers, environmental scientists, food safety experts, water quality managers, and students. It will help them better understand fecal bacteria and use their knowledge to protect human and environmental health. They can also apply many of the techniques and molecular tools discussed in this book to the study of a broad range of microorganisms in a variety of habitats.

  1. MOTILE MARINE BACTERIA I.

    PubMed Central

    Leifson, Einar; Cosenza, B. J.; Murchelano, R.; Cleverdon, R. C.

    1964-01-01

    Leifson, Einar (Loyola University, Chicago, Ill.), B. J. Cosenza, R. Murchelano, and R. C. Cleverdon. Motile marine bacteria. I. Techniques, ecology, and general characteristics. J. Bacteriol. 87:652–666. 1964.—Aerobic, heterotrophic bacteria were isolated from the waters of the Long Island Sound, Narragansett Bay, Atlantic Ocean, and from the intestine of a variety of marine animals found along the shore of the Long Island Sound. A total of about 600 cultures of motile bacteria were studied morphologically and physiologically, with special emphasis on flagellar characteristics. The great majority of the bacteria isolated from the water were polar flagellate, nonfermentative, nonpigmented, and gramnegative. Most of these were straight, capsulated rods, but a considerable number were curved like vibrios. Yellow-pigmented isolates were often nonmotile, and the motile forms were most frequently subpolar flagellate. Several rosette-forming bacteria, including Caulobacter species, were isolated. Two typical spirilla and one flagellated coccus were found. Peritrichous flagellate bacteria, both gram-positive and gram-negative, were rare except in bottom mud. The normal intestinal flora of marine animals, such as fish and shellfish, consisted of polar flagellate, fermentative, non-pigmented, gram-negative, straight rods. Curved forms, like vibrios, were less common. Polar multitrichous flagellate forms were not uncommon and included all the luminescent types isolated. A considerable proportion of the polar monotrichous flagellate rods swarmed over the surface of agar media. When grown on solid media, all of these showed mixed polar and lateral flagellation; in liquid media, mainly polar flagellation was found. The ecology and general taxonomy of marine bacteria are discussed. Images PMID:14129669

  2. Thermal control of virulence factors in bacteria: A hot topic

    PubMed Central

    Lam, Oliver; Wheeler, Jun; Tang, Christoph M

    2014-01-01

    Pathogenic bacteria sense environmental cues, including the local temperature, to control the production of key virulence factors. Thermal regulation can be achieved at the level of DNA, RNA or protein and although many virulence factors are subject to thermal regulation, the exact mechanisms of control are yet to be elucidated in many instances. Understanding how virulence factors are regulated by temperature presents a significant challenge, as gene expression and protein production are often influenced by complex regulatory networks involving multiple transcription factors in bacteria. Here we highlight some recent insights into thermal regulation of virulence in pathogenic bacteria. We focus on bacteria which cause disease in mammalian hosts, which are at a significantly higher temperature than the outside environment. We outline the mechanisms of thermal regulation and how understanding this fundamental aspect of the biology of bacteria has implications for pathogenesis and human health. PMID:25494856

  3. Natural soil reservoirs for human pathogenic and fecal indicator bacteria

    USGS Publications Warehouse

    Boschiroli, Maria L; Falkinham, Joseph; Favre-Bonte, Sabine; Nazaret, Sylvie; Piveteau, Pascal; Sadowsky, Michael J.; Byappanahalli, Muruleedhara; Delaquis, Pascal; Hartmann, Alain

    2016-01-01

    Soils receive inputs of human pathogenic and indicator bacteria through land application of animal manures or sewage sludge, and inputs by wildlife. Soil is an extremely heterogeneous substrate and contains meso- and macrofauna that may be reservoirs for bacteria of human health concern. The ability to detect and quantify bacteria of human health concern is important in risk assessments and in evaluating the efficacy of agricultural soil management practices that are protective of crop quality and protective of adjacent water resources. The present chapter describes the distribution of selected Gram-positive and Gram-negative bacteria in soils. Methods for detecting and quantifying soilborne bacteria including extraction, enrichment using immunomagnetic capture, culturing, molecular detection and deep sequencing of metagenomic DNA to detect pathogens are overviewed. Methods for strain phenotypic and genotypic characterization are presented, as well as how comparison with clinical isolates can inform the potential for human health risk.

  4. Light-dependent gene regulation in nonphototrophic bacteria.

    PubMed

    Elías-Arnanz, Montserrat; Padmanabhan, S; Murillo, Francisco J

    2011-04-01

    Bacteria sense and respond to light, a fundamental environmental factor, by employing highly evolved machineries and mechanisms. Cellular systems exist to harness light energy usefully as in phototrophic bacteria, to combat photo-oxidative damage stemming from the highly reactive species generated on absorption of light energy, and to link the light stimulus to DNA repair, taxis, development, and virulence. Recent findings on the genetic response to light in nonphototrophic bacteria highlight the ingenious transcriptional regulatory mechanisms and the panoply of factors that have evolved to perceive and transmit the signal, and to bring about finely tuned gene expression.

  5. Utilization of hexamethylenetetramine (urotropine) by bacteria and yeasts.

    PubMed

    Middelhoven, Wouter J; van Doesburg, Wim

    2007-02-01

    A slow growing bacterial population able to utilize hexamethylelenetetramine (urotropine) as sole source of carbon, nitrogen and energy was isolated from soil. From this crude enrichment culture two bacteria were isolated and identified as Brevundimonas diminuta and a Phyllobacterium sp. by sequencing of 16S ribosomal DNA. These bacteria also grew on urotropine but at a lower rate than the enrichment culture. Addition of glucose to the latter resulted in growth of some yeasts that overgrew the bacteria. Assimilation of urotropine as sole nitrogen source is very common among yeasts, 46 out of 60 species tested showed this characteristic.

  6. Amoeba-resisting bacteria and ventilator-associated pneumonia.

    PubMed

    La Scola, Bernard; Boyadjiev, Ioanna; Greub, Gilbert; Khamis, Atieh; Martin, Claude; Raoult, Didier

    2003-07-01

    To evaluate the role of amoeba-associated bacteria as agents of ventilator-associated pneumonia (VAP), we tested the water from an intensive care unit (ICU) every week for 6 months for such bacteria isolates; serum samples and bronchoalveolar lavage samples (BAL) were also obtained from 30 ICU patients. BAL samples were examined for amoeba-associated bacteria DNA by suicide-polymerase chain reaction, and serum samples were tested against ICU amoeba-associated bacteria. A total of 310 amoeba-associated bacteria from 10 species were isolated. Twelve of 30 serum samples seroconverted to one amoeba-associated bacterium isolated in the ICU, mainly Legionella anisa and Bosea massiliensis, the most common isolates from water (p=0.021). Amoeba-associated bacteria DNA was detected in BAL samples from two patients whose samples later seroconverted. Seroconversion was significantly associated with VAP and systemic inflammatory response syndrome, especially in patients for whom no etiologic agent was found by usual microbiologic investigations. Amoeba-associated bacteria might be a cause of VAP in ICUs, especially when microbiologic investigations are negative.

  7. Amoeba-Resisting Bacteria and Ventilator-Associated Pneumonia

    PubMed Central

    La Scola, Bernard; Boyadjiev, Ioanna; Greub, Gilbert; Khamis, Atieh; Martin, Claude

    2003-01-01

    To evaluate the role of amoeba-associated bacteria as agents of ventilator-associated pneumonia (VAP), we tested the water from an intensive care unit (ICU) every week for 6 months for such bacteria isolates; serum samples and bronchoalveolar lavage samples (BAL) were also obtained from 30 ICU patients. BAL samples were examined for amoeba-associated bacteria DNA by suicide-polymerase chain reaction, and serum samples were tested against ICU amoeba-associated bacteria. A total of 310 amoeba-associated bacteria from10 species were isolated. Twelve of 30 serum samples seroconverted to one amoeba-associated bacterium isolated in the ICU, mainly Legionella anisa and Bosea massiliensis, the most common isolates from water (p=0.021). Amoeba-associated bacteria DNA was detected in BAL samples from two patients whose samples later seroconverted. Seroconversion was significantly associated with VAP and systemic inflammatory response syndrome, especially in patients for whom no etiologic agent was found by usual microbiologic investigations. Amoeba-associated bacteria might be a cause of VAP in ICUs, especially when microbiologic investigations are negative. PMID:12890321

  8. Diversification of DnaA dependency for DNA replication in cyanobacterial evolution.

    PubMed

    Ohbayashi, Ryudo; Watanabe, Satoru; Ehira, Shigeki; Kanesaki, Yu; Chibazakura, Taku; Yoshikawa, Hirofumi

    2016-05-01

    Regulating DNA replication is essential for all living cells. The DNA replication initiation factor DnaA is highly conserved in prokaryotes and is required for accurate initiation of chromosomal replication at oriC. DnaA-independent free-living bacteria have not been identified. The dnaA gene is absent in plastids and some symbiotic bacteria, although it is not known when or how DnaA-independent mechanisms were acquired. Here, we show that the degree of dependency of DNA replication on DnaA varies among cyanobacterial species. Deletion of the dnaA gene in Synechococcus elongatus PCC 7942 shifted DNA replication from oriC to a different site as a result of the integration of an episomal plasmid. Moreover, viability during the stationary phase was higher in dnaA disruptants than in wild-type cells. Deletion of dnaA did not affect DNA replication or cell growth in Synechocystis sp. PCC 6803 or Anabaena sp. PCC 7120, indicating that functional dependency on DnaA was already lost in some nonsymbiotic cyanobacterial lineages during diversification. Therefore, we proposed that cyanobacteria acquired DnaA-independent replication mechanisms before symbiosis and such an ancestral cyanobacterium was the sole primary endosymbiont to form a plastid precursor.

  9. The RecQ DNA helicases in DNA Repair

    PubMed Central

    Bernstein, Kara A.; Gangloff, Serge; Rothstein, Rodney

    2014-01-01

    The RecQ helicases are conserved from bacteria to humans and play a critical role in genome stability. In humans, loss of RecQ gene function is associated with cancer predisposition and/or premature aging. Recent data have shown that the RecQ helicases function during two distinct steps during DNA repair; DNA end resection and resolution of double Holliday junctions (dHJs). RecQ functions in these different processing steps has important implications for its role in repair of double-strand breaks (DSBs) that occur during DNA replication, meiosis and at specific genomic loci such as telomeres. PMID:21047263

  10. Ice-Nucleating Bacteria

    NASA Astrophysics Data System (ADS)

    Obata, Hitoshi

    Since the discovery of ice-nucleating bacteria in 1974 by Maki et al., a large number of studies on the biological characteristics, ice-nucleating substance, ice nucleation gene and frost damage etc. of the bacteria have been carried out. Ice-nucleating bacteria can cause the freezing of water at relatively warm temperature (-2.3°C). Tween 20 was good substrates for ice-nucleating activity of Pseudomonas fluorescens KUIN-1. Major fatty acids of Isolate (Pseudomonas fluorescens) W-11 grown at 30°C were palmitic, cis-9-hexadecenoic and cis-11-octadecenoic which amounted to 90% of the total fatty acids. Sequence analysis shows that an ice nucleation gene from Pseudomonas fluorescens is related to the gene of Pseudomonas syringae.

  11. Intestinal Bacteria Composition and Translocation of Bacteria in Inflammatory Bowel Disease

    PubMed Central

    Vrakas, Spyros; Mountzouris, Konstantinos C.; Michalopoulos, George; Karamanolis, George; Papatheodoridis, George; Tzathas, Charalampos

    2017-01-01

    Background Live commensal intestinal bacteria are present in the peripheral blood where they can induce inflammation. Objective To evaluate the intestinal bacteria composition and translocation of bacteria in IBD. Methods Both blood and tissue biopsy samples were collected from adult patients with active/inactive Crohn’s disease (CD), active/inactive ulcerative colitis (UC) and healthy individuals. Most of the patients were newly diagnosed and none of them received antibiotics. Using a reverse transcription–quantitative real-time PCR (RT-qPCR) method, we determined the composition of microbiota. NOD2/CARD15 genotyping was also studied. Results Total bacterial DNA concentration was increased in tissue and blood samples of IBD patients compared to healthy controls. Furthermore, the active IBD cases had higher total bacterial DNA concentration levels compared to the inactive cases. Three species characterized dysbiosis in IBD, namely an increase of Bacteroides spp in active and inactive IBD samples, and a decrease in Clostridium leptum group (IV), and Faecalibacterium prausnitzi in both active and inactive IBD patients. No significant association between bacterial translocation and NOD2/CARD15 mutations was found. Conclusions The composition of the microbiota in IBD patients differs from that of healthy controls. The high rate of bacterial DNA in the blood samples indicates translocation in inflammatory bowel disease. PMID:28099495

  12. Conjugation in Gram-Positive Bacteria.

    PubMed

    Goessweiner-Mohr, Nikolaus; Arends, Karsten; Keller, Walter; Grohmann, Elisabeth

    2014-08-01

    Conjugative transfer is the most important means of spreading antibiotic resistance and virulence factors among bacteria. The key vehicles of this horizontal gene transfer are a group of mobile genetic elements, termed conjugative plasmids. Conjugative plasmids contain as minimum instrumentation an origin of transfer (oriT), DNA-processing factors (a relaxase and accessory proteins), as well as proteins that constitute the trans-envelope transport channel, the so-called mating pair formation (Mpf) proteins. All these protein factors are encoded by one or more transfer (tra) operons that together form the DNA transport machinery, the Gram-positive type IV secretion system. However, multicellular Gram-positive bacteria belonging to the streptomycetes appear to have evolved another mechanism for conjugative plasmid spread reminiscent of the machinery involved in bacterial cell division and sporulation, which transports double-stranded DNA from donor to recipient cells. Here, we focus on the protein key players involved in the plasmid spread through the two different modes and present a new secondary structure homology-based classification system for type IV secretion protein families. Moreover, we discuss the relevance of conjugative plasmid transfer in the environment and summarize novel techniques to visualize and quantify conjugative transfer in situ.

