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Sample records for 16s rrna-encoding reca

  1. Isolation of endophytic bacteria from arboreal species of the Amazon and identification by sequencing of the 16S rRNA encoding gene

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    Mariza M. Coêlho

    2011-01-01

    Full Text Available Endophytic bacteria from three arboreal species native to the Amazon (Carapa guianenses, Ceiba pentandra, and Swietenia macrophylla, were isolated and identified, through partial sequencing of the 16S rRNA encoding gene. From these, 16 isolates were obtained, although, when compared to sequences deposited in GenBank, only seven had produced identifiable fragments. Bacillus, Pantoea and two non-culturable samples were identified. Results obtained through sequence analysis revealed low genetic diversity across the isolates, even when analyzing different species and plant structures. This is the first report concerning the isolation and identification of endophytic bacteria in these plant species.

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

    Science.gov (United States)

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

    2016-03-01

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

  3. Rhizobia with 16S rRNA and nifH similar to Mesorhizobium huakuii but Novel recA, glnII, nodA and nodC genes are symbionts of New Zealand Carmichaelinae.

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    Heng Wee Tan

    Full Text Available New Zealand became geographically isolated about 80 million years ago and this separation gave rise to a unique native flora including four genera of legume, Carmichaelia, Clianthus and Montigena in the Carmichaelinae clade, tribe Galegeae, and Sophora, tribe Sophoreae, sub-family Papilionoideae. Ten bacterial strains isolated from NZ Carmichaelinae growing in natural ecosystems grouped close to the Mesorhizobium huakuii type strain in relation to their 16S rRNA and nifH gene sequences. However, the ten strains separated into four groups on the basis of their recA and glnII sequences: all groups were clearly distinct from all Mesorhizobium type strains. The ten strains separated into two groups on the basis of their nodA sequences but grouped closely together in relation to nodC sequences; all nodA and nodC sequences were novel. Seven strains selected and the M. huakuii type strain (isolated from Astragalus sinicus produced functional nodules on Carmichaelia spp., Clianthus puniceus and A. sinicus but did not nodulate two Sophora species. We conclude that rhizobia closely related to M. huakuii on the basis of 16S rRNA and nifH gene sequences, but with variable recA and glnII genes and novel nodA and nodC genes, are common symbionts of NZ Carmichaelinae.

  4. Identification of Lactic Acid Bacteria Ⅱ 32 by Sequence Analysis of 16S rDNA and RecA - gene%16S rDNA和recA-gene对乳酸菌Ⅱ32的鉴定

    Institute of Scientific and Technical Information of China (English)

    刘长建; 权春善; 范圣第

    2007-01-01

    对乳酸菌Ⅱ32进行了生化实验.以菌株Ⅱ32的总DNA为模板,采用细菌通用的引物,对其16S rDNA进行特异扩增,并进行序列测定,将测定结果与GenBank DNA数据库中已知菌种的16S rDNA序列通过BLAST软件进行分析比较,初步确定该菌株为戊糖乳酸菌、植物乳杆菌或类植物乳杆菌.采用recA-gene约300bp的特异扩增片段最终确定乳酸菌Ⅱ32为类植物乳杆菌.

  5. Molecular origin analysis of Shigella boydii Str. hn03 isolated from chicken based on 16S rRNA,gyrB,grpE and recA genes%16S rRNA基因联合gyrB、grpE、recA基因对鸡源鲍氏志贺菌hn03株进行分子起源分析

    Institute of Scientific and Technical Information of China (English)

