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Sample records for bacterial rrna extraction

  1. Effects of DNA extraction and universal primers on 16S rRNA gene-based DGGE analysis of a bacterial community from fish farming water

    NASA Astrophysics Data System (ADS)

    Luo, Peng; Hu, Chaoqun; Zhang, Lüping; Ren, Chunhua; Shen, Qi

    2007-07-01

    Among many reports investigating microbial diversity from environmental samples with denaturing gradient gel electrophoresis (DGGE), limited attention has been given to the effects of universal primers and DNA extraction on the outcome of DGGE analysis. In this study, these effects were tested with 16S rRNA gene-based DGGE on a bacterial community from farming water samples. The results indicate that the number of discernable bands in the DGGE fingerprint differed with the primer pairs used; the bands produced by 63f/518r, 341f/926r and 933f/1387r primer pairs were obviously fewer than those by 968f/1401r. Also, we found that each DNA extraction method resulted in different community profiles, reflected by the number and intensity of bands in the DGGE fingerprint. Furthermore, the main bands (theoretically representing dominant bacteria) differed with the extraction methods applied. It is therefore believed that the effects of universal primers and DNA extraction should be given more attention and carefully chosen before performing an investigation into a new environment with DGGE.

  2. Evaluating Bias of Illumina-Based Bacterial 16S rRNA Gene Profiles

    PubMed Central

    Kennedy, Katherine; Hall, Michael W.; Lynch, Michael D. J.; Moreno-Hagelsieb, Gabriel

    2014-01-01

    Massively parallel sequencing of 16S rRNA genes enables the comparison of terrestrial, aquatic, and host-associated microbial communities with sufficient sequencing depth for robust assessments of both alpha and beta diversity. Establishing standardized protocols for the analysis of microbial communities is dependent on increasing the reproducibility of PCR-based molecular surveys by minimizing sources of methodological bias. In this study, we tested the effects of template concentration, pooling of PCR amplicons, and sample preparation/interlane sequencing on the reproducibility associated with paired-end Illumina sequencing of bacterial 16S rRNA genes. Using DNA extracts from soil and fecal samples as templates, we sequenced pooled amplicons and individual reactions for both high (5- to 10-ng) and low (0.1-ng) template concentrations. In addition, all experimental manipulations were repeated on two separate days and sequenced on two different Illumina MiSeq lanes. Although within-sample sequence profiles were highly consistent, template concentration had a significant impact on sample profile variability for most samples. Pooling of multiple PCR amplicons, sample preparation, and interlane variability did not influence sample sequence data significantly. This systematic analysis underlines the importance of optimizing template concentration in order to minimize variability in microbial-community surveys and indicates that the practice of pooling multiple PCR amplicons prior to sequencing contributes proportionally less to reducing bias in 16S rRNA gene surveys with next-generation sequencing. PMID:25002428

  3. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

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

  4. Partial Bacterial Proteome Extraction Kit

    E-print Network

    Lebendiker, Mario

    -positive bacteria in the presence of different detergents and chaotropes. P-PEK is designed for a serial samplePartial Bacterial Proteome Extraction Kit Cat. No. 539780 Product Information FOR RESEARCH USE ONLY in the complex samples to be separated by 2DE. Additionally each biological sample requires special procedures e

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

    SciTech Connect

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

    2001-10-01

    ABSTRACT-We report on the development and validation of simple microarray method for the direct detection of intact 16S rRNA from un-purified soil extracts. Total RNA from Geobacter chapellei and Desulfovibrio desulfuricans was hybridized to an oligonculeotide array consisting of universal and species-specific 16S rRNA probes...

  6. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

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

  7. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development - Poster

    EPA Science Inventory

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

  8. Naïve Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy? †

    PubMed Central

    Wang, Qiong; Garrity, George M.; Tiedje, James M.; Cole, James R.

    2007-01-01

    The Ribosomal Database Project (RDP) Classifier, a naïve Bayesian classifier, can rapidly and accurately classify bacterial 16S rRNA sequences into the new higher-order taxonomy proposed in Bergey's Taxonomic Outline of the Prokaryotes (2nd ed., release 5.0, Springer-Verlag, New York, NY, 2004). It provides taxonomic assignments from domain to genus, with confidence estimates for each assignment. The majority of classifications (98%) were of high estimated confidence (?95%) and high accuracy (98%). In addition to being tested with the corpus of 5,014 type strain sequences from Bergey's outline, the RDP Classifier was tested with a corpus of 23,095 rRNA sequences as assigned by the NCBI into their alternative higher-order taxonomy. The results from leave-one-out testing on both corpora show that the overall accuracies at all levels of confidence for near-full-length and 400-base segments were 89% or above down to the genus level, and the majority of the classification errors appear to be due to anomalies in the current taxonomies. For shorter rRNA segments, such as those that might be generated by pyrosequencing, the error rate varied greatly over the length of the 16S rRNA gene, with segments around the V2 and V4 variable regions giving the lowest error rates. The RDP Classifier is suitable both for the analysis of single rRNA sequences and for the analysis of libraries of thousands of sequences. Another related tool, RDP Library Compare, was developed to facilitate microbial-community comparison based on 16S rRNA gene sequence libraries. It combines the RDP Classifier with a statistical test to flag taxa differentially represented between samples. The RDP Classifier and RDP Library Compare are available online at http://rdp.cme.msu.edu/. PMID:17586664

  9. Direct Amplification of rRNA Genes in Diagnosis of Bacterial Infections

    PubMed Central

    Rantakokko-Jalava, Kaisu; Nikkari, Simo; Jalava, Jari; Eerola, Erkki; Skurnik, Mikael; Meurman, Olli; Ruuskanen, Olli; Alanen, Anna; Kotilainen, Esa; Toivanen, Paavo; Kotilainen, Pirkko

    2000-01-01

    A broad-range bacterial PCR targeting rRNA genes (rDNAs) was used to directly analyze 536 clinical samples obtained from 459 hospitalized patients during a 4-year study period. The molecular diagnosis based on DNA sequencing of the PCR product was compared to that obtained by bacterial culture. The bacteriological diagnosis was concordant for 447 (83%) specimens. Broad-range rDNA PCR was the only method that yielded an etiologic diagnosis for 11 (2.4%) of 459 patients. Compared to culture and clinical assessment, the sensitivity of the PCR method combined with sequencing was 74.2%, and the specificity was between 98.7 and 99.6%. At present, the described molecular approach proved superior to bacterial culture in two clinical situations: infections caused by bacteria with unusual growth requirements and specimens taken during antimicrobial treatment of the patient. PMID:10618059

  10. 16S rRNA Gene Pyrosequencing Reveals Bacterial Dysbiosis in the Duodenum of Dogs with Idiopathic Inflammatory Bowel Disease

    PubMed Central

    Suchodolski, Jan S.; Dowd, Scot E.; Wilke, Vicky; Steiner, Jörg M.; Jergens, Albert E.

    2012-01-01

    Background Canine idiopathic inflammatory bowel disease (IBD) is believed to be caused by a complex interaction of genetic, immunologic, and microbial factors. While mucosa-associated bacteria have been implicated in the pathogenesis of canine IBD, detailed studies investigating the enteric microbiota using deep sequencing techniques are lacking. The objective of this study was to evaluate mucosa-adherent microbiota in the duodenum of dogs with spontaneous idiopathic IBD using 16 S rRNA gene pyrosequencing. Methodology/Principal Findings Biopsy samples of small intestinal mucosa were collected endoscopically from healthy dogs (n?=?6) and dogs with moderate IBD (n?=?7) or severe IBD (n?=?7) as assessed by a clinical disease activity index. Total RNA was extracted from biopsy specimens and 454-pyrosequencing of the 16 S rRNA gene was performed on aliquots of cDNA from each dog. Intestinal inflammation was associated with significant differences in the composition of the intestinal microbiota when compared to healthy dogs. PCoA plots based on the unweighted UniFrac distance metric indicated clustering of samples between healthy dogs and dogs with IBD (ANOSIM, p<0.001). Proportions of Fusobacteria (p?=?0.010), Bacteroidaceae (p?=?0.015), Prevotellaceae (p?=?0.022), and Clostridiales (p?=?0.019) were significantly more abundant in healthy dogs. In contrast, specific bacterial genera within Proteobacteria, including Diaphorobacter (p?=?0.044) and Acinetobacter (p?=?0.040), were either more abundant or more frequently identified in IBD dogs. Conclusions/Significance In conclusion, dogs with spontaneous IBD exhibit alterations in microbial groups, which bear resemblance to dysbiosis reported in humans with chronic intestinal inflammation. These bacterial groups may serve as useful targets for monitoring intestinal inflammation. PMID:22720094

  11. Development of a dual-internal-reference technique to improve accuracy when determining bacterial 16S rRNA:16S rRNA gene ratio with application to Escherichia coli liquid and aerosol samples.

    PubMed

    Zhen, Huajun; Krumins, Valdis; Fennell, Donna E; Mainelis, Gediminas

    2015-10-01

    Accurate enumeration of rRNA content in microbial cells, e.g. by using the 16S rRNA:16S rRNA gene ratio, is critical to properly understand its relationship to microbial activities. However, few studies have considered possible methodological artifacts that may contribute to the variability of rRNA analysis results. In this study, a technique utilizing genomic DNA and 16S rRNA from an exogenous species (Pseudomonas fluorescens) as dual internal references was developed to improve accuracy when determining the 16S rRNA:16S rRNA gene ratio of a target organism, Escherichia coli. This technique was able to adequately control the variability in sample processing and analysis procedures due to nucleic acid (DNA and RNA) losses, inefficient reverse transcription of RNA, and inefficient PCR amplification. The measured 16S rRNA:16S rRNA gene ratio of E. coli increased by 2-3 fold when E. coli 16S rRNA gene and 16S rRNA quantities were normalized to the sample-specific fractional recoveries of reference (P. fluorescens) 16S rRNA gene and 16S rRNA, respectively. In addition, the intra-sample variation of this ratio, represented by coefficients of variation from replicate samples, decreased significantly after normalization. This technique was applied to investigate the temporal variation of 16S rRNA:16S rRNA gene ratio of E. coli during its non-steady-state growth in a complex liquid medium, and to E. coli aerosols when exposed to particle-free air after their collection on a filter. The 16S rRNA:16S rRNA gene ratio of E. coli increased significantly during its early exponential phase of growth; when E. coli aerosols were exposed to extended filtration stress after sample collection, the ratio also increased. In contrast, no significant temporal trend in E. coli 16S rRNA:16S rRNA gene ratio was observed when the determined ratios were not normalized based on the recoveries of dual references. The developed technique could be widely applied in studies of relationship between cellular rRNA abundance and bacterial activity. PMID:26241659

  12. Using DGGE and 16S rRNA Gene Sequence Analysis to Evaluate Changes in Oral Bacterial Composition

    PubMed Central

    CHEN, Zhou; TRIVEDI, Harsh M.; CHHUN, Nok; BARNES, Virginia M.; SAXENA, Deepak; XU, Tao; LI, Yihong

    2015-01-01

    Objective To investigate whether a standard dental prophylaxis followed by tooth brushing with an antibacterial dentifrice will affect the oral bacterial community, as determined by denaturing gradient gel electrophoresis (DGGE) combined with 16S rRNA gene sequence analysis. Methods Twenty-four healthy adults were instructed to brush their teeth using commercial dentifrice for 1 week during a washout period. An initial set of pooled supragingival plaque samples was collected from each participant at baseline (0 h) before prophylaxis treatment. The subjects were given a clinical examination and dental prophylaxis and asked to brush for 1 min with a dentifrice containing 0.3% triclosan/2.0% PVM/MA copolymer/0.243% sodium fluoride (Colgate Total). On the following day, a second set of pooled supragingival plaque samples (24 h) was collected. Total bacterial genomic DNA was isolated from the samples. Differences in the microbial composition before and after the prophylactic procedure and tooth brushing were assessed by comparing the DGGE profiles of PCR-amplified and 16S rRNA gene segments sequence analysis. Results Two distinct clusters of DGGE profiles were found, suggesting that a shift in the microbial composition had occurred 24 h after the prophylaxis and brushing. A detailed sequencing analysis of 16S rRNA gene segments further identified six phyla and 29 genera, including known and unknown bacterial species. Importantly, an increase in bacterial diversity was observed after 24 h, including members of the Streptococcaceae family, Prevotella, Corynebacterium, TM7 and other commensal bacteria. Conclusion The results suggest that the use of a standard prophylaxis followed by the use of the dentifrice containing 0.3% triclosan/2.0% PVM/MA copolymer/0.243% sodium fluoride may promote a healthier composition within the oral bacterial community. PMID:22319750

  13. Bacterial Community Dynamics during Production of Registered Designation of Origin Salers Cheese as Evaluated by 16S rRNA Gene Single-Strand Conformation Polymorphism Analysis

    PubMed Central

    Duthoit, Frédérique; Godon, Jean-Jacques; Montel, Marie-Christine

    2003-01-01

    Microbial dynamics during processing and ripening of traditional cheeses such as registered designation of origin Salers cheese, an artisanal cheese produced in France, play an important role in the elaboration of sensory qualities. The aim of the present study was to obtain a picture of the dynamics of the microbial ecosystem of RDO Salers cheese by using culture-independent methods. This included DNA extraction, PCR, and single-strand conformation polymorphism (SSCP) analysis. Bacterial and high-GC% gram-positive bacterial primers were used to amplify V2 or V3 regions of the 16S rRNA gene. SSCP patterns revealed changes during the manufacturing of the cheese. Patterns of the ecosystems of cheeses that were provided by three farmers were also quite different. Cloning and sequencing of the 16S rRNA gene revealed sequences related to lactic acid bacteria (Lactococcus lactis, Streptococcus thermophilus, Enterococcus faecium, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Lactobacillus plantarum, and Lactobacillus pentosus), which were predominant during manufacturing and ripening. Bacteria belonging to the high-GC% gram-positive group (essentially corynebacteria) were found by using specific primers. The present molecular approach can effectively describe the ecosystem of artisanal dairy products. PMID:12839752

  14. Bacterial diversity of a Carolina Bay as determined by 16S rRNA gene analysis: Confirmation of novel taxa

    SciTech Connect

    Wise, M.G.; Shimkets, L.J.; McArthur, J.V.

    1997-04-01

    Carolina bays are naturally occurring shallow elliptical depressions largely fed by rain and shallow ground water. To identify members of the domain Bacteria which inhabit such an environment, we used PCR to construct a library of 16S rRNA genes (16S rDNAs) cloned from DNA extracted from the sediments of Rainbow Bay, located on the Savannah River Site, near Aiken, S.C. Oligonucleotides complementary to conserved regions of 16S rDNA were used as primers for PCR, and gel-purified PCR products were cloned into vector pGEM-T. Partial sequencing of the cloned 16S rDNAs revealed an extensive amount of phylogenetic diversity within this system. Of the 35 clones sequenced, 32 were affiliated with five bacterial groups: 11 clustered with the Proteobacteria division (including members of the alpha, beta, and delta subdivisions), 8 clustered with the Acidobacterium subdivision of the Fibrobacter division (as categorized by the Ribosomal Database Project`s taxonomic scheme, version 5.0), 7 clustered with the Verrucomicrobium subdivision of the Planctomyces division, 3 clustered with the gram-positive bacteria (Clostridium and relatives subdivision), and 3 clustered with the green nonsulfur bacteria. One sequence branched very deeply from the Bacteria and was found not to be associated with any of the major divisions when phylogenetic trees were constructed. Two clones did not consistently cluster with specific groups and may be chimeric sequences. None of the clones exhibited an exact match to any of the 16S rDNA sequences deposited in the databases, suggesting that most of the bacteria in Rainbow Bay are novel species. In particular, the clones related to the Acidobacterium subdivision and the Verrucomicrobium subdivision confirm the presence of novel taxa discovered previously in other molecular surveys of this type. 50 refs., 7 figs., 1 tab.

  15. DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure

    PubMed Central

    Rubin, Benjamin E R; Sanders, Jon G; Hampton-Marcell, Jarrad; Owens, Sarah M; Gilbert, Jack A; Moreau, Corrie S

    2014-01-01

    The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illumina 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied. PMID:25257543

  16. DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure

    DOE PAGESBeta

    None

    2014-12-01

    The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illuminamore »16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied.« less

  17. DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure

    SciTech Connect

    2014-12-01

    The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illumina 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied.

  18. Different bacterial communities in heat and gamma irradiation treated replant disease soils revealed by 16S rRNA gene analysis – contribution to improved aboveground apple plant growth?

    PubMed Central

    Yim, Bunlong; Winkelmann, Traud; Ding, Guo-Chun; Smalla, Kornelia

    2015-01-01

    Replant disease (RD) severely affects apple production in propagation tree nurseries and in fruit orchards worldwide. This study aimed to investigate the effects of soil disinfection treatments on plant growth and health in a biotest in two different RD soil types under greenhouse conditions and to link the plant growth status with the bacterial community composition at the time of plant sampling. In the biotest performed we observed that the aboveground growth of apple rootstock M26 plants after 8 weeks was improved in the two RD soils either treated at 50°C or with gamma irradiation compared to the untreated RD soils. Total community DNA was extracted from soil loosely adhering to the roots and quantitative real-time PCR revealed no pronounced differences in 16S rRNA gene copy numbers. 16S rRNA gene-based bacterial community analysis by denaturing gradient gel electrophoresis (DGGE) and 454-pyrosequencing revealed significant differences in the bacterial community composition even after 8 weeks of plant growth. In both soils, the treatments affected different phyla but only the relative abundance of Acidobacteria was reduced by both treatments. The genera Streptomyces, Bacillus, Paenibacillus, and Sphingomonas had a higher relative abundance in both heat treated soils, whereas the relative abundance of Mucilaginibacter, Devosia, and Rhodanobacter was increased in the gamma-irradiated soils and only the genus Phenylobacterium was increased in both treatments. The increased abundance of genera with potentially beneficial bacteria, i.e., potential degraders of phenolic compounds might have contributed to the improved plant growth in both treatments. PMID:26635733

  19. The Impact of Different DNA Extraction Kits and Laboratories upon the Assessment of Human Gut Microbiota Composition by 16S rRNA Gene Sequencing

    PubMed Central

    Kennedy, Nicholas A.; Walker, Alan W.; Berry, Susan H.; Duncan, Sylvia H.; Farquarson, Freda M.; Louis, Petra; Thomson, John M.; Ahmad, T; Anderson, CA; Barrett, JC; Drummond, H; Edwards, C; Hart, A; Hawkey, C; Henderson, P; Khan, M; Lamb, CA; Lee, JC; Mansfield, JC; Mathew, CG; Mowat, C; Newman, WG; Prescott, NJ; Simmons, A; Simpson, P; Taylor, K; Taylor, K; Wilson, DC; Satsangi, Jack; Flint, Harry J.; Parkhill, Julian

    2014-01-01

    Introduction Determining bacterial community structure in fecal samples through DNA sequencing is an important facet of intestinal health research. The impact of different commercially available DNA extraction kits upon bacterial community structures has received relatively little attention. The aim of this study was to analyze bacterial communities in volunteer and inflammatory bowel disease (IBD) patient fecal samples extracted using widely used DNA extraction kits in established gastrointestinal research laboratories. Methods Fecal samples from two healthy volunteers (H3 and H4) and two relapsing IBD patients (I1 and I2) were investigated. DNA extraction was undertaken using MoBio Powersoil and MP Biomedicals FastDNA SPIN Kit for Soil DNA extraction kits. PCR amplification for pyrosequencing of bacterial 16S rRNA genes was performed in both laboratories on all samples. Hierarchical clustering of sequencing data was done using the Yue and Clayton similarity coefficient. Results DNA extracted using the FastDNA kit and the MoBio kit gave median DNA concentrations of 475 (interquartile range 228-561) and 22 (IQR 9-36) ng/µL respectively (p<0.0001). Hierarchical clustering of sequence data by Yue and Clayton coefficient revealed four clusters. Samples from individuals H3 and I2 clustered by patient; however, samples from patient I1 extracted with the MoBio kit clustered with samples from patient H4 rather than the other I1 samples. Linear modelling on relative abundance of common bacterial families revealed significant differences between kits; samples extracted with MoBio Powersoil showed significantly increased Bacteroidaceae, Ruminococcaceae and Porphyromonadaceae, and lower Enterobacteriaceae, Lachnospiraceae, Clostridiaceae, and Erysipelotrichaceae (p<0.05). Conclusion This study demonstrates significant differences in DNA yield and bacterial DNA composition when comparing DNA extracted from the same fecal sample with different extraction kits. This highlights the importance of ensuring that samples in a study are prepared with the same method, and the need for caution when cross-comparing studies that use different methods. PMID:24586470

  20. Bacterial Community Diversity of Oil-Contaminated Soils Assessed by High Throughput Sequencing of 16S rRNA Genes

    PubMed Central

    Peng, Mu; Zi, Xiaoxue; Wang, Qiuyu

    2015-01-01

    Soil bacteria play a major role in ecological and biodegradable function processes in oil-contaminated soils. Here, we assessed the bacterial diversity and changes therein in oil-contaminated soils exposed to different periods of oil pollution using 454 pyrosequencing of 16S rRNA genes. No less than 24,953 valid reads and 6246 operational taxonomic units (OTUs) were obtained from all five studied samples. OTU richness was relatively higher in contaminated soils than clean samples. Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Planctomycetes and Proteobacteria were the dominant phyla among all the soil samples. The heatmap plot depicted the relative percentage of each bacterial family within each sample and clustered five samples into two groups. For the samples, bacteria in the soils varied at different periods of oil exposure. The oil pollution exerted strong selective pressure to propagate many potentially petroleum degrading bacteria. Redundancy analysis (RDA) indicated that organic matter was the highest determinant factor for explaining the variations in community compositions. This suggests that compared to clean soils, oil-polluted soils support more diverse bacterial communities and soil bacterial community shifts were mainly controlled by organic matter and exposure time. These results provide some useful information for bioremediation of petroleum contaminated soil in the future. PMID:26404329

  1. Use of 16S rRNA Gene for Identification of a Broad Range of Clinically Relevant Bacterial Pathogens

    PubMed Central

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; MacKichan, Joanna; Kato-Maeda, Midori; Miller, Steve; Nadarajan, Rohan; Brodie, Eoin L.; Lynch, Susan V.

    2015-01-01

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n = 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci. PMID:25658760

  2. Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens

    SciTech Connect

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; MacKichan, Joanna; Kato-Maeda, Midori; Miller, Steve; Nadarajan, Rohan; Brodie, Eoin L.; Lynch, Susan V.; Heimesaat, Markus M.

    2015-02-06

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n = 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci.

  3. Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens

    DOE PAGESBeta

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; MacKichan, Joanna; Kato-Maeda, Midori; Miller, Steve; Nadarajan, Rohan; Brodie, Eoin L.; Lynch, Susan V.; Heimesaat, Markus M.

    2015-02-06

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n =more »617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci.« less

  4. Description of Drinking Water Bacterial Communities Using 16S rRNA Gene Sequence Analyses

    EPA Science Inventory

    Descriptions of bacterial communities inhabiting water distribution systems (WDS) have mainly been accomplished using culture-based approaches. Due to the inherent selective nature of culture-based approaches, the majority of bacteria inhabiting WDS remain uncharacterized. The go...

  5. Macroalgal Extracts Induce Bacterial Assemblage Shifts and Sublethal Tissue Stress in Caribbean Corals

    PubMed Central

    Morrow, Kathleen M.; Ritson-Williams, Raphael; Ross, Cliff; Liles, Mark R.; Paul, Valerie J.

    2012-01-01

    Benthic macroalgae can be abundant on present-day coral reefs, especially where rates of herbivory are low and/or dissolved nutrients are high. This study investigated the impact of macroalgal extracts on both coral-associated bacterial assemblages and sublethal stress response of corals. Crude extracts and live algal thalli from common Caribbean macroalgae were applied onto the surface of Montastraea faveolata and Porites astreoides corals on reefs in both Florida and Belize. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene amplicons was used to examine changes in the surface mucus layer (SML) bacteria in both coral species. Some of the extracts and live algae induced detectable shifts in coral-associated bacterial assemblages. However, one aqueous extract caused the bacterial assemblages to shift to an entirely new state (Lobophora variegata), whereas other organic extracts had little to no impact (e.g. Dictyota sp.). Macroalgal extracts more frequently induced sublethal stress responses in M. faveolata than in P. astreoides corals, suggesting that cellular integrity can be negatively impacted in selected corals when comparing co-occurring species. As modern reefs experience phase-shifts to a higher abundance of macroalgae with potent chemical defenses, these macroalgae are likely impacting the composition of microbial assemblages associated with corals and affecting overall reef health in unpredicted and unprecedented ways. PMID:23028648

  6. Variability in abundance of the Bacterial and Archaeal 16S rRNA and amoA genes in water columns of northern South China Sea

    NASA Astrophysics Data System (ADS)

    Liu, H.; Yang, C.; Chen, S.; Xie, W.; Wang, P.; Zhang, C. L.

    2014-12-01

    Recent advances in marine microbial ecology have shown that ammonia-oxidizing Archaea (AOA) are more abundant than ammonia-oxidizing bacteria (AOB), although total Bacteria are more abundant than total Archaea in marine environments. This study aimed to examine the spatial distribution and abundance of planktonic archaeal and bacterial 16S rRNA- and amoA genes in the northern South China Sea. Water samples were collected at different depths at six stations (maximum depth ranging from 1800 m to 3200 m?with four stations (B2, B3, B6, B7) located along a transect from the northeastern continental slope to the Bashi Strait and the other two (D3, D5) located southwest of this transect. Quantitative PCR of the 16S rRNA- and amoA genes was used to estimate the abundances of total Archaea, total Bacteria, and AOA and AOB, respectively. At the B series stations, the abundance of bacterial 16S rRNA gene was twofold to 36fold higher than that of the archaeal 16S rRNA gene while fivefold lower to sixfold higher at the two D stations, with both genes showing peak values slightly below sea surface (5-75 m depths) at all stations. The archaeal amoA gene had similar variations with the archaeal 16S rRNA gene, but was 1-4 orders of magnitude lower than the archaeal 16S rRNA gene at all stations. Bacterial amoA gene was below the detection at all stations. Our results also show the difference in depth profiles among these stations, which may be caused by the difference in water movement between these regions. The non-detection of bacterial amoA gene indicates that ammonia-oxidizing Archaea are the dominant group of microorganisms in nitrification of the South China Sea, which is consistent with observations in other oceans.

  7. 23S rRNA as an a-Maz-ing new bacterial toxin target

    PubMed Central

    Schifano, Jason M; Woychik, Nancy A

    2014-01-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, is a bacterium with the unique ability to persist for years or decades as a latent infection. This latent state, during which bacteria have a markedly altered physiology and are thought to be dormant, is crucial for the bacteria to survive the stressful environments it encounters in the human host. Importantly, M. tuberculosis cells in the dormant state are generally refractory to antibiotics, most of which target cellular processes occurring in actively replicating bacteria. The molecular switches that enable M. tuberculosis to slow or stop its replication and become dormant remain unknown. However, the slow growth and dormant state that are hallmarks of latent tuberculosis infection have striking parallels to the “quasi-dormant” state of Escherichia coli cells caused by the toxin components of chromosomal toxin-antitoxin (TA) modules. An unusually large number of TA modules in M. tuberculosis, including nine in the mazEF family, may contribute to initiating this latent state or to adapting to stress conditions in the host. Toward filling the gap in our understanding of the physiological role of TA modules in M. tuberculosis, we are interested in identifying their molecular mechanisms to better understand how toxins impart growth control. Our recent publication1 uncovered a novel function of a MazF toxin in M. tuberculosis that had not been associated with any other MazF ortholog. This toxin, MazF-mt6, can disrupt protein synthesis by cleavage of 23S rRNA at a single location in an evolutionarily conserved five-base sequence in the ribosome active center. PMID:24525465

  8. Sequence-specific bacterial growth inhibition by peptide nucleic acid targeted to the mRNA binding site of 16S rRNA.

    PubMed

    Hatamoto, Masashi; Nakai, Kazufumi; Ohashi, Akiyoshi; Imachi, Hiroyuki

    2009-10-01

    Peptide nucleic acid (PNA) targeted to the functional domains of 23S rRNA can inhibit translation and cell growth. However, effective inhibition of translation and cell growth using 16S rRNA-targeted PNA has still not been achieved. Here, we report that PNA targeted to the functional site of 16S rRNA could inhibit both gene expression in vitro and bacterial growth in pure culture with sequence specificity. We used 10-mer PNAs conjugated with a cell-penetrating peptide, which targeted the mRNA binding site at the 3' end of 16S rRNA. Using 0.6 microM of the peptide-PNAs, cell-free ss-galactosidase production decreased by 50%, whereas peptide-PNAs with one or two mismatches to the target sequence showed much weaker inhibition effects. To determine the growth inhibition and bactericidal effects of the peptide-PNA conjugate, we performed OD measurement and viable cell counting. We observed dose- and sequence-dependent inhibition of cell growth and bactericidal effects. These growth inhibitory effects are observed both in the Gram-negative bacterium of Escherichia coli and the Gram-positive bacteria Bacillus subtilis and Corynebacterium efficiens, although inhibitory concentrations were different for each bacterial species. These results present possibilities for 16S rRNA sequence-based specific bacterial growth inhibition using a peptide-PNA conjugate. PMID:19578844

  9. Diversity of endophytic bacteria in Malaysian plants as revealed by 16S rRNA encoding gene sequence based method of bacterial identification?

    PubMed Central

    Loh, Chye Ying; Tan, Yin Yin; Rohani, Rahim; Weber, Jean-Frédéric F.; Bhore, Subhash Janardhan

    2013-01-01

    Bacterial endophytes do have several potential applications in pharmacy, medicine and agricultural biotech industry. The main objective of this study was to understand types of bacterial endophytes associated with dicotyledonous (dicot) and monocotyledonous (monocot) plant species. Isolation of the endophytic bacteria was performed using surface-sterilized various tissue samples, and identification of the endophytic bacterial isolates (EBIs) was completed using 16S rRNA encoding gene sequence similarity based method. In total, 996 EBIs were isolated and identified from 1055 samples of 31 monocot and 65 dicot plant species from Peninsular Malaysia. The 996 EBIs represented 71 different types of bacterial species. Twelve (12) out of 71 species are reported as endophytes for the first time. We conclude that diverse types of bacterial endophytes are associated with dicot and monocot plants, and could be useful in pharmacy, medicine and agricultural biotechnology for various potential applications. PMID:24396249

  10. Sulfur-oxidizing bacterial endosymbionts: analysis of phylogeny and specificity by 16S rRNA sequences. [Calyptogena magnifica; Bathymodiolus thermophilus; Lucinoma annulata; Lucinoma aequizonata; Codakia orbicularis

    SciTech Connect

    Distel, D.L.; Lane, D.J.; Olsen, G.J.; Giovannoni, S.J.; Pace, B.; Pace, N.R.; Stahl, D.A.; Felbeck, H.

    1988-06-01

    The 16S rRNAs from the bacterial endosymbionts of six marine invertebrates from diverse environments were isolated and partially sequenced. These symbionts included the trophosome symbiont of Riftia pachyptila, the gill symbionts of Calyptogena magnifica and Bathymodiolus thermophilus (from deep-sea hydrothermal vents), and the gill symbionts of Lucinoma annulata, Lucinoma aequizonata, and Codakia orbicularis (from relatively shallow coastal environments). Only one type of bacterial 16S rRNA was detected in each symbiosis. Using nucleotide sequence comparisons, we showed that each of the bacterial symbionts is distinct from the others and that all fall within a limited domain of the gamma subdivision of the purple bacteria (one of the major eubacterial divisions previously defined by 16S rRNA analysis. Two host specimens were analyzed in five of the symbioses; in each case, identical bacterial rRNA sequences were obtained from conspecific host specimens. These data indicate that the symbioses examined are species specific and that the symbiont species are unique to and invariant within their respective host species.

  11. [Characterizing Beijing's Airborne Bacterial Communities in PM2.5 and PM1 Samples During Haze Pollution Episodes Using 16S rRNA Gene Analysis Method].

    PubMed

    Wang, Bu-ying; Lang, Ji-dong; Zhang, Li-na; Fang, Jian-huo; Cao, Chen; Hao, Ji-ming; Zhu, Ting; Tian, Geng; Jiang, Jing-kun

    2015-08-01

    During 8th-14th Jan., 2013, severe particulate matter (PM) pollution episodes happened in Beijing. These air pollution events lead to high risks for public health. In addition to various PM chemical compositions, biological components in the air may also impose threaten. Little is known about airborne microbial community in such severe air pollution conditions. PM2.5 and PM10 samples were collected during that 7-day pollution period. The 16S rRNA gene V3 amplification and the MiSeq sequencing were performed for analyzing these samples. It is found that there is no significant difference at phylum level for PM2.5 bacterial communities during that 7-day pollution period both at phylum and at genus level. At genus level, Arthrobacter and Frankia are the major airborne microbes presented in Beijing winter.samples. At genus level, there are 39 common genera (combined by first 50 genera bacterial of the two analysis) between the 16S rRNA gene analysis and those are found by Metagenomic analysis on the same PM samples. Frankia and Paracoccus are relatively more abundant in 16S rRNA gene data, while Kocuria and Geodermatophilus are relatively more abundant in Meta-data. PM10 bacterial communities are similar to those of PM2.5 with some noticeable differences, i.e., at phylum level, more Firmicutes and less Actinobacteria present in PM10 samples than in PM2.5 samples, while at genus level, more Clostridium presents in PM10 samples. The findings in Beijing were compared with three 16S rRNA gene studies in other countries. Although the sampling locations and times are different from each other, compositions of bacterial community are similar for those sampled at the ground atmosphere. Airborne microbial communities near the ground surface are different from those sampled in the upper troposphere. PMID:26591997

  12. Sponge-associated actinobacterial diversity: validation of the methods of actinobacterial DNA extraction and optimization of 16S rRNA gene amplification.

    PubMed

    Yang, Qi; Franco, Christopher M M; Zhang, Wei

    2015-10-01

    Experiments were designed to validate the two common DNA extraction protocols (CTAB-based method and DNeasy Blood & Tissue Kit) used to effectively recover actinobacterial DNA from sponge samples in order to study the sponge-associated actinobacterial diversity. This was done by artificially spiking sponge samples with actinobacteria (spores, mycelia and a combination of the two). Our results demonstrated that both DNA extraction methods were effective in obtaining DNA from the sponge samples as well as the sponge samples spiked with different amounts of actinobacteria. However, it was noted that in the presence of the sponge, the bacterial 16S rRNA gene could not be amplified unless the combined DNA template was diluted. To test the hypothesis that the extracted sponge DNA contained inhibitors, dilutions of the DNA extracts were tested for six sponge species representing five orders. The results suggested that the inhibitors were co-extracted with the sponge DNA, and a high dilution of this DNA was required for the successful PCR amplification for most of the samples. The optimized PCR conditions, including primer selection, PCR reaction system and program optimization, further improved the PCR performance. However, no single PCR condition was found to be suitable for the diverse sponge samples using various primer sets. These results highlight for the first time that the DNA extraction methods used are effective in obtaining actinobacterial DNA and that the presence of inhibitors in the sponge DNA requires high dilution coupled with fine tuning of the PCR conditions to achieve success in the study of sponge-associated actinobacterial diversity. PMID:26245685

  13. Bacterial diversity among small-subunit rRNA gene clones and cellular isolates from the same seawater sample

    SciTech Connect

    Suzuki, M.T.; Rappe, M.S.; Haimberger, Z.W.

    1997-03-01

    Numerous investigations applying the cloning and sequencing of rRNA genes (rDNAs) to the study of marine bacterioplankton diversity have shown that the sequences of genes cloned directly from environmental DNA do not correspond to the genes of cultured marine taxa. These results have been interpreted as support for the hypothesis that the most abundant heterotrophic marine bacterioplankton species are not readily culturable by commonly used methods. However, an alternative explanation is that marine bacterioplankton can be easily cultured but are not well represented in sequence databases. To further examine this question, we compared the small-subunit (SSU) rDNAs of 127 cellular clones isolated from a water sample collected off the Oregon coast to 58 bacterial SSU rDNAs cloned from environmental DNAs from the same water sample. The results revealed little overlap between partial SSU rDNA sequences from the cellular clones and the environmental clone library. An exception was the SSU rDNA sequence recovered from a cellular clone belonging to the Pseudomonas subgroup of the {gamma} subclass of the class Proteobacteria, which was related to a single gene cloned directly from the same water sample (OCS181) (similarity, 94.6%). In addition, partial SSU rDNA sequences from three of the cultured strains matched a novel rDNA clone related to the {gamma} subclass of the Proteobacteria found previously in an environmental clone library from marine aggregates (AGG53) (similarity, 94.3 to 99.6%). Our results support the hypothesis that many of the most abundant bacterioplankton species are not readily culturable by standard methods but also show that heterotrophic bacterioplankton that are culturable on media with high organic contents include many strains for which SSU rDNA sequences are not available in sequence databases. 34 refs., 4 figs., 3 tabs.

  14. Ultradeep 16S rRNA Sequencing Analysis of Geographically Similar but Diverse Unexplored Marine Samples Reveal Varied Bacterial Community Composition

    PubMed Central

    Karutha Pandian, Shunmugiah

    2013-01-01

    Background Bacterial community composition in the marine environment differs from one geographical location to another. Reports that delineate the bacterial diversity of different marine samples from geographically similar location are limited. The present study aims to understand whether the bacterial community compositions from different marine samples harbour similar bacterial diversity since these are geographically related to each other. Methods and Principal Findings In the present study, 16S rRNA deep sequencing analysis targeting V3 region was performed using Illumina bar coded sequencing. A total of 22.44 million paired end reads were obtained from the metagenomic DNA of Marine sediment, Rhizosphere sediment, Seawater and the epibacterial DNA of Seaweed and Seagrass. Diversity index analysis revealed that Marine sediment has the highest bacterial diversity and the least bacterial diversity was observed in Rhizosphere sediment. Proteobacteria, Actinobacteria and Bacteroidetes were the dominant taxa present in all the marine samples. Nearly 62–71% of rare species were identified in all the samples and most of these rare species were unique to a particular sample. Further taxonomic assignment at the phylum and genus level revealed that the bacterial community compositions differ among the samples. Conclusion This is the first report that supports the fact that, bacterial community composition is specific for specific samples irrespective of its similar geographical location. Existence of specific bacterial community for each sample may drive overall difference in bacterial structural composition of each sample. Further studies like whole metagenomic sequencing will throw more insights to the key stone players and its interconnecting metabolic pathways. In addition, this is one of the very few reports that depicts the unexplored bacterial diversity of marine samples (Marine sediment, Rhizosphere sediment, Seawater) and the host associated marine samples (Seaweed and Seagrass) at higher depths from uncharacterised coastal region of Palk Bay, India using next generation sequencing technology. PMID:24167548

  15. Bacterial Diversity Studies Using the 16S rRNA Gene Provide a Powerful Research-Based Curriculum for Molecular Biology Laboratory

    PubMed Central

    BOOMER, SARAH M.; LODGE, DANIEL P.; DUTTON, BRYAN E.

    2002-01-01

    We have developed a ten-week curriculum for molecular biology that uses 16S ribosomal RNA genes to characterize and compare novel bacteria from hot spring communities in Yellowstone National Park. The 16S rRNA approach bypasses selective culture-based methods. Our molecular biology course offered the opportunity for students to learn broadly applicable methods while contributing to a long-term research project. Specifically, students isolated and characterized clones that contained novel 16S rRNA inserts using restriction enzyme, DNA sequencing, and computer-based phylogenetic methods. In both classes, students retrieved novel bacterial 16S rRNA genes, several of which were most similar to Green Nonsulfur bacterial isolates. During class, we evaluated student performance and mastery of skills and concepts using quizzes, formal lab notebooks, and a broad project assignment. For this report, we also assessed student performance alongside data quality and discussed the significance, our goal being to improve both research and teaching methods. PMID:23653546

  16. Isolation, Characterization, and Identification of Bacterial Contaminants in Semifinal Gelatin Extracts

    PubMed Central

    De Clerck, E.; Vanhoutte, T.; Hebb, T.; Geerinck, J.; Devos, J.; De Vos, P.

    2004-01-01

    Bacterial contamination of gelatin is of great concern. Indeed, this animal colloid has many industrial applications, mainly in food and pharmaceutical products. In a previous study (E. De Clerck and P. De Vos, Syst. Appl. Microbiol. 25:611-618), contamination of a gelatin production process with a variety of gram-positive and gram-negative bacteria was demonstrated. In this study, bacterial contamination of semifinal gelatin extracts from several production plants was examined. Since these extracts are subjected to harsh conditions during production and a final ultrahigh-temperature treatment, the bacterial load at this stage is expected to be greatly reduced. In total, 1,129 isolates were obtained from a total of 73 gelatin batches originating from six different production plants. Each of these batches was suspected of having bacterial contamination based on quality control testing at the production plant from which it originated. For characterization and identification of the 1,129 bacterial isolates, repetitive-element PCR was used to obtain manageable groups. Representative strains were identified by means of 16S rRNA genesequencing, species-specific gyrB PCR, and gyrA and rpoB sequencing and were tested for gelatinase activity. The majority of isolates belonged to members of Bacillus or related endospore-forming genera. Representative strains were identified as Bacillus cereus, Bacillus coagulans, Bacillus fumarioli, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus sonorensis, Bacillus subtilis, Bacillus gelatini, Bacillus thermoamylovorans, Anoxybacillus contaminans, Anoxybacillus flavithermus, Brevibacillus agri, Brevibacillus borstelensis, and Geobacillus stearothermophilus. The majority of these species include strains exhibiting gelatinase activity. Moreover, some of these species have known pathogenic properties. These findings are of great concern with regard to the safety and quality of gelatin and its applications. PMID:15184171

  17. Diversity and abundance of the bacterial 16S rRNA gene sequences in forestomach of alpacas (Lama pacos) and sheep (Ovis aries).

    PubMed

    Pei, Cai-Xia; Liu, Qiang; Dong, Chang-Sheng; Li, HongQuan; Jiang, Jun-Bing; Gao, Wen-Jun

    2010-08-01

    Two bacterial 16S rRNA gene clone libraries were constructed from the forestomach of alpacas and sheep fed alfalfa. After the amplification using the universal 16S rRNA gene primers, equal quantities of PCR products from the same species were mixed and used to construct the two libraries. Sequence analysis showed that the 60 clones from alpacas were divided into 27 phylotypes with 25% clones affiliated with Eubacterium sp. F1. The 60 clones from sheep were divided into 21 phylotypes with 7 phylotypes affiliated with Prevotella ruminicola (40% clones). Clones closely related to Clostridium proteoclasticum, Eubacterium sp. F1, Clostridium cellobioparum, Mogibacterium neglectum, Eubacterium ventriosum, Clostridiaceae bacterium WN011, Clostridium coccoides, Clostridium orbiscindens, Eubacterium sp. F1, Cytophaga sp. Dex80-37, Treponema bryantii and Pelotomaculum sp. FP were only found in the forestomach of alpacas, and those to Anaerovorax odorimutans, Treponema zioleckii, Bifidobacterium indicum, Paludibacter propionicigenes, Paraprevotella clara, Eubacterium siraeum, Desulfotomaculum sp. CYP1, Clostridium bolteae, Clostridium termitidis and Clostridiaceae bacterium DJF_LS40 only in the rumen of sheep. Quantitative real-time PCR revealed that the forestomach of alpacas had significantly lower density of bacteria, with bacterial 16S rRNA gene copies (6.89 [Log10 (copies per gram of wet weight)]), than that of sheep (7.71, P<0.01). The two clone libraries also appeared different in Shannon index (library from alpacas 3.30 and from sheep 3.04). Our results showed that there were apparent differences in the bacterial diversity and abundance in the forestomach between alpacas and sheep. PMID:20558310

  18. Presenter: Chengyong Yang E-mail: cyang01@cs.fiu.edu An Ecoinformatics Tool for Microbial Diversity Studies: Supervised Classification of ALH Profliles of 16S rRNA

    E-print Network

    Narasimhan, Giri

    Studies: Supervised Classification of ALH Profliles of 16S rRNA C. Yang1,Y. Wang1, D. Mills1, K. Mathee1 index of soil ecosystem health. Direct amplification of bacterial 16S rRNA genes from extracted soil DNA heterogeneity (ALH) profiles of the hypervariable regions from 16S rRNA have been used to rapidly analyze

  19. Bacterial Community Composition in the Gut Content and Ambient Sediment of Sea Cucumber Apostichopus japonicus Revealed by 16S rRNA Gene Pyrosequencing

    PubMed Central

    Gao, Fei; Li, Fenghui; Tan, Jie; Yan, Jingping; Sun, Huiling

    2014-01-01

    The composition of the bacterial communities in the contents of the foregut and hindgut of the sea cucumber Apostichopus japonicus and in the ambient surface sediment was surveyed by 16S rRNA gene 454-pyrosequencing. A total of 188,623 optimized reads and 15,527 operational taxonomic units (OTUs) were obtained from the ten gut contents samples and four surface sediment samples. The sequences in the sediments, foregut contents, and hindgut contents were assigned to 38.0±4.7, 31.2±6.2 and 27.8±6.5 phyla, respectively. The bacterial richness and Shannon diversity index were both higher in the ambient sediments than in the gut contents. Proteobacteria was the predominant phylum in both the gut contents and sediment samples. The predominant classes in the foregut, hindgut, and ambient sediment were Holophagae and Gammaproteobacteria, Deltaproteobacteria and Gammaproteobacteria, and Gammaproteobacteria and Deltaproteobacteria, respectively. The potential probiotics, including sequences related to Bacillus, lactic acid bacteria (Lactobacillus, Lactococcus, and Streptococcus) and Pseudomonas were detected in the gut of A. japonicus. Principle component analysis and heatmap figure showed that the foregut, hindgut, and ambient sediment respectively harbored different characteristic bacterial communities. Selective feeding of A. japonicus may be the primary source of the different bacterial communities between the foregut contents and ambient sediments. PMID:24967593

  20. Bacterial Population Changes in a Membrane Bioreactor for Graywater Treatment Monitored by Denaturing Gradient Gel Electrophoretic Analysis of 16S rRNA Gene Fragments

    PubMed Central

    Stamper, David M.; Walch, Marianne; Jacobs, Rachel N.

    2003-01-01

    The bacterial population of a graywater treatment system was monitored over the course of 100 days, along with several wastewater biochemical parameters. The graywater treatment system employed an 1,800-liter membrane bioreactor (MBR) to process the waste, with essentially 100% recycling of the biomass. Graywater feed consisting of 10% galley water and 90% laundry water, selected to approximate the graywater composition on board U.S. Navy ships, was collected offsite. Five-day biological oxygen demand (BOD5), oils and greases (O/G), nitrogen, and phosphorus were monitored in the feed and were found to vary greatly day to day. Changes in the bacterial population were monitored by PCR amplification of region 332 to 518 (Escherichia coli numbering) of the 16S rRNA gene and denaturing gradient gel electrophoresis (DGGE) analysis of the resultant PCR products. DGGE analysis indicated a diverse and unstable bacterial population throughout the 100-day period, with spikes in feed strength causing significant changes in community structure. Long-term similarity between the communities was 0 to 25%, depending on the method of analysis. In spite of the unstable bacterial population, the MBR system was able to meet effluent quality parameters approximately 90% of the time. PMID:12571004

  1. Bacterial community structure in Apis florea larvae analyzed by denaturing gradient gel electrophoresis and 16S rRNA gene sequencing.

    PubMed

    Saraithong, Prakaimuk; Li, Yihong; Saenphet, Kanokporn; Chen, Zhou; Chantawannakul, Panuwan

    2015-10-01

    This study characterizes the colonization and composition of bacterial flora in dwarf Asian honeybee (Apis florea) larvae and compares bacterial diversity and distribution among different sampling locations. A. florea larvae were collected from 3 locations in Chiang Mai province, Thailand. Bacterial DNA was extracted from each larva using the phenol-chloroform method. Denaturing gradient gel electrophoresis was performed, and the dominant bands were excised from the gels, cloned, and sequenced for bacterial species identification. The result revealed similarities of bacterial community profiles in each individual colony, but differences between colonies from the same and different locations. A. florea larvae harbor bacteria belonging to 2 phyla (Firmicutes and Proteobacteria), 5 classes (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacilli, and Clostridia), 6 genera (Clostridium, Gilliamella, Melissococcus, Lactobacillus, Saccharibacter, and Snodgrassella), and an unknown genus from uncultured bacterial species. The classes with the highest abundance of bacteria were Alphaproteobacteria (34%), Bacilli (25%), Betaproteobacteria (11%), Gammaproteobacteria (10%), and Clostridia (8%), respectively. Similarly, uncultured bacterial species were identified (12%). Environmental bacterial species, such as Saccharibacter floricola, were also found. This is the first study in which sequences closely related to Melissococcus plutonius, the causal pathogen responsible for European foulbrood, have been identified in Thai A. florea larvae. PMID:25393530

  2. Bacterial Community Composition in Central European Running Waters Examined by Temperature Gradient Gel Electrophoresis and Sequence Analysis of 16S rRNA Genes? †

    PubMed Central

    Beier, Sara; Witzel, Karl-Paul; Marxsen, Jürgen

    2008-01-01

    The bacterial community composition in small streams and a river in central Germany was examined by temperature gradient gel electrophoresis (TGGE) with PCR products of 16S rRNA gene fragments and sequence analysis. Complex TGGE band patterns suggested high levels of diversity of bacterial species in all habitats of these environments. Cluster analyses demonstrated distinct differences among the communities in stream and spring water, sandy sediments, biofilms on stones, degrading leaves, and soil. The differences between stream water and sediment were more significant than those between sites within the same habitat along the stretch from the stream source to the mouth. TGGE data from an entire stream course suggest that, in the upper reach of the stream, a special suspended bacterial community is already established and changes only slightly downstream. The bacterial communities in water and sediment in an acidic headwater with a pH below 5 were highly similar to each other but deviated distinctly from the communities at the other sites. As ascertained by nucleotide sequence analysis, stream water communities were dominated by Betaproteobacteria (one-third of the total bacteria), whereas sediment communities were composed mainly of Betaproteobacteria and members of the Fibrobacteres/Acidobacteria group (each accounting for about 25% of bacteria). Sequences obtained from bacteria from water samples indicated the presence of typical cosmopolitan freshwater organisms. TGGE bands shared between stream and soil samples, as well as sequences found in bacteria from stream samples that were related to those of soil bacteria, demonstrated the occurrence of some species in both stream and soil habitats. Changes in bacterial community composition were correlated with geographic distance along a stream, but in comparisons of different streams and rivers, community composition was correlated only with environmental conditions. PMID:18024682

  3. Base-specific fragmentation of amplified 16S rRNA genes analyzed by mass spectrometry: A tool for rapid bacterial identification

    PubMed Central

    von Wintzingerode, Friedrich; Böcker, Sebastian; Schlötelburg, Cord; Chiu, Norman H. L.; Storm, Niels; Jurinke, Christian; Cantor, Charles R.; Göbel, Ulf B.; van den Boom, Dirk

    2002-01-01

    A rapid approach to the 16S rRNA gene (16S rDNA)-based bacterial identification has been developed that combines uracil-DNA-glycosylase (UDG)-mediated base-specific fragmentation of PCR products with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). 16S rDNA signature sequences were PCR-amplified from both cultured and as-yet-uncultured bacteria in the presence of dUTP instead of dTTP. These PCR products then were immobilized onto a streptavidin-coated solid support to selectively generate either sense or antisense templates. Single-stranded amplicons were subsequently treated with uracil-DNA-glycosylase to generate T-specific abasic sites and fragmented by alkaline treatment. The resulting fragment patterns were analyzed by MALDI-TOF MS. Mass signals of 16S rDNA fragments were compared with patterns calculated from published 16S rDNA sequences. MS of base-specific fragments of amplified 16S rDNA allows reliable discrimination of sequences differing by only one nucleotide. This approach is fast and has the potential for high-throughput identification as required in clinical, pharmaceutical, or environmental microbiology. In contrast to identification by MS of intact whole bacterial cells, this technique allows for the characterization of both cultured and as-yet-uncultured bacteria. PMID:11983869

  4. [Dominant phylotypes in the 16S rRNA gene clone libraries from bacterial mats of the Uzon caldera (Kamchatka, Russia) hydrothermal springs].

    PubMed

    Akimov, V N; Podosokorskaia, O A; Shliapnikov, M G; Gal'chenko, V F

    2013-01-01

    In situ analysis of the 16S rRNA genes form bacterial mats of five hydrothermal springs (36-58 degrees C) in the Uzon caldera (Kamchatka, Russia) was carried out using clone libraries. Eight clone libraries contained 18 dominant phylotypes (over 4-5%). In most clone libraries, the phylotype of the green sulfur bacterium Chlorobaculum sp. was among the dominant ones. The phylotypes of the green nonsulfur bacteria Chloroflexus and Roseiflexus and of purple nonsulfur bacteria Rhodoblastus, Rhodopseudomonas, and Rhodoferax were also among the dominant ones. Cyanobacteria were represented by one dominant phylotype in a single spring. Among nonphototrophic bacteria, the dominant phylotypes belonged to Sulfyrihydrogenibium sp., Geothrixsp., Acidobacterium sp., Meiothermus sp., Thiomonas sp., Thiofaba sp., and Spirochaeta sp. Three phylotypes were not identified at the genus level. Most genera of phototrophic and nonphototrophic organisms corresponding to the phylotypes from Uzon hydrotherms have been previously revealed in the hydrotherms of volcanically active regions of America, Asia, and Europe. These results indicate predominance of bacterial mats carrying out anaerobic photosynthesis in the hydrotherms of the Uzon caldera. PMID:25509409

  5. Rapid 16S rRNA Next-Generation Sequencing of Polymicrobial Clinical Samples for Diagnosis of Complex Bacterial Infections

    PubMed Central

    Salipante, Stephen J.; Sengupta, Dhruba J.; Rosenthal, Christopher; Costa, Gina; Spangler, Jessica; Sims, Elizabeth H.; Jacobs, Michael A.; Miller, Samuel I.; Hoogestraat, Daniel R.; Cookson, Brad T.; McCoy, Connor; Matsen, Frederick A.; Shendure, Jay; Lee, Clarence C.; Harkins, Timothy T.; Hoffman, Noah G.

    2013-01-01

    Classifying individual bacterial species comprising complex, polymicrobial patient specimens remains a challenge for culture-based and molecular microbiology techniques in common clinical use. We therefore adapted practices from metagenomics research to rapidly catalog the bacterial composition of clinical specimens directly from patients, without need for prior culture. We have combined a semiconductor deep sequencing protocol that produces reads spanning 16S ribosomal RNA gene variable regions 1 and 2 (?360 bp) with a de-noising pipeline that significantly improves the fraction of error-free sequences. The resulting sequences can be used to perform accurate genus- or species-level taxonomic assignment. We explore the microbial composition of challenging, heterogeneous clinical specimens by deep sequencing, culture-based strain typing, and Sanger sequencing of bulk PCR product. We report that deep sequencing can catalog bacterial species in mixed specimens from which usable data cannot be obtained by conventional clinical methods. Deep sequencing a collection of sputum samples from cystic fibrosis (CF) patients reveals well-described CF pathogens in specimens where they were not detected by standard clinical culture methods, especially for low-prevalence or fastidious bacteria. We also found that sputa submitted for CF diagnostic workup can be divided into a limited number of groups based on the phylogenetic composition of the airway microbiota, suggesting that metagenomic profiling may prove useful as a clinical diagnostic strategy in the future. The described method is sufficiently rapid (theoretically compatible with same-day turnaround times) and inexpensive for routine clinical use. PMID:23734239

  6. Evaluation of bacterial communities by bacteriome analysis targeting 16S rRNA genes and quantitative analysis of ammonia monooxygenase gene in different types of compost.

    PubMed

    Kitamura, Rika; Ishii, Kazuo; Maeda, Isamu; Kozaki, Toshinori; Iwabuchi, Kazunori; Saito, Takahiro

    2016-01-01

    Biofiltration technology based on microbial degradation and assimilation is used for the removal of malodorous compounds, such as ammonia. Microbes that degrade malodorous and/or organic substances are involved in composting and are retained after composting; therefore, mature composts can serve as an ideal candidate for a biofilter medium. In this study, we focused on different types of raw compost materials, as these are important factors determining the bacterial community profile and the chemical component of the compost. Therefore, bacterial community profiles, the abundance of the bacterial ammonia monooxygenase gene (amoA), and the quantities of chemical components were analyzed in composts produced from either food waste or cattle manure. The community profiles with the lowest beta diversity were obtained from single type of cattle manure compost. However, cattle manure composts showed greater alpha diversity, contained higher amounts of various rRNA gene fragments than those of food waste composts and contained the amoA gene by relative quantification, and Proteobacteria were abundantly found and nitrifying bacteria were detected in it. Nitrifying bacteria are responsible for ammonia oxidation and mainly belong to the Proteobacteria or Nitrospira phyla. The quantities of chemical components, such as salt, phosphorus, and nitrogen, differed between the cattle manure and food waste composts, indicating that the raw materials provided different fermentation environments that were crucial for the formation of different community profiles. The results also suggest that cattle manure might be a more suitable raw material for the production of composts to be used in the biofiltration of ammonia. PMID:26111599

  7. Bacterial cellulose production from the litchi extract by Gluconacetobacter xylinus.

    PubMed

    Yang, Xiao-Yan; Huang, Chao; Guo, Hai-Jun; Xiong, Lian; Luo, Jun; Wang, Bo; Lin, Xiao-Qing; Chen, Xue-Fang; Chen, Xin-De

    2016-01-01

    Although litchi has both nutrient and edible value, the extremely short preservation time limited its further market promotion. To explore processed litchi products with longer preservation time, litchi extract was selected as an alternative feedstock for production of bacterial cellulose (BC). After 2 weeks of static fermentation, 2.53 g/L of the BC membrane was obtained. The trace elements including magnesium (Mg) and sodium (Na) in the litchi extract were partly absorbed in the BC membrane, but no potassium (K) element was detected in it, curiously. Scanning electron microscope (SEM) photographs exhibited an ultrafine network nanostructure for the BC produced in the litchi extract. Analysis of the fourier-transform infrared spectroscopy (FTIR) confirmed the pellicles to be a cellulosic material. Interestingly, X-ray diffraction (XRD) results showed the BC membrane obtained from litchi extract had higher crystallinity of 94.0% than that from HS medium. Overall, the work showed the potential of producing high value-added polymer from litchi resources. PMID:25181328

  8. Minimization of chloroplast contamination in 16S rRNA gene pyrosequencing of insect herbivore bacterial communities

    PubMed Central

    Hanshew, Alissa S.; Mason, Charles J.; Raffa, Kenneth F.; Currie, Cameron R.

    2014-01-01

    Chloroplast sequence contamination in 16S ribosomal RNA gene (16S) analyses can be particularly problematic when sampling microbial communities in plants and folivorous arthropods. We previously encountered high levels of plastid contamination in herbivorous insect samples when we used the predominant 454 pyrosequencing 16S methodologies described in the literature. 799F, a primer previously found to exclude chloroplast sequences, was modified to enhance its efficacy, and we describe, in detail, our methodology throughout amplicon pyrosequencing. Thirteen versions of 799F were assessed for the exclusion of chloroplast sequences from our samples. We found that a shift in the mismatch between 799F and chloroplast 16S resulted in significant reduction of chloroplast reads. Our results also indicate that amplifying sequences from environmental samples in a two-step PCR process, with the addition of the multiplex identifiers and 454 adapters in a second round of PCR, further improved primer specificity. Primers that included 3? phosphorothioate bonds, which were designed to block primer degradation, did not amplify consistently across samples. The different forward primers do not appear to bias the bacterial communities detected. We provide a methodological framework for reducing chloroplast reads in high-throughput sequencing data sets that can be applied to a number of environmental samples and sequencing techniques. PMID:23968645

  9. Identification of Bacterial Populations in Dairy Wastewaters by Use of 16S rRNA Gene Sequences and Other Genetic Markers

    PubMed Central

    McGarvey, Jeffery A.; Miller, William G.; Sanchez, Susan; Stanker, Larry

    2004-01-01

    Hydraulic flush waste removal systems coupled to solid/liquid separators and circulated treatment lagoons are commonly utilized to manage the large amounts of animal waste produced on high-intensity dairy farms. Although these systems are common, little is known about the microbial populations that inhabit them or how they change as they traverse the system. Using culture-based and non-culture-based methods, we characterized the microbial community structure of manure, water from the separator pit, and water from the circulated treatment lagoon from a large dairy in the San Joaquin Valley of California. Our results show that both total bacterial numbers and bacterial diversity are highest in manure, followed by the separator pit water and the lagoon water. The most prevalent phylum in all locations was the Firmicutes (low-G+C, gram-positive bacteria). The most commonly occurring operational taxonomic unit (OTU) had a 16S rRNA gene (rDNA) sequence 96 to 99% similar to that of Clostridium lituseburense and represented approximately 6% of the manure derived sequences, 14% of the separator pit-derived sequences and 20% of the lagoon-derived sequences. Also highly prevalent was an OTU with a 16S rDNA sequence 97 to 100% similar to that of Eubacterium tenue, comprising approximately 3% of the manure-derived sequences, 6% of the separator pit-derived sequences and 9% of the lagoon-derived sequences. Taken together, these sequences represent approximately one-third of the total organisms in the lagoon waters, suggesting that they are well adapted to this environment. PMID:15240310

  10. Deep 16S rRNA pyrosequencing reveals a bacterial community associated with Banana Fusarium Wilt disease suppression induced by bio-organic fertilizer application.

    PubMed

    Shen, Zongzhuan; Wang, Dongsheng; Ruan, Yunze; Xue, Chao; Zhang, Jian; Li, Rong; Shen, Qirong

    2014-01-01

    Our previous work demonstrated that application of a bio-organic fertilizer (BIO) to a banana mono-culture orchard with serious Fusarium wilt disease effectively decreased the number of soil Fusarium sp. and controlled the soil-borne disease. Because bacteria are an abundant and diverse group of soil organisms that responds to soil health, deep 16 S rRNA pyrosequencing was employed to characterize the composition of the bacterial community to investigate how it responded to BIO or the application of other common composts and to explore the potential correlation between bacterial community, BIO application and Fusarium wilt disease suppression. After basal quality control, 137,646 sequences and 9,388 operational taxonomic units (OTUs) were obtained from the 15 soil samples. Proteobacteria, Acidobacteria, Bacteroidetes, Gemmatimonadetes and Actinobacteria were the most frequent phyla and comprised up to 75.3% of the total sequences. Compared to the other soil samples, BIO-treated soil revealed higher abundances of Gemmatimonadetes and Acidobacteria, while Bacteroidetes were found in lower abundance. Meanwhile, on genus level, higher abundances compared to other treatments were observed for Gemmatimonas and Gp4. Correlation and redundancy analysis showed that the abundance of Gemmatimonas and Sphingomonas and the soil total nitrogen and ammonium nitrogen content were higher after BIO application, and they were all positively correlated with disease suppression. Cumulatively, the reduced Fusarium wilt disease incidence that was seen after BIO was applied for 1-year might be attributed to the general suppression based on a shift within the bacteria soil community, including specific enrichment of Gemmatimonas and Sphingomonas. PMID:24871319

  11. Deep 16S rRNA Pyrosequencing Reveals a Bacterial Community Associated with Banana Fusarium Wilt Disease Suppression Induced by Bio-Organic Fertilizer Application

    PubMed Central

    Ruan, Yunze; Xue, Chao; Zhang, Jian; Li, Rong; Shen, Qirong

    2014-01-01

    Our previous work demonstrated that application of a bio-organic fertilizer (BIO) to a banana mono-culture orchard with serious Fusarium wilt disease effectively decreased the number of soil Fusarium sp. and controlled the soil-borne disease. Because bacteria are an abundant and diverse group of soil organisms that responds to soil health, deep 16 S rRNA pyrosequencing was employed to characterize the composition of the bacterial community to investigate how it responded to BIO or the application of other common composts and to explore the potential correlation between bacterial community, BIO application and Fusarium wilt disease suppression. After basal quality control, 137,646 sequences and 9,388 operational taxonomic units (OTUs) were obtained from the 15 soil samples. Proteobacteria, Acidobacteria, Bacteroidetes, Gemmatimonadetes and Actinobacteria were the most frequent phyla and comprised up to 75.3% of the total sequences. Compared to the other soil samples, BIO-treated soil revealed higher abundances of Gemmatimonadetes and Acidobacteria, while Bacteroidetes were found in lower abundance. Meanwhile, on genus level, higher abundances compared to other treatments were observed for Gemmatimonas and Gp4. Correlation and redundancy analysis showed that the abundance of Gemmatimonas and Sphingomonas and the soil total nitrogen and ammonium nitrogen content were higher after BIO application, and they were all positively correlated with disease suppression. Cumulatively, the reduced Fusarium wilt disease incidence that was seen after BIO was applied for 1-year might be attributed to the general suppression based on a shift within the bacteria soil community, including specific enrichment of Gemmatimonas and Sphingomonas. PMID:24871319

  12. Survey of Culture, GoldenGate Assay, Universal Biosensor Assay, and 16S rRNA Gene Sequencing as Alternative Methods of Bacterial Pathogen Detection

    PubMed Central

    Pop, Mihai; Antonio, Martin; Walker, Alan W.; Mai, Volker; Ahmed, Dilruba; Oundo, Joseph; Tamboura, Boubou; Panchalingam, Sandra; Levine, Myron M.; Kotloff, Karen; Li, Shan; Magder, Laurence S.; Paulson, Joseph N.; Liu, Bo; Ikumapayi, Usman; Ebruke, Chinelo; Dione, Michel; Adeyemi, Mitchell; Rance, Richard; Stares, Mark D.; Ukhanova, Maria; Barnes, Bret; Lewis, Ian; Ahmed, Firoz; Alam, Meer Taifur; Amin, Ruhul; Siddiqui, Sabbir; Ochieng, John B.; Ouma, Emmanuel; Juma, Jane; Mailu, Eunice; Omore, Richard; O'Reilly, Ciara E.; Hannis, James; Manalili, Sheri; DeLeon, Jonna; Yasuda, Irene; Blyn, Lawrence; Ranken, Raymond; Li, Feng; Housley, Roberta; Ecker, David J.; Hossain, M. Anowar; Breiman, Robert F.; Morris, J. Glenn; McDaniel, Timothy K.; Parkhill, Julian; Saha, Debasish; Sampath, Rangarajan; Stine, O. Colin; Nataro, James P.

    2013-01-01

    Cultivation-based assays combined with PCR or enzyme-linked immunosorbent assay (ELISA)-based methods for finding virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies, new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya, and Bangladesh. The children were identified, using currently accepted clinical protocols, as either controls or cases with moderate to severe diarrhea. A total of 3,610 stool samples were tested by established clinical culture techniques: 3,179 DNA samples by the Universal Biosensor assay (Ibis Biosciences, Inc.), 1,466 DNA samples by the GoldenGate assay (Illumina), and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected different proportions of samples testing positive for each of seven enteric pathogens, enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shigella spp., Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. The comparisons among detection methods included the frequency of positive stool samples and kappa values for making pairwise comparisons. Overall, the standard culture methods detected Shigella spp., EPEC, ETEC, and EAEC in smaller proportions of the samples than either of the methods based on detection of the virulence genes from DNA in whole stools. The GoldenGate method revealed the greatest agreement with the other methods. The agreement among methods was higher in cases than in controls. The new molecular technologies have a high potential for highly sensitive identification of bacterial diarrheal pathogens. PMID:23884998

  13. Survey of culture, goldengate assay, universal biosensor assay, and 16S rRNA Gene sequencing as alternative methods of bacterial pathogen detection.

    PubMed

    Lindsay, Brianna; Pop, Mihai; Antonio, Martin; Walker, Alan W; Mai, Volker; Ahmed, Dilruba; Oundo, Joseph; Tamboura, Boubou; Panchalingam, Sandra; Levine, Myron M; Kotloff, Karen; Li, Shan; Magder, Laurence S; Paulson, Joseph N; Liu, Bo; Ikumapayi, Usman; Ebruke, Chinelo; Dione, Michel; Adeyemi, Mitchell; Rance, Richard; Stares, Mark D; Ukhanova, Maria; Barnes, Bret; Lewis, Ian; Ahmed, Firoz; Alam, Meer Taifur; Amin, Ruhul; Siddiqui, Sabbir; Ochieng, John B; Ouma, Emmanuel; Juma, Jane; Mailu, Eunice; Omore, Richard; O'Reilly, Ciara E; Hannis, James; Manalili, Sheri; Deleon, Jonna; Yasuda, Irene; Blyn, Lawrence; Ranken, Raymond; Li, Feng; Housley, Roberta; Ecker, David J; Hossain, M Anowar; Breiman, Robert F; Morris, J Glenn; McDaniel, Timothy K; Parkhill, Julian; Saha, Debasish; Sampath, Rangarajan; Stine, O Colin; Nataro, James P

    2013-10-01

    Cultivation-based assays combined with PCR or enzyme-linked immunosorbent assay (ELISA)-based methods for finding virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies, new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya, and Bangladesh. The children were identified, using currently accepted clinical protocols, as either controls or cases with moderate to severe diarrhea. A total of 3,610 stool samples were tested by established clinical culture techniques: 3,179 DNA samples by the Universal Biosensor assay (Ibis Biosciences, Inc.), 1,466 DNA samples by the GoldenGate assay (Illumina), and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected different proportions of samples testing positive for each of seven enteric pathogens, enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shigella spp., Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. The comparisons among detection methods included the frequency of positive stool samples and kappa values for making pairwise comparisons. Overall, the standard culture methods detected Shigella spp., EPEC, ETEC, and EAEC in smaller proportions of the samples than either of the methods based on detection of the virulence genes from DNA in whole stools. The GoldenGate method revealed the greatest agreement with the other methods. The agreement among methods was higher in cases than in controls. The new molecular technologies have a high potential for highly sensitive identification of bacterial diarrheal pathogens. PMID:23884998

  14. A new computational method for detection of chimeric 16S rRNA artifacts generated by PCR amplification from mixed bacterial populations.

    PubMed Central

    Komatsoulis, G A; Waterman, M S

    1997-01-01

    A new computational method (chimeric alignment) has been developed to detect chimeric 16S rRNA artifacts generated during PCR amplification from mixed bacterial populations. In contrast to other nearest-neighbor methods (e.g., CHECK_CHIMERA) that define sequence similarity by k-tuple matching, the chimeric alignment method uses the score from dynamic programming alignments. Further, the chimeric alignments are displayed to the user to assist in sequence classification. The distribution of improvement scores for 500 authentic, nonchimeric sequences and 300 artificial chimeras (constructed from authentic sequences) was used to study the sensitivity and accuracy of both chimeric alignment and CHECK_CHIMERA. At a constant rate of authentic sequence misclassification (5%), chimeric alignment incorrectly classified 13% of the artificial chimeras versus 14% for CHECK_CHIMERA. Interestingly, only 1% of nonchimeras and 10% of chimeras were misclassified by both programs, suggesting that optimum performance is obtained by using the two methods to assign sequences to three classes: high-probability nonchimeras, high-probability chimeras, and sequences that need further study by other means. This study suggests that k-tuple-based matching methods are more sensitive than alignment-based methods when there is significant parental sequence similarity, while the opposite becomes true as the sequences become more distantly related. The software and a World Wide Web-based server are available at http://www-hto.usc.edu/software/mglobal CHI. PMID:9172353

  15. Meta-barcoded evaluation of the ISO standard 11063 DNA extraction procedure to characterize soil bacterial and fungal community diversity and composition

    PubMed Central

    Terrat, Sebastien; Plassart, Pierre; Bourgeois, Emilie; Ferreira, Stéphanie; Dequiedt, Samuel; Adele-Dit-De-Renseville, Nathalie; Lemanceau, Philippe; Bispo, Antonio; Chabbi, Abad; Maron, Pierre-Alain; Ranjard, Lionel

    2015-01-01

    This study was designed to assess the influence of three soil DNA extraction procedures, namely the International Organization for Standardization (ISO-11063, GnS-GII and modified ISO procedure (ISOm), on the taxonomic diversity and composition of soil bacterial and fungal communities. The efficacy of each soil DNA extraction method was assessed on five soils, differing in their physico-chemical characteristics and land use. A meta-barcoded pyrosequencing approach targeting 16S and 18S rRNA genes was applied to characterize soil microbial communities. We first observed that the GnS-GII introduced some heterogeneity in bacterial composition between replicates. Then, although no major difference was observed between extraction procedures for soil bacterial diversity, we saw that the number of fungal genera could be underestimated by the ISO-11063. In particular, this procedure underestimated the detection in several soils of the genera Cryptococcus, Pseudallescheria, Hypocrea and Plectosphaerella, which are of ecological interest. Based on these results, we recommend using the ISOm method for studies focusing on both the bacterial and fungal communities. Indeed, the ISOm procedure provides a better evaluation of bacterial and fungal communities and is limited to the modification of the mechanical lysis step of the existing ISO-11063 standard. PMID:25195809

  16. Evaluation and optimization of nucleic acid extraction methods for the molecular analysis of bacterial communities associated with corroded carbon steel.

    PubMed

    Marty, Florence; Ghiglione, Jean-François; Païssé, Sandrine; Gueuné, Hervé; Quillet, Laurent; van Loosdrecht, Mark C M; Muyzer, Gerard

    2012-01-01

    Different DNA and RNA extraction approaches were evaluated and protocols optimized on in situ corrosion products from carbon steel in marine environments. Protocols adapted from the PowerSoil DNA/RNA Isolation methods resulted in the best nucleic acid (NA) extraction performances (ie combining high NA yield, quality, purity, representativeness of microbial community and processing time efficiency). The PowerSoil RNA Isolation Kit was the only method which resulted in amplifiable RNA of good quality (ie intact 16S/23S rRNA). Sample homogenization and hot chemical (SDS) cell lysis combined with mechanical (bead-beating) lysis in presence of a DNA competitor (skim milk) contributed to improving substantially (around 23 times) the DNA yield of the PowerSoil DNA Isolation Kit. Apart from presenting NA extraction strategies for optimizing extraction parameters with corrosion samples from carbon steel, this study proposes DNA and RNA extraction procedures suited for comparative molecular analysis of total and active fractions of bacterial communities associated with carbon steel corrosion events, thereby contributing to improved MIC diagnosis and control. PMID:22500778

  17. Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen suppressive soil

    SciTech Connect

    Hjort, K.; Bergstrom, M.; Adesina, M.F.; Jansson, J.K.; Smalla, K.; Sjoling, S.

    2009-09-01

    Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal-restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal-restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF{sup 103} of the isolate, Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.