  13. Hybrid origin of Japanese mice "Mus musculus molossinus": evidence from restriction analysis of mitochondrial DNA.

    PubMed

    Yonekawa, H; Moriwaki, K; Gotoh, O; Miyashita, N; Matsushima, Y; Shi, L M; Cho, W S; Zhen, X L; Tagashira, Y

    1988-01-01

    The Japanese mouse, Mus musculus molossinus, has long been considered an independent subspecies of the house mouse. A survey of restriction-site haplotypes of mitochondrial DNA (mtDNA) showed that Japanese mice have two main maternal lineages. The most common haplotype is closely related to the mtDNA of the European subspecies M. m. musculus. The other common haplotype and two minor ones are closely related to each other and to the mtDNA of an Asiatic subspecies, M. m. castaneus. Two other rare variants are probably the result of recent contamination by European M. m. domesticus. The musculus type of mtDNA is found in the southern two-thirds of Japan, whereas the common castaneus type is found in the northern third and the minor variants are found sporadically throughout Japan. The castaneus mtDNA lineage had a few minor variants, whereas the musculus lineage was completely monomorphic. By contrast, the native population of M. m. castaneus and the Chinese and Korean musculus populations were highly polymorphic. These results suggest that M. m. molossinus is a hybrid between ancestral colonies, possibly very small, of M. m. musculus and M. m. castaneus, rather than an independent subspecies.

  14. Have sex or not? Lessons from bacteria.

    PubMed

    Lodé, T

    2012-01-01

    Sex is one of the greatest puzzles in evolutionary biology. A true meiotic process occurs only in eukaryotes, while in bacteria, gene transcription is fragmentary, so asexual reproduction in this case really means clonal reproduction. Sex could stem from a signal that leads to increased reproductive output of all interacting individuals and could be understood as a secondary consequence of primitive metabolic reactions. Meiotic sex evolved in proto-eukaryotes to solve a problem that bacteria did not have, namely a large amount of DNA material, occurring in an archaic step of proto-cell formation and genetic exchanges. Rather than providing selective advantages through reproduction, sex could be thought of as a series of separate events which combines step-by-step some very weak benefits of recombination, meiosis, gametogenesis and syngamy.

  15. Enteric bacteria mandibular osteomyelitis.

    PubMed

    Scolozzi, Paolo; Lombardi, Tommaso; Edney, Timothy; Jaques, Bertrand

    2005-06-01

    Osteomyelitis of the mandible is a relatively rare inflammatory disease that usually stems from the odontogenic polymicrobial flora of the oral cavity. We are reporting 2 unusual cases of mandibular osteomyelitis resulting from enteric bacteria infection. In one patient, abundant clinical evidence suggested a diagnosis of a chronic factitious disease, whereas in the second patient no obvious etiology was found.

  16. Antibiotic-Resistant Bacteria.

    ERIC Educational Resources Information Center

    Longenecker, Nevin E.; Oppenheimer, Dan

    1982-01-01

    A study conducted by high school advanced bacteriology students appears to confirm the hypothesis that the incremental administration of antibiotics on several species of bacteria (Escherichia coli, Staphylococcus epidermis, Bacillus sublitus, Bacillus megaterium) will allow for the development of antibiotic-resistant strains. (PEB)

  17. Monoclonal antibodies against bacteria.

    PubMed

    Macario, A J; Conway de Macario, E

    1988-01-01

    Highlights are presented of most recent work in which monoclonal antibodies have been instrumental in the study of bacteria and their products. Topics summarized pertain to human and veterinary medicines, dentistry, phytopathology, ichthyology, and bacterial ecophysiology, differentiation, evolution and methanogenic biotechnology.

  18. Bacteria-surface interactions.

    PubMed

    Tuson, Hannah H; Weibel, Douglas B

    2013-05-14

    The interaction of bacteria with surfaces has important implications in a range of areas, including bioenergy, biofouling, biofilm formation, and the infection of plants and animals. Many of the interactions of bacteria with surfaces produce changes in the expression of genes that influence cell morphology and behavior, including genes essential for motility and surface attachment. Despite the attention that these phenotypes have garnered, the bacterial systems used for sensing and responding to surfaces are still not well understood. An understanding of these mechanisms will guide the development of new classes of materials that inhibit and promote cell growth, and complement studies of the physiology of bacteria in contact with surfaces. Recent studies from a range of fields in science and engineering are poised to guide future investigations in this area. This review summarizes recent studies on bacteria-surface interactions, discusses mechanisms of surface sensing and consequences of cell attachment, provides an overview of surfaces that have been used in bacterial studies, and highlights unanswered questions in this field.

  19. PATHOGENICITY OF BIOFILM BACTERIA

    EPA Science Inventory

    There is a paucity of information concerning any link between the microorganisms commonly found in biofilms of drinking water systems and their impacts on human health. For bacteria, culture-based techniques detect only a limited number of the total microorganisms associated wit...

  20. Antibiotic-Resistant Bacteria.

    ERIC Educational Resources Information Center

    Longenecker, Nevin E.; Oppenheimer, Dan

    1982-01-01

    A study conducted by high school advanced bacteriology students appears to confirm the hypothesis that the incremental administration of antibiotics on several species of bacteria (Escherichia coli, Staphylococcus epidermis, Bacillus sublitus, Bacillus megaterium) will allow for the development of antibiotic-resistant strains. (PEB)

  1. Bacteria-surface interactions

    PubMed Central

    Tuson, Hannah H.; Weibel, Douglas B.

    2013-01-01

    The interaction of bacteria with surfaces has important implications in a range of areas, including bioenergy, biofouling, biofilm formation, and the infection of plants and animals. Many of the interactions of bacteria with surfaces produce changes in the expression of genes that influence cell morphology and behavior, including genes essential for motility and surface attachment. Despite the attention that these phenotypes have garnered, the bacterial systems used for sensing and responding to surfaces are still not well understood. An understanding of these mechanisms will guide the development of new classes of materials that inhibit and promote cell growth, and complement studies of the physiology of bacteria in contact with surfaces. Recent studies from a range of fields in science and engineering are poised to guide future investigations in this area. This review summarizes recent studies on bacteria-surface interactions, discusses mechanisms of surface sensing and consequences of cell attachment, provides an overview of surfaces that have been used in bacterial studies, and highlights unanswered questions in this field. PMID:23930134

  2. Ribonucleotides in DNA: Origins, repair and consequences

    PubMed Central

    Williams, Jessica S.; Kunkel, Thomas A.

    2014-01-01

    While primordial life is thought to have been RNA-based (Cech, Cold Spring Harbor Perspect. Biol. 4 (2012) a006742), all living organisms store genetic information in DNA, which is chemically more stable. Distinctions between the RNA and DNA worlds and our views of “DNA” synthesis continue to evolve as new details emerge on the incorporation, repair and biological effects of ribonucleotides in DNA genomes of organisms from bacteria through humans. PMID:24794402

  3. Degradation of phenanthrene on plant leaves by phyllosphere bacteria.

    PubMed

    Waight, Karen; Pinyakong, Onruthai; Luepromchai, Ekawan

    2007-10-01

    The activity of phyllosphere bacteria in the degradation of phenanthrene was investigated as a mechanism for the removal of atmospheric phenanthrene after its deposition on plant leaves. Initially, leaf samples of six plant species were collected from two roadsides in Bangkok to determine the presence of phenanthrene-degrading bacteria. The numbers of phenanthrene-degrading phyllosphere bacteria were varied and ranged from 3.5 x 10(4) to 1.95 x 10(7) CFU/g, in which the highest number was found from Ixora sp. Further studies were carried out in the laboratory by spraying phenanthrene on Ixora sp. leaves and then monitoring the amount of deposited phenanthrene and number of phenanthrene-degrading bacteria after incubation. The results showed that the amount of phenanthrene was significantly reduced on leaves containing phenanthrene-degrading bacteria. These were detected along with a rapid increase in the number of bacteria on leaves. The results indicated that many phyllosphere bacteria could utilize phenanthrene to support their growth and thereby reduce the amount of deposited phenanthrene on leaf surfaces. Several phenanthrene-degrading bacteria were later isolated from the leaves and identified with a high 16S rDNA sequence similarity to the genera Pseudomonas, Microbacterium, Rhizobium, and Deinococcus.

  4. The first demonstration of the existence of reverse transcriptases in bacteria.

    PubMed

    Inouye, Masayori

    2017-01-15

    It has been long thought that reverse transcriptases are unique to the eukaryotes. However, through our research on a peculiar single stranded DNA called msDNA in Myxococcus xanthus, it was predicted that its synthesis requires reverse transcriptases. Subsequently, Lim and Maas as well as our group demonstrated the existence of reverse transcriptases for the production of msDNA. In this review, I describe how the discovery of msDNA led to the discovery of reverse transcriptases in bacteria and discuss the evolutionary significance of the discovery of revise transcriptases in bacteria.

  5. R-body-producing bacteria.

    PubMed Central

    Pond, F R; Gibson, I; Lalucat, J; Quackenbush, R L

    1989-01-01

    Until 10 years ago, R bodies were known only as diagnostic features by which endosymbionts of paramecia were identified as kappa particles. They were thought to be limited to the cytoplasm of two species in the Paramecium aurelia species complex. Now, R bodies have been found in free-living bacteria and other Paramecium species. The organisms now known to form R bodies include the cytoplasmic kappa endosymbionts of P. biaurelia and P. tetraurelia, the macronuclear kappa endosymbionts of P. caudatum, Pseudomonas avenae (a free-living plant pathogen), Pseudomonas taeniospiralis (a hydrogen-oxidizing soil microorganism), Rhodospirillum centenum (a photosynthetic bacterium), and a soil bacterium, EPS-5028, which is probably a pseudomonad. R bodies themselves fall into five distinct groups, distinguished by size, the morphology of the R-body ribbons, and the unrolling behavior of wound R bodies. In recent years, the inherent difficulties in studying the organization and assembly of R bodies by the obligate endosymbiont kappa, have been alleviated by cloning and expressing genetic determinants for these R bodies (type 51) in Escherichia coli. Type 51 R-body synthesis requires three low-molecular-mass polypeptides. One of these is modified posttranslationally, giving rise to 12 polypeptide species, which are the major structural subunits of the R body. R bodies are encoded in kappa species by extrachromosomal elements. Type 51 R bodies, produced in Caedibacter taeniospiralis, are encoded by a plasmid, whereas bacteriophage genomes probably control R-body synthesis in other kappa species. However, there is no evidence that either bacteriophages or plasmids are present in P. avenae or P. taeniospiralis. No sequence homology was detected between type 51 R-body-encoding DNA and DNA from any R-body-producing species, except C. varicaedens 1038. The evolutionary relatedness of different types of R bodies remains unknown. Images PMID:2651865

  6. DNA Charge Transport within the Cell

    PubMed Central

    Grodick, Michael A.; Muren, Natalie B.; Barton, Jacqueline K.

    2015-01-01

    The unique characteristics of DNA charge transport (CT) have prompted an examination of roles for this chemistry within a biological context. Not only can DNA CT facilitate long range oxidative damage of DNA, but redox-active proteins can couple to the DNA base stack and participate in long range redox reactions using DNA CT. DNA transcription factors with redox-active moieties such as SoxR and p53 can use DNA CT as a form of redox sensing. DNA CT chemistry also provides a means to monitor the integrity of the DNA, given the sensitivity of DNA CT to perturbations in base stacking as arise with mismatches and lesions. Enzymes that utilize this chemistry include an interesting and ever-growing class of DNA-processing enzymes involved in DNA repair, replication, and transcription that have been found to contain 4Fe-4S clusters. DNA repair enzymes containing 4Fe-4S clusters, that include Endonuclease III (EndoIII), MutY, and DinG from bacteria, as well as XPD from archaea, have been shown to be redox-active when bound to DNA, share a DNA-bound redox potential, and can be reduced and oxidized at long range via DNA CT. Interactions between DNA and these proteins in solution, in addition to genetics experiments within E. coli, suggest that DNA-mediated CT can be used as a means of cooperative signaling among DNA repair proteins that contain 4Fe-4S clusters as a first step in finding DNA damage, even within cells. Based on these data, we can consider also how DNA-mediated CT may be used as a means of signaling to coordinate DNA processing across the genome. PMID:25606780

  7. Seed-vectored endophytic bacteria modulate development of rice seedlings.

    PubMed

    Verma, S K; Kingsley, K; Irizarry, I; Bergen, M; Kharwar, R N; White, J F

    2017-06-01

    The aim of the present study was to evaluate the effects of the removal of indigenous bacteria from rice seeds on seedling growth and development. Here we report the presence of three indigenous endophytic bacteria in rice seeds that play important roles in modulating seedling development (shoot and root lengths, and formation of root hairs and secondary roots) and defence against pathogens. Seed-associated bacteria were removed using surface sterilization with NaOCl (bleach) followed by antibiotic treatment. When bacteria were absent, growth of seedlings in terms of root hair development and overall seedling size was less than that of seedlings that contained bacteria. Reactive oxygen staining of seedlings showed that endophytic bacteria became intracellular in root parenchyma cells and root hairs. Roots containing endophytic bacteria were seen to stain densely for reactive oxygen, while roots free of bacteria stained lightly for reactive oxygen. Bacteria were isolated and identified as Enterobacter asburiae (VWB1), Pantoea dispersa (VWB2) and Pseudomonas putida (VWB3) by 16S rDNA sequencing. Bacteria were found to produce indole acetic acid (auxins), inhibited the pathogen Fusarium oxysporum and solubilized phosphate. Reinoculation of bacteria onto seedlings derived from surface-disinfected rice and Bermuda grass seeds significantly restored seedling growth and development. Rice seeds harbour indigenous bacterial endophytes that greatly influence seedling growth and development, including root and shoot lengths, root hair formation and disease susceptibility of rice seedlings. This study shows that seeds of rice naturally harbour bacterial endophytes that play key roles in modulation of seedling development. © 2017 The Society for Applied Microbiology.