    杨霞; 陈陆; 赵军; 王新卫; 常洪涛; 刘红英; 姚慧霞; 王川庆

    2011-01-01

    The sequences of 16S rDNA, gyrB, grpE, recA of Shigella were analysed for the farther identification of newly found Shigella boydii strain isolated from chicken and the evolutionary position of the new strain was determined. Part of 16S rRNA and other three genes were amplified by polymerase chain reaction (PCR) from one identified Shigella boydii strains isolated from chicken and the PCR products were cloned and sequenced. These sequences obtained in this study were compared with the related sequneces reported in GenBank by software of DNA analyses. The similarity were gained and phylogenetic tree were constructed. Further evolutionary relationships among chicken Shigella boydii and other related strains were examined based on sequences. Shigella boydii isolated from chicken had the nearest homogeneity with Shigella boydii isolated from human and their homogeneity is 99.6 %-100 %. Shigella boydii isolated from chicken was in a small cluster with Shigella boydii isolated from human in four phylogenetic trees,and almost all Shigella strains are distributed among E. coli strains. Genes recA and gyrB shown a greater evolutionary divergence than 16S rRNA genes. Sequence analysis of 16S rDNA and gyrB,grpE, recA may be a reliable, rapid and low costs way for identification of Shigella strains. The homogeneity and the phylogenetic tree constructed by these four genes above revealed that the Shigella strain isolated from chicken may be a new species or subspecies of Shigella boydii.%为了从基因水平鉴定鸡源鲍氏志贺菌,分别根据GenBank中登陆的相应序列针对16srRNA、gyrB、grpE、recA4基因设计4对引物,用PCR扩增出鸡源鲍氏志贺菌4基因的部分片段并进行了克隆测序。将所获序列与GenBank中同源性较高的相关序列相比较,计算种间相似性,并构建志贺菌的系统发育树,对分离株进行分类与鉴定。研究发现,从4基因的序列分析结果总体上可知鸡

  6. RecA: A NOVEL TARGET FOR DRUG DELIVERY

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    Ramani Gade

    2013-04-01

    Full Text Available The RecA protein is a recombinase functioning in recombinational DNA repair in bacteria. RecA is regulated at many levels. The expression of the recA gene is regulated within the SOS response. The activity of the RecA protein itself is autoregulated by its own C-terminus. RecA responsible for the development of resistance by the microbes against antibiotic. Development of new medication against RecA focus light on the future newer antibiotics in the phrma sector.

  7. Regulation of bacterial RecA protein function.

    Science.gov (United States)

    Cox, Michael M

    2007-01-01

    The RecA protein is a recombinase functioning in recombinational DNA repair in bacteria. RecA is regulated at many levels. The expression of the recA gene is regulated within the SOS response. The activity of the RecA protein itself is autoregulated by its own C-terminus. RecA is also regulated by the action of other proteins. To date, these include the RecF, RecO, RecR, DinI, RecX, RdgC, PsiB, and UvrD proteins. The SSB protein also indirectly affects RecA function by competing for ssDNA binding sites. The RecO and RecR, and possibly the RecF proteins, all facilitate RecA loading onto SSB-coated ssDNA. The RecX protein blocks RecA filament extension, and may have other effects on RecA activity. The DinI protein stabilizes RecA filaments. The RdgC protein binds to dsDNA and blocks RecA access to dsDNA. The PsiB protein, encoded by F plasmids, is uncharacterized, but may inhibit RecA in some manner. The UvrD helicase removes RecA filaments from RecA. All of these proteins function in a network that determines where and how RecA functions. Additional regulatory proteins may remain to be discovered. The elaborate regulatory pattern is likely to be reprised for RecA homologues in archaeans and eukaryotes. PMID:17364684

  8. Isolation of the Helicobacter pylori recA gene and involvement of the recA region in resistance to low pH.

    OpenAIRE

    Thompson, S A; Blaser, M J

    1995-01-01

    To understand the potential roles of the important DNA repair protein RecA in Helicobacter pylori pathogenesis, we cloned the recA gene from H. pylori 84-183. Degenerate PCR primers based on conserved RecA protein regions were used to amplify a portion of H. pylori recA, which was used as a probe to isolate the full-length recA gene from H. pylori genomic libraries. The H. pylori recA gene encoded a protein of 347 amino acids with a molecular mass of 37.6 kDa. As expected, H. pylori RecA was ...

  9. Active displacement of RecA filaments by UvrD translocase activity

    OpenAIRE

    Petrova, Vessela; Chen, Stefanie H.; Molzberger, Eileen T.; Tomko, Eric; Chitteni-Pattu, Sindhu; Jia, Haifeng; Ordabayev, Yerdos; Lohman, Timothy M.; Cox, Michael M.