  18. Changes in the Composition of Drinking Water Bacterial Clone Libraries Introduced by Using Two Different 16S rRNA Gene PCR Primers

    EPA Science Inventory

    Sequence analysis of 16S rRNA gene clone libraries is a popular tool used to describe the composition of natural microbial communities. Commonly, clone libraries are developed by direct cloning of 16S rRNA gene PCR products. Different primers are often employed in the initial amp...

  19. Changes in the Composition of Drinking Water Bacterial Clone Libraries Introduced by Using Two Different 16S rRna Gene PCR Primers

    EPA Science Inventory

    Sequence analysis of 16S rRNA gene clone libraries is a popular tool used to describe the composition of natural microbial communities. Commonly, clone libraries are developed by direct cloning of 16S rRNA gene PCR products. Different primers are often employed in the initial amp...

  20. Profiling the Succession of Bacterial Communities throughout the Life Stages of a Higher Termite Nasutitermes arborum (Termitidae, Nasutitermitinae) Using 16S rRNA Gene Pyrosequencing

    PubMed Central

    Diouf, Michel; Roy, Virginie; Mora, Philippe; Frechault, Sophie; Lefebvre, Thomas; Hervé, Vincent; Rouland-Lefèvre, Corinne; Miambi, Edouard

    2015-01-01

    Previous surveys of the gut microbiota of termites have been limited to the worker caste. Termite gut microbiota has been well documented over the last decades and consists mainly of lineages specific to the gut microbiome which are maintained across generations. Despite this intimate relationship, little is known of how symbionts are transmitted to each generation of the host, especially in higher termites where proctodeal feeding has never been reported. The bacterial succession across life stages of the wood-feeding higher termite Nasutitermes arborum was characterized by 16S rRNA gene deep sequencing. The microbial community in the eggs, mainly affiliated to Proteobacteria and Actinobacteria, was markedly different from the communities in the following developmental stages. In the first instar and last instar larvae and worker caste termites, Proteobacteria and Actinobacteria were less abundant than Firmicutes, Bacteroidetes, Spirochaetes, Fibrobacteres and the candidate phylum TG3 from the last instar larvae. Most of the representatives of these phyla (except Firmicutes) were identified as termite-gut specific lineages, although their relative abundances differed. The most salient difference between last instar larvae and worker caste termites was the very high proportion of Spirochaetes, most of which were affiliated to the Treponema Ic, Ia and If subclusters, in workers. The results suggest that termite symbionts are not transmitted from mother to offspring but become established by a gradual process allowing the offspring to have access to the bulk of the microbiota prior to the emergence of workers, and, therefore, presumably through social exchanges with nursing workers. PMID:26444989

  1. Synthesis of bacterial cellulose using hot water extracted wood sugars.

    PubMed

    Erbas Kiziltas, Esra; Kiziltas, Alper; Gardner, Douglas J

    2015-06-25

    Bacterial cellulose (BC), a type of nanopolymer produced by Acetobacter xylinum is a nanostructured material with unique properties and wide applicability. However, a standard medium used for the cultivation of BC, the Hestrin-Schramm medium, is expensive and prevents wide scale extension of BC applications. In this research, a relatively low-cost culture media was successfully developed from wood hot water extracts for the Acetobacter xylinus 23769 strain. Hot water extract (HWE) is a residual material originating from pulp mills and lignocellulosic biorefineries and consists of mainly monomeric sugars, organic acids and organics. The effects of different pH (5, 6, 7 and 8) and temperatures (26, 28 and 30°C) were also examined in this research. There were no significant differences in the crystallinity and the recorded I? fraction of cellulose produced between Hestrin-Schramm and the HWE medium. The maximum production of 0.15g/l of BC was obtained at a pH of 8 and temperature of 28°C. Glucose and xylose in the HWE were the main nutrient sources utilized in all BC cultivations based on high-pressure liquid chromatography (HPLC) results. HWE was shown to be a suitable carbon source for BC production, and a process was established for BC production from lignocellulosic feedstocks without using any modification of the HWE. HWE is an abundant and relatively inexpensive forest by-product. Using HWE for BC production could reduce burdens on the environment and also, achieve the goal of large scale BC production at low cost without using added culture nutrients. PMID:25839803

  2. Investigation of antibacterial mechanism and identification of bacterial protein targets mediated by antibacterial medicinal plant extracts.

    PubMed

    Yong, Ann-Li; Ooh, Keng-Fei; Ong, Hean-Chooi; Chai, Tsun-Thai; Wong, Fai-Chu

    2015-11-01

    In this paper, we investigated the antibacterial mechanism and potential therapeutic targets of three antibacterial medicinal plants. Upon treatment with the plant extracts, bacterial proteins were extracted and resolved using denaturing gel electrophoresis. Differentially-expressed bacterial proteins were excised from the gels and subjected to sequence analysis by MALDI TOF-TOF mass spectrometry. From our study, seven differentially expressed bacterial proteins (triacylglycerol lipase, N-acetylmuramoyl-L-alanine amidase, flagellin, outer membrane protein A, stringent starvation protein A, 30S ribosomal protein s1 and 60 kDa chaperonin) were identified. Additionally, scanning electron microscope study indicated morphological damages induced on bacterial cell surfaces. To the best of our knowledge, this represents the first time these bacterial proteins are being reported, following treatments with the antibacterial plant extracts. Further studies in this direction could lead to the detailed understanding of their inhibition mechanism and discovery of target-specific antibacterial agents. PMID:25976788

  3. Simultaneous Extraction of Viral and Bacterial Nucleic Acids for Molecular Diagnostic Applications

    PubMed Central

    Kajiura, Lauren N.; Stewart, Scott D.; Dresios, John; Uyehara, Catherine F. T.

    2015-01-01

    Molecular detection of microbial pathogens in clinical samples requires the application of efficient sample lysis protocols and subsequent extraction and isolation of their nucleic acids. Here, we describe a simple and time-efficient method for simultaneous extraction of genomic DNA from gram-positive and -negative bacteria, as well as RNA from viral agents present in a sample. This method compared well with existing bacterial- and viral-specialized extraction protocols, worked reliably on clinical samples, and was not pathogen specific. This method may be used to extract DNA and RNA concurrently from viral and bacterial pathogens present in a sample and effectively detect coinfections in routine clinical diagnostics. PMID:26543438

  4. Simultaneous Extraction of Viral and Bacterial Nucleic Acids for Molecular Diagnostic Applications.

    PubMed

    Kajiura, Lauren N; Stewart, Scott D; Dresios, John; Uyehara, Catherine F T

    2015-12-01

    Molecular detection of microbial pathogens in clinical samples requires the application of efficient sample lysis protocols and subsequent extraction and isolation of their nucleic acids. Here, we describe a simple and time-efficient method for simultaneous extraction of genomic DNA from gram-positive and -negative bacteria, as well as RNA from viral agents present in a sample. This method compared well with existing bacterial- and viral-specialized extraction protocols, worked reliably on clinical samples, and was not pathogen specific. This method may be used to extract DNA and RNA concurrently from viral and bacterial pathogens present in a sample and effectively detect coinfections in routine clinical diagnostics. PMID:26543438

  5. Bacterial consortium for copper extraction from sulphide ore consisting mainly of chalcopyrite

    PubMed Central

    Romo, E.; Weinacker, D.F.; Zepeda, A.B.; Figueroa, C.A.; Chavez-Crooker, P.; Farias, J.G.

    2013-01-01

    The mining industry is looking forward for bacterial consortia for economic extraction of copper from low-grade ores. The main objective was to determine an optimal bacterial consortium from several bacterial strains to obtain copper from the leach of chalcopyrite. The major native bacterial species involved in the bioleaching of sulphide ore (Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Leptospirillum ferrooxidans and Leptospirillum ferriphilum) were isolated and the assays were performed with individual bacteria and in combination with At. thiooxidans. In conclusion, it was found that the consortium integrated by At. ferrooxidans and At. thiooxidans removed 70% of copper in 35 days from the selected ore, showing significant differences with the other consortia, which removed only 35% of copper in 35 days. To validate the assays was done an escalation in columns, where the bacterial consortium achieved a higher percentage of copper extraction regarding to control. PMID:24294251

  6. Broad-range PCR, cloning and sequencing of the full 16S rRNA gene for detection of bacterial DNA in synovial fluid samples of Tunisian patients with reactive and undifferentiated arthritis

    PubMed Central

    Siala, Mariam; Gdoura, Radhouane; Fourati, Hela; Rihl, Markus; Jaulhac, Benoit; Younes, Mohamed; Sibilia, Jean; Baklouti, Sofien; Bargaoui, Naceur; Sellami, Slaheddine; Sghir, Abdelghani; Hammami, Adnane

    2009-01-01

    Introduction Broad-range rDNA PCR provides an alternative, cultivation-independent approach for identifying bacterial DNA in reactive and other form of arthritis. The aim of this study was to use broad-range rDNA PCR targeting the 16S rRNA gene in patients with reactive and other forms of arthritis and to screen for the presence of DNA from any given bacterial species in synovial fluid (SF) samples. Methods We examined the SF samples from a total of 27 patients consisting of patients with reactive arthritis (ReA) (n = 5), undifferentiated arthritis (UA) (n = 9), rheumatoid arthritis (n = 7), and osteoarthritis (n = 6) of which the latter two were used as controls. Using broad-range bacterial PCR amplifying a 1400 bp fragment from the 16S rRNA gene, we identified and sequenced at least 24 clones from each SF sample. To identify the corresponding bacteria, DNA sequences were compared to the EMBL (European Molecular Biology Laboratory) database. Results Bacterial DNA was identified in 20 of the 27 SF samples (74, 10%). Analysis of a large number of sequences revealed the presence of DNA from more than one single bacterial species in the SF of all patients studied. The nearly complete sequences of the 1400 bp were obtained for most of the detected species. DNA of bacterial species including Shigella species, Escherichia species, and other coli-form bacteria as well as opportunistic pathogens such as Stenotrophomonas maltophilia and Achromobacter xylosoxidans were shared in all arthritis patients. Among pathogens described to trigger ReA, DNA from Shigella sonnei was found in ReA and UA patients. We also detected DNA from rarely occurring human pathogens such as Aranicola species and Pantoea ananatis. We also found DNA from bacteria so far not described in human infections such as Bacillus niacini, Paenibacillus humicus, Diaphorobacter species and uncultured bacterium genera incertae sedis OP10. Conclusions Broad-range PCR followed by cloning and sequencing the entire 16S rDNA, allowed the identification of the bacterial DNA environment in the SF samples of arthritic patients. We found a wide spectrum of bacteria including those known to be involved in ReA and others not previously associated with arthritis. PMID:19570210

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

    PubMed Central

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

    1992-01-01

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

  8. Wild Mushroom Extracts as Inhibitors of Bacterial Biofilm Formation

    PubMed Central

    Alves, Maria José; Ferreira, Isabel C. F. R.; Lourenço, Inês; Costa, Eduardo; Martins, Anabela; Pintado, Manuela

    2014-01-01

    Microorganisms can colonize a wide variety of medical devices, putting patients in risk for local and systemic infectious complications, including local-site infections, catheter-related bloodstream infections, and endocarditis. These microorganisms are able to grow adhered to almost every surface, forming architecturally complex communities termed biofilms. The use of natural products has been extremely successful in the discovery of new medicine, and mushrooms could be a source of natural antimicrobials. The present study reports the capacity of wild mushroom extracts to inhibit in vitro biofilm formation by multi-resistant bacteria. Four Gram-negative bacteria biofilm producers (Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, and Acinetobacter baumannii) isolated from urine were used to verify the activity of Russula delica, Fistulina hepatica, Mycena rosea, Leucopaxilus giganteus, and Lepista nuda extracts. The results obtained showed that all tested mushroom extracts presented some extent of inhibition of biofilm production. Pseudomonas aeruginosa was the microorganism with the highest capacity of biofilm production, being also the most susceptible to the extracts inhibition capacity (equal or higher than 50%). Among the five tested extracts against E. coli, Leucopaxillus giganteus (47.8%) and Mycenas rosea (44.8%) presented the highest inhibition of biofilm formation. The extracts exhibiting the highest inhibitory effect upon P. mirabilis biofilm formation were Sarcodon imbricatus (45.4%) and Russula delica (53.1%). Acinetobacter baumannii was the microorganism with the lowest susceptibility to mushroom extracts inhibitory effect on biofilm production (highest inhibition—almost 29%, by Russula delica extract). This is a pioneer study since, as far as we know, there are no reports on the inhibition of biofilm production by the studied mushroom extracts and in particular against multi-resistant clinical isolates; nevertheless, other studies are required to elucidate the mechanism of action. PMID:25438017

  9. Microfluidic device for bacterial genome extraction and analysis

    NASA Astrophysics Data System (ADS)

    Galajda, Peter; Riehn, Robert; Wang, Yan-Mei; Keymer, Juan; Golding, Ido; Cox, Edward C.; Austin, Robert H.

    2006-03-01

    Although single molecule DNA manipulation and analysis techniques are emerging, methods for whole genome extraction from single cells, genomic length DNA handling and analytics is still to be developed. Here we present a microfabricated device to address some of these needs. This microfluidic chip is suitable for culturing bacteria and subsequently retrieve their genetic content. As a next step, the extracted DNA can be introduced in a nanostructured segment of the chip for precise handling, stretching and analysis. We hope that similar microdevices can be useful in studying genetic aspects of the cell lifecycle in a variety of organisms.

  10. Influence of DNA Extraction Method, 16S rRNA Targeted Hypervariable Regions, and Sample Origin on Microbial Diversity Detected by 454 Pyrosequencing in Marine Chemosynthetic Ecosystems

    PubMed Central

    Cruaud, Perrine; Vigneron, Adrien; Lucchetti-Miganeh, Céline; Ciron, Pierre Emmanuel; Godfroy, Anne

    2014-01-01

    Next-generation sequencing (NGS) opens up exciting possibilities for improving our knowledge of environmental microbial diversity, allowing rapid and cost-effective identification of both cultivated and uncultivated microorganisms. However, library preparation, sequencing, and analysis of the results can provide inaccurate representations of the studied community compositions. Therefore, all these steps need to be taken into account carefully. Here we evaluated the effects of DNA extraction methods, targeted 16S rRNA hypervariable regions, and sample origins on the diverse microbes detected by 454 pyrosequencing in marine cold seep and hydrothermal vent sediments. To assign the reads with enough taxonomic precision, we built a database with about 2,500 sequences from Archaea and Bacteria from deep-sea marine sediments, affiliated according to reference publications in the field. Thanks to statistical and diversity analyses as well as inference of operational taxonomic unit (OTU) networks, we show that (i) while DNA extraction methods do not seem to affect the results for some samples, they can lead to dramatic changes for others; and (ii) the choice of amplification and sequencing primers also considerably affects the microbial community detected in the samples. Thereby, very different proportions of pyrosequencing reads were obtained for some microbial lineages, such as the archaeal ANME-1, ANME-2c, and MBG-D and deltaproteobacterial subgroups. This work clearly indicates that the results from sequencing-based analyses, such as pyrosequencing, should be interpreted very carefully. Therefore, the combination of NGS with complementary approaches, such as fluorescence in situ hybridization (FISH)/catalyzed reporter deposition (CARD)-FISH or quantitative PCR (Q-PCR), would be desirable to gain a more comprehensive picture of environmental microbial communities. PMID:24837380

  11. Antibacterial activity of plant extracts on foodborne bacterial pathogens and food spoilage bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial foodborne diseases are caused by consumption of foods contaminated with bacteria and/or their toxins. In this study, we evaluated antibacterial properties of twelve different extracts including turmeric, lemon and different kinds of teas against four major pathogenic foodborne bacteria inc...

  12. The bioactivity of plant extracts against representative bacterial pathogens of the lower respiratory tract

    PubMed Central

    Bocanegra-García, Virgilio; del Rayo Camacho-Corona, María; Ramírez-Cabrera, Mónica; Rivera, Gildardo; Garza-González, Elvira

    2009-01-01

    Background Lower respiratory tract infections are a major cause of illness and death. Such infections are common in intensive care units (ICU) and their lethality persists despite advances in diagnosis, treatment and prevention. In Mexico, some plants are used in traditional medicine to treat respiratory diseases or ailments such as cough, bronchitis, tuberculosis and other infections. Medical knowledge derived from traditional societies has motivated searches for new bioactive molecules derived from plants that show potent activity against bacterial pathogens. Therefore, the aim of this study was to evaluate the effect of hexanic, chloroformic (CLO), methanolic (MET) and aqueous extracts from various plants used in Mexican traditional medicine on various microorganisms associated with respiratory disease. Methods thirty-five extracts prepared from nine plants used in Mexican traditional medicine for the treatment of respiratory infections were evaluated against 15 control bacterial species and clinical isolates. Results Both chloroformic (CLO) and methanolic (MET) extracts of Larrea tridentata were active against Methicillin-resistant S. aureus, B. subtilis and L. monocytogenes. A MET extract of L. tridentata was also active against S. aureus, S. pneumoniae, S. maltophilia, E. faecalis and H. influenzae and the CLO extract was active against A. baumannii. An Aqueous extract of M. acumitata and a MET extract of N. officinale were active against S. pneumoniae. CLO and MET extracts of L. tridentata were active against clinical isolates of S. aureus, S. pneumoniae and E. faecalis. Conclusion Overall, our results support the potential use of L. tridentata as a source of antibacterial compounds. PMID:19486533

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

    PubMed

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

    2015-10-01

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

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

    PubMed Central

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

    2015-01-01

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

  15. Assessing the Fecal Microbiota: An Optimized Ion Torrent 16S rRNA Gene-Based Analysis Protocol

    PubMed Central

    Foroni, Elena; Duranti, Sabrina; Turroni, Francesca; Lugli, Gabriele Andrea; Sanchez, Borja; Martín, Rebeca; Gueimonde, Miguel; van Sinderen, Douwe; Margolles, Abelardo; Ventura, Marco

    2013-01-01

    Assessing the distribution of 16S rRNA gene sequences within a biological sample represents the current state-of-the-art for determination of human gut microbiota composition. Advances in dissecting the microbial biodiversity of this ecosystem have very much been dependent on the development of novel high-throughput DNA sequencing technologies, like the Ion Torrent. However, the precise representation of this bacterial community may be affected by the protocols used for DNA extraction as well as by the PCR primers employed in the amplification reaction. Here, we describe an optimized protocol for 16S rRNA gene-based profiling of the fecal microbiota. PMID:23869230

  16. 16S rRNA pyrosequencing-based investigation of the bacterial community in nukadoko, a pickling bed of fermented rice bran.

    PubMed

    Sakamoto, Naoshige; Tanaka, Shigemitsu; Sonomoto, Kenji; Nakayama, Jiro

    2011-01-01

    Nukadoko is a naturally fermented rice bran mash traditionally used for pickling vegetables in Japan; its refreshment and fermentation cycles sometimes continue for many years. Here, we investigated the structure and dynamics of the bacterial community in nukadoko by conducting pyrosequencing and quantitative polymerase chain reaction (PCR) analyses of 16S ribosomal RNA genes (rDNA). Of the 16 different samples studied, 13 showed Lactobacillus-dominated microbiota, suggesting that aged nukadoko samples tend to realize a niche, favorable Lactobacillus species. The lactic acid bacterial community of each of the 16 samples was classified into 3 types according to the presence or absence of 2 predominant species, Lactobacillus namurensis and Lactobacillus acetotolerans. The dynamics of the bacterial community during fermentation and the subsequent ripening process were examined using a laboratory model of nukadoko inoculated with an aged nukadoko sample (inoculated model). Lb. namurensis grew rapidly in the first 2 days, accompanied with a rapid decrease in pH and an increase in lactate levels, while Lb. acetotolerans grew with a longer doubling time and slow acidification during the 20 days after inoculation. On the other hand, spontaneous fermentation of the nukadoko model prepared from fresh rice bran without the nukadoko inoculation (inoculant-free model), showed the growth of some non-Lactobacillus species such as staphylococci and bacilli within the first 10 days; thereafter, Lb. namurensis was dominant, while Lb. acetotolerans was not detected during the 20-day experimental period. These results suggest that the naturally established Lactobacillus community in aged nukadoko is effectively involved in the biocontrol of the microbial community of nukadoko during the refreshment and fermentation cycles. PMID:21084126

  17. Critical factors influencing the recovery and integrity of rRNA extracted from environmental samples: use of an optimized protocol to measure depth-related biomass distribution in freshwater sediments.

    PubMed

    Alm, E W; Stahl, D A

    2000-04-01

    A protocol was developed for the efficient recovery of intact, high molecular weight rRNA from different environmental matrices. Critical variables were identified in sample processing that influenced yield and integrity of recovered nucleic acid. Most notably, the order of addition and the buffer to sample volume ratio profoundly influenced the efficiency of nucleic acid recovery from sediment material when utilizing a guanidine thiocyanate-beta-mercaptoethaol extraction buffer. Addition of one sample volume to five buffer volumes contributed to an order of magnitude increase in recovery relative to reverse order of addition (buffer addition to sample). An optimized extraction protocol was used to evaluate rRNA yield by seeding samples with whole cells and radiolabeled nucleic acid. Recovery of intact rRNA was confirmed by polyacrylamide gel electrophoresis, which was also used to provide another estimate of quantity. This optimized protocol was used to measure depth-related changes in biomass distribution in Lake Michigan deep-water sediments. This revealed a biomodal biomass distribution; a maximum near the water/sediment interface and a secondary peak associated with the oxic/suboxic boundary. A significant portion of the community at the oxic/suboxic boundary was composed of non-methanogenic Archaea. PMID:10699671

  18. Phylogenetic Clustering of Soil Microbial Communities by 16S rRNA but Not 16S rRNA Genes

    PubMed Central

    Firestone, Mary K.

    2012-01-01

    We evaluated phylogenetic clustering of bacterial and archaeal communities from redox-dynamic subtropical forest soils that were defined by 16S rRNA and rRNA gene sequences. We observed significant clustering for the RNA-based communities but not the DNA-based communities, as well as increasing clustering over time of the highly active taxa detected by only rRNA. PMID:22286992

  19. CHARACTERIZATION OF BACTERIAL BIOMASS IN MARINE SEDIMENTS BENEATH THE ROSS ICE SHEET, ANTARCTICA BY PHOSPHOLIPIDS ANALYSIS AND 16S RRNA GENE SEQUENCING

    NASA Astrophysics Data System (ADS)

    Carr, S. A.; Glossner, A. W.; Dunbar, R. B.; Vogel, S. W.; Brandes, J.; Sahl, J. W.; Pepe-Ranney, C.; Spear, J. R.; Naish, T.; Powell, R. D.; Mandernack, K. W.

    2009-12-01

    As concerns regarding climate change increase, so does the importance of understanding the biogeochemical cycling of elements such as carbon. In the marine sediments of the Ross Sea, Antarctica, the in situ microbial community plays a significant role in the decomposition, mineralization and recycling of both organic and inorganic carbon. In this study, viable biomass for the top 155 cm below seafloor of sediment cores in the Ross Sea were estimated based on microbial phospholipid concentrations and Acridine Orange direct cell counts (AODC). Results for the biomass estimates suggest that both methods are able to accurately estimate viable biomass. Structural and isotopic analyses of phospholipid fatty acids (PLFAs) and phospholipid ether lipids (PELs), as well as isotopic analyses of carbon sources within sediment porewaters were used to identify changes in microbial metabolic pathways. The ?13C values of dissolved inorganic carbon (DIC) in porewaters ranged from -2.52‰ to -3.72‰ while corresponding ?13C values for sedimentary organic carbon (OC) varied from -26.25‰ to -23.12‰ in the surface and 155cm porewaters, respectively. The ?13C values of PLFAs are slightly lighter than the ?13C values of the organic carbon, ranging between -29‰ to -35‰ throughout the sediment core. 16S ribosomal RNA gene sequencing was preformed to classify the microbial species present at various depths. 16S sequences revealed that members of this microbial community include ?, ?, ?, and ? proteobacteria, acitobacteria, acidobacteria, and flavobacteria, all of which have been previously sequenced from other Antarctic continental shelf sediments. Archaea represent 1 to 3% of the microbial community which is similar to comparable studies. Amongst the sequenced organisms, many have been reported to utilize organic carbon sources such as amino acids, oligosaccharides, and lactose. These heterotropic organisms compliment the constant lipid isotope values and suggest that heterotrophic organisms dominate these sediments, with the implication that primary productivity is derived from above. Integrating structural analyses and ?13C values of phospholipids, porewater chemistry, ?13CDIC and ?13CDIC values with 16S rRNA gene sequences provides a more comprehensive understanding of the biogeochemical influences of microbial carbon cycling that occur beneath marine sediments of Antarctica and elsewhere.

  20. Analysis of bacterial community structure in Saba-Narezushi (Narezushi of Mackerel) by 16S rRNA gene clone library.

    PubMed

    Matsui, Hiroki; Tsuchiya, Rie; Isobe, Yuka; Narita, Miyo

    2013-08-01

    Narezushi, a derivation of sushi, is a traditional Japanese food made by fermenting salted fish meat and cooked rice together. In this study, the microbial diversity of saba-narezushi (narezushi of mackerel, Scomber japonicus) was analyzed by the 16S ribosomal RNA gene clone library method. Chemical composition was also analyzed to compare with different kinds of narezushi. The chemical composition of the narezushi was similar to those obtained from samma-narezushi. Ninety-four clones were randomly selected and DNA sequences of cloned fragments (approx. 890 bp) were analyzed. The DNA sequences obtained were phylogenetically analyzed. The expected operational taxonomy units (OTUs) by Chao1 estimates and Shannon-Wiener index (H') at 97% identity threshold were 48 and 1.822, respectively. The sequence similarity of the cloned fragment was equal to or higher than 98% of the sequence of cultivated bacterial species in the public database. Most of the clones (85%) belonged to lactic acid bacteria (LAB). Lactobacillus curvatus was the most abundant species followed by Lactococcus piscium and Leuconostoc gasicomitatum, suggesting that these bacteria play important roles in the fermentation of saba-narezushi. PMID:24425983

  1. Structure and dynamics of the bacterial communities in fermentation of the traditional Chinese post-fermented pu-erh tea revealed by 16S rRNA gene clone library.

    PubMed

    Zhao, Ming; Xiao, Wei; Ma, Yan; Sun, Tingting; Yuan, Wenxia; Tang, Na; Zhang, Donglian; Wang, Yongxia; Li, Yali; Zhou, Hongjie; Cui, Xiaolong

    2013-10-01

    Microbes are thought to have key roles in the development of the special properties of post-fermented pu-erh tea (pu-erh shucha), a well-known traditional Chinese tea; however, little is known about the bacteria during the fermentation. In this work, the structure and dynamics of the bacterial community involved in the production of pu-erh shucha were investigated using 16S rRNA gene clone libraries constructed from samples collected on days zero (LD-0), 5 (LD-5), 10 (LD-10), 15 (LD-15) and 20 (LD-20) of the fermentation. A total of 747 sequences with individual clone library containing 115-174 sequences and 4-20 unique operational taxonomic units (OTUs) were obtained. These OTUs were grouped into four phyla (Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria) and further identified as members of 10 families, such as Alcaligenaceae, Bacillaceae, Enterobacteriaceae, etc. The dominant bacteria were Enterobacteriaceae in the raw material (LD-0) and in the initial stages of fermentation (LD-5 and LD-10), which changed to Bacillaceae at the last stages of fermentation (LD-15 and LD-20) at a temperature of 40-60 °C. It is interesting that the dominant OTUs in libraries LD-15 and LD-20 were very closely related to Bacillus coagulans, which is a safe thermoduric probiotic. Together the bacterial diversity and dynamics during a fermentation of pu-erh shucha were demonstrated, and a worthy clue for artificial inoculation of B. coagulans to improve the health benefits of pu-erh shucha or produce probiotic pu-erh tea were provided. PMID:23591759

  2. RNA Preservation Agents and Nucleic Acid Extraction Method Bias Perceived Bacterial Community Composition

    PubMed Central

    McCarthy, Ann; Chiang, Edna; Schmidt, Marian L.; Denef, Vincent J.

    2015-01-01

    Bias is a pervasive problem when characterizing microbial communities. An important source is the difference in lysis efficiencies of different populations, which vary depending on the extraction protocol used. To avoid such biases impacting comparisons between gene and transcript abundances in the environment, the use of one protocol that simultaneously extracts both types of nucleic acids from microbial community samples has gained popularity. However, knowledge regarding tradeoffs to combined nucleic acid extraction protocols is limited, particularly regarding yield and biases in the observed community composition. Here, we evaluated a commercially available protocol for simultaneous extraction of DNA and RNA, which we adapted for freshwater microbial community samples that were collected on filters. DNA and RNA yields were comparable to other commonly used, but independent DNA and RNA extraction protocols. RNA protection agents benefited RNA quality, but decreased DNA yields significantly. Choice of extraction protocol influenced the perceived bacterial community composition, with strong method-dependent biases observed for specific phyla such as the Verrucomicrobia. The combined DNA/RNA extraction protocol detected significantly higher levels of Verrucomicrobia than the other protocols, and those higher numbers were confirmed by microscopic analysis. Use of RNA protection agents as well as independent sequencing runs caused a significant shift in community composition as well, albeit smaller than the shift caused by using different extraction protocols. Despite methodological biases, sample origin was the strongest determinant of community composition. However, when the abundance of specific phylogenetic groups is of interest, researchers need to be aware of the biases their methods introduce. This is particularly relevant if different methods are used for DNA and RNA extraction, in addition to using RNA protection agents only for RNA samples. PMID:25798612

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

    SciTech Connect

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

    2009-03-01

    The bacterial diversity associated with citrus leaf midribs was characterized 1 from citrus groves that contained the Huanglongbing (HLB) pathogen, which has yet to be cultivated in vitro. We employed a combination of high-density phylogenetic 16S rDNA microarray and 16S rDNA clone library sequencing to determine the microbial community composition of symptomatic and asymptomatic citrus midribs. Our results revealed that citrus leaf midribs can support a diversity of microbes. PhyloChip analysis indicated that 47 orders of bacteria from 15 phyla were present in the citrus leaf midribs while 20 orders from phyla were observed with the cloning and sequencing method. PhyloChip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs compared to asymptomatic midribs. Candidatus Liberibacter asiaticus (Las) was detected at a very low level in asymptomatic plants, but was over 200 times more abundant in symptomatic plants. The PhyloChip analysis was further verified by sequencing 16S rDNA clone libraries, which indicated the dominance of Las in symptomatic leaves. These data implicate Las as the pathogen responsible for HLB disease. Citrus is the most important commercial fruit crop in Florida. In recent years, citrus Huanglongbing (HLB), also called citrus greening, has severely affected Florida's citrus production and hence has drawn an enormous amount of attention. HLB is one of the most devastating diseases of citrus (6,13), characterized by blotchy mottling with green islands on leaves, as well as stunting, fruit decline, and small, lopsided fruits with poor coloration. The disease tends to be associated with a phloem-limited fastidious {alpha}-proteobacterium given a provisional Candidatus status (Candidatus Liberobacter spp. later changed to Candidatus Liberibacter spp.) in nomenclature (18,25,34). Previous studies indicate that HLB infection causes disorder in the phloem and severely impairs the translocation of assimilates in host plants (5,27,40). Tatineni and colleagues discovered that the HLB bacteria were unevenly distributed in phloem of bark tissue, vascular tissue of the leaf midrib, roots, and different floral and fruit parts (43). Unsuccessful attempts in culturing the pathogen are notably hampering efforts to understand its biology and pathogenesis mechanism. Using a modified Koch's Postulates approach, Jagoueix and colleagues were able to re-infect periwinkle plants from a mixed microbial community harvested from HLB diseased plants (25). Emergence of the disease in otherwise healthy plants led to the conclusion that HLB was associated with Candidatus Liberibacter sp. based on its 16S rDNA sequence (18,25). Currently, three species of the pathogen are recognized from trees with HLB disease based on 16S rDNA sequence: Ca. Liberibacter asiaticus (Las), Ca. Liberibacter africanus (Laf), and Ca. Liberibacter americanus (Lam); Las is the most prevalent species among HLB diseased trees (5,12,18,25,44). Las is naturally transmitted to citrus by the psyllid, Diaphorina citri Kuwayama, and can be artificially transmitted by grafting from citrus to citrus and dodder (Cuscuta campestris) to periwinkle (Catharanthus roseus) or tobacco (Nicotiana tabacum Xanthi) (5). Based on current research regarding the associations of Liberibacter in planta there is not enough evidence to implicate Liberibacter as the definitive causal agent of HLB disease due to its resistance to cultivation in vitro. It is possible that HLB disease may be the result of complex etiology where Liberibacter interacts with other endophytic bacteria. However, there is not enough evidence regarding its association(s) in planta to make this conclusion, nor is it known whether associated microbial communities play a role in expression of pathogenic traits. The main objective of the study was to test the hypothesis that other bacteria besides Ca. Liberibacter spp. are associated with citrus greening disease. The differences between the relative abundance, species richness and phylogenetic diversity of the microbial communitie

  4. Leuconostoc pseudomesenteroides WCFur3 partial 16S rRNA gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  5. Electrochemical Biosensor for Rapid and Sensitive Detection of Magnetically Extracted Bacterial Pathogens

    PubMed Central

    Setterington, Emma B.; Alocilja, Evangelyn C.

    2012-01-01

    Biological defense and security applications demand rapid, sensitive detection of bacterial pathogens. This work presents a novel qualitative electrochemical detection technique which is applied to two representative bacterial pathogens, Bacillus cereus (as a surrogate for B. anthracis) and Escherichia coli O157:H7, resulting in detection limits of 40 CFU/mL and 6 CFU/mL, respectively, from pure culture. Cyclic voltammetry is combined with immunomagnetic separation in a rapid method requiring approximately 1 h for presumptive positive/negative results. An immunofunctionalized magnetic/polyaniline core/shell nano-particle (c/sNP) is employed to extract target cells from the sample solution and magnetically position them on a screen-printed carbon electrode (SPCE) sensor. The presence of target cells significantly inhibits current flow between the electrically active c/sNPs and SPCE. This method has the potential to be adapted for a wide variety of target organisms and sample matrices, and to become a fully portable system for routine monitoring or emergency detection of bacterial pathogens. PMID:25585629

  6. Prevention of Bacterial Biofilms Formation on Urinary Catheter by Selected Plant Extracts.

    PubMed

    Adesina, T D; Nwinyi, O C; Olugbuyiro, J A O

    2015-02-01

    In this study, we investigated the feasibility of using Psidium guajava, Mangifera indica and Ocimum gratissimum leaf extracts in preventing Escherichia coli biofilm formation. The plants extractions were done with methanol under cold extraction. The various concentrations 5.0, 10.0 and 20.0 mg mL(-1) were used to coat 63 catheters under mild heat from water bath. Biofilm formation on the catheter was induced using cultures of E. coli. Biofilm formation was evaluated using aerobic plate count and turbidity at 600 nm. From the obtained results, Psidium guajava, Mangifera indica and Ocimum gratissimum delayed the onset of biofilm formation for a week. Ocimum gratissimum coated catheter had the highest inhibitory effect at 5.0, 10.0 and 20.0 mg mL(-1) with bacterial count ranging from 2.2 x 10(5)-7.0 x 10(4) and 5.7 x 10(5)-3.7 x10(5) for 120 and 128 h, respectively. The Psidium guajava coated catheter had the lowest inhibitory effect at 5.0, 10.0 and 20.0 mg mL(-1), with bacterial count ranging between 4.3 x 10(5)-1.9 x 10(3) and 7.7 x 10(5)-3.8 x 10(5) for 120 and 128 h, respectively. Despite the antimicrobial activities, the differences in the activity of these plant extracts were statistically not significant (p < 0.05). PMID:26364356

  7. Induction of sister chromatid exchanges and bacterial revertants by organic extracts of airborne particles. [Humans

    SciTech Connect

    Lockard, J.M.; Viau, C.J.; Lee-Stephens, C.; Caldwell, J.C.; Wojciechowski, J.P.; Enoch, H.G.; Sabharwal, P.S.

    1981-01-01

    The genotoxicities of organic extracts of airborne particles have been studied extensively in the Salmonella/mammalian microsome (Ames) test, but in few other bioassays. In these studies, we tested benzene-acetone extracts of particulate pollutants collected in Lexington, Kentucky, for capacity to induce increases in sister chromatid exchanges (SCE) in human lumphocytes and V79 cells, as well as in the Ames assay. Extracts induced linear dose-related increases in SCE in human lumphocytes and in bacterial revertants.However, variable responses were observed in SCE assays in V79 cells with and without activation by rat liver S9 or feeder layers of irradiated Syrian hamster fetal cells. We conclude that the SCE assay in human lumphocytes may be a useful indicator of the potential risks to humans of airborne particulate pollutants, as it utilizes human cells recently taken from the host, is rapid and economical, and requires small quantities of test materials. However, thorough studies of the quantitative relationships between SCE induction and mutagenicity in human cells are needed.

  8. Polymeric Cryogel-Based Boronate Affinity Chromatography for Separation of Ribonucleic Acid from Bacterial Extracts.

    PubMed

    Shakya, Akhilesh Kumar; Srivastava, Akshay; Kumar, Ashok

    2015-01-01

    Three-dimensional monolithic columns are preferred stationary phase in column chromatography. Conventional columns based on silica or particles are efficient in bioseparation though associated with limitations of nonspecific interaction and uneven porosity that causes high mass transfer resistance for the movement of big molecules. Cryogels as a monolith column have shown promising application in bioseparation. Cryogels column can be synthesized in the form of a monolith at sub-zero temperature through gelation of pre-synthesized polymers or polymerization of monomers. Cryogels are macroporous and mechanically stable materials. They have open interconnected micron-sized pores with a wide range of porosity (10-200 ?m). Current protocol demonstrated the ability of poly(hydroxymethyl methacrylate)-co-vinylphenyl boronic acid p(HEMA-co-VPBA) cryogel matrix for selective separation of RNA from the bacterial crude extract. © 2015 by John Wiley & Sons, Inc. PMID:26623972

  9. metaxa2: improved identification and taxonomic classification of small and large subunit rRNA in metagenomic data.

    PubMed

    Bengtsson-Palme, Johan; Hartmann, Martin; Eriksson, Karl Martin; Pal, Chandan; Thorell, Kaisa; Larsson, Dan Göran Joakim; Nilsson, Rolf Henrik

    2015-11-01

    The ribosomal rRNA genes are widely used as genetic markers for taxonomic identification of microbes. Particularly the small subunit (SSU; 16S/18S) rRNA gene is frequently used for species- or genus-level identification, but also the large subunit (LSU; 23S/28S) rRNA gene is employed in taxonomic assignment. The metaxa software tool is a popular utility for extracting partial rRNA sequences from large sequencing data sets and assigning them to an archaeal, bacterial, nuclear eukaryote, mitochondrial or chloroplast origin. This study describes a comprehensive update to metaxa - metaxa2 - that extends the capabilities of the tool, introducing support for the LSU rRNA gene, a greatly improved classifier allowing classification down to genus or species level, as well as enhanced support for short-read (100 bp) and paired-end sequences, among other changes. The performance of metaxa2 was compared to other commonly used taxonomic classifiers, showing that metaxa2 often outperforms previous methods in terms of making correct predictions while maintaining a low misclassification rate. metaxa2 is freely available from http://microbiology.se/software/metaxa2/. PMID:25732605

  10. A hybrid DNA extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in th...

  11. COMPARISON OF RAPID METHODS FOR THE EXTRACTION OF BACTERIAL DNA FROM COLONIC AND CECAL LUMEN CONTENTS OF THE PIG

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The increasing use of DNA methodologies to study the microflora of the pig gastrointestinal tract requires an efficient recovery of bacterial DNA from intestinal samples. Thus, the objective of this study was to determine which extraction methods are most effective for colonic and cecal lumen sampl...

  12. Water Extract from Spent Mushroom Substrate of Hericium erinaceus Suppresses Bacterial Wilt Disease of Tomato

    PubMed Central

    Kwak, A Min; Min, Kyeong Jin; Lee, Sang Yeop

    2015-01-01

    Culture filtrates of six different edible mushroom species were screened for antimicrobial activity against tomato wilt bacteria Ralstonia solanacearum B3. Hericium erinaceus, Lentinula edodes (Sanjo 701), Grifola frondosa, and Hypsizygus marmoreus showed antibacterial activity against the bacteria. Water, n-butanol, and ethyl acetate extracts of spent mushroom substrate (SMS) of H. erinaceus exhibited high antibacterial activity against different phytopathogenic bacteria: Pectobacterium carotovorum subsp. carotovorum, Agrobacterium tumefaciens, R. solanacearum, Xanthomonas oryzae pv. oryzae, X. campestris pv. campestris, X. axonopodis pv. vesicatoria, X. axonopodis pv. citiri, and X. axonopodis pv. glycine. Quantitative real-time PCR revealed that water extracts of SMS (WESMS) of H. erinaceus induced expressions of plant defense genes encoding ?-1,3-glucanase (GluA) and pathogenesis-related protein-1a (PR-1a), associated with systemic acquired resistance. Furthermore, WESMS also suppressed tomato wilt disease caused by R. solanacearum by 85% in seedlings and promoted growth (height, leaf number, and fresh weight of the root and shoot) of tomato plants. These findings suggest the WESMS of H. erinaceus has the potential to suppress bacterial wilt disease of tomato through multiple effects including antibacterial activity, plant growth promotion, and defense gene induction. PMID:26539048

  13. Bacterial removal of quinolizidine alkaloids and other carbon sources from a Lupinus albus aqueous extract.

    PubMed

    Santana, Filomena M C; Pinto, Teresa; Fialho, Arsénio M; Sá-Correia, Isabel; Empis, José M A

    2002-04-10

    Two Gram-negative bacterial strains capable of using lupanine, the predominant quinolizidine alkaloid in Lupinus albus, as a sole carbon source were isolated from soil in which L. albus and L. luteus had been grown [Santana, F. M. et al. J. Ind. Microbiol. 1996, 17, 110-115]. In the present study, we present results suggesting that these isolates are of potential interest for removing lupanine and other quinolizidine alkaloids (QA) from the effluent resulting from the wet processing of Lupinus seeds, at temperatures within the range 20-34 degrees C. Growth in L. albus aqueous extract was diauxic, with a first period of rapid growth leading to the simultaneous consumption of a significant part of the initial concentration of QA (3 g L(-1), being 2 g L(-1) lupanine) and amino acids (1.5 g L(-1)). This period was followed by a second period of slower growth corresponding to the subsequent partial utilization (25%) of the carbohydrates (initial concentration of 20 g L(-1)) together with further removal of QA and amino acids. Despite the differences detected in the susceptibility of the two strains to lupanine toxicity, in particular at supraoptimal temperatures, and in the efficiency of lupanine catabolism, their performance on L. albus extract did not vary significantly. PMID:11929291

  14. Water Extract from Spent Mushroom Substrate of Hericium erinaceus Suppresses Bacterial Wilt Disease of Tomato.

    PubMed

    Kwak, A Min; Min, Kyeong Jin; Lee, Sang Yeop; Kang, Hee Wan

    2015-09-01

    Culture filtrates of six different edible mushroom species were screened for antimicrobial activity against tomato wilt bacteria Ralstonia solanacearum B3. Hericium erinaceus, Lentinula edodes (Sanjo 701), Grifola frondosa, and Hypsizygus marmoreus showed antibacterial activity against the bacteria. Water, n-butanol, and ethyl acetate extracts of spent mushroom substrate (SMS) of H. erinaceus exhibited high antibacterial activity against different phytopathogenic bacteria: Pectobacterium carotovorum subsp. carotovorum, Agrobacterium tumefaciens, R. solanacearum, Xanthomonas oryzae pv. oryzae, X. campestris pv. campestris, X. axonopodis pv. vesicatoria, X. axonopodis pv. citiri, and X. axonopodis pv. glycine. Quantitative real-time PCR revealed that water extracts of SMS (WESMS) of H. erinaceus induced expressions of plant defense genes encoding ?-1,3-glucanase (GluA) and pathogenesis-related protein-1a (PR-1a), associated with systemic acquired resistance. Furthermore, WESMS also suppressed tomato wilt disease caused by R. solanacearum by 85% in seedlings and promoted growth (height, leaf number, and fresh weight of the root and shoot) of tomato plants. These findings suggest the WESMS of H. erinaceus has the potential to suppress bacterial wilt disease of tomato through multiple effects including antibacterial activity, plant growth promotion, and defense gene induction. PMID:26539048

  15. Inhibitory effects of Olea ferruginea crude leaves extract against some bacterial and fungal pathogen.

    PubMed

    Amin, Adnan; Khan, Muhammad Ayaz; Shah, Swahid; Ahmad, Mushatq; Zafar, Muhammad; Hameed, Abdul

    2013-03-01

    This work aimed to evaluate the inhibitory effects of Olea ferruginea crude leaves extract that are commonly used as remedy to cure infections in the tribal (Khyber Agency) areas of Pakistan against some of bacterial and fungal pathogens. The crude n-hexane fraction was appreciably active against both gram positive and negative microorganisms (MIC ranged from 7.5 to 15 mg/ml) followed by butanol fraction (MIC 15 to 30 mg/ml). Conversely least biological activity was shown by chloroform (30mg/ml) and methanol (15 to 30mg/ml) crude fractions. The MBC observed for all crude fractions was same or 2 times higher when compared with MIC for all crude extract fractions. Likewise all the fractions showed activity against Aspergillus niger and maximum zones of inhibition were shown by the n-hexane fraction (14 ± (0.02), butanol (13 ± (0.02) followed by methanol (9 ± (0.05) and chloroform fractions (7 ± (0.02). These results clearly imitate the antibacterial and antifungal potential of Olea ferruginea and hence we recommend the whole plant for further futuristic studies. PMID:23455192

  16. Extraction of copper from an oxidized (lateritic) ore using bacterially catalysed reductive dissolution.

    PubMed

    Nancucheo, Ivan; Grail, Barry M; Hilario, Felipe; du Plessis, Chris; Johnson, D Barrie

    2014-01-01

    An oxidized lateritic ore which contained 0.8 % (by weight) copper was bioleached in pH- and temperature-controlled stirred reactors under acidic reducing conditions using pure and mixed cultures of the acidophilic chemolithotrophic bacterium Acidithiobacillus ferrooxidans. Sulfur was provided as the electron donor for the bacteria, and ferric iron present in goethite (the major ferric iron mineral present in the ore) acted as electron acceptor. Significantly more copper was leached by bacterially catalysed reductive dissolution of the laterite than in aerobic cultures or in sterile anoxic reactors, with up to 78 % of the copper present in the ore being extracted. This included copper that was leached from acid-labile minerals (chiefly copper silicates) and that which was associated with ferric iron minerals in the lateritic ore. In the anaerobic bioreactors, soluble iron in the leach liquors was present as iron (II) and copper as copper (I), but both metals were rapidly oxidized (to iron (III) and copper (II)) when the reactors were aerated. The number of bacteria added to the reactors had a critical role in dictating the rate and yield of copper solubilised from the ore. This work has provided further evidence that reductive bioprocessing, a recently described approach for extracting base metals from oxidized deposits, has the potential to greatly extend the range of metal ores that can be biomined. PMID:24687752

  17. Effect of Punica granatum L. Flower Water Extract on Five Common Oral Bacteria and Bacterial Biofilm Formation on Orthodontic Wire

    PubMed Central

    VAHID DASTJERDI, Elahe; ABDOLAZIMI, Zahra; GHAZANFARIAN, Marzieh; AMDJADI, Parisa; KAMALINEJAD, Mohammad; MAHBOUBI, Arash

    2014-01-01

    Background: Use of herbal extracts and essences as natural antibacterial compounds has become increasingly popular for the control of oral infectious diseases. Therefore, finding natural antimicrobial products with the lowest side effects seems necessary. The present study sought to assess the effect of Punica granatum L. water extract on five oral bacteria and bacterial biofilm formation on orthodontic wire. Methods: Antibacterial property of P. granatum L. water extract was primarily evaluated in brain heart infusion agar medium using well-plate method. The minimum inhibitory concentration and minimum bactericidal concentration were determined by macro-dilution method. The inhibitory effect on orthodontic wire bacterial biofilm formation was evaluated using viable cell count in biofilm medium. At the final phase, samples were fixed and analyzed by Scanning Electron Microscopy. Results: The growth inhibition zone diameter was proportional to the extract concentration. The water extract demonstrated the maximum antibacterial effect on Streptococcus sanguinis ATCC 10556 with a minimum inhibitory concentration of 6.25 mg/ml and maximum bactericidal effect on S. sanguinis ATCC 10556 and S. sobrinus ATCC 27607 with minimum bactericidal concentration of 25 mg/ml. The water extract decreased bacterial biofilm formation by S. sanguinis, S. sobrinus, S. salivarius, S. mutans ATCC 35608 and E. faecalis CIP 55142 by 93.7–100%, 40.6–99.9%, 85.2–86.5%, 66.4–84.4% and 35.5–56.3% respectively. Conclusion: Punica granatum L. water extract had significant antibacterial properties against 5 oral bacteria and prevented orthodontic wire bacterial biofilm formation. However, further investigations are required to generalize these results to the clinical setting. PMID:26171362

  18. Inhibitory activity of Salvadora persica extracts against oral bacterial strains associated with periodontitis: An in-vitro study

    PubMed Central

    Amir Alireza, Rasouli Ghahroudi; Afsaneh, Rezaei; Seied Hosein, Mohseni Salehifard; Siamak, Yaghoobee; Afshin, Khorsand; Zeinab, Kadkhoda; Mahvash, Moosavi Jazi; Amir Reza, Rokn

    2014-01-01

    Aims The use of natural plant extracts in pharmacology, medicine and dental hygiene has found a growing interest in modern scientific research. Salvadora persica is a natural tree whose fibrous branches have been approved by the World Health Organization for oral hygiene. Periodontitis is a highly prevalent adult gingival disease that leads to bone destruction and connective tissue attachment loss. The aim of this research was assessment the antimicrobial activities of methanolic extract of Salvadora persica (miswak) on isolated strains from the oral fluid. Methods In practical section, 50 female university students (21.4 ± 1 year) participated in the study. Based on examination by a periodontist, they were grouped into (Group I, n = 21) and (Group II, n = 29) i.e. with and without periodontitis respectively. Their un-stimulated saliva samples were obtained in sterile tubes. While three bacterial genera, Staphylococcus, Streptococcus and Lactobacillus were identified in all subjects, Enterococcus and Escherichia were only detected in Group I. Results A statistically significant difference in colonization levels between the two groups was observed. The effect of methanolic extract of S. persica against oral bacterial strains isolated from saliva was investigated using agar disc diffusion and microdilution methods. Although methanolic extract of S. persica was effective on growth inhibition of all strains, it was significantly more effective on Gram positive bacteria than Gram negative ones. Conclusions Effective substances present in S. persica extracts, exhibit a broad range of antibacterial activity and affect almost all bacterial species regardless of the Gram-staining nature. PMID:25737914

  19. Evaluation of antibacterial activity of crude protein extracts from seeds of six different medical plants against standard bacterial strains.

    PubMed

    Al Akeel, Raid; Al-Sheikh, Yazeed; Mateen, Ayesha; Syed, Rabbani; Janardhan, K; Gupta, V C

    2014-04-01

    A huge group of natural antimicrobial compounds are active against a large spectrum of bacterial strains causing infectious threat. The present study was conducted to investigate the crude extracts of antimicrobial protein and peptide efficacy from six medicinal plant seeds. Extraction was carried out in Sodium phosphate citrate buffer, and Sodium acetate buffer using different pH. Antimicrobial activities of these plants were determined by the microbiological technique using Agar well diffusion Assay. Extremely strong activity was observed in the seed extracts of Allium ascolinicum extracted in sodium phosphate citrate buffer at pH (5.8) against Proteus vulgaris, Escherichia coli and Staphylococcus aureus with zone of inhibition 17 mm, 17 mm and 15 mm and Rumex vesicarius at pH (7.6), Ammi majus at pH (6.8), Cichorium intybus at pH (7.4) and Cucumis sativus at pH (7.8) also showed better sensitivity against the bacterial strains with zone of inhibition ranges 16-10 mm and some of the strains were found to be resistant. Antibacterial activity pattern of different plant extracts prepared in sodium acetate buffer pH (6.5), among all the plant seed extracts used Foeniculum vulgare had shown good inhibition in all the bacterial strains used, with zone of inhibition ranges 11-12.5 mm, The extracts of C. intybus and C. sativus were found to be effective with zone of inhibition 11-6 mm and some of the strains were found to be resistant. Most of the strains found to have shown better sensitivity compared with the standard antibiotic Chloramphenicol (25 mcg). Our results showed that the plants used for our study are the richest source for antimicrobial proteins and peptides and they may be used for industrial extraction and isolation of antimicrobial compounds which may find a place in medicine industry as constituents of antibiotics. PMID:24600307

  20. Evaluation of antibacterial activity of crude protein extracts from seeds of six different medical plants against standard bacterial strains

    PubMed Central

    Al Akeel, Raid; Al-Sheikh, Yazeed; Mateen, Ayesha; Syed, Rabbani; Janardhan, K.; Gupta, V.C.

    2013-01-01

    A huge group of natural antimicrobial compounds are active against a large spectrum of bacterial strains causing infectious threat. The present study was conducted to investigate the crude extracts of antimicrobial protein and peptide efficacy from six medicinal plant seeds. Extraction was carried out in Sodium phosphate citrate buffer, and Sodium acetate buffer using different pH. Antimicrobial activities of these plants were determined by the microbiological technique using Agar well diffusion Assay. Extremely strong activity was observed in the seed extracts of Allium ascolinicum extracted in sodium phosphate citrate buffer at pH (5.8) against Proteus vulgaris, Escherichia coli and Staphylococcus aureus with zone of inhibition 17 mm, 17 mm and 15 mm and Rumex vesicarius at pH (7.6), Ammi majus at pH (6.8), Cichorium intybus at pH (7.4) and Cucumis sativus at pH (7.8) also showed better sensitivity against the bacterial strains with zone of inhibition ranges 16–10 mm and some of the strains were found to be resistant. Antibacterial activity pattern of different plant extracts prepared in sodium acetate buffer pH (6.5), among all the plant seed extracts used Foeniculum vulgare had shown good inhibition in all the bacterial strains used, with zone of inhibition ranges 11–12.5 mm, The extracts of C. intybus and C. sativus were found to be effective with zone of inhibition 11–6 mm and some of the strains were found to be resistant. Most of the strains found to have shown better sensitivity compared with the standard antibiotic Chloramphenicol (25 mcg). Our results showed that the plants used for our study are the richest source for antimicrobial proteins and peptides and they may be used for industrial extraction and isolation of antimicrobial compounds which may find a place in medicine industry as constituents of antibiotics. PMID:24600307

  1. Structure of ERA in complex with the 3 end of 16S rRNA: Implications for ribosome biogenesis

    E-print Network

    Structure of ERA in complex with the 3 end of 16S rRNA: Implications for ribosome biogenesis Chao-binding KH domain, is essential for bacterial cell viability. It binds to 16S rRNA and the 30S ribosomalS rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein

  2. Seasonal and Spatial Variability in Lake Michigan Sediment Small-Subunit rRNA Concentrations

    PubMed Central

    MacGregor, Barbara J.; Moser, Duane P.; Baker, Brett J.; Alm, Elizabeth W.; Maurer, Max; Nealson, Kenneth H.; Stahl, David A.

    2001-01-01

    We have used molecular biological methods to study the distribution of microbial small-subunit rRNAs (SSU rRNAs), in relation to chemical profiles, in offshore Lake Michigan sediments. The sampling site is at a depth of 100 m, with temperatures of 2 to 4°C year-round. RNA extracted from sediment was probed with radiolabeled oligonucleotides targeting bacterial, archaeal, and eukaryotic SSU rRNAs, as well as with a universal probe. The coverage of these probes in relation to the present sequence database is discussed. Because ribosome production is growth rate regulated, rRNA concentrations are an indicator of the microbial populations active in situ. Over a 1-year period, changes in sedimentary SSU rRNA concentrations followed seasonal changes in surface water temperature and SSU rRNA concentration. Sedimentary depth profiles of oxygen, reduced manganese and iron, and sulfate changed relatively little from season to season, but the nitrate concentration was approximately fivefold higher in April and June 1997 than at the other times sampling was done. We propose that sediment microbial SSU rRNA concentrations at our sampling site are influenced by seasonal inputs from the water column, particularly the settling of the spring diatom bloom, and that the timing of this input may be modulated by grazers, such that ammonia becomes available to sediment microbes sooner than fresh organic carbon. Nitrate production from ammonia by autotrophic nitrifying bacteria, combined with low activity of heterotrophic denitrifying bacteria in the absence of readily degradable organic carbon, could account for the cooccurrence of high nitrate and low SSU rRNA concentrations. PMID:11525985

  3. Identification of the Microbiota in Carious Dentin Lesions Using 16S rRNA Gene Sequencing

    PubMed Central

    Obata, Junko; Takeshita, Toru; Shibata, Yukie; Yamanaka, Wataru; Unemori, Masako; Akamine, Akifumi; Yamashita, Yoshihisa

    2014-01-01

    While mutans streptococci have long been assumed to be the specific pathogen responsible for human dental caries, the concept of a complex dental caries-associated microbiota has received significant attention in recent years. Molecular analyses revealed the complexity of the microbiota with the predominance of Lactobacillus and Prevotella in carious dentine lesions. However, characterization of the dentin caries-associated microbiota has not been extensively explored in different ethnicities and races. In the present study, the bacterial communities in the carious dentin of Japanese subjects were analyzed comprehensively with molecular approaches using the16S rRNA gene. Carious dentin lesion samples were collected from 32 subjects aged 4–76 years, and the 16S rRNA genes, amplified from the extracted DNA with universal primers, were sequenced with a pyrosequencer. The bacterial composition was classified into clusters I, II, and III according to the relative abundance (high, middle, low) of Lactobacillus. The bacterial composition in cluster II was composed of relatively high proportions of Olsenella and Propionibacterium or subdominated by heterogeneous genera. The bacterial communities in cluster III were characterized by the predominance of Atopobium, Prevotella, or Propionibacterium with Streptococcus or Actinomyces. Some samples in clusters II and III, mainly related to Atopobium and Propionibacterium, were novel combinations of microbiota in carious dentin lesions and may be characteristic of the Japanese population. Clone library analysis revealed that Atopobium sp. HOT-416 and P. acidifaciens were specific species associated with dentinal caries among these genera in a Japanese population. We summarized the bacterial composition of dentinal carious lesions in a Japanese population using next-generation sequencing and found typical Japanese types with Atopobium or Propionibacterium predominating. PMID:25083880

  4. Anti-bacterial activity and brine shrimp lethality bioassay of methanolic extracts of fourteen different edible vegetables from Bangladesh

    PubMed Central

    Ullah, M. Obayed; Haque, Mahmuda; Urmi, Kaniz Fatima; Zulfiker, Abu Hasanat Md.; Anita, Elichea Synthi; Begum, Momtaj; Hamid, Kaiser

    2013-01-01

    Objective To investigate the antibacterial and cytotoxic activity of fourteen different edible vegetables methanolic extract from Bangladesh. Methods The antibacterial activity was evaluated using disc diffusion assay method against 12 bacteria (both gram positive and gram negative). The plant extracts were also screened for cytotoxic activity using the brine shrimp lethality bioassay method and the lethal concentrations (LC50) were determined at 95% confidence intervals by analyzing the data on a computer loaded with “Finney Programme”. Results All the vegetable extracts showed low to elevated levels of antibacterial activity against most of the tested strains (zone of inhibition=5-28 mm). The most active extract against all bacterial strains was from Xanthium indicum which showed remarkable antibacterial activity having the diameter of growth inhibition zone ranging from 12 to 28 mm followed by Alternanthera sessilis (zone of inhibition=6-21 mm). All extracts exhibited considerable general toxicity towards brine shrimps. The LC50 value of the tested extracts was within the range of 8.447 to 60.323 µg/mL with respect to the positive control (vincristine sulphate) which was 0.91 µg/mL. Among all studied extracts, Xanthium indicum displayed the highest cytotoxic effect with LC50 value of 8.447 µg/mL. Conclusions The results of the present investigation suggest that most of the studied plants are potentially good source of antibacterial and anticancer agents. PMID:23570009

  5. Yeast cell wall extract induces disease resistance against bacterial and fungal pathogens in Arabidopsis thaliana and Brassica crop.

    PubMed

    Narusaka, Mari; Minami, Taichi; Iwabuchi, Chikako; Hamasaki, Takashi; Takasaki, Satoko; Kawamura, Kimito; Narusaka, Yoshihiro

    2015-01-01

    Housaku Monogatari (HM) is a plant activator prepared from a yeast cell wall extract. We examined the efficacy of HM application and observed that HM treatment increased the resistance of Arabidopsis thaliana and Brassica rapa leaves to bacterial and fungal infections. HM reduced the severity of bacterial leaf spot and anthracnose on A. thaliana and Brassica crop leaves with protective effects. In addition, gene expression analysis of A. thaliana plants after treatment with HM indicated increased expression of several plant defense-related genes. HM treatment appears to induce early activation of jasmonate/ethylene and late activation of salicylic acid (SA) pathways. Analysis using signaling mutants revealed that HM required SA accumulation and SA signaling to facilitate resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola and the fungal pathogen Colletotrichum higginsianum. In addition, HM-induced resistance conferred chitin-independent disease resistance to bacterial pathogens in A. thaliana. These results suggest that HM contains multiple microbe-associated molecular patterns that activate defense responses in plants. These findings suggest that the application of HM is a useful tool that may facilitate new disease control methods. PMID:25565273

  6. First Experience of a Multicenter External Quality Assessment of Molecular 16S rRNA Gene Detection in Bone and Joint Infections

    PubMed Central

    Bémer, Pascale; Valentin, Anne Sophie; Héry-Arnaud, Geneviève; Tandé, Didier; Jolivet-Gougeon, Anne; Vincent, Pascal; Kempf, Marie; Lemarié, Carole; Guinard, Jérôme; Bret, Laurent; Cognée, Anne Sophie; Gibaud, Sophie; Burucoa, Christophe; Corvec, Stéphane

    2014-01-01

    The objective of this study was to assess the performance of seven French laboratories for 16S rRNA gene detection by real-time PCR in the diagnosis of bone and joint infection (BJI) to validate a large multicenter study. External quality control (QC) was required owing to the differences in extraction procedures and the molecular equipment used in the different laboratories. Three proficiency sets were organized, including four bacterial DNA extracts and four bead mill-pretreated osteoarticular specimens. Extraction volumes, 16S rRNA gene primers, and sequencing interpretation rules were standardized. In order to assess each laboratory's ability to achieve the best results, scores were assigned, and each QC series was classified as optimal, acceptable, or to be improved. A total of 168 QCs were sent, and 160 responses were analyzed. The expected results were obtained for 93.8%, with the same proportion for extracts (75/80) and clinical specimens (75/80). For the specimens, there was no significant difference between manual and automated extraction. This QC demonstrated the ability to achieve good and homogeneous results using the same 16S rRNA gene PCR with different equipment and validates the possibility of high-quality multicenter studies using molecular diagnosis for BJI. PMID:25411177

  7. Estimating Bacterial Diversity for Ecological Studies: Methods, Metrics, and Assumptions

    PubMed Central

    Birtel, Julia; Walser, Jean-Claude; Pichon, Samuel; Bürgmann, Helmut; Matthews, Blake

    2015-01-01

    Methods to estimate microbial diversity have developed rapidly in an effort to understand the distribution and diversity of microorganisms in natural environments. For bacterial communities, the 16S rRNA gene is the phylogenetic marker gene of choice, but most studies select only a specific region of the 16S rRNA to estimate bacterial diversity. Whereas biases derived from from DNA extraction, primer choice and PCR amplification are well documented, we here address how the choice of variable region can influence a wide range of standard ecological metrics, such as species richness, phylogenetic diversity, ?-diversity and rank-abundance distributions. We have used Illumina paired-end sequencing to estimate the bacterial diversity of 20 natural lakes across Switzerland derived from three trimmed variable 16S rRNA regions (V3, V4, V5). Species richness, phylogenetic diversity, community composition, ?-diversity, and rank-abundance distributions differed significantly between 16S rRNA regions. Overall, patterns of diversity quantified by the V3 and V5 regions were more similar to one another than those assessed by the V4 region. Similar results were obtained when analyzing the datasets with different sequence similarity thresholds used during sequences clustering and when the same analysis was used on a reference dataset of sequences from the Greengenes database. In addition we also measured species richness from the same lake samples using ARISA Fingerprinting, but did not find a strong relationship between species richness estimated by Illumina and ARISA. We conclude that the selection of 16S rRNA region significantly influences the estimation of bacterial diversity and species distributions and that caution is warranted when comparing data from different variable regions as well as when using different sequencing techniques. PMID:25915756

  8. Secondary Metabolites Control the Associated Bacterial Communities of Saprophytic Basidiomycotina Fungi

    PubMed Central

    de Carvalho, Maira Peres; Türck, Patrick; Abraham, Wolf-Rainer

    2015-01-01

    Fungi grow under humid conditions and are, therefore, prone to biofilm infections. A 16S rRNA fingerprint analysis was performed on 49 sporocarps of Basidiomycotina in order to determine whether they are able to control these biofilms. Ninety-five bacterial phylotypes, comprising 4 phyla and 10 families, were identified. While ectomycorrhizal fungi harbored the highest bacterial diversity, saprophytic fungi showed little or no association with bacteria. Seven fungal species were screened for antimicrobial and antibiofilm activities. Biofilm formation and bacterial growth was inhibited by extracts obtained from saprophytic fungi, which confirmed the hypothesis that many fungi modulate biofilm colonization on their sporocarps. PMID:25904019

  9. Immunoglobulin isotype isolated from human placental extract does not interfere in complement-mediated bacterial opsonization within the wound milieu

    PubMed Central

    Sharma, Kanika; Bhattacharyya, Debasish

    2015-01-01

    The wound healing potency of an aqueous extract of placenta can be evaluated through the presence of numerous regulatory components. The presence of glycans was detected by thin layer chromatography and fluorophore-assisted carbohydrate electrophoresis. Mass spectrometric analysis revealed the existence of multiple fragments of immunoglobulin G (IgG). IgG was present in the extract at a concentration of 25.2 ± 3.97 ?g/ml. IgG possesses anti-complementary activity by diverting the complement activation from target surface. Thus, effect of placental IgG on complement–bacteria interaction was investigated through classical and alternative pathway and the preparation was ascertained to be safe with respect to their interference in the process of bacterial opsonization. PMID:25984442

  10. Phylogenetic differences in attached and free-living bacterial communities in a temperate coastal lagoon during summer, revealed via high-throughput 16S rRNA gene sequencing.

    PubMed

    Mohit, Vani; Archambault, Philippe; Toupoint, Nicolas; Lovejoy, Connie

    2014-04-01

    Most of what is known about coastal free-living and attached bacterial diversity is based on open coasts, with high particulate and nutrient riverine supply, terrestrial runoffs, and anthropogenic activities. The Magdalen Islands in the Gulf of St. Lawrence (Canada) are dominated by shallow lagoons with small, relatively pristine catchments and no freshwater input apart from rain. Such conditions provided an opportunity to investigate coastal free-living and attached marine bacterial diversity in the absence of confounding effects of steep freshwater gradients. We found significant differences between the two communities and marked temporal patterns in both. Taxonomic richness and diversity were greater in the attached than in the free-living community, increasing over summer, especially within the least abundant bacterial phyla. The highest number of reads fell within the SAR 11 clade (Pelagibacter, Alphaproteobacteria), which dominated free-living communities. The attached communities had deeper phylum-level diversity than the free-living fraction. Distance-based redundancy analysis indicated that the particulate organic matter (POM) concentration was the main variable separating early and late summer samples with salinity and temperature changes also significantly correlated to bacterial community structure. Our approach using high-throughput sequencing detected differences in free-living versus attached bacteria in the absence of riverine input, in keeping with the concept that marine attached communities are distinct from cooccurring free-living taxa. This diversity likely reflects the diverse microhabitats of available particles, implying that the total bacterial diversity in coastal systems is linked to particle supply and variability, with implications for understanding microbial biodiversity in marine systems. PMID:24463966

  11. Illumina Amplicon Sequencing of 16S rRNA Tag Reveals Bacterial Community Development in the Rhizosphere of Apple Nurseries at a Replant Disease Site and a New Planting Site

    PubMed Central

    Sun, Jian; Zhang, Qiang; Zhou, Jia; Wei, Qinping

    2014-01-01

    We used a next-generation, Illumina-based sequencing approach to characterize the bacterial community development of apple rhizosphere soil in a replant site (RePlant) and a new planting site (NewPlant) in Beijing. Dwarfing apple nurseries of ‘Fuji’/SH6/Pingyitiancha trees were planted in the spring of 2013. Before planting, soil from the apple rhizosphere of the replant site (ReSoil) and from the new planting site (NewSoil) was sampled for analysis on the Illumina MiSeq platform. In late September, the rhizosphere soil from both sites was resampled (RePlant and NewPlant). More than 16,000 valid reads were obtained for each replicate, and the community was composed of five dominant groups (Proteobacteria, Acidobacteria, Bacteroidetes, Gemmatimonadetes and Actinobacteria). The bacterial diversity decreased after apple planting. Principal component analyses revealed that the rhizosphere samples were significantly different among treatments. Apple nursery planting showed a large impact on the soil bacterial community, and the community development was significantly different between the replanted and newly planted soils. Verrucomicrobia were less abundant in RePlant soil, while Pseudomonas and Lysobacter were increased in RePlant compared with ReSoil and NewPlant. Both RePlant and ReSoil showed relatively higher invertase and cellulase activities than NewPlant and NewSoil, but only NewPlant soil showed higher urease activity, and this soil also had the higher plant growth. Our experimental results suggest that planting apple nurseries has a significant impact on soil bacterial community development at both replant and new planting sites, and planting on new site resulted in significantly higher soil urease activity and a different bacterial community composition. PMID:25360786

  12. Illumina amplicon sequencing of 16S rRNA tag reveals bacterial community development in the rhizosphere of apple nurseries at a replant disease site and a new planting site.

    PubMed

    Sun, Jian; Zhang, Qiang; Zhou, Jia; Wei, Qinping

    2014-01-01

    We used a next-generation, Illumina-based sequencing approach to characterize the bacterial community development of apple rhizosphere soil in a replant site (RePlant) and a new planting site (NewPlant) in Beijing. Dwarfing apple nurseries of 'Fuji'/SH6/Pingyitiancha trees were planted in the spring of 2013. Before planting, soil from the apple rhizosphere of the replant site (ReSoil) and from the new planting site (NewSoil) was sampled for analysis on the Illumina MiSeq platform. In late September, the rhizosphere soil from both sites was resampled (RePlant and NewPlant). More than 16,000 valid reads were obtained for each replicate, and the community was composed of five dominant groups (Proteobacteria, Acidobacteria, Bacteroidetes, Gemmatimonadetes and Actinobacteria). The bacterial diversity decreased after apple planting. Principal component analyses revealed that the rhizosphere samples were significantly different among treatments. Apple nursery planting showed a large impact on the soil bacterial community, and the community development was significantly different between the replanted and newly planted soils. Verrucomicrobia were less abundant in RePlant soil, while Pseudomonas and Lysobacter were increased in RePlant compared with ReSoil and NewPlant. Both RePlant and ReSoil showed relatively higher invertase and cellulase activities than NewPlant and NewSoil, but only NewPlant soil showed higher urease activity, and this soil also had the higher plant growth. Our experimental results suggest that planting apple nurseries has a significant impact on soil bacterial community development at both replant and new planting sites, and planting on new site resulted in significantly higher soil urease activity and a different bacterial community composition. PMID:25360786

  13. Phylogenetic Differences in Attached and Free-Living Bacterial Communities in a Temperate Coastal Lagoon during Summer, Revealed via High-Throughput 16S rRNA Gene Sequencing

    PubMed Central

    Mohit, Vani; Archambault, Philippe; Toupoint, Nicolas

    2014-01-01

    Most of what is known about coastal free-living and attached bacterial diversity is based on open coasts, with high particulate and nutrient riverine supply, terrestrial runoffs, and anthropogenic activities. The Magdalen Islands in the Gulf of St. Lawrence (Canada) are dominated by shallow lagoons with small, relatively pristine catchments and no freshwater input apart from rain. Such conditions provided an opportunity to investigate coastal free-living and attached marine bacterial diversity in the absence of confounding effects of steep freshwater gradients. We found significant differences between the two communities and marked temporal patterns in both. Taxonomic richness and diversity were greater in the attached than in the free-living community, increasing over summer, especially within the least abundant bacterial phyla. The highest number of reads fell within the SAR 11 clade (Pelagibacter, Alphaproteobacteria), which dominated free-living communities. The attached communities had deeper phylum-level diversity than the free-living fraction. Distance-based redundancy analysis indicated that the particulate organic matter (POM) concentration was the main variable separating early and late summer samples with salinity and temperature changes also significantly correlated to bacterial community structure. Our approach using high-throughput sequencing detected differences in free-living versus attached bacteria in the absence of riverine input, in keeping with the concept that marine attached communities are distinct from cooccurring free-living taxa. This diversity likely reflects the diverse microhabitats of available particles, implying that the total bacterial diversity in coastal systems is linked to particle supply and variability, with implications for understanding microbial biodiversity in marine systems. PMID:24463966

  14. Inhibition of Bacterial Quorum Sensing by Extracts from Aquatic Fungi: First Report from Marine Endophytes

    PubMed Central

    Martín-Rodríguez, Alberto J.; Reyes, Fernando; Martín, Jesús; Pérez-Yépez, Juan; León-Barrios, Milagros; Couttolenc, Alan; Espinoza, César; Trigos, Ángel; Martín, Víctor S.; Norte, Manuel; Fernández, José J.