  8. DNA recovery from soils of diverse composition.

    PubMed

    Zhou, J; Bruns, M A; Tiedje, J M

    1996-02-01

    A simple, rapid method for bacterial lysis and direct extraction of DNA from soils with minimal shearing was developed to address the risk of chimera formation from small template DNA during subsequent PCR. The method was based on lysis with a high-salt extraction buffer (1.5 M NaCl) and extended heating (2 to 3 h) of the soil suspension in the presence of sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide, and proteinase K. The extraction method required 6 h and was tested on eight soils differing in organic carbon, clay content, and pH, including ones from which DNA extraction is difficult. The DNA fragment size in crude extracts from all soils was > 23 kb. Preliminary trials indicated that DNA recovery from two soils seeded with gram-negative bacteria was 92 to 99%. When the method was tested on all eight unseeded soils, microscopic examination of indigenous bacteria in soil pellets before and after extraction showed variable cell lysis efficiency (26 to 92%). Crude DNA yields from the eight soils ranged from 2.5 to 26.9 micrograms of DNA g-1, and these were positively correlated with the organic carbon content in the soil (r = 0.73). DNA yields from gram-positive bacteria from pure cultures were two to six times higher when the high-salt-SDS-heat method was combined with mortar-and-pestle grinding and freeze-thawing, and most DNA recovered was of high molecular weight. Four methods for purifying crude DNA were also evaluated for percent recovery, fragment size, speed, enzyme restriction, PCR amplification, and DNA-DNA hybridization. In general, all methods produced DNA pure enough for PCR amplification. Since soil type and microbial community characteristics will influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods on the basis of experimental goals.

  9. DNA recovery from soils of diverse composition.

    PubMed Central

    Zhou, J; Bruns, M A; Tiedje, J M

    1996-01-01

    A simple, rapid method for bacterial lysis and direct extraction of DNA from soils with minimal shearing was developed to address the risk of chimera formation from small template DNA during subsequent PCR. The method was based on lysis with a high-salt extraction buffer (1.5 M NaCl) and extended heating (2 to 3 h) of the soil suspension in the presence of sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide, and proteinase K. The extraction method required 6 h and was tested on eight soils differing in organic carbon, clay content, and pH, including ones from which DNA extraction is difficult. The DNA fragment size in crude extracts from all soils was > 23 kb. Preliminary trials indicated that DNA recovery from two soils seeded with gram-negative bacteria was 92 to 99%. When the method was tested on all eight unseeded soils, microscopic examination of indigenous bacteria in soil pellets before and after extraction showed variable cell lysis efficiency (26 to 92%). Crude DNA yields from the eight soils ranged from 2.5 to 26.9 micrograms of DNA g-1, and these were positively correlated with the organic carbon content in the soil (r = 0.73). DNA yields from gram-positive bacteria from pure cultures were two to six times higher when the high-salt-SDS-heat method was combined with mortar-and-pestle grinding and freeze-thawing, and most DNA recovered was of high molecular weight. Four methods for purifying crude DNA were also evaluated for percent recovery, fragment size, speed, enzyme restriction, PCR amplification, and DNA-DNA hybridization. In general, all methods produced DNA pure enough for PCR amplification. Since soil type and microbial community characteristics will influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods on the basis of experimental goals. PMID:8593035

  10. Sequence signatures from DNA amplification fingerprints reveal fine population structure of the dogwood pathogen Discula destructiva.

    PubMed

    Caetano-Anollés, G; Trigiano, R N; Windham, M T

    1996-12-15

    Isolates of Discula destructiva Redlin and an undescribed species of Discula, the filamentous fungi that cause anthracnose of flowering (Cornus florida L.) and Pacific (Cornus nuttalli Aud.) dogwoods, were analyzed for genetic variation by nucleic acid scanning with arbitrary mini-hairpin oligonucleotide primers. While the fungal population was highly homogeneous throughout the disease range in eastern and western North America, the generation of arbitrary signatures from amplification profiles (ASAP) distinguished isolates from the northeast, middle and southern Appalachian mountain range, and western United States and Canada. ASAP involves a dual-step arbitrary primer-based amplification procedure that generates a large number of informative allelic characters by amplification of monomorphic DNA fingerprints. ASAP analyses showed a fine fungal population structure consistent with the recent and separate introduction of the pathogen on the west and east coasts of North America.

  11. Caenorhabditis elegans responses to bacteria from its natural habitats

    PubMed Central

    Rowedder, Holli; Braendle, Christian; Félix, Marie-Anne; Ruvkun, Gary

    2016-01-01

    Most Caenorhabditis elegans studies have used laboratory Escherichia coli as diet and microbial environment. Here we characterize bacteria of C. elegans' natural habitats of rotting fruits and vegetation to provide greater context for its physiological responses. By the use of 16S ribosomal DNA (rDNA)-based sequencing, we identified a large variety of bacteria in C. elegans habitats, with phyla Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria being most abundant. From laboratory assays using isolated natural bacteria, C. elegans is able to forage on most bacteria (robust growth on ∼80% of >550 isolates), although ∼20% also impaired growth and arrested and/or stressed animals. Bacterial community composition can predict wild C. elegans population states in both rotting apples and reconstructed microbiomes: alpha-Proteobacteria-rich communities promote proliferation, whereas Bacteroidetes or pathogens correlate with nonproliferating dauers. Combinatorial mixtures of detrimental and beneficial bacteria indicate that bacterial influence is not simply nutritional. Together, these studies provide a foundation for interrogating how bacteria naturally influence C. elegans physiology. PMID:27317746

  12. Replication-transcription conflicts in bacteria

    PubMed Central

    Merrikh, Houra; Zhang, Yan; Grossman, Alan D.; Wang, Jue D.

    2012-01-01

    Preface DNA replication and transcription use the same template and occur concurrently in bacteria. The lack of temporal and spatial separations of these two processes leads to their conflict. Failure to deal with these conflicts can result in genome alterations and reduced fitness. In recent years, significant advances have been made in understanding how cells avoid conflicts between replication and transcription, and how conflicts are resolved when they do occur. In this review, we summarize these findings, which shed light on the significance of the problem and on how cells deal with unwanted encounters between the replication and transcription machineries. PMID:22669220

  13. Radiobiological effects of heavy ions and protons. [on cells of mammals, bacteria and viruses

    NASA Technical Reports Server (NTRS)

    Ryzhov, N. I.; Vorozhtsova, S. V.; Krasavin, Y. A.; Mashinskaya, T. Y.; Savchenko, N. Y.; Fedorov, B. S.; Khlaponina, V. F.; Shelegedin, V. N.; Gut, L.; Sabo, L.

    1974-01-01

    Radiobiological effects of heavy ions and protons are studied on cells of mammals, bacteria, viruses and DNA of bacteria. Results show that the dose effect dependence bears an exponential character; the reduction of RBE as LET of particle increases reflects the different character of microdistribution of absorbed energy in biological objects with different levels of biological organization.

  14. Radiobiological effects of heavy ions and protons. [on cells of mammals, bacteria and viruses

    NASA Technical Reports Server (NTRS)

    Ryzhov, N. I.; Vorozhtsova, S. V.; Krasavin, Y. A.; Mashinskaya, T. Y.; Savchenko, N. Y.; Fedorov, B. S.; Khlaponina, V. F.; Shelegedin, V. N.; Gut, L.; Sabo, L.

    1974-01-01

    Radiobiological effects of heavy ions and protons are studied on cells of mammals, bacteria, viruses and DNA of bacteria. Results show that the dose effect dependence bears an exponential character; the reduction of RBE as LET of particle increases reflects the different character of microdistribution of absorbed energy in biological objects with different levels of biological organization.

  15. Bacterial identification and subtyping using DNA microarray and DNA sequencing.

    PubMed

    Al-Khaldi, Sufian F; Mossoba, Magdi M; Allard, Marc M; Lienau, E Kurt; Brown, Eric D

    2012-01-01

    The era of fast and accurate discovery of biological sequence motifs in prokaryotic and eukaryotic cells is here. The co-evolution of direct genome sequencing and DNA microarray strategies not only will identify, isotype, and serotype pathogenic bacteria, but also it will aid in the discovery of new gene functions by detecting gene expressions in different diseases and environmental conditions. Microarray bacterial identification has made great advances in working with pure and mixed bacterial samples. The technological advances have moved beyond bacterial gene expression to include bacterial identification and isotyping. Application of new tools such as mid-infrared chemical imaging improves detection of hybridization in DNA microarrays. The research in this field is promising and future work will reveal the potential of infrared technology in bacterial identification. On the other hand, DNA sequencing by using 454 pyrosequencing is so cost effective that the promise of $1,000 per bacterial genome sequence is becoming a reality. Pyrosequencing technology is a simple to use technique that can produce accurate and quantitative analysis of DNA sequences with a great speed. The deposition of massive amounts of bacterial genomic information in databanks is creating fingerprint phylogenetic analysis that will ultimately replace several technologies such as Pulsed Field Gel Electrophoresis. In this chapter, we will review (1) the use of DNA microarray using fluorescence and infrared imaging detection for identification of pathogenic bacteria, and (2) use of pyrosequencing in DNA cluster analysis to fingerprint bacterial phylogenetic trees.

  16. Flowthrough Bacteria-Detection System

    NASA Technical Reports Server (NTRS)

    Grana, D. C.; Wilkins, J. R.

    1983-01-01

    Online system allows repetitive cycling of sample intake, bacteria counting and sterilization. System measures bacteria count by using sample/incubate/ measure cycle. Steps in cycle are on/off operations to cycle automated easily.

  17. Manufacture of Probiotic Bacteria

    NASA Astrophysics Data System (ADS)

    Muller, J. A.; Ross, R. P.; Fitzgerald, G. F.; Stanton, C.

    Lactic acid bacteria (LAB) have been used for many years as natural biopreservatives in fermented foods. A small group of LAB are also believed to have beneficial health effects on the host, so called probiotic bacteria. Probiotics have emerged from the niche industry from Asia into European and American markets. Functional foods are one of the fastest growing markets today, with estimated growth to 20 billion dollars worldwide by 2010 (GIA, 2008). The increasing demand for probiotics and the new food markets where probiotics are introduced, challenges the industry to produce high quantities of probiotic cultures in a viable and stable form. Dried concentrated probiotic cultures are the most convenient form for incorporation into functional foods, given the ease of storage, handling and transport, especially for shelf-stable functional products. This chapter will discuss various aspects of the challenges associated with the manufacturing of probiotic cultures.

  18. Bacteria swimming in circles

    NASA Astrophysics Data System (ADS)

    Lauga, Eric; Diluzio, Willow; Garstecki, Piotr; Whitesides, George; Stone, Howard

    2004-11-01

    The bacteria E.coli, which lives in our stomach, swims in a viscous fluid by rotating its flagella together in a helical bundle. The rotation is due to the action of rotary motors embedded in the cell wall. Motivated by experimental observations (Frymier et al., 1995, PNAS vol. 92), as well as our own extensive experiments, that bacteria near solid surfaces do not swim in a straight line but swim in circles, we present a mechanical model for a swimming microorganism near a solid boundary. We show that the combination of a rotating helical bundle and a solid boundary leads to a circular motion to the right of the bacterium (as viewed from above), in accordance with experimental observations. Values of the radii of the circle and the rotation rate are predicted and compared with experimental data.

  19. Reanimation of Ancient Bacteria

    SciTech Connect

    Vreeland, Russell H.

    2009-01-09

    Recent highly publicized experiments conducted on salt crystals taken from the Permian Salado Formation in Southeastern New Mexico have shown that some ancient crystals contain viable microorganisms trapped within fluid inclusions. Stringent geological and microbiological selection criteria were used to select crystals and conduct all sampling. This talk will focus on how each of these lines of data support the conclusion that such isolated bacteria are as old as the rock in which they are trapped. In this case, the isolated microbes are salt tolerant bacilli that grow best in media containing 8% NaCl, and respond to concentrated brines by forming spores. One of the organisms is phylogenetically related to several bacilli, but does have several unique characteristics. This talk will trace the interdisciplinary data and procedures supporting these discoveries, and describe the various isolated bacteria.

  20. Reanimation of Ancient Bacteria

    SciTech Connect

    Vreeland, Russell H.

    2002-01-09

    Recent highly publicized experiments conducted on salt crystals taken from the Permian Salado Formation in Southeastern New Mexico have shown that some ancient crystals contain viable microorganisms trapped within fluid inclusions. Stringent geological and microbiological selection criteria were used to select crystals and conduct all sampling. This talk will focus on how each of these lines of data support the conclusion that such isolated bacteria are as old as the rock in which they are trapped. In this case, the isolated microbes are salt tolerant bacilli that grow best in media containing 8% NaCl, and respond to concentrated brines by forming spores. One of the organisms is phylogenetically related to several bacilli, but does have several unique characteristics. This talk will trace the interdisciplinary data and procedures supporting these discoveries, and describe the various isolated bacteria.