    2015-01-01

    The UvrD helicase has been implicated in the disassembly of RecA nucleoprotein filaments in vivo and in vitro. We demonstrate that UvrD utilizes an active mechanism to remove RecA from the DNA. Efficient RecA removal depends on the availability of DNA binding sites for UvrD and/or the accessibility of the RecA filament ends. The removal of RecA from DNA also requires ATP hydrolysis by the UvrD helicase but not by RecA protein. The RecA-removal activity of UvrD is slowed by RecA variants with ...

  10. 哈维弧菌16S rRNA基因拷贝数的种内变异%Intra-species variation of 16S rRNA gene copy number of Vibrio harveyi

    Institute of Scientific and Technical Information of China (English)

    郑嘉来; 阎永伟; 唐姝; 杨坤杰; 李长红; 王凯; 张德民

    2015-01-01

    利用荧光定量PCR法测定哈维弧菌( Vibrio harveyi)基因组内16S rRNA基因拷贝数. 基于16S rDNA、pyrH、rpoA和recA4个基因的系统发育学分析鉴定弧菌菌株,然后通过重叠PCR构建包含哈维弧菌模式菌株16S rD-NA和gyrB基因片段的标准质粒,建立了定量PCR测定细胞内16S rRNA基因拷贝数方法,并测定了6株测试菌株和哈维弧菌模式株的16S rRNA基因拷贝数,同时以已知拷贝数的菌株ATCC BAA-1116做对照. 6个测试菌株均为哈维弧菌. 菌株ATCC BAA-1116的16S rRNA基因拷贝数为11个,与已知值相符. 模式菌株MCCC 1H00031T和6个测试菌株SXB-C、SCB-4、NBV00029、NBV00035、NBV00039和NBV00269的拷贝数分别为9、12、13、12、11、13和9个. 利用荧光定量PCR法测定哈维弧菌基因组内16S rRNA基因拷贝数的方法简单可行. 哈维弧菌16S rRNA基因拷贝数的种内变异可能是其适应近岸多变生境的原因之一.%This study aims to determine intra-genomic 16S rRNA gene copy number of Vibrio harveyi strains by a method of fluorescent quantitative PCR. Vibrio strains were identified by multilocussequence analysis based on 16S rDNA, pyrH, rpoA and recA. A standard plasmid, harboring fragments of 16S rDNA and the single copy gene gyrB from V. harveyi type strain, was constructed. A fluorescent quantitative method was established and used to determine the intra-genomic 16S rRNA gene copy number of the tested 6 strains, taken the type strains of V. harveyi and V. harvei ATCC BAA-1116 as reference, whose 16S rRNA gene copy number is well known. All the 6 tested Vibrio strains were identified as V. harveyi. The copy number of 16S rRNA gene in strain ATCC BAA-1116 was 11, consistent with the known values. The copy number of strain MCCC 1H00031T, SXB-C, SCB-4, NBV00029, NBV00035, NBV00039 and NBV00269 was 9, 12, 13, 12, 11, 13 and 9, respectively. Fluorescent quantitative PCR method is simple and reliable for determi-ning 16S rRNA gene copy number

  11. The Walker A motif mutation recA4159 abolishes the SOS response and recombination in a recA730 mutant of Escherichia coli.

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    Šimatović, Ana; Mitrikeski, Petar T; Vlašić, Ignacija; Sopta, Mary; Brčić-Kostić, Krunoslav

    2016-01-01

    In bacteria, the RecA protein forms recombinogenic filaments required for the SOS response and DNA recombination. In order to form a recombinogenic filament, wild type RecA needs to bind ATP and to interact with mediator proteins. The RecA730 protein is a mutant version of RecA with superior catalytic abilities, allowing filament formation without the help of mediator proteins. The mechanism of RecA730 filament formation is not well understood, and the question remains as to whether the RecA730 protein requires ATP binding in order to become competent for filament formation. We examined two mutants, recA730,4159 (presumed to be defective for ATP binding) and recA730,2201 (defective for ATP hydrolysis), and show that they have different properties with respect to SOS induction, conjugational recombination and double-strand break repair. We show that ATP binding is essential for all RecA730 functions, while ATP hydrolysis is required only for double-strand break repair. Our results emphasize the similarity of the SOS response and conjugational recombination, neither of which requires ATP hydrolysis by RecA730.