    2014-01-01

    In our search for quorum-sensing (QS) disrupting molecules, 75 fungal isolates were recovered from reef organisms (endophytes), saline lakes and mangrove rhizosphere. Their QS inhibitory activity was evaluated in Chromobacterium violaceum CVO26. Four strains of endophytic fungi stood out for their potent activity at concentrations from 500 to 50 ?g mL?1. The molecular characterization, based on the internal transcribed spacer (ITS) region sequences (ITS1, 5.8S and ITS2) between the rRNA of 18S and 28S, identified these strains as belonging to four genera: Sarocladium (LAEE06), Fusarium (LAEE13), Epicoccum (LAEE14), and Khuskia (LAEE21). Interestingly, three came from coral species and two of them came from the same organism, the coral Diploria strigosa. Metabolic profiles obtained by Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) suggest that a combination of fungal secondary metabolites and fatty acids could be the responsible for the observed activities. The LC-HRMS analysis also revealed the presence of potentially new secondary metabolites. This is, to the best of our knowledge, the first report of QS inhibition by marine endophytic fungi. PMID:25415350

  15. Inhibition of bacterial quorum sensing by extracts from aquatic fungi: first report from marine endophytes.

    PubMed

    Martín-Rodríguez, Alberto J; Reyes, Fernando; Martín, Jesús; Pérez-Yépez, Juan; León-Barrios, Milagros; Couttolenc, Alan; Espinoza, César; Trigos, Angel; Martín, Víctor S; Norte, Manuel; Fernández, José J

    2014-11-01

    In our search for quorum-sensing (QS) disrupting molecules, 75 fungal isolates were recovered from reef organisms (endophytes), saline lakes and mangrove rhizosphere. Their QS inhibitory activity was evaluated in Chromobacterium violaceum CVO26. Four strains of endophytic fungi stood out for their potent activity at concentrations from 500 to 50 ?g mL-1. The molecular characterization, based on the internal transcribed spacer (ITS) region sequences (ITS1, 5.8S and ITS2) between the rRNA of 18S and 28S, identified these strains as belonging to four genera: Sarocladium (LAEE06), Fusarium (LAEE13), Epicoccum (LAEE14), and Khuskia (LAEE21). Interestingly, three came from coral species and two of them came from the same organism, the coral Diploria strigosa. Metabolic profiles obtained by Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) suggest that a combination of fungal secondary metabolites and fatty acids could be the responsible for the observed activities. The LC-HRMS analysis also revealed the presence of potentially new secondary metabolites. This is, to the best of our knowledge, the first report of QS inhibition by marine endophytic fungi. PMID:25415350

  16. EFFECT OF SOLVENT AND EXTRACTION METHODS ON THE BACTERIAL MUTAGENICITY OF SIDESTREAM CIGARETTE SMOKE

    EPA Science Inventory

    The mutagenic activity of sidestream cigarette smoke particles was estimated by testing sidestream cigarette smoke particles which had been collected under controlled burning conditions in the laboratory. Two different extraction methods (Soxhlet and ultrasonic agitation) and 3 d...

  17. Illumina sequencing of the V4 hypervariable region 16S rRNA gene reveals extensive changes in bacterial communities in the cecum following carbohydrate oral infusion and development of early-stage acute laminitis in the horse.

    PubMed

    Moreau, Michael M; Eades, Susan C; Reinemeyer, Craig R; Fugaro, Michael N; Onishi, Janet C

    2014-01-31

    In the equine carbohydrate overload model of acute laminitis, disease progression is associated with changes in bacteria found in the cecum. To date, research has focused on changes in specific Gram-positive bacteria in this portion of the intestinal tract. Metagenomic methods are now available making it possible to interrogate microbial communities using animal protocols that sufficiently power a study. In this study, the microbiota in cecal fluid collected from control, non-laminitic horses (n=8) and from horses with early-stage acute laminitis induced with either oligofructan (n=6) or cornstarch (n=6) were profiled. The microbiota were identified based on sequencing the V4 hypervariable region of the 16S rRNA gene. The results of the study show that the relative abundance of Lactobacillus sp. and Streptococcus sp. increased significantly (p<0.05) following OF and CS infusion. Other significant changes included an increase (p<0.05) in relative abundance of Veillonella sp. and Serratia sp., two potentially pathogenic, Gram-negative bacteria. Significant decreases in the relative abundance of presumptive normal flora were detected as well. Although changes in cecal microbiota described in this communication are from a pilot study, it is hypothesized that an overgrowth of pathogenic Gram-negative bacteria develops and contributes to enterocolitis, pyrexia and lameness in the carbohydrate overload model of acute laminitis. PMID:24355533

  18. An optimized method for the extraction of bacterial mRNA from plant roots infected with Escherichia coli O157:H7

    PubMed Central

    Holmes, Ashleigh; Birse, Louise; Jackson, Robert W.; Holden, Nicola J.

    2014-01-01

    Analysis of microbial gene expression during host colonization provides valuable information on the nature of interaction, beneficial or pathogenic, and the adaptive processes involved. Isolation of bacterial mRNA for in planta analysis can be challenging where host nucleic acid may dominate the preparation, or inhibitory compounds affect downstream analysis, e.g., quantitative reverse transcriptase PCR (qPCR), microarray, or RNA-seq. The goal of this work was to optimize the isolation of bacterial mRNA of food-borne pathogens from living plants. Reported methods for recovery of phytopathogen-infected plant material, using hot phenol extraction and high concentration of bacterial inoculation or large amounts of infected tissues, were found to be inappropriate for plant roots inoculated with Escherichia coli O157:H7. The bacterial RNA yields were too low and increased plant material resulted in a dominance of plant RNA in the sample. To improve the yield of bacterial RNA and reduce the number of plants required, an optimized method was developed which combines bead beating with directed bacterial lysis using SDS and lysozyme. Inhibitory plant compounds, such as phenolics and polysaccharides, were counteracted with the addition of high-molecular-weight polyethylene glycol and hexadecyltrimethyl ammonium bromide. The new method increased the total yield of bacterial mRNA substantially and allowed assessment of gene expression by qPCR. This method can be applied to other bacterial species associated with plant roots, and also in the wider context of food safety. PMID:25018749

  19. Structure-Activity Relationships of Antimicrobial Gallic Acid Derivatives from Pomegranate and Acacia Fruit Extracts against Potato Bacterial Wilt Pathogen.

    PubMed

    Farag, Mohamed A; Al-Mahdy, Dalia A; Salah El Dine, Riham; Fahmy, Sherifa; Yassin, Aymen; Porzel, Andrea; Brandt, Wolfgang

    2015-06-01

    Bacterial wilts of potato, tomato, pepper, and or eggplant caused by Ralstonia solanacearum are among the most serious plant diseases worldwide. In this study, the issue of developing bactericidal agents from natural sources against R. solanacearum derived from plant extracts was addressed. Extracts prepared from 25 plant species with antiseptic relevance in Egyptian folk medicine were screened for their antimicrobial properties against the potato pathogen R. solancearum by using the disc-zone inhibition assay and microtitre plate dilution method. Plants exhibiting notable antimicrobial activities against the tested pathogen include extracts from Acacia arabica and Punica granatum. Bioactivity-guided fractionation of A. arabica and P. granatum resulted in the isolation of bioactive compounds 3,5-dihydroxy-4-methoxybenzoic acid and gallic acid, in addition to epicatechin. All isolates displayed significant antimicrobial activities against R. solanacearum (MIC values 0.5-9 mg/ml), with 3,5-dihydroxy-4-methoxybenzoic acid being the most effective one with a MIC value of 0.47 mg/ml. We further performed a structure-activity relationship (SAR) study for the inhibition of R. solanacearum growth by ten natural, structurally related benzoic acids. PMID:26080741

  20. Micelle PCR reduces chimera formation in 16S rRNA profiling of complex microbial DNA mixtures

    PubMed Central

    Boers, Stefan A.; Hays, John P.; Jansen, Ruud

    2015-01-01

    16S rRNA gene profiling has revolutionized the field of microbial ecology. Many researchers in various fields have embraced this technology to investigate bacterial compositions of samples derived from many different ecosystems. However, it is important to acknowledge the current limitations and drawbacks of 16S rRNA gene profiling. Although sample handling, DNA extraction methods and the choice of universal 16S rRNA gene PCR primers are well known factors that could seriously affect the final results of microbiota profiling studies, inevitable amplification artifacts, such as chimera formation and PCR competition, are seldom appreciated. Here we report on a novel micelle based amplification strategy, which overcomes these limitations via the clonal amplification of targeted DNA molecules. Our results show that micelle PCR drastically reduces chimera formation by a factor of 38 (1.5% vs. 56.9%) compared with traditional PCR, resulting in improved microbial diversity estimates. In addition, compartmentalization during micelle PCR prevents PCR competition due to unequal amplification rates of different 16S template molecules, generating robust and accurate 16S microbiota profiles required for comparative studies (e.g. longitudinal surveys). PMID:26373611

  1. Folding of 16S rRNA in a signal-producing structure for the detection of bacteria.

    PubMed

    Gerasimova, Yulia V; Kolpashchikov, Dmitry M

    2013-09-27

    Sixty-four DNA strands hybridize to 16S rRNA to form 32 deoxyribozyme catalytic cores that produce a fluorescent signal. The approach allows detection of 0.6 pM 16S rRNA, or about 3×10(4) bacterial cells in a PCR-free format. PMID:24038733

  2. Inhibition of bacterial, fungal and plant growth by testa extracts of Citrullus genotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Watermelon (Citrullus lanatus var. lanatus (Thunb.) Matsum & Nakai) seed exudates inhibit germination and seedling growth of several plant species and growth of pathogenic fungi and bacteria. This study was conducted to determine if extractable components in testae contribute to the inhibition. T...

  3. Optimization of isolation and cultivation of bacterial endophytes through addition of plant extract to nutrient media

    PubMed Central

    Eevers, N; Gielen, M; Sánchez-López, A; Jaspers, S; White, J C; Vangronsveld, J; Weyens, N

    2015-01-01

    Many endophytes have beneficial effects on plants and can be exploited in biotechnological applications. Studies hypothesize that only 0.001–1% of all plant-associated bacteria are cultivable. Moreover, even after successful isolations, many endophytic bacteria often show reduced regrowth capacity. This research aimed to optimize isolation processes and culturing these bacteria afterwards. We compared several minimal and complex media in a screening. Beside the media themselves, two gelling agents and adding plant extract to media were investigated to enhance the number and diversity of endophytes as well as the growth capacity when regrown after isolation. In this work, 869 medium delivered the highest numbers of cultivable bacteria, as well as the highest diversity. When comparing gelling agents, no differences were observed in the numbers of bacteria. Adding plant extract to the media lead to a slight increase in diversity. However, when adding plant extract to improve the regrowth capacity, sharp increases of viable bacteria occurred in both rich and minimal media. PMID:25997013

  4. Tools for Characterizing Bacterial Protein Synthesis Inhibitors

    PubMed Central

    Orelle, Cédric; Carlson, Skylar; Kaushal, Bindiya; Almutairi, Mashal M.; Liu, Haipeng; Ochabowicz, Anna; Quan, Selwyn; Pham, Van Cuong; Squires, Catherine L.; Murphy, Brian T.

    2013-01-01

    Many antibiotics inhibit the growth of sensitive bacteria by interfering with ribosome function. However, discovery of new protein synthesis inhibitors is curbed by the lack of facile techniques capable of readily identifying antibiotic target sites and modes of action. Furthermore, the frequent rediscovery of known antibiotic scaffolds, especially in natural product extracts, is time-consuming and expensive and diverts resources that could be used toward the isolation of novel lead molecules. In order to avoid these pitfalls and improve the process of dereplication of chemically complex extracts, we designed a two-pronged approach for the characterization of inhibitors of protein synthesis (ChIPS) that is suitable for the rapid identification of the site and mode of action on the bacterial ribosome. First, we engineered antibiotic-hypersensitive Escherichia coli strains that contain only one rRNA operon. These strains are used for the rapid isolation of resistance mutants in which rRNA mutations identify the site of the antibiotic action. Second, we show that patterns of drug-induced ribosome stalling on mRNA, monitored by primer extension, can be used to elucidate the mode of antibiotic action. These analyses can be performed within a few days and provide a rapid and efficient approach for identifying the site and mode of action of translation inhibitors targeting the bacterial ribosome. Both techniques were validated using a bacterial strain whose culture extract, composed of unknown metabolites, exhibited protein synthesis inhibitory activity; we were able to rapidly detect the presence of the antibiotic chloramphenicol. PMID:24041905

  5. Protective Effect of Polygonum orientale L. Extracts against Clavibater michiganense subsp. sepedonicum, the Causal Agent of Bacterial Ring Rot of Potato

    PubMed Central

    Cai, Jin; Xie, Shulian; Feng, Jia; Wang, Feipeng; Xu, Qiufeng

    2013-01-01

    The Polygonum orientale L. extracts were investigated for antibacterial activity against Clavibater michiganense subsp. sepedonicum (Spieckermann & Kotthoff) Davis et al., the causal agent of a serious disease called bacterial ring rot of potato. The results showed that the leaf extracts of P. orientale had significantly (p<0.05) greater antibacterial activity against C. michiganense subsp. sepedonicum than root, stem, flower extracts in vitro. According to the results of single factor experiments and L273(13) orthogonal experiments, optimum extraction conditions were A1B3C1, extraction time 6 h, temperature 80°C, solid to liquid ratio 1?10 (g:mL). The highest (p<0.05) antibacterial activity was observed when pH was 5, excluding the effect of control. The extracts were stable under ultraviolet (UV). In vivo analysis revealed that 50 mg/mL of P. orientale leaf extracts was effective in controlling decay. Under field conditions, 50 mg/mL of P. orientale leaf extracts also improved growth parameters (whole plant length, shoot length, root length, plant fresh weight, shoot fresh weight, root fresh weight, dry weight, and number of leaves), in the 2010 and 2011 two growing seasons. Further solvent partition assays showed that the most active compounds were in the petroleum ether fractionation. Transmission electron microscopy (TEM) showed drastic ultrastructural changes caused by petroleum ether fractionation, including bacterial deformation, electron-dense particles, formation of vacuoles and lack of cytoplasmic materials. These results indicated that P. orientale extracts have strong antibacterial activity against C. michiganense subsp. sepedonicum and a promising effect in control of bacterial ring rot of potato disease. PMID:23861908

  6. Mosquitocidal properties of Calotropis gigantea (Family: Asclepiadaceae) leaf extract and bacterial insecticide, Bacillus thuringiensis, against the mosquito vectors.

    PubMed

    Kovendan, Kalimuthu; Murugan, Kadarkarai; Prasanna Kumar, Kanagarajan; Panneerselvam, Chellasamy; Mahesh Kumar, Palanisamy; Amerasan, Duraisamy; Subramaniam, Jayapal; Vincent, Savariar

    2012-08-01

    Calotropis gigantea leaf extract and Bacillus thuringiensis were tested first to fourth-instar larvae and pupae of Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus. The medicinal plants were collected from the area around Bharathiar University, Coimbatore, India. Calotropis gigantea leaf was washed with tap water and shade-dried at room temperature. An electrical blender powdered the dried plant materials (leaves). The powder 500 g of the leaf was extracted with 1.5 L of organic solvents of methanol for 8 h using a Soxhlet apparatus and filtered. The crude leaf extracts were evaporated to dryness in a rotary vacuum evaporator. The plant extract showed larvicidal and pupicidal effects after 24 h of exposure; no mortality was observed in the control group. For Calotropis gigantea, the median lethal concentration values (LC(50)) observed for the larvicidal and pupicidal activities against mosquito vector species Anopheles stephensi I to IV larval instars and pupae were 73.77, 89.64, 121.69, 155.49, and 213.79 ppm; Aedes aegypti values were 92.27, 106.60, 136.48, 164.01, and 202.56 ppm; and Culex quinquefasciatus values were 104.66, 127.71, 173.75, 251.65, and 314.70 ppm, respectively. For B. thuringiensis, the LC(50) values of I to IV larval instars and pupae of Anopheles stephensi were 37.24, 45.41, 57.82, 80.09, and 98.34 ppm; Aedes aegypti values were 42.38, 51.90, 71.02, 96.17, and 121.59 ppm; and Culex quinquefasciatus values were 55.85, 68.07, 94.11, 113.35, and 133.87 ppm, respectively. The study proved that the methanol leaf extract of Calotropis gigantea and bacterial insecticide B. thuringiensis has mosquitocidal property and was evaluated as target species of mosquito vectors. This is an ideal ecofriendly approach for the control of vector control programs. PMID:22382205

  7. Comparison of the Rhizosphere Bacterial Communities of Zigongdongdou Soybean and a High-Methionine Transgenic Line of This Cultivar

    PubMed Central

    Ji, Jun; Wu, Haiying; Meng, Fang; Zhang, Mingrong; Zheng, Xiaobo; Wu, Cunxiang; Zhang, Zhengguang

    2014-01-01

    Previous studies have shown that methionine from root exudates affects the rhizosphere bacterial population involved in soil nitrogen fixation. A transgenic line of Zigongdongdou soybean cultivar (ZD91) that expresses Arabidopsis cystathionine ?-synthase resulting in an increased methionine production was examined for its influence to the rhizosphere bacterial population. Using 16S rRNA gene-based pyrosequencing analysis of the V4 region and DNA extracted from bacterial consortia collected from the rhizosphere of soybean plants grown in an agricultural field at the pod-setting stage, we characterized the populational structure of the bacterial community involved. In total, 87,267 sequences (approximately 10,908 per sample) were analyzed. We found that Acidobacteria, Proteobacteria, Bacteroidetes, Actinobacteria, Chloroflexi, Planctomycetes, Gemmatimonadetes, Firmicutes, and Verrucomicrobia constitute the dominant taxonomic groups in either the ZD91 transgenic line or parental cultivar ZD, and that there was no statistically significant difference in the rhizosphere bacterial community structure between the two cultivars. PMID:25079947

  8. Influence of First-Line Antibiotics on the Antibacterial Activities of Acetone Stem Bark Extract of Acacia mearnsii De Wild. against Drug-Resistant Bacterial Isolates.

    PubMed

    Olajuyigbe, Olufunmiso O; Coopoosamy, Roger M

    2014-01-01

    Background. This study was aimed at evaluating the antibacterial activity of the acetone extract of A. mearnsii and its interactions with antibiotics against some resistant bacterial strains. Methods. The antibacterial susceptibility testing was determined by agar diffusion and macrobroth dilution methods while the checkerboard method was used for the determination of synergy between the antibiotics and the extract. Results. The results showed that the susceptibility of the different bacterial isolates was concentration dependent for the extract and the different antibiotics. With the exception of S. marcescens, the inhibition zones of the extract produced by 20?mg/mL ranged between 18 and 32?mm. While metronidazole did not inhibit any of the bacterial isolates, all the antibiotics and their combinations, except for ciprofloxacin and its combination, did not inhibit Enterococcus faecalis. The antibacterial combinations were more of being antagonistic than of being synergistic in the agar diffusion assay. From the macrobroth dilution, the extract and the antibiotics exerted a varied degree of inhibitory effect on the test organisms. The MIC values of the acetone extract which are in mg/mL are lower than those of the different antibiotics which are in ?g/mL. From the checkerboard assay, the antibacterial combinations showed varied degrees of interactions including synergism, additive, indifference, and antagonism interactions. While antagonistic and additive interactions were 14.44%, indifference interaction was 22.22% and synergistic interaction was 37.78% of the antibacterial combinations against the test isolates. While the additivity/indifference interactions indicated no interactions, the antagonistic interaction may be considered as a negative interaction that could result in toxicity and suboptimal bioactivity. Conclusion. The synergistic effects of the herbal-drug combinations may be harnessed for the discovery and development of more rational evidence-based drug combinations with optimized efficiency in the prevention of multidrug resistance and therapy of multifactorial diseases. PMID:25101132

  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. Inhibitory activity of a standardized elderberry liquid extract against clinically-relevant human respiratory bacterial pathogens and influenza A and B viruses

    PubMed Central

    2011-01-01

    Background Black elderberries (Sambucus nigra L.) are well known as supportive agents against common cold and influenza. It is further known that bacterial super-infection during an influenza virus (IV) infection can lead to severe pneumonia. We have analyzed a standardized elderberry extract (Rubini, BerryPharma AG) for its antimicrobial and antiviral activity using the microtitre broth micro-dilution assay against three Gram-positive bacteria and one Gram-negative bacteria responsible for infections of the upper respiratory tract, as well as cell culture experiments for two different strains of influenza virus. Methods The antimicrobial activity of the elderberry extract was determined by bacterial growth experiments in liquid cultures using the extract at concentrations of 5%, 10%, 15% and 20%. The inhibitory effects were determined by plating the bacteria on agar plates. In addition, the inhibitory potential of the extract on the propagation of human pathogenic H5N1-type influenza A virus isolated from a patient and an influenza B virus strain was investigated using MTT and focus assays. Results For the first time, it was shown that a standardized elderberry liquid extract possesses antimicrobial activity against both Gram-positive bacteria of Streptococcus pyogenes and group C and G Streptococci, and the Gram-negative bacterium Branhamella catarrhalis in liquid cultures. The liquid extract also displays an inhibitory effect on the propagation of human pathogenic influenza viruses. Conclusion Rubini elderberry liquid extract is active against human pathogenic bacteria as well as influenza viruses. The activities shown suggest that additional and alternative approaches to combat infections might be provided by this natural product. PMID:21352539

  11. Inhibitory effect of pomegranate (Punica granatum L.) polyphenol extracts on the bacterial growth and survival of clinical isolates of pathogenic Staphylococcus aureus and Escherichia coli.

    PubMed

    Pagliarulo, Caterina; De Vito, Valentina; Picariello, Gianluca; Colicchio, Roberta; Pastore, Gabiria; Salvatore, Paola; Volpe, Maria Grazia

    2016-01-01

    In the present study major polyphenols of pomegranate arils and peel by-products were extracted in 50% (v/v) aqueous ethanol, characterized and used in microbiological assays in order to test antimicrobial activity against clinically isolated human pathogenic microorganisms. Total concentration of polyphenols and in vitro antioxidant properties were determined by the Folin-Ciocalteu and DPPH methods, respectively. The most abundant bioactive molecules, including anthocyanins, catechins, tannins, gallic and ellagic acids were identified by RP-HPLC-DAD, also coupled to off-line matrix assisted laser desorption/ionization (MALDI-TOF) mass spectrometry (MS). The inhibitory spectrum of extracts against test microorganisms was assessed by the agar well-diffusion method. Data herein indicated that both pomegranate aril and peel extracts have an effective antimicrobial activity, as evidenced by the inhibitory effect on the bacterial growth of two important human pathogens, including Staphylococcus aureus and Escherichia coli, which are often involved in foodborne illness. PMID:26213044

  12. Benthic Bacterial Diversity in Submerged Sinkhole Ecosystems? †

    PubMed Central

    Nold, Stephen C.; Pangborn, Joseph B.; Zajack, Heidi A.; Kendall, Scott T.; Rediske, Richard R.; Biddanda, Bopaiah A.

    2010-01-01

    Physicochemical characterization, automated ribosomal intergenic spacer analysis (ARISA) community profiling, and 16S rRNA gene sequencing approaches were used to study bacterial communities inhabiting submerged Lake Huron sinkholes inundated with hypoxic, sulfate-rich groundwater. Photosynthetic cyanobacterial mats on the sediment surface were dominated by Phormidium autumnale, while deeper, organically rich sediments contained diverse and active bacterial communities. PMID:19880643

  13. Benthic bacterial diversity in submerged sinkhole ecosystems.

    PubMed

    Nold, Stephen C; Pangborn, Joseph B; Zajack, Heidi A; Kendall, Scott T; Rediske, Richard R; Biddanda, Bopaiah A

    2010-01-01

    Physicochemical characterization, automated ribosomal intergenic spacer analysis (ARISA) community profiling, and 16S rRNA gene sequencing approaches were used to study bacterial communities inhabiting submerged Lake Huron sinkholes inundated with hypoxic, sulfate-rich groundwater. Photosynthetic cyanobacterial mats on the sediment surface were dominated by Phormidium autumnale, while deeper, organically rich sediments contained diverse and active bacterial communities. PMID:19880643

  14. The anti-adhesive mode of action of a purified mushroom (Lentinus edodes) extract with anticaries and antigingivitis properties in two oral bacterial phatogens

    PubMed Central

    2014-01-01

    Background In previous works we have shown that a low-molecular-mass (LMM) fraction from mushroom (Lentinus edodes) homogenate interferes with binding of Streptococcus mutans to hydroxyapatite and Prevotella intermedia to gingival cells. Additionally, inhibition of biofilm formation of both odonto- and periodonto-pathogenic bacteria and detachment from preformed biofilms have been described for this compound. Further purification of mushroom extract has been recently achieved and a sub-fraction (i.e. # 5) has been identified as containing the majority of the mentioned biological activities. The aim of this study was to characterise the bacterial receptors for the purified mushroom sub-fraction #5 in order to better elucidate the mode of action of this compound when interfering with bacterial adhesion to host surfaces or with bacteria-bacteria interactions in the biofilm state. Methods Candidate bacterial molecules to act as target of this compound were bacterial surface molecules involved in cell adhesion and biofilm formation, and, thus, we have considered cell wall associated proteins (CWPs), teichoic acid (TA) and lipoteichoic acid (LTA) of S. mutans, and outer membrane proteins (OMPs) and lipopolysaccharide (LPS) of P. intermedia. Results Fifteen S. mutans CWPs and TA were capable of binding sub-fraction #5, while LTA did not. As far as P. intermedia is concerned, we show that five OMPs interact with sub-fraction # 5. Capacity of binding to P. intermedia LPS was also studied but in this case negative results were obtained. Conclusions Binding sub-fraction # 5 to surface molecules of S. mutans or P. intermedia may result in inactivation of their physiological functions. As a whole, these results indicate, at molecular level, the bacterial surface alterations affecting adhesion and biofim formation. For these antimicrobial properties, the compound may find use in daily oral hygiene. PMID:24564835

  15. Large Variations in Bacterial Ribosomal RNA Genes

    PubMed Central

    Lim, Kyungtaek; Furuta, Yoshikazu; Kobayashi, Ichizo

    2012-01-01

    Ribosomal RNA (rRNA) genes, essential to all forms of life, have been viewed as highly conserved and evolutionarily stable, partly because very little is known about their natural variations. Here, we explored large-scale variations of rRNA genes through bioinformatic analyses of available complete bacterial genomic sequences with an emphasis on formation mechanisms and biological significance. Interestingly, we found bacterial genomes in which no 16S rRNA genes harbor the conserved core of the anti–Shine-Dalgarno sequence (5?-CCTCC-3?). This loss was accompanied by elimination of Shine-Dalgarno–like sequences upstream of their protein-coding genes. Those genomes belong to 1 or 2 of the following categories: primary symbionts, hemotropic Mycoplasma, and Flavobacteria. We also found many rearranged rRNA genes and reconstructed their history. Conjecturing the underlying mechanisms, such as inversion, partial duplication, transposon insertion, deletion, and substitution, we were able to infer their biological significance, such as co-orientation of rRNA transcription and chromosomal replication, lateral transfer of rRNA gene segments, and spread of rRNA genes with an apparent structural defect through gene conversion. These results open the way to understanding dynamic evolutionary changes of rRNA genes and the translational machinery. PMID:22446745

  16. Bioefficacy of larvicdial and pupicidal properties of Carica papaya (Caricaceae) leaf extract and bacterial insecticide, spinosad, against chikungunya vector, Aedes aegypti (Diptera: Culicidae).

    PubMed

    Kovendan, Kalimuthu; Murugan, Kadarkarai; Naresh Kumar, Arjunan; Vincent, Savariar; Hwang, Jiang-Shiou

    2012-02-01

    The present study was carried out to establish the properties of Carica papaya leaf extract and bacterial insecticide, spinosad on larvicidal and pupicidal activity against the chikungunya vector, Aedes aegypti. The medicinal plants were collected from the area around Bharathiar University, Coimbatore, India. C. papaya leaf was washed with tap water and shade-dried at room temperature. An electrical blender powdered the dried plant materials (leaves). The powder (500 g) of the leaf was extracted with 1.5 l of organic solvents of methanol for 8 h using a Soxhlet apparatus and then filtered. The crude leaf extracts were evaporated to dryness in a rotary vacuum evaporator. The plant extract showed larvicidal and pupicidal effects after 24 h of exposure; however, the highest larval and pupal mortality was found in the leaf extract of methanol C. papaya against the first- to fourth-instar larvae and pupae of values LC(50)?=?I instar was 51.76 ppm, II instar was 61.87 ppm, III instar was 74.07 ppm, and IV instar was 82.18 ppm, and pupae was 440.65 ppm, respectively, and bacterial insecticide, spinosad against the first to fourth instar larvae and pupae of values LC(50)?=?I instar was 51.76 ppm, II instar was 61.87 ppm, III instar was 74.07 ppm, and IV instar was 82.18 ppm, and pupae was 93.44 ppm, respectively. Moreover, combined treatment of values of LC(50)?=?I instar was 55.77 ppm, II instar was 65.77 ppm, III instar was 76.36 ppm, and IV instar was 92.78 ppm, and pupae was 107.62 ppm, respectively. No mortality was observed in the control. The results that the leaves extract of C. papaya and bacterial insecticide, Spinosad is promising as good larvicidal and pupicidal properties of against chikungunya vector, A. aegypti. This is an ideal eco-friendly approach for the control of chikungunya vector, A. aegypti as target species of vector control programs. PMID:21750871

  17. rRNA subtraction protocol Stewart, Ottesen, and DeLong (2010) rRNA subtraction protocol for metatranscriptomics

    E-print Network

    Stewart, Frank

    2010-01-01

    rRNA subtraction protocol ­ Stewart, Ottesen, and DeLong (2010) 1 rRNA subtraction protocol the availability of all downstream reagents before proceeding to the next step. #12;rRNA subtraction protocol ­ Stewart, Ottesen, and DeLong (2010) 2 rRNA subtraction A. PCR amplification of rRNA genes This step

  18. Pyrosequencing of 16S rRNA genes in fecal samples reveals high diversity of hindgut microflora in horses and potential links to chronic laminitis

    PubMed Central

    2012-01-01

    Background The nutrition and health of horses is closely tied to their gastrointestinal microflora. Gut bacteria break down plant structural carbohydrates and produce volatile fatty acids, which are a major source of energy for horses. Bacterial communities are also essential for maintaining gut homeostasis and have been hypothesized to contribute to various diseases including laminitis. We performed pyrosequencing of 16S rRNA bacterial genes isolated from fecal material to characterize hindgut bacterial communities in healthy horses and those with chronic laminitis. Results Fecal samples were collected from 10 normal horses and 8 horses with chronic laminitis. Genomic DNA was extracted and the V4-V5 segment of the 16S rRNA gene was PCR amplified and sequenced on the 454 platform generating a mean of 2,425 reads per sample after quality trimming. The bacterial communities were dominated by Firmicutes (69.21% control, 56.72% laminitis) and Verrucomicrobia (18.13% control, 27.63% laminitis), followed by Bacteroidetes, Proteobacteria, and Spirochaetes. We observed more OTUs per individual in the laminitis group than the control group (419.6 and 355.2, respectively, P = 0.019) along with a difference in the abundance of two unassigned Clostridiales genera (P = 0.03 and P = 0.01). The most abundant bacteria were Streptococcus spp., Clostridium spp., and Treponema spp.; along with unassigned genera from Subdivision 5 of Verrucomicrobia, Ruminococcaceae, and Clostridiaceae, which together constituted ~ 80% of all OTUs. There was a high level of individual variation across all taxonomic ranks. Conclusions Our exploration of the equine fecal microflora revealed higher bacterial diversity in horses with chronic laminitis and identification of two Clostridiales genera that differed in abundance from control horses. There was large individual variation in bacterial communities that was not explained in our study. The core hindgut microflora was dominated by Streptococcus spp., several cellulytic genera, and a large proportion of uncharacterized OTUs that warrant further investigation regarding their function. Our data provide a foundation for future investigations of hindgut bacterial factors that may influence the development and progression of chronic laminitis. PMID:23186268

  19. Phylogenetic analysis of rumen bacteria by comparative sequence analysis of cloned 16S rRNA genes.

    PubMed

    Whitford, M F; Forster, R J; Beard, C E; Gong, J; Teather, R M

    1998-06-01

    Comparative DNA sequence analysis of 16S rRNA genes (rDNA) was undertaken to further our understanding of the make-up of bacterial communities in the rumen fluid of dairy cattle. Total DNA was extracted from the rumen fluid of 10 cattle fed haylage/corn silage/concentrate rations at two different times. Rumen samples were collected on two separate occasions from five cows each. In experiment 1, 31 cloned rDNA sequences were analysed. In experiment 2, DNA extractions were amplified using either 12 or 30 cycles of PCR in order to examine biases introduced during the reactions. A set of 53 sequences were analysed in experiment 2 from DNA amplified using 12 cycles and 49 sequences from PCR using 30 cycles. Sequences from the 5' end of 16S rRNA gene were compared with existing sequences in the Ribosomal Database Project. Clones from experiment 1 produced a data set in which 55% of the sequences were similar to low G+C Gram-positive bacteria related to the genus Clostridia, the majority of which were closely related to bacteria in Cluster XIV. Approximately 30% of the cloned sequences were related to bacteria in the Prevotella-Bacteroides group. Clones from experiment 2 produced a data set in which the majority of sequences were related to the Prevotella-Bacteroides group, regardless of the number of cycles of PCR. The remaining sequences clustered with members of the genus Clostridia. The majority of rDNA sequences analysed in this study represent novel rumen bacteria which have not yet been isolated. PMID:16887636

  20. Taxonomy of bacterial fish pathogens

    PubMed Central

    2011-01-01

    Bacterial taxonomy has progressed from reliance on highly artificial culture-dependent techniques involving the study of phenotype (including morphological, biochemical and physiological data) to the modern applications of molecular biology, most recently 16S rRNA gene sequencing, which gives an insight into evolutionary pathways (= phylogenetics). The latter is applicable to culture-independent approaches, and has led directly to the recognition of new uncultured bacterial groups, i.e. "Candidatus", which have been associated as the cause of some fish diseases, including rainbow trout summer enteritic syndrome. One immediate benefit is that 16S rRNA gene sequencing has led to increased confidence in the accuracy of names allocated to bacterial pathogens. This is in marked contrast to the previous dominance of phenotyping, and identifications, which have been subsequently challenged in the light of 16S rRNA gene sequencing. To date, there has been some fluidity over the names of bacterial fish pathogens, with some, for example Vibrio anguillarum, being divided into two separate entities (V. anguillarum and V. ordalii). Others have been combined, for example V. carchariae, V. harveyi and V. trachuri as V. harveyi. Confusion may result with some organisms recognized by more than one name; V. anguillarum was reclassified as Beneckea and Listonella, with Vibrio and Listonella persisting in the scientific literature. Notwithstanding, modern methods have permitted real progress in the understanding of the taxonomic relationships of many bacterial fish pathogens. PMID:21314902

  1. Bacterial clearance, heterophil function, and hematological parameters of transport stressed turkey poults supplemented with dietary yeast extract

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Yeast extracts contain biological response modifiers that may be useful as alternatives to antibiotics for controlling pathogens in poultry production and mitigating the deleterious effects of production stressors. A standardized yeast extract feed supplement, Alphamune™ (YE), was added to turkey po...

  2. Medicinal plant extracts as anti-Escherichia coli O157:H7 agents and their effects on bacterial cell aggregation.

    PubMed

    Voravuthikunchai, Supayang Piyawan; Limsuwan, Surasak

    2006-10-01

    Ethanolic extracts of eight Thai medicinal plants (representing five families) that are used as traditional remedies for treating diarrhea were examined with a salt aggregation test for their ability to modulate cell surface hydrophobicity of enterohemorrhagic Escherichia coli strains, including E. coli O157:H7. Four of these medicinal plants, Acacia catechu, Peltophorum pterocarpum, Punica granatum, and Quercus infectoria, have high bacteriostatic and bactericidal activities. The ethanolic extract of Q. infectoria was the most effective against all strains of E. coli, with MICs of 0.12 to 0.98 mg/ml and MBCs of 0.98 to 3.91 mg/ml. The ethanolic extract of P. granatum had MICs of 0.49 to 1.95 mg/ml and MBCs of 1.95 to 3.91 mg/ml. Ethanolic extracts of Q. infectoria, P. pterocarpum, and P. granatum were among the most effective extracts against the two strains of E. coli O157:H7. The other four plants, Andrographis paniculata, Pluchia indica, Tamarindus indica, and Walsura robusta, did not have high bacteriostatic and bactericidal activities but were able to affect hydrophobicity characteristics on their outermost surface. All plants except Q. infectoria had some ability to increase cell surface hydrophobicity. There appears to be no correlation between antibacterial activity and cell aggregative properties. PMID:17066910

  3. Integrated DNA and RNA extraction and purification on an automated microfluidic cassette from bacterial and viral pathogens causing community-acquired lower respiratory tract infections.

    PubMed

    Van Heirstraeten, Liesbet; Spang, Peter; Schwind, Carmen; Drese, Klaus S; Ritzi-Lehnert, Marion; Nieto, Benjamin; Camps, Marta; Landgraf, Bryan; Guasch, Francesc; Corbera, Antoni Homs; Samitier, Josep; Goossens, Herman; Malhotra-Kumar, Surbhi; Roeser, Tina

    2014-05-01

    In this paper, we describe the development of an automated sample preparation procedure for etiological agents of community-acquired lower respiratory tract infections (CA-LRTI). The consecutive assay steps, including sample re-suspension, pre-treatment, lysis, nucleic acid purification, and concentration, were integrated into a microfluidic lab-on-a-chip (LOC) cassette that is operated hands-free by a demonstrator setup, providing fluidic and valve actuation. The performance of the assay was evaluated on viral and Gram-positive and Gram-negative bacterial broth cultures previously sampled using a nasopharyngeal swab. Sample preparation on the microfluidic cassette resulted in higher or similar concentrations of pure bacterial DNA or viral RNA compared to manual benchtop experiments. The miniaturization and integration of the complete sample preparation procedure, to extract purified nucleic acids from real samples of CA-LRTI pathogens to, and above, lab quality and efficiency, represent important steps towards its application in a point-of-care test (POCT) for rapid diagnosis of CA-LRTI. PMID:24615272

  4. Microbial rRNA: rDNA gene ratios may be unexpectedly low due to extracellular DNA preservation in soils

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We tested a method of estimating the activity of detectable individual bacterial and archaeal OTUs within a community by calculating ratios of absolute 16S rRNA to rDNA copy numbers. We investigated phylogenetically coherent patterns of activity among soil prokaryotes in non-growing soil communitie...

  5. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and relevance to genomovar assignment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flavobacterium columnare is the causative agent of columnaris disease which severely impacts channel catfish production in the USA and may be emerging as an important pathogen in the rainbow trout industry. The 16S rRNA gene is a housekeeping gene commonly used for bacterial taxonomy and genotyping...

  6. The intestinal bacterial community in the food waste-reducing larvae of Hermetia illucens.

    PubMed

    Jeon, Hyunbum; Park, Soyoung; Choi, Jiyoung; Jeong, Gilsang; Lee, Sang-Beom; Choi, Youngcheol; Lee, Sung-Jae

    2011-05-01

    As it is known that food waste can be reduced by the larvae of Hermetia illucens (Black soldier fly, BSF), the scientific and commercial value of BSF larvae has increased recently. We hypothesised that the ability of catabolic degradation by BSF larvae might be due to intestinal microorganisms. Herein, we analysed the bacterial communities in the gut of BSF larvae by pyrosequencing of extracting intestinal metagenomic DNA from larvae that had been fed three different diets. The 16S rRNA sequencing results produced 9737, 9723 and 5985 PCR products from larval samples fed food waste, cooked rice and calf forage, respectively. A BLAST search using the EzTaxon program showed that the bacterial community in the gut of larvae fed three different diets was mainly composed of the four phyla with dissimilar proportions. Although the composition of the bacterial communities depended on the different nutrient sources, the identified bacterial strains in the gut of BSF larvae represented unique bacterial species that were unlike the intestinal microflora of other insects. Thus, our study analysed the structure of the bacterial communities in the gut of BSF larvae after three different feedings and assessed the application of particular bacteria for the efficient degradation of organic compounds. PMID:21267722

  7. Group-specific 16S rRNA hybridization probes for determinative and community structure studies of Butyrivibrio fibrisolvens in the rumen.

    PubMed

    Forster, R J; Gong, J; Teather, R M

    1997-04-01

    Oligonucleotide probes covering three phylogenetically defined groups of Butyrivibrio spp. were successfully designed and tested. The specificity of each probe was examined by hybridization to rRNAs from an assortment of B. fibrisolvens isolates as well as additional ruminal and nonruminal bacteria. The sensitivity of the hybridization method was determined by using one of the probes (probe 156). When RNA was extracted from a culture of OB156, the probe was able to detect target cells at densities as low as 10(4) cells/ml. When 10(4) or more target cells/ml were added to cattle rumen samples, detectable hybridization signals were obtained with 1,000 ng of total RNA loaded onto the nylon membrane. In contrast, the sensitivity was reduced to 10(6) target cells/ml at 100 ng of RNA per slot. The probes were used to type 19 novel Butyrivibrio isolates. The phylogenetic placement was confirmed by partial 16S rRNA gene sequencing. The use of the probes in community-based studies indicated that the Butyrivibrio groups examined in this paper did not represent a significant portion of the bacterial 16S rRNA pool in the rumen of the cattle, sheep, and deer examined. PMID:9097421

  8. Bacterial protein signals are associated with Crohn’s disease

    PubMed Central

    Juste, Catherine; Kreil, David P; Beauvallet, Christian; Guillot, Alain; Vaca, Sebastian; Carapito, Christine; Mondot, Stanislas; Sykacek, Peter; Sokol, Harry; Blon, Florence; Lepercq, Pascale; Levenez, Florence; Valot, Benoît; Carré, Wilfrid; Loux, Valentin; Pons, Nicolas; David, Olivier; Schaeffer, Brigitte; Lepage, Patricia; Martin, Patrice; Monnet, Véronique; Seksik, Philippe; Beaugerie, Laurent; Ehrlich, S Dusko; Gibrat, Jean-François; Van Dorsselaer, Alain; Doré, Joël

    2014-01-01

    Objective No Crohn’s disease (CD) molecular maker has advanced to clinical use, and independent lines of evidence support a central role of the gut microbial community in CD. Here we explore the feasibility of extracting bacterial protein signals relevant to CD, by interrogating myriads of intestinal bacterial proteomes from a small number of patients and healthy controls. Design We first developed and validated a workflow—including extraction of microbial communities, two-dimensional difference gel electrophoresis (2D-DIGE), and LC-MS/MS—to discover protein signals from CD-associated gut microbial communities. Then we used selected reaction monitoring (SRM) to confirm a set of candidates. In parallel, we used 16S rRNA gene sequencing for an integrated analysis of gut ecosystem structure and functions. Results Our 2D-DIGE-based discovery approach revealed an imbalance of intestinal bacterial functions in CD. Many proteins, largely derived from Bacteroides species, were over-represented, while under-represented proteins were mostly from Firmicutes and some Prevotella members. Most overabundant proteins could be confirmed using SRM. They correspond to functions allowing opportunistic pathogens to colonise the mucus layers, breach the host barriers and invade the mucosae, which could still be aggravated by decreased host-derived pancreatic zymogen granule membrane protein GP2 in CD patients. Moreover, although the abundance of most protein groups reflected that of related bacterial populations, we found a specific independent regulation of bacteria-derived cell envelope proteins. Conclusions This study provides the first evidence that quantifiable bacterial protein signals are associated with CD, which can have a profound impact on future molecular diagnosis. PMID:24436141

  9. Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

    PubMed Central

    Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.; Bælum, Jacob; Ta?, Neslihan; Elberling, Bo; Jansson, Janet K.; Semenchuk, Philipp; Priemé, Anders

    2015-01-01

    The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78°N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy number of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below ?10°C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface. PMID:25983731

  10. Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses.

    PubMed

    Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S; Bælum, Jacob; Ta?, Neslihan; Elberling, Bo; Jansson, Janet K; Semenchuk, Philipp; Priemé, Anders

    2015-01-01

    The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78°N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy number of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below -10°C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface. PMID:25983731

  11. Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

    DOE PAGESBeta

    Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.; Bælum, Jacob; Ta?, Neslihan; Elberling, Bo; Jansson, Janet K.; Semenchuk, Philipp; Priemé, Anders

    2015-04-30

    The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78°N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy numbermore »of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below -10°C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface.« less

  12. Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

    SciTech Connect

    Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.; Bælum, Jacob; Ta?, Neslihan; Elberling, Bo; Jansson, Janet K.; Semenchuk, Philipp; Priemé, Anders

    2015-04-30

    The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78°N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy number of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below -10°C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface.

  13. Survival of free-living Acholeplasma in aerated pig manure slurry revealed by 13C-labeled bacterial biomass probing

    PubMed Central

    Hanajima, Dai; Aoyagi, Tomo; Hori, Tomoyuki

    2015-01-01

    Many studies have been performed on microbial community succession and/or predominant taxa during the composting process; however, the ecophysiological roles of microorganisms are not well understood because microbial community structures are highly diverse and dynamic. Bacteria are the most important contributors to the organic-waste decomposition process, while decayed bacterial cells can serve as readily digested substrates for other microbial populations. In this study, we investigated the active bacterial species responsible for the assimilation of dead bacterial cells and their components in aerated pig manure slurry by using 13C-labeled bacterial biomass probing. After 3 days of forced aeration, 13C-labeled and unlabeled dead Escherichia coli cell suspensions were added to the slurry. The suspensions contained 13C-labeled and unlabeled bacterial cell components, possibly including the cell wall and membrane, as well as intracellular materials. RNA extracted from each slurry sample 2 h after addition of E. coli suspension was density-resolved by isopycnic centrifugation and analyzed by terminal restriction fragment length polymorphism, followed by cloning and sequencing of bacterial 16S rRNA genes. In the heavy isotopically labeled RNA fraction, the predominant 13C-assimilating population was identified as belonging to the genus Acholeplasma, which was not detected in control heavy RNA. Acholeplasma spp. have limited biosynthetic capabilities and possess a wide variety of transporters, resulting in their metabolic dependence on external carbon and energy sources. The prevalence of Acholeplasma spp. was further confirmed in aerated pig manure slurry from four different pig farms by pyrosequencing of 16S rRNA genes; their relative abundance was ?4.4%. Free-living Acholeplasma spp. had a competitive advantage for utilizing dead bacterial cells and their components more rapidly relative to other microbial populations, thus allowing the survival and prevalence of Acholeplasma spp. in pig manure slurry. PMID:26583009

  14. Determining Fungi rRNA Copy Number by PCR

    PubMed Central

    Black, Jonathan; Dean, Timothy; Byfield, Grace; Foarde, Karin; Menetrez, Marc

    2013-01-01

    The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within a standard qPCR reaction. The first control developed was the internal standard control gene, benA. This gene encodes for ?-tubulin and was selected based on its single-copy nature. The second control developed was the standard control plasmid, which contained a fragment of the ribosomal RNA (rRNA) gene and produced a specific PCR product. The results confirm the multicopy nature of the rRNA region in several filamentous fungi and show that we can quantify fungi of unknown genome size over a range of spore extractions by inclusion of these two standard controls. Advances in qPCR have led to extremely sensitive and quantitative methods for single-copy genes; however, it has not been well established that the rRNA can be used to quantitate fungal contamination. We report on the use of qPCR, combined with two controls, to identify and quantify indoor fungal contaminants with a greater degree of confidence than has been achieved previously. Advances in indoor environmental health have demonstrated that contamination of the built environment by the filamentous fungi has adverse impacts on the health of building occupants. This study meets the need for more accurate and reliable methods for fungal identification and quantitation in the indoor environment. PMID:23543828

  15. Antibiotic drugs targeting bacterial RNAs

    PubMed Central

    Hong, Weiling; Zeng, Jie; Xie, Jianping

    2014-01-01

    RNAs have diverse structures that include bulges and internal loops able to form tertiary contacts or serve as ligand binding sites. The recent increase in structural and functional information related to RNAs has put them in the limelight as a drug target for small molecule therapy. In addition, the recognition of the marked difference between prokaryotic and eukaryotic rRNA has led to the development of antibiotics that specifically target bacterial rRNA, reduce protein translation and thereby inhibit bacterial growth. To facilitate the development of new antibiotics targeting RNA, we here review the literature concerning such antibiotics, mRNA, riboswitch and tRNA and the key methodologies used for their screening.

  16. Molecular analysis of bacterial population structure and dynamics during cold storage of untreated and treated milk.

    PubMed

    Rasolofo, Eric Andriamahery; St-Gelais, Daniel; LaPointe, Gisele; Roy, Denis

    2010-03-31

    Spoilage bacteria in milk are controlled by treatments such as thermization, microfiltration and addition of carbon dioxide. However, little information is known about the changes in microbial communities during subsequent cold storage of treated milk. Culture-dependent methods and a direct molecular approach combining 16S rRNA gene clone libraries and quantitative PCR (Q-PCR) were applied to obtain a better overview of the structure and the dynamics of milk microbiota. Raw milk samples were treated by the addition of carbon dioxide (CO(2)), thermization (TH) or microfiltration (MF) and stored at 4 degrees C or 8 degrees C up to 7d. Untreated milk (UT) was used as a control. Psychrotrophic and staphylococci bacteria were enumerated in the milk samples by culture methods. For the molecular approach, DNA was extracted from milk samples and 16S rRNA gene was amplified by PCR with universal primers prior to cloning. The Q-PCR method was used to evaluate the dynamics of dominant bacterial species revealed by clone library analysis of 16S rRNA gene. Comparison of the 16S rRNA gene sequence indicated that the two most abundant operational taxonomic units (OTU), determined at 97% identity, belonged to the class Gammaproteobacteria (40.3% of the 1415 sequences) and Bacilli (40%). Dominant bacterial species in UT, CO(2) and TH milk samples at day 3 were affiliated with Staphylococcus, Streptococcus, Clostridia, Aerococcus, Facklamia, Corynebacterium, Acinetobacter and Trichococcus. Dominant bacterial species detected in MF milk were Stenotrophomonas, Pseudomonas and Delftia, while Pseudomonas species dominated the bacterial population of UT, CO(2) and MF milk samples at day 7. Staphylococcus and Delftia were the dominant bacterial species in thermized milk. Q-PCR results showed that populations of S. aureus, A. viridans, A. calcoaceticus, C. variabile and S. uberis were stable during 7d of storage at 4 degrees C. Populations of P. fluorescens, S. uberis and total bacteria increased in UT and CO(2) milk samples during 7d of storage at 8 degrees C and were noticeable from day 3. This study shows new microbial species which can develop during cold storage after milk treatment and contributes to identifying causes of reduced shelf life and deterioration of technological properties of milk during storage. PMID:20137820

  17. Assessing the viability of bacterial species in drinking water by combined cellular and molecular analyses.

    PubMed

    Kahlisch, Leila; Henne, Karsten; Gröbe, Lothar; Brettar, Ingrid; Höfle, Manfred G

    2012-02-01

    The question which bacterial species are present in water and if they are viable is essential for drinking water safety but also of general relevance in aquatic ecology. To approach this question we combined propidium iodide/SYTO9 staining ("live/dead staining" indicating membrane integrity), fluorescence-activated cell sorting (FACS) and community fingerprinting for the analysis of a set of tap water samples. Live/dead staining revealed that about half of the bacteria in the tap water had intact membranes. Molecular analysis using 16S rRNA and 16S rRNA gene-based single-strand conformation polymorphism (SSCP) fingerprints and sequencing of drinking water bacteria before and after FACS sorting revealed: (1) the DNA- and RNA-based overall community structure differed substantially, (2) the community retrieved from RNA and DNA reflected different bacterial species, classified as 53 phylotypes (with only two common phylotypes), (3) the percentage of phylotypes with intact membranes or damaged cells were comparable for RNA- and DNA-based analyses, and (4) the retrieved species were primarily of aquatic origin. The pronounced difference between phylotypes obtained from DNA extracts (dominated by Betaproteobacteria, Bacteroidetes, and Actinobacteria) and from RNA extracts (dominated by Alpha-, Beta-, Gammaproteobacteria, Bacteroidetes, and Cyanobacteria) demonstrate the relevance of concomitant RNA and DNA analyses for drinking water studies. Unexpected was that a comparable fraction (about 21%) of phylotypes with membrane-injured cells was observed for DNA- and RNA-based analyses, contradicting the current understanding that RNA-based analyses represent the actively growing fraction of the bacterial community. Overall, we think that this combined approach provides an interesting tool for a concomitant phylogenetic and viability analysis of bacterial species of drinking water. PMID:21845446

  18. The Majority of In Vitro Macrophage Activation Exhibited by Extracts of Some Immune Enhancing Botanicals is Due to Bacterial Lipoproteins and Lipopolysaccharides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have identified potent monocyte/macrophage activating bacterial lipoproteins within commonly used immune enhancing botanicals such as Echinacea, American ginseng and alfalfa sprouts. These bacterial lipoproteins, along with lipopolysaccharides, were substantially more potent than other bacteriall...

  19. Bacterial communities in the phylloplane of Prunus species.

    PubMed

    Jo, Yeonhwa; Cho, Jin Kyong; Choi, Hoseong; Chu, Hyosub; Lian, Sen; Cho, Won Kyong

    2015-04-01

    Bacterial populations in the phylloplane of four different Prunus species were investigated by 16 S rRNA pyrosequencing. Bioinformatic analysis identified an average of 510 operational taxonomic units belonging to 159 genera in 76 families. The two genera, Sphingomonas and Methylobacterium, were dominant in the phylloplane of four Prunus species. Twenty three genera were commonly identified in the four Prunus species, indicating a high level of bacterial diversity dependent on the plant species. Our study based on 16 S rRNA sequencing reveals the complexity of bacterial diversity in the phylloplane of Prunus species in detail. PMID:25515303

  20. Direct 5S rRNA assay for monitoring mixed-culture bioprocesses

    SciTech Connect

    Stoner, D.L.; Bulmer, D.K.; Ward, T.E.

    1996-06-01

    This study demonstrates the efficacy of a direct 5S rRNA assay for the characterization of mixed microbial populations by using as an example the bacteria associated with acidic mining environments. The direct 5S rRNA assay described herein represents a nonselective, direct molecular method for monitoring and characterizing the predominant, metabolically active members of a microbial population. The foundation of the assay is high-resolution denaturing gradient gel electrophoresis in denaturing gradient gel electrophoresis (DGGE), which is used to separate 5S rRNA species during electrophoresis in denaturing gradient gels. With mixtures of RNA extracted from laboratory cultures, the upper practical limit for detection in the current experimental system has been estimated to be greater than 15 different species. With this method, the resolution was demonstrated to be effective at least to the species level. The strength of this approach was demonstrated by the ability to discriminate between Thiobacillus ferrooxidans ATCC 19859 and Thiobacillus thiooxidans ATCC 8085, two very closely related species. Migration patterns for the 5S rRNA from members of the genus Thiobacillus were readily distinguishable from those of the general Acidiphilium and Leptospirillum. In conclusion, the 5S rRNA assay represents a powerful method by which the structure of a microbial population within acidic environments can be assessed. 40 refs., 12 figs., 1 tab.

  1. Molecular analysis of the bacterial drinking water community with respect to live/dead status.

    PubMed

    Kahlisch, L; Henne, K; Groebe, L; Draheim, J; Höfle, M G; Brettar, I

    2010-01-01

    The assessment of the physiological state of the bacteria in drinking water is a critical issue, especially with respect to the presence of pathogenic bacteria. Though molecular methods can provide insight into the taxonomic composition of the drinking water microflora, the question if a bacterial species is alive or dead still needs to be addressed. To distinguish live and dead bacteria at the taxonomic level, we combined three methods: i) a staining procedure indicating membrane-injured cells (using SYTO9 and Propidium Iodide) that is considered to distinguish between live and dead cells, ii) Fluorescence Activated Cell Sorting (FACS) of the membrane injured and intact bacteria, and iii) molecular analyses of the RNA extracted from the bacteria before and after sorting to analyse the bacterial community at the species level. By staining and FACS analysis the drinking water bacteria could be separated according to their different membrane integrities, and RNA could be extracted from the live and dead sorted bacterial fractions. 16S rRNA based fingerprints revealed a diverse bacterial community in the drinking water samples with the majority being represented by 31 identified phylotypes. Most of the phylotypes referenced belonged to the phyla Proteobacteria (Alpha-, Beta-, Gamma-), Cyanobacteria and Bacteroidetes, and were mostly related to freshwater bacteria. 90% of the total phylotypes could be recovered after FAC-Sorting; 32% of the phylotypes occurred only in the "live" sorted fraction, 21% only in the "dead" sorted fraction, and 46% occurred in both fractions. PMID:20057086

  2. The murine lung microbiome in relation to the intestinal and vaginal bacterial communities

    PubMed Central

    2013-01-01

    Background This work provides the first description of the bacterial population of the lung microbiota in mice. The aim of this study was to examine the lung microbiome in mice, the most used animal model for inflammatory lung diseases such as COPD, cystic fibrosis and asthma. Bacterial communities from broncho-alveolar lavage fluids and lung tissue were compared to samples taken from fecal matter (caecum) and vaginal lavage fluid from female BALB/cJ mice. Results Using a customized 16S rRNA sequencing protocol amplifying the V3-V4 region our study shows that the mice have a lung microbiome that cluster separately from mouse intestinal microbiome (caecum). The mouse lung microbiome is dominated by Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Cyanobacteria overlapping the vaginal microbiome. We also show that removal of host tissue or cells from lung fluid during the DNA extraction step has an impact on the resulting bacterial community profile. Sample preparation needs to be considered when choosing an extraction method and interpreting data. Conclusions We have consistently amplified bacterial DNA from mouse lungs that is distinct from the intestinal microbiome in these mice. The gut microbiome has been extensively studied for its links to development of disease. Here we suggest that also the lung microbiome could be important in relation to inflammatory lung diseases. Further research is needed to understand the contribution of the lung microbiome and the gut-lung axis to the development of lung diseases such as COPD and asthma. PMID:24373613

  3. Mulberry leaf extract mediated synthesis of gold nanoparticles and its anti-bacterial activity against human pathogens

    NASA Astrophysics Data System (ADS)

    Adavallan, K.; Krishnakumar, N.

    2014-06-01

    Gold nanoparticles (Au-NPs) were synthesized at room temperature using Morus alba (mulberry) leaf extract as reducing and stabilizing agent. The development of plant mediated synthesis of nanoparticles is gaining importance due to its simplicity, low cost, non-toxicity, eco-friendliness, long term stability and reproducible aqueous synthesis method to obtain a self-assembly of nearly monodispersed Au-NPs. The formation and morphology of biosynthesized nanoparticles are investigated with the help of UV-Vis spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), atomic force microscopy (AFM), x-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FT-IR) techniques. Au-NPs formation was screened by UV-Vis spectroscopy through color conversion due to surface plasmon resonance band at 538 nm for Au-NPs. DLS studies revealed that the average size of Au-NPs was 50 nm. TEM studies showed the particles to be nearly spherical with few irregular shapes and particle size ranges 15-53 nm. The AFM image clearly shows the surface morphology of the well-dispersed Au-NPs with less than 50 nm. The high crystallinity of nanoparticles is evident from bright circular spots in the selected area electron diffraction (SAED) pattern. X-ray diffraction pattern showed high purity and face-centered cubic structure of Au-NPs. The FT-IR results indicate the presence of different functional groups present in the biomolecule capping the nanoparticles. Further, biosynthesized Au-NPs show strong zone of inhibition against Vibrio cholera (gram-negative) and Staphylococcus aureus (gram-positive) whereas, chemically synthesized Au-NPs and mulberry leaf extract exhibit a fair zone of inhibition.

  4. Fecal Bacterial Diversity in a Wild Gorilla†

    PubMed Central

    Frey, Julie C.; Rothman, Jessica M.; Pell, Alice N.; Nizeyi, John Bosco; Cranfield, Michael R.; Angert, Esther R.

    2006-01-01

    We describe the bacterial diversity in fecal samples of a wild gorilla by use of a 16S rRNA gene clone library and terminal-restriction fragment length polymorphism (T-RFLP). Clones were classified as Firmicutes, Verrucomicrobia, Actinobacteria, Lentisphaerae, Bacteroidetes, Spirochetes, and Planctomycetes. Our data suggest that fecal populations did not change temporally, as determined by T-RFLP. PMID:16672537

  5. Variable rRNA gene copies in extreme halobacteria

    SciTech Connect

    Sanz, J.L.; Marin, I.; Ramirez, L.; Amils, R. ); Abad, J.P.; Smith, C.L. )

    1988-08-25

    Using PFG electrophoresis techniques, the authors have examined the organization of rRNA gene in halobacterium species. The results show that the organization of rRNA genes among closely related halobacteria is quite heterogeneous. This contrasts with the high degree of conservation of rRNA sequence. The possible mechanism of such rRNA gene amplification and its evolutionary implications are discussed.

  6. Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis.

    PubMed

    Bahl, Martin Iain; Bergström, Anders; Licht, Tine Rask

    2012-04-01

    Freezing stool samples prior to DNA extraction and downstream analysis is widely used in metagenomic studies of the human microbiota but may affect the inferred community composition. In this study, DNA was extracted either directly or following freeze storage of three homogenized human fecal samples using three different extraction methods. No consistent differences were observed in DNA yields between extractions on fresh and frozen samples; however, differences were observed between extraction methods. Quantitative PCR analysis was subsequently performed on all DNA samples using six different primer pairs targeting 16S rRNA genes of significant bacterial groups, and the community composition was evaluated by comparing specific ratios of the calculated abundances. In seven of nine cases, the Firmicutes to Bacteroidetes 16S rRNA gene ratio was significantly higher in fecal samples that had been frozen compared to identical samples that had not. This effect was further supported by qPCR analysis of bacterial groups within these two phyla. The results demonstrate that storage conditions of fecal samples may adversely affect the determined Firmicutes to Bacteroidetes ratio, which is a frequently used biomarker in gut microbiology. PMID:22325006

  7. Pilot-scale chitin extraction from shrimp shell waste by deproteination and decalcification with bacterial enrichment cultures.

    PubMed

    Bajaj, Mini; Freiberg, Andrea; Winter, Josef; Xu, Youmei; Gallert, Claudia

    2015-11-01

    Extraction of chitin from mechanically pre-purified shrimp shells can be achieved by successive NaOH/HCl treatment, protease/HCl treatment or by environmentally friendly fermentation with proteolytic/lactic acid bacteria (LAB). For the last mentioned alternative, scale-up of shrimp shell chitin purification was investigated in 0.25 L (F1), 10 L (F2), and 300 L (F3) fermenters using an anaerobic, chitinase-deficient, proteolytic enrichment culture from ground meat for deproteination and a mixed culture of LAB from bio-yoghurt for decalcification. Protein removal in F1, F2, and F3 proceeded in parallel within 40 h at an efficiency of 89-91 %. Between 85 and 90 % of the calcit was removed from the shells by LAB in another 40 h in F1, F2, and F3. After deproteination of shrimp shells in F3, spent fermentation liquor was re-used for a next batch of 30-kg shrimp shells in F4 (300 L) which eliminated 85.5 % protein. The purity of the resulting chitin was comparable in F1, F2, F3, and F4. Viscosities of chitosan, obtained after chitin deacetylation and of chitin, prepared biologically or chemically in the laboratory, were much higher than those of commercially available chitin and chitosan. PMID:26227412

  8. Multiple self-splicing introns in the 16S rRNA genes of giant sulfur bacteria.

    PubMed

    Salman, Verena; Amann, Rudolf; Shub, David A; Schulz-Vogt, Heide N

    2012-03-13

    The gene encoding the small subunit rRNA serves as a prominent tool for the phylogenetic analysis and classification of Bacteria and Archaea owing to its high degree of conservation and its fundamental function in living organisms. Here we show that the 16S rRNA genes of not-yet-cultivated large sulfur bacteria, among them the largest known bacterium Thiomargarita namibiensis, regularly contain numerous self-splicing introns of variable length. The 16S rRNA genes can thus be enlarged to up to 3.5 kb. Remarkably, introns have never been identified in bacterial 16S rRNA genes before, although they are the most frequently sequenced genes today. This may be caused in part by a bias during the PCR amplification step that discriminates against longer homologs, as we show experimentally. Such length heterogeneity of 16S rRNA genes has so far never been considered when constructing 16S rRNA-based clone libraries, even though an elongation of rRNA genes due to intervening sequences has been reported previously. The detection of elongated 16S rRNA genes has profound implications for common methods in molecular ecology and may cause systematic biases in several techniques. In this study, catalyzed reporter deposition-fluorescence in situ hybridization on both ribosomes and rRNA precursor molecules as well as in vitro splicing experiments were performed and confirmed self-splicing of the introns. Accordingly, the introns do not inhibit the formation of functional ribosomes. PMID:22371583

  9. Bacteriocin from Bacillus subtilis as a novel drug against diabetic foot ulcer bacterial pathogens

    PubMed Central

    Joseph, Baby; Dhas, Berlina; Hena, Vimalin; Raj, Justin

    2013-01-01

    Objective To isolate and identify Bacillus subtilis (B. subtilis) from soil and to characterize and partially purify the bacteriocin. To evaluate the antimicrobial activity against four diabetic foot ulcer bacterial pathogens. Methods Genotypic identification was done based on Bergey's manual of systemic bacteriology. Antimicrobial susceptibility test was done by Kirby-Bauer disc diffusion method. Colonies were identified by colony morphology and biochemical characterization and also compared with MTCC 121 strain. Further identification was done by 16S rRNA sequencing. Inhibitory activities of partially purified bacteriocin on all the DFU isolates were done by agar well diffusion method. The strain was identified to produce bacteriocin by stab overlay assay. Bacteriocin was extracted by organic solvent extraction using chloroform, further purified by HPLC and physical, and chemical characterization was performed. Results The four isolates showed high level of resistance to amoxyclav and sensitivity to ciprofloxacin. HPLC purification revealed that the extracts are bacteriocin. The phylogenetic tree analysis results showed that the isolate was 99% related to B. subtilis BSF01. The results reveled activity to all the four isolates and high level of activity was seen in case of Klebsiella sp. Conclusions Partially purified bacteriocin was found to have antimicrobial activity against the four diabetic foot ulcer bacterial pathogens, which can thus be applied as a better drug molecule on further studies. The strain B. subtilis are found to be safe for use and these antimicrobial peptides can be used as an antimicrobial in humans to treat DFU bacterial pathogens. PMID:24093784

  10. Bacterial populations colonizing and degrading rice straw in anoxic paddy soil.

    PubMed

    Weber, S; Stubner, S; Conrad, R

    2001-03-01

    Rice straw is a major substrate for the production of methane, a greenhouse gas, in flooded rice fields. The bacterial community degrading rice straw under anoxic conditions was investigated with molecular methods. Rice straw was incubated in paddy soil anaerobically for 71 days. Denaturing gradient gel electrophoresis (DGGE) of the amplified bacterial 16S rRNA genes showed that the composition of the bacterial community changed during the first 15 days but then was stable until the end of incubation. Fifteen DGGE bands with different signal intensities were excised, cloned, and sequenced. In addition, DNA was extracted from straw incubated for 1 and 29 days and the bacterial 16S rRNA genes were amplified and cloned. From these clone libraries 16 clones with different electrophoretic mobilities on a DGGE gel were sequenced. From a total of 31 clones, 20 belonged to different phylogenetic clusters of the clostridia, i.e., clostridial clusters I (14 clones), III (1 clone), IV (1 clone), and XIVa (4 clones). One clone fell also within the clostridia but could not be affiliated to one of the clostridial clusters. Ten clones grouped closely with the genera Bacillus (3 clones), Nitrosospira (1 clone), Fluoribacter (1 clones), and Acidobacterium (2 clones) and with clone sequences previously obtained from rice field soil (3 clones). The relative abundances of various phylogenetic groups in the rice straw-colonizing community were determined by fluorescence in situ hybridization (FISH). Bacteria were detached from the incubated rice straw with an efficiency of about 80 to 90%, as determined by dot blot hybridization of 16S rRNA in extract and residue. The number of active (i.e., a sufficient number of ribosomes) Bacteria detected with a general eubacterial probe (Eub338) after 8 days of incubation was 61% of the total cell counts. This percentage decreased to 17% after 29 days of incubation. Most (55%) of the active cells on day 8 belonged to the genus Clostridium, mainly to clostridial clusters I (24%), III (6%), and XIVa (24%). An additional 5% belonged to the Cytophaga-Flavobacterium cluster of the Cytophaga-Flavobacterium-Bacteroides phylum, 4% belonged to the alpha, beta, and gamma Proteobacteria, and 1.3% belonged to the Bacillus subbranch of the gram-positive bacteria with a low G+C content. The results show that the bacterial community colonizing and decomposing rice straw developed during the first 15 days of incubation and was dominated by members of different clostridial clusters, especially clusters I, III, and XIVa. PMID:11229927

  11. Bacterial gastroenteritis

    MedlinePLUS

    Infectious diarrhea - bacterial gastroenteritis; Acute gastroenteritis; Gastroenteritis - bacterial ... the sickness. All types of food poisoning cause diarrhea . Other symptoms include: Abdominal cramps Abdominal pain Bloody ...

  12. Patterns of bacterial diversity across a range of Antarctic terrestrial habitats.

    PubMed

    Yergeau, Etienne; Newsham, Kevin K; Pearce, David A; Kowalchuk, George A

    2007-11-01

    Although soil-borne bacteria represent the world's greatest source of biological diversity, it is not well understood whether extreme environmental conditions, such as those found in Antarctic habitats, result in reduced soil-borne microbial diversity. To address this issue, patterns of bacterial diversity were studied in soils sampled along a > 3200 km southern polar transect spanning a gradient of increased climate severity over 27 degrees of latitude. Vegetated and fell-field plots were sampled at the Falkland (51 degrees S), South Georgia (54 degrees S), Signy (60 degrees S) and Anchorage Islands (67 degrees S), while bare frost-sorted soil polygons were examined at Fossil Bluff (71 degrees S), Mars Oasis (72 degrees S), Coal Nunatak (72 degrees S) and the Ellsworth Mountains (78 degrees S). Bacterial 16S rRNA gene sequences were recovered subsequent to direct DNA extraction from soil, polymerase chain reaction amplification and cloning. Although bacterial diversity was observed to decline with increased latitude, habitat-specific patterns appeared to also be important. Namely, a negative relationship was found between bacterial diversity and latitude for fell-field soils, but no such pattern was observed for vegetated sites. The Mars Oasis site, previously identified as a biodiversity hotspot within this region, proved exceptional within the study transect, with unusually high bacterial diversity. In independent analyses, geographical distance and vegetation cover were found to significantly influence bacterial community composition. These results provide insight into the factors shaping the composition of bacterial communities in Antarctic terrestrial habitats and support the notion that bacterial diversity declines with increased climatic severity. PMID:17922752

  13. Automated Identification of Medically Important Bacteria by 16S rRNA Gene Sequencing Using a Novel Comprehensive Database, 16SpathDB?

    PubMed Central

    Woo, Patrick C. Y.; Teng, Jade L. L.; Yeung, Juilian M. Y.; Tse, Herman; Lau, Susanna K. P.; Yuen, Kwok-Yung

    2011-01-01

    Despite the increasing use of 16S rRNA gene sequencing, interpretation of 16S rRNA gene sequence results is one of the most difficult problems faced by clinical microbiologists and technicians. To overcome the problems we encountered in the existing databases during 16S rRNA gene sequence interpretation, we built a comprehensive database, 16SpathDB (http://147.8.74.24/16SpathDB) based on the 16S rRNA gene sequences of all medically important bacteria listed in the Manual of Clinical Microbiology and evaluated its use for automated identification of these bacteria. Among 91 nonduplicated bacterial isolates collected in our clinical microbiology laboratory, 71 (78%) were reported by 16SpathDB as a single bacterial species having >98.0% nucleotide identity with the query sequence, 19 (20.9%) were reported as more than one bacterial species having >98.0% nucleotide identity with the query sequence, and 1 (1.1%) was reported as no match. For the 71 bacterial isolates reported as a single bacterial species, all results were identical to their true identities as determined by a polyphasic approach. For the 19 bacterial isolates reported as more than one bacterial species, all results contained their true identities as determined by a polyphasic approach and all of them had their true identities as the “best match in 16SpathDB.” For the isolate (Gordonibacter pamelaeae) reported as no match, the bacterium has never been reported to be associated with human disease and was not included in the Manual of Clinical Microbiology. 16SpathDB is an automated, user-friendly, efficient, accurate, and regularly updated database for 16S rRNA gene sequence interpretation in clinical microbiology laboratories. PMID:21389154

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

    SciTech Connect

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

    2010-06-01

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

  15. Effects on enteric methane production and bacterial and archaeal communities by the addition of cashew nut shell extract or glycerol-an in vitro evaluation.

    PubMed

    Danielsson, Rebecca; Werner-Omazic, Anna; Ramin, Mohammad; Schnürer, Anna; Griinari, Mikko; Dicksved, Johan; Bertilsson, Jan

    2014-09-01

    The objective of the study was to evaluate the effect of cashew nut shell extract (CNSE) and glycerol (purity >99%) on enteric methane (CH4) production and microbial communities in an automated gas in vitro system. Microbial communities from the in vitro system were compared with samples from the donor cows, in vivo. Inoculated rumen fluid was mixed with a diet with a 60:40 forage:concentrate ratio and, in total, 5 different treatments were set up: 5mg of CNSE (CNSE-L), 10mg of CNSE (CNSE-H), 15mmol of glycerol/L (glycerol-L), and 30mmol of glycerol/L (glycerol-H), and a control without feed additive. Gas samples were taken at 2, 4, 8, 24, 32, and 48h of incubation, and the CH4 concentration was measured. Samples of rumen fluid were taken for volatile fatty acid analysis and for microbial sequence analyses after 8, 24, and 48h of incubation. In vivo rumen samples from the cows were taken 2h after the morning feeding at 3 consecutive days to compare the in vitro system with in vivo conditions. The gas data and data from microbial sequence analysis (454 sequencing) were analyzed using a mixed model and principal components analysis. These analyses illustrated that CH4 production was reduced with the CNSE treatment, by 8 and 18%, respectively, for the L and H concentration. Glycerol instead increased CH4 production by 8 and 12%, respectively, for the L and H concentration. The inhibition with CNSE could be due to the observed shift in bacterial population, possibly resulting in decreased production of hydrogen or formate, the methanogenic substrates. Alternatively the response could be explained by a shift in the methanogenic community. In the glycerol treatments, no main differences in bacterial or archaeal population were detected compared with the in vivo control. Thus, the increase in CH4 production may be explained by the increase in substrate in the in vitro system. The reduced CH4 production in vitro with CNSE suggests that CNSE can be a promising inhibitor of CH4 formation in the rumen of dairy cows. PMID:24996274

  16. Profiling of ornithine lipids in bacterial extracts of Rhodobacter sphaeroides by reversed-phase liquid chromatography with electrospray ionization and multistage mass spectrometry (RPLC-ESI-MS(n)).