  1. Computation by Bacteria

    DTIC Science & Technology

    2011-01-03

    inversion symmetry and time reversal symmetry by dissipat - ing energy , and breaking both these symmetries allows ratcheting. The ability of...durations. All of these devices take advantage of the conversion of chemical energy into propulsion that occurs within bacteria. These devices break spatial...micromachines relying on energy that microorganisms would dissipate “anyway” even in the absence of ratchet structures suggests that researchers could

  2. Elevated Rate of Genome Rearrangements in Radiation-Resistant Bacteria

    PubMed Central

    Repar, Jelena; Supek, Fran; Klanjscek, Tin; Warnecke, Tobias; Zahradka, Ksenija; Zahradka, Davor

    2017-01-01

    A number of bacterial, archaeal, and eukaryotic species are known for their resistance to ionizing radiation. One of the challenges these species face is a potent environmental source of DNA double-strand breaks, potential drivers of genome structure evolution. Efficient and accurate DNA double-strand break repair systems have been demonstrated in several unrelated radiation-resistant species and are putative adaptations to the DNA damaging environment. Such adaptations are expected to compensate for the genome-destabilizing effect of environmental DNA damage and may be expected to result in a more conserved gene order in radiation-resistant species. However, here we show that rates of genome rearrangements, measured as loss of gene order conservation with time, are higher in radiation-resistant species in multiple, phylogenetically independent groups of bacteria. Comparison of indicators of selection for genome organization between radiation-resistant and phylogenetically matched, nonresistant species argues against tolerance to disruption of genome structure as a strategy for radiation resistance. Interestingly, an important mechanism affecting genome rearrangements in prokaryotes, the symmetrical inversions around the origin of DNA replication, shapes genome structure of both radiation-resistant and nonresistant species. In conclusion, the opposing effects of environmental DNA damage and DNA repair result in elevated rates of genome rearrangements in radiation-resistant bacteria. PMID:28188144

  3. How honey kills bacteria.

    PubMed

    Kwakman, Paulus H S; te Velde, Anje A; de Boer, Leonie; Speijer, Dave; Vandenbroucke-Grauls, Christina M J E; Zaat, Sebastian A J

    2010-07-01

    With the rise in prevalence of antibiotic-resistant bacteria, honey is increasingly valued for its antibacterial activity. To characterize all bactericidal factors in a medical-grade honey, we used a novel approach of successive neutralization of individual honey bactericidal factors. All bacteria tested, including Bacillus subtilis, methicillin-resistant Staphylococcus aureus, extended-spectrum beta-lactamase producing Escherichia coli, ciprofloxacin-resistant Pseudomonas aeruginosa, and vancomycin-resistant Enterococcus faecium, were killed by 10-20% (v/v) honey, whereas > or = 40% (v/v) of a honey-equivalent sugar solution was required for similar activity. Honey accumulated up to 5.62 +/- 0.54 mM H(2)O(2) and contained 0.25 +/- 0.01 mM methylglyoxal (MGO). After enzymatic neutralization of these two compounds, honey retained substantial activity. Using B. subtilis for activity-guided isolation of the additional antimicrobial factors, we discovered bee defensin-1 in honey. After combined neutralization of H(2)O(2), MGO, and bee defensin-1, 20% honey had only minimal activity left, and subsequent adjustment of the pH of this honey from 3.3 to 7.0 reduced the activity to that of sugar alone. Activity against all other bacteria tested depended on sugar, H(2)O(2), MGO, and bee defensin-1. Thus, we fully characterized the antibacterial activity of medical-grade honey.

  4. Growing Unculturable Bacteria

    PubMed Central

    2012-01-01

    The bacteria that can be grown in the laboratory are only a small fraction of the total diversity that exists in nature. At all levels of bacterial phylogeny, uncultured clades that do not grow on standard media are playing critical roles in cycling carbon, nitrogen, and other elements, synthesizing novel natural products, and impacting the surrounding organisms and environment. While molecular techniques, such as metagenomic sequencing, can provide some information independent of our ability to culture these organisms, it is essentially impossible to learn new gene and pathway functions from pure sequence data. A true understanding of the physiology of these bacteria and their roles in ecology, host health, and natural product production requires their cultivation in the laboratory. Recent advances in growing these species include coculture with other bacteria, recreating the environment in the laboratory, and combining these approaches with microcultivation technology to increase throughput and access rare species. These studies are unraveling the molecular mechanisms of unculturability and are identifying growth factors that promote the growth of previously unculturable organisms. This minireview summarizes the recent discoveries in this area and discusses the potential future of the field. PMID:22661685

  5. Growing unculturable bacteria.

    PubMed

    Stewart, Eric J

    2012-08-01

    The bacteria that can be grown in the laboratory are only a small fraction of the total diversity that exists in nature. At all levels of bacterial phylogeny, uncultured clades that do not grow on standard media are playing critical roles in cycling carbon, nitrogen, and other elements, synthesizing novel natural products, and impacting the surrounding organisms and environment. While molecular techniques, such as metagenomic sequencing, can provide some information independent of our ability to culture these organisms, it is essentially impossible to learn new gene and pathway functions from pure sequence data. A true understanding of the physiology of these bacteria and their roles in ecology, host health, and natural product production requires their cultivation in the laboratory. Recent advances in growing these species include coculture with other bacteria, recreating the environment in the laboratory, and combining these approaches with microcultivation technology to increase throughput and access rare species. These studies are unraveling the molecular mechanisms of unculturability and are identifying growth factors that promote the growth of previously unculturable organisms. This minireview summarizes the recent discoveries in this area and discusses the potential future of the field.

  6. Biocide tolerance in bacteria.

    PubMed

    Ortega Morente, Elena; Fernández-Fuentes, Miguel Angel; Grande Burgos, Maria José; Abriouel, Hikmate; Pérez Pulido, Rubén; Gálvez, Antonio

    2013-03-01

    Biocides have been employed for centuries, so today a wide range of compounds showing different levels of antimicrobial activity have become available. At the present time, understanding the mechanisms of action of biocides has also become an important issue with the emergence of bacterial tolerance to biocides and the suggestion that biocide and antibiotic resistance in bacteria might be linked. While most of the mechanisms providing antibiotic resistance are agent specific, providing resistance to a single antimicrobial or class of antimicrobial, there are currently numerous examples of efflux systems that accommodate and, thus, provide tolerance to a broad range of structurally unrelated antimicrobials, both antibiotics and biocides. If biocide tolerance becomes increasingly common and it is linked to antibiotic resistance, not only resistant (even multi-resistant) bacteria could be passed along the food chain, but also there are resistance determinants that can spread and lead to the emergence of new resistant microorganisms, which can only be detected and monitored when the building blocks of resistance traits are understood on the molecular level. This review summarizes the main advances reached in understanding the mechanism of action of biocides, the mechanisms of bacterial resistance to both biocides and antibiotics, and the incidence of biocide tolerance in bacteria of concern to human health and the food industry.

  7. Recovering glycoside hydrolase genes from active tundra cellulolytic bacteria.

    PubMed

    Pinnell, Lee J; Dunford, Eric; Ronan, Patrick; Hausner, Martina; Neufeld, Josh D

    2014-07-01

    Bacteria responsible for cellulose hydrolysis in situ are poorly understood, largely because of the relatively recent development of cultivation-independent methods for their detection and characterization. This study combined DNA stable-isotope probing (DNA-SIP) and metagenomics for identifying active bacterial communities that assimilated carbon from glucose and cellulose in Arctic tundra microcosms. Following DNA-SIP, bacterial fingerprint analysis of gradient fractions confirmed isotopic enrichment. Sequenced fingerprint bands and clone library analysis of 16S rRNA genes identified active bacterial taxa associated with cellulose-associated labelled DNA, including Bacteroidetes (Sphingobacteriales), Betaproteobacteria (Burkholderiales), Alphaproteobacteria (Caulobacteraceae), and Chloroflexi (Anaerolineaceae). We also compared glycoside hydrolase metagenomic profiles from bulk soil and heavy DNA recovered from DNA-SIP incubations. Active populations consuming [(13)C]glucose and [(13)C]cellulose were distinct, based on ordinations of light and heavy DNA. Metagenomic analysis demonstrated a ∼3-fold increase in the relative abundance of glycoside hydrolases in DNA-SIP libraries over bulk-soil libraries. The data also indicate that multiple displacement amplification introduced bias into the resulting metagenomic analysis. This research identified DNA-SIP incubation conditions for glucose and cellulose that were suitable for Arctic tundra soil and confirmed that DNA-SIP enrichment can increase target gene frequencies in metagenomic libraries.

  8. REVIEW ARTICLE: DNA protein interactions and bacterial chromosome architecture

    NASA Astrophysics Data System (ADS)

    Stavans, Joel; Oppenheim, Amos

    2006-12-01

    Bacteria, like eukaryotic organisms, must compact the DNA molecule comprising their genome and form a functional chromosome. Yet, bacteria do it differently. A number of factors contribute to genome compaction and organization in bacteria, including entropic effects, supercoiling and DNA-protein interactions. A gamut of new experimental techniques have allowed new advances in the investigation of these factors, and spurred much interest in the dynamic response of the chromosome to environmental cues, segregation, and architecture, during both exponential and stationary phases. We review these recent developments with emphasis on the multifaceted roles that DNA-protein interactions play.

  9. Small Talk: Cell-to-Cell Communication in Bacteria

    SciTech Connect

    Bassler, Bonnie

    2008-05-14

    Cell-cell communication in bacteria involves the production, release, and subsequent detection of chemical signaling molecules called autoinducers. This process, called quorum sensing, allows bacteria to regulate gene expression on a population-wide scale. Processes controlled by quorum sensing are usually ones that are unproductive when undertaken by an individual bacterium but become effective when undertaken by the group. For example, quorum sensing controls bioluminescence, secretion of virulence factors, biofilm formation, sporulation, and the exchange of DNA. Thus, quorum sensing is a mechanism that allows bacteria to function as multi-cellular organisms. Bacteria make, detect, and integrate information from multiple autoinducers, some of which are used exclusively for intra-species communication while others enable communication between species. Research is now focused on the development of therapies that interfere with quorum sensing to control bacterial virulence.

  10. Small Talk: Cell-to-Cell Communication in Bacteria

    SciTech Connect

    Bassler, Bonnie

    2008-12-03

    Cell-cell communication in bacteria involves the production, release, and subsequent detection of chemical signaling molecules called autoinducers. This process, called quorum sensing, allows bacteria to regulate gene expression on a population-wide scale. Processes controlled by quorum sensing are usually ones that are unproductive when undertaken by an individual bacterium but become effective when undertaken by the group. For example, quorum sensing controls bioluminescence, secretion of virulence factors, biofilm formation, sporulation, and the exchange of DNA. Thus, quorum sensing is a mechanism that allows bacteria to function as multi-cellular organisms. Bacteria make, detect, and integrate information from multiple autoinducers, some of which are used exclusively for intra-species communication while others enable communication between species. Research is now focused on the development of therapies that interfere with quorum sensing to control bacterial virulence.

  11. Phylogenetic analysis on the soil bacteria distributed in karst forest

    PubMed Central

    Zhou, JunPei; Huang, Ying; Mo, MingHe

    2009-01-01

    Phylogenetic composition of bacterial community in soil of a karst forest was analyzed by culture-independent molecular approach. The bacterial 16S rRNA gene was amplified directly from soil DNA and cloned to generate a library. After screening the clone library by RFLP, 16S rRNA genes of representative clones were sequenced and the bacterial community was analyzed phylogenetically. The 16S rRNA gene inserts of 190 clones randomly selected were analyzed by RFLP and generated 126 different RFLP types. After sequencing, 126 non-chimeric sequences were obtained, generating 113 phylotypes. Phylogenetic analysis revealed that the bacteria distributed in soil of the karst forest included the members assigning into Proteobacteria, Acidobacteria, Planctomycetes, Chloroflexi (Green nonsulfur bacteria), Bacteroidetes, Verrucomicrobia, Nitrospirae, Actinobacteria (High G+C Gram-positive bacteria), Firmicutes (Low G+C Gram-positive bacteria) and candidate divisions (including the SPAM and GN08). PMID:24031430

  12. Small Talk: Cell-to-Cell Communication in Bacteria

    ScienceCinema

    Bassler, Bonnie [Princeton University, Princeton, New Jersey, United States

    2016-07-12

    Cell-cell communication in bacteria involves the production, release, and subsequent detection of chemical signaling molecules called autoinducers. This process, called quorum sensing, allows bacteria to regulate gene expression on a population-wide scale. Processes controlled by quorum sensing are usually ones that are unproductive when undertaken by an individual bacterium but become effective when undertaken by the group. For example, quorum sensing controls bioluminescence, secretion of virulence factors, biofilm formation, sporulation, and the exchange of DNA. Thus, quorum sensing is a mechanism that allows bacteria to function as multi-cellular organisms. Bacteria make, detect, and integrate information from multiple autoinducers, some of which are used exclusively for intra-species communication while others enable communication between species. Research is now focused on the development of therapies that interfere with quorum sensing to control bacterial virulence.

  13. Bacteria-mediated delivery of nanoparticles and cargo into cells

    NASA Astrophysics Data System (ADS)

    Akin, Demir; Sturgis, Jennifer; Ragheb, Kathy; Sherman, Debby; Burkholder, Kristin; Robinson, J. Paul.; Bhunia, Arun K.; Mohammed, Sulma; Bashir, Rashid

    2007-07-01

    Nanoparticles and bacteria can be used, independently, to deliver genes and proteins into mammalian cells for monitoring or altering gene expression and protein production. Here, we show the simultaneous use of nanoparticles and bacteria to deliver DNA-based model drug molecules in vivo and in vitro. In our approach, cargo (in this case, a fluorescent or a bioluminescent gene) is loaded onto the nanoparticles, which are carried on the bacteria surface. When incubated with cells, the cargo-carrying bacteria (`microbots') were internalized by the cells, and the genes released from the nanoparticles were expressed in the cells. Mice injected with microbots also successfully expressed the genes as seen by the luminescence in different organs. This new approach may be used to deliver different types of cargo into live animals and a variety of cells in culture without the need for complicated genetic manipulations.

  14. How-to-Do-It: Recombinant DNA Made Easy II. Gene, Gene, Who's Got the Gene?

    ERIC Educational Resources Information Center

    Thomson, Robert G.

    1989-01-01

    Described is an activity in which students are able to determine that DNA can be transferred between bacteria and should be able to predict the type of DNA transferred. Methods, materials, and results are discussed. (CW)

  15. Denitrification by extremely halophilic bacteria

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.; Tomlinson, G. A.