  12. La Palabra de Lucio Reca: El Sector Agropecuario

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    Marcelo Coser

    2007-12-01

    Full Text Available Marcelo Coser entrevista al Profesor Lucio Reca en el marco del programa“Prisma Económico” del Instituto de Economía y Finanzas (IEF de la UNC (Entrevista desarrollada el 30 de abril de 2008

  13. Phylogeny of Vibrio cholerae Based on recA Sequence

    OpenAIRE

    Stine, O. Colin; Sozhamannan, Shanmuga; Gou, Qing; Zheng, Siqen; Morris, J. Glenn; Johnson, Judith A.

    2000-01-01

    We sequenced a 705-bp fragment of the recA gene from 113 Vibrio cholerae strains and closely related species. One hundred eighty-seven nucleotides were phylogenetically informative, 55 were phylogenetically uninformative, and 463 were invariant. Not unexpectedly, Vibrio parahaemolyticus and Vibrio vulnificus strains formed out-groups; we also identified isolates which resembled V. cholerae biochemically but which did not cluster with V. cholerae. In many instances, V. cholerae serogroup desig...

  14. Suppression of constitutive SOS expression by recA4162 (I298V) and recA4164 (L126V) requires UvrD and RecX in Escherichia coli K-12.

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    Long, Jarukit E; Renzette, Nicholas; Sandler, Steven J

    2009-07-01

    Sensing DNA damage and initiation of genetic responses to repair DNA damage are critical to cell survival. In Escherichia coli, RecA polymerizes on ssDNA produced by DNA damage creating a RecA-DNA filament that interacts with the LexA repressor inducing the SOS response. RecA filament stability is negatively modulated by RecX and UvrD. recA730 (E38K) and recA4142 (F217Y) constitutively express the SOS response. recA4162 (I298V) and recA4164 (L126V) are intragenic suppressors of the constitutive SOS phenotype of recA730. Herein, it is shown that these suppressors are not allele specific and can suppress SOS(C) expression of recA730 and recA4142 in cis and in trans. recA4162 and recA4164 single mutants (and the recA730 and recA4142 derivatives) are Rec(+), UV(R) and are able to induce the SOS response after UV treatment like wild-type. UvrD and RecX are required for the suppression in two (recA730,4164 and recA4142,4162) of the four double mutants tested. To explain the data, one model suggests that recA(C) alleles promote SOS(C) expression by mimicking RecA filament structures that induce SOS and the suppressor alleles mimic RecA filament at end of SOS. UvrD and RecX are attracted to these latter structures to help dismantle or destabilize the RecA filament. PMID:19555451

  15. Suppression of Constitutive SOS Expression by recA4162 (I298V) and recA4164 (L126V) requires UvrD and RecX in Escherichia coli K-12

    OpenAIRE

    Long, Jarukit Edward; Renzette, Nicholas; Sandler, Steven J.

    2009-01-01

    Sensing DNA damage and initiation of genetic responses to repair DNA damage are critical to cell survival. In E. coli, RecA polymerizes on ssDNA produced by DNA damage creating a RecA-DNA filament that interacts with the LexA repressor inducing the SOS Response. RecA filament stability is negatively modulated by RecX and UvrD. recA730 (E38K) and recA4142 (F217Y) constitutively express the SOS Response. recA4162 (I298V) and recA4164 (L126V) are intragenic suppressors of the constitutive SOS ph...

  16. Active displacement of RecA filaments by UvrD translocase activity.

    Science.gov (United States)

    Petrova, Vessela; Chen, Stefanie H; Molzberger, Eileen T; Tomko, Eric; Chitteni-Pattu, Sindhu; Jia, Haifeng; Ordabayev, Yerdos; Lohman, Timothy M; Cox, Michael M

    2015-04-30

    The UvrD helicase has been implicated in the disassembly of RecA nucleoprotein filaments in vivo and in vitro. We demonstrate that UvrD utilizes an active mechanism to remove RecA from the DNA. Efficient RecA removal depends on the availability of DNA binding sites for UvrD and/or the accessibility of the RecA filament ends. The removal of RecA from DNA also requires ATP hydrolysis by the UvrD helicase but not by RecA protein. The RecA-removal activity of UvrD is slowed by RecA variants with enhanced DNA-binding properties. The ATPase rate of UvrD during RecA removal is much slower than the ATPase activity of UvrD when it is functioning either as a translocase or a helicase on DNA in the absence of RecA. Thus, in this context UvrD may operate in a specialized disassembly mode. PMID:25824953