    PubMed

    Granafei, Sara; Losito, Ilario; Trotta, Massimo; Italiano, Francesca; de Leo, Vincenzo; Agostiano, Angela; Palmisano, Francesco; Cataldi, Tommaso R I

    2016-01-15

    Ornithine lipids (OLs), a sub-group of the large (and of emerging interest) family of lipoamino acids of bacterial origin, contain a 3-hydroxy fatty acyl chain linked via an amide bond to the ?-amino group of ornithine and via an ester bond to a second fatty acyl chain. OLs in extracts of Rhodobacter sphaeroides (R. sphaeroides) were investigated by high-performance reversed phase liquid chromatography (RPLC) with electrospray ionization mass spectrometry (ESI-MS) in negative ion mode using a linear ion trap (LIT). The presence of OLs bearing both saturated (i.e, 16:0, 17:0, 18:0, 19:0 and 20:0) and unsaturated chains (i.e., 18:1, 19:1, 19:2 and 20:1) was ascertained and their identification, even for isomeric, low abundance and partially co-eluting species, was achieved by low-energy collision induced dissociation (CID) multistage mass spectrometry (MS(n), n = 2-4). OLs signatures found in two R. sphaeroides strains, i.e., wild type 2.4.1 and mutant R26, were examined and up to 16 and 17 different OL species were successfully identified, respectively. OLs in both bacterial strains were characterized by several combinations of fatty chains on ester-linked and amide-linked 3-OH fatty acids. Multistage MS spectra of monoenoic amide-linked 3-OH acyl chains, allowed the identification of positional isomer of OL containing 18:1 (i.e. 9-octadecenoic) and 20:1 (i.e. 11-eicosenoic) fatty acids. The most abundant OL ([M-H](-) at m/z 717.5) in R. sphaeroides R26 was identified as OL 3-OH 20:1/19:1 (i.e., 3-OH-eicosenoic acid amide-linked to ornithine and esterified to a nonadecenoic chain containing a cyclopropane ring). An unusual OL (m/z 689.5 for the [M-H](-) ion), most likely containing a cyclopropene ester-linked acyl chain (i.e., OL 3-OH 18:0/19:2), was retrieved only in the carotenoidless mutant strain R26. Based on the biosynthetic pathways already known for cyclopropa(e)ne ring-including acyl chains, a plausible explanation was invoked for the enzymatic generation of this ester-linked chain in R. sphaeroides. PMID:26709304

  17. STUDIES ON BACTERIAL NUTRITION

    PubMed Central

    Thjötta, Theodor

    1921-01-01

    From the data presented in the foregoing experiments it is evident that Bacillus influenzæ will grow in a fluid medium consisting of plain broth to which have been added small amounts of emulsions or extracts of mucoid bacilli or of Bacillus proteus. The bacterial extracts may be made by simple boiling of the bacillary emulsions in broth or saline solution and centrifuging out the bacterial bodies; they may be filtered without losing their growth-inducing property. Cultures of Bacillus influenzæ in bacterial extract broth, if not too small doses of the extracts were employed, always showed heavier growth than the control cultures in blood broth, and growth occurred at a considerably earlier period than in blood broth. In many instances growth could be seen after 3 to 4 hours, and a bacterial whirl was always visible after 6 hours incubation. When the nature of the culture used for seeding is not stated, this was 0.1 cc. of the supernatant fluid of a blood broth culture. All cultures were made in fluid medium; solid medium is much more difficult to use in connection with the extracts. In explanation of the remarkable growth of Bacillus influenzæ in this blood-free medium, the idea is proposed that the growth-stimulating effect of the bacterial extracts is due possibly to substances of the same nature as the so called vitamines. Further investigations on this principle of bacterial nutrition will appear in subsequent papers, together with a more thorough study of the sources and character of the growth-inducing substances. PMID:19868534

  18. Nested PCR Biases in Interpreting Microbial Community Structure in 16S rRNA Gene Sequence Datasets

    PubMed Central

    Yu, Guoqin; Fadrosh, Doug; Goedert, James J.; Ravel, Jacques; Goldstein, Alisa M.

    2015-01-01

    Background Sequencing of the PCR-amplified 16S rRNA gene has become a common approach to microbial community investigations in the fields of human health and environmental sciences. This approach, however, is difficult when the amount of DNA is too low to be amplified by standard PCR. Nested PCR can be employed as it can amplify samples with DNA concentration several-fold lower than standard PCR. However, potential biases with nested PCRs that could affect measurement of community structure have received little attention. Results In this study, we used 17 DNAs extracted from vaginal swabs and 12 DNAs extracted from stool samples to study the influence of nested PCR amplification of the 16S rRNA gene on the estimation of microbial community structure using Illumina MiSeq sequencing. Nested and standard PCR methods were compared on alpha- and beta-diversity metrics and relative abundances of bacterial genera. The effects of number of cycles in the first round of PCR (10 vs. 20) and microbial diversity (relatively low in vagina vs. high in stool) were also investigated. Vaginal swab samples showed no significant difference in alpha diversity or community structure between nested PCR and standard PCR (one round of 40 cycles). Stool samples showed significant differences in alpha diversity (except Shannon’s index) and relative abundance of 13 genera between nested PCR with 20 cycles in the first round and standard PCR (P<0.01), but not between nested PCR with 10 cycles in the first round and standard PCR. Operational taxonomic units (OTUs) that had low relative abundance (sum of relative abundance <0.167) accounted for most of the distortion (>27% of total OTUs in stool). Conclusions Nested PCR introduced bias in estimated diversity and community structure. The bias was more significant for communities with relatively higher diversity and when more cycles were applied in the first round of PCR. We conclude that nested PCR could be used when standard PCR does not work. However, rare taxa detected by nested PCR should be validated by other technologies. PMID:26196512

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

    PubMed

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

    2015-02-25

    The human oral microbiome is known to play a significant role in human health and disease. While less well studied, the feline oral microbiome is thought to play a similarly important role. To determine roles oral bacteria play in health and disease, one first has to be able to accurately identify bacterial species present. 16S rRNA gene sequence information is widely used for molecular identification of bacteria and is also useful for establishing the taxonomy of novel species. The objective of this research was to obtain full 16S rRNA gene reference sequences for feline oral bacteria, place the sequences in species-level phylotypes, and create a curated 16S rRNA based taxonomy for common feline oral bacteria. Clone libraries were produced using "universal" and phylum-selective PCR primers and DNA from pooled subgingival plaque from healthy and periodontally diseased cats. Bacteria in subgingival samples were also cultivated to obtain isolates. Full-length 16S rDNA sequences were determined for clones and isolates that represent 171 feline oral taxa. A provisional curated taxonomy was developed based on the position of each taxon in 16S rRNA phylogenetic trees. The feline oral microbiome curated taxonomy and 16S rRNA gene reference set will allow investigators to refer to precisely defined bacterial taxa. A provisional name such as "Propionibacterium sp. feline oral taxon FOT-327" is an anchor to which clone, strain or GenBank names or accession numbers can point. Future next-generation-sequencing studies of feline oral bacteria will be able to map reads to taxonomically curated full-length 16S rRNA gene sequences. PMID:25523504

  20. Phylogenetic analysis of a biofilm bacterial population in a water pipeline in the Gulf of Mexico.

    PubMed

    López, Miguel A; Zavala-Díaz de la Serna, F Javier; Jan-Roblero, Janet; Romero, Juan M; Hernández-Rodríguez, César

    2006-10-01

    The aim of this study was to assess the bacterial diversity associated with a corrosive biofilm in a steel pipeline from the Gulf of Mexico used to inject marine water into the oil reservoir. Several aerobic and heterotrophic bacteria were isolated and identified by 16S rRNA gene sequence analysis. Metagenomic DNA was also extracted to perform a denaturing gradient gel electrophoresis analysis of ribosomal genes and to construct a 16S rRNA gene metagenomic library. Denaturing gradient gel electrophoresis profiles and ribosomal libraries exhibited a limited bacterial diversity. Most of the species detected in the ribosomal library or isolated from the pipeline were assigned to Proteobacteria (Halomonas spp., Idiomarina spp., Marinobacter aquaeolei, Thalassospira sp., Silicibacter sp. and Chromohalobacter sp.) and Bacilli (Bacillus spp. and Exiguobacterium spp.). This is the first report that associates some of these bacteria with a corrosive biofilm. It is relevant that no sulfate-reducing bacteria were isolated or detected by a PCR-based method. The diversity and relative abundance of bacteria from water pipeline biofilms may contribute to an understanding of the complexity and mechanisms of metal corrosion during marine water injection in oil secondary recovery. PMID:16958915

  1. Common 5S rRNA variants are likely to be accepted in many sequence contexts

    NASA Technical Reports Server (NTRS)

    Zhang, Zhengdong; D'Souza, Lisa M.; Lee, Youn-Hyung; Fox, George E.

    2003-01-01

    Over evolutionary time RNA sequences which are successfully fixed in a population are selected from among those that satisfy the structural and chemical requirements imposed by the function of the RNA. These sequences together comprise the structure space of the RNA. In principle, a comprehensive understanding of RNA structure and function would make it possible to enumerate which specific RNA sequences belong to a particular structure space and which do not. We are using bacterial 5S rRNA as a model system to attempt to identify principles that can be used to predict which sequences do or do not belong to the 5S rRNA structure space. One promising idea is the very intuitive notion that frequently seen sequence changes in an aligned data set of naturally occurring 5S rRNAs would be widely accepted in many other 5S rRNA sequence contexts. To test this hypothesis, we first developed well-defined operational definitions for a Vibrio region of the 5S rRNA structure space and what is meant by a highly variable position. Fourteen sequence variants (10 point changes and 4 base-pair changes) were identified in this way, which, by the hypothesis, would be expected to incorporate successfully in any of the known sequences in the Vibrio region. All 14 of these changes were constructed and separately introduced into the Vibrio proteolyticus 5S rRNA sequence where they are not normally found. Each variant was evaluated for its ability to function as a valid 5S rRNA in an E. coli cellular context. It was found that 93% (13/14) of the variants tested are likely valid 5S rRNAs in this context. In addition, seven variants were constructed that, although present in the Vibrio region, did not meet the stringent criteria for a highly variable position. In this case, 86% (6/7) are likely valid. As a control we also examined seven variants that are seldom or never seen in the Vibrio region of 5S rRNA sequence space. In this case only two of seven were found to be potentially valid. The results demonstrate that changes that occur multiple times in a local region of RNA sequence space in fact usually will be accepted in any sequence context in that same local region.

  2. Genotoxicity evaluation of Guibi-Tang extract using an in vitro bacterial reverse mutation assay, chromosome aberration assay, and in vivo micronucleus test

    PubMed Central

    2014-01-01

    Background Guibi-Tang is a traditional herbal prescription made from 12 different herbs that is used in the treatment of amnesia and poor memory. Methods In the present study, we evaluated the acute oral toxicity and genotoxic potential of Guibi-Tang water extract (GBT) at doses up to 2000 ?g/plate an using a bacterial reverse mutation test (Ames test) with Salmonella typhimurium strains TA100, TA1535, TA98, and TA1537, and Escherichia coli strain WP2uvrA. Acute toxicity and genotoxic potential were measured in the presence and absence of an exogenous source of metabolic activation, in an in vitro chromosome aberration assay with Chinese hamster lung (CHL) cells, and in an in vivo micronucleus test using ICR mice bone marrow as recommended by the Korean Food and Drug Administration. An acute oral toxicity test of GBT was performed in Sprague Dawley rats. The Ames test showed that GBT did not induce gene mutations in S. typhimurium or in E. coli in the presence or absence of S9 activation. Results GBT did not significantly increase the number of structural aberrations in CHL cells with or without S9 activation. The oral administration of GBT at a dose of up to 2000 mg/kg caused no significant increase in the number of micronucleated polychromatic erythrocytes or in the mean ratio of polychromatic to total erythrocytes. Conclusions However, as we did not identify the components of GBT responsible for these effects, other assays are needed to confirm its genotoxicity. PMID:24985139

  3. Dynamics of bacterial and fungal communities associated with eggshells during incubation

    PubMed Central

    Grizard, Stéphanie; Dini-Andreote, Francisco; Tieleman, B Irene; Salles, Joana F

    2014-01-01

    Microorganisms are closely associated with eggs and may play a determinant role in embryo survival. Yet, the majority of studies focusing on this association relied on culture-based methodology, eventually leading to a skewed assessment of microbial communities. By targeting the 16S rRNA gene and internal transcribed spacer (ITS) region, we, respectively, described bacterial and fungal communities on eggshells of the homing pigeon Columba livia. We explored their structure, abundance, and composition. Firstly, we showed that sampling technique affected the outcome of the results. While broadly used, the egg swabbing procedure led to a lower DNA extraction efficiency and provided different profiles of bacterial communities than those based on crushed eggshell pieces. Secondly, we observed shifts in bacterial and fungal communities during incubation. At late incubation, bacterial communities showed a reduction in diversity, while their abundance increased, possibly due to the competitive advantage of some species. When compared to their bacterial counterparts, fungal communities also decreased in diversity at late incubation. In that case, however, the decline was associated with a diminution of their overall abundance. Conclusively, our results showed that although incubation might inhibit microbial growth when compared to unincubated eggs, we observed the selective growth of specific bacterial species during incubation. Moreover, we showed that fungi are a substantial component of the microbial communities associated with eggshells and require further investigations in avian ecology. Identifying the functional roles of these microorganisms is likely to provide news insights into the evolutionary strategies that control embryo survival. We aimed to describe the dynamics of bacterial and fungal communities on homing pigeon eggshell surfaces. We investigated these communities at early and late incubation stages. PMID:24772289

  4. Dynamics of bacterial and fungal communities associated with eggshells during incubation.

    PubMed

    Grizard, Stéphanie; Dini-Andreote, Francisco; Tieleman, B Irene; Salles, Joana F

    2014-04-01

    Microorganisms are closely associated with eggs and may play a determinant role in embryo survival. Yet, the majority of studies focusing on this association relied on culture-based methodology, eventually leading to a skewed assessment of microbial communities. By targeting the 16S rRNA gene and internal transcribed spacer (ITS) region, we, respectively, described bacterial and fungal communities on eggshells of the homing pigeon Columba livia. We explored their structure, abundance, and composition. Firstly, we showed that sampling technique affected the outcome of the results. While broadly used, the egg swabbing procedure led to a lower DNA extraction efficiency and provided different profiles of bacterial communities than those based on crushed eggshell pieces. Secondly, we observed shifts in bacterial and fungal communities during incubation. At late incubation, bacterial communities showed a reduction in diversity, while their abundance increased, possibly due to the competitive advantage of some species. When compared to their bacterial counterparts, fungal communities also decreased in diversity at late incubation. In that case, however, the decline was associated with a diminution of their overall abundance. Conclusively, our results showed that although incubation might inhibit microbial growth when compared to unincubated eggs, we observed the selective growth of specific bacterial species during incubation. Moreover, we showed that fungi are a substantial component of the microbial communities associated with eggshells and require further investigations in avian ecology. Identifying the functional roles of these microorganisms is likely to provide news insights into the evolutionary strategies that control embryo survival. We aimed to describe the dynamics of bacterial and fungal communities on homing pigeon eggshell surfaces. We investigated these communities at early and late incubation stages. PMID:24772289

  5. Determinants of Acidobacteria activity inferred from the relative abundances of 16S rRNA transcripts in German grassland and forest soils.

    PubMed

    Foesel, Bärbel U; Nägele, Verena; Naether, Astrid; Wüst, Pia K; Weinert, Jan; Bonkowski, Michael; Lohaus, Gertrud; Polle, Andrea; Alt, Fabian; Oelmann, Yvonne; Fischer, Markus; Friedrich, Michael W; Overmann, Jörg

    2014-03-01

    16S rRNA genes and transcripts of Acidobacteria were investigated in 57 grassland and forest soils of three different geographic regions. Acidobacteria contributed 9-31% of bacterial 16S rRNA genes whereas the relative abundances of the respective transcripts were 4-16%. The specific cellular 16S rRNA content (determined as molar ratio of rRNA : rRNA genes) ranged between 3 and 80, indicating a low in situ growth rate. Correlations with flagellate numbers, vascular plant diversity and soil respiration suggest that biotic interactions are important determinants of Acidobacteria 16S rRNA transcript abundances in soils. While the phylogenetic composition of Acidobacteria differed significantly between grassland and forest soils, high throughput denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism fingerprinting detected 16S rRNA transcripts of most phylotypes in situ. Partial least squares regression suggested that chemical soil conditions such as pH, total nitrogen, C : N ratio, ammonia concentrations and total phosphorus affect the composition of this active fraction of Acidobacteria. Transcript abundance for individual Acidobacteria phylotypes was found to correlate with particular physicochemical (pH, temperature, nitrogen or phosphorus) and, most notably, biological parameters (respiration rates, abundances of ciliates or amoebae, vascular plant diversity), providing culture-independent evidence for a distinct niche specialization of different Acidobacteria even from the same subdivision. PMID:23802854

  6. Characterization of bacterial community associated to biofilms of corroded oil pipelines from the southeast of Mexico.

    PubMed

    Neria-González, Isabel; Wang, En Tao; Ramírez, Florina; Romero, Juan M; Hernández-Rodríguez, César

    2006-06-01

    Microbial communities associated to biofilms promote corrosion of oil pipelines. The community structure of bacteria in the biofilm formed in oil pipelines is the basic knowledge to understand the complexity and mechanisms of metal corrosion. To assess bacterial diversity, biofilm samples were obtained from X52 steel coupons corroded after 40 days of exposure to normal operation and flow conditions. The biofilm samples were directly used to extract metagenomic DNA, which was used as template to amplify 16S ribosomal gene by PCR. The PCR products of 16S ribosomal gene were also employed as template for sulfate-reducing bacteria (SRB) specific nested-PCR and both PCR products were utilized for the construction of gene libraries. The V3 region of the 16S rRNA gene was also amplified to analyse the bacterial diversity by analysis of denaturing gradient gel electrophoresis (DGGE). Ribosomal library and DGGE profiles exhibited limited bacterial diversity, basically including Citrobacter spp., Enterobacter spp. and Halanaerobium spp. while Desulfovibrio alaskensis and a novel clade within the genus Desulfonatronovibrio were detected from the nested PCR library. The biofilm samples were also taken for the isolation of SRB. Desulfovibrio alaskensis and Desulfovibrio capillatus, as well as some strains related to Citrobacter were isolated. SRB consists in a very small proportion of the community and Desulfovibrio spp. were the relatively abundant groups among the SRB. This is the first study directly exploring bacterial diversity in corrosive biofilms associated to steel pipelines subjected to normal operation conditions. PMID:16765858

  7. Bacteria evade immune recognition via TLR13 and binding of their 23S rRNA by MLS antibiotics by the same mechanisms

    PubMed Central

    Hochrein, Hubertus; Kirschning, Carsten J.

    2013-01-01

    The immune system recognizes pathogens and other danger by means of pattern recognition receptors. Recently, we have demonstrated that the orphan Toll-like receptor 13 (TLR13) senses a defined sequence of the bacterial rRNA and that bacteria use specific mechanisms to evade macrolide lincosamide streptogramin (MLS) antibiotics detection via TLR13. PMID:23802068

  8. Turkey fecal microbial community structure and functional gene diversity revealed by 16S rRNA gene and metagenomic sequences.

    PubMed

    Lu, Jingrang; Domingo, Jorge Santo

    2008-10-01

    The primary goal of this study was to better understand the microbial composition and functional genetic diversity associated with turkey fecal communities. To achieve this, 16S rRNA gene and metagenomic clone libraries were sequenced from turkey fecal samples. The analysis of 382 16S rRNA gene sequences showed that the most abundant bacteria were closely related to Lactobacillales (47%), Bacillales (31%), and Clostridiales (11%). Actinomycetales, Enterobacteriales, and Bacteroidales sequences were also identified, but represented a smaller part of the community. The analysis of 379 metagenomic sequences showed that most clones were similar to bacterial protein sequences (58%). Bacteriophage (10%) and avian viruses (3%) sequences were also represented. Of all metagenomic clones potentially encoding for bacterial proteins, most were similar to low G+C Gram-positive bacterial proteins, particularly from Lactobacillales (50%), Bacillales (11%), and Clostridiales (8%). Bioinformatic analyses suggested the presence of genes encoding for membrane proteins, lipoproteins, hydrolases, and functional genes associated with the metabolism of nitrogen and sulfur containing compounds. The results from this study further confirmed the predominance of Firmicutes in the avian gut and highlight the value of coupling 16S rRNA gene and metagenomic sequencing data analysis to study the microbial composition of avian fecal microbial communities. PMID:18974945

  9. Bacterial Sialidase

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Data shows that elevated sialidase in bacterial vaginosis patients correlates to premature births in women. Bacterial sialidase also plays a significant role in the unusual colonization of Pseudomonas aeruginosa in cystic fibrosis patients. Crystals of Salmonella sialidase have been reproduced and are used for studying the inhibitor-enzyme complexes. These inhibitors may also be used to inhibit a trans-sialidase of Trypanosome cruzi, a very similar enzyme to bacterial sialidase, therefore preventing T. cruzi infection, the causitive agent of Chagas' disease. The Center for Macromolecular Crystallography suggests that inhibitors of bacterial sialidases can be used as prophylactic drugs to prevent bacterial infections in these critical cases.

  10. Bacterial community composition in Brazilian Anthrosols and adjacent soils characterized using culturing and molecular identification.

    PubMed

    O'Neill, B; Grossman, J; Tsai, M T; Gomes, J E; Lehmann, J; Peterson, J; Neves, E; Thies, J E

    2009-07-01

    Microbial community composition was examined in two soil types, Anthrosols and adjacent soils, sampled from three locations in the Brazilian Amazon. The Anthrosols, also known as Amazonian dark earths, are highly fertile soils that are a legacy of pre-Columbian settlement. Both Anthrosols and adjacent soils are derived from the same parent material and subject to the same environmental conditions, including rainfall and temperature; however, the Anthrosols contain high levels of charcoal-like black carbon from which they derive their dark color. The Anthrosols typically have higher cation exchange capacity, higher pH, and higher phosphorus and calcium contents. We used culture media prepared from soil extracts to isolate bacteria unique to the two soil types and then sequenced their 16S rRNA genes to determine their phylogenetic placement. Higher numbers of culturable bacteria, by over two orders of magnitude at the deepest sampling depths, were counted in the Anthrosols. Sequences of bacteria isolated on soil extract media yielded five possible new bacterial families. Also, a higher number of families in the bacteria were represented by isolates from the deeper soil depths in the Anthrosols. Higher bacterial populations and a greater diversity of isolates were found in all of the Anthrosols, to a depth of up to 1 m, compared to adjacent soils located within 50-500 m of their associated Anthrosols. Compared to standard culture media, soil extract media revealed diverse soil microbial populations adapted to the unique biochemistry and physiological ecology of these Anthrosols. PMID:19381712

  11. Evaluation of Microbial Bacterial and Fungal Diversity in Cerebrospinal Fluid Shunt Infection

    PubMed Central

    Simon, Tamara D.; Pope, Christopher E.; Browd, Samuel R.; Ojemann, Jeffrey G.; Riva-Cambrin, Jay; Mayer-Hamblett, Nicole; Rosenfeld, Margaret; Zerr, Danielle M.; Hoffman, Lucas

    2014-01-01

    Background Cerebrospinal fluid shunt infection can be recalcitrant. Recurrence is common despite appropriate therapy for the pathogens identified by culture. Improved diagnostic and therapeutic approaches are required, and culture-independent molecular approaches to cerebrospinal fluid shunt infections have not been described. Objectives To identify the bacteria and fungi present in cerebrospinal fluid from children with cerebrospinal fluid shunt infection using a high-throughput sequencing approach, and to compare those results to those from negative controls and conventional culture. Methods This descriptive study included eight children ?18 years old undergoing treatment for culture-identified cerebrospinal fluid shunt infection. After routine aerobic culture of each cerebrospinal fluid sample, deoxyribonucleic acid (DNA) extraction was followed by amplification of the bacterial 16S rRNA gene and the fungal ITS DNA region tag-encoded FLX-Titanium amplicon pyrosequencing and microbial phylogenetic analysis. Results The microbiota analyses for the initial cerebrospinal fluid samples from all eight infections identified a variety of bacteria and fungi, many of which did not grow in conventional culture. Detection by conventional culture did not predict the relative abundance of an organism by pyrosequencing, but in all cases, at least one bacterial taxon was detected by both conventional culture and pyrosequencing. Individual bacterial species fluctuated in relative abundance but remained above the limits of detection during infection treatment. Conclusions Numerous bacterial and fungal organisms were detected in these cerebrospinal fluid shunt infections, even during and after treatment, indicating diverse and recalcitrant shunt microbiota. In evaluating cerebrospinal fluid shunt infection, fungal and anaerobic bacterial cultures should be considered in addition to aerobic bacterial cultures, and culture-independent approaches offer a promising alternative diagnostic approach. More effective treatment of cerebrospinal fluid shunt infections is needed to reduce unacceptably high rates of reinfection, and this work suggests that one effective strategy may be reduction of the diverse microbiota present in infection. PMID:24421877

  12. Bacterial Proteasomes

    PubMed Central

    Jastrab, Jordan B.; Darwin, K. Heran

    2015-01-01

    Interest in bacterial proteasomes was sparked by the discovery that proteasomal degradation is required for the pathogenesis of Mycobacterium tuberculosis, one of the world's deadliest pathogens. Although bacterial proteasomes are structurally similar to their eukaryotic and archaeal homologs, there are key differences in their mechanisms of assembly, activation, and substrate targeting for degradation. In this article, we compare and contrast bacterial proteasomes with their archaeal and eukaryotic counterparts, and we discuss recent advances in our understanding of how bacterial proteasomes function to influence microbial physiology. PMID:26488274

  13. Bacterial population structure and dynamics during the development of almond drupes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aims: To describe the bacterial populations and their dynamics during the development of almond drupes. Methods and Results: We examined 16S rRNA gene libraries derived from the bacterial populations on almond drupes at three stages of development: 1) when the drupes were full sized, but before embr...

  14. Rapid in situ hybridization technique using 16S rRNA segments for detecting and differentiating the closely related gram-positive organisms Bacillus polymyxa and Bacillus macerans

    NASA Technical Reports Server (NTRS)

    Jurtshuk, R. J.; Blick, M.; Bresser, J.; Fox, G. E.; Jurtshuk, P. Jr

    1992-01-01

    A rapid, sensitive, inexpensive in situ hybridization technique, using 30-mer 16S rRNA probes, can specifically differentiate two closely related Bacillus spp., B. polymyxa and B. macerans. The 16S rRNA probes were labeled with a rhodamine derivative (Texas Red), and quantitative fluorescence measurements were made on individual bacterial cells. The microscopic fields analyzed were selected by phase-contrast microscopy, and the fluorescence imaging analyses were performed on 16 to 67 individual cells. The labeled 16S rRNA probe, POL, whose sequence was a 100% match with B. polymyxa 16S rRNA but only a 60% match with B. macerans 16S rRNA, gave quantitative fluorescence ratio measurements that were 34.8-fold higher for B. polymyxa cells than for B. macerans cells. Conversely, the labeled probe, MAC, which matched B. polymyxa 16S rRNA in 86.6% of its positions and B. macerans 16S rRNA in 100% of its positions, gave quantitative fluorescence measurements that were 59.3-fold higher in B. macerans cells than in B. polymyxa cells. Control probes, whose 16S rRNA sequence segment (P-M) was present in both B. polymyxa and B. macerans as well as a panprokaryotic probe (16S), having a 100% match with all known bacteria, hybridized equally well with both organisms. These latter hybridizations generated very high fluorescence signals, but their comparative fluorescence ratios (the differences between two organisms) were low. The control paneukaryotic probe (28S), which had less than 30% identity for both B. macerans and B. polymyxa, did not hybridize with either organism.

  15. Biomonitoring of the genotoxic potential of aqueous extracts of soils and bottom ash resulting from municipal solid waste incineration, using the comet and micronucleus tests on amphibian (Xenopus laevis) larvae and bacterial assays (Mutatox and Ames tests).

    PubMed

    Mouchet, F; Gauthier, L; Mailhes, C; Jourdain, M J; Ferrier, V; Triffault, G; Devaux, A

    2006-02-15

    The management of contaminated soils and wastes is a matter of considerable human concern. The present study evaluates the genotoxic potential of aqueous extracts of two soils (leachates) and of bottom ash resulting from municipal solid waste incineration (MSWIBA percolate), using amphibian larvae (Xenopus laevis). Soil A was contaminated by residues of solvents and metals and Soil B by polycyclic aromatic hydrocarbons and metals. MSWIBA was predominantly contaminated by metals. Two genotoxic endpoints were analysed in circulating erythrocytes taken from larvae: clastogenic and/or aneugenic effects (micronucleus induction) after 12 days of exposure and DNA-strand-breaking potency (comet assay) after 1 and 12 days of exposure. In addition, in vitro bacterial assays (Mutatox and Ames tests) were carried out and the results were compared with those of the amphibian test. Physicochemical analyses were also taken into account. Results obtained with the amphibians established the genotoxicity of the aqueous extracts and the comet assay revealed that they were genotoxic from the first day of exposure. The latter test could thus be considered as a genotoxicity-screening tool. Although genotoxicity persisted after 12 days' exposure, DNA damage decreased overall between days 1 and 12 in the MSWIBA percolate, in contrast to the soil leachates. Bacterial tests detected genotoxicity only for the leachate of soil A (Mutatox). The results confirm the ecotoxicological relevance of the amphibian model and underscore the importance of bioassays, as a complement to physico-chemical data, for risk evaluation. PMID:16442436

  16. Assessing the effects of salmon farming seabed enrichment using bacterial community diversity and high-throughput sequencing.

    PubMed

    Dowle, Eddy; Pochon, Xavier; Keeley, Nigel; Wood, Susanna A

    2015-08-01

    Aquaculture is an extremely valuable and rapidly expanding sector of the seafood industry. The sediment below active aquaculture farms receives inputs of organic matter from uneaten food and faecal material and this has led to concerns related to environmental sustainability. The impacts of organic enrichment on macrobenthic infauna are well characterized; however, much less is known about effect on bacterial communities. In this study, sediment, macrobenthic infauna samples and environmental data were collected along an enrichment gradient radiating out from a Chinook salmon (Oncorhynchus tshawytscha) farm (Marlborough Sounds; New Zealand). DNA and RNA were extracted and 16S rRNA metabarcodes from bacterial communities characterized using high-throughput sequencing. Desulfobacterales dominated at the cage (DNA and RNA), and at sites 50 m (DNA and RNA) and 150 m (RNA) from the farm. In contrast, unclassified bacteria from the class Gammaproteobacteria were the most abundant taxa at control sites (625 and 4000 m). Pronounced differences among DNA and RNA samples occurred at the cage site where Desulfobacterales abundance was markedly higher in RNA samples. There were strong correlations between shifts in bacterial communities and total organic matter and redox. This suggests that bacterial composition is strongly influenced by organic enrichment, a trait that may make them useful for assessing impacts associated with aquaculture farms. PMID:26207046

  17. Bacterial Diversity at an Acid Mine Drainage Site in Maine

    NASA Astrophysics Data System (ADS)

    Gaynor, J.; Sawyer, T.; Riley, F. E.; Moulton, K. D.; Rothschild, L. J.; Duboise, S. M.

    2010-04-01

    Bacterial diversity in acidic mine drainage at a historic Maine iron mining site was investigated by isolation of environmental DNA, PCR amplification of the V3 region of the 16S rRNA gene, denaturing gradient gel electrophoresis, and DNA sequencing.

  18. Effects of Functionalized and Raw Multi-Walled Carbon Nanotubes on Soil Bacterial Community Composition

    PubMed Central

    Kerfahi, Dorsaf; Tripathi, Binu M.; Singh, Dharmesh; Kim, Hyoki; Lee, Sujin; Lee, Junghoon; Adams, Jonathan M.

    2015-01-01

    Carbon nanotubes (CNTs) are widely used in industry, but their environmental impacts on soil microbial communities are poorly known. In this paper, we compare the effect of both raw and acid treated or functionalized (fCNTs) multi-walled carbon nanotubes (MWCNTs) on soil bacterial communities, applying different concentrations of MWCNTs (0 µg/g, 50 µg/g, 500 µg/g and 5000 µg/g) to a soil microcosm system. Soil DNA was extracted at 0, 2 and 8 weeks and the V3 region of the 16S rRNA gene was PCR-amplified and sequenced using paired-end Illumina bar-coded sequencing. The results show that bacterial diversity was not affected by either type of MWCNT. However, overall soil bacterial community composition, as illustrated by NMDS, was affected only by fMWCNT at high concentrations. This effect, detectable at 2 weeks, remained equally strong by 8 weeks. In the case of fMWCNTs, overall changes in relative abundance of the dominant phyla were also found. The stronger effect of fMWCNTs could be explained by their intrinsically acidic nature, as the soil pH was lower at higher concentrations of fMWCNTs. Overall, this study suggests that fMWCNTs may at least temporarily alter microbial community composition on the timescale of at least weeks to months. It appears, by contrast, that raw MWCNTs do not affect soil microbial community composition. PMID:25825905

  19. The impact of date palm fruits and their component polyphenols, on gut microbial ecology, bacterial metabolites and colon cancer cell proliferation.

    PubMed

    Eid, Noura; Enani, Sumia; Walton, Gemma; Corona, Giulia; Costabile, Adele; Gibson, Glenn; Rowland, Ian; Spencer, Jeremy P E

    2014-01-01

    The fruit of the date palm (Phoenix dactylifera L.) is a rich source of dietary fibre and polyphenols. We have investigated gut bacterial changes induced by the whole date fruit extract (digested date extract; DDE) and its polyphenol-rich extract (date polyphenol extract; DPE) using faecal, pH-controlled, mixed batch cultures mimicking the distal part of the human large intestine, and utilising an array of microbial group-specific 16S rRNA oligonucleotide probes. Fluorescence microscopic enumeration indicated that there was a significant increase in the growth of bifidobacteria in response to both treatments, whilst whole dates also increased bacteroides at 24 h and the total bacterial counts at later fermentation time points when compared with DPE alone. Bacterial metabolism of whole date fruit led to the production of SCFA, with acetate significantly increasing following bacterial incubation with DDE. In addition, the production of flavonoid aglycones (myricetin, luteolin, quercetin and apigenin) and the anthocyanidin petunidin in less than 1 h was also observed. Lastly, the potential of DDE, DPE and metabolites to inhibit Caco-2 cell growth was investigated, indicating that both were capable of potentially acting as antiproliferative agents in vitro, following a 48 h exposure. This potential to inhibit growth was reduced following fermentation. Together these data suggest that consumption of date fruits may enhance colon health by increasing beneficial bacterial growth and inhibiting the proliferation of colon cancer cells. This is an early suggestion that date intake by humans may aid in the maintenance of bowel health and even the reduction of colorectal cancer development. PMID:26101614

  20. Identification of bacteria in a biodegraded wall painting by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.

    PubMed

    Rölleke, S; Muyzer, G; Wawer, C; Wanner, G; Lubitz, W

    1996-06-01

    Medieval wall paintings are often affected by biodecay. An inventory of the existing microorganisms associated with the damage to the paintings is not yet an integral part of the restoration process. This stems from the lack of effective means for such a stocktaking. Nevertheless, fungi and bacteria cause severe damage through mechanical processes from growth into the painting and its grounding and through their metabolism. Detailed information on the bacterial colonization of ancient wall paintings is essential for the protection of the paintings. We used a molecular approach based on the detection and identification of DNA sequences encoding rRNA (rDNA) to identify bacteria present on an ancient wall painting without prior cultivation of the organisms, since it has been shown that most of these bacteria cannot be cultivated under laboratory conditions. To trace the noncultivated fraction of bacteria, total DNA from a biodegraded wall painting sample from a 13th century fresco was extracted and 194-bp fragments of the 16S rDNA were amplified with eubacterial primers. The 16S rDNA fragments of uniform length obtained from the different bacterial species were separated according to their sequence differences by denaturing gradient gel electrophoresis (DGGE). By sequencing excised and reamplified individual DNA bands, we characterized the phylogenetic affiliation of the corresponding bacteria. Using this approach, we identified members or close relatives of the genera Halomonas, Clostridium, and Frankia. To our knowledge, these groups of bacteria have not yet been isolated and implicated by conventional microbiological techniques as contributing to the biodegradation of wall paintings. PMID:8787403

  1. Different Bacterial Populations Associated with the Roots and Rhizosphere of Rice Incorporate Plant-Derived Carbon

    PubMed Central

    Hernández, Marcela; Yuan, Quan; Conrad, Ralf

    2015-01-01

    Microorganisms associated with the roots of plants have an important function in plant growth and in soil carbon sequestration. Rice cultivation is the second largest anthropogenic source of atmospheric CH4, which is a significant greenhouse gas. Up to 60% of fixed carbon formed by photosynthesis in plants is transported below ground, much of it as root exudates that are consumed by microorganisms. A stable isotope probing (SIP) approach was used to identify microorganisms using plant carbon in association with the roots and rhizosphere of rice plants. Rice plants grown in Italian paddy soil were labeled with 13CO2 for 10 days. RNA was extracted from root material and rhizosphere soil and subjected to cesium gradient centrifugation followed by 16S rRNA amplicon pyrosequencing to identify microorganisms enriched with 13C. Thirty operational taxonomic units (OTUs) were labeled and mostly corresponded to Proteobacteria (13 OTUs) and Verrucomicrobia (8 OTUs). These OTUs were affiliated with the Alphaproteobacteria, Betaproteobacteria, and Deltaproteobacteria classes of Proteobacteria and the “Spartobacteria” and Opitutae classes of Verrucomicrobia. In general, different bacterial groups were labeled in the root and rhizosphere, reflecting different physicochemical characteristics of these locations. The labeled OTUs in the root compartment corresponded to a greater proportion of the 16S rRNA sequences (?20%) than did those in the rhizosphere (?4%), indicating that a proportion of the active microbial community on the roots greater than that in the rhizosphere incorporated plant-derived carbon within the time frame of the experiment. PMID:25616793

  2. Characterization of the Fecal Microbial Communities of Duroc Pigs Using 16S rRNA Gene Pyrosequencing

    PubMed Central

    Pajarillo, Edward Alain B.; Chae, Jong Pyo; Balolong, Marilen P.; Kim, Hyeun Bum; Seo, Kang-Seok; Kang, Dae-Kyung

    2015-01-01

    This study characterized the fecal bacterial community structure and inter-individual variation in 30-week-old Duroc pigs, which are known for their excellent meat quality. Pyrosequencing of the V1–V3 hypervariable regions of the 16S rRNA genes generated 108,254 valid reads and 508 operational taxonomic units at a 95% identity cut-off (genus level). Bacterial diversity and species richness as measured by the Shannon diversity index were significantly greater than those reported previously using denaturation gradient gel electrophoresis; thus, this study provides substantial information related to both known bacteria and the untapped portion of unclassified bacteria in the population. The bacterial composition of Duroc pig fecal samples was investigated at the phylum, class, family, and genus levels. Firmicutes and Bacteroidetes predominated at the phylum level, while Clostridia and Bacteroidia were most abundant at the class level. This study also detected prominent inter-individual variation starting at the family level. Among the core microbiome, which was observed at the genus level, Prevotella was consistently dominant, as well as a bacterial phylotype related to Oscillibacter valericigenes, a valerate producer. This study found high bacterial diversity and compositional variation among individuals of the same breed line, as well as high abundance of unclassified bacterial phylotypes that may have important functions in the growth performance of Duroc pigs. PMID:25656184

  3. Phylogenetic Relationship of Phosphate Solubilizing Bacteria according to 16S rRNA Genes

    PubMed Central

    Javadi Nobandegani, Mohammad Bagher; Saud, Halimi Mohd; Yun, Wong Mui

    2015-01-01

    Phosphate solubilizing bacteria (PSB) can convert insoluble form of phosphorous to an available form. Applications of PSB as inoculants increase the phosphorus uptake by plant in the field. In this study, isolation and precise identification of PSB were carried out in Malaysian (Serdang) oil palm field (University Putra Malaysia). Identification and phylogenetic analysis of 8 better isolates were carried out by 16S rRNA gene sequencing in which as a result five isolates belong to the Beta subdivision of Proteobacteria, one isolate was related to the Gama subdivision of Proteobacteria, and two isolates were related to the Firmicutes. Bacterial isolates of 6upmr, 2upmr, 19upmnr, 10upmr, and 24upmr were identified as Alcaligenes faecalis. Also, bacterial isolates of 20upmnr and 17upmnr were identified as Bacillus cereus and Vagococcus carniphilus, respectively, and bacterial isolates of 31upmr were identified as Serratia plymuthica. Molecular identification and characterization of oil palm strains as the specific phosphate solubilizer can reduce the time and cost of producing effective inoculate (biofertilizer) in an oil palm field. PMID:25632387

  4. Influence of Commonly Used Primer Systems on Automated Ribosomal Intergenic Spacer Analysis of Bacterial Communities in Environmental Samples

    PubMed Central

    Francioli, Davide; Reitz, Thomas; Buscot, François; Schloter, Michael; Krüger, Dirk

    2015-01-01

    Due to the high diversity of bacteria in many ecosystems, their slow generation times, specific but mostly unknown nutrient requirements and syntrophic interactions, isolation based approaches in microbial ecology mostly fail to describe microbial community structure. Thus, cultivation independent techniques, which rely on directly extracted nucleic acids from the environment, are a well-used alternative. For example, bacterial automated ribosomal intergenic spacer analysis (B-ARISA) is one of the widely used methods for fingerprinting bacterial communities after PCR-based amplification of selected regions of the operon coding for rRNA genes using community DNA. However, B-ARISA alone does not provide any taxonomic information and the results may be severely biased in relation to the primer set selection. Furthermore, amplified DNA stemming from mitochondrial or chloroplast templates might strongly bias the obtained fingerprints. In this study, we determined the applicability of three different B-ARISA primer sets to the study of bacterial communities. The results from in silico analysis harnessing publicly available sequence databases showed that all three primer sets tested are specific to bacteria but only two primers sets assure high bacterial taxa coverage (1406f/23Sr and ITSF/ITSReub). Considering the study of bacteria in a plant interface, the primer set ITSF/ITSReub was found to amplify (in silico) sequences of some important crop species such as Sorghum bicolor and Zea mays. Bacterial genera and plant species potentially amplified by different primer sets are given. These data were confirmed when DNA extracted from soil and plant samples were analyzed. The presented information could be useful when interpreting existing B-ARISA results and planning B-ARISA experiments, especially when plant DNA can be expected. PMID:25749323

  5. 16S rRNA gene phylogenesis of culturable predominant bacteria from diseased Apostichopus japonicus (Holothuroidea, Echinodermata)

    NASA Astrophysics Data System (ADS)

    Ma, Haiyan; Jiang, Guoliang; Wu, Zhiqiang; Wang, Xin

    2009-06-01

    Cultured Apostichopus japonicus in China suffers from a kind of skin ulceration disease that has caused severe economic loss in recent years. The disease, pathogens of which are supposed to be bacteria by most researchers, is highly infectious and can often cause all individuals in the same culture pool to die in a very short time. The 16S rRNA gene phylogenesis of the culturable bacteria from the lesions of diseased individuals was conducted to study the biodiversity of the bacterial communities in the lesions and to identify probable pathogen(s) associated with this kind of disease. S. japonica samples were selected from a hatchery located in the eastern part of Qingdao, China. Bacterial universal primers GM5F and DS907R were used to amplify the 16S rRNA gene of bacteria colonies, and touchdown PCR was performed to amplify the target sequences. The results suggest that ?- proteobacteria (Alteromonadales and Vibrionales) of CFB group, many strains of which have been also determined as pathogens in other marine species, are the predominant bacterial genera of the diseased Apostichopus japonicus individuals.

  6. Changes in rRNA Levels during Stress Invalidates Results from mRNA Blotting: Fluorescence In Situ rRNA Hybridization Permits Renormalization for Estimation of Cellular mRNA Levels

    PubMed Central

    Hansen, Martin C.; Nielsen, Allan K.; Molin, Søren; Hammer, Karin; Kilstrup, Mogens

    2001-01-01

    Regulation of gene expression can be analyzed by a number of different techniques. Some techniques monitor the level of specific mRNA directly, and others monitor indirectly by determining the level of enzymes encoded by the mRNA. Each method has its own inherent way of normalization. When results obtained by these techniques are compared between experiments in which differences in growth rates, strains, or stress treatments occur, the normalization procedure may have a significant impact on the results. In this report we present a solution to the normalization problem in RNA slot blotting experiments, in which mRNA levels routinely are normalized to a fixed amount of extracted total RNA. The cellular levels of specific mRNA species were estimated using a renormalization with the total RNA content per cell. By a combination of fluorescence in situ rRNA hybridization, which estimates the relative level of rRNA per cell, and slot blotting to rRNA probes, which estimates the level of rRNA per extracted total RNA, the amount of RNA per cell was calculated in a series of heat shock experiments with the gram-positive bacterium Lactococcus lactis. It was found that the level of rRNA per cell decreased to 30% in the course of the heat shock. This lowered ribosome level led to a decrease in the total RNA content, resulting in a gradually increasing overestimation of the mRNA levels throughout the experiment. Using renormalized cellular mRNA levels, the HrcA-mediated regulation of the genes in the hrcA-grpE-dnaK operon was analyzed. The hybridization data suggested a complex heat shock regulation indicating that the mRNA levels continued to rise after 30 min, but after renormalization the calculated average cellular levels exhibited a much simpler induction pattern, eventually attaining a moderately increased value. PMID:11466277

  7. Standardized ginger (Zingiber officinale) extract reduces bacterial load and suppresses acute and chronic inflammation in Mongolian gerbils infected with cagA+Helicobacter pylori

    PubMed Central

    Gaus, Kristen; Huang, Yue; Israel, Dawn A.; Pendland, Susan L.; Adeniyi, Bolanle A.; Mahady, Gail B.

    2010-01-01

    Previous investigations demonstrated that a standardized extract of ginger rhizome inhibited the growth of Helicobacter pylori in vitro with a minimum inhibitory concentration in the range 0.78 to 12.5 ?g/mL. In the present work, the extract was tested in a rodent model of H. pylori-induced disease, the Mongolian gerbil, to examine the effects of the extract on both prevention and eradication of infection. The extract was administered to Mongolian gerbils at a daily dose of 100 mg/kg body weight in rations either 3 weeks prior to infection or 6 weeks post-infection. Treatment with the standardized ginger extract reduced H. pylori load as compared with controls and significantly (P<0.05) reduced both acute and chronic muscosal and submucosal inflammation, cryptitis, as well as epithelial cell degeneration and erosion induced by H. pylori. Importantly, the extract did not increase morbidity or mortality. Further investigations of the mechanism demonstrated that the ginger extract inhibited the activity of cyclooxygenase-2, with 50% inhibitory concentration (IC50) of 8.5 ?g/mL in vitro, inhibited the nuclear factor-?B transcriptional response in kBZ Jurkat cells (human T lymphocytes) with an IC50 of 24.6 ?g/mL, and significantly inhibited the release of interleukin (IL)-1?, IL-6, IL-8, and tumor necrosis factor-? from lipopolysaccharide-stimulated human peripheral blood mononuclear cells with IC50 values of 3.89, 7.7, 8.5, and 8.37 ?g/mL, respectively. These results suggest ginger extracts may be useful for development as agents to reduce H. pylori-induced inflammation and as for gastric cancer chemoprevention. PMID:20376296

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

    PubMed Central

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

    2015-01-01

    Background Rodents are major reservoirs of pathogens responsible for numerous zoonotic diseases in humans and livestock. Assessing their microbial diversity at both the individual and population level is crucial for monitoring endemic infections and revealing microbial association patterns within reservoirs. Recently, NGS approaches have been employed to characterize microbial communities of different ecosystems. Yet, their relative efficacy has not been assessed. Here, we compared two NGS approaches, RNA-Sequencing (RNA-Seq) and 16S-metagenomics, assessing their ability to survey neglected zoonotic bacteria in rodent populations. Methodology/Principal Findings We first extracted nucleic acids from the spleens of 190 voles collected in France. RNA extracts were pooled, randomly retro-transcribed, then RNA-Seq was performed using HiSeq. Assembled bacterial sequences were assigned to the closest taxon registered in GenBank. DNA extracts were analyzed via a 16S-metagenomics approach using two sequencers: the 454 GS-FLX and the MiSeq. The V4 region of the gene coding for 16S rRNA was amplified for each sample using barcoded universal primers. Amplicons were multiplexed and processed on the distinct sequencers. The resulting datasets were de-multiplexed, and each read was processed through a pipeline to be taxonomically classified using the Ribosomal Database Project. Altogether, 45 pathogenic bacterial genera were detected. The bacteria identified by RNA-Seq were comparable to those detected by 16S-metagenomics approach processed with MiSeq (16S-MiSeq). In contrast, 21 of these pathogens went unnoticed when the 16S-metagenomics approach was processed via 454-pyrosequencing (16S-454). In addition, the 16S-metagenomics approaches revealed a high level of coinfection in bank voles. Conclusions/Significance We concluded that RNA-Seq and 16S-MiSeq are equally sensitive in detecting bacteria. Although only the 16S-MiSeq method enabled identification of bacteria in each individual reservoir, with subsequent derivation of bacterial prevalence in host populations, and generation of intra-reservoir patterns of bacterial interactions. Lastly, the number of bacterial reads obtained with the 16S-MiSeq could be a good proxy for bacterial prevalence. PMID:26284930

  9. Cisplatin Targeting of Bacterial Ribosomal RNA Hairpins.

    PubMed

    Dedduwa-Mudalige, Gayani N P; Chow, Christine S

    2015-01-01

    Cisplatin is a clinically important chemotherapeutic agent known to target purine bases in nucleic acids. In addition to major deoxyribonucleic acid (DNA) intrastrand cross-links, cisplatin also forms stable adducts with many types of ribonucleic acid (RNA) including siRNA, spliceosomal RNAs, tRNA, and rRNA. All of these RNAs play vital roles in the cell, such as catalysis of protein synthesis by rRNA, and therefore serve as potential drug targets. This work focused on platination of two highly conserved RNA hairpins from E. coli ribosomes, namely pseudouridine-modified helix 69 from 23S rRNA and the 790 loop of helix 24 from 16S rRNA. RNase T1 probing, MALDI mass spectrometry, and dimethyl sulfate mapping revealed platination at GpG sites. Chemical probing results also showed platination-induced RNA structural changes. These findings reveal solvent and structural accessibility of sites within bacterial RNA secondary structures that are functionally significant and therefore viable targets for cisplatin as well as other classes of small molecules. Identifying target preferences at the nucleotide level, as well as determining cisplatin-induced RNA conformational changes, is important for the design of more potent drug molecules. Furthermore, the knowledge gained through studies of RNA-targeting by cisplatin is applicable to a broad range of organisms from bacteria to human. PMID:26370969

  10. Cisplatin Targeting of Bacterial Ribosomal RNA Hairpins

    PubMed Central

    Dedduwa-Mudalige, Gayani N. P.; Chow, Christine S.

    2015-01-01

    Cisplatin is a clinically important chemotherapeutic agent known to target purine bases in nucleic acids. In addition to major deoxyribonucleic acid (DNA) intrastrand cross-links, cisplatin also forms stable adducts with many types of ribonucleic acid (RNA) including siRNA, spliceosomal RNAs, tRNA, and rRNA. All of these RNAs play vital roles in the cell, such as catalysis of protein synthesis by rRNA, and therefore serve as potential drug targets. This work focused on platination of two highly conserved RNA hairpins from E. coli ribosomes, namely pseudouridine-modified helix 69 from 23S rRNA and the 790 loop of helix 24 from 16S rRNA. RNase T1 probing, MALDI mass spectrometry, and dimethyl sulfate mapping revealed platination at GpG sites. Chemical probing results also showed platination-induced RNA structural changes. These findings reveal solvent and structural accessibility of sites within bacterial RNA secondary structures that are functionally significant and therefore viable targets for cisplatin as well as other classes of small molecules. Identifying target preferences at the nucleotide level, as well as determining cisplatin-induced RNA conformational changes, is important for the design of more potent drug molecules. Furthermore, the knowledge gained through studies of RNA-targeting by cisplatin is applicable to a broad range of organisms from bacteria to human. PMID:26370969

  11. A randomized, double-blind, placebo-controlled trial to assess the bacterial anti-adhesion effects of cranberry extract beverages.

    PubMed

    Kaspar, Kerrie L; Howell, Amy B; Khoo, Christina

    2015-04-01

    In this study, we examined the ex vivo urinary anti-adhesion activity of low-calorie cranberry extract beverages in both a pilot study (n = 10) and a randomized, double-blind, placebo controlled clinical trial (n = 59). In the pilot study, subjects consumed a cranberry extract beverage (CEB) or a cranberry extract and juice beverage (CEJB), compared to placebo. Both cranberry beverages utilized a standardized cranberry extract powder at a level equivalent to low-calorie cranberry juice cocktail (LCJC) on a PAC content basis. Clean-catch urine samples collected at baseline and post intervention were tested for anti-adhesion activity utilizing a mannose-resistant human red blood cell hemagglutination assay specific for P-fimbriated E. coli. Results from the pilot study indicated that ex vivo anti-adhesion activity for both cranberry treatments were higher (p < 0.05) than placebo. In the clinical trial, we compared CEJB to LCJC and a placebo beverage. Post-consumption urine from both cranberry treatment groups showed significantly higher (p < 0.05) anti-adhesion activity compared to placebo. There were no differences observed in anti-adhesion activity between CJEB and LCJC, indicating similar bioactivity. Therefore, acute beverage consumption of cranberry extract and/or juice provides ex vivo anti-adhesion activity, which may help to improve urinary tract health. PMID:25723356

  12. Development and evaluation of immunochromatography to detect Gram-negative bacteria producing ArmA 16S rRNA methylase responsible for aminoglycoside resistance.

    PubMed

    Oshiro, Satoshi; Tada, Tatsuya; Kameoka, Yousuke; Suzuki, Kazuo; Ohmagari, Norio; Miyoshi-Akiyama, Tohru; Kirikae, Teruo

    2015-11-01

    Rapid and reliable detection of aminoglycoside-resistant bacteria is an important infection-control measure and a crucial aspect of antimicrobial chemotherapy. The enzyme 16S rRNA methylase has been shown to mediate aminoglycoside resistance in bacteria. This study describes a newly developed immunochromatographic assay using novel monoclonal antibodies (mAbs) that recognize ArmA 16S rRNA methylase. Epitope mapping showed that these mAbs recognized amino acids 1-93 of ArmA, which consists of 257 amino acids. Evaluation of the assay using ArmA producing and non-producing bacterial species, as well as bacteria producing other types of 16S rRNA methylases, indicated that immunochromatographic detection of the ArmA-type 16S rRNA methylase was fully consistent with PCR analysis for armA genes, with all immunochromatographically positive strains being resistant to aminoglycosides (MIC?128?g/mL). The detection limit of the assay was 12ng ArmA. These findings indicate that this assay can be used for the rapid and reliable detection of the production of ArmA 16S rRNA methylase by Gram-negative bacteria, including Acinetobacter baumannii and Escherichia coli. PMID:26381663

  13. A multiphasic approach for the identification of endophytic bacterial in strawberry fruit and their potential for plant growth promotion.

    PubMed

    de Melo Pereira, Gilberto Vinícius; Magalhães, Karina Teixeira; Lorenzetii, Emi Rainildes; Souza, Thiago Pereira; Schwan, Rosane Freitas

    2012-02-01

    This study used a multiphasic approach, characterized by the simultaneous use of culture-dependent and culture-independent methods, to investigate endophytic bacterial communities in strawberry (Fragaria ananassa) fruit. A total of 92 bacterial endophytes were isolated and initially grouped by their repetitive extragenic palindromic (rep)-PCR banding pattern and biochemical features. Phylogenetic analysis of the 16S rRNA gene sequences of 45 representatives showed that the isolates belonged to the species Bacillus subtilis (eight isolates), Bacillus sp. (seven isolates), Enterobacter sp. (seven isolates), Enterobacter ludwigii (six isolates), Lactobacillus plantarum (six isolates), Pseudomonas sp. (five isolates), Pantoea punctata (three isolates), and Curtobacterium citreum (three isolates). Nucleic acids were extracted from the strawberry fruit and subjected to 16S rRNA gene directed polymerase chain reaction denaturing gradient gel electrophoresis (16S rRNA PCR-DGGE). The species B. subtilis, Enterobacter sp., and Pseudomonas sp. were detected both by isolation and DGGE. The DGGE fingerprints of total bacterial DNA did not exhibit bands corresponding to several of the representative species isolated in the extinction dilution (L. plantarum, C. citreum, and P. punctata). In contrast, bands in the DGGE profile that were identified as relatives of Arthrobacter sp. and one uncultivable Erythrobacter sp. were not recovered by cultivation techniques. After isolation, the nitrogen fixation ability and the in vitro production of indole-3-acetic acid (IAA) equivalents and siderophores were evaluated. A high percentage of isolates were found to possess the ability to produce siderophores and IAA equivalents; however, only a few isolates belonging to the genera Pseudomonas and Enterobacter showed the ability to fix nitrogen. Plant growth promotion was evaluated under greenhouse conditions and revealed the ability of the Bacillus strains to enhance the number of leaves, shoot length, root dry weight, and shoot dry weight. The activity of the bacterial isolate identified as B. subtilis NA-108 exerted the greatest influence on strawberry growth and showed a 42.8% increase in number of leaves, 15.26% for high shoot, 43.5% increase in root dry weight, and a 77% increase in shoot dry weight when compared with untreated controls. PMID:21837472

  14. Effect of Elevated Tropospheric Ozone on the Structure of Bacterial Communities Inhabiting the Rhizosphere of Herbaceous Plants Native to Germany†

    PubMed Central

    Dohrmann, Anja B.; Tebbe, Christoph C.

    2005-01-01

    Current elevated concentrations of ozone in the atmosphere, as they are observed during summer seasons, can cause severe effects on plant vegetation. This study was initiated to analyze whether ozone-stressed plants also transfer signals below ground and thereby alter the bacterial community composition in their rhizospheres. Herbaceous plants, native to Germany, with tolerance (Anthoxanthum odoratum, Achillea millefolium, Poa pratensis, Rumex acetosa, and Veronica chamaedrys) and sensitivity (Matricaria chamomilla, Sonchus asper, and Tanacetum vulgare) to ozone, raised in the greenhouse, were exposed in open-top chambers to two different ozone regimes, i.e., “summer stress” and a normal ozone background. DNA of bacterial cells from the rhizospheres was directly extracted, and partial sequences of the 16S rRNA genes were PCR amplified with primers targeting the following phylogenetic groups: Bacteria, ?-Proteobacteria, Actinobacteria, and Pseudomonas, respectively. The diversity of the amplified products was analyzed by genetic profiling based on single-strand conformation polymorphism (SSCP). Neither the tolerant nor the sensitive plants, the latter with visible above-ground damage, showed ozone-induced differences in any of the SSCP profiles, with the single exception of Actinobacteria-targeted profiles from S. asper. To increase the stress, S. asper was germinated and raised in the continuous presence of an elevated level of ozone. SSCP profiles with Bacteria-specific primers combined with gene probe hybridizations indicated an ozone-related increase in a Xanthomonas-related 16S rRNA gene and a decrease in the respective gene from the plant plastids. The fact that only this latter unrealistic scenario caused a detectable effect demonstrated that ozone stress has a surprisingly small effect on the structural diversity of the bacterial community in rhizospheres. PMID:16332747

  15. Extract from Rumex acetosa L. for Prophylaxis of Periodontitis: Inhibition of Bacterial In Vitro Adhesion and of Gingipains of Porphyromonas gingivalis by Epicatechin-3-O-(4??8)-Epicatechin-3-O-Gallate (Procyanidin-B2-Di-Gallate)

    PubMed Central

    Schmuch, Jana; Beckert, Sabine; Brandt, Simone; Löhr, Gesine; Hermann, Fabian; Schmidt, Thomas J.; Beikler, Thomas; Hensel, Andreas

    2015-01-01

    Background The aerial parts of Rumex acetosa L. have been used in traditional European medicine for inflammatory diseases of the mouth epithelial tissue. The following study aimed to investigate the influence of a proanthocyanidin-enriched extract from R. acetosa extract against the adhesion of Porphyromonas gingivalis (P. gingivalis), a pathogen strongly involved in chronic and aggressive periodontitis. A further goal was to define the bioactive lead structures responsible for a potential antiadhesive activity and to characterize the underlying molecular mechanisms of the antiadhesive effects. Methodology An extract of R. acetosa (RA1) with a defined mixture of flavan-3-ols, oligomeric proanthocyanidins and flavonoids, was used. Its impact on P. gingivalis adhesion to KB cells was studied by flow cytometry, confocal laser scanning microscopy and in situ adhesion assay using murine buccal tissue. RA1 and its compounds 1 to 15 were further investigated for additional effects on gingipain activity, hemagglutination and gene expression by RT-PCR. Principal Findings RA1 (5 to 15 ?g/mL) reduced P. gingivalis adhesion in a dose-dependent manner to about 90%. Galloylated proanthocyanidins were confirmed to be responsible for this antiadhesive effect with epicatechin-3-O-gallate-(4?,8)-epicatechin-3’-O-gallate (syn. procyanidin B2-di-gallate) being the lead compound. Ungalloylated flavan-3-ols and oligomeric proanthocyanidins were inactive. RA1 and the galloylated proanthocyanidins strongly interact with the bacterial virulence factor Arg-gingipain, while the corresponding Lys-gingipain was hardly influenced. RA1 inhibited also hemagglutination. In silico docking studies indicated that epicatechin-3-O-gallate-(4?,8)-epicatechin-3’-O-gallate interacts with the active side of Arg-gingipain and hemaglutinin from P. gingivalis; the galloylation of the molecule seems to be responsible for fixation of the ligand to the protein. In conclusion, the proanthocyanidin-enriched extract RA1 and its main active constituent procyanidin B2-di-gallate protect cells from P. gingivalis infection by inhibiting bacterial adhesion to the host cell. RA1 and procyanidin B2-di-gallate appear to be promising candidates for future cytoprotective preparations for oral mouth care products. PMID:25803708

  16. Effects of a dietary yeast extract on hematological parameters, heterophil function, and bacterial clearance in turkey poults challenged with Escherichia coli and subjected to transport stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There is a need to develop nutritional methods for controlling pathogens in poultry production. A standardized yeast extract supplement, Alphamune™ (YE), was added to turkey poult diets. Male poults were challenged by air sac injection with 60 cfu of E. coli at 1 week of age. At 3 weeks of age chal...

  17. Pyrosequencing-based analysis of bacterial community and metabolites profiles in Korean traditional seafood fermentation: a flatfish-fermented seafood.

    PubMed

    Jung, Jaejoon; Lee, Se Hee; Jin, Hyun Mi; Jeon, Che Ok; Park, Woojun

    2014-01-01

    Bacterial community and metabolites were analyzed in a flatfish jeotgal, a Korean fermented seafood. Inverse relationship of pH and 16S rRNA gene copy number was identified during fermentation. Lactobacillus was the predominant bacterial genus. Increase of Firmicutes was a common characteristic shared by other fermented seafood. Fructose, glucose, and maltose were the major metabolites. PMID:25035997

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

    PubMed

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

    2006-01-01

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

  19. Small-Scale Vertical Distribution of Bacterial Biomass and Diversity in Biological Soil Crusts from Arid Lands in the Colorado Plateau

    USGS Publications Warehouse

    Garcia-Pichel, F.; Johnson, S.L.; Youngkin, D.; Belnap, J.

    2003-01-01

    We characterized, at millimeter resolution, bacterial biomass, diversity, and vertical stratification of biological soil crusts in arid lands from the Colorado Plateau. Microscopic counts, extractable DNA, and plate counts of viable aerobic copiotrophs (VAC) revealed that the top centimeter of crusted soils contained atypically large bacterial populations, tenfold larger than those in uncrusted, deeper soils. The plate counts were not always consistent with more direct estimates of microbial biomass. Bacterial populations peaked at the immediate subsurface (1-2 mm) in light-appearing, young crusts, and at the surface (0-1 mm) in well-developed, dark crusts, which corresponds to the location of cyanobacterial populations. Bacterial abundance decreased with depth below these horizons. Spatially resolved DGGE fingerprints of Bacterial 16S rRNA genes demonstrated the presence of highly diverse natural communities, but we could detect neither trends with depth in bacterial richness or diversity, nor a difference in diversity indices between crust types. Fingerprints, however, revealed the presence of marked stratification in the structure of the microbial communities, probably a result of vertical gradients in physicochemical parameters. Sequencing and phylogenetic analyses indicated that most of the naturally occurring bacteria are novel types, with low sequence similarity (83-93%) to those available in public databases. DGGE analyses of the VAC populations indicated communities of lower diversity, with most types having sequences more than 94% similar to those in public databases. Our study indicates that soil crusts represent small-scale mantles of fertility in arid ecosystems, harboring vertically structured, little-known bacterial populations that are not well represented by standard cultivation methods.

  20. Phylogenetic mapping of bacterial morphology

    NASA Technical Reports Server (NTRS)

    Siefert, J. L.; Fox, G. E.

    1998-01-01

    The availability of a meaningful molecular phylogeny for bacteria provides a context for examining the historical significance of various developments in bacterial evolution. Herein, the classical morphological descriptions of selected members of the domain Bacteria are mapped upon the genealogical ancestry deduced from comparison of small-subunit rRNA sequences. For the species examined in this study, a distinct pattern emerges which indicates that the coccus shape has arisen and accumulated independently multiple times in separate lineages and typically survived as a persistent end-state morphology. At least two other morphologies persist but have evolved only once. This study demonstrates that although bacterial morphology is not useful in defining bacterial phylogeny, it is remarkably consistent with that phylogeny once it is known. An examination of the experimental evidence available for morphogenesis as well as microbial fossil evidence corroborates these findings. It is proposed that the accumulation of persistent morphologies is a result of the biophysical properties of peptidoglycan and their genetic control, and that an evolved body-plan strategy based on peptidoglycan may have been a fate-sealing step in the evolution of Bacteria. More generally, this study illustrates that significant evolutionary insights can be obtained by examining biological and biochemical data in the context of a reliable phylogenetic structure.

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

    PubMed Central

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

    2006-01-01

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

  2. Structure of ERA in complex with the 3? end of 16S rRNA: Implications for ribosome biogenesis

    SciTech Connect

    Tu, Chao; Zhou, Xiaomei; Tropea, Joseph E.; Austin, Brian P.; Waugh, David S.; Court, Donald L.; Ji, Xinhua

    2009-10-09

    ERA, composed of an N-terminal GTPase domain followed by an RNA-binding KH domain, is essential for bacterial cell viability. It binds to 16S rRNA and the 30S ribosomal subunit. However, its RNA-binding site, the functional relationship between the two domains, and its role in ribosome biogenesis remain unclear. We have determined two crystal structures of ERA, a binary complex with GDP and a ternary complex with a GTP-analog and the {sub 1531}AUCACCUCCUUA{sub 1542} sequence at the 3' end of 16S rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein. We show that GTP binding is a prerequisite for RNA recognition by ERA and that RNA recognition stimulates its GTP-hydrolyzing activity. Based on these and other data, we propose a functional cycle of ERA, suggesting that the protein serves as a chaperone for processing and maturation of 16S rRNA and a checkpoint for assembly of the 30S ribosomal subunit. The AUCA sequence is highly conserved among bacteria, archaea, and eukaryotes, whereas the CCUCC, known as the anti-Shine-Dalgarno sequence, is conserved in noneukaryotes only. Therefore, these data suggest a common mechanism for a highly conserved ERA function in all three kingdoms of life by recognizing the AUCA, with a 'twist' for noneukaryotic ERA proteins by also recognizing the CCUCC.

  3. Molecular phylogenetic analysis of bacterial community and characterization of Cr(VI) reducers from the sediments of Tantloi hot spring, India

    PubMed Central

    2014-01-01

    Background A geothermal ecosystem located at Tantloi, India has been found to be an interesting habitat for microbes of diverse nature. However, the microbial diversity of this habitat is poorly explored. In this study, a detailed phylogenetic study has been carried out to understand the bacterial diversity of this habitat and to identify prospective metal reducers using culture independent approach. The bacterial diversity of the sediments, which contain undetectable levels of Cr(VI), was analysed with respect to chromium reduction and the strains highly resistant to and efficiently reducing chromium under aerobic conditions were isolated and characterized. Results 16S rRNA gene sequence analysis of Tantloi hot spring microbial community revealed a significant bacterial diversity represented by at least ten taxonomic divisions of Bacteria with clear predominance of Thermus. Similar sequence analysis of rRNA gene library clones derived from bacterial consortia enriched from sediments in presence of Cr(VI) revealed the abundance of the family Bacillaceae. Under aerobic conditions at 65°C, the consortia reduced 1 mM of Cr(VI) completely within 24 h and 5 mM in 6 days. A complete reduction of 1 mM Cr(VI) has been shown by five of our isolates within 36 h. 16S rRNA gene sequences of all the isolates showed high degree of similarity (97-99%) to Bacillaceae with ten of them being affiliated to Anoxybacillus. Crude extract as well as the soluble fraction from isolates TSB-1 and TSB-9 readily reduced Cr(VI); TSB-1 showed higher chromium reductase activity. Conclusion Most of the Tantloi Spring Bacterial (TSB) sequences analyzed in different taxonomic divisions could be related to representatives with known metabolic traits which indicated presence of organisms involved in redox processes of a variety of elements including iron, sulphur and chromium. Approximately 80% of the sequences obtained in this study represented novel phylotypes indicating the possibility of discovery of bacteria with biotechnologically important new biomolecules. Again, highly chromium-resistant and remarkably active Cr(VI)-reducing Anoxybacillus strains isolated in this study could serve as potential candidates for designing chromium bioremediation strategies at high temperatures and also at high chromium concentrations. PMID:25243065

  4. Spatial distribution, diversity and composition of bacterial communities in sub-seafloor fluids at a deep-sea hydrothermal field of the Suiyo Seamount

    NASA Astrophysics Data System (ADS)

    Kato, Shingo; Hara, Kurt; Kasai, Hiroko; Teramura, Takashi; Sunamura, Michinari; Ishibashi, Jun-ichiro; Kakegawa, Takeshi; Yamanaka, Toshiro; Kimura, Hiroyuki; Marumo, Katsumi; Urabe, Tetsuro; Yamagishi, Akihiko

    2009-10-01

    Spatial distribution, diversity, and composition of bacterial communities within the shallow sub-seafloor at the deep-sea hydrothermal field of the Suiyo Seamount, Izu-Bonin Arc, Western Pacific Ocean, were investigated. Fluids were sampled from four boreholes in this area. Each borehole was located near or away from active vents, the distance ranging 2-40 m from active vents. In addition, fluids discharging from a natural vent and ambient seawater were sampled in this area. We extracted DNA from each sample, amplified bacterial 16S rRNA genes by PCR, cloned the PCR products and sequenced. The total number of clones analyzed was 348. Most of the detected phylotypes were affiliated with the phylum Proteobacteria, of which the detection frequency in each clone library ranged from 84.6% to 100%. The bacterial community diversity and composition were different between hydrothermal fluids and seawater, between fluids from the boreholes and the vent, and even among fluids from each borehole. The relative abundances of the phylotypes related to Thiomicrospira, Methylobacterium and Sphingomonas were significantly different among fluids from each borehole. The phylotypes related to Thiomicrospira and Alcanivorax were detected in all of the boreholes and vent samples. Our findings provide insights into bacterial communities in the shallow sub-seafloor environments at active deep-sea hydrothermal vent fields.

  5. Characterization of rumen bacterial strains isolated from enrichments of rumen content in the presence of propolis.

    PubMed

    de Aguiar, Sílvia Cristina; Zeoula, Lucia Maria; do Prado, Odimari Pricila Pires; Arcuri, Pedro Braga; Forano, Evelyne

    2014-11-01

    Propolis presents many biological properties, including antibacterial activities, and has been proposed as an additive in ruminant nutrition. Twenty bacterial strains, previously isolated from enrichments of Brazilian cow rumen contents in the presence of different propolis extracts (LLOS), were characterized using phenotyping and 16S rRNA identification. Seven strains were assigned to Streptococcus sp., most likely S. bovis, and were all degrading starch. One amylolytic lactate-utilizing strain of Selenomonas ruminantium was also found. Two strains of Clostridium bifermentans were identified and showed proteolytic activity. Two strains were assigned to Mitsuokella jalaludinii and were saccharolytic. One strain belonged to a Bacillus species and seven strains were affiliated with Escherichia coli. All of the 20 strains were able to use many sugars, but none of them were able to degrade the polysaccharides carboxymethylcellulose and xylans. The effect of three propolis extracts (LLOS B1, C1 and C3) was tested on the in vitro growth of four representative isolates of S. bovis, E. coli, M. jalaludinii and C. bifermentans. The growth of S. bovis, E. coli and M. jalaludinii was not affected by the three propolis extracts at 1 mg ml(-1). C. bifermentans growth was completely inhibited at this LLOS concentration, but this bacterium was partially resistant at lower concentrations. LLOS C3, with the lower concentration of phenolic compounds, was a little less inhibitory than B1 and C1 on this strain. PMID:25172217

  6. Pyrosequencing-Derived Bacterial, Archaeal, and Fungal Diversity of Spacecraft Hardware Destined for Mars

    PubMed Central

    Vaishampayan, Parag; Nilsson, Henrik R.; Torok, Tamas; Venkateswaran, Kasthuri

    2012-01-01

    Spacecraft hardware and assembly cleanroom surfaces (233 m2 in total) were sampled, total genomic DNA was extracted, hypervariable regions of the 16S rRNA gene (bacteria and archaea) and ribosomal internal transcribed spacer (ITS) region (fungi) were subjected to 454 tag-encoded pyrosequencing PCR amplification, and 203,852 resulting high-quality sequences were analyzed. Bioinformatic analyses revealed correlations between operational taxonomic unit (OTU) abundance and certain sample characteristics, such as source (cleanroom floor, ground support equipment [GSE], or spacecraft hardware), cleaning regimen applied, and location about the facility or spacecraft. National Aeronautics and Space Administration (NASA) cleanroom floor and GSE surfaces gave rise to a larger number of diverse bacterial communities (619 OTU; 20 m2) than colocated spacecraft hardware (187 OTU; 162 m2). In contrast to the results of bacterial pyrosequencing, where at least some sequences were generated from each of the 31 sample sets examined, only 13 and 18 of these sample sets gave rise to archaeal and fungal sequences, respectively. As was the case for bacteria, the abundance of fungal OTU in the GSE surface samples dramatically diminished (9× less) once cleaning protocols had been applied. The presence of OTU representative of actinobacteria, deinococci, acidobacteria, firmicutes, and proteobacteria on spacecraft surfaces suggests that certain bacterial lineages persist even following rigorous quality control and cleaning practices. The majority of bacterial OTU observed as being recurrent belonged to actinobacteria and alphaproteobacteria, supporting the hypothesis that the measures of cleanliness exerted in spacecraft assembly cleanrooms (SAC) inadvertently select for the organisms which are the most fit to survive long journeys in space. PMID:22729532

  7. Biodegradation of Leonardite by an alkali-producing bacterial community and characterization of the degraded products.