    1985-01-01

    Extremely halophilic bacteria were isolated from widely separated sites by anaerobic enrichment in the presence of nitrate. The anaerobic growth of several of these isolates was accompanied by the production of nitrite, nitrous oxide, and dinitrogen. These results are a direct confirmation of the existence of extremely halophilic denitrifying bacteria, and suggest that such bacteria may be common inhabitants of hypersaline environments.

  16. Denitrification by extremely halophilic bacteria

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.; Tomlinson, G. A.

    1985-01-01

    Extremely halophilic bacteria were isolated from widely separated sites by anaerobic enrichment in the presence of nitrate. The anaerobic growth of several of these isolates was accompanied by the production of nitrite, nitrous oxide, and dinitrogen. These results are a direct confirmation of the existence of extremely halophilic denitrifying bacteria, and suggest that such bacteria may be common inhabitants of hypersaline environments.

  17. Recombinant Salmonella Bacteria Vectoring HIV/AIDS Vaccines.

    PubMed

    Chin'ombe, Nyasha; Ruhanya, Vurayai

    2013-01-01

    HIV/AIDS is an important public health problem globally. An affordable, easy-to-deliver and protective HIV vaccine is therefore required to curb the pandemic from spreading further. Recombinant Salmonella bacteria can be harnessed to vector HIV antigens or DNA vaccines to the immune system for induction of specific protective immunity. These are capable of activating the innate, humoral and cellular immune responses at both mucosal and systemic compartments. Several studies have already demonstrated the utility of live recombinant Salmonella in delivering expressed foreign antigens as well as DNA vaccines to the host immune system. This review gives an overview of the studies in which recombinant Salmonella bacteria were used to vector HIV/AIDS antigens and DNA vaccines. Most of the recombinant Salmonella-based HIV/AIDS vaccines developed so far have only been tested in animals (mainly mice) and are yet to reach human trials.

  18. DNA phosphorothioate modifications influence the global transcriptional response and protect DNA from double-stranded breaks

    PubMed Central

    Gan, Rui; Wu, Xiaolin; He, Wei; Liu, Zhenhua; Wu, Shuangju; Chen, Chao; Chen, Si; Xiang, Qianrong; Deng, Zixin; Liang, Dequan; Chen, Shi; Wang, Lianrong

    2014-01-01

    The modification of DNA by phosphorothioate (PT) occurs when the non-bridging oxygen in the sugar-phosphate backbone of DNA is replaced with sulfur. This DNA backbone modification was recently discovered and is governed by the dndABCDE genes in a diverse group of bacteria and archaea. However, the biological function of DNA PT modifications is poorly understood. In this study, we employed the RNA-seq analysis to characterize the global transcriptional changes in response to PT modifications. Our results show that DNA without PT protection is susceptible to DNA damage caused by the dndFGHI gene products. The DNA double-stranded breaks then trigger the SOS response, cell filamentation and prophage induction. Heterologous expression of dndBCDE conferring DNA PT modifications at GPSA and GPST prevented the damage in Salmonella enterica. Our data provide insights into the physiological role of the DNA PT system. PMID:25319634

  19. Pepsin homologues in bacteria

    PubMed Central

    Rawlings, Neil D; Bateman, Alex

    2009-01-01

    Background Peptidase family A1, to which pepsin belongs, had been assumed to be restricted to eukaryotes. The tertiary structure of pepsin shows two lobes with similar folds and it has been suggested that the gene has arisen from an ancient duplication and fusion event. The only sequence similarity between the lobes is restricted to the motif around the active site aspartate and a hydrophobic-hydrophobic-Gly motif. Together, these contribute to an essential structural feature known as a psi-loop. There is one such psi-loop in each lobe, and so each lobe presents an active Asp. The human immunodeficiency virus peptidase, retropepsin, from peptidase family A2 also has a similar fold but consists of one lobe only and has to dimerize to be active. All known members of family A1 show the bilobed structure, but it is unclear if the ancestor of family A1 was similar to an A2 peptidase, or if the ancestral retropepsin was derived from a half-pepsin gene. The presence of a pepsin homologue in a prokaryote might give insights into the evolution of the pepsin family. Results Homologues of the aspartic peptidase pepsin have been found in the completed genomic sequences from seven species of bacteria. The bacterial homologues, unlike those from eukaryotes, do not possess signal peptides, and would therefore be intracellular acting at neutral pH. The bacterial homologues have Thr218 replaced by Asp, a change which in renin has been shown to confer activity at neutral pH. No pepsin homologues could be detected in any archaean genome. Conclusion The peptidase family A1 is found in some species of bacteria as well as eukaryotes. The bacterial homologues fall into two groups, one from oceanic bacteria and one from plant symbionts. The bacterial homologues are all predicted to be intracellular proteins, unlike the eukaryotic enzymes. The bacterial homologues are bilobed like pepsin, implying that if no horizontal gene transfer has occurred the duplication and fusion event might be

  20. DNA Nanotechnology

    NASA Astrophysics Data System (ADS)

    Taniguchi, Masateru; Kawai, Tomoji

    2002-11-01

    DNA is one candidate of promising molecules for molecular electronic devices, since it has the double helix structure with pi-electron bases for electron transport, the address at 0.4 nm intervals, and the self-assembly. Electrical conductivity and nanostructure of DNA and modified DNA molecules are investigated in order to research the application of DNA in nanoelectronic devices. It has been revealed that DNA is a wide-gap semiconductor in the absence of doping. The conductivity of DNA has been controlled by chemical doping, electric field doping, and photo-doping. It has found that Poly(dG)[middle dot]Poly(dC) has the best conductivity and can function as a conducting nanowire. The pattern of DNA network is controlled by changing the concentration of the DNA solution.

  1. Mitochondrial DNA.

    ERIC Educational Resources Information Center

    Wright, Russell G.; Bottino, Paul J.

    1986-01-01

    Provides background information for teachers on mitochondrial DNA, pointing out that it may have once been a free-living organism. Includes a ready-to-duplicate exercise titled "Using Microchondrial DNA to Measure Evolutionary Distance." (JN)

  2. Mitochondrial DNA.

    ERIC Educational Resources Information Center

    Wright, Russell G.; Bottino, Paul J.

    1986-01-01

    Provides background information for teachers on mitochondrial DNA, pointing out that it may have once been a free-living organism. Includes a ready-to-duplicate exercise titled "Using Microchondrial DNA to Measure Evolutionary Distance." (JN)

  3. Nucleoid restructuring in stationary-state bacteria.

    PubMed

    Frenkiel-Krispin, Daphna; Ben-Avraham, Irit; Englander, Joseph; Shimoni, Eyal; Wolf, Sharon G; Minsky, Abraham

    2004-01-01

    The textbook view of the bacterial cytoplasm as an unstructured environment has been overturned recently by studies that highlighted the extent to which non-random organization and coherent motion of intracellular components are central for bacterial life-sustaining activities. Because such a dynamic order critically depends on continuous consumption of energy, it cannot be perpetuated in starved, and hence energy-depleted, stationary-state bacteria. Here, we show that, at the onset of the stationary state, bacterial chromatin undergoes a massive reorganization into ordered toroidal structures through a process that is dictated by the intrinsic properties of DNA and by the ubiquitous starvation-induced DNA-binding protein Dps. As starvation proceeds, the toroidal morphology acts as a structural template that promotes the formation of DNA-Dps crystalline assemblies through epitaxial growth. Within the resulting condensed assemblies, DNA is effectively protected by means of structural sequestration. We thus conclude that the transition from bacterial active growth to stationary phase entails a co-ordinated process, in which the energy-dependent dynamic order of the chromatin is sequentially substituted with an equilibrium crystalline order.

  4. Structural and Thermodynamic Signatures of DNA Recognition by Mycobacterium tuberculosis DnaA

    SciTech Connect

    Tsodikov, Oleg V.; Biswas, Tapan

    2011-09-06

    An essential protein, DnaA, binds to 9-bp DNA sites within the origin of replication oriC. These binding events are prerequisite to forming an enigmatic nucleoprotein scaffold that initiates replication. The number, sequences, positions, and orientations of these short DNA sites, or DnaA boxes, within the oriCs of different bacteria vary considerably. To investigate features of DnaA boxes that are important for binding Mycobacterium tuberculosis DnaA (MtDnaA), we have determined the crystal structures of the DNA binding domain (DBD) of MtDnaA bound to a cognate MtDnaA-box (at 2.0 {angstrom} resolution) and to a consensus Escherichia coli DnaA-box (at 2.3 {angstrom}). These structures, complemented by calorimetric equilibrium binding studies of MtDnaA DBD in a series of DnaA-box variants, reveal the main determinants of DNA recognition and establish the [T/C][T/A][G/A]TCCACA sequence as a high-affinity MtDnaA-box. Bioinformatic and calorimetric analyses indicate that DnaA-box sequences in mycobacterial oriCs generally differ from the optimal binding sequence. This sequence variation occurs commonly at the first 2 bp, making an in vivo mycobacterial DnaA-box effectively a 7-mer and not a 9-mer. We demonstrate that the decrease in the affinity of these MtDnaA-box variants for MtDnaA DBD relative to that of the highest-affinity box TTGTCCACA is less than 10-fold. The understanding of DnaA-box recognition by MtDnaA and E. coli DnaA enables one to map DnaA-box sequences in the genomes of M. tuberculosis and other eubacteria.

  5. Anaerobic cellulolytic bacteria from wetwood of living trees

    SciTech Connect

    Warshaw, J.E.; Leschine, S.B.; Canale-Parola, E.

    1985-10-01

    Obligately anaerobic, mesophilic, cellulolytic bacteria were isolated from the wetwood of elm and maple trees. The isolation of these bacteria involved inoculation of selective enrichment cultures with increment cores taken from trees showing evidence of wetwood. Cellulolytic bacteria were present in the cores from seven of nine trees sampled, as indicated by the disappearance of cellulose from enrichment cultures. With two exceptions, cellulolytic activity was confined to the darker, wetter, inner section of the cores. Cellulolytic bacteria were also present in the fluid from core holes. The cellulolytic isolates were motile rods that stained gram negative. Endospores were formed by some strains. The physiology of one of the cellulolytic isolates (strain JW2) was studied in detail. Strain JW2 fermented cellobiose, D-glucose, glycerol, L-arabinose, D-xylose, and xylan in addition to cellulose. In a defined medium, p-aminobenzoic acid and biotin were the only exogenous growth factors required by strain JW2 for the fermentation of cellobiose or cellulose. Acetate and ethanol were the major nongaseous end products of cellulose fermentation. The guanine-plus-cytosine content of the DNA of strain JW2 was 33.7 mol%. Cellulolytic bacteria have not previously been reported to occur in wetwood. The isolation of such bacteria indicates that cellulolytic bacteria are inhabitants of wetwood environments and suggests that they may be involved in wetwood development.

  6. Growth rates and rRNA content of four marine bacteria in pure cultures and in the Delaware estuary.

    PubMed

    Lankiewicz, Thomas S; Cottrell, Matthew T; Kirchman, David L

    2016-04-01

    Interpretation of 16S ribosomal RNA (rRNA) to 16S rRNA gene ratios (rRNA:rDNA) is based on a limited number of studies with rapidly growing copiotrophic bacteria. The most abundant bacteria in the ocean are oligotrophs, which probably grow more slowly than those bacteria whose rRNA:rDNA versus growth rate relationships are known. To examine whether rRNA:rDNA varies differently in oligotrophic marine bacteria than in copiotrophic bacteria, we used quantitative PCR and reverse transcriptase quantitative PCR to measure rRNA:rDNA in two marine copiotrophs and in two marine oligotrophs, including Candidatus Pelagibacter ubique HTCC1062, a coastal isolate of SAR11, the most abundant bacterial clade in the ocean. The rRNA:rDNA ratios for the two copiotrophs were similar to those expected on the basis of an analysis of previously studied copiotrophic bacteria, while the ratios for the two oligotrophs were substantially lower than predicted even given their slow growth rates. The rRNA:rDNA ratios determined along a transect in the Delaware estuary suggested that SAR11 bacteria grow at rates close to the growth rate in culture, while rates of the two copiotrophs were far below those observed in laboratory cultures. Our results have implications for interpreting rRNA:rDNA from natural communities, understanding growth strategies and comparing regulatory mechanisms in copiotrophs and oligotrophs.

  7. Living bacteria in silica gels

    NASA Astrophysics Data System (ADS)

    Nassif, Nadine; Bouvet, Odile; Noelle Rager, Marie; Roux, Cécile; Coradin, Thibaud; Livage, Jacques

    2002-09-01

    The encapsulation of enzymes within silica gels has been extensively studied during the past decade for the design of biosensors and bioreactors. Yeast spores and bacteria have also been recently immobilized within silica gels where they retain their enzymatic activity, but the problem of the long-term viability of whole cells in an inorganic matrix has never been fully addressed. It is a real challenge for the development of sol-gel processes. Generic tests have been performed to check the viability of Escherichia coli bacteria in silica gels. Surprisingly, more bacteria remain culturable in the gel than in an aqueous suspension. The metabolic activity of the bacteria towards glycolysis decreases slowly, but half of the bacteria are still viable after one month. When confined within a mineral environment, bacteria do not form colonies. The exchange of chemical signals between isolated bacteria rather than aggregates can then be studied, a point that could be very important for 'quorum sensing'.