  17. Characterization of the Pseudomonas aeruginosa recA analog and its protein product: rec-102 is a mutant allele of the P. aeruginosa PAO recA gene

    International Nuclear Information System (INIS)

    We cloned a 2.3-kilobase-pair fragment of the Pseudomonas aeruginosa PAO chromosome which is capable of complementing recA mutations of Escherichia coli. The recA-complementing activity was further localized to a 1.5-kilobase-pair PvuII-HindIII fragment. Southern blot analysis under conditions of high stringency indicated that DNA sequence homology is shared by the E. coli recA gene and the P. aeruginosa recA analog. The cloned recA analog was shown to restore resistance to methyl methanesulfonate, nitrofurantoin, and UV irradiation to E. coli recA mutants. Upon introduction of the cloned P. aeruginosa gene, these mutants regained recombination proficiency in HfrH-mediated conjugation and the ability to induce lambda prophages and SOS functions (din gene transcription) after exposure to DNA-damaging agents. Lambda prophage carrying a cI ind mutation was not inducible, suggesting that the mechanism of induction of these SOS functions by the P. aeruginosa RecA analog is similar to that by the activated E. coli RecA protein. The product of the recA analog was identified in minicells as a protein of approximately 47,000 daltons. Western blot analysis using anti-E. coli RecA antibody demonstrated that this protein is antigenically cross-reactive with the E. coli recA protein. The recA-containing fragment was cloned into the broad-host-range vector pCP13 and introduced into Rec- strains of P. aeruginosa containing the rec-102 allele. The plasmid was shown to restore recombination proficiency in FP5-mediated conjugations and to restore resistance to UV irradiation and methyl methanesulfonate to these Rec- mutants. It was shown that a wild-type allele of rec-102 is necessary for UV-mediated induction of D3 and F116 prophages. The cloned recA analog restored the UV inducibility of these prophages in rec-102 mutants

  18. Specificity in suppression of SOS expression by recA4162 and uvrD303

    OpenAIRE

    Massoni, Shawn C.; Sandler, Steven J.

    2013-01-01

    Detection and repair of DNA damage is essential in all organisms and depends on the ability of proteins recognizing and processing specific DNA substrates. In E. coli, the RecA protein forms a filament on single-stranded DNA (ssDNA) produced by DNA damage and induces the SOS response. Previous work has shown that one type of recA mutation (e.g., recA4162 (I298V)) and one type of uvrD mutation (e.g., uvrD303 (D403A, D404A)) can differentially decrease SOS expression depending on the type of in...

  19. Autogenous regulation and kinetics of induction of Pseudomonas aeruginosa recA transcription as analyzed with operon fusions

    International Nuclear Information System (INIS)

    A promoterless chloramphenicol acetyltransferase gene (cat) was used to construct recA-cat operon fusions to quantitatively examine the transcriptional regulation of the Pseudomonas aeruginosa recA gene in P. aeruginosa PAO. Wild-type P. aeruginosa containing the recA8-cat fusion was treated with methyl methanesulfonate (MMS) and showed immediate induction of chloramphenicol acetyltransferase (CAT) specific activity, whereas a recA::Tn501 mutant of P. aeruginosa containing recA8-cat showed no induction with MMS. This indicated that a functional copy of recA was required for derepression of recA transcription and that P. aeruginosa recA protein was a positive regulatory factor promoting its own expression. Compared with that in the wild type, the uninduced level of CAT in recA8-cat-containing cells was reduced by approximately one-half in the recA::Tn501 mutant, indicating that recA+-dependent spontaneous induction contributes to the uninduced levels of recA expression in P. aeruginosa. MMS (0.012%) caused recA-directed CAT synthesis to increase almost immediately, with maximum CAT activity, fourfold higher than uninduced levels, attained at 60 min postinduction. The kinetics of recA8-cat fusion activity were shown to be directly related to the MMS doses used. Another fusion called recAa1-cat, where cat was located between the two transcriptional terminators of the P. aeruginosa recA gene, also showed dose-dependent induction by MMS, but the CAT activity from recAa1-cat was only one-half of that obtained with recA8-cat under the same conditions. Treatment of recA+ P. aeruginosa containing recA8-cat with UV irradiation produced an immediate effect on recA8-cat transcription and showed little UV dose dependency at doses of 5 J/m2 or greater

  20. Accuracy of Conventional PCR Targeting the 16S rRNA Gene with the Ot-16sRF1 and Ot-16sRR1 Primers for Diagnosis of Scrub Typhus: a Case-Control Study.