    PubMed

    Gao, Tong-Guo; Jiang, Feng; Yang, Jin-Shui; Li, Bao-Zhen; Yuan, Hong-Li

    2012-03-01

    In this study, three bacterial communities were obtained from 12 Leonardite samples with the aim of identifying a clean, effective, and economic technique for the dissolution of Leonardite, a type of low-grade coal, in the production of humic acid (HA). The biodegradation ability and characteristics of the degraded products of the most effective bacterial community (MCSL-2), which degraded 50% of the Leonardite within 21 days, were further investigated. Analyses of elemental composition, (13)C NMR, and Fourier transform infrared revealed that the contents of C, O, and aliphatic carbon were similar in biodegraded humic acid (bHA) and chemically (alkali) extracted humic acid (cHA). However, the N and carboxyl carbon contents of bHA was higher than that of cHA. Furthermore, a positive correlation was identified between the degradation efficiency and the increasing pH of the culture medium, while increases of manganese peroxidase and esterase activities were also observed. These data demonstrated that both alkali production and enzyme reactions were involved in Leonardite solubilization by MCSL-2, although the former mechanism predominated. No fungus was observed by microscopy. Only four bacterial phylotypes were recognized, and Bacillus licheniformis-related bacteria were identified as the main group in MCSL-2 by analysis of amplified 16S rRNA genes, thus demonstrating that Leonardite degradation ability has a limited distribution in bacteria. Hormone-like bioactivities of bHA were also detected. In this study, a bacterial community capable of Leonardite degradation was identified and the products characterized. These data implicate the use of such bacteria for the exploitation of Leonardite as a biofertilizer. PMID:22075634

  8. Curcuma and Scutellaria plant extracts protect chickens against inflammation and Salmonella Enteritidis infection.

    PubMed

    Varmuzova, Karolina; Matulova, Marta Elsheimer; Gerzova, Lenka; Cejkova, Darina; Gardan-Salmon, Delphine; Panhéleux, Marina; Robert, Fabrice; Sisak, Frantisek; Havlickova, Hana; Rychlik, Ivan

    2015-09-01

    After a ban on the use of antibiotics as growth promoters in farm animals in the European Union in 2006, an interest in alternative products with antibacterial or anti-inflammatory properties has increased. In this study, we therefore tested the effects of extracts from Curcuma longa and Scutellaria baicalensis used as feed additives against cecal inflammation induced by heat stress or Salmonella Enteritidis (S. Enteritidis) infection in chickens. Curcuma extract alone was not enough to decrease gut inflammation induced by heat stress. However, a mixture of Curcuma and Scutellaria extracts used as feed additives decreased gut inflammation induced by heat or S. Enteritidis, decreased S. Enteritidis counts in the cecum but was of no negative effect on BW or humoral immune response. Using next-generation sequencing of 16S rRNA we found out that supplementation of feed with the 2 plant extracts had no effect on microbiota diversity. However, if the plant extract supplementation was provided to the chickens infected with S. Enteritidis, Faecalibacterium, and Lactobacillus, both bacterial genera with known positive effects on gut health were positively selected. The supplementation of chicken feed with extracts from Curcuma and Scutelleria thus may be used in poultry production to effectively decrease gut inflammation and increase chicken performance. PMID:26188032

  9. Atmospheric cloud water contains a diverse bacterial community

    NASA Astrophysics Data System (ADS)

    Kourtev, Peter S.; Hill, Kimberly A.; Shepson, Paul B.; Konopka, Allan

    2011-09-01

    Atmospheric cloud water contains an active microbial community which can impact climate, human health and ecosystem processes in terrestrial and aquatic systems. Most studies on the composition of microbial communities in clouds have been performed with orographic clouds that are typically in direct contact with the ground. We collected water samples from cumulus clouds above the upper U.S. Midwest. The cloud water was analyzed for the diversity of bacterial phylotypes by denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S rRNA gene amplicons. DGGE analyses of bacterial communities detected 17-21 bands per sample. Sequencing confirmed the presence of a diverse bacterial community; sequences from seven bacterial phyla were retrieved. Cloud water bacterial communities appeared to be dominated by members of the cyanobacteria, proteobacteria, actinobacteria and firmicutes.

  10. Atmospheric cloud water contains a diverse bacterial community

    SciTech Connect

    Kourtev, P. S.; Hill, Kimberly A.; Shepson, Paul B.; Konopka, Allan

    2011-06-15

    Atmospheric cloud water contains an active microbial community which can impact climate, human health and ecosystem processes in terrestrial and aquatic systems. Most studies on the composition of microbial communities in clouds have been performed with orographic clouds that are typically in direct contact with the ground. We collected water samples from cumulus clouds above the upper U.S. Midwest. The cloud water was analyzed for the diversity of bacterial phylotypes by denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S rRNA gene amplicons. DGGE analyses of bacterial communities detected 17e21 bands per sample. Sequencing confirmed the presence of a diverse bacterial community; sequences from seven bacterial phyla were retrieved. Cloud water bacterial communities appeared to be dominated by members of the cyanobacteria, proteobacteria, actinobacteria and firmicutes.

  11. Molecular diversity of drinking water bacterial communities using 16S rRNA gene sequence analyses

    EPA Science Inventory

    Our understanding of the microbial community structure of drinking water distribution system has relied on culture-based methods. However, recent studies have suggested that the majority of bacteria inhabiting distribution systems are unable to grow on artificial media. The goal ...

  12. Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective

    NASA Technical Reports Server (NTRS)

    Gutell, R. R.; Larsen, N.; Woese, C. R.

    1994-01-01

    The 16S and 23S rRNA higher-order structures inferred from comparative analysis are now quite refined. The models presented here differ from their immediate predecessors only in minor detail. Thus, it is safe to assert that all of the standard secondary-structure elements in (prokaryotic) rRNAs have been identified, with approximately 90% of the individual base pairs in each molecule having independent comparative support, and that at least some of the tertiary interactions have been revealed. It is interesting to compare the rRNAs in this respect with tRNA, whose higher-order structure is known in detail from its crystal structure (36) (Table 2). It can be seen that rRNAs have as great a fraction of their sequence in established secondary-structure elements as does tRNA. However, the fact that the former show a much lower fraction of identified tertiary interactions and a greater fraction of unpaired nucleotides than the latter implies that many of the rRNA tertiary interactions remain to be located. (Alternatively, the ribosome might involve protein-rRNA rather than intramolecular rRNA interactions to stabilize three-dimensional structure.) Experimental studies on rRNA are consistent to a first approximation with the structures proposed here, confirming the basic assumption of comparative analysis, i.e., that bases whose compositions strictly covary are physically interacting. In the exhaustive study of Moazed et al. (45) on protection of the bases in the small-subunit rRNA against chemical modification, the vast majority of bases inferred to pair by covariation are found to be protected from chemical modification, both in isolated small-subunit rRNA and in the 30S subunit. The majority of the tertiary interactions are reflected in the chemical protection data as well (45). On the other hand, many of the bases not shown as paired in Fig. 1 are accessible to chemical attack (45). However, in this case a sizeable fraction of them are also protected against chemical modification (in the isolated rRNA), which suggests that considerable higher-order structure remains to be found (although all of it may not involve base-base interactions and so may not be detectable by comparative analysis). The agreement between the higher-order structure of the small-subunit rRNA and protection against chemical modification is not perfect, however; some bases shown to covary canonically are accessible to chemical modification (45).(ABSTRACT TRUNCATED AT 400 WORDS).

  13. Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective.

    PubMed Central

    Gutell, R R; Larsen, N; Woese, C R

    1994-01-01

    The 16S and 23S rRNA higher-order structures inferred from comparative analysis are now quite refined. The models presented here differ from their immediate predecessors only in minor detail. Thus, it is safe to assert that all of the standard secondary-structure elements in (prokaryotic) rRNAs have been identified, with approximately 90% of the individual base pairs in each molecule having independent comparative support, and that at least some of the tertiary interactions have been revealed. It is interesting to compare the rRNAs in this respect with tRNA, whose higher-order structure is known in detail from its crystal structure (36) (Table 2). It can be seen that rRNAs have as great a fraction of their sequence in established secondary-structure elements as does tRNA. However, the fact that the former show a much lower fraction of identified tertiary interactions and a greater fraction of unpaired nucleotides than the latter implies that many of the rRNA tertiary interactions remain to be located. (Alternatively, the ribosome might involve protein-rRNA rather than intramolecular rRNA interactions to stabilize three-dimensional structure.) Experimental studies on rRNA are consistent to a first approximation with the structures proposed here, confirming the basic assumption of comparative analysis, i.e., that bases whose compositions strictly covary are physically interacting. In the exhaustive study of Moazed et al. (45) on protection of the bases in the small-subunit rRNA against chemical modification, the vast majority of bases inferred to pair by covariation are found to be protected from chemical modification, both in isolated small-subunit rRNA and in the 30S subunit. The majority of the tertiary interactions are reflected in the chemical protection data as well (45). On the other hand, many of the bases not shown as paired in Fig. 1 are accessible to chemical attack (45). However, in this case a sizeable fraction of them are also protected against chemical modification (in the isolated rRNA), which suggests that considerable higher-order structure remains to be found (although all of it may not involve base-base interactions and so may not be detectable by comparative analysis). The agreement between the higher-order structure of the small-subunit rRNA and protection against chemical modification is not perfect, however; some bases shown to covary canonically are accessible to chemical modification (45).(ABSTRACT TRUNCATED AT 400 WORDS) PMID:8177168

  14. Marine mesocosm bacterial colonisation of volcanic ash

    NASA Astrophysics Data System (ADS)

    Witt, Verena; Cimarelli, Corrado; Ayris, Paul; Kueppers, Ulrich; Erpenbeck, Dirk; Dingwell, Donald; Woerheide, Gert

    2015-04-01

    Volcanic eruptions regularly eject large quantities of ash particles into the atmosphere, which can be deposited via fallout into oceanic environments. Such fallout has the potential to alter pH, light and nutrient availability at local scales. Shallow-water coral reef ecosystems - "rainforests of the sea" - are highly sensitive to disturbances, such as ocean acidification, sedimentation and eutrophication. Therefore, wind-delivered volcanic ash may lead to burial and mortality of such reefs. Coral reef ecosystem resilience may depend on pioneer bacterial colonisation of the ash layer, supporting subsequent establishment of the micro- and ultimately the macro-community. However, which bacteria are involved in pioneer colonisation remain unknown. We hypothesize that physico-chemical properties (i.e., morphology, mineralogy) of the ash may dictate bacterial colonisation. The effect of substrate properties on bacterial colonisation was tested by exposing five substrates: i) quartz sand ii) crystalline ash (Sakurajima, Japan) iii) volcanic glass iv) carbonate reef sand and v) calcite sand of similar grain size, in controlled marine coral reef aquaria under low light conditions for six months. Bacterial communities were screened every month by Automated Ribosomal Intergenic Spacer Analysis of the 16S-23S rRNA Internal Transcribed Spacer region. Multivariate statistics revealed discrete groupings of bacterial communities on substrates of volcanic origin (ash and glass) and reef origin (three sands). Analysis of Similarity supported significantly different communities associated with all substrates (p=0.0001), only quartz did not differ from both carbonate and calcite sands. The ash substrate exhibited the most diverse bacterial community with the most substrate-specific bacterial operational taxonomic units. Our findings suggest that bacterial diversity and community composition during colonisation of volcanic ash in a coral reef-like environment is controlled by the physico-chemical composition of the substrate. Knowledge on pioneer bacterial colonisation may increase our understanding on the resilience of coral reefs to natural "catastrophes", such as volcanic ash fallout.

  15. Temporal dynamics of fibrolytic and methanogenic rumen microorganisms during in situ incubation of switchgrass determined by 16S rRNA gene profiling

    PubMed Central

    Piao, Hailan; Lachman, Medora; Malfatti, Stephanie; Sczyrba, Alexander; Knierim, Bernhard; Auer, Manfred; Tringe, Susannah G.; Mackie, Roderick I.; Yeoman, Carl J.; Hess, Matthias

    2014-01-01

    The rumen microbial ecosystem is known for its biomass-degrading and methane-producing phenotype. Fermentation of recalcitrant plant material, comprised of a multitude of interwoven fibers, necessitates the synergistic activity of diverse microbial taxonomic groups that inhabit the anaerobic rumen ecosystem. Although interspecies hydrogen (H2) transfer, a process during which bacterially generated H2 is transferred to methanogenic Archaea, has obtained significant attention over the last decades, the temporal variation of the different taxa involved in in situ biomass-degradation, H2 transfer and the methanogenesis process remains to be established. Here we investigated the temporal succession of microbial taxa and its effect on fiber composition during rumen incubation using 16S rRNA amplicon sequencing. Switchgrass filled nylon bags were placed in the rumen of a cannulated cow and collected at nine time points for DNA extraction and 16S pyrotag profiling. The microbial community colonizing the air-dried and non-incubated (0 h) switchgrass was dominated by members of the Bacilli (recruiting 63% of the pyrotag reads). During in situ incubation of the switchgrass, two major shifts in the community composition were observed: Bacilli were replaced within 30 min by members belonging to the Bacteroidia and Clostridia, which recruited 34 and 25% of the 16S rRNA reads generated, respectively. A second significant shift was observed after 16 h of rumen incubation, when members of the Spirochaetes and Fibrobacteria classes became more abundant in the fiber-adherent community. During the first 30 min of rumen incubation ~13% of the switchgrass dry matter was degraded, whereas little biomass degradation appeared to have occurred between 30 min and 4 h after the switchgrass was placed in the rumen. Interestingly, methanogenic members of the Euryarchaeota (i.e., Methanobacteria) increased up to 3-fold during this period of reduced biomass-degradation, with peak abundance just before rates of dry matter degradation increased again. We hypothesize that during this period microbial-mediated fibrolysis was temporarily inhibited until H2 was metabolized into CH4 by methanogens. Collectively, our results demonstrate the importance of inter-species interactions for the biomass-degrading and methane-producing phenotype of the rumen microbiome—both microbially facilitated processes with global significance. PMID:25101058

  16. Pyrosequencing 16S rRNA genes of bacteria associated with wild tiger mosquito Aedes albopictus: a pilot study

    PubMed Central

    Minard, Guillaume; Tran, Florence-Hélène; Dubost, Audrey; Tran-Van, Van; Mavingui, Patrick; Valiente Moro, Claire

    2014-01-01

    The Asian tiger mosquito Aedes (Stegomya) albopictus is an invasive species that has spread across the world in the last two decades, showing a great capacity to adapt to contrasting climates and environments. While demonstrated in many insects, the contribution of bacterial symbionts in Aedes ecology is a challenging aspect that needs to be investigated. Also some bacterial species have already been identified in Ae. albopictus using classical methods, but a more accurate survey of mosquito-associated bacterial diversity is needed to decipher the potential biological functions of bacterial symbionts in mediating or constraining insect adaptation. We surveyed the bacteria associated with field populations of Ae. albopictus from Madagascar by pyrosequencing 16S rRNA gene amplicons. Different aspects of amplicon preparation and sequencing depth were tested to optimize the breadth of bacterial diversity identified. The results revealed that all mosquitoes collected from different sites have a bacterial microbiota dominated by a single taxon, Wolbachia pipientis, which accounted for about 99% of all 92,615 sequences obtained. As Ae. albopictus is known to harbor two Wolbachia strains (wAlbA and wAlbB), a quantitative PCR was used to estimate the relative densities, (i.e., the bacteria-to-host gene ratios) of each strains in individual mosquitoes. Relative densities were between 6.25 × 100.01 and 5.47 × 100.1 for wAlbA and between 2.03 × 100.1 and 1.4 × 101 for wAlbB. Apart from Wolbachia, a total of 31 bacterial taxa were identified at the genus level using different method variations. Diversity index values were low and probably underestimated the true diversity due to the high abundance of Wolbachia sequences vastly outnumbering sequences from other taxa. Further studies should implement alternative strategies to specifically discard from analysis any sequences from Wolbachia, the dominant endosymbiotic bacterium in Ae. albopictus from this area. PMID:24860790

  17. 23S RRNA OPERON DIFFERENCES IN ISOLATES OF CAMPYLOBACTER SPP.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lateral transfer of genes appears to occur between Campylobacter species, but the extent of that transfer is not fully appreciated. The presence or absence of an intervening sequence (IVS) in the 23S rRNA has been noted in Campylobacter coli and Campylobacter jejuni. As part of a survey, we used P...

  18. RNA–Stable-Isotope Probing Shows Utilization of Carbon from Inulin by Specific Bacterial Populations in the Rat Large Bowel

    PubMed Central

    Lawley, Blair; Munro, Karen; Sims, Ian M.; Lee, Julian; Butts, Christine A.; Roy, Nicole

    2014-01-01

    Knowledge of the trophisms that underpin bowel microbiota composition is required in order to understand its complex phylogeny and function. Stable-isotope (13C)-labeled inulin was added to the diet of rats on a single occasion in order to detect utilization of inulin-derived substrates by particular members of the cecal microbiota. Cecal digesta from Fibruline-inulin-fed rats was collected prior to (0 h) and at 6, 12, 18 and 24 h following provision of the [13C]inulin diet. RNA was extracted from these cecal specimens and fractionated in isopycnic buoyant density gradients in order to detect 13C-labeled nucleic acid originating in bacterial cells that had metabolized the labeled dietary constituent. RNA extracted from specimens collected after provision of the labeled diet was more dense than 0-h RNA. Sequencing of 16S rRNA genes amplified from cDNA obtained from these fractions showed that Bacteroides uniformis, Blautia glucerasea, Clostridium indolis, and Bifidobacterium animalis were the main users of the 13C-labeled substrate. Culture-based studies of strains of these bacterial species enabled trophisms associated with inulin and its hydrolysis products to be identified. B. uniformis utilized Fibruline-inulin for growth, whereas the other species used fructo-oligosaccharide and monosaccharides. Thus, RNA–stable-isotope probing (RNA-SIP) provided new information about the use of carbon from inulin in microbiota metabolism. PMID:24487527

  19. Wide distribution and diversity of members of the bacterial kingdom Acidobacterium in the environment

    SciTech Connect

    Barns, S.M.; Takala, S.L.; Kuske, C.R.

    1999-04-01

    The assess the distribution and diversity of members of the recently identified bacterial kingdom Acidobacterium, members of this kingdom present in 43 environmental samples were surveyed by PCR amplification. A primer designated to amplify rRNA gene sequences (ribosomal DNAs [rDNAs]) from most known members of the kingdom was used to interrogate bulk DNA extracted from the samples. Positive PCR results were obtained with all temperate soil and sediment samples tested, as well as some hot spring samples, indicating that members of this kingdom are very widespread in terrestrial environments. PCR primers specific for four phylogenetic subgroups within the kingdom were used in similar surveys. All four subgroups were detected in most neutral soils and some sediments, while only two of the groups were seen in most low-pH environments. The combined use of these primers allowed identification of a novel lineage within the kingdom in a hot spring environment. Phylogenetic analysis of rDNA sequences from the survey and the literature outlines at least six major subgroups within the kingdom. Taken together, these data suggest that members of the Acidobacterium kingdom are as genetically and metabolically diverse, environmentally widespread and perhaps as ecologically important as the well-known Proteobacteria and gram-positive bacterial kingdoms.

  20. Hot-Alkaline DNA Extraction Method for Deep-Subseafloor Archaeal Communities

    PubMed Central

    Terada, Takeshi; Hoshino, Tatsuhiko; Inagaki, Fumio

    2014-01-01

    A prerequisite for DNA-based microbial community analysis is even and effective cell disruption for DNA extraction. With a commonly used DNA extraction kit, roughly two-thirds of subseafloor sediment microbial cells remain intact on average (i.e., the cells are not disrupted), indicating that microbial community analyses may be biased at the DNA extraction step, prior to subsequent molecular analyses. To address this issue, we standardized a new DNA extraction method using alkaline treatment and heating. Upon treatment with 1 M NaOH at 98°C for 20 min, over 98% of microbial cells in subseafloor sediment samples collected at different depths were disrupted. However, DNA integrity tests showed that such strong alkaline and heat treatment also cleaved DNA molecules into short fragments that could not be amplified by PCR. Subsequently, we optimized the alkaline and temperature conditions to minimize DNA fragmentation and retain high cell disruption efficiency. The best conditions produced a cell disruption rate of 50 to 80% in subseafloor sediment samples from various depths and retained sufficient DNA integrity for amplification of the complete 16S rRNA gene (i.e., ?1,500 bp). The optimized method also yielded higher DNA concentrations in all samples tested compared with extractions using a conventional kit-based approach. Comparative molecular analysis using real-time PCR and pyrosequencing of bacterial and archaeal 16S rRNA genes showed that the new method produced an increase in archaeal DNA and its diversity, suggesting that it provides better analytical coverage of subseafloor microbial communities than conventional methods. PMID:24441163

  1. A bacterial enrichment study and overview of the extractable lipids from paleosols in the Dry Valleys, Antarctica: implications for future Mars reconnaissance.

    PubMed

    Hart, Kris M; Szpak, Michal T; Mahaney, William C; Dohm, James M; Jordan, Sean F; Frazer, Andrew R; Allen, Christopher C R; Kelleher, Brian P

    2011-05-01

    The Dry Valleys of Antarctica are one of the coldest and driest environments on Earth with paleosols in selected areas that date to the emplacement of tills by warm-based ice during the Early Miocene. Cited as an analogue to the martian surface, the ability of the Antarctic environment to support microbial life-forms is a matter of special interest, particularly with the upcoming NASA/ESA 2018 ExoMars mission. Lipid biomarkers were extracted and analyzed by gas chromatography--mass spectrometry to assess sources of organic carbon and evaluate the contribution of microbial species to the organic matter of the paleosols. Paleosol samples from the ice-free Dry Valleys were also subsampled and cultivated in a growth medium from which DNA was extracted with the explicit purpose of the positive identification of bacteria. Several species of bacteria were grown in solution and the genus identified. A similar match of the data to sequenced DNA showed that Alphaproteobacteria, Gammaproteobacteria, Bacteriodetes, and Actinobacteridae species were cultivated. The results confirm the presence of bacteria within some paleosols, but no assumptions have been made with regard to in situ activity at present. These results underscore the need not only to further investigate Dry Valley cryosols but also to develop reconnaissance strategies to determine whether such likely Earth-like environments on the Red Planet also contain life. PMID:21545270

  2. Bacterial ?-glucosidase function and metabolic activity depend on soil management in semiarid rainfed agriculture

    PubMed Central

    Cañizares, Rosa; Moreno, Beatriz; Benitez, Emilio

    2012-01-01

    Genomic and transcriptomic approaches were used to gain insights into the relationship between soil management and bacterial-mediated functions in an olive orchard agroecosystem. Four management practices were assessed in a 30-year trial in a semiarid Mediterranean region. Transcriptional activity of bacterial 16S rRNA genes increased in noncovered soils, indicating higher microbial maintenance requirements to thrive in less favorable environmental conditions. The 16S rRNA transcript:gene copy ratio confirmed this assumption and pointed toward a much higher constitutive expression from rRNA operons in noncovered soils and to even higher expression levels when spontaneous vegetation was removed chemically. As described for 16S rRNA, potential transcription did not reveal the real transcription of bacterial ?-glucosidase genes, and higher gene expression in noncovered soils plus herbicides was evidenced. Since no relationship between total or soluble organic carbon and bacterial ?-glucosidase transcription was found, the above hypothesis could indicate either that soluble organic carbon is not the main pool of enzyme-inducing substrates or that constitutive production of bacterial ?-glucosidase enzymes increases as soil conditions worsen. PMID:22837821

  3. Salinity and Bacterial Diversity: To What Extent Does the Concentration of Salt Affect the Bacterial Community in a Saline Soil?

    PubMed Central

    Canfora, Loredana; Bacci, Giovanni; Pinzari, Flavia; Lo Papa, Giuseppe; Dazzi, Carmelo; Benedetti, Anna

    2014-01-01

    In this study, the evaluation of soil characteristics was coupled with a pyrosequencing analysis of the V2-V3 16S rRNA gene region in order to investigate the bacterial community structure and diversity in the A horizon of a natural saline soil located in Sicily (Italy). The main aim of the research was to assess the organisation and diversity of microbial taxa using a spatial scale that revealed physical and chemical heterogeneity of the habitat under investigation. The results provided information on the type of distribution of different bacterial groups as a function of spatial gradients of soil salinity and pH. The analysis of bacterial 16S rRNA showed differences in bacterial composition and diversity due to a variable salt concentration in the soil. The bacterial community showed a statistically significant spatial variability. Some bacterial phyla appeared spread in the whole area, whatever the salinity gradient. It emerged therefore that a patchy saline soil can not contain just a single microbial community selected to withstand extreme osmotic phenomena, but many communities that can be variously correlated to one or more environmental parameters. Sequences have been deposited to the SRA database and can be accessed on ID Project PRJNA241061. PMID:25188357

  4. Jellyfish Modulate Bacterial Dynamic and Community Structure

    PubMed Central

    Tinta, Tinkara; Kogovšek, Tjaša; Malej, Alenka; Turk, Valentina

    2012-01-01

    Jellyfish blooms have increased in coastal areas around the world and the outbreaks have become longer and more frequent over the past few decades. The Mediterranean Sea is among the heavily affected regions and the common bloom - forming taxa are scyphozoans Aurelia aurita s.l., Pelagia noctiluca, and Rhizostoma pulmo. Jellyfish have few natural predators, therefore their carcasses at the termination of a bloom represent an organic-rich substrate that supports rapid bacterial growth, and may have a large impact on the surrounding environment. The focus of this study was to explore whether jellyfish substrate have an impact on bacterial community phylotype selection. We conducted in situ jellyfish - enrichment experiment with three different jellyfish species. Bacterial dynamic together with nutrients were monitored to assess decaying jellyfish-bacteria dynamics. Our results show that jellyfish biomass is characterized by protein rich organic matter, which is highly bioavailable to ‘jellyfish - associated’ and ‘free - living’ bacteria, and triggers rapid shifts in bacterial population dynamics and composition. Based on 16S rRNA clone libraries and denaturing gradient gel electrophoresis (DGGE) analysis, we observed a rapid shift in community composition from unculturable Alphaproteobacteria to culturable species of Gammaproteobacteria and Flavobacteria. The results of sequence analyses of bacterial isolates and of total bacterial community determined by culture independent genetic analysis showed the dominance of the Pseudoalteromonadaceae and the Vibrionaceae families. Elevated levels of dissolved proteins, dissolved organic and inorganic nutrient release, bacterial abundance and carbon production as well as ammonium concentrations characterized the degradation process. The biochemical composition of jellyfish species may influence changes in the amount of accumulated dissolved organic and inorganic nutrients. Our results can contribute insights into possible changes in bacterial population dynamics and nutrient pathways following jellyfish blooms which have important implications for ecology of coastal waters. PMID:22745726

  5. Molecular Analysis of Bacterial Microbiota on Brazilian Currency Note Surfaces

    PubMed Central

    Pereira da Fonseca, Tairacan Augusto; Pessôa, Rodrigo; Sanabani, Sabri Saeed

    2015-01-01

    Currency notes have been implicated as a vehicle for transmitting community-acquired bacterial infections. However, the overall diversity of the bacterial population residing on banknotes is still unknown in Brazil. In this study, we aimed to investigate the overall bacterial population from 150 different Brazilian Rial (R$) notes in circulation using a culture-independent Illumina massively parallel sequencing approach of the 16S rRNA genes. Samples were randomly collected from three different street markets or “feiras” in the metropolitan region of São Paulo. Taxonomical composition revealed the abundance of Proteobacteria phyla, followed by Firmicutes and Streptophyta, with a total of 1193 bacterial families and 3310 bacterial genera. Most of these bacterial genera are of human, animal, and environmental origins. Also, our analysis revealed the presence of some potential pathogenic bacterial genera including Salmonella, Staphylococcus, and Klebsiella. The results demonstrate that there is a tremendous diversity of bacterial contamination on currency notes, including organisms known to be opportunistic pathogens. One of the factors that may contribute to the richness of bacterial diversity in currency notes is personal hygiene. Thus, our results underscore the need to increase public awareness of the importance of personal hygiene of money handlers who also handle food. PMID:26506368

  6. Molecular Analysis of Bacterial Microbiota on Brazilian Currency Note Surfaces.

    PubMed

    Pereira da Fonseca, Tairacan Augusto; Pessôa, Rodrigo; Sanabani, Sabri Saeed

    2015-10-01

    Currency notes have been implicated as a vehicle for transmitting community-acquired bacterial infections. However, the overall diversity of the bacterial population residing on banknotes is still unknown in Brazil. In this study, we aimed to investigate the overall bacterial population from 150 different Brazilian Rial (R$) notes in circulation using a culture-independent Illumina massively parallel sequencing approach of the 16S rRNA genes. Samples were randomly collected from three different street markets or "feiras" in the metropolitan region of São Paulo. Taxonomical composition revealed the abundance of Proteobacteria phyla, followed by Firmicutes and Streptophyta, with a total of 1193 bacterial families and 3310 bacterial genera. Most of these bacterial genera are of human, animal, and environmental origins. Also, our analysis revealed the presence of some potential pathogenic bacterial genera including Salmonella, Staphylococcus, and Klebsiella. The results demonstrate that there is a tremendous diversity of bacterial contamination on currency notes, including organisms known to be opportunistic pathogens. One of the factors that may contribute to the richness of bacterial diversity in currency notes is personal hygiene. Thus, our results underscore the need to increase public awareness of the importance of personal hygiene of money handlers who also handle food. PMID:26506368

  7. Diversity and distribution of 16S rRNA and phenol monooxygenase genes in the rhizosphere and endophytic bacteria isolated from PAH-contaminated sites

    PubMed Central

    Peng, Anping; Liu, Juan; Ling, Wanting; Chen, Zeyou; Gao, Yanzheng

    2015-01-01

    This is the first investigation of the diversity and distribution of 16S rRNA and phenol monooxygenase (PHE) genes in endophytic and rhizosphere bacteria of plants at sites contaminated with different levels of PAHs. Ten PAHs at concentrations from 34.22 to 55.29 and 45.79 to 97.81?mg·kg?1 were measured in rhizosphere soils of Alopecurus aequalis Sobol and Oxalis corniculata L., respectively. The diversity of 16S rRNA and PHE genes in rhizosphere soils or plants changed with varying PAH pollution levels, as shown based on PCR-DGGE data. Generally, higher Shannon-Weiner indexes were found in mild or moderate contaminated areas. A total of 82 different bacterial 16S rRNA gene sequences belonging to five phyla; namely, Acfinobacteria, Proteobacteria, Chloroflexi, Cyanophyta, and Bacteroidetes, were obtained from rhizosphere soils. For the 57 identified PHE gene sequences, 18 were excised from rhizosphere bacteria and 39 from endophytic bacteria. The copy numbers of 16S rRNA and PHE genes in rhizosphere and endophytic bacteria varied from 3.83?×?103 to 2.28?×?106 and 4.17?×?102 to 1.99?×?105, respectively. The copy numbers of PHE genes in rhizosphere bacteria were significantly higher than in endophytic bacteria. Results increase our understanding of the diversity of rhizosphere and endophytic bacteria from plants grown in PAH-contaminated sites. PMID:26184609

  8. Diversity and distribution of 16S rRNA and phenol monooxygenase genes in the rhizosphere and endophytic bacteria isolated from PAH-contaminated sites

    NASA Astrophysics Data System (ADS)

    Peng, Anping; Liu, Juan; Ling, Wanting; Chen, Zeyou; Gao, Yanzheng

    2015-07-01

    This is the first investigation of the diversity and distribution of 16S rRNA and phenol monooxygenase (PHE) genes in endophytic and rhizosphere bacteria of plants at sites contaminated with different levels of PAHs. Ten PAHs at concentrations from 34.22 to 55.29 and 45.79 to 97.81?mg·kg-1 were measured in rhizosphere soils of Alopecurus aequalis Sobol and Oxalis corniculata L., respectively. The diversity of 16S rRNA and PHE genes in rhizosphere soils or plants changed with varying PAH pollution levels, as shown based on PCR-DGGE data. Generally, higher Shannon-Weiner indexes were found in mild or moderate contaminated areas. A total of 82 different bacterial 16S rRNA gene sequences belonging to five phyla; namely, Acfinobacteria, Proteobacteria, Chloroflexi, Cyanophyta, and Bacteroidetes, were obtained from rhizosphere soils. For the 57 identified PHE gene sequences, 18 were excised from rhizosphere bacteria and 39 from endophytic bacteria. The copy numbers of 16S rRNA and PHE genes in rhizosphere and endophytic bacteria varied from 3.83?×?103 to 2.28?×?106 and 4.17?×?102 to 1.99?×?105, respectively. The copy numbers of PHE genes in rhizosphere bacteria were significantly higher than in endophytic bacteria. Results increase our understanding of the diversity of rhizosphere and endophytic bacteria from plants grown in PAH-contaminated sites.

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

    PubMed

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

    2015-01-01

    Because of human population growth, increased food production and alternatives to conventional methods of biocontrol and development of plants such as the use of endophytic bacteria and fungi are required. One of the methods used to study microorganism diversity is sequencing of the 16S rRNA gene, which has several advantages, including universality, size, and availability of databases for comparison. The objective of this study was to analyze endophytic bacterial diversity in agricultural crops using published papers, sequence databases, and phylogenetic analysis. Fourteen papers were selected in which the ribosomal 16S rRNA gene was used to identify endophytic bacteria, in important agricultural crops, such as coffee, sugar cane, beans, corn, soybean, tomatoes, and grapes, located in different geographical regions (America, Europe, and Asia). The corresponding 16S rRNA gene sequences were selected from the NCBI database, aligned using the Mega 5.2 program, and phylogenetic analysis was undertaken. The most common orders present in the analyzed cultures were Bacillales, Enterobacteriales, and Actinomycetales and the most frequently observed genera were Bacillus, Pseudomonas, and Microbacterium. Phylogenetic analysis showed that only approximately 1.56% of the total sequences were not properly grouped, demonstrating reliability in the identification of microorganisms. This study identified the main genera found in endophytic bacterial cultures from plants, providing data for future studies on improving plant agriculture, biotechnology, endophytic bacterium prospecting, and to help understand relationships between endophytic bacteria and their interactions with plants. PMID:26345903

  10. [Atopobium vaginae: characterization and association with pathogenesis of bacterial vaginosis].

    PubMed

    Romanik, Ma?gorzata; Friedek, Daniela; Wojciechowska-Wieja, Anna; Martirosian, Gayane

    2006-05-01

    Atopobium vaginae was described in 1999 by Rodriguez et al. It is a Gram-positive bacterium producing organic acids (lactic acid, acetic acid, formic acid) as a results of glucose fermentation. It was first found in vaginal swab taken from a healthy woman using 16S rRNA analysis. A. vaginae is associated with bacterial vaginosis and its consequences in upper part of women reproductive organs. PMID:16958231

  11. Transcriptional control of a rRNA promoter of the nodulating symbiont Sinorhizobium meliloti

    E-print Network

    Gage, Daniel J.

    Transcriptional control of a rRNA promoter of the nodulating symbiont Sinorhizobium meliloti-copy-number plasmid containing a fusion between a Sinorhizobium meliloti rRNA promoter and gfp(mut3). When transformed. The explanation for this appears to be that transcription from the rRNA promoter was shut off in mid

  12. Back to Basics – The Influence of DNA Extraction and Primer Choice on Phylogenetic Analysis of Activated Sludge Communities

    PubMed Central

    Kirkegaard, Rasmus H.; Nielsen, Per H.

    2015-01-01

    DNA extraction and primer choice have a large effect on the observed community structure in all microbial amplicon sequencing analyses. Although the biases are well known, no comprehensive analysis has been conducted in activated sludge communities. In this study we systematically explored the impact of a number of parameters on the observed microbial community: bead beating intensity, primer choice, extracellular DNA removal, and various PCR settings. In total, 176 samples were subjected to 16S rRNA amplicon sequencing, and selected samples were investigated through metagenomics and metatranscriptomics. Quantitative fluorescence in situ hybridization was used as a DNA extraction-independent method for qualitative comparison. In general, an effect on the observed community was found on all parameters tested, although bead beating and primer choice had the largest effect. The effect of bead beating intensity correlated with cell-wall strength as seen by a large increase in DNA from Gram-positive bacteria (up to 400%). However, significant differences were present at lower phylogenetic levels within the same phylum, suggesting that additional factors are at play. The best primer set based on in silico analysis was found to underestimate a number of important bacterial groups. For 16S rRNA gene analysis in activated sludge we recommend using the FastDNA SPIN Kit for Soil with four times the normal bead beating and V1-3 primers. PMID:26182345

  13. Characterization of Halophilic Bacterial Communities in Turda Salt Mine (Romania)

    NASA Astrophysics Data System (ADS)

    Carpa, Rahela; Keul, Anca; Muntean, Vasile; Dobrot?, Cristina

    2014-09-01

    Halophilic organisms are having adaptations to extreme salinity, the majority of them being Archaean, which have the ability to grow at extremely high salt concentrations, (from 3 % to 35 %). Level of salinity causes natural fluctuations in the halophilic populations that inhabit this particular habitat, raising problems in maintaining homeostasis of the osmotic pressure. Samples such as salt and water taken from Turda Salt Mine were analyzed in order to identify the eco-physiological bacterial groups. Considering the number of bacteria of each eco-physiological group, the bacterial indicators of salt quality (BISQ) were calculated and studied for each sample. The phosphatase, catalase and dehydrogenases enzymatic activities were quantitatively determined and the enzymatic indicators of salt quality (EISQ) were calculated. Bacterial isolates were analyzed using 16S rRNA gene sequence analysis. Universal bacterial primers, targeting the consensus region of the bacterial 16S rRNA gene were used. Analysis of a large fragment, of 1499 bp was performed to improve discrimination at the species level.

  14. Diversity of bacterial isolates during full scale rotary drum composting.

    PubMed

    Bhatia, Akansha; Madan, Sangeeta; Sahoo, Jitendra; Ali, Muntjeer; Pathania, Ranjana; Kazmi, Absar Ahmed

    2013-07-01

    Bacterial diversity of full scale rotary drum composter from biodegradable organic waste samples were analyzed through two different approaches, i.e., Culture dependent and independent techniques. Culture-dependent enumerations for indigenous population of bacterial isolates mainly total heterotrophic bacteria (Bacillus species, Pseudomonas species and Enterobacter species), Fecal Coliforms, Fecal Streptococci, Escherichia coli, Salmonella species and Shigella species showed reduction during the composting period. On the other hand, Culture-independent method using PCR amplification of specific 16S rRNA sequences identified the presence of Acinetobacter species, Actinobacteria species, Bacillus species, Clostridium species, Hydrogenophaga species, Butyrivibrio species, Pedobacter species, Empedobactor species and Flavobacterium species by sequences clustering in the phylogenetic tree. Furthermore, correlating physico-chemical analysis of samples with bacterial diversity revealed the bacterial communities have undergone changes, possibly linked to the variations in temperature and availability of new metabolic substrates while decomposing organics at different stages of composting. PMID:23663960

  15. Bacterial polyhydroxyalkanoates.

    PubMed

    Lee, S Y

    1996-01-01

    Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates (HAs) synthesized by numerous bacteria as intracellular carbon and energy storage compounds and accumulated as granules in the cytoplasm of cells. More than 80 HAs have been detected as constituents of PHAs, which allows these thermoplastic materials to have various mechanical properties resembling hard crystalline polymer or elastic rubber depending on the incorporated monomer units. Even though PHAs have been recognized as good candidates for biodegradable plastics, their high price compared with conventional plastics has limited their use in a wide range of applications. A number of bacteria including Alcaligenes eutrophus, Alcaligenes latus, Azotobacter vinelandii, methylotrophs, pseudomonads, and recombinant Escherichia coli have been employed for the production of PHAs, and the productivity of greater than 2 g PHA/L/h has been achieved. Recent advances in understanding metabolism, molecular biology, and genetics of the PHA-synthesizing bacteria and cloning of more than 20 different PHA biosynthesis genes allowed construction of various recombinant strains that were able to synthesize polyesters having different monomer units and/or to accumulate much more polymers. Also, genetically engineered plants harboring the bacterial PHA biosynthesis genes are being developed for the economical production of PHAs. Improvements in fermentation/separation technology and the development of bacterial strains or plants that more efficiently synthesize PHAs will bring the costs down to make PHAs competitive with the conventional plastics. PMID:18623547

  16. Drivers shaping the diversity and biogeography of total and active bacterial communities in the South China Sea

    PubMed Central

    Zhang, Yao; Zhao, Zihao; Dai, Minhan; Jiao, Nianzhi; Herndl, Gerhard J

    2014-01-01

    To test the hypothesis that different drivers shape the diversity and biogeography of the total and active bacterial community, we examined the bacterial community composition along two transects, one from the inner Pearl River estuary to the open waters of the South China Sea (SCS) and the other from the Luzon Strait to the SCS basin, using 454 pyrosequencing of the 16S rRNA and 16S rRNA gene (V1-3 regions) and thereby characterizing the active and total bacterial community, respectively. The diversity and biogeographic patterns differed substantially between the active and total bacterial communities. Although the composition of both the total and active bacterial community was strongly correlated with environmental factors and weakly correlated with geographic distance, the active bacterial community displayed higher environmental sensitivity than the total community and particularly a greater distance effect largely caused by the active assemblage from deep waters. The 16S rRNA vs. rDNA relationships indicated that the active bacteria were low in relative abundance in the SCS. This might be due to a high competition between active bacterial taxa as indicated by our community network models. Based on these analyses, we speculate that high competition could cause some dispersal limitation of the active bacterial community resulting in a distinct distance-decay relationship. Altogether, our results indicated that the biogeographic distribution of bacteria in the SCS is the result of both environmental control and distance decay. PMID:24684298

  17. Metagenomic evaluation of bacterial and archaeal diversity in the geothermal hot springs of manikaran, India.

    PubMed

    Bhatia, Sonu; Batra, Navneet; Pathak, Ashish; Green, Stefan J; Joshi, Amit; Chauhan, Ashvini

    2015-01-01

    Bacterial and archaeal diversity in geothermal spring water were investigated using 16S rRNA gene amplicon metagenomic sequencing. This revealed the dominance of Firmicutes, Aquificae, and the Deinococcus-Thermus group in this thermophilic environment. A number of sequences remained taxonomically unresolved, indicating the presence of potentially novel microbes in this unique habitat. PMID:25700403

  18. Metagenomic Evaluation of Bacterial and Archaeal Diversity in the Geothermal Hot Springs of Manikaran, India

    PubMed Central

    Pathak, Ashish; Green, Stefan J.; Joshi, Amit; Chauhan, Ashvini

    2015-01-01

    Bacterial and archaeal diversity in geothermal spring water were investigated using 16S rRNA gene amplicon metagenomic sequencing. This revealed the dominance of Firmicutes, Aquificae, and the Deinococcus-Thermus group in this thermophilic environment. A number of sequences remained taxonomically unresolved, indicating the presence of potentially novel microbes in this unique habitat. PMID:25700403

  19. Antarctic ice core samples: culturable bacterial diversity.

    PubMed

    Shivaji, Sisinthy; Begum, Zareena; Shiva Nageswara Rao, Singireesu Soma; Vishnu Vardhan Reddy, Puram V; Manasa, Poorna; Sailaja, Buddi; Prathiba, Mambatta S; Thamban, Meloth; Krishnan, Kottekkatu P; Singh, Shiv M; Srinivas, Tanuku N R

    2013-01-01

    Culturable bacterial abundance at 11 different depths of a 50.26 m ice core from the Tallaksenvarden Nunatak, Antarctica, varied from 0.02 to 5.8 × 10(3) CFU ml(-1) of the melt water. A total of 138 bacterial strains were recovered from the 11 different depths of the ice core. Based on 16S rRNA gene sequence analyses, the 138 isolates could be categorized into 25 phylotypes belonging to phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. All isolates had 16S rRNA sequences similar to previously determined sequences (97.2-100%). No correlation was observed in the distribution of the isolates at the various depths either at the phylum, genus or species level. The 25 phylotypes varied in growth temperature range, tolerance to NaCl, growth pH range and ability to produce eight different extracellular enzymes at either 4 or 18 °C. Iso-, anteiso-, unsaturated and saturated fatty acids together constituted a significant proportion of the total fatty acid composition. PMID:23041141

  20. Growth rate regulation of rRNA content of a marine Synechococcus (cyanobacterium) strain

    SciTech Connect

    Binder, B.J.; Liu, Y.C.

    1998-09-01

    The relationship between growth rate and rRNA content in a marine Synechococcus strain was examined. A combination of flow cytometry and whole-cell hybridization with fluorescently labeled 16S rRNA-targeted oligonucleotide probes was used to measure the rRNA content of Synechococcus strain WH8101 cells grown at a range of light-limited growth rates. The sensitivity of this approach was sufficient for the analysis of rRNA even in very slowly growing Synechococcus cells. The relationship between growth rate and cellular rRNA content comprised three phases: (1) at low growth rates, rRNA cell{sup {minus}1} remained approximately constant; (2) at intermediate rates, rRNA cell{sup {minus}1} increased proportionally with growth rate; and (3) at the highest, light-saturated rates, rRNA cell{sup {minus}1} dropped abruptly. Total cellular RNA was well correlated with the probe-based measure of rRNA and varied in a similar manner with growth rate. Mean cell volume and rRNA concentration were related to growth rate in a manner similar to rRNA cell{sup {minus}1}, although the overall magnitude linear increase in ribosome efficiency with increasing growth rate, which is consistent with the prevailing prokaryotic model at low growth rates. Taken together, these results support the notion that measurements of cellular rRNA content might be useful for estimating in situ growth rates in natural Synechococcus populations.

  1. Bacterial hydrodynamics

    E-print Network

    Eric Lauga

    2015-09-07

    Bacteria predate plants and animals by billions of years. Today, they are the world's smallest cells yet they represent the bulk of the world's biomass, and the main reservoir of nutrients for higher organisms. Most bacteria can move on their own, and the majority of motile bacteria are able to swim in viscous fluids using slender helical appendages called flagella. Low-Reynolds-number hydrodynamics is at the heart of the ability of flagella to generate propulsion at the micron scale. In fact, fluid dynamic forces impact many aspects of bacteriology, ranging from the ability of cells to reorient and search their surroundings to their interactions within mechanically and chemically-complex environments. Using hydrodynamics as an organizing framework, we review the biomechanics of bacterial motility and look ahead to future challenges.

  2. Bacterial hydrodynamics

    E-print Network

    Lauga, Eric

    2015-01-01

    Bacteria predate plants and animals by billions of years. Today, they are the world's smallest cells yet they represent the bulk of the world's biomass, and the main reservoir of nutrients for higher organisms. Most bacteria can move on their own, and the majority of motile bacteria are able to swim in viscous fluids using slender helical appendages called flagella. Low-Reynolds-number hydrodynamics is at the heart of the ability of flagella to generate propulsion at the micron scale. In fact, fluid dynamic forces impact many aspects of bacteriology, ranging from the ability of cells to reorient and search their surroundings to their interactions within mechanically and chemically-complex environments. Using hydrodynamics as an organizing framework, we review the biomechanics of bacterial motility and look ahead to future challenges.

  3. Bacterial Cholangitis.

    PubMed

    Lum, Donald F.; Leung, Joseph W.

    2001-04-01

    The treatment of acute bacterial cholangitis requires broad-spectrum antibiotics to cover against gram-negative aerobic enteric organisms (Escherichia coli, Klebsiella species, and Enterobacter species), gram-positive Enterococcus and anaerobic bacteria (Bacteroides fragilis and Clostridium perfringens). Approximately 20% of patients with acute cholangitis fail to respond to conservative treatment with antibiotic therapy and require urgent biliary decompression, which is the mainstay of therapy. This is best accomplished by endoscopic retrograde cholangiopancreatography (ERCP) and placement of a nasobiliary drainage tube or a large bore (10 F or larger) indwelling plastic stent. Alternative therapy includes percutaneous transhepatic biliary drainage or surgical biliary decompression, but these carry a significantly higher morbidity and mortality. Supportive care includes intravenous fluid hydration to prevent renal failure and close monitoring of vital signs for determination of potential septicemia. PMID:11469972

  4. Beyond Streptococcus mutans: dental caries onset linked to multiple species by 16S rRNA community analysis.

    PubMed

    Gross, Erin L; Beall, Clifford J; Kutsch, Stacey R; Firestone, Noah D; Leys, Eugene J; Griffen, Ann L

    2012-01-01

    Dental caries in very young children may be severe, result in serious infection, and require general anesthesia for treatment. Dental caries results from a shift within the biofilm community specific to the tooth surface, and acidogenic species are responsible for caries. Streptococcus mutans, the most common acid producer in caries, is not always present and occurs as part of a complex microbial community. Understanding the degree to which multiple acidogenic species provide functional redundancy and resilience to caries-associated communities will be important for developing biologic interventions. In addition, microbial community interactions in health and caries pathogenesis are not well understood. The purpose of this study was to investigate bacterial community profiles associated with the onset of caries in the primary dentition. In a combination cross-sectional and longitudinal design, bacterial community profiles at progressive stages of caries and over time were examined and compared to those of health. 16S rRNA gene sequencing was used for bacterial community analysis. Streptococcus mutans was the dominant species in many, but not all, subjects with caries. Elevated levels of S. salivarius, S. sobrinus, and S. parasanguinis were also associated with caries, especially in subjects with no or low levels of S. mutans, suggesting these species are alternative pathogens, and that multiple species may need to be targeted for interventions. Veillonella, which metabolizes lactate, was associated with caries and was highly correlated with total acid producing species. Among children without previous history of caries, Veillonella, but not S. mutans or other acid-producing species, predicted future caries. Bacterial community diversity was reduced in caries as compared to health, as many species appeared to occur at lower levels or be lost as caries advanced, including the Streptococcus mitis group, Neisseria, and Streptococcus sanguinis. This may have implications for bacterial community resilience and the restoration of oral health. PMID:23091642

  5. Beyond Streptococcus mutans: Dental Caries Onset Linked to Multiple Species by 16S rRNA Community Analysis

    PubMed Central

    Gross, Erin L.; Beall, Clifford J.; Kutsch, Stacey R.; Firestone, Noah D.; Leys, Eugene J.; Griffen, Ann L.

    2012-01-01

    Dental caries in very young children may be severe, result in serious infection, and require general anesthesia for treatment. Dental caries results from a shift within the biofilm community specific to the tooth surface, and acidogenic species are responsible for caries. Streptococcus mutans, the most common acid producer in caries, is not always present and occurs as part of a complex microbial community. Understanding the degree to which multiple acidogenic species provide functional redundancy and resilience to caries-associated communities will be important for developing biologic interventions. In addition, microbial community interactions in health and caries pathogenesis are not well understood. The purpose of this study was to investigate bacterial community profiles associated with the onset of caries in the primary dentition. In a combination cross-sectional and longitudinal design, bacterial community profiles at progressive stages of caries and over time were examined and compared to those of health. 16S rRNA gene sequencing was used for bacterial community analysis. Streptococcus mutans was the dominant species in many, but not all, subjects with caries. Elevated levels of S. salivarius, S. sobrinus, and S. parasanguinis were also associated with caries, especially in subjects with no or low levels of S. mutans, suggesting these species are alternative pathogens, and that multiple species may need to be targeted for interventions. Veillonella, which metabolizes lactate, was associated with caries and was highly correlated with total acid producing species. Among children without previous history of caries, Veillonella, but not S. mutans or other acid-producing species, predicted future caries. Bacterial community diversity was reduced in caries as compared to health, as many species appeared to occur at lower levels or be lost as caries advanced, including the Streptococcus mitis group, Neisseria, and Streptococcus sanguinis. This may have implications for bacterial community resilience and the restoration of oral health. PMID:23091642

  6. Extremely Acidophilic Protists from Acid Mine Drainage Host Rickettsiales-Lineage Endosymbionts That Have Intervening Sequences in Their 16S rRNA Genes

    PubMed Central

    Baker, Brett J.; Hugenholtz, Philip; Dawson, Scott C.; Banfield, Jillian F.

    2003-01-01

    During a molecular phylogenetic survey of extremely acidic (pH < 1), metal-rich acid mine drainage habitats in the Richmond Mine at Iron Mountain, Calif., we detected 16S rRNA gene sequences of a novel bacterial group belonging to the order Rickettsiales in the Alphaproteobacteria. The closest known relatives of this group (92% 16S rRNA gene sequence identity) are endosymbionts of the protist Acanthamoeba. Oligonucleotide 16S rRNA probes were designed and used to observe members of this group within acidophilic protists. To improve visualization of eukaryotic populations in the acid mine drainage samples, broad-specificity probes for eukaryotes were redesigned and combined to highlight this component of the acid mine drainage community. Approximately 4% of protists in the acid mine drainage samples contained endosymbionts. Measurements of internal pH of the protists showed that their cytosol is close to neutral, indicating that the endosymbionts may be neutrophilic. The endosymbionts had a conserved 273-nucleotide intervening sequence (IVS) in variable region V1 of their 16S rRNA genes. The IVS does not match any sequence in current databases, but the predicted secondary structure forms well-defined stem loops. IVSs are uncommon in rRNA genes and appear to be confined to bacteria living in close association with eukaryotes. Based on the phylogenetic novelty of the endosymbiont sequences and initial culture-independent characterization, we propose the name “Candidatus Captivus acidiprotistae.” To our knowledge, this is the first report of an endosymbiotic relationship in an extremely acidic habitat. PMID:12957940

  7. The phylogenetic position of Siboglinidae (Annelida) inferred from 18S rRNA, 28S rRNA and morphological data

    E-print Network

    Siddall, Mark E.

    The phylogenetic position of Siboglinidae (Annelida) inferred from 18S rRNA, 28S r and Vestimentifera, but now referred to Annelida) in parsimony analyses of 1100 bp from 18S rRNA, 320 bp from the D1). For a while, they were regarded as not being closely related to Annelida, but referred to as deuterostomes

  8. Bacterial biodiversity from anthropogenic extreme environments: a hyper-alkaline and hyper-saline industrial residue contaminated by chromium and iron.

    PubMed

    Brito, Elcia M S; Piñón-Castillo, Hilda A; Guyoneaud, Rémy; Caretta, César A; Gutiérrez-Corona, J Félix; Duran, Robert; Reyna-López, Georgina E; Nevárez-Moorillón, G Virginia; Fahy, Anne; Goñi-Urriza, Marisol

    2013-01-01

    Anthropogenic extreme environments are among the most interesting sites for the bioprospection of extremophiles since the selection pressures may favor the presence of microorganisms of great interest for taxonomical and astrobiological research as well as for bioremediation technologies and industrial applications. In this work, T-RFLP and 16S rRNA gene library analyses were carried out to describe the autochthonous bacterial populations from an industrial waste characterized as hyper-alkaline (pH between 9 and 14), hyper-saline (around 100 PSU) and highly contaminated with metals, mainly chromium (from 5 to 18 g kg(-1)) and iron (from 2 to 108 g kg(-1)). Due to matrix interference with DNA extraction, a protocol optimization step was required in order to carry out molecular analyses. The most abundant populations, as evaluated by both T-RFLP and 16S rRNA gene library analyses, were affiliated to Bacillus and Lysobacter genera. Lysobacter related sequences were present in the three samples: solid residue and lixiviate sediments from both dry and wet seasons. Sequences related to Thiobacillus were also found; although strains affiliated to this genus are known to have tolerance to metals, they have not previously been detected in alkaline environments. Together with Bacillus (already described as a metal reducer), such organisms could be of use in bioremediation technologies for reducing chromium, as well as for the prospection of enzymes of biotechnological interest. PMID:22350256

  9. Complete nucleotide sequence of the 16S rRNA from Lactobacillus paracasei HS-05 isolated from women's hands.

    PubMed

    Choi, Woon Yong; Lee, Hyeon Yong

    2015-12-01

    We determined the complete nucleotide sequence of the 16S rRNA from a new bacterium collected from the surfaces of women's hands. We also compared the presence of various bacteria based on the subjects' sex and age. The number of colonies isolated from the hand surface was larger for women than men, and the largest number of isolates was confirmed to be present for the women in their 30 s and men in their 40 s (147 and 34 isolates, respectively). The morphology of an isolated bacterial strain was found to be rod type, and the bacterium was identified as Lactobacillaceae species based on the GenBank database, through a phylogenetic analysis using the 16S rRNA sequence. Based on the results of a homology search, the isolated strain was 99 % identical to Lactobacillus paracasei, so it was designated Lactobacillus paracasei HS-05 and was registered in the Korea Culture Center of Microorganisms (KCCM) database as [KCCM11349P]. PMID:26660786

  10. Negamycin interferes with decoding and translocation by simultaneous interaction with rRNA and tRNA

    PubMed Central

    Polikanov, Yury S.; Szal, Teresa; Jiang, Fuyan; Gupta, Pulkit; Matsuda, Ryoichi; Shiozuka, Masataka; Steitz, Thomas A.; Vázquez-Laslop, Nora; Mankin, Alexander S.

    2014-01-01

    SUMMARY Negamycin (NEG) is a ribosome targeting antibiotic which exhibits clinically promising activity. Its binding site and mode of action have remained unknown. We solved the structure of the Thermus thermophilus ribosome bound to mRNA and three tRNAs, in complex with NEG. The drug binds to both small and large ribosomal subunits at 9 independent sites. Resistance mutations in the 16S rRNA unequivocally identified the binding site in the vicinity of the conserved helix 34 (h34) in the small subunit as the primary site of antibiotic action in the bacterial and, possibly, eukaryotic ribosome. At this site, NEG contacts 16S rRNA as well as the anticodon loop of the A-site tRNA. Although the NEG site of action overlaps with that of tetracycline (TET), the two antibiotics exhibit different activities: while TET sterically hinders binding of aminoacyl-tRNA to the ribosome, NEG stabilizes its binding thereby inhibiting translocation and stimulating miscoding. PMID:25306922

  11. [Diversity of 16S rRNA genes in metagenomic community of the freshwater sponge Lubomirskia baicalensis].

    PubMed

    Kaliuzhnaia, O V; Krivich, A A; Itskovich, V B

    2012-08-01

    Phylogenetic analysis of the nucleotide sequences of 16S rRNA genes in the metagenomic community of Lubomirskia baicalensis has revealed taxonomic diversity of bacteria associated with the endemic freshwater sponge. Fifty-four operational taxonomic units (OTUs) belonging to six bacterial phyla (Actinobacteria, alpha-Proteobacteria, beta-Proteobacteria, Verrucomicrobia, Bacteroidetes, Cyanobacteria, and Nitrospiracea) have been identified. Actinobacteria, whose representatives are known as antibiotic producers, is the dominant phylum of the community (37%, 20 OTUs). All sequences detected shared the maximal homology with unculturable microorganisms from freshwater habitats. The wide diversity of bacteria closely coexisting with the Baikal sponge indicate the complex ecological relationships in the community formed under the unique conditions of Lake Baikal. PMID:23035553

  12. Bacterial census of poultry intestinal microbiome.

    PubMed

    Wei, S; Morrison, M; Yu, Z

    2013-03-01

    The objective of this study was to generate a phylogenetic diversity census of bacteria identified in the intestinal tract of chickens and turkeys using a naïve analysis of all the curated 16S rRNA gene sequences archived in public databases. High-quality sequences of chicken and turkey gastrointestinal origin (3,184 and 1,345, respectively) were collected from the GenBank, Ribosomal Database Project, and Silva comprehensive ribosomal RNA database. Through phylogenetic and statistical analysis, 915 and 464 species-equivalent operational taxonomic units (defined at 0.03 phylogenetic distance) were found in the chicken and the turkey sequence collections, respectively. Of the 13 bacterial phyla identified in both bird species, Firmicutes, Bacteroidetes, and Proteobacteria were the largest phyla, accounting for >90% of all the sequences. The chicken sequences represent 117 established bacterial genera, and the turkey sequences represent 69 genera. The most predominant genera found in both the chicken and the turkey sequence data sets were Clostridium, Ruminococcus, Lactobacillus, and Bacteroides, but with different distribution between the 2 bird species. The estimated coverage of bacterial diversity of chicken and turkey reached 89 and 68% at species-equivalent and 93 and 73% at genus-equivalent levels, respectively. Less than 7,000 bacterial sequences from each bird species from various locations would be needed to reach 99% coverage for either bird species. Based on annotation of the sequence records, cecum was the most sampled gut segment. Chickens and turkeys were shown to have distinct intestinal microbiomes, sharing only 16% similarity at the species-equivalent level. Besides identifying gaps in knowledge on bacterial diversity in poultry gastrointestinal tract, the bacterial census generated in this study may serve as a framework for future studies and development of analytic tools. PMID:23436518

  13. A Markovian analysis of bacterial genome sequence constraints

    PubMed Central

    Skewes, Aaron D.

    2013-01-01

    The arrangement of nucleotides within a bacterial chromosome is influenced by numerous factors. The degeneracy of the third codon within each reading frame allows some flexibility of nucleotide selection; however, the third nucleotide in the triplet of each codon is at least partly determined by the preceding two. This is most evident in organisms with a strong G + C bias, as the degenerate codon must contribute disproportionately to maintaining that bias. Therefore, a correlation exists between the first two nucleotides and the third in all open reading frames. If the arrangement of nucleotides in a bacterial chromosome is represented as a Markov process, we would expect that the correlation would be completely captured by a second-order Markov model and an increase in the order of the model (e.g., third-, fourth-…order) would not capture any additional uncertainty in the process. In this manuscript, we present the results of a comprehensive study of the Markov property that exists in the DNA sequences of 906 bacterial chromosomes. All of the 906 bacterial chromosomes studied exhibit a statistically significant Markov property that extends beyond second-order, and therefore cannot be fully explained by codon usage. An unrooted tree containing all 906 bacterial chromosomes based on their transition probability matrices of third-order shares ?25% similarity to a tree based on sequence homologies of 16S rRNA sequences. This congruence to the 16S rRNA tree is greater than for trees based on lower-order models (e.g., second-order), and higher-order models result in diminishing improvements in congruence. A nucleotide correlation most likely exists within every bacterial chromosome that extends past three nucleotides. This correlation places significant limits on the number of nucleotide sequences that can represent probable bacterial chromosomes. Transition matrix usage is largely conserved by taxa, indicating that this property is likely inherited, however some important exceptions exist that may indicate the convergent evolution of some bacteria. PMID:24010012

  14. Ice formation and growth shape bacterial community structure in Baltic Sea drift ice.

    PubMed

    Eronen-Rasimus, Eeva; Lyra, Christina; Rintala, Janne-Markus; Jürgens, Klaus; Ikonen, Vilma; Kaartokallio, Hermanni

    2015-02-01

    Drift ice, open water and under-ice water bacterial communities covering several developmental stages from open water to thick ice were studied in the northern Baltic Sea. The bacterial communities were assessed with 16S rRNA gene terminal-restriction fragment length polymorphism and cloning, together with bacterial abundance and production measurements. In the early stages, open water and pancake ice were dominated by Alphaproteobacteria and Actinobacteria, which are common bacterial groups in Baltic Sea wintertime surface waters. The pancake ice bacterial communities were similar to the open-water communities, suggesting that the parent water determines the sea-ice bacterial community in the early stages of sea-ice formation. In consolidated young and thick ice, the bacterial communities were significantly different from water bacterial communities as well as from each other, indicating community development in Baltic Sea drift ice along with ice-type changes. The thick ice was dominated by typical sea-ice genera from classes Flavobacteria and Gammaproteobacteria, similar to those in polar sea-ice bacterial communities. Since the thick ice bacterial community was remarkably different from that of the parent seawater, results indicate that thick ice bacterial communities were recruited from the rarer members of the seawater bacterial community. PMID:25764550

  15. Bacterial vaginosis.

    PubMed Central

    Spiegel, C A

    1991-01-01

    Bacterial vaginosis (BV) is the most common of the vaginitides affecting women of reproductive age. It appears to be due to an alteration in the vaginal ecology by which Lactobacillus spp., the predominant organisms in the healthy vagina, are replaced by a mixed flora including Prevotella bivia, Prevotella disiens, Porphyromonas spp., Mobiluncus spp., and Peptostreptococcus spp. All of these organisms except Mobiluncus spp. are also members of the endogenous vaginal flora. While evidence from treatment trials does not support the notion that BV is sexually transmitted, recent studies have shown an increased risk associated with multiple sexual partners. It has also been suggested that the pathogenesis of BV may be similar to that of urinary tract infections, with the rectum serving as a reservoir for some BV-associated flora. The organisms associated with BV have also been recognized as agents of female upper genital tract infection, including pelvic inflammatory disease, and the syndrome BV has been associated with adverse outcome of pregnancy, including premature rupture of membranes, chorioamnionitis, and fetal loss; postpartum endometritis; cuff cellulitis; and urinary tract infections. The mechanisms by which the BV-associated flora causes the signs of BV are not well understood, but a role for H2O2-producing Lactobacillus spp. in protecting against colonization by catalase-negative anaerobic bacteria has been recognized. These and other aspects of BV are reviewed. PMID:1747864

  16. Identification and Quantification of Abundant Species from Pyrosequences of 16S rRNA by Consensus Alignment

    E-print Network

    Ye, Yuzhen

    Identification and Quantification of Abundant Species from Pyrosequences of 16S rRNA by Consensus@indiana.edu Abstract--16S rRNA gene profiling has recently been boosted by the development of pyrosequencing methodsS rRNA pyrosequences may be inflated because the reads sampled from the same 16S rRNA gene may

  17. Nucleolin Is Required for DNA Methylation State and the Expression of rRNA Gene Variants in Arabidopsis thaliana

    E-print Network

    Pikaard, Craig

    Nucleolin Is Required for DNA Methylation State and the Expression of rRNA Gene Variants Investigacion Cienti´ficas, Madrid, Spain Abstract In eukaryotes, 45S rRNA genes are arranged in tandem arrays the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants

  18. Effect of Mutations in the A Site of 16 S rRNA on Aminoglycoside Antibiotic-Ribosome Interaction

    E-print Network

    Dahlquist, Kam D.

    Effect of Mutations in the A Site of 16 S rRNA on Aminoglycoside Antibiotic-Ribosome Interaction. The ribosomal decoding region is associated with highly conserved sequences near the 3H end of 16 S rRNA of rRNA. Mutations of certain nucleotides in rRNA reduce amino- glycoside binding af

  19. PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species

    PubMed Central

    2010-01-01

    Background The genus Vibrio is a diverse group of Gram-negative bacteria comprised of 74 species. Furthermore, the genus has and is expected to continue expanding with the addition of several new species annually. Consequently, it is of paramount importance to have a method which is able to reliably and efficiently differentiate the numerous Vibrio species. Results In this study, a novel and rapid polymerase chain reaction (PCR)-based intergenic spacer (IGS)-typing system for vibrios was developed that is based on the well-known IGS regions located between the 16S and 23S rRNA genes on the bacterial chromosome. The system was optimized to resolve heteroduplex formation as well as to take advantage of capillary gel electrophoresis technology such that reproducible analyses could be achieved in a rapid manner. System validation was achieved through testing of 69 archetypal Vibrio strains, representing 48 Vibrio species, from which an 'IGS-type' profile database was generated. These data, presented here in several cluster analyses, demonstrated successful differentiation of the 69 type strains showing that this PCR-based fingerprinting method easily discriminates bacterial strains at the species level among Vibrio. Furthermore, testing 36 strains each of V. parahaemolyticus and V. vulnificus, important food borne pathogens, isolated from a variety of geographical locations with the IGS-typing method demonstrated distinct IGS-typing patterns indicative of subspecies divergence in both populations making this technique equally useful for intraspecies differentiation, as well. Conclusion This rapid, reliable and efficient IGS-typing system, especially in combination with 16S rRNA gene sequencing, has the capacity to not only discern and identify vibrios at the species level but, in some cases, at the sub-species level, as well. This procedure is particularly well-suited for preliminary species identification and, lends itself nicely to epidemiological investigations providing information more quickly than other time-honoured methods traditionally used in these types of analyses. PMID:20331883

  20. Higher-order structure of rRNA

    NASA Technical Reports Server (NTRS)

    Gutell, R. R.; Woese, C. R.

    1986-01-01

    A comparative search for phylogenetically covarying basepair replacements within potential helices has been the only reliable method to determine the correct secondary structure of the 3 rRNAs, 5S, 16S, and 23S. The analysis of 16S from a wide phylogenetic spectrum, that includes various branches of the eubacteria, archaebacteria, eucaryotes, in addition to the mitochondria and chloroplast, is beginning to reveal the constraints on the secondary structures of these rRNAs. Based on the success of this analysis, and the assumption that higher order structure will also be phylogenetically conserved, a comparative search was initiated for positions that show co-variation not involved in secondary structure helices. From a list of potential higher order interactions within 16S rRNA, two higher-order interactions are presented. The first of these interactions involves positions 570 and 866. Based on the extent of phylogenetic covariation between these positions while maintaining Watson-Crick pairing, this higher-order interaction is considered proven. The other interaction involves a minimum of six positions between the 1400 and 1500 regions of the 16S rRNA. Although these patterns of covariation are not as striking as the 570/866 interaction, the fact that they all exist in an anti-parallel fashion and that experimental methods previously implicated these two regions of the molecule in tRNA function suggests that these interactions be given serious consideration.

  1. The rRNA evolution and procaryotic phylogeny

    NASA Technical Reports Server (NTRS)

    Fox, G. E.

    1986-01-01

    Studies of ribosomal RNA primary structure allow reconstruction of phylogenetic trees for prokaryotic organisms. Such studies reveal major dichotomy among the bacteria that separates them into eubacteria and archaebacteria. Both groupings are further segmented into several major divisions. The results obtained from 5S rRNA sequences are essentially the same as those obtained with the 16S rRNA data. In the case of Gram negative bacteria the ribosomal RNA sequencing results can also be directly compared with hybridization studies and cytochrome c sequencing studies. There is again excellent agreement among the several methods. It seems likely then that the overall picture of microbial phylogeny that is emerging from the RNA sequence studies is a good approximation of the true history of these organisms. The RNA data allow examination of the evolutionary process in a semi-quantitative way. The secondary structures of these RNAs are largely established. As a result it is possible to recognize examples of local structural evolution. Evolutionary pathways accounting for these events can be proposed and their probability can be assessed.

  2. Different bacterial communities in ectomycorrhizae and surrounding soil.

    PubMed

    Vik, Unni; Logares, Ramiro; Blaalid, Rakel; Halvorsen, Rune; Carlsen, Tor; Bakke, Ingrid; Kolstø, Anne-Brit; Økstad, Ole Andreas; Kauserud, Håvard

    2013-01-01

    Several eukaryotic symbioses have shown to host a rich diversity of prokaryotes that interact with their hosts. Here, we study bacterial communities associated with ectomycorrhizal root systems of Bistorta vivipara compared to bacterial communities in bulk soil using pyrosequencing of 16S rRNA amplicons. A high richness of Operational Taxonomic Units (OTUs) was found in plant roots (3,571 OTUs) and surrounding soil (3,476 OTUs). The community composition differed markedly between these two environments. Actinobacteria, Armatimonadetes, Chloroflexi and OTUs unclassified at phylum level were significantly more abundant in plant roots than in soil. A large proportion of the OTUs, especially those in plant roots, presented low similarity to Sanger 16S rRNA reference sequences, suggesting novel bacterial diversity in ectomycorrhizae. Furthermore, the bacterial communities of the plant roots were spatially structured up to a distance of 60?cm, which may be explained by bacteria using fungal hyphae as a transport vector. The analyzed ectomycorrhizae presents a distinct microbiome, which likely influence the functioning of the plant-fungus symbiosis. PMID:24326907

  3. Different bacterial communities in ectomycorrhizae and surrounding soil

    PubMed Central

    Vik, Unni; Logares, Ramiro; Blaalid, Rakel; Halvorsen, Rune; Carlsen, Tor; Bakke, Ingrid; Kolstø, Anne-Brit; Økstad, Ole Andreas; Kauserud, Håvard

    2013-01-01

    Several eukaryotic symbioses have shown to host a rich diversity of prokaryotes that interact with their hosts. Here, we study bacterial communities associated with ectomycorrhizal root systems of Bistorta vivipara compared to bacterial communities in bulk soil using pyrosequencing of 16S rRNA amplicons. A high richness of Operational Taxonomic Units (OTUs) was found in plant roots (3,571?OTUs) and surrounding soil (3,476?OTUs). The community composition differed markedly between these two environments. Actinobacteria, Armatimonadetes, Chloroflexi and OTUs unclassified at phylum level were significantly more abundant in plant roots than in soil. A large proportion of the OTUs, especially those in plant roots, presented low similarity to Sanger 16S rRNA reference sequences, suggesting novel bacterial diversity in ectomycorrhizae. Furthermore, the bacterial communities of the plant roots were spatially structured up to a distance of 60?cm, which may be explained by bacteria using fungal hyphae as a transport vector. The analyzed ectomycorrhizae presents a distinct microbiome, which likely influence the functioning of the plant-fungus symbiosis. PMID:24326907

  4. Biomineralization and formulation of endosulfan degrading bacterial and fungal consortiums.

    PubMed

    Abraham, Jayanthi; Silambarasan, Sivagnanam

    2014-11-01

    Microbial degradation offers an effective approach to remove toxicants and in this study, a microbial consortium consisting of bacterial strains and fungal strains were originally obtained from endosulfan contaminated agricultural soils. Identification of the bacterial isolates by 16S rRNA sequences revealed the isolates to be Halophilic bacterium JAS4, Klebsiella pneumoniae JAS8, Enterobacter asburiae JAS5, and Enterobacter cloacae JAS7, whereas the fungal isolates were identified by 18S rRNA sequences and the isolates were Botryosphaeria laricina JAS6, Aspergillus tamarii JAS9 and Lasiodiplodia sp. JAS12. The biodegradation of endosulfan was monitored by using HPLC and FTIR analysis. The bacterial and fungal consortium could degrade 1000?mg l(-1) of endosulfan efficiently in aqueous medium and in soil. The infrared spectrum of endosulfan degraded samples in the aqueous medium by bacterial and fungal consortium showed bands at 1400 and 950?cm(-1) which are the characteristics of COOH group and acid dimer band respectively. In the present investigation, low cost solid materials such as sawdust, soil, fly ash, molasses and nutrients were used for the formulation of microbial consortium and to achieve greater multiplication and survival of the microbial strains. PMID:25454517

  5. In Vitro Culture of Previously Uncultured Oral Bacterial Phylotypes.

    PubMed

    Thompson, Hayley; Rybalka, Alexandra; Moazzez, Rebecca; Dewhirst, Floyd E; Wade, William G

    2015-12-15

    Around a third of oral bacteria cannot be grown using conventional bacteriological culture media. Community profiling targeting 16S rRNA and shotgun metagenomics methods have proved valuable in revealing the complexity of the oral bacterial community. Studies investigating the role of oral bacteria in health and disease require phenotypic characterizations that are possible only with live cultures. The aim of this study was to develop novel culture media and use an in vitro biofilm model to culture previously uncultured oral bacteria. Subgingival plaque samples collected from subjects with periodontitis were cultured on complex mucin-containing agar plates supplemented with proteose peptone (PPA), beef extract (BEA), or Gelysate (GA) as well as on fastidious anaerobe agar plus 5% horse blood (FAA). In vitro biofilms inoculated with the subgingival plaque samples and proteose peptone broth (PPB) as the growth medium were established using the Calgary biofilm device. Specific PCR primers were designed and validated for the previously uncultivated oral taxa Bacteroidetes bacteria HOT 365 and HOT 281, Lachnospiraceae bacteria HOT 100 and HOT 500, and Clostridiales bacterium HOT 093. All agar media were able to support the growth of 10 reference strains of oral bacteria. One previously uncultivated phylotype, Actinomyces sp. HOT 525, was cultivated on FAA. Of 93 previously uncultivated phylotypes found in the inocula, 26 were detected in in vitro-cultivated biofilms. Lachnospiraceae bacterium HOT 500 was successfully cultured from biofilm material harvested from PPA plates in coculture with Parvimonas micra or Veillonella dispar/parvula after colony hybridization-directed enrichment. The establishment of in vitro biofilms from oral inocula enables the cultivation of previously uncultured oral bacteria and provides source material for isolation in coculture. PMID:26407883

  6. Active bacterial community structure along vertical redox gradients in Baltic Sea sediment

    SciTech Connect

    Jansson, Janet; Edlund, Anna; Hardeman, Fredrik; Jansson, Janet K.; Sjoling, Sara

    2008-05-15

    Community structures of active bacterial populations were investigated along a vertical redox profile in coastal Baltic Sea sediments by terminal-restriction fragment length polymorphism (T-RFLP) and clone library analysis. According to correspondence analysis of T-RFLP results and sequencing of cloned 16S rRNA genes, the microbial community structures at three redox depths (179 mV, -64 mV and -337 mV) differed significantly. The bacterial communities in the community DNA differed from those in bromodeoxyuridine (BrdU)-labeled DNA, indicating that the growing members of the community that incorporated BrdU were not necessarily the most dominant members. The structures of the actively growing bacterial communities were most strongly correlated to organic carbon followed by total nitrogen and redox potentials. Bacterial identification by sequencing of 16S rRNA genes from clones of BrdU-labeled DNA and DNA from reverse transcription PCR (rt-PCR) showed that bacterial taxa involved in nitrogen and sulfur cycling were metabolically active along the redox profiles. Several sequences had low similarities to previously detected sequences indicating that novel lineages of bacteria are present in Baltic Sea sediments. Also, a high number of different 16S rRNA gene sequences representing different phyla were detected at all sampling depths.