  8. Metagenomic Evaluation of Bacteria from Voles.

    PubMed

    Koskela, Katja A; Kalin-Mänttäri, Laura; Hemmilä, Heidi; Smura, Teemu; Kinnunen, Paula M; Niemimaa, Jukka; Henttonen, Heikki; Nikkari, Simo

    2017-02-01

    Voles (Arvicolinae, Rodentia) are known carriers of zoonotic bacteria such as Bartonella spp. and Francisella tularensis. However, apart from F. tularensis, the bacterial microbiome of voles has not previously been determined in Finland and rarely elsewhere. Therefore, we studied liver samples from 61 voles using 16S ribosomal RNA gene PCR analysis, followed by Sanger sequencing. Twenty-three of these samples were also studied with tag-encoded pyrosequencing. The samples originated from 21 field voles (Microtus agrestis), 37 tundra voles (Microtus oeconomus), and 3 bank voles (Myodes glareolus). With the more conventional 16S rDNA PCR analysis, 90 (33%) of the recovered 269 sequence types could be identified to genus level, including Bartonella, Francisella, Mycoplasma, Anaplasma, and Acinetobacter in 31, 15, 9, 9, and 9 sequences, respectively. Seventy-five (28%) matched best with sequences of uncultured bacteria, of which 40/75 could be classified to the order Clostridiales and, more specifically, to families Lachnospiraceae and Ruminococcaceae. Pyrosequencing from 23 samples revealed comparable and similar results: clinically relevant bacterial families such as Mycoplasmataceae, Bartonellaceae, Anaplasmataceae, and Francisellaceae were recognized. These analyses revealed significant bacterial diversity in vole livers, consisting of distinct and constant sequence patterns reflecting bacteria found in the intestinal gut, but including some known zoonotic pathogens as well. The molecular bacterial sequence types determined with the two different techniques shared major similarities and verified remarkable congruency between the methods.

  9. Dissemination of 6S RNA among Bacteria

    PubMed Central

    Wehner, Stefanie; Damm, Katrin; Hartmann, Roland K; Marz, Manja

    2014-01-01

    6S RNA is a highly abundant small non-coding RNA widely spread among diverse bacterial groups. By competing with DNA promoters for binding to RNA polymerase (RNAP), the RNA regulates transcription on a global scale. RNAP produces small product RNAs derived from 6S RNA as template, which rearranges the 6S RNA structure leading to dissociation of 6S RNA:RNAP complexes. Although 6S RNA has been experimentally analysed in detail for some species, such as Escherichia coli and Bacillus subtilis, and was computationally predicted in many diverse bacteria, a complete and up-to-date overview of the distribution among all bacteria is missing. In this study we searched with new methods for 6S RNA genes in all currently available bacterial genomes. We ended up with a set of 1,750 6S RNA genes, of which 1,367 are novel and bona fide, distributed among 1,610 bacteria, and had a few tentative candidates among the remaining 510 assembled bacterial genomes accessible. We were able to confirm two tentative candidates by Northern blot analysis. We extended 6S RNA genes of the Flavobacteriia significantly in length compared to the present Rfam entry. We describe multiple homologs of 6S RNAs (including split 6S RNA genes) and performed a detailed synteny analysis. PMID:25483037

  10. Bacteria counting method based on polyaniline/bacteria thin film.

    PubMed

    Zhihua, Li; Xuetao, Hu; Jiyong, Shi; Xiaobo, Zou; Xiaowei, Huang; Xucheng, Zhou; Tahir, Haroon Elrasheid; Holmes, Mel; Povey, Malcolm

    2016-07-15

    A simple and rapid bacteria counting method based on polyaniline (PANI)/bacteria thin film was proposed. Since the negative effects of immobilized bacteria on the deposition of PANI on glass carbon electrode (GCE), PANI/bacteria thin films containing decreased amount of PANI would be obtained when increasing the bacteria concentration. The prepared PANI/bacteria film was characterized with cyclic voltammetry (CV) technique to provide quantitative index for the determination of the bacteria count, and electrochemical impedance spectroscopy (EIS) was also performed to further investigate the difference in the PANI/bacteria films. Good linear relationship of the peak currents of the CVs and the log total count of bacteria (Bacillus subtilis) could be established using the equation Y=-30.413X+272.560 (R(2)=0.982) over the range of 5.3×10(4) to 5.3×10(8)CFUmL(-1), which also showed acceptable stability, reproducibility and switchable ability. The proposed method was feasible for simple and rapid counting of bacteria. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Films of bacteria at interfaces.

    PubMed

    Vaccari, Liana; Molaei, Mehdi; Niepa, Tagbo H R; Lee, Daeyeon; Leheny, Robert L; Stebe, Kathleen J

    2017-09-01

    Bacteria are often discussed as active colloids, self-propelled organisms whose collective motion can be studied in the context of non-equilibrium statistical mechanics. In such studies, the behavior of bacteria confined to interfaces or in the proximity of an interface plays an important role. For instance, many studies have probed collective behavior of bacteria in quasi two-dimensional systems such as soap films. Since fluid interfaces can adsorb surfactants and other materials, the stress and velocity boundary conditions at interfaces can alter bacteria motion; hydrodynamic studies of interfaces with differing boundary conditions are reviewed. Also, bacteria in bulk can become trapped at or near fluid interfaces, where they colonize and form structures comprising secretions like exopolysaccharides, surfactants, living and dead bacteria, thereby creating Films of Bacteria at Interfaces (FBI). The formation of FBI is discussed at air-water, oil-water, and water-water interfaces, with an emphasis on film mechanics, and with some allusion to genetic functions guiding bacteria to restructure fluid interfaces. At air-water interfaces, bacteria form pellicles or interfacial biofilms. Studies are reviewed that reveal that pellicle material properties differ for different strains of bacteria, and that pellicle physicochemistry can act as a feedback mechanism to regulate film formation. At oil-water interfaces, a range of FBI form, depending on bacteria strain. Some bacteria-laden interfaces age from an initial active film, with dynamics dominated by motile bacteria, through viscoelastic states, to form an elastic film. Others remain active with no evidence of elastic film formation even at significant interface ages. Finally, bacteria can adhere to and colonize ultra-low surface tension interfaces such as aqueous-aqueous systems common in food industries. Relevant literature is reviewed, and areas of interest for potential application are discussed, ranging from health

  12. Dna Sequencing

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  13. Bacteria in solitary confinement.

    PubMed

    Mullineaux, Conrad W

    2015-02-15

    Even in clonal bacterial cultures, individual bacteria can show substantial stochastic variation, leading to pitfalls in the interpretation of data derived from millions of cells in a culture. In this issue of the Journal of Bacteriology, as part of their study on osmoadaptation in a cyanobacterium, Nanatani et al. describe employing an ingenious microfluidic device that gently cages individual cells (J Bacteriol 197:676-687, 2015, http://dx.doi.org/10.1128/JB.02276-14). The device is a welcome addition to the toolkit available to probe the responses of individual cells to environmental cues.

  14. Surface layers of bacteria.

    PubMed Central

    Beveridge, T J; Graham, L L

    1991-01-01

    Since bacteria are so small, microscopy has traditionally been used to study them as individual cells. To this end, electron microscopy has been a most powerful tool for studying bacterial surfaces; the viewing of macromolecular arrangements of some surfaces is now possible. This review compares older conventional electron-microscopic methods with new cryotechniques currently available and the results each has produced. Emphasis is not placed on the methodology but, rather, on the importance of the results in terms of our perception of the makeup and function of bacterial surfaces and their interaction with the surrounding environment. Images PMID:1723487

  15. Phylogeny and molecular fingerprinting of green sulfur bacteria.

    PubMed

    Overmann, J; Tuschak, C

    1997-05-01

    The 16S rDNA sequences of nine strains of green sulfur bacteria (Chlorobiaceae) were determined and compared to the four known sequences of Chlorobiaceae and to sequences representative for all eubacterial phyla. The sequences of the Chlorobiaceae strains were consistent with the secondary structure model proposed earlier for Chlorobium vibrioforme strain 6030. Similarity values > 90.1% and Knuc values < 0.11 indicate a close phylogenetic relatedness among the green sulfur bacteria. As a group, these bacteria represent an isolated branch within the eubacterial radiation. In Chlorobiaceae, a similar morphology does not always reflect a close phylogenetic relatedness. While ternary fission is a morphological trait of phylogenetic significance, gas vesicle formation occurs also in distantly related species. Pigment composition is not an indicator of phylogenetic relatedness since very closely related species contain different bacteriochlorophylls and carotenoids. Two different molecular fingerprinting techniques for the rapid differentiation of Chlorobiaceae species were investigated. The 16S rDNA fragments of several species could not be separated by denaturing gradient gel electrophoresis. In contrast, all strains investigated during the present work gave distinct banding patterns when dispersed repetitive DNA sequences were used as targets in PCR. The latter technique is, therefore, well suited for the rapid screening of isolated pure cultures of green sulfur bacteria.

  16. Dissimilatory Nitrite Reductase Genes from Autotrophic Ammonia-Oxidizing Bacteria

    PubMed Central

    Casciotti, Karen L.; Ward, Bess B.

    2001-01-01

    The presence of a copper-containing dissimilatory nitrite reductase gene (nirK) was discovered in several isolates of β-subdivision ammonia-oxidizing bacteria using PCR and DNA sequencing. PCR primers Cunir3 and Cunir4 were designed based on published nirK sequences from denitrifying bacteria and used to amplify a 540-bp fragment of the nirK gene from Nitrosomonas marina and five additional isolates of ammonia-oxidizing bacteria. Amplification products of the expected size were cloned and sequenced. Alignment of the nucleic acid and deduced amino acid (AA) sequences shows significant similarity (62 to 75% DNA, 58 to 76% AA) between nitrite reductases present in these nitrifiers and the copper-containing nitrite reductase found in classic heterotrophic denitrifiers. While the presence of a nitrite reductase in Nitrosomonas europaea is known from early biochemical work, preliminary sequence data from its genome indicate a rather low similarity to the denitrifier nirKs. Phylogenetic analysis of the partial nitrifier nirK sequences indicates that the topology of the nirK tree corresponds to the 16S rRNA and amoA trees. While the role of nitrite reduction in the metabolism of nitrifying bacteria is still uncertain, these data show that the nirK gene is present in closely related nitrifying isolates from many oceanographic regions and suggest that nirK sequences retrieved from the environment may include sequences from ammonia-oxidizing bacteria. PMID:11319103

  17. Plasmid incidence in bacteria from deep subsurface sediments

    SciTech Connect

    Fredrickson, J.K.; Hicks, R.J.; Li, S.W.; Brockman, F.J. )

    1988-12-01

    Bacteria were isolated from deep terrestrial subsurface sediments underlying the coastal plain of South Carolina. A total of 163 isolates from deep sediments, surface soil, and return drill muds were examined for plasmid DNA content and resistance to the antibiotics penicillin, ampicillin, carbenicillin, streptomycin, kanamycin, and tetracycline. MICs of Cu{sup 2+}, Cr{sup 3+}, and Hg{sup 2+} for each isolate were also determined. The overall frequency of plasmid occurrence in the subsurface bacteria was 33%. Resistance was most frequent to penicillin (70% of all isolates), ampicillin (49%), and carbenicillin (32%) and was concluded to be related to the concentrations of the individual antibiotics in the disks used for assaying resistance and to the production of low levels of {beta}-lactamase. The frequencies of resistance to penicillin and ampicillin were significantly greater for isolates bearing plasmids than for plasmidless isolates; however, resistance was not transferable to penicillin-sensitive Escherichia coli. Hybridization of subsurface bacterial plasmids and chromosomal DNA with a whole-TOL-plasmid (pWWO) probe revealed some homology of subsurface bacterial plasmid and chromosomal DNAs, indicating a potential for those bacterial to harbor catabolic genes on plasmids or chromosomes. The incidences of antibiotic resistance and MICs of metals for subsurface bacteria were significantly different from those drill mud bacteria, ruling out the possibility that bacteria from sediments were derived from drill muds.

  18. Recovery of periodontopathogenic bacteria from embalmed human cadavers.

    PubMed

    Wood, Nelson; Johnson, Roger B

    2005-01-01

    There is recent interest in recovery of periodontopathogenic bacteria from arterial and bronchial tissues to identify a link between periodontal and cardiovascular or pulmonary diseases. This interest could provide a useful clinical correlation exercise for gross anatomy. Our objective was to perform a feasibility study to determine whether these bacteria could be recovered from two sites within eight (4 dentate, 4 edentulous) human embalmed cadavers from an anatomical dissection laboratory. Bacterial samples were collected from the right coronary artery and the right superior secondary bronchus and assayed for the presence and concentrations of the DNA of A. actinomycetemcomitans, E. corrodens, C. rectus, P. intermedia, P. gingivalis, B. forsythus, T. denticola, and F. nucleatum. Frequencies were compared using a Kruskal-Wallis H-test. Correlations between the presence of teeth, bacterial species, and site were determined by a Spearman's rho correlation test. A. actinomycetemcomitans and B. forsythus frequencies were different between the sites in edentulous subjects (P <0.05); the frequency of B. forsythus was different in dentate and edentulous subjects at the bronchus site (P <0.05). Numerous significant correlations were identified between strains of bacteria, site, and presence of teeth. Thus, it is possible for the DNA of periodontopathogenic bacteria to be recovered from human embalmed cadavers. Collection and identification of these bacteria from these cadavers could be a useful clinical correlation exercise for dental students in a gross anatomy class.

  19. Molecular identification of bacteria from aseptically loose implants.

    PubMed

    Kobayashi, Naomi; Procop, Gary W; Krebs, Viktor; Kobayashi, Hideo; Bauer, Thomas W

    2008-07-01

    Polymerase chain reaction (PCR) assays have been used to detect bacteria adherent to failed orthopaedic implants, but some PCR assays have had problems with probable false-positive results. We used a combination of a Staphylococcus species-specific PCR and a universal PCR followed by DNA sequencing to identify bacteria on implants retrieved from 52 patients (92 implants) at revision arthroplasty. We addressed two questions in this study: (1) Is this method able to show the existence of bacterial DNA on presumed aseptic loosed implants?; and (2) What proportion of presumed aseptic or culture-negative implants was positive for bacterial DNA by PCR? Fourteen implants (15%) were believed infected, whereas 74 implants (85%) were believed aseptic. Each implant was sonicated and the resulting solution was submitted for dual real-time PCR assay and culture. All implants believed aseptically loose were culture-negative, but nine of the 74 (12%) had bacterial DNA by PCR; two (2.7%) were PCR-positive and also showed histologic findings suggestive of infection. Uniquely developed PCR and bacterial sequencing assays showed bacterial DNA on 12% of implants removed for presumed aseptic loosening. Additional studies are needed to determine the clinical importance of bacterial DNA detected by PCR but not by conventional culture. Level III, diagnostic study.