    Science.gov (United States)

    Kim, Choon-Mee; Cho, Min Keun; Kim, Dong-Min; Yun, Na-Ra; Kim, Seok Won; Jang, Sook Jin; Ahn, Young-Joon; Lim, Donghoon

    2016-01-01

    We retrospectively evaluated the accuracy of conventional PCR targeting the 16S rRNA gene (16S C-PCR) using the Ot-16sRF1/Ot-16sRR1 primers for diagnosing scrub typhus. The diagnosis of Orientia tsutsugamushi infection by 16S C-PCR presented an increased sensitivity of 87.0% and specificity of 100% compared with those obtained with other targets and is thus a simple and clinically useful method with good diagnostic accuracy.

  1. The RecA Protein of Helicobacter pylori Requires a Posttranslational Modification for Full Activity

    OpenAIRE

    Fischer, Wolfgang; Haas, Rainer

    2004-01-01

    The RecA protein is a central component of the homologous recombination machinery and of the SOS system in most bacteria. In performing these functions, it is involved in DNA repair processes and plays an important role in natural transformation competence. This may be especially important in Helicobacter pylori, where an unusually high degree of microdiversity among strains is generated by homologous recombination. We have suggested previously that the H. pylori RecA protein is subject to po...

  2. Enhanced radiative Auger emission from lithiumlike 16S13+

    International Nuclear Information System (INIS)

    The radiative Auger emission (RAE) from 0.94--6.25-MeV/u 16S13+ (lithiumlike) projectiles excited in collisions with He target atoms has been measured. For these highly stripped ions the intensity of RAE photons relative to Kα x-ray emission is enhanced by about a factor of five compared with theoretical calculations and an earlier experimental measurement for S ions with few electron vacancies. The enhancement of RAE for S13+ is qualitatively similar to results reported previously for lithiumlike 23V20+; however, some differences between S and V are evident

  3. The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

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    C.W. Galvão

    2012-12-01

    Full Text Available DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs can interact with the H. seropedicaeRecA protein (RecA Hs and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA Hs. RecA Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX Hs was present before the formation of RecA filaments (RecA-ssDNA, inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX Hs protein negatively modulates the RecA Hs activities by protein-protein interactions and also by DNA-protein interactions.

  4. A genomic island integrated into recA of Vibrio cholerae contains a divergent recA and provides multi-pathway protection from DNA damage.

    Science.gov (United States)

    Rapa, Rita A; Islam, Atiqul; Monahan, Leigh G; Mutreja, Ankur; Thomson, Nicholas; Charles, Ian G; Stokes, Harold W; Labbate, Maurizio

    2015-04-01

    Lateral gene transfer (LGT) has been crucial in the evolution of the cholera pathogen, Vibrio cholerae. The two major virulence factors are present on two different mobile genetic elements, a bacteriophage containing the cholera toxin genes and a genomic island (GI) containing the intestinal adhesin genes. Non-toxigenic V. cholerae in the aquatic environment are a major source of novel DNA that allows the pathogen to morph via LGT. In this study, we report a novel GI from a non-toxigenic V. cholerae strain containing multiple genes involved in DNA repair including the recombination repair gene recA that is 23% divergent from the indigenous recA and genes involved in the translesion synthesis pathway. This is the first report of a GI containing the critical gene recA and the first report of a GI that targets insertion into a specific site within recA. We show that possession of the island in Escherichia coli is protective against DNA damage induced by UV-irradiation and DNA targeting antibiotics. This study highlights the importance of genetic elements such as GIs in the evolution of V. cholerae and emphasizes the importance of environmental strains as a source of novel DNA that can influence the pathogenicity of toxigenic strains.