  7. Environmental Factors Shape Sediment Anammox Bacterial Communities in Hypernutrified Jiaozhou Bay, China? †

    PubMed Central

    Dang, Hongyue; Chen, Ruipeng; Wang, Lin; Guo, Lizhong; Chen, Pingping; Tang, Zuwang; Tian, Fang; Li, Shaozheng; Klotz, Martin G.

    2010-01-01

    Bacterial anaerobic ammonium oxidation (anammox) is an important process in the marine nitrogen cycle. Because ongoing eutrophication of coastal bays contributes significantly to the formation of low-oxygen zones, monitoring of the anammox bacterial community offers a unique opportunity for assessment of anthropogenic perturbations in these environments. The current study used targeting of 16S rRNA and hzo genes to characterize the composition and structure of the anammox bacterial community in the sediments of the eutrophic Jiaozhou Bay, thereby unraveling their diversity, abundance, and distribution. Abundance and distribution of hzo genes revealed a greater taxonomic diversity in Jiaozhou Bay, including several novel clades of anammox bacteria. In contrast, the targeting of 16S rRNA genes verified the presence of only “Candidatus Scalindua,” albeit with a high microdiversity. The genus “Ca. Scalindua” comprised the apparent majority of active sediment anammox bacteria. Multivariate statistical analyses indicated a heterogeneous distribution of the anammox bacterial assemblages in Jiaozhou Bay. Of all environmental parameters investigated, sediment organic C/organic N (OrgC/OrgN), nitrite concentration, and sediment median grain size were found to impact the composition, structure, and distribution of the sediment anammox bacterial community. Analysis of Pearson correlations between environmental factors and abundance of 16S rRNA and hzo genes as determined by fluorescent real-time PCR suggests that the local nitrite concentration is the key regulator of the abundance of anammox bacteria in Jiaozhou Bay sediments. PMID:20833786

  8. Metagenomic analysis of bacterial and archaeal assemblages in the soil-mousse surrounding a geothermal spring.

    PubMed

    Bhatia, Sonu; Batra, Navneet; Pathak, Ashish; Joshi, Amit; Souza, Leila; Almeida, Paulo; Chauhan, Ashvini

    2015-09-01

    The soil-mousse surrounding a geothermal spring was analyzed for bacterial and archaeal diversity using 16S rRNA gene amplicon metagenomic sequencing which revealed the presence of 18 bacterial phyla distributed across 109 families and 219 genera. Firmicutes, Actinobacteria, and the Deinococcus-Thermus group were the predominant bacterial assemblages with Crenarchaeota and Thaumarchaeota as the main archaeal assemblages in this largely understudied geothermal habitat. Several metagenome sequences remained taxonomically unassigned suggesting the presence of a repertoire of hitherto undescribed microbes in this geothermal soil-mousse econiche. PMID:26484255

  9. Metagenomic analysis of bacterial and archaeal assemblages in the soil-mousse surrounding a geothermal spring

    PubMed Central

    Bhatia, Sonu; Batra, Navneet; Pathak, Ashish; Joshi, Amit; Souza, Leila; Almeida, Paulo; Chauhan, Ashvini

    2015-01-01

    The soil-mousse surrounding a geothermal spring was analyzed for bacterial and archaeal diversity using 16S rRNA gene amplicon metagenomic sequencing which revealed the presence of 18 bacterial phyla distributed across 109 families and 219 genera. Firmicutes, Actinobacteria, and the Deinococcus-Thermus group were the predominant bacterial assemblages with Crenarchaeota and Thaumarchaeota as the main archaeal assemblages in this largely understudied geothermal habitat. Several metagenome sequences remained taxonomically unassigned suggesting the presence of a repertoire of hitherto undescribed microbes in this geothermal soil-mousse econiche. PMID:26484255

  10. Initial insights into bacterial succession during human decomposition.

    PubMed

    Hyde, Embriette R; Haarmann, Daniel P; Petrosino, Joseph F; Lynne, Aaron M; Bucheli, Sibyl R

    2015-05-01

    Decomposition is a dynamic ecological process dependent upon many factors such as environment, climate, and bacterial, insect, and vertebrate activity in addition to intrinsic properties inherent to individual cadavers. Although largely attributed to microbial metabolism, very little is known about the bacterial basis of human decomposition. To assess the change in bacterial community structure through time, bacterial samples were collected from several sites across two cadavers placed outdoors to decompose and analyzed through 454 pyrosequencing and analysis of variable regions 3-5 of the bacterial 16S ribosomal RNA (16S rRNA) gene. Each cadaver was characterized by a change in bacterial community structure for all sites sampled as time, and decomposition, progressed. Bacteria community structure is variable at placement and before purge for all body sites. At bloat and purge and until tissues began to dehydrate or were removed, bacteria associated with flies, such as Ignatzschineria and Wohlfahrtimonas, were common. After dehydration and skeletonization, bacteria associated with soil, such as Acinetobacter, were common at most body sites sampled. However, more cadavers sampled through multiple seasons are necessary to assess major trends in bacterial succession. PMID:25431049

  11. The bacterial communities in plant phloem-sap-feeding insects.

    PubMed

    Jing, Xiangfeng; Wong, Adam C-N; Chaston, John M; Colvin, John; McKenzie, Cindy L; Douglas, Angela E

    2014-03-01

    The resident microbiota of animals represents an important contribution to the global microbial diversity, but it is poorly known in many animals. This study investigated the bacterial diversity in plant phloem-sap-feeding whiteflies, aphids and psyllids by pyrosequencing bacterial 16S rRNA gene amplicons. After correction for sequencing error, just 3-7 bacterial operational taxonomic units were recovered from each insect sample sequenced to sufficient depth for saturation of rarefaction curves. Most samples were dominated by primary and secondary symbionts, which are localized to insect cells or the body cavity, indicative of a dearth of bacterial colonists of the gut lumen. Diversity indices of the bacterial communities (Shannon's index: 0.40-1.46, Simpson's index: 0.15-0.74) did not differ significantly between laboratory and field samples of the phloem-feeding insects, but were significantly lower than in drosophilid flies quantified by the same methods. Both the low bacterial content of the phloem sap diet and biological processes in the insect may contribute to the apparently low bacterial diversity in these phloem-feeding insects. PMID:24350573

  12. Marine Mesocosm Bacterial Colonisation of Volcanic Ash

    NASA Astrophysics Data System (ADS)

    Witt, V.; Cimarelli, C.; Ayris, P. M.; Kueppers, U.; Erpenbeck, D.; Dingwell, D. B.; Woerheide, G.

    2014-12-01

    Explosive volcanic eruptions regularly eject large quantities of ash particles into the atmosphere, which can be deposited via fallout into oceanic environments. Such fallout has the potential to alter pH, light and nutrient availability at local or regional scales. Shallow-water coral reef ecosystems - "rainforests of the sea" - are highly sensitive to disturbances, such as ocean acidification, sedimentation and eutrophication. Therefore, ash deposition may lead to burial and mortality of such reefs. Coral reef ecosystem resilience may depend on pioneer bacterial colonisation of the ash layer, supporting subsequent establishment of the micro- and ultimately the macro-community. However, it is currently unknown which bacteria are involved in pioneer colonisation. We hypothesize that physico-chemical properties (i.e., morphology, chemistry, mineralogy) of the ash may dictate bacterial colonisation. We have tested the effect of substrate properties on bacterial diversity and abundance colonising five substrates: i) quartz sand ii) crystalline ash from the Sakurajima volcano (Japan) iii) volcanic glass iv) carbonate reef sand and v) calcite sand of similar grain size - by incubation in a controlled marine mesocosm (coral reef aquarium) under low light conditions for three months. Bacterial communities were screened every month by Automated Ribosomal Intergenic Spacer Analysis of the 16S-23S rRNA Internal Transcribed Spacer region. Multivariate statistics revealed discrete groupings of bacterial communities on substrates of volcanic origin (ash and glass) and reef origin (three sands). Analysis Of Similarity supports significantly different communities associated with all substrates (p=0.0001), only quartz did not differ from both carbonate and calcite sands. The ash substrate exhibited the most diverse bacterial community and carried the most substrate-specific bacterial operational taxonomic units. Our findings suggest that bacterial diversity and community composition during colonisation of volcanic ash in a coral reef-like environment is controlled by the physico-chemical composition of the substrate. Knowledge on pioneer bacterial colonisation may increase our understanding on the resilience of coral reefs to natural "catastrophes", such as volcanic ash fallout.

  13. Mycoplasmas (Mollicutes) have a low number of rRNA genes.

    PubMed Central

    Amikam, D; Glaser, G; Razin, S

    1984-01-01

    DNA from Mycoplasma, Ureaplasma, Acholeplasma, and Spiroplasma species digested by restriction endonucleases was hybridized with probes consisting of portions of the rrnB rRNA operon of Escherichia coli and the rRNA operon of Mycoplasma capricolum. The results indicate the presence of only one or two sets of rRNA genes in the genome of Mollicutes linked in the procaryotic fashion, 16S-23S-5S. PMID:6201476

  14. Optimisation of DNA extraction and validation of PCR assays to detect Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Timms, Verlaine J; Mitchell, Hazel M; Neilan, Brett A

    2015-05-01

    The aim of this study was to investigate DNA extraction methods and PCR assays suitable for the detection of Mycobacterium paratuberculosis in bovine tissue. The majority of methods currently used to detect M. paratuberculosis have been developed using bovine samples, such as faeces, blood or tissue and, in many cases, have been based on detection from pooled samples from a herd. However most studies have not compared PCR results to culture results. In order to address this problem, four DNA extraction protocols and three PCR assays were employed to detect M. paratuberculosis in bovine tissue. Given that culture is reliable from cows, the results were then compared with the known M. paratuberculosis culture status. The following DNA extractions were included, two commercial kits, a boiling method, an in house extraction based on a published method and enrichment by sonication. The three PCR assays used included single round IS900 and f57 assays and a nested IS900 assay. In addition, another PCR assay was validated for the detection of any Mycobacterial species and a universal bacterial 16S rRNA gene assay was used to detect sample inhibition. The in-house DNA extraction was the most consistent in extracting good quality DNA compared to all other methods. The use of two PCR markers, IS900 and f57, and a universal PCR enabled the correct samples to be identified as M. paratuberculosis positive. In addition, when compared to the culture result, false-positives did not occur and PCR inhibition was readily identified. Using an in house DNA extraction coupled with the IS900 and f57 PCR markers, this study provides a reliable and simple method to detect M. paratuberculosis in both veterinary and spill over infections. PMID:25797305

  15. 30S Subunit-Dependent Activation of the Sorangium cellulosum So ce56 Aminoglycoside Resistance-Conferring 16S rRNA Methyltransferase Kmr

    PubMed Central

    Savic, Miloje; Sunita, S.; Zelinskaya, Natalia; Desai, Pooja M.; Macmaster, Rachel; Vinal, Kellie

    2015-01-01

    Methylation of bacterial 16S rRNA within the ribosomal decoding center confers exceptionally high resistance to aminoglycoside antibiotics. This resistance mechanism is exploited by aminoglycoside producers for self-protection while functionally equivalent methyltransferases have been acquired by human and animal pathogenic bacteria. Here, we report structural and functional analyses of the Sorangium cellulosum So ce56 aminoglycoside resistance-conferring methyltransferase Kmr. Our results demonstrate that Kmr is a 16S rRNA methyltransferase acting at residue A1408 to confer a canonical aminoglycoside resistance spectrum in Escherichia coli. Kmr possesses a class I methyltransferase core fold but with dramatic differences in the regions which augment this structure to confer substrate specificity in functionally related enzymes. Most strikingly, the region linking core ?-strands 6 and 7, which forms part of the S-adenosyl-l-methionine (SAM) binding pocket and contributes to base flipping by the m1A1408 methyltransferase NpmA, is disordered in Kmr, correlating with an exceptionally weak affinity for SAM. Kmr is unexpectedly insensitive to substitutions of residues critical for activity of other 16S rRNA (A1408) methyltransferases and also to the effects of by-product inhibition by S-adenosylhomocysteine (SAH). Collectively, our results indicate that adoption of a catalytically competent Kmr conformation and binding of the obligatory cosubstrate SAM must be induced by interaction with the 30S subunit substrate. PMID:25733511

  16. Culture independent molecular analysis of bacterial communities in the mangrove sediment of Sundarban, India

    PubMed Central

    2010-01-01

    Background Sundarban is the world's largest coastal sediment comprising of mangrove forest which covers about one million hectares in the south-eastern parts of India and southern parts of Bangladesh. The microbial diversity in this sediment is largely unknown till date. In the present study an attempt has been made to understand the microbial diversity in this sediment using a cultivation-independent molecular approach. Results Two 16 S rRNA gene libraries were constructed and partial sequencing of the selected clones was carried out to identify bacterial strains present in the sediment. Phylogenetic analysis of partially sequenced 16 S rRNA gene sequences revealed the diversity of bacterial strains in the Sundarban sediment. At least 8 different bacterial phyla were detected. The major divisions of detected bacterial phyla were Proteobacteria (alpha, beta, gamma, and delta), Flexibacteria (CFB group), Actinobacteria, Acidobacteria, Chloroflexi, Firmicutes, Planctomycetes and Gammatimonadates. Conclusion The gammaproteobacteria were found to be the most abundant bacterial group in Sundarban sediment. Many clones showed similarity with previously reported bacterial lineages recovered from various marine sediments. The present study indicates a probable hydrocarbon and oil contamination in this sediment. In the present study, a number of clones were identified that have shown similarity with bacterial clones or isolates responsible for the maintenance of the S-cycle in the saline environment. PMID:20163727

  17. Transformation of tetrahymena thermophila with hypermethylated rRNA genes

    SciTech Connect

    Karrer, K.M.; Yao, M.C.

    1988-04-01

    The extrachromosomal rRNA genes (rDNA) of Tetrahymena thermophila contain 0.4% N/sup 6/-methyladenine. C3 strain rDNA was isolated, hypermethylated in vitro, and microinjected into B strain host cells. Clonal cell lines were established, and transformants were selected on the basis of resistance to paromomycin, conferred by the injected rDNA. The effects of methylation by three enzymes which methylate the sequence 5'-NAT-3'', the dam, EcoRI, and ClaI methylases, were tested. Hypermethylation of the injected rDNA had no effect on transformation efficiency relative to mock-methylated controls. The injected C3 strain rDNA efficiently replaced host rDNA as the major constituent of the population of rDNA molecules. Hypermethylation of the injected DNA was not maintained through 20 to 25 cell generations.

  18. Exchange of Spacer Regions between Rrna Operons in Escherichia Coli

    PubMed Central

    Harvey, S.; Hill, C. W.

    1990-01-01

    The Escherichia coli rRNA operons each have one of two types of spacer separating the 16S and 23S coding regions. The spacers of four operons encode tRNA(Glu2) and the other three encode both tRNA(Ile) and tRNA(Ala 1 B). We have prepared a series of mutants in which the spacer region of a particular rrn operon has been replaced by the opposite type. Included among these were a mutant retaining only a single copy of the tRNA(Glu2) spacer (at rrnG) and another retaining only a single copy of the tRNA(Ile)-tRNA(Ala 1 B) spacer (at rrnA). While both mutants grew more slowly than controls, the mutant deficient in tRNA(Glu2) spacers was more severely affected. At a frequency of 6 X 10(-5), these mutants phenotypically reverted to faster growing types by increasing the copy number of the deficient spacer. In most of these phenotypic revertants, the deficient spacer type appeared in a rrn operon which previously contained the surplus type, bringing the ratio of spacer types closer to normal. In a few cases, these spacer changes were accompanied by an inversion of the chromosomal material between the donor and recipient rrn operons. Two examples of inversion of one-half of the E. coli chromosome between rrnG and rrnH were observed. The correlation of spacer change with inversion indicated that, in these particular cases, the change was due to an intrachromatid gene conversion event accompanied by a reciprocal crossover rather than reciprocal exchange between sister chromatids. PMID:2168847

  19. Toolbox Approaches Using Molecular Markers and 16S rRNA Gene Amplicon Data Sets for Identification of Fecal Pollution in Surface Water.

    PubMed

    Ahmed, W; Staley, C; Sadowsky, M J; Gyawali, P; Sidhu, J P S; Palmer, A; Beale, D J; Toze, S

    2015-10-01

    In this study, host-associated molecular markers and bacterial 16S rRNA gene community analysis using high-throughput sequencing were used to identify the sources of fecal pollution in environmental waters in Brisbane, Australia. A total of 92 fecal and composite wastewater samples were collected from different host groups (cat, cattle, dog, horse, human, and kangaroo), and 18 water samples were collected from six sites (BR1 to BR6) along the Brisbane River in Queensland, Australia. Bacterial communities in the fecal, wastewater, and river water samples were sequenced. Water samples were also tested for the presence of bird-associated (GFD), cattle-associated (CowM3), horse-associated, and human-associated (HF183) molecular markers, to provide multiple lines of evidence regarding the possible presence of fecal pollution associated with specific hosts. Among the 18 water samples tested, 83%, 33%, 17%, and 17% were real-time PCR positive for the GFD, HF183, CowM3, and horse markers, respectively. Among the potential sources of fecal pollution in water samples from the river, DNA sequencing tended to show relatively small contributions from wastewater treatment plants (up to 13% of sequence reads). Contributions from other animal sources were rarely detected and were very small (<3% of sequence reads). Source contributions determined via sequence analysis versus detection of molecular markers showed variable agreement. A lack of relationships among fecal indicator bacteria, host-associated molecular markers, and 16S rRNA gene community analysis data was also observed. Nonetheless, we show that bacterial community and host-associated molecular marker analyses can be combined to identify potential sources of fecal pollution in an urban river. This study is a proof of concept, and based on the results, we recommend using bacterial community analysis (where possible) along with PCR detection or quantification of host-associated molecular markers to provide information on the sources of fecal pollution in waterways. PMID:26231650

  20. Biodegradation of crude oil by individual bacterial strains and a mixed bacterial consortium

    PubMed Central

    Santisi, Santina; Cappello, Simone; Catalfamo, Maurizio; Mancini, Giuseppe; Hassanshahian, Mehdi; Genovese, Lucrezia; Giuliano, Laura; Yakimov, Michail M.

    2015-01-01

    Three bacterial isolates identified as Alcanivorax borkumensis SK2, Rhodococcus erythropolis HS4 and Pseudomonas stutzeri SDM, based on 16S rRNA gene sequences, were isolated from crude oil enrichments of natural seawater. Single strains and four bacterial consortia designed by mixing the single bacterial cultures respectively in the following ratios: (Alcanivorax: Pseudomonas, 1:1), (Alcanivorax: Rhodococcus, 1:1), (Pseudomonas: Rhodococcus, 1:1), and (Alcanivorax: Pseudomonas: Rhodococcus, 1:1:1), were analyzed in order to evaluate their oil degrading capability. All experiments were carried out in microcosms systems containing seawater (with and without addition of inorganic nutrients) and crude oil (unique carbon source). Measures of total and live bacterial abundance, Card-FISH and quali-, quantitative analysis of hydrocarbons (GC-FID) were carried out in order to elucidate the co-operative action of mixed microbial populations in the process of biodegradation of crude oil. All data obtained confirmed the fundamental role of bacteria belonging to Alcanivorax genus in the degradation of linear hydrocarbons in oil polluted environments. PMID:26273252

  1. Biodegradation of crude oil by individual bacterial strains and a mixed bacterial consortium.

    PubMed

    Santisi, Santina; Cappello, Simone; Catalfamo, Maurizio; Mancini, Giuseppe; Hassanshahian, Mehdi; Genovese, Lucrezia; Giuliano, Laura; Yakimov, Michail M

    2015-06-01

    Three bacterial isolates identified as Alcanivorax borkumensis SK2, Rhodococcus erythropolis HS4 and Pseudomonas stutzeri SDM, based on 16S rRNA gene sequences, were isolated from crude oil enrichments of natural seawater. Single strains and four bacterial consortia designed by mixing the single bacterial cultures respectively in the following ratios: (Alcanivorax: Pseudomonas, 1:1), (Alcanivorax: Rhodococcus, 1:1), (Pseudomonas: Rhodococcus, 1:1), and (Alcanivorax: Pseudomonas: Rhodococcus, 1:1:1), were analyzed in order to evaluate their oil degrading capability. All experiments were carried out in microcosms systems containing seawater (with and without addition of inorganic nutrients) and crude oil (unique carbon source). Measures of total and live bacterial abundance, Card-FISH and quali-, quantitative analysis of hydrocarbons (GC-FID) were carried out in order to elucidate the co-operative action of mixed microbial populations in the process of biodegradation of crude oil. All data obtained confirmed the fundamental role of bacteria belonging to Alcanivorax genus in the degradation of linear hydrocarbons in oil polluted environments. PMID:26273252

  2. Effects of Fertilization and Sampling Time on Composition and Diversity of Entire and Active Bacterial Communities in German Grassland Soils

    PubMed Central

    Herzog, Sarah; Wemheuer, Franziska; Wemheuer, Bernd; Daniel, Rolf

    2015-01-01

    Soil bacteria are major players in driving and regulating ecosystem processes. Thus, the identification of factors shaping the diversity and structure of these communities is crucial for understanding bacterial-mediated processes such as nutrient transformation and cycling. As most studies only target the entire soil bacterial community, the response of active community members to environmental changes is still poorly understood. The objective of this study was to investigate the effect of fertilizer application and sampling time on structure and diversity of potentially active (RNA-based) and the entire (DNA-based) bacterial communities in German grassland soils. Analysis of more than 2.3 million 16S rRNA transcripts and gene sequences derived from amplicon-based sequencing of 16S rRNA genes revealed that fertilizer application and sampling time significantly altered the diversity and composition of entire and active bacterial communities. Although the composition of both the entire and the active bacterial community was correlated with environmental factors such as pH or C/N ratio, the active community showed a higher sensitivity to environmental changes than the entire community. In addition, functional analyses were performed based on predictions derived from 16S rRNA data. Genes encoding the uptake of nitrate/nitrite, nitrification, and denitrification were significantly more abundant in fertilized plots compared to non-fertilized plots. Hence, this study provided novel insights into changes in dynamics and functions of soil bacterial communities as response to season and fertilizer application. PMID:26694644

  3. Nitrate treatment effects on bacterial community biofilm formed on carbon steel in produced water stirred tank bioreactor.

    PubMed

    Marques, Joana Montezano; de Almeida, Fernando Pereira; Lins, Ulysses; Seldin, Lucy; Korenblum, Elisa

    2012-06-01

    To better understand the impact of nitrate in Brazilian oil reservoirs under souring processes and corrosion, the goal of this study was to analyse the effect of nitrate on bacterial biofilms formed on carbon steel coupons using reactors containing produced water from a Brazilian oil platform. Three independent experiments were carried out (E1, E2 and E3) using the same experimental conditions and different incubation times (5, 45 and 80 days, respectively). In every experiment, two biofilm-reactors were operated: one was treated with continuous nitrate flow (N reactor), and the other was a control reactor without nitrate (C reactor). A Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis approach using the 16S rRNA gene was performed to compare the bacterial groups involved in biofilm formation in the N and C reactors. DGGE profiles showed remarkable changes in community structure only in experiments E2 and E3. Five bands extracted from the gel that represented the predominant bacterial groups were identified as Bacillus aquimaris, B. licheniformis, Marinobacter sp., Stenotrophomonas maltophilia and Thioclava sp. A reduction in the sulfate-reducing bacteria (SRB) most probable number counts was observed only during the longer nitrate treatment (E3). Carbon steel coupons used for biofilm formation had a slightly higher weight loss in N reactors in all experiments. When the coupon surfaces were analysed by scanning electron microscopy, an increase in corrosion was observed in the N reactors compared with the C reactors. In conclusion, nitrate reduced the viable SRB counts. Nevertheless, the nitrate dosing increased the pitting of coupons. PMID:22806109

  4. Five-week dietary exposure to dry diets alters the faecal bacterial populations in the domestic cat (Felis catus).

    PubMed

    Bermingham, Emma N; Kittelmann, Sandra; Henderson, Gemma; Young, Wayne; Roy, Nicole C; Thomas, David G

    2011-10-01

    The effects of wet (canned) or dry (kibbled) diets on faecal bacterial populations in the cat were investigated in eight domestic short-haired cats (four males and four females; averaging 6 years of age and 3.4 kg) in a nested design. The cats were fed ad libitum a commercially available wet diet (moisture 82.0 %, crude protein 51.7 %, fat 28.9 %, carbohydrate (CHO) 8.9 % and ash 10.6 % DM) for 5 weeks. On the fifth week, individual feed intakes and faecal outputs were determined. Fresh faecal samples were collected twice daily, mixed for homogeneity, subsampled and stored at - 85 °C until analysis. The cats were then switched to a commercially available dry diet (moisture 8.5 %, crude protein 33.0 %, fat 11.0 %, CHO 49.4 % and ash 6.6 % DM) for 5 weeks, and fresh faeces were sampled as described previously. Energy intake tended to be higher in cats fed dry diets (P < 0.10), but body weight was similar between the two feeding periods (P>0.05). Denaturing gradient gel electrophoresis (DGGE) of bacterial 16S rRNA genes amplified from DNA extracted from faeces was performed. The unweighted pair group method with arithmetic mean cluster analysis of bacterial community profiles using Pearson's correlation revealed diet-specific clustering when the same cats were fed on either a dry or a wet diet (dissimilarity between the groups, 88.6 %; P < 0.001). Subsequent cloning and sequencing of five selected distinct DGGE bands indicated that members of the Pelomonas and Fusobacteriaceae were influenced by a short-term change in diet format. This suggests that 5-week dietary exposure is sufficient to alter gastrointestinal microflora. PMID:22005435

  5. Solution Structure of an Alternate Conformation of Helix 27 from Escherichia Coli 16S rRNA

    PubMed Central

    Spano, Meredith Newby; Walter, Nils G.

    2011-01-01

    Helix (H)27 of 16S ribosomal (r)RNA from Escherichia coli was dubbed the “switch helix” when mutagenesis suggested that two alternative base pair registers may have distinct functional roles in the bacterial ribosome. Although more recent genetic analyses suggest that H27 conformational switching is not required for translation, previous solution studies demonstrated that the isolated E. coli H27 can dynamically convert between the 885 and 888 conformations. Here, we have solved the NMR solution structure of a locked 888 conformation. NOE and residual dipolar coupling restraints reveal an architecture that markedly differs from that of the 885 conformation found in crystal structures of the bacterial ribosome. In place of the loop E motif that characterizes the 885 conformer and that the 888 conformer cannot adopt, we find evidence for an asymmetrical A-rich internal loop stabilized by stacking interactions among the unpaired A’s. Comparison of the isolated H27 888 solution structure with the 885 crystal structure within the context of the ribosome suggests a difference in overall length of H27 that presents one plausible reason for the absence of H27 conformational switching within the sterically confining ribosome. PMID:21442607

  6. Life history correlates of fecal bacterial species richness in a wild population of the blue tit Cyanistes caeruleus

    E-print Network

    MacDonald, Mark

    University, Lancaster LA1 4YQ, UK Keywords 16S rRNA, bacterial species richness, fecal microbial community, UK 2 Centre for Ecology and Hydrology, Lancaster Environment Centre, Library Avenue, Bailrigg most likely to transmit bacteria to humans or acquire bacteria from human sources, such as gulls (Larus

  7. Direct 16S rRNA Gene Sequencing from Clinical Specimens, with Special Focus on Polybacterial Samples and Interpretation of Mixed DNA Chromatograms ?

    PubMed Central

    Kommedal, Øyvind; Kvello, Kristine; Skjåstad, Rune; Langeland, Nina; Wiker, Harald G.

    2009-01-01

    RipSeq (iSentio, Bergen, Norway) is a web-based application for the analysis of mixed DNA chromatograms. It opens the possibility to analyze chromatograms obtained by direct 16S rRNA gene sequencing from polybacterial human clinical samples. In this study, we used direct 16S rRNA gene sequencing to investigate 264 samples from a wide range of suspected human bacterial infections. The sequence-based identification was compared with the results from routine culture-based identification. A total of 151 samples were positive by the first PCR, producing 85 pure and 66 mixed DNA chromatograms. All mixed chromatograms were analyzed by RipSeq, although seven were so complex that only the dominant bacterial sequences could be identified. In general, sequence-based identification detected a larger number of species than did culture for samples from patients who had received antibiotics prior to sample collection and for samples containing anaerobic bacteria. RipSeq made it possible to apply this supplementary diagnostic tool to typical polybacterial specimens, such as internal abscesses, pleural fluids, and bile. PMID:19741089

  8. Plaque Bacterial Microbiome Diversity in Children Younger than 30 Months with or without Caries Prior to Eruption of Second Primary Molars

    PubMed Central

    Xu, He; Hao, Wenjing; Zhou, Qiong; Wang, Wenhong; Xia, Zhongkui; Liu, Chuan; Chen, Xiaochi; Qin, Man; Chen, Feng

    2014-01-01

    Objective Our primary objective is to phylogenetically characterize the supragingival plaque bacterial microbiome of children prior to eruption of second primary molars by pyrosequencing method for studying etiology of early childhood caries. Methods Supragingival plaque samples were collected from 10 caries children and 9 caries-free children. Plaque DNA was extracted, used to generate DNA amplicons of the V1–V3 hypervariable region of the bacterial 16S rRNA gene, and subjected to 454-pyrosequencing. Results On average, over 22,000 sequences per sample were generated. High bacterial diversity was noted in the plaque of children with caries [170 operational taxonomical units (OTU) at 3% divergence] and caries-free children (201 OTU at 3% divergence) with no significant difference. A total of 8 phyla, 15 classes, 21 orders, 30 families, 41 genera and 99 species were represented. In addition, five predominant phyla (Firmicute, Fusobacteria, Proteobacteria, Bacteroidetes and Actinobacteria) and seven genera (Leptotrichia, Streptococcus, Actinomyces, Prevotella, Porphyromonas, Neisseria, and Veillonella) constituted a majority of contents of the total microbiota, independent of the presence or absence of caries. Principal Component Analysis (PCA) presented that caries-related genera included Streptococcus and Veillonella; while Leptotrichia, Selenomonas, Fusobacterium, Capnocytophaga and Porphyromonas were more related to the caries-free samples. Neisseria and Prevotella presented approximately in between. In both groups, the degree of shared organism lineages (as defined by species-level OTUs) among individual supragingival plaque microbiomes was minimal. Conclusion Our study represented for the first time using pyrosequencing to elucidate and monitor supragingival plaque bacterial diversity at such young age with second primary molar unerrupted. Distinctions were revealed between caries and caries-free microbiomes in terms of microbial community structure. We observed differences in abundance for several microbial groups between the caries and caries-free host populations, which were consistent with the ecological plaque hypothesis. Our approach and findings could be extended to correlating microbiomic changes after occlusion establishment and caries treatment. PMID:24586647

  9. Temporal stability of bacterial symbionts in a temperate ascidian

    PubMed Central

    López-Legentil, Susanna; Turon, Xavier; Espluga, Roger; Erwin, Patrick M.

    2015-01-01

    In temperate seas, both bacterioplankton communities and invertebrate lifecycles follow a seasonal pattern. To investigate whether the bacterial community associated with the Mediterranean ascidian Didemnum fulgens exhibited similar variations, we monitored its bacterial community structure monthly for over a year using terminal restriction fragment length polymorphism and clone library analyses based on a nearly full length fragment of the 16S rRNA gene. D. fulgens harbored a bacterial consortium typical of ascidians, including numerous members of the phylum Proteobacteria, and a few members of the phyla Cyanobacteria and Acidobacteria. The overall bacterial community in D. fulgens had a distinct signature from the surrounding seawater and was stable over time and across seasonal fluctuations in temperature. Bacterial symbionts were also observed around animal cells in the tunic of adult individuals and in the inner tunic of D. fulgens larvae by transmission electron microscopy. Our results suggest that, as seen for sponges and corals, some species of ascidians host stable and unique bacterial communities that are at least partially inherited by their progeny by vertical transmission. PMID:26441944

  10. Spatial structuring of bacterial communities within individual Ginkgo biloba trees.

    PubMed

    Leff, Jonathan W; Del Tredici, Peter; Friedman, William E; Fierer, Noah

    2015-07-01

    Plant-associated microorganisms affect the health of their hosts in diverse ways, yet the distribution of these organisms within individual plants remains poorly understood. To address this knowledge gap, we assessed the spatial variability in bacterial community diversity and composition found on and in aboveground tissues of individual Ginkgo biloba trees. We sampled bacterial communities from >?100 locations per tree, including leaf, branch and trunk samples and used high-throughput sequencing of the 16S rRNA gene to determine the diversity and composition of these communities. Bacterial community structure differed strongly between bark and leaf samples, with bark samples harbouring much greater bacterial diversity and a community composition distinct from leaves. Within sample types, we observed clear spatial patterns in bacterial diversity and community composition that corresponded to the samples' proximity to the exterior of the tree. The composition of the bacterial communities found on trees is highly variable, but this variability is predictable and dependent on sampling location. Moreover, this work highlights the importance of carefully considering plant spatial structure when characterizing the microbial communities associated with plants and their impacts on plant hosts. PMID:25367625

  11. CONFLICTING PHYLOGEOGRAPHIC PATTERNS IN RRNA AND NIFD INDICATE REGIONALLY RESTRICTED GENE TRANSFER IN BRADYRHIZOBIUM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Major differences in evolutionary relationships of the 16S rRNA gene and the nitrogenase alpha-subunit gene (nifD) were observed among 38 strains of Bradyrhizobium sp. nodule bacteria from North America, Central America, Asia and Australia. Two lineages were evident in the 16S rRNA phylogeny represe...

  12. J. Phycol. 39, 527534 (2003) GROWTH REGULATION OF rRNA CONTENT IN PROCHLOROCOCCUS AND

    E-print Network

    Worden, Alexandra Z.

    2003-01-01

    527 J. Phycol. 39, 527­534 (2003) GROWTH REGULATION OF rRNA CONTENT IN PROCHLOROCOCCUS AND SYNECHOCOCCUS (MARINE CYANOBACTERIA) MEASURED BY WHOLE-CELL HYBRIDIZATION OF rRNA-TARGETED PEPTIDE NUCLEIC ACIDS in the field, we have been exploring the feasi- bility of using quantitative rRNA measurements as the basis

  13. AZOSPIRA ORYZAE (A SELENIUM OXYANION-REDUCING BACTERIUM) 16S RRNA GENE COMPLETE SEQUENCE.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study used 1535 base pair 16S rRNA gene sequence methods to confirm the identification of a bacterium that reduces selenite to elemental red selenium as Azospira oryzae. Morphological and biochemical characteristics are consistent with the 16S rRNA gene sequence identification of the bacterium....

  14. Pregnancy Complications: Bacterial Vaginosis

    MedlinePLUS

    ... Global Map Premature birth report card Careers Archives Pregnancy Before or between pregnancies Nutrition, weight & fitness Prenatal ... complications > Bacterial vaginosis and pregnancy Bacterial vaginosis and pregnancy Now playing: E-mail to a friend Please ...

  15. PyroTRF-ID: a novel bioinformatics methodology for the affiliation of terminal-restriction fragments using 16S rRNA gene pyrosequencing data

    PubMed Central

    2012-01-01

    Background In molecular microbial ecology, massive sequencing is gradually replacing classical fingerprinting techniques such as terminal-restriction fragment length polymorphism (T-RFLP) combined with cloning-sequencing for the characterization of microbiomes. Here, a bioinformatics methodology for pyrosequencing-based T-RF identification (PyroTRF-ID) was developed to combine pyrosequencing and T-RFLP approaches for the description of microbial communities. The strength of this methodology relies on the identification of T-RFs by comparison of experimental and digital T-RFLP profiles obtained from the same samples. DNA extracts were subjected to amplification of the 16S rRNA gene pool, T-RFLP with the HaeIII restriction enzyme, 454 tag encoded FLX amplicon pyrosequencing, and PyroTRF-ID analysis. Digital T-RFLP profiles were generated from the denoised full pyrosequencing datasets, and the sequences contributing to each digital T-RF were classified to taxonomic bins using the Greengenes reference database. The method was tested both on bacterial communities found in chloroethene-contaminated groundwater samples and in aerobic granular sludge biofilms originating from wastewater treatment systems. Results PyroTRF-ID was efficient for high-throughput mapping and digital T-RFLP profiling of pyrosequencing datasets. After denoising, a dataset comprising ca. 10?000 reads of 300 to 500 bp was typically processed within ca. 20 minutes on a high-performance computing cluster, running on a Linux-related CentOS 5.5 operating system, enabling parallel processing of multiple samples. Both digital and experimental T-RFLP profiles were aligned with maximum cross-correlation coefficients of 0.71 and 0.92 for high- and low-complexity environments, respectively. On average, 63±18% of all experimental T-RFs (30 to 93 peaks per sample) were affiliated to phylotypes. Conclusions PyroTRF-ID profits from complementary advantages of pyrosequencing and T-RFLP and is particularly adapted for optimizing laboratory and computational efforts to describe microbial communities and their dynamics in any biological system. The high resolution of the microbial community composition is provided by pyrosequencing, which can be performed on a restricted set of selected samples, whereas T-RFLP enables simultaneous fingerprinting of numerous samples at relatively low cost and is especially adapted for routine analysis and follow-up of microbial communities on the long run. PMID:23270314

  16. Advanced computational algorithms for microbial community analysis using massive 16S rRNA sequence data

    PubMed Central

    Sun, Yijun; Cai, Yunpeng; Mai, Volker; Farmerie, William; Yu, Fahong; Li, Jian; Goodison, Steve

    2010-01-01

    With the aid of next-generation sequencing technology, researchers can now obtain millions of microbial signature sequences for diverse applications ranging from human epidemiological studies to global ocean surveys. The development of advanced computational strategies to maximally extract pertinent information from massive nucleotide data has become a major focus of the bioinformatics community. Here, we describe a novel analytical strategy including discriminant and topology analyses that enables researchers to deeply investigate the hidden world of microbial communities, far beyond basic microbial diversity estimation. We demonstrate the utility of our approach through a computational study performed on a previously published massive human gut 16S rRNA data set. The application of discriminant and topology analyses enabled us to derive quantitative disease-associated microbial signatures and describe microbial community structure in far more detail than previously achievable. Our approach provides rigorous statistical tools for sequence-based studies aimed at elucidating associations between known or unknown organisms and a variety of physiological or environmental conditions. PMID:20929878

  17. Analysis of a 5S rRNA gene cloned from Euplotes eurstomus

    SciTech Connect

    Roberson, A.E.; Wolffe, A.; Olins, D.E.

    1987-05-01

    The macronucleus of the hypotrichous ciliated protozoan Euplotes eurystomus lends itself to the study of eukaryotic gene and chromatin structure because native macronuclear DNA exists as linear, gene-sized fragments between 400 and 20,000 bp in length. The macronuclear chromatin, while arranged in a typical nucleosomal structure, is freely soluble in low ionic strength buffers without treatment by nucleases. Thus, specific genes may be enriched as native, intact chromatin molecules. The 5S rRNA gene from Euplotes has been cloned to facilitate investigation of 5S gene-chromatin following characterization of the gene at the DNA level. It has been demonstrated that the gene, while in circular or linear form, can be transcribed in vitro by a Xenopus oocyte nuclear extract. The transcript generated in vitro is 120 nucleotides in length and is synthesized by RNA polymerase III. Anti-Xenopus TFIIIA antibodies recognize a Euplotes macronuclear chromatin-associated protein which is approx. 80 KD in size. It has been established that the sequence of the telomere flanking the 5S gene in Euplotes eurystomus is the same telomeric sequence published for Euplotes aediculatus.

  18. In Vitro Processing of the 16S rRNA of the Thermophilic Archaeon Sulfolobus solfataricus

    PubMed Central

    Ciammaruconi, Andrea; Londei, Paola

    2001-01-01

    In this paper we have analyzed the processing in vitro of the 16S rRNA of the thermophilic archaeon Sulfolobus solfataricus, using pre-rRNA substrates transcribed in vitro and different protein preparations as the source of processing enzymes. We show that the 5? external transcribed spacer of the S. solfataricus pre-rRNA transcript contains a target site for a specific endonuclease, which recognizes a conserved sequence also existing in the early A0 and 0 processing sites of Saccharomyces cerevisiae and vertebrates. This site is present in other members of the kingdom Crenarchaeota but apparently not in the Euryarchaeota. Furthermore, S. solfataricus pre-16S RNA is processed within the double-helical stem formed by the inverted repeats flanking the 16S RNA sequence, in correspondence with a bulge-helix-bulge motif. The endonuclease responsible for this cleavage is present in both the Crenarchaeota and the Euryarchaeota. The processing pattern remained the same when the substrate was a 30S ribonucleoprotein particle instead of the naked RNA. Maturation of either the 5? or the 3? end of the 16S RNA molecule was not observed, suggesting either that maturation requires conditions not easily reproducible in vitro or that the responsible endonucleases are scarcely represented in cell extracts. PMID:11395449

  19. Functional profiling and distribution of the forest soil bacterial communities along the soil mycorrhizosphere continuum.

    PubMed

    Uroz, S; Courty, P E; Pierrat, J C; Peter, M; Buée, M; Turpault, M P; Garbaye, J; Frey-Klett, P

    2013-08-01

    An ectomycorrhiza is a multitrophic association between a tree root, an ectomycorrhizal fungus, free-living fungi and the associated bacterial communities. Enzymatic activities of ectomycorrhizal root tips are therefore result of the contribution from different partners of the symbiotic organ. However, the functional potential of the fungus-associated bacterial communities remains unknown. In this study, a collection of 80 bacterial strains randomly selected and isolated from a soil-ectomycorrhiza continuum (oak-Scleroderma citrinum ectomycorrhizas, the ectomycorrhizosphere and the surrounding bulk soil) were characterized. All the bacterial isolates were identified by partial 16S rRNA gene sequences as members of the genera Burkholderia, Collimonas, Dyella, Mesorhizobium, Pseudomonas, Rhizobium and Sphingomonas. The bacterial strains were then assayed for ?-xylosidase, ?-glucosidase, N-acetyl-hexosaminidase, ?-glucuronidase, cellobiohydrolase, phosphomonoesterase, leucine-aminopeptidase and laccase activities, chitin solubilization and auxin production. Using these bioassays, we demonstrated significant differences in the functional distribution of the bacterial communities living in the different compartments of the soil-ectomycorrhiza continuum. The surrounding bulk soil was significantly enriched in bacterial isolates capable of hydrolysing cellobiose and N-acetylglucosamine. In contrast, the ectomycorrhizosphere appeared significantly enriched in bacterial isolates capable of hydrolysing glucopyranoside and chitin. Notably, chitinase and laccase activities were found only in bacterial isolates belonging to the Collimonas and Pseudomonas genera. Overall, the results suggest that the ectomycorrhizal fungi favour specific bacterial communities with contrasting functional characteristics from the surrounding soil. PMID:23455431

  20. Diversity of arsenite oxidizing bacterial communities in arsenic-rich deltaic aquifers in West Bengal, India

    PubMed Central

    Ghosh, Devanita; Bhadury, Punyasloke; Routh, Joyanto

    2014-01-01

    High arsenic (As) concentration in groundwater has affected human health, particularly in South-East Asia putting millions of people at risk. Biogeochemical cycling of As carried out by different bacterial groups are suggested to control the As fluxes in aquifers. A functional diversity approach in link with As precipitation was adopted to study bacterial community structures and their variation within the As contaminated Bengal Delta Plain (BDP) aquifers of India. Groundwater samples collected from two shallow aquifers in Karimpur II (West Bengal, India), during years 2010 and 2011, were investigated to trace the effects immediately after monsoon period (precipitation) on community structure and diversity of bacterial assemblages with a focus on arsenite oxidizing bacterial phyla for two successive years. The study focused on amplification, clone library generation and sequencing of the arsenite oxidase large sub-unit gene aioA and 16S rRNA marker, with respect to changes in elemental concentrations. New set of primers were designed to amplify the aioA gene as a phylogenetic marker to study taxonomically diverse arsenite oxidizing bacterial groups in these aquifers. The overall narrow distribution of bacterial communities based on aioA and 16S rRNA sequences observed was due to poor nutrient status and anoxic conditions in these As contaminated aquifers. Proteobacteria was the dominant phylum detected, within which Acidovorax, Hydrogenophaga, Albidiferax, Bosea, and Polymorphum were the major arsenite oxidizing bacterial genera based on the number of clones sequenced. The structure of bacterial assemblages including those of arsenite oxidizing bacteria seems to have been affected by increase in major elemental concentrations (e.g., As, Fe, S, and Si) within two sampling sessions, which was supported by statistical analyses. One of the significant findings of this study is detection of novel lineages of 16S rRNA-like bacterial sequences indicating presence of indigenous bacterial communities BDP wells that can play important role in biogeochemical cycling of elements including As. PMID:25484877

  1. The bacteriology of pouchitis: a molecular phylogenetic analysis using 16S rRNA gene cloning and sequencing

    PubMed Central

    McLaughlin, Simon D; Walker, Alan W; Churcher, Carol; Clark, Susan K; Tekkis, Paris P; Johnson, Matthew W; Parkhill, Julian; Ciclitira, Paul J; Dougan, Gordon; Nicholls, R John; Petrovska, Liljana

    2012-01-01

    Objective To identify, compare and contrast the microbiota in patients with and without pouchitis after RPC for UC and FAP. Summary Background Data Pouchitis is the most common complication following restorative proctocolectomy (RPC). An abnormal host-microbial interaction has been implicated. We investigated the pouch microbiota in patients with and without pouchitis undergoing restorative proctocolectomy for UC and familial adenomatous polyposis (FAP). Methods Mucosal pouch biopsies, taken from 16 UC (pouchitis 8) and 8 FAP (pouchitis 3) patients were analysed to the species (or phylotype) level by cloning and sequencing of 3,184 full-length bacterial 16S rRNA genes. Results There was a significant increase in Proteobacteria (p= 0.019) and a significant decrease in Bacteroidetes (p= 0.001) and Faecalibacterium prausnitzii (p=0.029) in the total UC compared to the total FAP cohort, but only limited differences were found between the UC non-pouchitis and pouchitis groups and the FAP pouchitis and non-pouchitis groups. Bacterial diversity in the FAP non-pouchitis group was significantly greater than in UC non-pouchitis (p= 0.019) and significantly greater in UC non-pouchitis compared to UC pouchitis (p= 0.009). No individual species or phylotype specifically associated with either UC or FAP pouchitis was found. Conclusions UC pouch patients have a different, less diverse, gut microbiota than FAP patients. A further reduction in bacterial diversity but no significant dysbiosis occurs in those with pouchitis. The study suggests that a dysbiosis occurs in the ileal pouch of UC RPC patients which predisposes to, but may not directly cause, pouchitis. PMID:20562611

  2. Microbial methane turnover at Marmara Sea cold seeps: a combined 16S rRNA and lipid biomarker investigation.

    PubMed

    Chevalier, N; Bouloubassi, I; Birgel, D; Taphanel, M-H; López-García, P

    2013-01-01

    Lipid biomarkers and their stable carbon isotopic composition, as well as 16S rRNA gene sequences, were investigated in sediment cores from active seepage zones in the Sea of Marmara (Turkey) located on the active North Anatolian Fault, to assess processes associated with methane turnover by indigenous microbial communities. Diagnostic (13) C-depleted archaeal lipids of anaerobic methane oxidizers were only found in one core from the South of Çinarcik Basin and consist mainly of archaeol, sn-2 hydroxyarchaeol and various unsaturated pentamethylicosenes. Concurrently, abundant fatty acids (FAs) and a substantial amount of monoalkylglycerolethers (MAGEs), assigned to sulphate-reducing bacteria, were detected with strong (13) C-depletions. Both microbial lipids and their ?(13) C values suggest that anaerobic oxidation of methane with sulphate reduction (AOM/SR) occurs, specially in the 10- to 12-cm depth interval. Lipid biomarker results accompanied by 16S rRNA-based microbial diversity analyses showed that ANME-2 (ANME-2a and -2c) archaea and Desulfosarcina/Desulfococcus and Desulfobulbus deltaproteobacterial clades are the major AOM assemblages, which indicate a shallow AOM community at high methane flux. Apart from the typical AOM lipid biomarker pattern, a (13) C-depleted diunsaturated hydrocarbon, identified as 7,14-tricosadiene, occurred in the inferred maximum AOM interval at 10-12 cm depth. Its isotopic fingerprint implies that its microbial precursor occurs in close association with the AOM communities. Interestingly, the presence of 7,14-tricosadiene coincides with the presence of the so-far uncultured bacterial Candidate Division JS1, often detected in AOM areas. We propose the hypothesis that the JS1 bacterial group could be the potential source of (13) C-depleted tricosadiene. Future testing of this hypothesis is essential to fully determine the role of this bacterial group in AOM. PMID:23205581

  3. Bacterial communities associated with the ctenophores Mnemiopsis leidyi and Beroe ovata.

    PubMed

    Daniels, Camille; Breitbart, Mya

    2012-10-01

    Residing in a phylum of their own, ctenophores are gelatinous zooplankton that drift through the ocean's water column. Although ctenophores are known to be parasitized by a variety of eukaryotes, no studies have examined their bacterial associates. This study describes the bacterial communities associated with the lobate ctenophore Mnemiopsis leidyi and its natural predator Beroe ovata in Tampa Bay, Florida, USA. Investigations using terminal restriction fragment length polymorphism (T-RFLP) and cloning and sequencing of 16S rRNA genes demonstrated that ctenophore bacterial communities were distinct from the surrounding water. In addition, each ctenophore genus contained a unique microbiota. Ctenophore samples contained fewer bacterial operational taxonomic units (OTUs) by T-RFLP and lower diversity communities by 16S rRNA gene sequencing than the water column. Both ctenophore genera contained sequences related to bacteria previously described in marine invertebrates, and sequences similar to a sea anemone pathogen were abundant in B. ovata. Temporal sampling revealed that the ctenophore-associated bacterial communities varied over time, with no single OTU detected at all time points. This is the first report of distinct and dynamic bacterial communities associated with ctenophores, suggesting that these microbial consortia may play important roles in ctenophore ecology. Future work needs to elucidate the functional roles and mode of acquisition of these bacteria. PMID:22571334

  4. Characteristic archaebacterial 16S rRNA oligonucleotides

    NASA Technical Reports Server (NTRS)

    McGill, T. J.; Jurka, J.; Sobieski, J. M.; Pickett, M. H.; Woese, C. R.; Fox, G. E.

    1986-01-01

    A method of analyzing 16S rRNA catalog data has been developed in which groupings at various taxonomic levels can be characterized in terms of specific "signature" oligonucleotides. This approach provides an alternative means for evaluating higher order branching possibilities and can be used to assess the phylogenetic position of isolates that are poorly placed by the usual clustering procedures. This signature approach has been applied to forty archaebacterial catalogs and every oligonucleotide with significant signature value has been identified. Sets of specific oligonucleotides were identified for every major group on a dendrogram produced by cluster analysis procedures. Signatures that would establish between group relationships were also sought and found. In the case of the Methanobacteriaceae the clustering methods suggest a specific relationship to the Methanococcaceae. This inclusion is in fact supported by six strong signature oligonucleotides. However there are also significant numbers of signature oligonucleotides supporting a specific relationship of the Methanobacteriaceae to either the Halobacteriaceae or the Methanomicrobiaceae. Thus the placement of the Methanobacteriaceae is less certain than the usual dendrograms imply. The signature approach also was used to assess the phylogenetic position of Thermoplasma acidophilum which is found to be more closely related to the methanogen/halophile Division than to the sulfur dependent Division of the archaebacteria. This does not imply however that Thermoplasma acidophilum is properly regarded as being in the methanogen/halophile Division.

  5. Feature extraction Feature extraction

    E-print Network

    Giger, Christine

    Feature extraction #12;Feature extraction ! · Image interpretation: extract information from images · but the desired information may not be explicit in the raw observed pixel intensities · Transform image to make (hyperspectral sensors) Meteosat thermal IR channel hyperspectral "image cube" #12;Raw intensities ! · Pros

  6. Feature extraction Feature extraction

    E-print Network

    Giger, Christine

    Feature extraction #12;Feature extraction · Image interpretation: extract information from images · but the desired information may not be explicit in the raw observed pixel intensities · Transform image to make (hyperspectral sensors) Meteosat thermal IR channel hyperspectral "image cube" #12;Raw intensities · Pros

  7. Cospeciation of albatrosses and their chewing lice: COI and 12S rRNA sequence data reveal different

    E-print Network

    Page, Roderic

    Cospeciation of albatrosses and their chewing lice: COI and 12S rRNA sequence data reveal different was produced using combined data from two mitochondrial genes; COI and 12S rRNA. Using this phylogeny, four

  8. Identification and assessment of genetic similarity of soil bacterial isolates of Pseudomonas spp. using molecular techniques.

    PubMed

    Lisek, Anna; Sas Paszt, Lidia; Trzci?ski, Pawe?

    2014-01-01

    Bacteria of the genus Pseudomonas are often components of bioproducts designed to enhance the condition of the soil and plants. The use of Pseudomonas bacteria in bioproducts must be preceded by the acquisition, characterization and selection of beneficial strains living in the soil. A prerequisite for the selection of bacterial strains for use in bioproducts is to be able to identify the isolates rapidly and accurately. To identify and differentiate 15 bacterial isolates obtained from the soil surrounding the roots of sour cherry trees and to assess their genetic similarity, the rep-PCR technique and restriction analysis of the 16S rRNA gene and the 16S-ITS-23S rRNA operon were used. In addition, a sequence analysis of the 16S rRNA gene was performed. The analyses made it possible to divide the isolates into four clusters and to confirm their affiliation with the Pseudomonas species. RFLP analysis of the 16S-ITS-23S rRNA operon enabled greater differentiation of the isolates than RFLP of the 16S rRNA gene. The greatest differentiation of isolates within the clusters was obtained after using the rep-PCR technique. However, none of the techniques was able to discriminate all the isolates, which indicates very high genetic similarity of the Pseudomonas isolates found in the same sample of soil from around the roots of sour cherry trees. The tests performed will find application for distinguishing and identifying Pseudomonas strains collected from the soil in order to select the most valuable bacterial strains that produce beneficial effects on plants. PMID:25546939

  9. Comparative Metagenomic Profiling of Symbiotic Bacterial Communities Associated with Ixodes persulcatus, Ixodes pavlovskyi and Dermacentor reticulatus Ticks.

    PubMed

    Kurilshikov, Alexander; Livanova, Natalya N; Fomenko, Nataliya V; Tupikin, Alexey E; Rar, Vera A; Kabilov, Marsel R; Livanov, Stanislav G; Tikunova, Nina V

    2015-01-01

    Ixodes persulcatus, Ixodes pavlovskyi, and Dermacentor reticulatus ticks inhabiting Western Siberia are responsible for the transmission of a number of etiological agents that cause human and animal tick-borne diseases. Because these ticks are abundant in the suburbs of large cities, agricultural areas, and popular tourist sites and frequently attack people and livestock, data regarding the microbiomes of these organisms are required. Using metagenomic 16S profiling, we evaluate bacterial communities associated with I. persulcatus, I. pavlovskyi, and D. reticulatus ticks collected from the Novosibirsk region of Russia. A total of 1214 ticks were used for this study. DNA extracted from the ticks was pooled according to tick species and sex. Sequencing of the V3-V5 domains of 16S rRNA genes was performed using the Illumina Miseq platform. The following bacterial genera were prevalent in the examined communities: Acinetobacter (all three tick species), Rickettsia (I. persulcatus and D. reticulatus) and Francisella (D. reticulatus). B. burgdorferi sensu lato and B. miyamotoi sequences were detected in I. persulcatus and I. pavlovskyi but not in D. reticulatus ticks. The pooled samples of all tick species studied contained bacteria from the Anaplasmataceae family, although their occurrence was low. DNA from A. phagocytophilum and Candidatus Neoehrlichia mikurensis was first observed in I. pavlovskyi ticks. Significant inter-species differences in the number of bacterial taxa as well as intra-species diversity related to tick sex were observed. The bacterial communities associated with the I. pavlovskyi ticks displayed a higher biodiversity compared with those of the I. persulcatus and D. reticulatus ticks. Bacterial community structure was also diverse across the studied tick species, as shown by permutational analysis of variance using the Bray-Curtis dissimilarity metric (p = 0.002). Between-sex variation was confirmed by PERMANOVA testing in I. persulcatus (p = 0.042) and I. pavlovskyi (p = 0.042) ticks. Our study indicated that 16S metagenomic profiling could be used for rapid assessment of the occurrence of medically important bacteria in tick populations inhabiting different natural biotopes and therefore the epidemic danger of studied foci. PMID:26154300

  10. Comparative Metagenomic Profiling of Symbiotic Bacterial Communities Associated with Ixodes persulcatus, Ixodes pavlovskyi and Dermacentor reticulatus Ticks

    PubMed Central

    Kurilshikov, Alexander; Livanova, Natalya N.; Fomenko, Nataliya V.; Tupikin, Alexey E.; Rar, Vera A.; Kabilov, Marsel R.; Livanov, Stanislav G.; Tikunova, Nina V.

    2015-01-01

    Ixodes persulcatus, Ixodes pavlovskyi, and Dermacentor reticulatus ticks inhabiting Western Siberia are responsible for the transmission of a number of etiological agents that cause human and animal tick-borne diseases. Because these ticks are abundant in the suburbs of large cities, agricultural areas, and popular tourist sites and frequently attack people and livestock, data regarding the microbiomes of these organisms are required. Using metagenomic 16S profiling, we evaluate bacterial communities associated with I. persulcatus, I. pavlovskyi, and D. reticulatus ticks collected from the Novosibirsk region of Russia. A total of 1214 ticks were used for this study. DNA extracted from the ticks was pooled according to tick species and sex. Sequencing of the V3-V5 domains of 16S rRNA genes was performed using the Illumina Miseq platform. The following bacterial genera were prevalent in the examined communities: Acinetobacter (all three tick species), Rickettsia (I. persulcatus and D. reticulatus) and Francisella (D. reticulatus). B. burgdorferi sensu lato and B. miyamotoi sequences were detected in I. persulcatus and I. pavlovskyi but not in D. reticulatus ticks. The pooled samples of all tick species studied contained bacteria from the Anaplasmataceae family, although their occurrence was low. DNA from A. phagocytophilum and Candidatus Neoehrlichia mikurensis was first observed in I. pavlovskyi ticks. Significant inter-species differences in the number of bacterial taxa as well as intra-species diversity related to tick sex were observed. The bacterial communities associated with the I. pavlovskyi ticks displayed a higher biodiversity compared with those of the I. persulcatus and D. reticulatus ticks. Bacterial community structure was also diverse across the studied tick species, as shown by permutational analysis of variance using the Bray-Curtis dissimilarity metric (p = 0.002). Between-sex variation was confirmed by PERMANOVA testing in I. persulcatus (p = 0.042) and I. pavlovskyi (p = 0.042) ticks. Our study indicated that 16S metagenomic profiling could be used for rapid assessment of the occurrence of medically important bacteria in tick populations inhabiting different natural biotopes and therefore the epidemic danger of studied foci. PMID:26154300

  11. 18S rRNA degradation is not accompanied by altered rRNA transport at early times following irradiation of HeLa cells

    SciTech Connect

    Fuchs, P.; Krolak, J.M.; McClain, D.; Minton, K.W.

    1990-01-01

    In recent investigations on the effects of radiation on rRNA processing in HeLa S3 cells, the authors pulse-labeled the cells with uridine immediately prior to irradiation. The 45 S rRNA precursor, which undergoes nuclear processing to form one each of its major daughter species, 28S and 18S rRNA, was separated from the daughter species by gel electrophoresis and the radiolabel in each species determined at various times after irradiation. By pulse-labeling the cells prior to irradiation, superimposed effects caused by radiation-induced alterations of rRNA transcription and Refs. therein were minimized, permitting selective analysis of the processing of that fraction of 45S precursor that had been synthesized (radiolabeled) predominantly prior to irradiation. They now report more detailed studies on 45S rRNA processing within the first 2 h following irradiation in which they have found a maximum 28 S:18 S ratio of 2:1 that is observed about 1 h following irradiation of 5 or 10 Gy.

  12. Trans-splicing and RNA editing of LSU rRNA in Diplonema mitochondria

    PubMed Central

    Valach, Matus; Moreira, Sandrine; Kiethega, Georgette N.; Burger, Gertraud

    2014-01-01

    Mitochondrial ribosomal RNAs (rRNAs) often display reduced size and deviant secondary structure, and sometimes are fragmented, as are their corresponding genes. Here we report a mitochondrial large subunit rRNA (mt-LSU rRNA) with unprecedented features. In the protist Diplonema, the rnl gene is split into two pieces (modules 1 and 2, 534- and 352-nt long) that are encoded by distinct mitochondrial chromosomes, yet the rRNA is continuous. To reconstruct the post-transcriptional maturation pathway of this rRNA, we have catalogued transcript intermediates by deep RNA sequencing and RT-PCR. Gene modules are transcribed separately. Subsequently, transcripts are end-processed, the module-1 transcript is polyuridylated and the module-2 transcript is polyadenylated. The two modules are joined via trans-splicing that retains at the junction ?26 uridines, resulting in an extent of insertion RNA editing not observed before in any system. The A-tail of trans-spliced molecules is shorter than that of mono-module 2, and completely absent from mitoribosome-associated mt-LSU rRNA. We also characterize putative antisense transcripts. Antisense-mono-modules corroborate bi-directional transcription of chromosomes. Antisense-mt-LSU rRNA, if functional, has the potential of guiding concomitantly trans-splicing and editing of this rRNA. Together, these findings open a window on the investigation of complex regulatory networks that orchestrate multiple and biochemically diverse post-transcriptional events. PMID:24259427

  13. Bacterial populations in samples of bioleached copper ore as revealed by analysis of DNA obtained before and after cultivation.

    PubMed Central

    Pizarro, J; Jedlicki, E; Orellana, O; Romero, J; Espejo, R T

    1996-01-01

    The composition of bacterial populations in copper bioleaching systems was investigated by analysis of DNA obtained either directly from ores or leaching solutions or after laboratory cultures. This analysis consisted of the characterization of the spacer regions between the 16 and 23S genes in the bacterial rRNA genetic loci after PCR amplification. The sizes of the spacer regions, amplified from DNAs obtained from samples, were compared with the sizes of those obtained from cultures of the main bacterial species isolated from bioleaching systems. This allowed a preliminary assessment of the bacterial species present in the samples. Identification of the bacteria was achieved by partial sequencing of the 16S rRNA genes adjacent to the spacer regions. The spacer regions observed in DNA from columns leached at different iron concentrations indicated the presence of a mixture of different bacteria. The spacer region corresponding to Thiobacillus ferrooxidans was the main product observed at high ferrous iron concentration. At low ferrous iron concentration, spacer regions of different lengths, corresponding to Thiobacillus thiooxidans and "Leptospirillum ferrooxidans" were observed. However, T. ferrooxidans appeared to predominate after culture of these samples in medium containing ferrous iron as energy source. Although some of these strains contained singular spacer regions, they belonged within previously described groups of T. ferrooxidans according to the nucleotide sequence of the neighbor 16S rRNA. These results illustrate the bacterial diversity in bioleaching systems and the selective pressure generated by different growth conditions. PMID:8919792

  14. An Assessment of Urea-Formaldehyde Fertilizer on the Diversity of Bacterial Communities in Onion and Sugar Beet

    PubMed Central

    Ikeda, Seishi; Suzuki, Keijiro; Kawahara, Makoto; Noshiro, Masao; Takahashi, Naokazu

    2014-01-01

    The impact of a urea-formaldehyde (UF) fertilizer on bacterial diversity in onion bulbs and main roots of sugar beet were examined using a 16S rRNA gene clone library. The UF fertilizer markedly increased bacterial diversity in both plants. The results of principal coordinates analysis (PCoA) revealed that nearly 30% of the variance observed in bacterial diversity in both the onion and sugar beet was attributed to the fertilization conditions and also that the community structures in both plants shifted unidirectionally in response to the UF fertilizer. PMID:24882062

  15. An assessment of urea-formaldehyde fertilizer on the diversity of bacterial communities in onion and sugar beet.

    PubMed

    Ikeda, Seishi; Suzuki, Keijiro; Kawahara, Makoto; Noshiro, Masao; Takahashi, Naokazu

    2014-01-01

    The impact of a urea-formaldehyde (UF) fertilizer on bacterial diversity in onion bulbs and main roots of sugar beet were examined using a 16S rRNA gene clone library. The UF fertilizer markedly increased bacterial diversity in both plants. The results of principal coordinates analysis (PCoA) revealed that nearly 30% of the variance observed in bacterial diversity in both the onion and sugar beet was attributed to the fertilization conditions and also that the community structures in both plants shifted unidirectionally in response to the UF fertilizer. PMID:24882062

  16. New screening software shows most recent large 16S rRNA gene clone libraries1 contain chimeras.2

    E-print Network

    Jones, Antonia J.

    1 New screening software shows most recent large 16S rRNA gene clone libraries1 contain chimeras.2 3 Running title4 Detecting chimeras within 16S rRNA gene libraries.5 6 Authors7 Kevin E. Ashelford for screening entire 16S rRNA gene2 libraries, of up to 1,000 sequences, for chimeras and other artifacts

  17. Mutagenesis of 16 S rRNA C1409-G1491 Base-pair Differentiates Between 60

    E-print Network

    Westhof, Eric

    Mutagenesis of 16 S rRNA C1409-G1491 Base-pair Differentiates Between 60 OH and 60 NH3 C Universite´ Louis Pasteur 15 rue Rene´ Descartes, 67084 Strasbourg Cedex, France Using a single rRNA allelic Gram-positive model system, we system- atically mutagenized 16 S rRNA positions 1409 and 1491 to probe

  18. Solution Structure of an Alternate Conformation of Helix 27 from Escherichia coli 16S rRNA

    E-print Network

    Walter, Nils G.

    Solution Structure of an Alternate Conformation of Helix 27 from Escherichia coli 16S rRNA Meredith ribosomal subunit, comprising nucleotides (nt) 885-912 in E. coli 16S ribosomal (r)RNA (Figure 1A% conserved among bacteria.1 Early sequence alignment of 16S rRNA predicted a base pair between C912 and G888

  19. Novel methods for rRNA removal and directional, ligation-free RNA-seq library preparation

    E-print Network

    Cai, Long

    Novel methods for rRNA removal and directional, ligation-free RNA-seq library preparation Existing that are dependent on intact (nondegraded) total RNA samples, efficient removal of ribosomal RNA (rRNA), ligation methods for rRNA removal from intact and fragmented RNA (Ribo-ZeroTM technology) and for rapid preparation

  20. Complete rRNA Gene Sequences Reveal that the Microsporidium Nosema bombi Infects Diverse Bumblebee (Bombus spp.) Hosts and

    E-print Network

    Paxton, Robert

    Complete rRNA Gene Sequences Reveal that the Microsporidium Nosema bombi Infects Diverse Bumblebee and differentiation among genetic variants of the same species has typically relied on ribosomal RNA (rRNA) gene sequences. We characterised the entire rRNA gene of a microsporidium from 11 isolates rep- resenting eight

  1. The 18S rRNA from Odontophrynus americanus 2n and 4n (Amphibia, Anura) reveals unusual extra

    E-print Network

    Gent, Universiteit

    The 18S rRNA from Odontophrynus americanus 2n and 4n (Amphibia, Anura) reveals unusual extra and used to predict the secondary structure of the corresponding 18S rRNA molecules. Comparison of the Odontophrynus 18S rRNA was the presence of unusual extra sequences in the V2 region, between helices 9 and 11

  2. 23S rRNA base pair 20572611 determines ketolide susceptibility and fitness cost of the macrolide

    E-print Network

    Yonath, Ada E.

    23S rRNA base pair 2057­2611 determines ketolide susceptibility and fitness cost of the macrolide, Weizmann Institute, Rehovot 76100, Israel Contributed by Ada Yonath, February 27, 2005 The 23S rRNA A2058G. We used site-directed mutagenesis of nucleotides within domain V of 23S rRNA to study the molecular

  3. Rapid in vivo exploration of a 5S rRNA neutral network Zhengdong D. Zhang a,

    E-print Network

    Fox, George

    Rapid in vivo exploration of a 5S rRNA neutral network Zhengdong D. Zhang a, , Madhavi Nayar b: 5S rRNA E. coli knockout strains Neutral network In vivo mutagenesis A partial knockout compensation an Escherichia coli strain in which five of eight genomic 5S rRNA genes were deleted in conjunction

  4. Copyright 0 1988 by the Genetics Society of America Chromosomal Organizationof rRNA Operonsin Bacillus subtilis

    E-print Network

    Jarvis, Erich D.

    Copyright 0 1988 by the Genetics Society of America Chromosomal Organizationof rRNA Operonsin rRNA gene sets in the endospore forming bacterium Bacillus subtilis. Southern hybridizationsallowed by transductional crosses. Nine ofthe ten rRNA gene sets are located between 0 and 70" on thegenomic map

  5. E. Braga R. Zardoya A. Meyer J. Yen Mitochondrial and nuclear rRNA based copepod phylogeny

    E-print Network

    Yen, Jeannette

    sequences of the mitochondrial 16S rRNA and the nu- clear 28S rRNA genes. DNA isolation, polymerase chain mitochondrial DNA sequence data to address systematic relationships among copepods, primarily within the familyE. Braga á R. Zardoya á A. Meyer á J. Yen Mitochondrial and nuclear rRNA based copepod phylogeny

  6. Molecular Survey of Bacterial Communities Associated with Bacterial Chondronecrosis with Osteomyelitis (BCO) in Broilers

    PubMed Central

    Jiang, Tieshan; Mandal, Rabindra K.; Wideman, Robert F.; Khatiwara, Anita; Pevzner, Igal; Min Kwon, Young

    2015-01-01

    Bacterial chondronecrosis with osteomyelitis (BCO) is recognized as an important cause of lameness in commercial broiler chickens (meat-type chickens). Relatively little is known about the microbial communities associated with BCO. This study was conducted to increase our understanding of the microbial factors associated with BCO using a culture-independent approach. Using Illumina sequencing of the hyper-variable region V6 in the 16S rRNA gene, we characterized the bacterial communities in 97 femoral or tibial heads from normal and lame broilers carefully selected to represent diverse variations in age, line, lesion type, floor type, clinical status and bone type. Our in-depth survey based on 14 million assembled sequence reads revealed that complex bacterial communities exist in all samples, including macroscopically normal bones from clinically healthy birds. Overall, Proteobacteria (mean 90.9%) comprised the most common phylum, followed by Firmicutes (6.1%) and Actinobacteria (2.6%), accounting for more than 99% of all reads. Statistical analyses demonstrated that there are differences in bacterial communities in different types of bones (femur vs. tibia), lesion types (macroscopically normal femora or tibiae vs. those with pathognomonic BCO lesions), and among individual birds. This analysis also showed that BCO samples overrepresented genera Staphylococcus, whose species have been frequently isolated in BCO samples in previous studies. Rarefaction analysis demonstrated the general tendency that increased severities of BCO lesions were associated with reduced species diversity in both femoral and tibial samples when compared to macroscopically normal samples. These observations suggest that certain bacterial subgroups are preferentially selected in association with the development of BCO lesions. Understanding the microbial species associated with BCO will identify opportunities for understanding and modulating the pathogenesis of this form of lameness in broilers. PMID:25881241

  7. Soil bacterial diversity patterns and drivers along an elevational gradient on Shennongjia Mountain, China

    PubMed Central

    Zhang, Yuguang; Cong, Jing; Lu, Hui; Li, Guangliang; Xue, Yadong; Deng, Ye; Li, Hui; Zhou, Jizhong; Li, Diqiang

    2015-01-01

    Understanding biological diversity elevational pattern and the driver factors are indispensable to develop the ecological theories. Elevational gradient may minimize the impact of environmental factors and is the ideal places to study soil microbial elevational patterns. In this study, we selected four typical vegetation types from 1000 to 2800?m above the sea level on the northern slope of Shennongjia Mountain in central China, and analysed the soil bacterial community composition, elevational patterns and the relationship between soil bacterial diversity and environmental factors by using the 16S rRNA Illumina sequencing and multivariate statistical analysis. The results revealed that the dominant bacterial phyla were Acidobacteria, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Verrucomicrobia, which accounted for over 75% of the bacterial sequences obtained from tested samples, and the soil bacterial operational taxonomic unit (OTU) richness was a significant monotonous decreasing (P?bacterial population composition decreased significantly (P?bacterial community. Therefore, the soil bacterial diversity on Shennongjia Mountain had a significant and different elevational pattern, and plant diversity and soil pH may be the key factors in shaping the soil bacterial spatial pattern. PMID:26032124

  8. Bacterial diversity in different regions of gastrointestinal tract of Giant African snail (Achatina fulica).

    PubMed

    Pawar, Kiran D; Banskar, Sunil; Rane, Shailendra D; Charan, Shakti S; Kulkarni, Girish J; Sawant, Shailesh S; Ghate, Hemant V; Patole, Milind S; Shouche, Yogesh S

    2012-12-01

    The gastrointestinal (GI) tract of invasive land snail Achatina fulica is known to harbor metabolically active bacterial communities. In this study, we assessed the bacterial diversity in the different regions of GI tract of Giant African snail, A. fulica by culture-independent and culture-dependent methods. Five 16S rRNA gene libraries from different regions of GI tract of active snails indicated that sequences affiliated to phylum ?-Proteobacteria dominated the esophagus, crop, intestine, and rectum libraries, whereas sequences affiliated to Tenericutes dominated the stomach library. On phylogenetic analysis, 30, 27, 9, 27, and 25 operational taxonomic units (OTUs) from esophagus, crop, stomach, intestine, and rectum libraries were identified, respectively. Estimations of the total bacterial diversity covered along with environmental cluster analysis showed highest bacterial diversity in the esophagus and lowest in the stomach. Thirty-three distinct bacterial isolates were obtained, which belonged to 12 genera of two major bacterial phyla namely ?-Proteobacteria and Firmicutes. Among these, Lactococcus lactis and Kurthia gibsonii were the dominant bacteria present in all GI tract regions. Quantitative real-time polymerase chain reaction (qPCR) analysis indicated significant differences in bacterial load in different GI tract regions of active and estivating snails. The difference in the bacterial load between the intestines of active and estivating snail was maximum. Principal component analysis (PCA) of terminal restriction fragment length polymorphism suggested that bacterial community structure changes only in intestine when snail enters estivation state. PMID:23233413

  9. Evaluating variation in human gut microbiota profiles due to DNA extraction method and inter-subject differences

    PubMed Central

    Wagner Mackenzie, Brett; Waite, David W.; Taylor, Michael W.