  20. Identification and Characterization of the fis Operon in Enteric Bacteria

    PubMed Central

    Beach, Michael B.; Osuna, Robert

    1998-01-01

    The small DNA binding protein Fis is involved in several different biological processes in Escherichia coli. It has been shown to stimulate DNA inversion reactions mediated by the Hin family of recombinases, stimulate integration and excision of phage λ genome, regulate the transcription of several different genes including those of stable RNA operons, and regulate the initiation of DNA replication at oriC. fis has also been isolated from Salmonella typhimurium, and the genomic sequence of Haemophilus influenzae reveals its presence in this bacteria. This work extends the characterization of fis to other organisms. Very similar fis operon structures were identified in the enteric bacteria Klebsiella pneumoniae, Serratia marcescens, Erwinia carotovora, and Proteus vulgaris but not in several nonenteric bacteria. We found that the deduced amino acid sequences for Fis are 100% identical in K. pneumoniae, S. marcescens, E. coli, and S. typhimurium and 96 to 98% identical when E. carotovora and P. vulgaris Fis are considered. The deduced amino acid sequence for H. influenzae Fis is about 80% identical and 90% similar to Fis in enteric bacteria. However, in spite of these similarities, the E. carotovora, P. vulgaris, and H. influenzae Fis proteins are not functionally identical. An open reading frame (ORF1) preceding fis in E. coli is also found in all these bacteria, and their deduced amino acid sequences are also very similar. The sequence preceding ORF1 in the enteric bacteria showed a very strong similarity to the E. coli fis P region from −53 to +27 and the region around −116 containing an ihf binding site. Both β-galactosidase assays and primer extension assays showed that these regions function as promoters in vivo and are subject to growth phase-dependent regulation. However, their promoter strengths vary, as do their responses to Fis autoregulation and integration host factor stimulation. PMID:9811652

  1. Identification and characterization of the fis operon in enteric bacteria.

    PubMed

    Beach, M B; Osuna, R

    1998-11-01

    The small DNA binding protein Fis is involved in several different biological processes in Escherichia coli. It has been shown to stimulate DNA inversion reactions mediated by the Hin family of recombinases, stimulate integration and excision of phage lambda genome, regulate the transcription of several different genes including those of stable RNA operons, and regulate the initiation of DNA replication at oriC. fis has also been isolated from Salmonella typhimurium, and the genomic sequence of Haemophilus influenzae reveals its presence in this bacteria. This work extends the characterization of fis to other organisms. Very similar fis operon structures were identified in the enteric bacteria Klebsiella pneumoniae, Serratia marcescens, Erwinia carotovora, and Proteus vulgaris but not in several nonenteric bacteria. We found that the deduced amino acid sequences for Fis are 100% identical in K. pneumoniae, S. marcescens, E. coli, and S. typhimurium and 96 to 98% identical when E. carotovora and P. vulgaris Fis are considered. The deduced amino acid sequence for H. influenzae Fis is about 80% identical and 90% similar to Fis in enteric bacteria. However, in spite of these similarities, the E. carotovora, P. vulgaris, and H. influenzae Fis proteins are not functionally identical. An open reading frame (ORF1) preceding fis in E. coli is also found in all these bacteria, and their deduced amino acid sequences are also very similar. The sequence preceding ORF1 in the enteric bacteria showed a very strong similarity to the E. coli fis P region from -53 to +27 and the region around -116 containing an ihf binding site. Both beta-galactosidase assays and primer extension assays showed that these regions function as promoters in vivo and are subject to growth phase-dependent regulation. However, their promoter strengths vary, as do their responses to Fis autoregulation and integration host factor stimulation.

  2. Mitochondrial DNA pattern of the fine shrimp Metapenaeus elegans (De Man, 1907) in the lagoon of Segara Anakan, Central Java, using Hind III

    NASA Astrophysics Data System (ADS)

    Nugraha, Fitra Arya Dwi; Holil, Kholifah; Kurniawan, Nia

    2017-05-01

    Ecological damages to the Lagoon of Segara Anakan, Central Java, as well as large-scale and continuous exploitation are threatening the sustainability of fine shrimp, Metapenaeus elegans, and resources. Information in regards to genetic resources is crucial to establish long-term conservation programs and to preserve germplasm quality. This study aims to evaluate the number and size of the fragment which is digested with restriction enzyme Hind III. Seven individuals of Metapenaeus elegans from the Lagoon of Segara Anakan were examined using Hind III. Amplification of mitochondrial DNA resulted in 950 bp, and the digestion using Hind III generated four fragments consisting of 114 bp, 200 bp, 250 bp, and 386 bp, which formed a monomorphic pattern. The restriction pattern showed the probability of homozygosity of alleles that restricted using Hind III. Homozygosity indicates no variation of DNA sequence.

  3. Chemical communication in bacteria

    NASA Astrophysics Data System (ADS)

    Suravajhala, Srinivasa Sandeep; Saini, Deepak; Nott, Prabhu

    Luminescence in Vibrio fischeri is a model for quorum-sensing-gene-regulation in bacteria. We study luminescence response of V. fischeri to both internal and external cues at the single cell and population level. Experiments with ES114, a wild-type strain, and ainS mutant show that luminescence induction in cultures is not always proportional to cell-density and there is always a basal level of luminescence. At any given concentration of the exogenously added signals, C6-HSL and C8-HSL, luminescence per cell reaches a maximum during the exponential phase and decreases thereafter. We hypothesize that (1) C6-HSL production and LuxR activity are not proportional to cell-density, and (2) there is a shift in equilibrium from C6-HSL to C8-HSL during the later stages of growth of the culture. RT-PCR analysis of luxI and luxR shows that the expression of these genes is maximum corresponding to the highest level of luminescence. The shift in equilibrium is shown by studying competitive binding of C6-HSL and C8-HSL to LuxR. We argue that luminescence is a unicellular behaviour, and an intensive property like per cell luminescence is more important than gross luminescence of the population in understanding response of bacteria to chemical signalling. Funding from the Department of Science and Technology, India is acknowledged.

  4. Beneficial bacteria inhibit cachexia

    PubMed Central

    Varian, Bernard J.; Goureshetti, Sravya; Poutahidis, Theofilos; Lakritz, Jessica R.; Levkovich, Tatiana; Kwok, Caitlin; Teliousis, Konstantinos; Ibrahim, Yassin M.; Mirabal, Sheyla; Erdman, Susan E.

    2016-01-01

    Muscle wasting, known as cachexia, is a debilitating condition associated with chronic inflammation such as during cancer. Beneficial microbes have been shown to optimize systemic inflammatory tone during good health; however, interactions between microbes and host immunity in the context of cachexia are incompletely understood. Here we use mouse models to test roles for bacteria in muscle wasting syndromes. We find that feeding of a human commensal microbe, Lactobacillus reuteri, to mice is sufficient to lower systemic indices of inflammation and inhibit cachexia. Further, the microbial muscle-building phenomenon extends to normal aging as wild type animals exhibited increased growth hormone levels and up-regulation of transcription factor Forkhead Box N1 [FoxN1] associated with thymus gland retention and longevity. Interestingly, mice with a defective FoxN1 gene (athymic nude) fail to inhibit sarcopenia after L. reuteri therapy, indicating a FoxN1-mediated mechanism. In conclusion, symbiotic bacteria may serve to stimulate FoxN1 and thymic functions that regulate inflammation, offering possible alternatives for cachexia prevention and novel insights into roles for microbiota in mammalian ontogeny and phylogeny. PMID:26933816

  5. RNA localization in bacteria

    PubMed Central

    Buskila, Avi-ad Avraam; Kannaiah, Shanmugapriya; Amster-Choder, Orna

    2014-01-01

    One of the most important discoveries in the field of microbiology in the last two decades is that bacterial cells have intricate subcellular organization. This understanding has emerged mainly from the depiction of spatial and temporal organization of proteins in specific domains within bacterial cells, e.g., midcell, cell poles, membrane and periplasm. Because translation of bacterial RNA molecules was considered to be strictly coupled to their synthesis, they were not thought to specifically localize to regions outside the nucleoid. However, the increasing interest in RNAs, including non-coding RNAs, encouraged researchers to explore the spatial and temporal localization of RNAs in bacteria. The recent technological improvements in the field of fluorescence microscopy allowed subcellular imaging of RNAs even in the tiny bacterial cells. It has been reported by several groups, including ours that transcripts may specifically localize in such cells. Here we review what is known about localization of RNA and of the pathways that determine RNA fate in bacteria, and discuss the possible cues and mechanisms underlying these distribution patterns. PMID:25482897

  6. Functional amyloids in bacteria.

    PubMed

    Romero, Diego; Kolter, Roberto

    2014-06-01

    The term amyloidosis is used to refer to a family of pathologies altering the homeostasis of human organs. Despite having a name that alludes to starch content, the amyloid accumulations are made up of proteins that polymerize as long and rigid fibers. Amyloid proteins vary widely with respect to their amino acid sequences but they share similarities in their quaternary structure; the amyloid fibers are enriched in β-sheets arranged perpendicular to the axis of the fiber. This structural feature provides great robustness, remarkable stability, and insolubility. In addition, amyloid proteins specifically stain with certain dyes such as Congo red and thioflavin-T. The aggregation into amyloid fibers, however, it is not restricted to pathogenic processes, rather it seems to be widely distributed among proteins and polypeptides. Amyloid fibers are present in insects, fungi and bacteria, and they are important in maintaining the homeostasis of the organism. Such findings have motivated the use of the term "functional amyloid" to differentiate these amyloid proteins from their toxic siblings. This review focuses on systems that have evolved in bacteria that control the expression and assembly of amyloid proteins on cell surfaces, such that the robustness of amyloid proteins are used towards a beneficial end.

  7. DNA Flexibility

    NASA Astrophysics Data System (ADS)

    Widom, Jonathan

    2005-03-01

    Classic experimental and theoretical analyses led to a unified view of DNA as a semiflexible polymer, characterized by a bending persistence length, P, ˜50 nm (˜150 bp). In this view, DNA lengths that are greater than P are, on average, spontaneously gently bent, and require relatively little force to bend significantly, while DNA lengths that are shorter than P are nearly straight, and require great force to bend significantly. Nevertheless, sharply bent DNA plays important roles in biology. We used the method of ligase catalyzed DNA cyclization to investigate the spontaneous looping of short DNAs. Remarkably, DNAs as short as 84 bp spontaneously bend into circles, and 94 bp DNA sequences cyclize up to 10^5 times more easily than predicted from classic theories of DNA bending. In subsequent studies we find that the twistability of sharply looped DNAs exceeds the prediction of classic theories by up to 400-fold. These results can only be understood by greatly enhanced DNA flexibility, not by permanent bends. Our results provide striking support for two new theories of DNA mechanics based on local melted or kinked regions, and they establish DNA as an active participant in the formation and function of looped regulatory complexes in vivo.

  8. Local and global regulation of transcription initiation in bacteria.

    PubMed

    Browning, Douglas F; Busby, Stephen J W

    2016-10-01

    Gene expression in bacteria relies on promoter recognition by the DNA-dependent RNA polymerase and subsequent transcription initiation. Bacterial cells are able to tune their transcriptional programmes to changing environments, through numerous mechanisms that regulate the activity of RNA polymerase, or change the set of promoters to which the RNA polymerase can bind. In this Review, we outline our current understanding of the different factors that direct the regulation of transcription initiation in bacteria, whether by interacting with promoters, with RNA polymerase or with both, and we discuss the diverse molecular mechanisms that are used by these factors to regulate gene expression.

  9. Methanogenic bacteria: presence in foodstuffs.

    PubMed

    Brusa, T; Ferrari, F; Canzi, E

    1998-01-01

    Methanogenic bacteria are anaerobic, oxygen-intolerant microorganisms, and it is only by studying the different habitats of such bacteria that fundamental information about their ecology becomes available. This research has evaluated methanogenic bacteria in apparently aerobic ecosystems, in foodstuffs not subjected to chemical-physical reclamation processes, where the presence of methanogenic bacteria has never been investigated. Methanogenic bacteria, ascribable to the Methanogenium, Methanobacterium and Methanosarcina genera, were found in vegetables, meat, fish and cheese but were generally absent in confectionery products and fruit. The microorganisms appear to be chance contaminants, usually being present in only very low numbers. It should be noted that none of the tested foods showed the presence of Methanobrevibacter smithii, M. oralis or Methanosphaera stadtmaneae, methanogenic bacteria sometimes present in the human digestive tract.

  10. Biotechnology of Anoxygenic Phototrophic Bacteria.

    PubMed

    Frigaard, Niels-Ulrik

    Anoxygenic phototrophic bacteria are a diverse collection of organisms that are defined by their ability to grow using energy from light without evolving oxygen. The dominant groups are purple sulfur bacteria, purple nonsulfur bacteria, green sulfur bacteria, and green and red filamentous anoxygenic phototrophic bacteria. They represent several bacterial phyla but they all have bacteriochlorophylls and carotenoids and photochemical reaction centers which generate ATP and cellular reductants used for CO2 fixation. They typically have an anaerobic lifestyle in the light, although some grow aerobically in the dark. Some of them oxidize inorganic sulfur compounds for light-dependent CO2 fixation; this ability can be exploited for photobiological removal of hydrogen sulfide from wastewater and biogas. The anoxygenic phototrophic bacteria also perform bioremediation of recalcitrant dyes, pesticides, and heavy metals under anaerobic conditions. Finally, these organisms may be useful for overexpression of membrane proteins and photobiological production of H2 and other valuable compounds.

  11. DNA Camouflage

    DTIC Science & Technology

    2016-01-08

    1 DNA Camouflage Supplementary Information Bijan Zakeri1,2*, Timothy K. Lu1,2*, Peter A. Carr2,3* 1Department of Electrical Engineering and...ll.mit.edu). Distribution A: Public Release   2 Supplementary Figure 1 DNA camouflage with the 2-state device. (a) In the presence of Cre, DSD-2[α...Supplementary Figure 2 DNA shuffling does not comprise sequencing outside of DSDs. (a) Sequencing of 1 kb downstream of DSD-2[α] produces high quality

  12. DNA Immunization

    PubMed Central

    Wang, Shixia; Lu, Shan

    2013-01-01

    DNA immunization was discovered in early 1990s and its use has been expanded from vaccine studies to a broader range of biomedical research, such as the generation of high quality polyclonal and monoclonal antibodies as research reagents. In this unit, three common DNA immunization methods are described: needle injection, electroporation and gene gun. In addition, several common considerations related to DNA immunization are discussed. PMID:24510291

  13. Authentication of medicinal plant botanical identity by amplified fragmented length polymorphism dominant DNA marker: inferences from the Plectranthus genus.

    PubMed

    Passinho-Soares, Helna; Felix, Durvalina; Kaplan, Maria Auxiliadora; Margis-Pinheiro, Marcia; Margis, Rogério

    2006-08-01

    In Brazil, Plectranthus species are known as "boldo" and have been used in popular medicine for analgesic and dyspeptic purposes. Plectranthus need to be well identified in order to be used as commercially genuine medicinal plants. Here we describe AFLP DNA patterns able to distinguish among different Pectranthus species. The genetic variability of P. grandis Cramer, P. barbatus Andr. and P. ornatus Codd was analyzed with two sets of AFLP primers allowing detection of 241 loci. A total of 22 monomorphic loci were identified in P. barbatus, 15 in P. grandis and 30 in P. ornatus. Among these, 5 loci were informative and species-specific to P. barbatus, 3 to P. grandis and 2 loci were unique to P. ornatus. The AFLP pattern analyzed by different clustering methods assembled individuals according to their species. So far, AFLP represents a genuine and strong method to certify medicinal plant materials.

  14. Kin Recognition in Bacteria.

    PubMed

    Wall, Daniel

    2016-09-08

    The ability of bacteria to recognize kin provides a means to form social groups. In turn these groups can lead to cooperative behaviors that surpass the ability of the individual. Kin recognition involves specific biochemical interactions between a receptor(s) and an identification molecule(s). Recognition specificity, ensuring that nonkin are excluded and kin are included, is critical and depends on the number of loci and polymorphisms involved. After recognition and biochemical perception, the common ensuing cooperative behaviors include biofilm formation, quorum responses, development, and swarming motility. Although kin recognition is a fundamental mechanism through which cells might interact, microbiologists are only beginning to explore the topic. This review considers both molecular and theoretical aspects of bacterial kin recognition. Consideration is also given to bacterial diversity, genetic relatedness, kin selection theory, and mechanisms of recognition.

  15. Phosphonate utilization by bacteria.

    PubMed Central

    Cook, A M; Daughton, C G; Alexander, M

    1978-01-01

    Bacteria able to use at least one of 13 ionic alkylphosphonates of O-alkyl or O,O-dialkyl alkylphosphonates as phosphorus sources were isolated from sewage and soil. Four of these isolates used 2-aminoethylphosphonic acid (AEP) as a sole carbon, nitrogen, and phosphorus source. None of the other phosphonates served as a carbon source for the organisms. One isolate, identified as Pseudomonas putida, grew with AEP as its sole carbon, nitrogen, and phosphorus source and released nearly all of the organic phosphorus as orthophosphate and 72% of the AEP nitrogen as ammonium. This is the first demonstration of utilization of a phosphonoalkyl moiety as a sole carbon source. Cell-free extracts of P. putida contained an inducible enzyme system that required pyruvate and pyridoxal phosphate to release orthophosphate from AEP; acetaldehyde was tentatively identified as a second product. Phosphite inhibited the enzyme system. PMID:618850

  16. Acoustofluidic bacteria separation

    NASA Astrophysics Data System (ADS)

    Li, Sixing; Ma, Fen; Bachman, Hunter; Cameron, Craig E.; Zeng, Xiangqun; Huang, Tony Jun

    2017-01-01

    Bacterial separation from human blood samples can help with the identification of pathogenic bacteria for sepsis diagnosis. In this work, we report an acoustofluidic device for label-free bacterial separation from human blood samples. In particular, we exploit the acoustic radiation force generated from a tilted-angle standing surface acoustic wave (taSSAW) field to separate Escherichia coli from human blood cells based on their size difference. Flow cytometry analysis of the E. coli separated from red blood cells shows a purity of more than 96%. Moreover, the label-free electrochemical detection of the separated E. coli displays reduced non-specific signals due to the removal of blood cells. Our acoustofluidic bacterial separation platform has advantages such as label-free separation, high biocompatibility, flexibility, low cost, miniaturization, automation, and ease of in-line integration. The platform can be incorporated with an on-chip sensor to realize a point-of-care sepsis diagnostic device.

  17. Toxic effects of gold nanoparticles on Salmonella typhimurium bacteria

    PubMed Central

    Wang, Shuguang; Lawson, Rasheeda; Ray, Paresh C; Yu, Hongtao

    2013-01-01

    Nanometer-sized gold, due to its beautiful and bountiful color and unique optical properties, is a versatile material for many industrial and societal applications. We have studied the effect of gold nanoparticles on Salmonella typhimurium strain TA 102. The gold nanoparticles in solution prepared using the citrate reduction method is found not to be toxic or mutagenic but photomutagenic to the bacteria; however, careful control experiments indicate that the photomutagenicity is due to the co-existing citrate and Au3+ ions, not due to the gold nanoparticle itself. Au3+ is also found to be photomutagenic to the bacteria at concentrations lower than 1 µM, but toxic at higher concentrations. The toxicity of Au3+ is enhanced by light irradiation. The photomutagenicity of both citrate and Au3+ is likely due to the formation of free radicals, as a result of light-induced citrate decarboxylation or Au3+ oxidation of co-existing molecules. Both processes can generate free radicals that may cause DNA damage and mutation. Studies of the interaction of gold nanoparticles with the bacteria indicate that gold nanoparticles can be absorbed onto the bacteria surface but not able to penetrate the bacteria wall to enter the bacteria. PMID:21415096

  18. Bacteria associated with the bleached and cave coral Oculina patagonica.

    PubMed

    Koren, Omry; Rosenberg, Eugene

    2008-04-01

    The relative abundance of bacteria in the mucus and tissues of Oculina patagonica taken from bleached and cave (azooxanthellae) corals was determined by analyses of the 16S rRNA genes from cloned libraries of extracted DNA and from isolated colonies. The results were compared to previously published data on healthy O. patagonica. The bacterial community of bleached, cave, and healthy corals were completely different from each other. A tight cluster (>99.5% identity) of bacteria, showing 100% identity to Acinetobacter species, dominated bleached corals, comprising 25% of the 316 clones sequenced. The dominant bacterial cluster found in cave corals, representing 29% of the 97 clones sequenced, showed 98% identity to an uncultured bacterium from the Great Barrier Reef. Vibrio splendidus was the most dominant species in healthy O. patagonica. The culturable bacteria represented 0.1-1.0% of the total bacteria (SYBR Gold staining) of the corals. The most abundant culturable bacteria in bleached, cave, and healthy corals were clusters that most closely matched Microbulbifer sp., an alpha-proteobacterium previously isolated from healthy corals and an alpha-protobacterium (AB026194), respectively. Three generalizations emerge from this study on O. patagonica: (1) More bacteria are associated with coral tissue than mucus; (2) tissue and mucus populations are different; (3) bacterial populations associated with corals change dramatically when corals lack their symbiotic zooxanthellae, either as a result of the bleaching disease or when growing in the absence of light.

  19. Bacteria Responsive Antibacterial Surfaces for Indwelling Device Infections

    PubMed Central

    Traba, Christian; Liang, Jun F.

    2014-01-01

    Indwelling device infections now represents life-threatening circumstances as a result of the biofilms’ tolerance to antibiotic treatments. Current antibiotic impregnation approaches through sustained antibiotic release have some unsolved problems which include short life-span, narrowed antibacterial spectrum, ineffectiveness towards resistant mutants, and the potential to hasten the antibiotic resistance process. In this study, bacteria responsive anti-biofilm surfaces were developed using bioactive peptides with proved activity to antibiotic resistant bacteria and biofilms. Resulting surfaces were stable under physiological conditions and in the presence of high concentrations of salts (0.5 M NaCl) and biomacromolcules (1.0% DNA and 2.0% alginate), and thus showed good biocompatibility to various tissue cells. However, lytic peptide immobilized surfaces could sense bacteria adhesion and kill attached bacteria effectively and specifically, so biofilms were unable to develop on the lytic peptide immobilized surfaces. Bacteria responsive catheters remained biofilm free for up to a week. Therefore, the bacteria responsive antibacterial surfaces developed in this study represent new opportunities for indwelling device infections. PMID:25481445

  20. DNA extraction protocol for rapid PCR detection of pathogenic bacteria

    USDA-ARS?s Scientific Manuscript database

    Virtually all current assays for foodborne pathogens, including PCR assays, are conducted after lengthy cultural enrichment of the sample to increase the concentration of the target organism. This delays detection by many hours, prevents quantitation, and limits the ability to detect multiple organ...

  1. A nontoxic and versatile protein salting-out method for isolation of DNA.

    PubMed

    Laitinen, J; Samarut, J; Hölttä, E

    1994-08-01

    A pivotal technique in basic and applied molecular biology is the isolation of DNA. However, the present DNA extraction methods are either toxic, expensive, time-consuming and laborious or restricted to certain applications. Here we describe a nontoxic and versatile protein salting-out method for convenient and rapid extraction of large as well as small DNA molecules from vertebrate cells and plasmid DNA from bacteria. Easy and relatively imprecise manipulations of a large number of samples result in high yields of pure mammalian and plasmid DNA that are suitable for transformation of bacteria, restriction enzyme analyses, Southern blotting, end labeling of DNA, PCR and sequencing.

  2. Visualization of yeast chromosomal DNA

    NASA Technical Reports Server (NTRS)

    Lubega, Seth

    1990-01-01

    The DNA molecule is the most significant life molecule since it codes the blue print for other structural and functional molecules of all living organisms. Agarose gel electrophoresis is now being widely used to separate DNA of virus, bacteria, and lower eukaryotes. The task was undertaken of reviewing the existing methods of DNA fractionation and microscopic visualization of individual chromosonal DNA molecules by gel electrophoresis as a basis for a proposed study to investigate the feasibility of separating DNA molecules in free fluids as an alternative to gel electrophoresis. Various techniques were studied. On the molecular level, agarose gel electrophoresis is being widely used to separate chromosomal DNA according to molecular weight. Carl and Olson separate and characterized the entire karyotype of a lab strain of Saccharomyces cerevisiae. Smith et al. and Schwartz and Koval independently reported the visualization of individual DNA molecules migrating through agarose gel matrix during electrophoresis. The techniques used by these researchers are being reviewed in the lab as a basis for the proposed studies.

  3. Evolution of Eukaryotic DNA Polymerases via Interaction Between Cells and Large DNA Viruses.

    PubMed

    Takemura, Masaharu; Yokobori, Shin-ichi; Ogata, Hiroyuki

    2015-08-01

    B-family DNA-directed DNA polymerases are DNA replication enzymes found in Eukaryota, Archaea, large DNA viruses, and in some, but not all, bacteria. Several polymerase domains are conserved among the B-family DNA polymerases from these organisms, suggesting that the B-family DNA polymerases evolved from a common ancestor. Eukaryotes retain at least three replicative B-family DNA polymerases, DNA polymerase α, δ, and ε, and one translesion B-family DNA polymerase, DNA polymerase ζ. Here, we present molecular evolutionary evidence that suggests DNA polymerase genes evolved through horizontal gene transfer between the viral and archaeal-eukaryotic lineages. Molecular phylogenetic analyses of the B-family DNA polymerases from nucleo-cytoplasmic large DNA viruses (NCLDVs), eukaryotes, and archaea suggest that different NCLDV lineages such as Poxviridae and Mimiviridae were involved in the evolution of different DNA polymerases (pol-α-, δ-, ε-, and ζ-like genes) in archaeal-eukaryotic cell lineages, putatively through horizontal gene transfer. These results support existing theories that link the evolution of NCLDVs and the origin of the eukaryotic nucleus.

  4. [Screening and functional properties of cholesterol-degrading lactic acid bacteria from Jiangshui].

    PubMed

    Li, Xueping; Li, Jianhong; Li, Minquan; Meng, Xiangang

    2015-08-04

    We intended to obtain and characterize lactic acid bacteria with high capacity of cholesterol-degrading. We chose Jiangshui as the experimental material, screened lactic acid bacteria by the culture medium with high cholesterol, and studied other features of lactic acid bacteria like salt-tolerant, acid resistance, then identified the species of lactic acid bacteria by combining physiological and biochemical methods and 16S rDNA sequence. All lactic acid bacteria isolated had the capacity of cholesterol-degrading to some extent. There were 4 strains had high cholesterol-degrading rate (> 75%). Four strains were Lactococcus lactis subsp. lactis, two were Brevibacterium casei, and one was Lactococcus raffinolactis. Cholesterol-degrading lactic acid bacteria were screened from Jiangshui, with application potential for cholesterol degradation.

  5. Scarce Evidence of Yogurt Lactic Acid Bacteria in Human Feces after Da