    2015-01-01

    The human gut contains dense and diverse microbial communities which have profound influences on human health. Gaining meaningful insights into these communities requires provision of high quality microbial nucleic acids from human fecal samples, as well as an understanding of the sources of variation and their impacts on the experimental model. We present here a systematic analysis of commonly used microbial DNA extraction methods, and identify significant sources of variation. Five extraction methods (Human Microbiome Project protocol, MoBio PowerSoil DNA Isolation Kit, QIAamp DNA Stool Mini Kit, ZR Fecal DNA MiniPrep, phenol:chloroform-based DNA isolation) were evaluated based on the following criteria: DNA yield, quality and integrity, and microbial community structure based on Illumina amplicon sequencing of the V4 region of bacterial and archaeal 16S rRNA genes. Our results indicate that the largest portion of variation within the model was attributed to differences between subjects (biological variation), with a smaller proportion of variation associated with DNA extraction method (technical variation) and intra-subject variation. A comprehensive understanding of the potential impact of technical variation on the human gut microbiota will help limit preventable bias, enabling more accurate diversity estimates. PMID:25741335

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

    SciTech Connect

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

    2004-08-01

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

  11. BactQuant: An enhanced broad-coverage bacterial quantitative real-time PCR assay

    PubMed Central

    2012-01-01

    Background Bacterial load quantification is a critical component of bacterial community analysis, but a culture-independent method capable of detecting and quantifying diverse bacteria is needed. Based on our analysis of a diverse collection of 16?S rRNA gene sequences, we designed a broad-coverage quantitative real-time PCR (qPCR) assay—BactQuant—for quantifying 16?S rRNA gene copy number and estimating bacterial load. We further utilized in silico evaluation to complement laboratory-based qPCR characterization to validate BactQuant. Methods The aligned core set of 4,938 16?S rRNA gene sequences in the Greengenes database were analyzed for assay design. Cloned plasmid standards were generated and quantified using a qPCR-based approach. Coverage analysis was performed computationally using >670,000 sequences and further evaluated following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Results A bacterial TaqMan® qPCR assay targeting a 466?bp region in V3-V4 was designed. Coverage analysis showed that 91% of the phyla, 96% of the genera, and >80% of the 89,537 species analyzed contained at least one perfect sequence match to the BactQuant assay. Of the 106 bacterial species evaluated, amplification efficiencies ranged from 81 to 120%, with r2-value of >0.99, including species with sequence mismatches. Inter- and intra-run coefficient of variance was <3% and <16% for Ct and copy number, respectively. Conclusions The BactQuant assay offers significantly broader coverage than a previously reported universal bacterial quantification assay BactQuant in vitro performance was better than the in silico predictions. PMID:22510143

  12. Detecting human bacterial pathogens in wastewater treatment plants by a high-throughput shotgun sequencing technique.

    PubMed

    Cai, Lin; Zhang, Tong

    2013-05-21

    Human pathogens are one of the major threats to global public health. Wastewater treatment plants (WWTPs) serve as city guts to receive and digest various human pathogens. Several techniques have been developed to detect human pathogens in WWTPs and to assess potential environmental risks. In this study, we employed 24 metagenomic DNA data sets derived from a high-throughput shotgun sequencing technique to more accurately and efficiently detect human bacterial pathogens in influent, activated sludge, and effluent of two Hong Kong WWTPs. Each data set was quality-filtered and normalized to 12,500,000 DNA sequences with a length of 150-190 bp. Then, a BLASTN search against Greengenes general 16S rRNA gene database and human pathogenic bacteria 16S rRNA gene database, a BLASTX search against human pathogenic bacteria virulence factor database, as well as MetaPhlAn analysis were conducted to survey the distribution, diversity, and abundance of human bacterial pathogens. The results revealed that (i) nine bacterial pathogens were detected; (ii) the overall pathogenic bacteria abundance was estimated as 0.06-3.20% in the total bacteria population using 16S rRNA gene fingerprinting; (iii) pathogenic bacteria detected in activated sludge and effluent shared similar profiles but were different from influent based on both 16S rRNA gene and virulence factor fingerprintings; (iv) Mycobacterium tuberculosis -like species may present potential pathogenic risks because it was detected with high abundance in both activated sludge and effluent. These findings provided a comprehensive profile of commonly concerned human pathogens in two Hong Kong WWTPs and demonstrated that the high-throughput shotgun sequencing technique is a feasible and effectual approach for environmental detection of human bacterial pathogens. PMID:23594284

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

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

  14. Extraction of inhibitor-free metagenomic DNA from polluted sediments, compatible with molecular diversity analysis using adsorption and ion-exchange treatments.

    PubMed

    Desai, Chirayu; Madamwar, Datta

    2007-03-01

    PCR inhibitor-free metagenomic DNA of high quality and high yield was extracted from highly polluted sediments using a simple remediation strategy of adsorption and ion-exchange chromatography. Extraction procedure was optimized with series of steps, which involved gentle mechanical lysis, treatment with powdered activated charcoal (PAC) and ion-exchange chromatography with amberlite resin. Quality of the extracted DNA for molecular diversity analysis was tested by amplifying bacterial 16S rDNA (16S rRNA gene) with eubacterial specific universal primers (8f and 1492r), cloning of the amplified 16S rDNA and ARDRA (amplified rDNA restriction analysis) of the 16S rDNA clones. The presence of discrete differences in ARDRA banding profiles provided evidence for expediency of the DNA extraction protocol in molecular diversity studies. A comparison of the optimized protocol with commercial Ultraclean Soil DNA isolation kit suggested that method described in this report would be more efficient in removing metallic and organic inhibitors, from polluted sediment samples. PMID:16806911

  15. Bacterial community diversity in the Brazilian Atlantic forest soils.

    PubMed

    Bruce, Thiago; Martinez, Ivana B; Maia Neto, Oswaldo; Vicente, Ana Carolina P; Kruger, Ricardo H; Thompson, Fabiano L

    2010-11-01

    The aim of this study was to characterize the bacterial community diversity of the Brazilian Atlantic forest soil by means of both cultivation and 16S rRNA clone libraries. A collection of 86 representative isolates, obtained from six samples of Atlantic forest soils from the National Park of Serra dos Órgãos (PARNASO), belonged to the genera Arthrobacter, Bacillus, Burkholderia, Leifsonia, Paenibacillus, Pseudomonas, Ralstonia, Serratia, and Streptomyces according to the 16S rRNA sequences. Representative isolates from the different genera degraded cellulose and lignin. The culture-independent analysis based on 894 partial 16S rRNA gene sequences revealed that the most frequently retrieved groups belonged to the phyla Acidobacteria (29-54%), Proteobacteria (16-38%), and Verrucomicrobia (0.6-14%). The majority of the sequences (82.6%) were unidentified singletons and doubletons, indicating a high diversity of rare unique sequences. Chao1 estimator disclosed a high number of phyla (41-152) and species (263-446). This is the first survey on the Atlantic Forest soils using a combination of cultivation and culture-independent approaches. We conclude that the Brazilian Atlantic Forest soil represents a vast source of novel bacteria. PMID:20886336

  16. Identification of Bacteriology and Risk Factor Analysis of Asymptomatic Bacterial Colonization in Pacemaker Replacement Patients

    PubMed Central

    Chu, Xian-Ming; Yu, Hua; Sun, Xue-Xia; An, Yi; Li, Bing; Li, Xue-Bin

    2015-01-01

    Background Recent researches revealed that asymptomatic bacterial colonization on PMs might be ubiquitous and increase the risk of clinical PM infection. Early diagnosis of patients with asymptomatic bacterial colonization could provide opportunity for targeted preventive measures. Objective The present study explores the incidence of bacterial colonization of generator pockets in pacemaker replacement patients without signs of infection, and to analyze risk factors for asymptomatic bacterial colonization. Methods From June 2011 to December 2013, 118 patients underwent pacemaker replacement or upgrade. Identification of bacteria was carried out by bacterial culture and 16S rRNA sequencing. Clinical risk characteristics were analyzed. Results The total bacterial positive rate was 37.3% (44 cases), and the coagulase-negative Staphylococcus aureus detection rate was the highest. Twenty two (18.6%) patients had positive bacterial culture results, of which 50% had coagulase-negative staphylococcus. The bacterial DNA detection rate was 36.4 % (43 cases). Positive bacterial DNA results from pocket tissues and the surface of the devices were 22.0% and 29.7%, respectively. During follow-up (median, 27.0 months), three patients (6.8%, 3/44) became symptomatic with the same genus of microorganism, S. aureus (n=2) and S. epidermidis (n=1). Multivariable logistic regression analysis showed that history of bacterial infection, use of antibiotics, application of antiplatelet drugs, replacement frequency were independent risk factors for asymptomatic bacterial colonization. Conclusion There was a high incidence of asymptomatic bacterial colonization in pacemaker patients with independent risk factors. Bacterial culture combined genetic testing could improve the detection rate. PMID:25768661

  17. Asphalt Showing Bacterial Degeneration 

    E-print Network

    Unknown

    2011-08-17

    , has been expanded through a new project, Development of a Synergistic, Comprehensive Statewide Lone Star Healthy Streams Program. The LSHS Program initially developed and tested educational opportunities for cattlemen focusing on bacterial... contami- nation of watersheds by grazing animals and how that bacterial contamination can be reduced. The program also encouraged adoption of best management practices designed to reduce bacterial loading to Texas streams and waterways...

  18. Facial bacterial infections: folliculitis.

    PubMed

    Laureano, Ana Cristina; Schwartz, Robert A; Cohen, Philip J

    2014-01-01

    Facial bacterial infections are most commonly caused by infections of the hair follicles. Wherever pilosebaceous units are found folliculitis can occur, with the most frequent bacterial culprit being Staphylococcus aureus. We review different origins of facial folliculitis, distinguishing bacterial forms from other infectious and non-infectious mimickers. We distinguish folliculitis from pseudofolliculitis and perifolliculitis. Clinical features, etiology, pathology, and management options are also discussed. PMID:25441463

  19. Global dispersion of bacterial cells on Asian dust

    PubMed Central

    Yamaguchi, Nobuyasu; Ichijo, Tomoaki; Sakotani, Akiko; Baba, Takashi; Nasu, Masao

    2012-01-01

    The atmospheric dispersion of bacteria over long distances is an important facet of microbial ecology. Certain groups of dispersed bacteria can adapt to their new location and affect established ecosystems. Aeolian dust particles are known to be carriers of microbes but further research is needed to expand our understanding of this field of microbiology. Here we showed the potential of aeolian dust to global migration of bacterial cells. We demonstrated the presence of microbial cells on dust particles directly by bio-imaging. Bacterial abundance on dust particles declined from 105 to less than 103 cells/m3 as the dust event subsided. Taxonomically diverse bacteria were identified by 16S rRNA gene sequencing and some of these bacteria retained growth potential. Our results confirm that bacteria can attach to aeolian dust particles and they have the potential to migrate globally during dust events and thus can contribute to the diversity of downwind ecosystems. PMID:22826803

  20. BacDive – The Bacterial Diversity Metadatabase in 2016

    PubMed Central

    Söhngen, Carola; Podstawka, Adam; Bunk, Boyke; Gleim, Dorothea; Vetcininova, Anna; Reimer, Lorenz Christian; Ebeling, Christian; Pendarovski, Cezar; Overmann, Jörg

    2016-01-01

    BacDive–the Bacterial Diversity Metadatabase (http://bacdive.dsmz.de) provides strain-linked information about bacterial and archaeal biodiversity. The range of data encompasses taxonomy, morphology, physiology, sampling and concomitant environmental conditions as well as molecular biology. The majority of data is manually annotated and curated. Currently (with release 9/2015), BacDive covers 53 978 strains. Newly implemented RESTful web services provide instant access to the content in machine-readable XML and JSON format. Besides an overall increase of data content, BacDive offers new data fields and features, e.g. the search for gene names, plasmids or 16S rRNA in the advanced search, as well as improved linkage of entries to external life science web resources. PMID:26424852

  1. BacDive - The Bacterial Diversity Metadatabase in 2016.

    PubMed

    Söhngen, Carola; Podstawka, Adam; Bunk, Boyke; Gleim, Dorothea; Vetcininova, Anna; Reimer, Lorenz Christian; Ebeling, Christian; Pendarovski, Cezar; Overmann, Jörg

    2016-01-01

    BacDive-the Bacterial Diversity Metadatabase (http://bacdive.dsmz.de) provides strain-linked information about bacterial and archaeal biodiversity. The range of data encompasses taxonomy, morphology, physiology, sampling and concomitant environmental conditions as well as molecular biology. The majority of data is manually annotated and curated. Currently (with release 9/2015), BacDive covers 53 978 strains. Newly implemented RESTful web services provide instant access to the content in machine-readable XML and JSON format. Besides an overall increase of data content, BacDive offers new data fields and features, e.g. the search for gene names, plasmids or 16S rRNA in the advanced search, as well as improved linkage of entries to external life science web resources. PMID:26424852

  2. An Archaea 5S rRNA analog is stably expressed in Escherichia coli

    NASA Technical Reports Server (NTRS)

    Yang, Y.; Fox, G. E.

    1996-01-01

    Mini-genes for 5S-like rRNA were constructed. These genes had a sequence which largely resembles that of the naturally occurring 5S rRNA of a bacterium, Halococcus morrhuae, which phylogenetically belongs to the Archaea. Plasmids carrying the mini-genes were transformed into Escherichia coli (Ec). Ribosomal incorporation was not a prerequisite for stable accumulation of the RNA product. However, only those constructs with a well-base-paired helix I accumulated RNA product. This result strongly implies that this aspect of the structure is likely to be an important condition for stabilizing 5S rRNA-like products. The results are consistent with our current understanding of 5S rRNA processing in Ec. When used in conjunction with rRNA probe technology, the resulting chimeric RNA may be useful as a monitoring tool for genetically engineered microorganisms or naturally occurring organisms that are released into the environment.

  3. Phylogenetically specific separation of rRNA from prokaryotes for isotopic analysis

    E-print Network

    Sessions, Alex L.

    Phylogenetically specific separation of rRNA from prokaryotes for isotopic analysis Ann Pearsona the course of 1 year, the carbon isotopic composition of the mixed prokaryotic community varied between À33x

  4. Dinoflagellate 17S rRNA sequence inferred from the gene sequence: Evolutionary implications

    PubMed Central

    Herzog, Michel; Maroteaux, Luc

    1986-01-01

    We present the complete sequence of the nuclear-encoded small-ribosomal-subunit RNA inferred from the cloned gene sequence of the dinoflagellate Prorocentrum micans. The dinoflagellate 17S rRNA sequence of 1798 nucleotides is contained in a family of 200 tandemly repeated genes per haploid genome. A tentative model of the secondary structure of P. micans 17S rRNA is presented. This sequence is compared with the small-ribosomal-subunit rRNA of Xenopus laevis (Animalia), Saccharomyces cerevisiae (Fungi), Zea mays (Planta), Dictyostelium discoideum (Protoctista), and Halobacterium volcanii (Monera). Although the secondary structure of the dinoflagellate 17S rRNA presents most of the eukaryotic characteristics, it contains sufficient archaeobacterial-like structural features to reinforce the view that dinoflagellates branch off very early from the eukaryotic lineage. PMID:16578795

  5. Dinoflagellate 17S rRNA sequence inferred from the gene sequence: Evolutionary implications.

    PubMed

    Herzog, M; Maroteaux, L

    1986-11-01

    We present the complete sequence of the nuclear-encoded small-ribosomal-subunit RNA inferred from the cloned gene sequence of the dinoflagellate Prorocentrum micans. The dinoflagellate 17S rRNA sequence of 1798 nucleotides is contained in a family of 200 tandemly repeated genes per haploid genome. A tentative model of the secondary structure of P. micans 17S rRNA is presented. This sequence is compared with the small-ribosomal-subunit rRNA of Xenopus laevis (Animalia), Saccharomyces cerevisiae (Fungi), Zea mays (Planta), Dictyostelium discoideum (Protoctista), and Halobacterium volcanii (Monera). Although the secondary structure of the dinoflagellate 17S rRNA presents most of the eukaryotic characteristics, it contains sufficient archaeobacterial-like structural features to reinforce the view that dinoflagellates branch off very early from the eukaryotic lineage. PMID:16578795

  6. Identification of bacterial endophytes associated with traditional medicinal plant Tridax procumbens Linn.

    PubMed Central

    Preveena, Jagadesan; Bhore, Subhash J.

    2013-01-01

    Background: In traditional medicine, Tridax procumbens Linn. is used in the treatment of injuries and wounds. The bacterial endophytes (BEs) of medicinal plants could produce medicinally important metabolites found in their hosts; and hence, the involvement of BEs in conferring wound healing properties to T. Procumbens cannot be ruled out. But, we do not know which types of BEs are associated with T. Procumbens. Objective: The objective of this study was to investigate the fast growing and cultivable BEs associated with T. procumbens. Materials and Methods: Leaves and stems of healthy T. Procumbens plants were collected and cultivable BEs were isolated from surface-sterilized leaf and stem tissue samples using Luria-Bertani (LB) agar (medium) at standard conditions. A polymerase chain reaction was employed to amplify 16S rRNA coding gene fragments from the isolates. Cultivable endophytic bacterial isolates (EBIs) were identified using 16S rRNA gene nucleotide sequence similarity based method of bacterial identification. Results: Altogether, 50 culturable EBIs were isolated. 16S rRNA gene nucleotide sequences analysis using the Basic Local Alignment Search Tool (BLAST) revealed identities of the EBIs. Analysis reveals that cultivable Bacillus spp., Cronobacter sakazakii, Enterobacter spp., Lysinibacillus sphaericus, Pantoea spp., Pseudomonas spp. and Terribacillus saccharophilus are associated with T. Procumbens. Conclusion: Based on the results, we conclude that 24 different types of culturable BEs are associated with traditionally used medicinal plant, T. Procumbens, and require further study. PMID:24501447

  7. Quantification of host-specific Bacteroides-Prevotella 16S rRNA genetic markers for assessment of fecal pollution in freshwater.

    PubMed

    Okabe, Satoshi; Okayama, Noriko; Savichtcheva, Olga; Ito, Tsukasa

    2007-03-01

    Based on the comparative 16S rRNA gene sequence analysis of fecal DNAs, we identified one human-, three cow-, and two pig-specific Bacteroides-Prevotella 16S rRNA genetic markers, designed host-specific real-time polymerase chain reaction (real-time PCR) primer sets, and successfully developed real-time PCR assay to quantify the fecal contamination derived from human, cow, and pig in natural river samples. The specificity of each newly designed host-specific primer pair was evaluated on fecal DNAs extracted from these host feces. All three cow-specific and two pig-specific primer sets amplified only target fecal DNAs (in the orders of 9-11 log(10) copies per gram of wet feces), showing high host specificity. This real-time PCR assay was then applied to the river water samples with different fecal contamination sources and levels. It was confirmed that this assay could sufficiently discriminate and quantify human, cow, and pig fecal contamination. There was a moderate level of correlation between the Bacteroides-Prevotella group-specific 16S rRNA gene markers with fecal coliforms (r (2) = 0.49), whereas no significant correlation was found between the human-specific Bacteroides 16S rRNA gene with total and fecal coliforms. Using a simple filtration method, the minimum detection limits of this assay were in the range of 50-800 copies/100 ml. With a combined sample processing and analysis time of less than 8 h, this real-time PCR assay is useful for monitoring or identifying spatial and temporal distributions of host-specific fecal contaminations in natural water environments. PMID:17139508

  8. Prevalence of Mitochondrial 12S rRNA Mutations Associated with Aminoglycoside Ototoxicity

    ERIC Educational Resources Information Center

    Guan, Min-Xin

    2005-01-01

    The mitochondrial DNA (mtDNA) 12S rRNA is a hot spot for mutations associated with both aminoglycoside-induced and nonsyndromic hearing loss. Of those, the homoplasmic A1555G and C1494T mutations at a highly conserved decoding region of the 12S rRNA have been associated with hearing loss. These two mutations account for a significant number of…

  9. Intragenomic heterogeneity of 16S rRNA genes causes overestimation of prokaryotic diversity.

    PubMed

    Sun, Dong-Lei; Jiang, Xuan; Wu, Qinglong L; Zhou, Ning-Yi

    2013-10-01

    Ever since Carl Woese introduced the use of 16S rRNA genes for determining the phylogenetic relationships of prokaryotes, this method has been regarded as the "gold standard" in both microbial phylogeny and ecology studies. However, intragenomic heterogeneity within 16S rRNA genes has been reported in many investigations and is believed to bias the estimation of prokaryotic diversity. In the current study, 2,013 completely sequenced genomes of bacteria and archaea were analyzed and intragenomic heterogeneity was found in 952 genomes (585 species), with 87.5% of the divergence detected being below the 1% level. In particular, some extremophiles (thermophiles and halophiles) were found to harbor highly divergent 16S rRNA genes. Overestimation caused by 16S rRNA gene intragenomic heterogeneity was evaluated at different levels using the full-length and partial 16S rRNA genes usually chosen as targets for pyrosequencing. The result indicates that, at the unique level, full-length 16S rRNA genes can produce an overestimation of as much as 123.7%, while at the 3% level, an overestimation of 12.9% for the V6 region may be introduced. Further analysis showed that intragenomic heterogeneity tends to concentrate in specific positions, with the V1 and V6 regions suffering the most intragenomic heterogeneity and the V4 and V5 regions suffering the least intragenomic heterogeneity in bacteria. This is the most up-to-date overview of the diversity of 16S rRNA genes within prokaryotic genomes. It not only provides general guidance on how much overestimation can be introduced when applying 16S rRNA gene-based methods, due to its intragenomic heterogeneity, but also recommends that, for bacteria, this overestimation be minimized using primers targeting the V4 and V5 regions. PMID:23872556

  10. Surveying the Microbiome of Ants: Comparing 454 Pyrosequencing with Traditional Methods To Uncover Bacterial Diversity

    PubMed Central

    Rubin, Benjamin E. R.; Russell, Jacob A.; Moreau, Corrie S.

    2013-01-01

    We are only beginning to understand the depth and breadth of microbial associations across the eukaryotic tree of life. Reliably assessing bacterial diversity is a key challenge, and next-generation sequencing approaches are facilitating this endeavor. In this study, we used 16S rRNA amplicon pyrosequencing to survey microbial diversity in ants. We compared 454 libraries with Sanger-sequenced clone libraries as well as cultivation of live bacteria. Pyrosequencing yielded 95,656 bacterial 16S rRNA reads from 19 samples derived from four colonies of one ant species. The most dominant bacterial orders in the microbiome of the turtle ant Cephalotes varians were Rhizobiales, Burkholderiales, Opitutales, Xanthomonadales, and Campylobacterales, as revealed through both 454 sequencing and cloning. Even after stringent quality filtering, pyrosequencing recovered 445 microbe operational taxonomic units (OTUs) not detected with traditional techniques. In comparing bacterial communities associated with specific tissues, we found that gut tissues had significantly higher diversity than nongut tissues, and many of the OTUs identified from these groups clustered within ant-specific lineages, indicating a deep coevolutionary history of Cephalotes ants and their associated microbes. These lineages likely function as nutritional symbionts. One of four ant colonies investigated was infected with a Spiroplasma sp. (order Entomoplasmatales), a potential ant pathogen. Our work shows that the microbiome associated with Cephalotes varians is dominated by a few dozen bacterial lineages and that 454 sequencing is a cost-efficient tool to screen ant symbiont diversity. PMID:23124239

  11. Grape berry bacterial microbiota: impact of the ripening process and the farming system.

    PubMed

    Martins, Guilherme; Miot-Sertier, Cécile; Lauga, Béatrice; Claisse, Olivier; Lonvaud-Funel, Aline; Soulas, Guy; Masneuf-Pomarède, Isabelle

    2012-08-17

    Wine grapes are a primary source of microbial communities that play a prominent role in the quality of grapes prior to harvesting, as well as in the winemaking process. This study investigated the dynamics and diversity of the epiphytic bacteria on the grape berry surface during maturation. The quantitative and qualitative effects of conventional and organic farming systems on this microbial community were investigated, using both cultivation-dependent and independent approaches. Analyses of grape berry bacterial microbiota revealed changes in the size and structure of the population during the berry ripening process, with levels rising gradually and reaching their highest value when the berries were over ripe. As the season progressed to maturity, Gram-negative bacterial communities (mostly Pseudomonas spp.) declined whereas Gram-positive communities (mostly Micrococcus spp.) increased. The 16S rRNA gene sequences of cultured isolates were analysed and over 44 species were identified from 21 genera in the Proteobacteria, Actinobacteria, and Firmicutes phyla. Copper concentrations originating from phytosanitary treatments varied according to the vineyard and farming system. A negative correlation between copper concentrations and cell densities provided clear evidence that copper inhibited bacterial communities. The bacterial community structure was analysed by targeting the 16S rRNA genes, using PCR-DGGE on cultivable populations and T-RFLP on whole communities in cell suspension. The results suggest that the farming system has a clear impact on the bacterial community structure. PMID:22809638

  12. [Effects of transgenic cotton expressing chitinase and glucanase genes on the diversity of soil bacterial community].

    PubMed

    Li, Zhifang; Feng, Zili; Zhao, Lihong; Shi, Yongqiang; Feng, Hongjie; Zhu, Heqin

    2015-08-01

    The transgenic cotton expressing chitinase and glucanase genes was studied using nontransgenic cotton as a control. Specifically, the effects of exogenous genes on bacterial community diversity in rhizospheres of cotton at stages of seedling, budding, boll forming and boll opening were evaluated through comparing the number of cultivable bacteria and analyzing 16S rRNA gene clone libraries. The results showed that the number of cultivable bacteria was not affected by exogenous genes but the cotton growth period, and the number peaked at the stage of boll forming with vigorous metabolism. The 16S rRNA gene clone library prepared from soil bacteria in rhizospheres of transgenic and nontransgenic cotton at different stages contained 2400 clones which covered 283 genera. Among them, Acidobacterium was the most dominant group which contained 642 clones, followed by unclassified bacterium and Flavisolibacter. Compared with nontransgenic cotton, the rhizosphere bacterial diversity of transgenic cotton exhibited lower level at the same growth stage, however, their common bacterial communities increased with growth and development. Our results suggest that chitinase and glucanase genes decrease the rhizosphere bacterial diversity at distinct degrees, however, the difference of bacterial diversity between transgenic and nontransgenic cotton reduces gradually with the extension of cultivation period. PMID:26266785

  13. Spatial and Vertical Variability in Bacterial Community Structure in the Sediment of the South China Sea

    NASA Astrophysics Data System (ADS)

    Wang, P.; Xie, W.; Chen, S.; Zhang, C. L.

    2014-12-01

    The ocean subsurface contains one of the largest pools of reactive carbon and nitrogen on earth, and thus serves as the largest realm for microbial life. However, the microbial communities that drive deep-subsurface geochemical processes are vastly unexplored. In this study, the bacterial community structure in the subsurface of the South China Sea were examined using sediment cores collected from shelf (water depth 667 m) to slope (water depth 3840 m). High-throughput sequencing of the bacterial 16S rRNA genes from the sediment samples resulted in a total of 270,000 sequences with each sample averaging about 10,000 sequences. In all sediment cores, the 16S rRNA gene copies of bacteria were highest in the surface sediment and decreased with the core depth. The bacterial community was dominated by Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes. In most of the sediment cores, Proteobacteria dominated surface sediment samples and decreased with depth. The community structure showed no significant difference among the stations at different water depths, which indicates that bacterial distribution in the sediment is not influenced by the water column above. However, stations along the transect from Pearl River canyon to the deep basin were grouped together by cluster analysis, which indicates that bacterial community structure at these stations may bear the same consequence of sedimentary processes of the deep South China Sea.

  14. Namib Desert dune/interdune transects exhibit habitat-specific edaphic bacterial communities

    PubMed Central

    Ronca, Sandra; Ramond, Jean-Baptiste; Jones, Brian E.; Seely, Mary; Cowan, Don A.

    2015-01-01

    The sand dunes and inter-dune zones of the hyper-arid central Namib Desert represent heterogeneous soil habitats. As little is known about their indigenous edaphic bacterial communities, we aimed to evaluate their diversity and factors of assembly and hypothesized that soil physicochemistry gradients would strongly shape dune/interdune communities. We sampled a total of 125 samples from 5 parallel dune/interdune transects and characterized 21 physico-chemical edaphic parameters coupled with 16S rRNA gene bacterial community fingerprinting using T-RFLP and 454 pyrosequencing. Multivariate analyses of T-RFLP data showed significantly different bacterial communities, related to physico-chemical gradients, in four distinct dune habitats: the dune top, slope, base and interdune zones. Pyrosequencing of 16S rRNA gene amplicon sets showed that each dune zone presented a unique phylogenetic profile, suggesting a high degree of environmental selection. The combined results strongly infer that habitat filtering is an important factor shaping Namib Desert dune bacterial communities, with habitat stability, soil texture and mineral and nutrient contents being the main environmental drivers of bacterial community structures. PMID:26388839

  15. Characterization of Bacterial Communities Associated with the Tyrian Purple Producing Gland in a Marine Gastropod

    PubMed Central

    Ngangbam, Ajit Kumar; Baten, Abdul; Waters, Daniel L. E.; Whalan, Steve; Benkendorff, Kirsten

    2015-01-01

    Dicathais orbita is a marine mollusc recognised for the production of anticancer compounds that are precursors to Tyrian purple. This study aimed to assess the diversity and identity of bacteria associated with the Tyrian purple producing hypobranchial gland, in comparison with foot tissue, using a high-throughput sequencing approach. Taxonomic and phylogenetic analysis of variable region V1-V3 of 16S rRNA bacterial gene amplicons in QIIME and MEGAN were carried out. This analysis revealed a highly diverse bacterial assemblage associated with the hypobranchial gland and foot tissues of D. orbita. The dominant bacterial phylum in the 16S rRNA bacterial profiling data set was Proteobacteria followed by Bacteroidetes, Tenericutes and Spirochaetes. In comparison to the foot, the hypobranchial gland had significantly lower bacterial diversity and a different community composition, based on taxonomic assignment at the genus level. A higher abundance of indole producing Vibrio spp. and the presence of bacteria with brominating capabilities in the hypobranchial gland suggest bacteria have a potential role in biosynthesis of Tyrian purple in D. orbita. PMID:26488885

  16. Shift of Bacterial Community in Synanthropic Mite Tyrophagus putrescentiae Induced by Fusarium Fungal Diet

    PubMed Central

    Hubert, Jan; Nesvorná, Marta; Ságová-Mare?ková, Markéta; Kopecký, Jan

    2012-01-01

    Background Tyrophagus putrescentiae (Acari: Astigmata) and Fusarium sp. co-occur in poorly managed grain. In a laboratory experiment, mite grazing resulted in significant reduction of fungal mycelium on cultivation plates. The destruction of mycelium appeared to be a result of an interaction between the mites, fungi and associated bacteria. Methodology and Principal Findings A laboratory experiment was performed to simulate a situation of grain multiinfested by mites and Fusarium fungi. Changes of mite-associated bacterial community in T. putrescentiae were described in 3 habitats: (i) T. putrescentiae mites from a rearing diet prior to their transfer to fungal diet; (ii) fungal mycelium before mite introduction; (iii) mites after 7 day diet of each Fusarium avenaceum, F. culmorum, F. poae and F. verticillioides. Bacterial communities were characterized by 16 S rRNA gene sequencing. In total, 157 nearly full-length 16 S rRNA gene sequences from 9 samples representing selected habitats were analyzed. In the mites, the shift from rearing to fungal diet caused changes in mite associated bacterial community. A diverse bacterial community was associated with mites feeding on F. avenaceum, while feeding on the other three Fusarium spp. led to selection of a community dominated by Bacillaceae. Conclusions/Significance The work demonstrated changes of bacterial community associated with T. putrescentiae after shift to fungal diets suggesting selection for Bacillaceae species known as chitinase producers, which might participate in the fungal mycelium hydrolysis. PMID:23119013

  17. [Analysis of bacterial communities of two Lake Baikal endemic sponge species].

    PubMed

    Gladkikh, A S; kaluyzhnaya, Ok V; Belykh, O I; Ahn, T S; Parfenova, V V

    2014-01-01

    Bacterial diversity of two Lake Baikal endemic sponges characterized by different life forms, branching Lubomirskia baicalensis and cortical Baikalospongia sp., was studied using the method of 454 pyrosequencing of the 16S rRNA gene fragments. In the communities associated with L. baicalensis and Baikalospongia sp., 426 and 428 OTUs, respectively, were identified. In microbial associations of these spong- es, 24 bacterial phyla were identified, with predominance of Bacteroidetes, Proteobacteria, and Actinobacteria. Analysis of the taxonomic composition of bacterial communities of the sponges was carried out by search of the dominant phylotypes within the phylum level cluster. Comparison of bacterial associations of the sponges with Lake Baikal bacterioplankton revealed both the shared OTUs and the unique ones characteristic of the studied species. PMID:25941718

  18. Removal of 2,4,5-trichlorophenol by bacterial isolates from the secondary sludge of pulp and paper mill.

    PubMed

    Karn, Santosh Kr; Reddy, M Sudhakara

    2013-09-01

    2,4,5-trichlorophenol (2,4,5-TCP) mineralizing bacteria were isolated from the secondary sludge of pulp and paper industry. These isolates used 2,4,5-TCP as a source of carbon and energy and were capable of degrading this compound, as indicated by stoichimetric release of chloride and biomass formation. Based on 16S rRNA sequence analysis, these bacteria were identified as Kocuria sp. (CL2), Bacillus pumillus (CL5), Pseudomonas stutzeri (CL7). HPLC analysis revealed that these isolates were able to degrade 2,4,5-TCP at higher concentrations (600?mg/l or 3.0?mM). A consortia of these isolates completely removed 2,4,5-TCP from the sludge obtained from pulp and paper mill within 2 weeks when supplemented at a rate of 100?mg l(-1) . Bacterial consortium also significantly reduced absorbable organic halogen (AOX) and extractable organic halogen (EOX) by 61% and 63%, respectively from the sludge. These isolates have high potential to remove 2,4,5-TCP and may be used for remediation of pulp paper mill waste containing 2,4,5-TCP. PMID:22961799

  19. Differential assembly of 16S rRNA domains during 30S subunit formation.

    PubMed

    Xu, Zhili; Culver, Gloria M

    2010-10-01

    Rapid and accurate assembly of the ribosomal subunits, which are responsible for protein synthesis, is required to sustain cell growth. Our best understanding of the interaction of 30S ribosomal subunit components (16S ribosomal RNA [rRNA] and 20 ribosomal proteins [r-proteins]) comes from in vitro work using Escherichia coli ribosomal components. However, detailed information regarding the essential elements involved in the assembly of 30S subunits still remains elusive. Here, we defined a set of rRNA nucleotides that are critical for the assembly of the small ribosomal subunit in E. coli. Using an RNA modification interference approach, we identified 54 nucleotides in 16S rRNA whose modification prevents the formation of a functional small ribosomal subunit. The majority of these nucleotides are located in the head and interdomain junction of the 30S subunit, suggesting that these regions are critical for small subunit assembly. In vivo analysis of specific identified sites, using engineered mutations in 16S rRNA, revealed defective protein synthesis capability, aberrant polysome profiles, and abnormal 16S rRNA processing, indicating the importance of these residues in vivo. These studies reveal that specific segments of 16S rRNA are more critical for small subunit assembly than others, and suggest a hierarchy of importance. PMID:20736336

  20. Vineyard soil bacterial diversity and composition revealed by 16S rRNA genes: differentiation by geographic features

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here, we examine soil-borne microbial biogeography as a function of the features that 31 define an American Viticultural Area (AVA), a geographically delimited American wine grape32 growing region, defined for its distinguishing features of climate, geology, soils, physical 33 features (topography a...

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

    EPA Science Inventory

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

  2. The bacterial community composition of the bovine rumen detected using pyrosequencing of 16S rRNA genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rumen as a complex microbial ecosystem plays a critical role in sustainable agriculture. Rumen microorganisms perform important biochemical conversions, including the fermentation of plant fiber to small molecules such as short-chain fatty acids for meat and dairy production. In this study, we s...

  3. Rumen Bacterial Diversity of 80 to 110-Day-Old Goats Using 16S rRNA Sequencing

    PubMed Central

    Han, Xufeng; Yang, Yuxin; Yan, Hailong; Wang, Xiaolong; Qu, Lei; Chen, Yulin

    2015-01-01

    The ability of rumen microorganisms to use fibrous plant matter plays an important role in ruminant animals; however, little information about rumen colonization by microbial populations after weaning has been reported. In this study, high-throughput sequencing was used to investigate the establishment of this microbial population in 80 to 110-day-old goats. Illumina sequencing of goat rumen samples yielded 101,356,610 nucleotides that were assembled into 256,868 reads with an average read length of 394 nucleotides. Taxonomic analysis of metagenomic reads indicated that the predominant phyla were distinct at different growth stages. The phyla Firmicutes and Synergistetes were predominant in samples taken from 80 to 100-day-old goats, but Bacteroidetes and Firmicutes became the most abundant phyla in samples from 110-day-old animals. There was a remarkable variation in the microbial populations with age; Firmicutes and Synergistetes decreased after weaning, but Bacteroidetes and Proteobacteria increased from 80 to 110 day of age. These findings suggested that colonization of the rumen by microorganisms is related to their function in the rumen digestive system. These results give a better understanding of the role of rumen microbes and the establishment of the microbial population, which help to maintain the host’s health and improve animal performance. PMID:25700157

  4. Higher-Level Snake Phylogeny Inferred from Mitochondrial DNA Sequences of 12s rRNA and 16s rRNA Genes

    E-print Network

    Hedges, Blair

    that snakes underwent a subterranean period early in their evolution. Caenophidians (advanced snakes oceanic islands. Despite this ecological diversity and a long evolution- ary history, snakesHigher-Level Snake Phylogeny Inferred from Mitochondrial DNA Sequences of 12s rRNA and 16s r

  5. Effect of postharvest UV-C treatment on the bacterial diversity of Ataulfo mangoes by PCR-DGGE, survival of E. coli and antimicrobial activity.

    PubMed

    Fernández-Suárez, Rocío; Ramírez-Villatoro, Guadalupe; Díaz-Ruiz, Gloria; Eslava, Carlos; Calderón, Montserrat; Navarro-Ocaña, Arturo; Trejo-Márquez, Andrea; Wacher, Carmen

    2013-01-01

    Since Mexico is the second largest exporter of mangoes, its safety assurance is essential. Research in microbial ecology and knowledge of complex interactions among microbes must be better understood to achieve maximal control of pathogens. Therefore, we investigated the effect of UV-C treatments on bacterial diversity of the Ataulfo mangoes surface using PCR-DGGE analysis of variable region V3 of 16S rRNA genes, and the survival of E. coli, by plate counting. The UV-C irradiation reduced the microbial load on the surface of mangoes immediately after treatment and the structure of bacterial communities was modified during storage. We identified the key members of the bacterial communities on the surface of fruits, predominating Enterobacter genus. Genera as Lactococcus and Pantoea were only detected on the surface of non-treated (control) mangoes. This could indicate that these genera were affected by the UV-C treatment. On the other hand, the treatment did not have a significant effect on survival of E. coli. However, genera that have been recognized as antagonists against foodborne pathogens were identified in the bands patterns. Also, phenolic compounds were determined by HPLC and antimicrobial activity was assayed according to the agar diffusion method. The main phenolic compounds were chlorogenic, gallic, and caffeic acids. Mango peel methanol extracts (UV-C treated and control mangoes) showed antimicrobial activity against strains previously isolated from mango, detecting significant differences (P < 0.05) among treated and control mangoes after 4 and 12 days of storage. Ps. fluorescens and Ps. stutszeri were the most sensitive. PMID:23761788

  6. Effect of postharvest UV-C treatment on the bacterial diversity of Ataulfo mangoes by PCR-DGGE, survival of E. coli and antimicrobial activity

    PubMed Central

    Fernández-Suárez, Rocío; Ramírez-Villatoro, Guadalupe; Díaz-Ruiz, Gloria; Eslava, Carlos; Calderón, Montserrat; Navarro-Ocaña, Arturo; Trejo-Márquez, Andrea; Wacher, Carmen

    2013-01-01

    Since Mexico is the second largest exporter of mangoes, its safety assurance is essential. Research in microbial ecology and knowledge of complex interactions among microbes must be better understood to achieve maximal control of pathogens. Therefore, we investigated the effect of UV-C treatments on bacterial diversity of the Ataulfo mangoes surface using PCR-DGGE analysis of variable region V3 of 16S rRNA genes, and the survival of E. coli, by plate counting. The UV-C irradiation reduced the microbial load on the surface of mangoes immediately after treatment and the structure of bacterial communities was modified during storage. We identified the key members of the bacterial communities on the surface of fruits, predominating Enterobacter genus. Genera as Lactococcus and Pantoea were only detected on the surface of non-treated (control) mangoes. This could indicate that these genera were affected by the UV-C treatment. On the other hand, the treatment did not have a significant effect on survival of E. coli. However, genera that have been recognized as antagonists against foodborne pathogens were identified in the bands patterns. Also, phenolic compounds were determined by HPLC and antimicrobial activity was assayed according to the agar diffusion method. The main phenolic compounds were chlorogenic, gallic, and caffeic acids. Mango peel methanol extracts (UV-C treated and control mangoes) showed antimicrobial activity against strains previously isolated from mango, detecting significant differences (P < 0.05) among treated and control mangoes after 4 and 12 days of storage. Ps. fluorescens and Ps. stutszeri were the most sensitive. PMID:23761788

  7. Differential growth responses of soil bacterial taxa to carbon substrates of varying chemical recalcitrance

    SciTech Connect

    Goldfarb, K.C.; Karaoz, U.; Hanson, C.A.; Santee, C.A.; Bradford, M.A.; Treseder, K.K.; Wallenstein, M.D.; Brodie, E.L.

    2011-04-18

    Soils are immensely diverse microbial habitats with thousands of co-existing bacterial, archaeal, and fungal species. Across broad spatial scales, factors such as pH and soil moisture appear to determine the diversity and structure of soil bacterial communities. Within any one site however, bacterial taxon diversity is high and factors maintaining this diversity are poorly resolved. Candidate factors include organic substrate availability and chemical recalcitrance, and given that they appear to structure bacterial communities at the phylum level, we examine whether these factors might structure bacterial communities at finer levels of taxonomic resolution. Analyzing 16S rRNA gene composition of nucleotide analog-labeled DNA by PhyloChip microarrays, we compare relative growth rates on organic substrates of increasing chemical recalcitrance of >2,200 bacterial taxa across 43 divisions/phyla. Taxa that increase in relative abundance with labile organic substrates (i.e., glycine, sucrose) are numerous (>500), phylogenetically clustered, and occur predominantly in two phyla (Proteobacteria and Actinobacteria) including orders Actinomycetales, Enterobacteriales, Burkholderiales, Rhodocyclales, Alteromonadales, and Pseudomonadales. Taxa increasing in relative abundance with more chemically recalcitrant substrates (i.e., cellulose, lignin, or tannin-protein) are fewer (168) but more phylogenetically dispersed, occurring across eight phyla and including Clostridiales, Sphingomonadalaes, Desulfovibrionales. Just over 6% of detected taxa, including many Burkholderiales increase in relative abundance with both labile and chemically recalcitrant substrates. Estimates of median rRNA copy number per genome of responding taxa demonstrate that these patterns are broadly consistent with bacterial growth strategies. Taken together, these data suggest that changes in availability of intrinsically labile substrates may result in predictable shifts in soil bacterial composition.

  8. Molecular Characterization of the Bacterial Community in a Potato Phytosphere

    PubMed Central

    Someya, Nobutaka; Kobayashi, Yuki Ohdaira; Tsuda, Shogo; Ikeda, Seishi

    2013-01-01

    The bacterial community of a potato phytosphere at the flowering stage was examined using both culture-dependent and -independent methods. Tissues (leaves, stems, roots and tubers) were sampled from field-grown potato plants (cultivar Matilda), and the clone libraries of 16S rRNA genes and the isolate collections using R2A medium were constructed. By analyzing the combined data set of 16S rRNA gene sequences from both clone libraries and isolate collections, 82 genera from 8 phyla were found and 237 OTUs (?97% identity) at species level were identified across the potato phytosphere. The statistical analyses of clone libraries suggested that stems harbor the lowest diversity among the tissues examined. The phylogenetic analyses revealed that the most dominant phylum was shown to be Proteobacteria for all tissues (62.0%–89.7% and 57.7%–72.9%, respectively), followed by Actinobacteria (5.0%–10.7% and 14.6%–39.4%, respectively). The results of principal coordinates analyses of both clone libraries and isolate collections indicated that distinct differences were observed between above- and below-ground tissues for bacterial community structures. The results also revealed that leaves harbored highly similar community structures to stems, while the tuber community was shown to be distinctly different from the stem and root communities. PMID:23748858

  9. Detection and Capturing of (14)C Radioactively-Labeled Small Subunit rRNA from Mixed Microbial Communities of a Microbial Mat Using Magnetic Beads.

    PubMed

    Abed, Raeid M M

    2012-03-01

    Carbon cycling in the hypersaline microbial mats from Chiprana Lake, Spain is primarily dependent on phototrophic microorganisms with the ability to fix CO2 into organics that can be further utilized by aerobic as well as anaerobic heterotrophic bacteria. Here, mat pieces were incubated in seawater amended with (14)C sodium bicarbonate and the incorporation of the radiocarbon in the small subunit ribosomal RNA (SSU rRNA) of mat organisms was followed using scintillation counter and autoradiography. Different domains of SSU rRNA were separated from the total RNA by means of streptavidin-coated magnetic beads and biotin-labeled oligonucleotide probes. The (14)C label was detected in isolated RNA by both scintillation counter and autoradiography, however the latter technique was less sensitive. Using scintillation counter, the radiolabel incorporation increased with time with a maximum rate of 0.18 Bq ng(-1) detected after 25 days. The bacterial SSU rRNA could be captured using the magnetic beads, however the hybridization efficiency was around 20%. The captured RNA was radioactively labeled, which could be mainly due to the fixation of radiocarbon by phototrophic organisms. In conclusion, the incubation of microbial mats in the presence of radiolabeled bicarbonate leads to the incorporation of the (14)C label into RNA molecules through photosynthesis and this label can be detected using scintillation counter. The used approach could be useful in studying the fate of fixed carbon and its uptake by other microorganisms in complex microbial mats, particularly when species-specific probes are used and the hybridization efficiency and RNA yield are further optimized. PMID:23449550

  10. Comparison of bacteroides-prevotella 16S rRNA genetic markers for fecal samples from different animal species.

    PubMed

    Fogarty, Lisa R; Voytek, Mary A

    2005-10-01

    To effectively manage surface and ground waters it is necessary to improve our ability to detect and identify sources of fecal contamination. We evaluated the use of the anaerobic bacterial group Bacteroides-Prevotella as a potential fecal indicator. Terminal restriction length polymorphism (T-RFLP) of the 16S rRNA genes from this group was used to determine differences in populations and to identify any unique populations in chickens, cows, deer, dogs, geese, horses, humans, pigs, and seagulls. The group appears to be a good potential fecal indicator in all groups tested except for avians. Cluster analysis of Bacteroides-Prevotella community T-RFLP profiles indicates that Bacteroides-Prevotella populations from samples of the same host species are much more similar to each other than to samples from different source species. We were unable to identify unique peaks that were exclusive to any source species; however, for most host species, at least one T-RFLP peak was identified to be more commonly found in that species, and a combination of peaks could be used to identify the source. T-RFLP profiles obtained from water spiked with known-source feces contained the expected diagnostic peaks from the source. These results indicate that the approach of identifying Bacteroides-Prevotella molecular markers associated with host species might be useful in identifying sources of fecal contamination in the environment. PMID:16204514

  11. Characterization by 16S rRNA Sequence Analysis of Pseudomonads Causing Blotch Disease of Cultivated Agaricus bisporus

    PubMed Central

    Godfrey, S. A. C.; Harrow, S. A.; Marshall, J. W.; Klena, J. D.

    2001-01-01

    Bacterial blotch of Agaricus bisporus has typically been identified as being caused by either Pseudomonas tolaasii (brown blotch) or Pseudomonas gingeri (ginger blotch). To address the relatedness of pseudomonads able to induce blotch, a pilot study was initiated in which pseudomonads were selectively isolated from mushroom farms throughout New Zealand. Thirty-three pseudomonad isolates were identified as being capable of causing different degrees of discoloration (separable into nine categories) of A. bisporus tissue in a bioassay. These isolates were also identified as unique using repetitive extragenic palindromic PCR and biochemical analysis. Relationships between these 33 blotch-causing organisms (BCO) and a further 22 selected pseudomonad species were inferred by phylogenetic analyses of near-full-length 16S rRNA gene nucleotide sequences. The 33 BCO isolates were observed to be distributed throughout the Pseudomonas fluorescens intrageneric cluster. These results show that in addition to known BCO (P. tolaasii, P. gingeri, and Pseudomonas reactans), a number of diverse pseudomonad species also have the ability to cause blotch diseases with various discolorations. Furthermore, observation of ginger blotch discoloration of A. bisporus being independently caused by many different pseudomonad species impacts on the homogeneity and classification of the previously described P. gingeri. PMID:11526038

  12. Comparison of Bacteroides-Prevotella 16S rRNA genetic markers for fecal samples from different animal species

    USGS Publications Warehouse

    Fogarty, L.R.; Voytek, M.A.

    2005-01-01

    To effectively manage surface and ground waters it is necessary to improve our ability to detect and identify sources of fecal contamination. We evaluated the use of the anaerobic bacterial group Bacteroides-Prevotella as a potential fecal indicator. Terminal restriction length polymorphism (T-RFLP) of the 16S rRNA genes from this group was used to determine differences in populations and to identify any unique populations in chickens, cows, deer, dogs, geese, horses, humans, pigs, and seagulls. The group appears to be a good potential fecal indicator in all groups tested except for avians. Cluster analysis of Bacteroides-Prevotella community T-RFLP profiles indicates that Bacteroides-Prevotella populations from samples of the same host species are much more similar to each other than to samples from different source species. We were unable to identify unique peaks that were exclusive to any source species; however, for most host species, at least one T-RFLP peak was identified to be more commonly found in that species, and a combination of peaks could be used to identify the source. T-RFLP profiles obtained from water spiked with known-source feces contained the expected diagnostic peaks from the source. These results indicate that the approach of identifying Bacteroides-Prevotella molecular markers associated with host species might be useful in identifying sources of fecal contamination in the environment.

  13. Novel bacterial and archaeal lineages from an in situ growth chamber deployed at a Mid-Atlantic Ridge hydrothermal vent.

    PubMed

    Reysenbach, A L; Longnecker, K; Kirshtein, J

    2000-09-01

    The phylogenetic diversity was determined for a microbial community obtained from an in situ growth chamber placed on a deep-sea hydrothermal vent on the Mid-Atlantic Ridge (23 degrees 22' N, 44 degrees 57' W). The chamber was deployed for 5 days, and the temperature within the chamber gradually decreased from 70 to 20 degrees C. Upon retrieval of the chamber, the DNA was extracted and the small-subunit rRNA genes (16S rDNA) were amplified by PCR using primers specific for the Archaea or Bacteria domain and cloned. Unique rDNA sequences were identified by restriction fragment length polymorphisms, and 38 different archaeal and bacterial phylotypes were identified from the 85 clones screened. The majority of the archaeal sequences were affiliated with the Thermococcales (71%) and Archaeoglobales (22%) orders. A sequence belonging to the Thermoplasmales confirms that thermoacidophiles may have escaped enrichment culturing attempts of deep-sea hydrothermal vent samples. Additional sequences that represented deeply rooted lineages in the low-temperature eurarchaeal (marine group II) and crenarchaeal clades were obtained. The majority of the bacterial sequences obtained were restricted to the Aquificales (18%), the epsilon subclass of the Proteobacteria (epsilon-Proteobacteria) (40%), and the genus Desulfurobacterium (25%). Most of the clones (28%) were confined to a monophyletic clade within the epsilon-Proteobacteria with no known close relatives. The prevalence of clones related to thermophilic microbes that use hydrogen as an electron donor and sulfur compounds (S(0), SO(4), thiosulfate) indicates the importance of hydrogen oxidation and sulfur metabolism at deep-sea hydrothermal vents. The presence of sequences that are related to sequences from hyperthermophiles, moderate thermophiles, and mesophiles suggests that the diversity obtained from this analysis may reflect the microbial succession that occurred in response to the shift in temperature and possible associated changes in the chemistry of the hydrothermal fluid. PMID:10966393

  14. Buccal Swabbing as a Noninvasive Method To Determine Bacterial, Archaeal, and Eukaryotic Microbial Community Structures in the Rumen.

    PubMed

    Kittelmann, Sandra; Kirk, Michelle R; Jonker, Arjan; McCulloch, Alan; Janssen, Peter H

    2015-11-01

    Analysis of rumen microbial community structure based on small-subunit rRNA marker genes in metagenomic DNA samples provides important insights into the dominant taxa present in the rumen and allows assessment of community differences between individuals or in response to treatments applied to ruminants. However, natural animal-to-animal variation in rumen microbial community composition can limit the power of a study considerably, especially when only subtle differences are expected between treatment groups. Thus, trials with large numbers of animals may be necessary to overcome this variation. Because ruminants pass large amounts of rumen material to their oral cavities when they chew their cud, oral samples may contain good representations of the rumen microbiota and be useful in lieu of rumen samples to study rumen microbial communities. We compared bacterial, archaeal, and eukaryotic community structures in DNAs extracted from buccal swabs to those in DNAs from samples collected directly from the rumen by use of a stomach tube for sheep on four different diets. After bioinformatic depletion of potential oral taxa from libraries of samples collected via buccal swabs, bacterial communities showed significant clustering by diet (R = 0.37; analysis of similarity [ANOSIM]) rather than by sampling method (R = 0.07). Archaeal, ciliate protozoal, and anaerobic fungal communities also showed significant clustering by diet rather than by sampling method, even without adjustment for potentially orally associated microorganisms. These findings indicate that buccal swabs may in future allow quick and noninvasive sampling for analysis of rumen microbial communities in large numbers of ruminants. PMID:26276109

  15. Diversity of Bacterial Communities of Fitness Center Surfaces in a U.S. Metropolitan Area

    PubMed Central

    Mukherjee, Nabanita; Dowd, Scot E.; Wise, Andy; Kedia, Sapna; Vohra, Varun; Banerjee, Pratik

    2014-01-01

    Public fitness centers and exercise facilities have been implicated as possible sources for transmitting community-acquired bacterial infections. However, the overall diversity of the bacterial community residing on the surfaces in these indoor environments is still unknown. In this study, we investigated the overall bacterial ecology of selected fitness centers in a metropolitan area (Memphis, TN, USA) utilizing culture-independent pyrosequencing of the 16S rRNA genes. Samples were collected from the skin-contact surfaces (e.g., exercise instruments, floor mats, handrails, etc.) within fitness centers. Taxonomical composition revealed the abundance of Firmicutes phyla, followed by Proteobacter and Actinobacteria, with a total of 17 bacterial families and 25 bacterial genera. Most of these bacterial genera are of human and environmental origin (including, air, dust, soil, and water). Additionally, we found the presence of some pathogenic or potential pathogenic bacterial genera including Salmonella, Staphylococcus, Klebsiella, and Micrococcus. Staphylococcus was found to be the most prevalent genus. Presence of viable forms of these pathogens elevates risk of exposure of any susceptible individuals. Several factors (including personal hygiene, surface cleaning and disinfection schedules of the facilities) may be the reasons for the rich bacterial diversity found in this study. The current finding underscores the need to increase public awareness on the importance of personal hygiene and sanitation for public gym users. PMID:25479039

  16. The arthropod, but not the vertebrate host or its environment, dictates bacterial community composition of fleas and ticks

    PubMed Central

    Hawlena, Hadas; Rynkiewicz, Evelyn; Toh, Evelyn; Alfred, Andrew; Durden, Lance A; Hastriter, Michael W; Nelson, David E; Rong, Ruichen; Munro, Daniel; Dong, Qunfeng; Fuqua, Clay; Clay, Keith

    2013-01-01

    Bacterial community composition in blood-sucking arthropods can shift dramatically across time and space. We used 16S rRNA gene amplification and pyrosequencing to investigate the relative impact of vertebrate host-related, arthropod-related and environmental factors on bacterial community composition in fleas and ticks collected from rodents in southern Indiana (USA). Bacterial community composition was largely affected by arthropod identity, but not by the rodent host or environmental conditions. Specifically, the arthropod group (fleas vs ticks) determined the community composition of bacteria, where bacterial communities of ticks were less diverse and more dependent on arthropod traits—especially tick species and life stage—than bacterial communities of fleas. Our data suggest that both arthropod life histories and the presence of arthropod-specific endosymbionts may mask the effects of the vertebrate host and its environment. PMID:22739493

  17. Bacterial and archaeal communities in long-term contaminated surface and subsurface soil evaluated through coextracted RNA and DNA.

    PubMed

    Mikkonen, Anu; Santalahti, Minna; Lappi, Kaisa; Pulkkinen, Anni-Mari; Montonen, Leone; Suominen, Leena

    2014-10-01

    Soil RNA and DNA were coextracted along a contamination gradient at a landfarming field with aged crude oil contamination to investigate pollution-dependent differences in 16S rRNA and rRNA gene pools. Microbial biomass correlated with nucleic acid yields as well as bacterial community change, indicating that the same factors controlled community size and structure. In surface soil, bacterial community evenness, estimated through length heterogeneity PCR (LH-PCR) fingerprinting, appeared higher for RNA-based than for DNA-based communities. The RNA-based community profiles resembled the DNA-based communities of soil with a lower contamination level. Cloning-based identification of bacterial hydrocarbon-degrading taxa in the RNA pool, representing the viable community with high protein synthesis potential, indicated that decontamination processes still continue. Analyses of archaea revealed that only Thaumarchaeota were present in the aerobic samples, whereas more diverse communities were found in the compacted subsurface soil with more crude oil. For subsurface bacteria, hydrocarbon concentration explained neither the community structure nor the difference between RNA-based and DNA-based communities. However, rRNA of bacterial taxa associated with syntrophic and sulphate-reducing alkane degradation was detected. Although the same prokaryotic taxa were identified in DNA and RNA, comparison of the two nucleic acid pools can aid in the assessment of past and future restoration success. PMID:24986450

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

    USGS Publications Warehouse

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

    2007-01-01

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

  19. Identity and geometry of a base triple in 16S rRNA determined by comparative sequence analysis and molecular modeling.

    PubMed Central

    Babin, P; Dolan, M; Wollenzien, P; Gutell, R R

    1999-01-01

    Comparative sequence analysis complements experimental methods for the determination of RNA three-dimensional structure. This approach is based on the concept that different sequences within the same gene family form similar higher-order structures. The large number of rRNA sequences with sufficient variation, along with improved covariation algorithms, are providing us with the opportunity to identify new base triples in 16S rRNA. The three-dimensional conformations for one of our strongest candidates involving U121 (C124:G237) and/or U121 (U125:A236) (Escherichia coli sequence and numbering) are analyzed here with different molecular modeling tools. Molecular modeling shows that U121 interacts with C124 in the U121 (C124:G237) base triple. This arrangement maintains isomorphic structures for the three most frequent sequence motifs (approximately 93% of known bacterial and archaeal sequences), is consistent with chemical reactivity of U121 in E. coli ribosomes, and is geometrically favorable. Further, the restricted set of observed canonical (GU, AU, GC) base-pair types at positions 124:237 and 125:236 is consistent with the fact that the canonical base-pair sets (for both base pairs) that are not observed in nature prevent the formation of the 121 (124:237) base triple. The analysis described here serves as a general scheme for the prediction of specific secondary and tertiary structure base pairing where there is a network of correlated base changes. PMID:10580471

  20. Simultaneous 16S and 18S rRNA fluorescence in situ hybridization (FISH) on LR White sections demonstrated in Vestimentifera (Siboglinidae) tubeworms.

    PubMed

    Schimak, Mario P; Toenshoff, Elena R; Bright, Monika

    2012-02-01

    Traditional morphological identification of invertebrate marine species is limited in early life history stages for many taxa. In this study, we demonstrate, by example of Vestimentiferan tubeworms (Siboglinidae, Polychaeta), that the simultaneous fluorescence in situ hybridization (FISH) of both eukaryotic host and bacterial symbiont cells is possible on a single semi-thin (1 ?m) section. This allows the identification of host specimens to species level as well as offering visualization of bacteria distributed within the host tissue. Previously published 18S rRNA host-specific oligonucleotide probes for Riftia pachyptila, Tevnia jerichonana and a newly designed Oasisia alvinae probe, as well as a 16S rRNA probe targeting symbionts found in all host species, were applied. A number of standard fixation and hybridization parameters were tested and optimized for the best possible signal intensity and cellular resolution. Ethanol conserved samples embedded in LR White low viscosity resin yielded the best results with regard to both signal intensity and resolution. We show that extended storage times of specimens does not affect the quality of signals attained by FISH and use our protocol to identify morphologically unidentifiable tubeworm individuals from a small data set, conforming to previous findings in succession studies of the Siboglinidae family. PMID:21507466

  1. Events during eucaryotic rRNA transcription initiation and elongation: Conversion from the closed to the open promoter complex requires nucleotide substrates

    SciTech Connect

    Bateman, E.; Paule, M.R.

    1988-05-01

    Chemical footprinting and topological analysis were carried out on the Acanthamoeba castellanii rRNA transcription initiation factor (TIF) and RNA polymerase I complexes with DNA during transcription initiation and elongation. The results show that the binding of TIF and polymerase to the promoter does not alter the supercoiling of the DNA template and the template does not become sensitive to modification by diethylpyro-carbonate, which can identify melted DNA regions. Thus, in contrast to bacterial RNA polymerase, the eucaryotic RNA polymerase I-promoter complex is in a closed configuration preceding addition of nucleotides in vitro. Initiation and 3'-O-methyl CTP-limited translocation by RNA polymerase I results in separation of the polymerase-TIF footprints, leaving the TIF footprint unaltered. In contrast, initiation and translocation result in a significant change in the conformation of the polymerase-DNA complex, culminating in an unwound DNA region of at least 10 base pairs.

  2. B chromosomes showing active ribosomal RNA genes contribute insignificant amounts of rRNA in the grasshopper Eyprepocnemis plorans.

    PubMed

    Ruiz-Estévez, Mercedes; Badisco, Liesbeth; Broeck, Jozef Vanden; Perfectti, Francisco; López-León, María Dolores; Cabrero, Josefa; Camacho, Juan Pedro M

    2014-12-01

    The genetic inertness of supernumerary (B) chromosomes has recently been called into question after finding several cases of gene activity on them. The grasshopper Eyprepocnemis plorans harbors B chromosomes containing large amounts of ribosomal DNA (rDNA) units, some of which are eventually active, but the amount of rRNA transcripts contributed by B chromosomes, compared to those of the standard (A) chromosomes, is unknown. Here, we address this question by means of quantitative PCR (qPCR) for two different ITS2 amplicons, one coming from rDNA units located in both A and B chromosomes (ITS2(A+B)) and the other being specific to B chromosomes (ITS2(B)). We analyzed six body parts in nine males showing rDNA expression in their B chromosomes in the testis. Amplification of the ITS2(B) amplicon was successful in RNA extracted from all six body parts analyzed, but showed relative quantification (RQ) values four orders of magnitude lower than those obtained for the ITS(A+B) amplicon. RQ values differed significantly between body parts for the two amplicons, with testis, accessory gland and wing muscle showing threefold higher values than head, gastric cecum and hind leg. We conclude that the level of B-specific rDNA expression is extremely low even in individuals where B chromosome rDNA is not completely silenced. Bearing in mind that B chromosomes carry the largest rDNA cluster in the E. plorans genome, we also infer that the relative contribution of B chromosome rRNA genes to ribosome biogenesis is insignificant, at least in the body parts analyzed. PMID:24997085

  3. Bacterial media LB media (1L)

    E-print Network

    Abou Elela, Sherif

    Bacterial media LB media (1L) In 800mL ddH2O, dissolve (Bacto) Tryptone 10g Yeast Extract 5g NaCl 5 Autoclave #12;Store at RT SOB media (1L) In 800mL ddH2O, dissolve Tryptone 20g Yeast Extract 5.0g NaCl 0.5g separately. Just before using the media, add 5.0mL MgCl2 2M to 1L SOB. #12;

  4. Changes in bacterial community structure associated with coastal copper enrichment.

    PubMed

    Morán, Ana C; Hengst, Martha B; De La Iglesia, Rodrigo; Andrade, Santiago; Correa, Juan A; González, Bernardo

    2008-11-01

    Marine bacterial communities isolated from the water column, sediment, the rock surface, and the green seaweed Ulva compressa were studied in an intertidal ecosystem. The study area included a coastal zone chronically affected by copper mine waste disposals. Bacterial community composition was analyzed by terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes, and multivariate analyses of T-RFLP data sets were used for comparisons. Results showed that diversity and richness indexes were not able to detect differences among compartments. However, comparisons within the same compartment clearly showed that copper enrichment was associated with changes in the composition of the bacterial communities and revealed that the magnitude of the effect depends on the compartment being considered. In this context, communities from sediments appeared as the most affected by copper enrichment. The present study also demonstrated that intertidal bacterial communities were dominated by Gammaproteobacteria, Firmicutes, and Actinobacteria and the changes in these communities were mainly due to changes in their relative abundances. PMID:18522451

  5. Bacterial colonization of a fumigated alkaline saline soil.

    PubMed

    Bello-López, Juan M; Domínguez-Mendoza, Cristina A; de León-Lorenzana, Arit S; Delgado-Balbuena, Laura; Navarro-Noya, Yendi E; Gómez-Acata, Selene; Rodríguez-Valentín, Analine; Ruíz-Valdiviezo, Victor M; Luna-Guido, Marco; Verhulst, Nele; Govaerts, Bram; Dendooven, Luc

    2014-07-01

    After chloroform fumigating an arable soil, the relative abundance of phylotypes belonging to only two phyla (Actinobacteria and Firmicutes) and two orders [Actinomycetales and Bacillales (mostly Bacillus)] increased in a subsequent aerobic incubation, while it decreased for a wide range of bacterial groups. It remained to be seen if similar bacterial groups were affected when an extreme alkaline saline soil was fumigated. Soil with electrolytic conductivity between 139 and 157 dS m(-1), and pH 10.0 and 10.3 was fumigated and the bacterial community structure determined after 0, 1, 5 and 10 days by analysis of the 16S rRNA gene, while an unfumigated soil served as control. The relative abundance of the Firmicutes increased in the fumigated soil (52.8%) compared to the unfumigated soil (34.2%), while that of the Bacteroidetes decreased from 16.2% in the unfumigated soil to 8.8% in the fumigated soil. Fumigation increased the relative abundance of the genus Bacillus from 14.7% in the unfumigated soil to 25.7%. It was found that phylotypes belonging to the Firmicutes, mostly of the genus Bacillus, were dominant in colonizing the fumigated alkaline saline as found in the arable soil, while the relative abundance of a wide range of bacterial groups decreased. PMID:24846742

  6. Determining the culturability of the rumen bacterial microbiome

    PubMed Central

    Creevey, Christopher J; Kelly, William J; Henderson, Gemma; Leahy, Sinead C

    2014-01-01

    The goal of the Hungate1000 project is to generate a reference set of rumen microbial genome sequences. Toward this goal we have carried out a meta-analysis using information from culture collections, scientific literature, and the NCBI and RDP databases and linked this with a comparative study of several rumen 16S rRNA gene-based surveys. In this way we have attempted to capture a snapshot of rumen bacterial diversity to examine the culturable fraction of the rumen bacterial microbiome. Our analyses have revealed that for cultured rumen bacteria, there are many genera without a reference genome sequence. Our examination of culture-independent studies highlights that there are few novel but many uncultured taxa within the rumen bacterial microbiome. Taken together these results have allowed us to compile a list of cultured rumen isolates that are representative of abundant, novel and core bacterial species in the rumen. In addition, we have identified taxa, particularly within the phylum Bacteroidetes, where further cultivation efforts are clearly required. This information is being used to guide the isolation efforts and selection of bacteria from the rumen microbiota for sequencing through the Hungate1000. PMID:24986151

  7. Bacterial succession in a glacier foreland of the High Arctic

    PubMed Central

    Schütte, Ursel M.E.; Abdo, Zaid; Bent, Stephen J.; Williams, Christopher J.; Schneider, G. Maria; Solheim, Bjørn; Forney, Larry J.

    2009-01-01

    Succession is defined as changes in biological communities over time. It has been extensively studied in plant communities, but little is known about bacterial succession, in particular in environments such as High Arctic glacier forelands. Bacteria carry out key processes in the development of soil, biogeochemical cycling, and facilitating plant colonization. In this study we sampled two roughly parallel chronosequences in the foreland of Midre Lovén glacier on Svalbard, Norway and tested whether any of several factors were associated with changes in the structure of bacterial communities, including time after glacier retreat, horizontal variation caused by the distance between chronosequences, and vertical variation at two soil depths. The structures of soil bacterial communities at different locations were compared using terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes, and the data were analyzed by sequential analysis of log-linear statistical models. While no significant differences in community structure were detected between the two chronosequences, statistically significant differences between sampling locations in the surface and mineral soils could be demonstrated even though glacier forelands are patchy and dynamic environments. These findings suggest bacterial succession occurs in High Arctic glacier forelands but may differ in different soil depths. PMID:19587774

  8. Successional Trajectories of Rhizosphere Bacterial Communities over Consecutive Seasons

    PubMed Central

    Shi, Shengjing; Nuccio, Erin; Herman, Donald J.; Rijkers, Ruud; Estera, Katerina; Li, Jiabao; da Rocha, Ulisses Nunes; He, Zhili; Pett-Ridge, Jennifer; Brodie, Eoin L.; Zhou, Jizhong

    2015-01-01

    ABSTRACT It is well known that rhizosphere microbiomes differ from those of surrounding soil, and yet we know little about how these root-associated microbial communities change through the growing season and between seasons. We analyzed the response of soil bacteria to roots of the common annual grass Avena fatua over two growing seasons using high-throughput sequencing of 16S rRNA genes. Over the two periods of growth, the rhizosphere bacterial communities followed consistent successional patterns as plants grew, although the starting communities were distinct. Succession in the rhizosphere was characterized by a significant decrease in both taxonomic and phylogenetic diversity relative to background soil communities, driven by reductions in both richness and evenness of the bacterial communities. Plant roots selectively stimulated the relative abundance of Alphaproteobacteria, Betaproteobacteria, and Bacteroidetes but reduced the abundance of Acidobacteria, Actinobacteria, and Firmicutes. Taxa that increased in relative abundance in the rhizosphere soil displayed phylogenetic clustering, suggesting some conservation and an evolutionary basis for the response of complex soil bacterial communities to the presence of plant roots. The reproducibility of rhizosphere succession and the apparent phylogenetic conservation of rhizosphere competence traits suggest adaptation of the indigenous bacterial community to this common grass over the many decades of its presence. PMID:26242625

  9. Defining 5S rRNA Structure Space: Point Mutation Data Can Be Used to Predict the Phenotype of Multichange Variants

    E-print Network

    Fox, George

    Defining 5S rRNA Structure Space: Point Mutation Data Can Be Used to Predict the PhenotypeS ribosomal RNA (rRNA) structure space in the vicinity of the Vibrio proteolyticus 5S rRNA sequence sequence variants differing from the V. proteolyticus 5S rRNA wild-type sequence in 1­7 positions were

  10. Evidence of two lineages of the symbiont 'Candidatus Erwinia dacicola' in Italian populations of Bactrocera oleae (Rossi) based on 16S rRNA gene sequences.

    PubMed

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

    2012-01-01

    The close association between the olive fly Bactrocera oleae (Rossi) (Diptera: Tephritidae) and bacteria has been known for more than a century. Recently, the presence of a host-specific, hereditary, unculturable symbiotic bacterium, designated 'Candidatus Erwinia dacicola', has been described inside the cephalic organ of the fly, called the oesophageal bulb. In the present study, the 16S rRNA gene sequence variability of 'Ca. E. dacicola' was examined within and between 26 Italian olive fly populations sampled across areas where olive trees occur in the wild and areas where cultivated olive trees have been introduced through history. The bacterial contents of the oesophageal bulbs of 314 olive flies were analysed and a minimum of 781 bp of the 16S rRNA gene was sequenced. The corresponding host fly genotype was assessed by sequencing a 776 bp portion of the mitochondrial genome. Two 'Ca. E. dacicola' haplotypes were found (htA and htB), one being slightly more prevalent than the other (57%). The two haplotypes did not co-exist in the same individuals, as confirmed by cloning. Interestingly, the olive fly populations of the two main Italian islands, Sicily and Sardinia, appeared to be represented exclusively by the htB and htA haplotypes, respectively, while peninsular populations showed both bacterial haplotypes in different proportions. No significant correlation emerged between the two symbiont haplotypes and the 16 host fly haplotypes observed, suggesting evidence for a mixed model of vertical and horizontal transmission of the symbiont during the fly life cycle. PMID:21378134

  11. Bacterial aerosols in the dental clinic: a review.

    PubMed

    Leggat, P A; Kedjarune, U

    2001-02-01

    A number of sources of bacterial aerosols exist within and outside the dental clinic. The concentration of bacterial aerosols and splatters appears to be highest during dental procedures, especially those generated by some procedures such as ultrasonic scaling, or using a high speed drill. Several infectious diseases could be transmitted to staff and patients by airborne bacterial and other contaminants in the dental clinic. Air-conditioning and ventilation systems should be regularly maintained to reduce environmental contaminants and to prevent recirculation of bacterial aerosols. Pre-procedural rinsing by patients with mouthwashes as well as vacuum and electrostatic extraction of aerosols during dental procedures could also be employed. Dental staff should also consider appropriate immunizations and continue to use personal protective measures, which reduce contact with bacterial aerosols and splatters in the dental clinic. PMID:11326448

  12. Improved statistical methods reveal direct interactions between 16S and 23S rRNA

    PubMed Central

    Kelley, Scott T.; Akmaev, Viatcheslav R.; Stormo, Gary D.

    2000-01-01

    Recent biochemical studies have indicated a number of regions in both the 16S and 23S rRNA that are exposed on the ribosomal subunit surface. In order to predict potential interactions between these regions we applied novel phylogenetically-based statistical methods to detect correlated nucleotide changes occurring between the rRNA molecules. With these methods we discovered a number of highly significant correlated changes between different sets of nucleotides in the two ribosomal subunits. The predictions with the highest correlation values belong to regions of the rRNA subunits that are in close proximity according to recent crystal structures of the entire ribosome. We also applied a new statistical method of detecting base triple interactions within these same rRNA subunit regions. This base triple statistic predicted a number of new base triples not detected by pair-wise interaction statistics within the rRNA molecules. Our results suggest that these statistical methods may enhance the ability to detect novel structural elements both within and between RNA molecules. PMID:11121485

  13. Substrate Binding Analysis of the 23S rRNA Methyltransferase RrmJ

    PubMed Central

    Hager, Jutta; Staker, Bart L.; Jakob, Ursula

    2004-01-01

    The 23S rRNA methyltransferase RrmJ (FtsJ) is responsible for the 2?-O methylation of the universally conserved U2552 in the A loop of 23S rRNA. This 23S rRNA modification appears to be critical for ribosome stability, because the absence of functional RrmJ causes the cellular accumulation of the individual ribosomal subunits at the expense of the functional 70S ribosomes. To gain insight into the mechanism of substrate recognition for RrmJ, we performed extensive site-directed mutagenesis of the residues conserved in RrmJ and characterized the mutant proteins both in vivo and in vitro. We identified a positively charged, highly conserved ridge in RrmJ that appears to play a significant role in 23S rRNA binding and methylation. We provide a structural model of how the A loop of the 23S rRNA binds to RrmJ. Based on these modeling studies and the structure of the 50S ribosome, we propose a two-step model where the A loop undocks from the tightly packed 50S ribosomal subunit, allowing RrmJ to gain access to the substrate nucleotide U2552, and where U2552 undergoes base flipping, allowing the enzyme to methylate the 2?-O position of the ribose. PMID:15375145

  14. Diversity and composition of the bacterial community in Amphioxus feces.

    PubMed

    Pan, Minming; Yuan, Dongjuan; Chen, Shangwu; Xu, Anlong

    2015-11-01

    Amphioxus is a typical filter feeder animal and is confronted with a complex bacterial community in the seawater of its habitat. It has evolved a strong innate immune system to cope with the external bacterial stimulation, however, the ecological system of the bacterial community in Amphioxus remains unknown. Through massive parallel 16S rRNA gene tag pyrosequencing, the investigation indicated that the composition of wild and lab-cultured Amphioxus fecal bacteria was complex with more than 85,000 sequence tags being assigned to 12/13 phyla. The bacterial diversity between the two fecal samples was similar according to OTU richness of V4 tag, Chao1 index, Shannon index and Rarefaction curves, however, the most prominent bacteria in wild feces were genera Pseudoalteromonas (gamma Proteobacteria) and Arcobacter (epsilon Proteobacteria); the highly abundant bacteria in lab-cultured feces were other groups, including Leisingera, Phaeobacter (alpha Proteobacteria), and Vibrio (gamma Proteobacteria). Such difference indicates the complex fecal bacteria with the potential for multi-stability. The bacteria of habitat with 28 assigned phyla had the higher bacterial diversity and species richness than both fecal bacteria. Shared bacteria between wild feces and its habitat reached to approximately 90% (153/169 genera) and 28% (153/548 genera), respectively. As speculative, the less diversity of both fecal bacteria compared to its habitat partly because Amphioxus lives buried and the feces will ultimately end up in the sediment. Therefore, our study comprehensively investigates the complex bacterial community of Amphioxus and provides evidence for understanding the relationship of this basal chordate with the environment. PMID:26173442

  15. Differential resistance of drinking water bacterial populations to monochloramine disinfection.

    PubMed

    Chiao, Tzu-Hsin; Clancy, Tara M; Pinto, Ameet; Xi, Chuanwu; Raskin, Lutgarde

    2014-04-01

    The impact of monochloramine disinfection on the complex bacterial community structure in drinking water systems was investigated using culture-dependent and culture-independent methods. Changes in viable bacterial diversity were monitored using culture-independent methods that distinguish between live and dead cells based on membrane integrity, providing a highly conservative measure of viability. Samples were collected from lab-scale and full-scale drinking water filters exposed to monochloramine for a range of contact times. Culture-independent detection of live cells was based on propidium monoazide (PMA) treatment to selectively remove DNA from membrane-compromised cells. Quantitative PCR (qPCR) and pyrosequencing of 16S rRNA genes was used to quantify the DNA of live bacteria and characterize the bacterial communities, respectively. The inactivation rate determined by the culture-independent PMA-qPCR method (1.5-log removal at 664 mg·min/L) was lower than the inactivation rate measured by the culture-based methods (4-log removal at 66 mg·min/L). Moreover, drastic changes in the live bacterial community structure were detected during monochloramine disinfection using PMA-pyrosequencing, while the community structure appeared to remain stable when pyrosequencing was performed on samples that were not subject to PMA treatment. Genera that increased in relative abundance during monochloramine treatment include Legionella, Escherichia, and Geobacter in the lab-scale system and Mycobacterium, Sphingomonas, and Coxiella in the full-scale system. These results demonstrate that bacterial populations in drinking water exhibit differential resistance to monochloramine, and that the disinfection process selects for resistant bacterial populations. PMID:24625288

  16. Histophagous ciliate Pseudocollinia brintoni and bacterial assemblage interaction with krill Nyctiphanes simplex. I. Transmission process.

    PubMed

    Gómez-Gutiérrez, Jaime; López-Cortés, Alejandro; Aguilar-Méndez, Mario J; Angel-Rodríguez, Jorge A; Tremblay, Nelly; Zenteno-Savín, Tania; Robinson, Carlos J

    2015-10-27

    Histophagous ciliates of the genus Pseudocollinia cause epizootic events that kill adult female krill (Euphausiacea), but their mode of transmission is unknown. We compared 16S rRNA sequences of bacterial strains isolated from stomachs of healthy krill Nyctiphanes simplex specimens with sequences of bacterial isolates and sequences of natural bacterial communities from the hemocoel of N. simplex specimens infected with P. brintoni to determine possible transmission pathways. All P. brintoni endoparasitic life stages and the transmission tomite stage (outside the host) were associated with bacterial assemblages. 16S rRNA sequences from isolated bacterial strains showed that Photobacterium spp. and Pseudoalteromonas spp. were dominant members of the bacterial assemblages during all life phases of P. brintoni and potential pathobionts. They were apparently unaffected by the krill's immune system or the histophagous activity of P. brintoni. However, other bacterial strains were found only in certain P. brintoni life phases, indicating that as the infection progressed, microhabitat conditions and microbial interactions may have become unfavorable for some strains of bacteria. Trophic infection is the most parsimonious explanation for how P. brintoni infects krill. We estimated N. simplex vulnerability to P. brintoni infection during more than three-fourths of their life span, infecting mostly adult females. The ciliates have relatively high prevalence levels (albeit at <10% of sampled stations) and a short life cycle (estimated <7 d). Histophagous ciliate-krill interactions may occur in other krill species, particularly those that form dense swarms and attain high population densities that potentially enhance trophic transmission and allow completion of the Pseudocollinia spp. life cycle. PMID:26503776

  17. Spatial distribution of bacterial communities driven by multiple environmental factors in a beach wetland of the largest freshwater lake in China

    PubMed Central

    Ding, Xia; Peng, Xiao-Jue; Jin, Bin-Song; Xiao, Ming; Chen, Jia-Kuan; Li, Bo; Fang, Chang-Ming; Nie, Ming

    2015-01-01

    The spatial distributions of bacterial communities may be driven by multiple environmental factors. Thus, understanding the relationships between bacterial distribution and environmental factors is critical for understanding wetland stability and the functioning of freshwater lakes. However, little research on the bacterial communities in deep sediment layers exists. In this study, thirty clone libraries of 16S rRNA were constructed from a beach wetland of the Poyang Lake along both horizontal (distance to the water-land junction) and vertical (sediment depth) gradients to assess the effects of sediment properties on bacterial community structure and diversity. Our results showed that bacterial diversity increased along the horizontal gradient and decreased along the vertical gradient. The heterogeneous sediment properties along gradients substantially affected the dominant bacterial groups at the phylum and species levels. For example, the NH+4 concentration decreased with increasing depth, which was positively correlated with the relative abundance of Alphaproteobacteria. The changes in bacterial diversity and dominant bacterial groups showed that the top layer had a different bacterial community structure than the deeper layers. Principal component analysis revealed that both gradients, not each gradient independently, contributed to the shift in the bacterial community structure. A multiple linear regression model explained the changes in bacterial diversity and richness along the depth and distance gradients. Overall, our results suggest that spatial gradients associated with sediment properties shaped the bacterial communities in the Poyang Lake beach wetland. PMID:25767466

  18. Contrasting spatial patterns and ecological attributes of soil bacterial and archaeal taxa across a landscape

    PubMed Central

    Constancias, Florentin; Saby, Nicolas P A; Terrat, Sébastien; Dequiedt, Samuel; Horrigue, Wallid; Nowak, Virginie; Guillemin, Jean-Philippe; Biju-Duval, Luc; Chemidlin Prévost-Bouré, Nicolas; Ranjard, Lionel

    2015-01-01

    Even though recent studies have clarified the influence and hierarchy of environmental filters on bacterial community structure, those constraining bacterial populations variations remain unclear. In consequence, our ability to understand to ecological attributes of soil bacteria and to predict microbial community response to environmental stress is therefore limited. Here, we characterized the bacterial community composition and the various bacterial taxonomic groups constituting the community across an agricultural landscape of 12 km2, by using a 215 × 215 m systematic grid representing 278 sites to precisely decipher their spatial distribution and drivers at this scale. The bacterial and Archaeal community composition was characterized by applying 16S rRNA gene pyrosequencing directly to soil DNA from samples. Geostatistics tools were used to reveal the heterogeneous distribution of bacterial composition at this scale. Soil physical parameters and land management explained a significant amount of variation, suggesting that environmental selection is the major process shaping bacterial composition. All taxa systematically displayed also a heterogeneous and particular distribution patterns. Different relative influences of soil characteristics, land use and space were observed, depending on the taxa, implying that selection and spatial processes might be differentially but not exclusively involved for each bacterial phylum. Soil pH was a major factor determining the distribution of most of the bacterial taxa and especially the most important factor explaining the spatial patterns of ?-Proteobacteria and Planctomycetes. Soil texture, organic carbon content and quality were more specific to a few number of taxa (e.g., ?-Proteobacteria and Chlorobi). Land management also influenced the distribution of bacterial taxa across the landscape and revealed different type of response to cropping intensity (positive, negative, neutral or hump-backed relationships) according to phyla. Altogether, this study provided valuable clues about the ecological behavior of soil bacterial and archaeal taxa at an agricultural landscape scale and could be useful for developing sustainable strategies of land management. PMID:25922908

  19. Bacterial Skin Infections

    MedlinePLUS

    ... Quiz) Structure and Function of the Skin (Video) Skin Cancer (News) Health Tip: Recognizing Signs of Nail Fungus (News) Health Tip: Easing Hives Additional Content Medical News Overview of Bacterial Skin Infections by A. Damian Dhar, MD, JD NOTE: ...

  20. Microfluidics for bacterial chemotaxis

    E-print Network

    Ahmed, Tanvir, Ph. D. Massachusetts Institute of Technology

    2011-01-01

    Bacterial chemotaxis, a remarkable behavioral trait which allows bacteria to sense and respond to chemical gradients in the environment, has implications in a broad range of fields including but not limited to disease ...

  1. Bacterial surface adaptation

    NASA Astrophysics Data System (ADS)

    Utada, Andrew

    2014-03-01

    Biofilms are structured multi-cellular communities that are fundamental to the biology and ecology of bacteria. Parasitic bacterial biofilms can cause lethal infections and biofouling, but commensal bacterial biofilms, such as those found in the gut, can break down otherwise indigestible plant polysaccharides and allow us to enjoy vegetables. The first step in biofilm formation, adaptation to life on a surface, requires a working knowledge of low Reynolds number fluid physics, and the coordination of biochemical signaling, polysaccharide production, and molecular motility motors. These crucial early stages of biofilm formation are at present poorly understood. By adapting methods from soft matter physics, we dissect bacterial social behavior at the single cell level for several prototypical bacterial species, including Pseudomonas aeruginosa and Vibrio cholerae.

  2. Molecular phylogeny of pneumocystis based on 5.8S rRNA gene and the internal transcribed spacers of rRNA gene sequences.

    PubMed

    Li, ZiHui; Feng, XianMin; Lu, SiQi; Zhang, Fan; Wang, FengYun; Huang, Song

    2008-05-01

    To clarify the phylogenetic relationships and species status of Pneumocystis, the 5.8S rRNA gene and the internal transcribed spacers (ITS, 1 and 2) of Pneumocystis rRNA derived from rat, gerbil and human were amplified, cloned and sequenced. The genetic distance matrix of six Pneumocystis species compared with other fungi like Taphrina and Saccharomyces indicated that the Pneumocystis genus contained multiple species including Pneumocystis from gerbil. The phylogenetic tree also showed that Pneumocystis from human and monkey formed one group and four rodent Pneumocystis formed another group. Among the four members, Pneumocystis wakefieldiae was most closely related to Pneumocystis murina and Pneumocystis carinii, and was least related to gerbil Pneumocystis. PMID:18785590

  3. Dynamics of bacterial community succession in a salt marsh chronosequence: evidences for temporal niche partitioning.

    PubMed

    Dini-Andreote, Francisco; de Cássia Pereira e Silva, Michele; Triadó-Margarit, Xavier; Casamayor, Emilio O; van Elsas, Jan Dirk; Salles, Joana Falcão

    2014-10-01

    The mechanisms underlying community assembly and promoting temporal succession are often overlooked in microbial ecology. Here, we studied an undisturbed salt marsh chronosequence, spanning over a century of ecosystem development, to understand bacterial succession in soil. We used 16S rRNA gene-based quantitative PCR to determine bacterial abundance and multitag 454 pyrosequencing for community composition and diversity analyses. Despite 10-fold lower 16S rRNA gene abundances, the initial stages of soil development held higher phylogenetic diversities than the soil at late succession. Temporal variations in phylogenetic ?-diversity were greater at initial stages of soil development, possibly as a result of the great dynamism imposed by the daily influence of the tide, promoting high immigration rates. Allogenic succession of bacterial communities was mostly driven by shifts in the soil physical structure, as well as variations in pH and salinity, which collectively explained 84.5% of the variation concerning community assemblage. The community assembly data for each successional stage were integrated into a network co-occurrence analysis, revealing higher complexity at initial stages, coinciding with great dynamism in turnover and environmental variability. Contrary to a spatial niche-based perspective of bacterial community assembly, we suggest temporal niche partitioning as the dominant mechanism of assembly (promoting more phylotype co-occurrence) in the initial stages of succession, where continuous environmental change results in the existence of multiple niches over short periods of time. PMID:24739625

  4. Evolution of bacterial consortia in spontaneously started rye sourdoughs during two months of daily propagation.

    PubMed

    Bessmeltseva, Marianna; Viiard, Ene; Simm, Jaak; Paalme, Toomas; Sarand, Inga

    2014-01-01

    The evolution of bacterial consortia was studied in six semi-solid rye sourdoughs during long-term backslopping at different temperatures. Each rye sourdough was started spontaneously in a laboratory (dough yield 200), propagated at either 20°C or 30°C, and renewed daily at an inoculation rate of 1?10 for 56 days. The changes in bacterial diversity over time were followed by both DGGE coupled with partial 16S rRNA gene sequencing and pyrosequencing of bar-coded 16S rRNA gene amplicons. Four species from the genus Lactobacillus (brevis, crustorum, plantarum, and paralimentarius) were detected in different combinations in all sourdoughs after 56 propagation cycles. Facultative heterofermentative lactic acid bacteria dominated in sourdoughs fermented at 30°C, while both obligate and facultative heterofermentative LAB were found to dominate in sourdoughs fermented at 20°C. After 56 propagation cycles, Kazachstania unispora (formerly Saccharomyces unisporus) was identified as the only yeast species that dominated in sourdoughs fermented at 20°C, while different combinations of strains from four yeast species (Kazachstania unispora, Saccharomyces cerevisiae, Candida krusei and Candida glabrata) were detected in sourdoughs propagated at 30°C. The evolution of bacterial communities in sourdoughs fermented at the same temperature did not follow the same time course and changes in the composition of dominant and subdominant bacterial communities occurred even after six weeks of backslopping. PMID:24748058

  5. Effect of dietary monensin on the bacterial population structure of dairy cattle colonic contents.

    PubMed

    McGarvey, Jeffery A; Hamilton, Scott W; DePeters, Edward J; Mitloehner, Frank M

    2010-02-01

    To determine the effect of monensin, a carboxylic polyether ionophore antibiotic, on the bacterial population structure of dairy cattle colonic contents, we fed six lactating Holstein cows a diet containing monensin (600 mg day(-1)) or an identical diet without monensin. Fresh waste samples were taken directly from the animals once a month for 3 months and assayed for their bacterial population structure via 16S rRNA gene sequence analysis. In total 6,912 16S rRNA genes were examined, comprising 345 and 315 operational taxonomic units (OTUs) from the monensin fed and control animals, respectively. Coverage estimates of the OTUs identified were 87.6% for the monensin fed and 88.3% for the control colonic content derived library. Despite this high level of coverage, no significant difference was found between the libraries down to the genus level. Thus we concluded that although monensin is believed to increase milk production in dairy cattle by altering the bacterial population structure within the bovine gastrointestinal tract, we were unable to identify any significant difference in the bacterial population structure of the colonic contents of monensin fed vs. the control dairy cattle, down to the genus level. PMID:19784643

  6. Effects of bacterial inoculants on the indigenous microbiome and secondary metabolites of chamomile plants

    PubMed Central

    Schmidt, Ruth; Köberl, Martina; Mostafa, Amr; Ramadan, Elshahat M.; Monschein, Marlene; Jensen, Kenneth B.; Bauer, Rudolf; Berg, Gabriele

    2014-01-01

    Plant-associated bacteria fulfill important functions for plant growth and health. However, our knowledge about the impact of bacterial treatments on the host's microbiome and physiology is limited. The present study was conducted to assess the impact of bacterial inoculants on the microbiome of chamomile plants Chamomilla recutita (L.) Rauschert grown in a field under organic management in Egypt. Chamomile seedlings were inoculated with three indigenous Gram-positive strains (Streptomyces subrutilus Wbn2-11, Bacillus subtilis Co1-6, Paenibacillus polymyxa Mc5Re-14) from Egypt and three European Gram-negative strains (Pseudomonas fluorescens L13-6-12, Stenotrophomonas rhizophila P69, Serratia plymuthica 3Re4-18) already known for their beneficial plant-microbe interaction. Molecular fingerprints of 16S rRNA gene as well as real-time PCR analyses did not show statistically significant differences for all applied bacterial antagonists compared to the control. In contrast, a pyrosequencing analysis of the 16S rRNA gene libraries revealed significant differences in the community structure of bacteria between the treatments. These differences could be clearly shown by a shift within the community structure and corresponding beta-diversity indices. Moreover, B. subtilis Co1-6 and P. polymyxa Mc5Re-14 showed an enhancement of the bioactive secondary metabolite apigenin-7-O-glucoside. This indicates a possible new function of bacterial inoculants: to interact with the plant microbiome as well as to influence the plant metabolome. PMID:24600444

  7. Evolution of Bacterial Consortia in Spontaneously Started Rye Sourdoughs during Two Months of Daily Propagation

    PubMed Central

    Simm, Jaak; Paalme, Toomas; Sarand, Inga

    2014-01-01

    The evolution of bacterial consortia was studied in six semi-solid rye sourdoughs during long-term backslopping at different temperatures. Each rye sourdough was started spontaneously in a laboratory (dough yield 200), propagated at either 20°C or 30°C, and renewed daily at an inoculation rate of 1?10 for 56 days. The changes in bacterial diversity over time were followed by both DGGE coupled with partial 16S rRNA gene sequencing and pyrosequencing of bar-coded 16S rRNA gene amplicons. Four species from the genus Lactobacillus (brevis, crustorum, plantarum, and paralimentarius) were detected in different combinations in all sourdoughs after 56 propagation cycles. Facultative heterofermentative lactic acid bacteria dominated in sourdoughs fermented at 30°C, while both obligate and facultative heterofermentative LAB were found to dominate in sourdoughs fermented at 20°C. After 56 propagation cycles, Kazachstania unispora (formerly Saccharomyces unisporus) was identified as the only yeast species that dominated in sourdoughs fermented at 20°C, while different combinations of strains from four yeast species (Kazachstania unispora, Saccharomyces cerevisiae, Candida krusei and Candida glabrata) were detected in sourdoughs propagated at 30°C. The evolution of bacterial communities in sourdoughs fermented at the same temperature did not follow the same time course and changes in the composition of dominant and subdominant bacterial communities occurred even after six weeks of backslopping. PMID:24748058

  8. Diagnostic assay for Helicobacter hepaticus based on nucleotide sequence of its 16S rRNA gene.

    PubMed Central

    Battles, J K; Williamson, J C; Pike, K M; Gorelick, P L; Ward, J M; Gonda, M A

    1995-01-01

    Conserved primers were used to PCR amplify 95% of the Helicobacter hepaticus 16S rRNA gene. Its sequence was determined and aligned to those of related bacteria, enabling the selection of primers to highly diverged regions of the 16S rRNA gene and an oligonucleotide probe for the development of a PCR-liquid hybridization assay. This assay was shown to be both sensitive and specific for H. hepaticus 16S rRNA gene sequences. PMID:7542270

  9. Distinct Soil Bacterial Communities Revealed under a Diversely Managed Agroecosystem

    PubMed Central

    Shange, Raymon S.; Ankumah, Ramble O.; Ibekwe, Abasiofiok M.; Zabawa, Robert; Dowd, Scot E.

    2012-01-01

    Land-use change and management practices are normally enacted to manipulate environments to improve conditions that relate to production, remediation, and accommodation. However, their effect on the soil microbial community and their subsequent influence on soil function is still difficult to quantify. Recent applications of molecular techniques to soil biology, especially the use of 16S rRNA, are helping to bridge this gap. In this study, the influence of three land-use systems within a demonstration farm were evaluated with a view to further understand how these practices may impact observed soil bacterial communities. Replicate soil samples collected from the three land-use systems (grazed pine forest, cultivated crop, and grazed pasture) on a single soil type. High throughput 16S rRNA gene pyrosequencing was used to generate sequence datasets. The different land use systems showed distinction in the structure of their bacterial communities with respect to the differences detected in cluster analysis as well as diversity indices. Specific taxa, particularly Actinobacteria, Acidobacteria, and classes of Proteobacteria, showed significant shifts across the land-use strata. Families belonging to these taxa broke with notions of copio- and oligotrphy at the class level, as many of the less abundant groups of families of Actinobacteria showed a propensity for soil environments with reduced carbon/nutrient availability. Orders Actinomycetales and Solirubrobacterales showed their highest abundance in the heavily disturbed cultivated system despite the lowest soil organic carbon (SOC) values across the site. Selected soil properties ([SOC], total nitrogen [TN], soil texture, phosphodiesterase [PD], alkaline phosphatase [APA], acid phosphatase [ACP] activity, and pH) also differed significantly across land-use regimes, with SOM, PD, and pH showing variation consistent with shifts in community structure and composition. These results suggest that use of pyrosequencing along with traditional analysis of soil physiochemical properties may provide insight into the ecology of descending taxonomic groups in bacterial communities. PMID:22844402

  10. Bacterial Diversity in Bohai Bay Solar Saltworks, China.

    PubMed

    Zhang, Jiaojiao; Ma, Guannan; Deng, Yuangao; Dong, Jinggang; Van Stappen, Gilbert; Sui, Liying

    2016-01-01

    The microbiota in solar salterns plays an important role in salt production quantitatively and qualitatively. Bohai Bay coast is the major sea salt producing area in China. However, few ecological characterization studies of the Bohai Bay salt ponds, particularly of their microbial diversity, have been conducted. This study investigated the structure and diversity of the bacterial community in Hangu saltworks in response to environmental factors. The brine water was sampled from five selected saltponds within a salinity range of 5.0-19.3 % in May, July, and October, 2012. Phylogenetic analysis based on the denaturing gradient gel electrophoresis (DGGE) patterns of the PCR-amplified 16S rRNA gene fragment showed that, rather than pond salinity, especially the month of sampling influenced the structure of the bacterial community in the saltponds, which may be related to the water temperature or other factors fluctuating over the months. Moreover, canonical correspondence analysis of biological and physico-chemical parameters indicated that especially other environmental factors such as nitrogenous and phosphorous nutrient contents and pH structured the microbial community. The relatively high range-weighted richness index and Shannon-Wiener index (H') observed in this study reflect the high level of richness and biodiversity present, though there were substantial fluctuations over the months and salinities of sampling. The fragment of 16S rRNA gene sequence recovered from DGGE bands indicated that the bacterial assemblage in Hangu Saltworks was dominated by members of ?-Proteobacteria (34 % of total sequences obtained), followed by Firmicutes (14 %) and Bacteroidetes (9 %). PMID:26424140

  11. Bacterial Community Analysis of Drinking Water Biofilms in Southern Sweden

    PubMed Central

    Lührig, Katharina; Canbäck, Björn; Paul, Catherine J.; Johansson, Tomas; Persson, Kenneth M.; Rådström, Peter

    2015-01-01

    Next-generation sequencing of the V1–V2 and V3 variable regions of the 16S rRNA gene generated a total of 674,116 reads that described six distinct bacterial biofilm communities from both water meters and pipes. A high degree of reproducibility was demonstrated for the experimental and analytical work-flow by analyzing the communities present in parallel water meters, the rare occurrence of biological replicates within a working drinking water distribution system. The communities observed in water meters from households that did not complain about their drinking water were defined by sequences representing Proteobacteria (82–87%), with 22–40% of all sequences being classified as Sphingomonadaceae. However, a water meter biofilm community from a household with consumer reports of red water and flowing water containing elevated levels of iron and manganese had fewer sequences representing Proteobacteria (44%); only 0.6% of all sequences were classified as Sphingomonadaceae; and, in contrast to the other water meter communities, markedly more sequences represented Nitrospira and Pedomicrobium. The biofilm communities in pipes were distinct from those in water meters, and contained sequences that were identified as Mycobacterium, Nocardia, Desulfovibrio, and Sulfuricurvum. The approach employed in the present study resolved the bacterial diversity present in these biofilm communities as well as the differences that occurred in biofilms within a single distribution system, and suggests that next-generation sequencing of 16S rRNA amplicons can show changes in bacterial biofilm communities associated with different water qualities. PMID:25739379

  12. pRNA: NoRC-associated RNA of rRNA operons.

    PubMed

    Wehner, Stefanie; Dörrich, Anja K; Ciba, Philipp; Wilde, Annegret; Marz, Manja

    2014-01-01

    Promoter-associated RNAs (pRNAs) are a family of ~90-100 nt-long divergent RNAs overlapping the promoter of the rRNA (rDNA) operon. pRNA transcripts interact with TIP5, a component of the chromatin remodeling complex NoRC, which recruits enzymes for heterochromatin formation and mediates silencing of rRNA genes. Here we present a comprehensive analysis of pRNA homologs, including different versions per species, as result of in silico studies in available metazoan genome assemblies. Comparative sequence analysis and secondary structure prediction ended up in two possible secondary structures, which let us assume a possible dual function of pRNAs for regulation of rRNA operons. Furthermore, we validated parts of our computational predictions experimentally by RT-PCR and sequencing. A representative seed alignment of the pRNA family, annotated with possible secondary structures was released to the Rfam database. PMID:24440945

  13. The human 18S rRNA base methyltransferases DIMT1L and WBSCR22-TRMT112 but not rRNA modification are required for ribosome biogenesis

    PubMed Central

    Zorbas, Christiane; Nicolas, Emilien; Wacheul, Ludivine; Huvelle, Emmeline; Heurgué-Hamard, Valérie; Lafontaine, Denis L. J.

    2015-01-01

    At the heart of the ribosome lie rRNAs, whose catalytic function in translation is subtly modulated by posttranscriptional modifications. In the small ribosomal subunit of budding yeast, on the 18S rRNA, two adjacent adenosines (A1781/A1782) are N6-dimethylated by Dim1 near the decoding site, and one guanosine (G1575) is N7-methylated by Bud23-Trm112 at a ridge between the P- and E-site tRNAs. Here we establish human DIMT1L and WBSCR22-TRMT112 as the functional homologues of yeast Dim1 and Bud23-Trm112. We report that these enzymes are required for distinct pre-rRNA processing reactions leading to synthesis of 18S rRNA, and we demonstrate that in human cells, as in budding yeast, ribosome biogenesis requires the presence of the modification enzyme rather than its RNA-modifying catalytic activity. We conclude that a quality control mechanism has been conserved from yeast to human by which binding of a methyltransferase to nascent pre-rRNAs is a prerequisite to processing, so that all cleaved RNAs are committed to faithful modification. We further report that 18S rRNA dimethylation is nuclear in human cells, in contrast to yeast, where it is cytoplasmic. Yeast and human ribosome biogenesis thus have both conserved and distinctive features. PMID:25851604

  14. Bacterial dynamics within the mucus, tissue and skeleton of the coral Porites lutea during different seasons

    PubMed Central

    Li, Jie; Chen, Qi; Long, Li-Juan; Dong, Jun-De; Yang, Jian; Zhang, Si

    2014-01-01

    Investigation of the response of coral microbial communities to seasonal ecological environment at the microscale will advance our understanding of the relationship between coral-associated bacteria community and coral health. In this study, we examined bacteria community composition from mucus, tissue and skeleton of Porites lutea and surrounding seawater every three months for 1 year on Luhuitou fringing reef. The bacterial communities were analyzed using pyrosequencing of the V1-V2 region of the 16S rRNA gene, which demonstrated diverse bacterial consortium profiles in corals. The bacterial communities in all three coral compartments studied were significantly different from the surrounding seawater. Moreover, they had a much more dynamic seasonal response compared to the seawater communities. The bacterial communities in all three coral compartments collected in each seasonal sample tended to cluster together. Analysis of the relationship between bacterial assemblages and the environmental parameters showed that the bacterial community correlated to dissolved oxygen and rainfall significantly at our study site. This study highlights a dynamic relationship between the high complexity of coral associated bacterial community and seasonally varying ecosystem parameters. PMID:25475855

  15. Dynamic bacterial communities on reverse-osmosis membranes in a full-scale desalination plant.

    PubMed

    Manes, C-L de O; West, N; Rapenne, S; Lebaron, P

    2011-01-01

    To better understand biofouling of seawater reverse osmosis (SWRO) membranes, bacterial diversity was characterized in the intake water, in subsequently pretreated water and on SWRO membranes from a full-scale desalination plant (FSDP) during a 9 month period. 16S rRNA gene fingerprinting and sequencing revealed that bacterial communities in the water samples and on the SWRO membranes were very different. For the different sampling dates, the bacterial diversity of the active and the total bacterial fractions of the water samples remained relatively stable over the sampling period whereas the bacterial community structure on the four SWRO membrane samples was significantly different. The richness and evenness of the SWRO membrane bacterial communities increased with usage time with an increase in the Shannon diversity index of 2.2 to 3.7. In the oldest SWRO membrane (330 days), no single operational taxonomic unit (OTU) dominated and the majority of the OTUs fell into the Alphaproteobacteria or the Planctomycetes. In striking contrast, a Betaproteobacteria OTU affiliated to the genus Ideonella was dominant and exclusively found in the membrane used for the shortest time (10 days). This suggests that bacteria belonging to this genus could be one of the primary colonizers of the SWRO membrane. Knowledge of the dominant bacterial species on SWRO membranes and their dynamics should help guide culture studies for physiological characterization of biofilm forming species. PMID:21108068

  16. Bacterial dynamics within the mucus, tissue and skeleton of the coral Porites lutea during different seasons

    NASA Astrophysics Data System (ADS)

    Li, Jie; Chen, Qi; Long, Li-Juan; Dong, Jun-De; Yang, Jian; Zhang, Si

    2014-12-01

    Investigation of the response of coral microbial communities to seasonal ecological environment at the microscale will advance our understanding of the relationship between coral-associated bacteria community and coral health. In this study, we examined bacteria community composition from mucus, tissue and skeleton of Porites lutea and surrounding seawater every three months for 1 year on Luhuitou fringing reef. The bacterial communities were analyzed using pyrosequencing of the V1-V2 region of the 16S rRNA gene, which demonstrated diverse bacterial consortium profiles in corals. The bacterial communities in all three coral compartments studied were significantly different from the surrounding seawater. Moreover, they had a much more dynamic seasonal response compared to the seawater communities. The bacterial communities in all three coral compartments collected in each seasonal sample tended to cluster together. Analysis of the relationship between bacterial assemblages and the environmental parameters showed that the bacterial community correlated to dissolved oxygen and rainfall significantly at our study site. This study highlights a dynamic relationship between the high complexity of coral associated bacterial community and seasonally varying ecosystem parameters.

  17. Assessment of Bacterial Community Assembly Patterns and Processes in Pig Manure Slurry

    PubMed Central

    Kumari, Priyanka; Choi, Hong L.; Sudiarto, Sartika I. A.

    2015-01-01

    The bacterial community assembly patterns and processes are poorly understood in pig manure slurry. We collected pig manure slurry samples during the winter and summer seasons from eight commercial pig farms in South Korea. The V3 region of 16S rRNA genes was PCR amplified and sequenced using paired-end Illumina technology for in-depth characterization of bacterial community. Firmicutes, Bacteroidetes, Proteobacteria, Spirochaetes, and Tenericutes were the predominant bacterial phyla present in slurry samples. Bacterial taxonomic community composition was not influenced by the season; however, phylogenetic community composition was affected by seasonal variations. The community composition and diversity patterns were strongly influenced by pH. The bacterial diversity indices showed a unimodal relationship with pH. Phylogenetic signals were detected over only short phylogenetic distances, revealing that closely related bacterial operational taxonomic units (OTUs) tend to co-occur in the same environment; hence, they are ecologically similar. Across all samples, a niche-based process, through strong environmental filtering imposed by pH, primarily governed bacterial community assembly; however, in samples close to the neutral pH range, the role of environmental filtering was decreased due to neutral community assembly. In summary, pH emerged as the major physico-chemical variable in pig manure slurry that regulates the relative importance of niche-based and neutral processes in shaping the community assembly of bacteria. PMID:26422375

  18. Co-occurrence patterns in aquatic bacterial communities across changing permafrost landscapes

    NASA Astrophysics Data System (ADS)

    Comte, J.; Lovejoy, C.; Crevecoeur, S.; Vincent, W. F.

    2015-07-01

    Permafrost thaw ponds and lakes are widespread across the northern landscape and may play a central role in global biogeochemical cycles, yet knowledge about their microbial ecology is limited. We sampled a set of thaw ponds and lakes as well as shallow rock-basin lakes that are located in distinct valleys along a North-South permafrost degradation gradient. We applied high-throughput sequencing of the 16S rRNA gene to determine co-occurrence patterns among bacterial taxa, and then analyzed these results relative to environmental variables to identify factors controlling bacterial community structure. Network analysis was applied to identify possible ecological linkages among the bacterial taxa and with abiotic and biotic variables. The results showed an overall high level of shared taxa among bacterial communities within each valley, however the bacterial co-occurrence patterns were non-random, with evidence of habitat preferences. There were taxonomic differences in bacterial assemblages among the different valleys that were statistically related to dissolved organic carbon concentration, conductivity and phytoplankton biomass. Co-occurrence networks revealed complex interdependencies within the bacterioplankton communities and showed contrasting linkages to environmental conditions among the main bacterial phyla. The thaw pond networks were composed of a limited number of highly connected taxa. This "small world network" property would render the communities more robust to environmental change but vulnerable to the loss of microbial keystone species.

  19. Bacterial Communities in Malagasy Soils with Differing Levels of Disturbance Affecting Botanical Diversity

    PubMed Central

    Blasiak, Leah C.; Schmidt, Alex W.; Andriamiarinoro, Honoré; Mulaw, Temesgen; Rasolomampianina, Rado; Applequist, Wendy L.; Birkinshaw, Chris; Rejo-Fienena, Félicitée; Lowry, Porter P.; Schmidt, Thomas M.; Hill, Russell T.

    2014-01-01

    Madagascar is well-known for the exceptional biodiversity of its macro-flora and fauna, but the biodiversity of Malagasy microbial communities remains relatively unexplored. Understanding patterns of bacterial diversity in soil and their correlations with above-ground botanical diversity could influence conservation planning as well as sampling strategies to maximize access to bacterially derived natural products. We present the first detailed description of Malagasy soil bacterial communities from a targeted 16S rRNA gene survey of greater than 290,000 sequences generated using 454 pyrosequencing. Two sampling plots in each of three forest conservation areas were established to represent different levels of disturbance resulting from human impact through agriculture and selective exploitation of trees, as well as from natural impacts of cyclones. In parallel, we performed an in-depth characterization of the total vascular plant morphospecies richness within each plot. The plots representing different levels of disturbance within each forest did not differ significantly in bacterial diversity or richness. Changes in bacterial community composition were largest between forests rather than between different levels of impact within a forest. The largest difference in bacterial community composition with disturbance was observed at the Vohibe forest conservation area, and this difference was correlated with changes in both vascular plant richness and soil pH. These results provide the first survey of Malagasy soil bacterial diversity and establish a baseline of botanical diversity within important conservation areas. PMID:24465484

  20. Sequence variation of human 28S rRNA and expression of variants in different tissues

    SciTech Connect

    Sylvester, J.E.; Gonzalez, I.L.; Gillespie, D.A.

    1994-09-01

    There are 400 or so copies of the ribosomal RNA (rRNA) gene in humans. Previous studies have shown that human 28S encoding rDNA, and therefore rRNAs, vary in sequence at specific locations termed variable regions; these variations appear in a population and within an individual. This study asked the following questions regarding one variable region (V5) within 28S rRNA gene sequence variation in a single individual: (1) what is the extent and nature of the sequence variation at the DNA level?; (2) are these variant rRNA genes expressed?; (3) does rRNA gene sequence variation result in rRNA structural variation?; (4) is there evidence of differential expression of variant rRNA genes?. To address these four questions, genomic copies of the V5 region were cloned from the individual and sequenced. Six unique sequences were found among seven V5 clones, each differing only in the number of repeats of simple sequences (CGG), (TG), (T), and (C) at a specific site of the V5 region. Computer-generated secondary structure models of each of these variants indicated that the basic structure was not compromised. To test for V5 variant expression in different tissues of an individual, RNAs isolated from different tissues were used to protect each of the cloned V5 variant probes in the RNase protection assay (RPA). We conclude from the RPA results that different variants are present in proportionately different amounts in each tissue tested; however, the relative proportion of variants are similar in these tissues.

  1. Taxonomic Resolutions Based on 18S rRNA Genes: A Case Study of Subclass Copepoda

    PubMed Central

    Wu, Shu; Xiong, Jie; Yu, Yuhe

    2015-01-01

    Biodiversity studies are commonly conducted using 18S rRNA genes. In this study, we compared the inter-species divergence of variable regions (V1–9) within the copepod 18S rRNA gene, and tested their taxonomic resolutions at different taxonomic levels. Our results indicate that the 18S rRNA gene is a good molecular marker for the study of copepod biodiversity, and our conclusions are as follows: 1) 18S rRNA genes are highly conserved intra-species (intra-species similarities are close to 100%); and could aid in species-level analyses, but with some limitations; 2) nearly-whole-length sequences and some partial regions (around V2, V4, and V9) of the 18S rRNA gene can be used to discriminate between samples at both the family and order levels (with a success rate of about 80%); 3) compared with other regions, V9 has a higher resolution at the genus level (with an identification success rate of about 80%); and 4) V7 is most divergent in length, and would be a good candidate marker for the phylogenetic study of Acartia species. This study also evaluated the correlation between similarity thresholds and the accuracy of using nuclear 18S rRNA genes for the classification of organisms in the subclass Copepoda. We suggest that sample identification accuracy should be considered when a molecular sequence divergence threshold is used for taxonomic identification, and that the lowest similarity threshold should be determined based on a pre-designated level of acceptable accuracy. PMID:26107258

  2. Different bulk and active bacterial communities in cryoconite from the margin and interior of the Greenland ice sheet.

    PubMed

    Stibal, Marek; Schostag, Morten; Cameron, Karen A; Hansen, Lars H; Chandler, David M; Wadham, Jemma L; Jacobsen, Carsten S

    2015-04-01

    Biological processes in the supraglacial ecosystem, including cryoconite, contribute to nutrient cycling within the cryosphere and may affect surface melting, yet little is known of the diversity of the active microbes in these environments. We examined the bacterial abundance and community composition of cryoconite over a melt season at two contrasting sites at the margin and in the interior of the Greenland ice sheet, using sequence analysis and quantitative polymerase chain reaction of coextracted 16S rDNA and rRNA. Significant differences were found between bulk (rDNA) and potentially active (rRNA) communities, and between communities sampled from the two sites. Higher concentrations of rRNA than rDNA were detected at the interior site, whereas at the margin several orders of magnitude less rRNA was found compared with rDNA, which may be explained by a lower proportion of active bacteria at the margin site. The rRNA communities at both sites were dominated by a few taxa of Cyanobacteria and Alpha- and/or Betaproteobacteria. The bulk alpha diversity was higher in the margin site community, suggesting that local sources may be contributing towards the gene pool in addition to long distance transport. PMID:25405749

  3. Comparison of rpoB gene sequencing, 16S rRNA gene sequencing, gyrB multiplex PCR, and the VITEK2 system for identification of Acinetobacter clinical isolates.

    PubMed

    Lee, Min Jung; Jang, Sook Jin; Li, Xue Min; Park, Geon; Kook, Joong-Ki; Kim, Min Jung; Chang, Young-Hyo; Shin, Jong Hee; Kim, Soo Hyun; Kim, Dong-Min; Kang, Seong-Ho; Moon, Dae-Soo

    2014-01-01

    Since accurate identification of species is necessary for proper treatment of Acinetobacter infections, we compared the performances of 4 bacterial identification methods using 167 Acinetobacter clinical isolates to identify the best identification method. To secure more non-baumannii Acinetobacter (NBA) strains as target strains, we first identified Acinetobacter baumannii in a total of 495 Acinetobacter clinical isolates identified using the VITEK 2 system. Because 371 of 495 strains were identified as A. baumannii using gyrB multiplex 1 PCR and blaOXA51-like PCR, we performed rpoB gene sequencing and 16S rRNA gene sequencing on remaining 124 strains belonging to NBA and 52 strains of A. baumannii. For identification of Acinetobacter at the species level, the accuracy rates of rpoB gene sequencing, 16S rRNA gene sequencing, gyrB multiplex PCR, and the VITEK 2 were 98.2%, 93.4%, 77.2%, and 35.9%, respectively. The gyrB multiplex PCR seems to be very useful for the detection of ACB complex because its concordance rates to the final identification of strains of ACB complex were 100%. Both the rpoB gene sequencing and the 16S rRNA gene sequencing may be useful in identifying Acinetobacter. PMID:24157058

  4. Object extraction Object extraction

    E-print Network

    Giger, Christine

    ("house", "lake") · usually solved jointly as detection: identify all objects of a certain class · object methods · for well-defined corners ­ least-squares matching pixel ­ human (stereoscopic) >0.3 pixel ­ least-squares matching pixel ­ human (stereoscopic) >0.3 pixel #12;Semi-automatic extraction

  5. Bacterial loads of Ureaplasma parvum contribute to the development of inflammatory responses in the male urethra.

    PubMed

    Deguchi, Takashi; Shimada, Yasushi; Horie, Kengo; Mizutani, Kohsuke; Seike, Kensaku; Tsuchiya, Tomohiro; Yokoi, Shigeaki; Yasuda, Mitsuru; Ito, Shin

    2015-12-01

    Ureaplasma parvum, which has been recognised as a coloniser in the male urethra, is detected in some men with non-gonococcal urethritis. In this study, we quantified the 16?S rRNA genes of U. parvum by a real-time polymerase chain reaction-based assay in first-voided urine from 15 symptomatic and 38 asymptomatic men who were positive only for U. parvum. We also determined the leukocyte counts by automated quantitative urine particle analysis in their first-voided urine. Positive correlations were observed between copies of the 16?S rRNA genes of U. parvum/ml and the leukocyte counts/µl in first-voided urine (p?=?0.0019). The loads of ?10(4) copies of the 16?S rRNA gene/ml, corresponding to ?5?×?10(3) cells of U. parvum/ml, were significantly associated with the presence of ?12.5 leukocytes/µl in first-voided urine that might document the presence of inflammatory responses in the urethra. However, a large portion of the subjects (83.0%) had bacterial loads of <5?×?10(3) cells of U. parvum/ml, and 79.5% of them showed <12.5 leukocytes/µl. The ambiguity of the pathogenic role of U. parvum in non-gonococcal urethritis could, in part, be due to its low bacterial loads, which might not give rise to inflammatory responses in the male urethra. PMID:25525054

  6. Onset and establishment of diazotrophs and other bacterial associates in the early life history stages of the coral Acropora millepora.

    PubMed

    Lema, Kimberley A; Bourne, David G; Willis, Bette L

    2014-10-01

    Early establishment of coral-microbial symbioses is fundamental to the fitness of corals, but comparatively little is known about the onset and succession of bacterial communities in their early life history stages. In this study, bacterial associates of the coral Acropora millepora were characterized throughout the first year of life, from larvae and 1-week-old juveniles reared in laboratory conditions in the absence of the dinoflagellate endosymbiont Symbiodinium to field-outplanted juveniles with established Symbiodinium symbioses, and sampled at 2 weeks and at 3, 6 and 12 months. Using an amplicon pyrosequencing approach, the diversity of both nitrogen-fixing bacteria and of bacterial communities overall was assessed through analysis of nifH and 16S rRNA genes, respectively. The consistent presence of sequences affiliated with diazotrophs of the order Rhizobiales (23-58% of retrieved nifH sequences; 2-12% of 16S rRNA sequences), across all samples from larvae to 12-month-old coral juveniles, highlights the likely functional importance of this nitrogen-fixing order to the coral holobiont. Dominance of Roseobacter-affiliated sequences (>55% of retrieved 16S rRNA sequences) in larvae and 1-week-old juveniles, and the consistent presence of sequences related to Oceanospirillales and Altermonadales throughout all early life history stages, signifies their potential importance as coral associates. Increased diversity of bacterial communities once juveniles were transferred to the field, particularly of Cyanobacteria and Deltaproteobacteria, demonstrates horizontal (environmental) uptake of coral-associated bacterial communities. Although overall bacterial communities were dynamic, bacteria with likely important functional roles remain stable throughout early life stages of Acropora millepora. PMID:25156176

  7. 99mTc-MORF oligomers specific for bacterial ribosomal RNA as potential specific infection imaging agents

    PubMed Central

    Chen, Ling; Wang, Yi; Cheng, Dengfeng; Liu, Xinrong; Dou, Shuping; Liu, Guozheng; Hnatowich, Donald J.; Rusckowski, Mary

    2013-01-01

    Purpose Radiolabeled oligomers complementary to the 16S rRNA in bacteria were investigated as bacterial infection imaging agents. Methods and Results Identical sequences with backbones phosphorodiamidate morpholino (MORF), peptide nucleic acid (PNA), and phosphorothioate DNA (PS-DNA) were 99mTc-labeled and evaluated for binding to bacterial RNA. MORF binding to RNA from E. coli strains SM101 and K12 was 4-fold and 150-fold higher compared to PNA, and PS-DNA, respectively. Subsequently MORF oligomer in fluorescence in situ hybridization showed a stronger signal with study MORF compared to control in fixed preparations of two E. coli strains and K. pneumoniae. Flow cytometry analysis showed study MORF accumulation to be 8- and 80-fold higher compared to the control in live K. pneumoniae and S. aureus, respectively. Further, fluorescence microscopy showed increased accumulation of study MORF over control in live E. coli and K. pneumonia. Binding of 99mTc-study MORF to RNA from E. coli SM101 and K12 was 30.4 pmoles and 117.8 pmoles, respectively, per 1010 cells. Mice with K. pneumoniae live or heat-killed (sterile inflammation) in one thigh at 90 min for both 99mTc-study MORF and control showed higher accumulation in target thighs than in blood and all organs other than kidney and small intestines. Whereas accumulation of 99mTc-study MORF was significantly higher (p=0.009) than that of the control in the thigh with sterile inflammation. Conclusion A 99mTc-MORF oligomer complimentary to the bacterial 16S rRNA demonstrated binding to bacterial RNA in vitro with specific accumulation into live bacteria. Radiolabeled MORF oligomers antisense to the bacterial rRNA may be useful to image bacterial infection. PMID:24054488

  8. Pyrosequencing of Bacterial Symbionts within Axinella corrugata Sponges: Diversity and Seasonal Variability

    PubMed Central

    White, James R.; Patel, Jignasa; Ottesen, Andrea; Arce, Gabriela; Blackwelder, Patricia; Lopez, Jose V.

    2012-01-01

    Background Marine sponge species are of significant interest to many scientific fields including marine ecology, conservation biology, genetics, host-microbe symbiosis and pharmacology. One of the most intriguing aspects of the sponge “holobiont” system is the unique physiology, interaction with microbes from the marine environment and the development of a complex commensal microbial community. However, intraspecific variability and temporal stability of sponge-associated bacterial symbionts remain relatively unknown. Methodology/Principal Findings We have characterized the bacterial symbiont community biodiversity of seven different individuals of the Caribbean reef sponge Axinella corrugata, from two different Florida reef locations during variable seasons using multiplex 454 pyrosequencing of 16 S rRNA amplicons. Over 265,512 high-quality 16 S rRNA sequences were generated and analyzed. Utilizing versatile bioinformatics methods and analytical software such as the QIIME and CloVR packages, we have identified 9,444 distinct bacterial operational taxonomic units (OTUs). Approximately 65,550 rRNA sequences (24%) could not be matched to bacteria at the class level, and may therefore represent novel taxa. Differentially abundant classes between seasonal Axinella communities included Gammaproteobacteria, Flavobacteria, Alphaproteobacteria, Cyanobacteria, Acidobacter and Nitrospira. Comparisons with a proximal outgroup sponge species (Amphimedon compressa), and the growing sponge symbiont literature, indicate that this study has identified approximately 330 A. corrugata-specific symbiotic OTUs, many of which are related to the sulfur-oxidizing Ectothiorhodospiraceae. This family appeared exclusively within A. corrugata, comprising >34.5% of all sequenced amplicons. Other A. corrugata symbionts such as Deltaproteobacteria, Bdellovibrio, and Thiocystis among many others are described. Conclusions/Significance Slight shifts in several bacterial taxa were observed between communities sampled during spring and fall seasons. New 16 S rDNA sequences and concomitant identifications greatly expand the microbial community profile for this model reef sponge, and will likely be useful as a baseline for any future comparisons regarding sponge microbial community dynamics. PMID:22701613

  9. Evaluating the Assignment of alkB Terminal Restriction Fragments and Sequence Types to Distinct Bacterial Taxa

    PubMed Central

    Giebler, Julia; Wick, Lukas Y.; Schloter, Michael; Harms, Hauke

    2013-01-01

    Sequence and terminal restriction fragment length polymorphism (T-RFLP) analyses revealed multiple alkB gene copies/cell in soil bacterial isolates and an apparently high genetic mobility among various phylogenetic groups. Identifying alkane degraders by alkB terminal restriction fragments (T-RFs) and sequences is strongly biased, as the phylogenetic trees based on 16S rRNA and alkB gene sequences were highly inconsistent. PMID:23455350

  10. Sulfur minimization in bacterial leaching

    SciTech Connect

    Seth, R.; Prasad, D.; Henry, J.G.

    1996-11-01

    The production of sewage biosolids in Ontario in 1989 was estimated to be 7 million m{sup 3} of wet sludge per year. Of this amount, land application accounts for between 20 and 30% of the total. Unfortunately, the use of sewage biosolids on agricultural land is often prohibited because of heavy metal contamination of the biosolids. High cost and operational problems have made chemical methods of metal extraction unattractive. Consequently, microbiological methods of leaching of heavy metals have been studied for over a decade. A relatively simple microbiological process has been investigated in recent years in flask level experiments and recently in a semicontinuous system. The process exploits nonacidophilic and acidophilic indigenous thiobacilli to extract heavy metals from sewage biosolids. These thiobacilli use elemental sulfur as the energy source, producing sulfuric acid. However, the resulting decontaminated biosolids can cause environmental problems like acidification of the soil, when acid is generated from the residual sulfur in the biosolids. The present study examines the possibility of reducing the amount of sulfur added in batch and semicontinuous bacterial leaching systems, and maximizing sulfur oxidation efficiency, thereby reducing the residual sulfur in leached biosolids.

  11. Biofuel components change the ecology of bacterial volatile petroleum hydrocarbon degradation in aerobic sandy soil.

    PubMed

    Elazhari-Ali, Abdulmagid; Singh, Arvind K; Davenport, Russell J; Head, Ian M; Werner, David

    2013-02-01

    We tested the hypothesis that the biodegradation of volatile petroleum hydrocarbons (VPHs) in aerobic sandy soil is affected by the blending with 10 percent ethanol (E10) or 20 percent biodiesel (B20). When inorganic nutrients were scarce, competition between biofuel and VPH degraders temporarily slowed monoaromatic hydrocarbon degradation. Ethanol had a bigger impact than biodiesel, reflecting the relative ease of ethanol compared to methyl ester biodegradation. Denaturing gradient gel electrophoresis (DGGE) of bacterial 16S rRNA genes revealed that each fuel mixture selected for a distinct bacterial community, each dominated by Pseudomonas spp. Despite lasting impacts on soil bacterial ecology, the overall effects on VHP biodegradation were minor, and average biomass yields were comparable between fuel types, ranging from 0.40 ± 0.16 to 0.51 ± 0.22 g of biomass carbon per gram of fuel carbon degraded. Inorganic nutrient availability had a greater impact on petroleum hydrocarbon biodegradation than fuel composition. PMID:23202642

  12. Bacterial species associated with sound and Botrytis-infected grapes from a Greek vineyard.

    PubMed

    Nisiotou, Aspasia A; Rantsiou, Kalliopi; Iliopoulos, Vassilios; Cocolin, Luca; Nychas, George-John E

    2011-02-28

    Grape bacterial microbiota plays central roles in the quality of grapes and wine, yet its diversity remains poorly described. In the present study, bacterial species associated with sound and Botrytis-infected grapes of two cultivars originating from the same vineyard were assessed. Isolates were identified by PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and sequence analysis of partial 16S rRNA gene. Comparable counts were recorded between Botrytis-infected and sound grape samples. In all cases, the majority of isolates belonged to different species of Enterobacteriaceae. The dominant species in the vineyard was Klebsiella oxytoca that was found in different combinations with Citrobacter freundii, Enterobacter spp., Erwinia sp., Pantoea dispersa, Tatumella ptyseos or other species. In fermenting musts, those populations declined while other species evolved, like Lactobacillus plantarum and Enterobacter ludwigii. Populations in botrytised samples persisted longer during spontaneous fermentations. Present study suggests that bacterial diversity on grapes may be wider than previously described. PMID:21315469

  13. Initial community and environment determine the response of bacterial communities to dispersant and oil contamination.

    PubMed

    Ortmann, Alice C; Lu, YueHan

    2015-01-15

    Bioremediation of seawater by natural bacterial communities is one potential response to coastal oil spills, but the success of the approach may vary, depending on geographical location, oil composition and the timing of spill. The short term response of coastal bacteria to dispersant, oil and dispersed oil was characterized using 16S rRNA gene tags in two mesocosm experiments conducted two months apart. Despite differences in the amount of oil-derived alkanes across the treatments and experiments, increases in the contributions of hydrocarbon degrading taxa and decreases in common estuarine bacteria were observed in response to dispersant and/or oil. Between the two experiments, the direction and rates of changes in particulate alkane concentrations differed, as did the magnitude of the bacterial response to oil and/or dispersant. Together, our data underscore large variability in bacterial responses to hydrocarbon pollutants, implying that bioremediation success varies with starting biological and environmental conditions. PMID:25487088

  14. A Cross-Taxon Analysis of Insect-Associated Bacterial Diversity

    PubMed Central

    Jones, Ryan Thomas; Sanchez, Leticia Gonzales; Fierer, Noah

    2013-01-01

    Although it is well known that plants and animals harbor microbial symbionts that can influence host traits, the factors regulating the structure of these microbial communities often remain largely undetermined. This is particularly true for insect-associated microbial communities, as few cross-taxon comparisons have been conducted to date. To address this knowledge gap and determine how host phylogeny and ecology affect insect-associated microbial communities, we collected 137 insect specimens representing 39 species, 28 families, and 8 orders, and characterized the bacterial communities associated with each specimen via 16S rRNA gene sequencing. Bacterial taxa within the phylum Proteobacteria were dominant in nearly all insects sampled. On average, the insect-associated bacterial communities were not very diverse, with individuals typically harboring fewer than 8 bacterial phylotypes. Bacterial communities also tended to be dominated by a single phylotype; on average, the most abundant phylotype represented 54.7% of community membership. Bacterial communities were significantly more similar among closely related insects than among less-related insects, a pattern driven by within-species community similarity but detected at every level of insect taxonomy tested. Diet was a poor predictor of bacterial community composition. Individual insect species harbored remarkably unique communities: the distribution of 69.0% of bacterial phylotypes was limited to unique insect species, whereas only 5.7% of phylotypes were detected in more than five insect species. Together these results suggest that host characteristics strongly regulate the colonization and assembly of bacterial communities across insect lineages, patterns that are driven either by co-evolution between insects and their symbionts or by closely related insects sharing conserved traits that directly select for similar bacterial communities. PMID:23613815

  15. Longitudinal Changes in the Bacterial Community Composition of the Danube River: a Whole-River Approach? †

    PubMed Central

    Winter, Christian; Hein, Thomas; Kavka, Gerhard; Mach, Robert L.; Farnleitner, Andreas H.

    2007-01-01

    The Danube River is the second longest river in Europe, and its bacterial community composition has never been studied before over its entire length. In this study, bacterial community composition was determined by denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified portions of the bacterial 16S rRNA gene from a total of 98 stations on the Danube River (73 stations) and its major tributaries (25 stations), covering a distance of 2,581 km. Shifts in the bacterial community composition were related to changes in environmental conditions found by comparison with physicochemical parameters (e.g., temperature and concentration of nutrients) and the concentration of chlorophyll a (Chl a). In total, 43 distinct DGGE bands were detected. Sequencing of selected bands revealed that the phylotypes were associated with typical freshwater bacteria. Apparent bacterial richness in the Danube varied between 18 and 32 bands and correlated positively with the concentration of P-PO4 (r = 0.56) and negatively with Chl a (r = ?0.52). An artificial neural network-based model explained 90% of the variation of apparent bacterial richness using the concentrations of N-NO2 and P-PO4 and the distance to the Black Sea as input parameters. Between the cities of Budapest and Belgrade, apparent bacterial richness was significantly lower than that of other regions of the river, and Chl a showed a pronounced peak. Generally, the bacterial community composition developed gradually; however, an abrupt and clear shift was detected in the section of the phytoplankton bloom. Large impoundments did not have a discernible effect on the bacterial community of the water column. In conclusion, the riverine bacterial community was largely influenced by intrinsic factors. PMID:17085708

  16. Bop1 Is a Mouse WD40 Repeat Nucleolar Protein Involved in 28S and 5.8S rRNA Processing and 60S Ribosome Biogenesis

    PubMed Central

    Strezoska, Žaklina; Pestov, Dimitri G.; Lau, Lester F.

    2000-01-01

    We have identified and characterized a novel mouse protein, Bop1, which contains WD40 repeats and is highly conserved through evolution. bop1 is ubiquitously expressed in all mouse tissues examined and is upregulated during mid-G1 in serum-stimulated fibroblasts. Immunofluorescence analysis shows that Bop1 is localized predominantly to the nucleolus. In sucrose density gradients, Bop1 from nuclear extracts cosediments with the 50S-80S ribonucleoprotein particles that contain the 32S rRNA precursor. RNase A treatment disrupts these particles and releases Bop1 into a low-molecular-weight fraction. A mutant form of Bop1, Bop1?, which lacks 231 amino acids in the N- terminus, is colocalized with wild-type Bop1 in the nucleolus and in ribonucleoprotein complexes. Expression of Bop1? leads to cell growth arrest in the G1 phase and results in a specific inhibition of the synthesis of the 28S and 5.8S rRNAs without affecting 18S rRNA formation. Pulse-chase analyses show that Bop1? expression results in a partial inhibition in the conversion of the 36S to the 32S pre-rRNA and a complete inhibition of the processing of the 32S pre-rRNA to form the mature 28S and 5.8S rRNAs. Concomitant with these defects in rRNA processing, expression of Bop1? in mouse cells leads to a deficit in the cytosolic 60S ribosomal subunits. These studies thus identify Bop1 as a novel, nonribosomal mammalian protein that plays a key role in the formation of the mature 28S and 5.8S rRNAs and in the biogenesis of the 60S ribosomal subunit. PMID:10891491

  17. Ribosome origins: The relative age of 23S rRNA Domains

    NASA Astrophysics Data System (ADS)

    Hury, James; Nagaswamy, Uma; Larios-Sanz, Maia; Fox, George E.

    2006-08-01

    The modern ribosome and its component RNAs are quite large and it is likely that at an earlier time they were much smaller. Hence, not all regions of the modern ribosomal RNAs (rRNA) are likely to be equally old. In the work described here, it is hypothesized that the oldest regions of the RNAs will usually be highly integrated into the machinery. When this is the case, an examination of the interconnectivity between local RNA regions can provide insight to the relative age of the various regions. Herein, we describe an analysis of all known long-range RNA/RNA interactions within the 23S rRNA and between the 23S rRNA and the 16S rRNA in order to assess the interconnectivity between the usual Domains as defined by secondary structure. Domain V, which contains the peptidyl transferase center is centrally located, extensively connected, and therefore likely to be the oldest region. Domain IV and Domain II are extensively interconnected with both themselves and Domain V. A portion of Domain IV is also extensively connected with the 30S subunit and hence Domain IV may be older than Domain II. These results are consistent with other evidence relating to the relative age of RNA regions. Although the relative time of addition of the GTPase center can not be reliably deduced it is pointed out that the development of this may have dramatically affected the progenotes that preceded the last common ancestor.

  18. Spt6 Is Essential for rRNA Synthesis by RNA Polymerase I.

    PubMed

    Engel, Krysta L; French, Sarah L; Viktorovskaya, Olga V; Beyer, Ann L; Schneider, David A

    2015-07-01

    Spt6 (suppressor of Ty6) has many roles in transcription initiation and elongation by RNA polymerase (Pol) II. These effects are mediated through interactions with histones, transcription factors, and the RNA polymerase. Two lines of evidence suggest that Spt6 also plays a role in rRNA synthesis. First, Spt6 physically associates with a Pol I subunit (Rpa43). Second, Spt6 interacts physically and genetically with Spt4/5, which directly affects Pol I transcription. Utilizing a temperature-sensitive allele, spt6-1004, we show that Spt6 is essential for Pol I occupancy of the ribosomal DNA (rDNA) and rRNA synthesis. Our data demonstrate that protein levels of an essential Pol I initiation factor, Rrn3, are reduced when Spt6 is inactivated, leading to low levels of Pol I-Rrn3 complex. Overexpression of RRN3 rescues Pol I-Rrn3 complex formation; however, rRNA synthesis is not restored. These data suggest that Spt6 is involved in either recruiting the Pol I-Rrn3 complex to the rDNA or stabilizing the preinitiation complex. The findings presented here identify an unexpected, essential role for Spt6 in synthesis of rRNA. PMID:25918242

  19. Peculiar feature of the organization of rRNA genes of the Chlorella chloroplast DNA.

    PubMed Central

    Yamada, T; Shimaji, M

    1986-01-01

    The organization of a cloned rRNA gene cluster from Chlorella ellipsoidea chloroplast DNA (cpDNA) has been analyzed. Southern hybridization experiments with labelled chloroplast rRNAs as probes revealed an extraordinarily large size of the 16S-23S rRNA spacer region, ca. 4.8 kbp, almost twice as large as those of most higher plants. The nucleotide sequence determined on this region has shown that: (1) The tRNAIle gene locating in this region is similar to those of higher plant chloroplasts, blue-green algae and E. coli but does not contain any introns in contrast to higher plant chloroplasts. (2) The tRNAAla gene is absent from this region. (3) There are four open reading frames (ORFs) coding for 55, 102, 107 and 110 amino acids, respectively. (4) A few sets of unique sequence were found repeatedly in this region. (5) The 23S rRNA gene is coded on the opposite strand in the reverse order. This arrangement of the 16S-23S rRNA region of Chlorella cpDNA is quite different from any of those reported so far for various organisms. Images PMID:3714498

  20. Complex Patterns of Plastid 16S rRNA Gene Evolution in Nonphotosynthetic Green Algae

    E-print Network

    Nedelcu, Aurora M.

    Complex Patterns of Plastid 16S rRNA Gene Evolution in Nonphotosynthetic Green Algae Aurora M AT values are increased in nonphotosynthetic green algae compared to their closest photosynthetic relatives "green plants," both land plants and green algae, are known. Among such lineages are the parasitic

  1. Diagnostic Accuracy of Presepsin (sCD14-ST) for Prediction of Bacterial Infection in Cerebrospinal Fluid Samples from Children with Suspected Bacterial Meningitis or Ventriculitis

    PubMed Central

    Stubljar, David; Kopitar, Andreja Natasa; Groselj-Grenc, Mojca; Suhadolc, Kristina; Fabjan, Teja

    2015-01-01

    Children with temporary external ventricular drains (EVD) are prone to nosocomial infections. Diagnosis of bacterial meningitis and ventriculitis in these children is challenging due to frequent blood contamination of cerebrospinal fluid (CSF) and the presence of chemical ventriculitis. The aim of this study was to compare diagnostic accuracy of presepsin (sCD14-ST), a novel biomarker of bacterial infection in CSF, to predict bacterial infection in comparison to the accuracy of established biomarkers like those demonstrated in biochemical analysis of CSF. We conducted a prospective study with 18 children with suspected bacterial meningitis or ventriculitis who had 66 episodes of disease. CSF samples were taken from external ventricular drainage. We measured presepsin in CSF, as well as CSF leukocyte count, glucose, and proteins. CSF was also taken to prove bacterial infection with culture methods or with 16S rRNA gene broad-range PCR (SepsiTest; Molzym, Germany). Infection was clinically confirmed in 57 (86%) episodes of suspected meningitis or ventriculitis. Chemical ventriculitis was diagnosed in 9 (14%) episodes of suspected meningitis or ventriculitis. Diagnostic accuracies presented as area under the curve (AUC) for sCD14-ST, leukocytes, and proteins measured in CSF were 0.877 (95% confidence interval [CI], 0.793 to 0.961), 0.798 (95% CI, 0.677 to 0.920), and 0.857 (95% CI, 0.749 to 0.964), respectively. With CSF culture, we detected bacteria in 17 samples, compared to 37 detected with broad-range PCR. It was found that presepsin was present at a significantly higher level in children with clinically proven ventriculitis than in those without meningitis or ventriculitis. Diagnostic accuracies of presepsin were superior to those of leukocytes or proteins in CSF. Presepsin-guided 16S rRNA gene PCR could be used in everyday clinical practice to improve etiological diagnosis of meningitis and ventriculitis and to prescribe more appropriate antibiotics. PMID:25653398

  2. Active Marine Subsurface Bacterial Population Composition in Low Organic Carbon Environments from IODP Expedition 320

    NASA Astrophysics Data System (ADS)

    Shepard, A.; Reese, B. K.; Mills, H. J.; IODP Expedition 320 Shipboard Science Party

    2011-12-01

    The marine subsurface environment contains abundant and active microorganisms. These microbial populations are considered integral players in the marine subsurface biogeochemical system with significance in global geochemical cycles and reservoirs. However, variations in microbial community structure, activity and function associated with the wide-ranging sedimentary and geochemical environments found globally have not been fully resolved. Integrated Ocean Drilling Program Expedition 320 recovered sediments from site U1332. Two sampling depths were selected for analysis that spanned differing lithological units in the sediment core. Sediments were composed of mostly clay with zeolite minerals at 8 meters below sea floor (mbsf). At 27 mbsf, sediments were composed of alternating clayey radiolarian ooze and nannofossil ooze. The concentration of SO42- had little variability throughout the core and the concentration of Fe2+ remained close to, or below, detection limits (0.4 ?M). Total organic carbon content ranged from a low of 0.03 wt% to a high of 0.07 wt% between 6 and 30 mbsf providing an opportunity to evaluate marine subsurface microbial communities under extreme electron donor limiting conditions. The metabolically active fraction of the bacterial population was isolated by the extraction and amplification of 16S ribosomal RNA. Pyrosequencing of 16S rRNA transcripts and subsequent bioinformatic analyses provided a robust data set (15,931 total classified sequences) to characterize the community at a high resolution. As observed in other subsurface environments, the overall diversity of active bacterial populations decreased with depth. The population shifted from a diverse but evenly distributed community at approximately 8 mbsf to a Firmicutes dominated population at 27 mbsf (80% of sequences). A total of 95% of the sequences at 27 mbsf were grouped into three genera: Lactobacillus (phylum Firmicutes) at 80% of the total sequences, Marinobacter (phylum Proteobacteria) at 8%, and Formosa (phylum Bacteroidetes) at 7%. These lineages support a paradigm suggesting the importance of fermentation in the subsurface. However, this study extends the predicted range for fermentation below the shallow subsurface and into organic carbon limited marine sediments. Other previously characterized subsurface active populations from environments with higher organic carbon concentrations do not show similar levels of reduced diversity or predominance of fermentative populations. This study further emphasizes the spatial variability of microbial populations in the deep subsurface and highlights the need for continued exploration.

  3. Methanotrophic activity and bacterial diversity in volcanic-geothermal soils at Pantelleria island (Italy)

    NASA Astrophysics Data System (ADS)

    Gagliano, A. L.; D'Alessandro, W.; Tagliavia, M.; Parello, F.; Quatrini, P.

    2014-04-01

    Volcanic and geothermal systems emit endogenous gases by widespread degassing from soils, including CH4, a greenhouse gas twenty-five times as potent as CO2. Recently, it has been demonstrated that volcanic/geothermal soils are source of methane, but also sites of methanotrophic activity. Methanotrophs are able to consume 10-40 Tg of CH4 a-1 and to trap more than 50% of the methane degassing through the soils. We report on methane microbial oxidation in the geothermally most active site of Pantelleria island (Italy), Favara Grande, whose total methane emission was previously estimated in about 2.5 t a-1. Laboratory incubation experiments with three top-soil samples from Favara Grande indicated methane consumption values up to 950 ng g-1 dry soil h-1. One of the three sites, FAV2, where the highest oxidation rate was detected, was further analysed on a vertical soil profile and the maximum methane consumption was measured in the top-soil layer but values > 100 ng g-1 h-1 were maintained up to a depth of 15 cm. The highest consumption rate was measured at 37 °C, but a still recognizable consumption at 80 °C (> 20 ng g-1 h-1) was recorded. In order to estimate the bacterial diversity, total soil DNA was extracted from Favara Grande and analysed using a Temporal Temperature Gradient gel Electrophoresis (TTGE) analysis of the amplified bacterial 16S rRNA gene. The three soil samples were probed by PCR using standard proteobacterial primers and newly designed verrucomicrobial primers targeting the unique methane monooxygenase gene pmoA; the presence of methanotrophs was detected in sites FAV2 and FAV3, but not in FAV1, where harsher chemical-physical conditions and negligible methane oxidation were detected. The pmoA gene libraries from the most active site FAV2 pointed out a high diversity of gammaproteobacterial methanotrophs distantly related to Methylococcus/Methylothermus genera and the presence of the newly discovered acido-thermophilic methanotrophs Verrucomicrobia. Alphaproteobacteria of the genus Methylocystis were isolated from enrichment cultures, under a methane containing atmosphere at 37 °C. The isolates grow at pH 3.5-8 and temperatures of 18-45 °C, and show a methane oxidation rate of ~ 450 ?mol mol-1 h-1. Soils from Favara Grande showed the largest diversity of methanotrophic bacteria until now detected in a geothermal soil. While methanotrophic Verrucomicrobia are reported to dominate highly acidic geothermal sites, our results suggest that slightly acidic soils, in high enthalpy geothermal systems, host a more diverse group of both culturable and uncultivated methanotrophs.

  4. The role of bacterial biomineralization on the pedogenesis of magnetic Apennines paleosols

    NASA Astrophysics Data System (ADS)

    Mocali, Stefano; Chiellini, Carolina; Cardelli, Valeria; Cocco, Stefania; Corti, Giuse