  5. Specificity in suppression of SOS expression by recA4162 and uvrD303.

    Science.gov (United States)

    Massoni, Shawn C; Sandler, Steven J

    2013-12-01

    Detection and repair of DNA damage is essential in all organisms and depends on the ability of proteins recognizing and processing specific DNA substrates. In E. coli, the RecA protein forms a filament on single-stranded DNA (ssDNA) produced by DNA damage and induces the SOS response. Previous work has shown that one type of recA mutation (e.g., recA4162 (I298V)) and one type of uvrD mutation (e.g., uvrD303 (D403A, D404A)) can differentially decrease SOS expression depending on the type of inducing treatments (UV damage versus RecA mutants that constitutively express SOS). Here it is tested using other SOS inducing conditions if there is a general feature of ssDNA generated during these treatments that allows recA4162 and uvrD303 to decrease SOS expression. The SOS inducing conditions tested include growing cells containing temperature-sensitive DNA replication mutations (dnaE486, dnaG2903, dnaN159, dnaZ2016 (at 37°C)), a del(polA)501 mutation and induction of Double-Strand Breaks (DSBs). uvrD303 could decrease SOS expression under all conditions, while recA4162 could decrease SOS expression under all conditions except in the polA strain or when DSBs occur. It is hypothesized that recA4162 suppresses SOS expression best when the ssDNA occurs at a gap and that uvrD303 is able to decrease SOS expression when the ssDNA is either at a gap or when it is generated at a DSB (but does so better at a gap).

  6. Specificity in suppression of SOS expression by recA4162 and uvrD303.

    Science.gov (United States)

    Massoni, Shawn C; Sandler, Steven J

    2013-12-01

    Detection and repair of DNA damage is essential in all organisms and depends on the ability of proteins recognizing and processing specific DNA substrates. In E. coli, the RecA protein forms a filament on single-stranded DNA (ssDNA) produced by DNA damage and induces the SOS response. Previous work has shown that one type of recA mutation (e.g., recA4162 (I298V)) and one type of uvrD mutation (e.g., uvrD303 (D403A, D404A)) can differentially decrease SOS expression depending on the type of inducing treatments (UV damage versus RecA mutants that constitutively express SOS). Here it is tested using other SOS inducing conditions if there is a general feature of ssDNA generated during these treatments that allows recA4162 and uvrD303 to decrease SOS expression. The SOS inducing conditions tested include growing cells containing temperature-sensitive DNA replication mutations (dnaE486, dnaG2903, dnaN159, dnaZ2016 (at 37°C)), a del(polA)501 mutation and induction of Double-Strand Breaks (DSBs). uvrD303 could decrease SOS expression under all conditions, while recA4162 could decrease SOS expression under all conditions except in the polA strain or when DSBs occur. It is hypothesized that recA4162 suppresses SOS expression best when the ssDNA occurs at a gap and that uvrD303 is able to decrease SOS expression when the ssDNA is either at a gap or when it is generated at a DSB (but does so better at a gap). PMID:24084169

  7. Two-component suppression of recF143 by recA441 in Escherichia coli K-12.

    OpenAIRE

    Volkert, M R; Margossian, L J; Clark, A. J.

    1984-01-01

    Sensitivity to UV irradiation conferred by recF143 was partially suppressed by recA441 (also known as tif-1). A temperature-conditional component depended on uvrA function and is thought to involve thermal induction of excision repair enzymes. In a uvrA6 mutant, a temperature-independent component of suppression was seen. This is thought to indicate that recA441 also caused temperature-independent changes in recA activity. Two hypotheses are offered to explain how recA441 produced both thermo...

  8. An integrative approach to the study of filamentous oligomeric assemblies, with application to RecA.

    Science.gov (United States)

    Boyer, Benjamin; Ezelin, Johann; Poulain, Pierre; Saladin, Adrien; Zacharias, Martin; Robert, Charles H; Prévost, Chantal

    2015-01-01

    Oligomeric macromolecules in the cell self-organize into a wide variety of geometrical motifs such as helices, rings or linear filaments. The recombinase proteins involved in homologous recombination present many such assembly motifs. Here, we examine in particular the polymorphic characteristics of RecA, the most studied member of the recombinase family, using an integrative approach that relates local modes of monomer/monomer association to the global architecture of their screw-type organization. In our approach, local modes of association are sampled via docking or Monte Carlo simulations. This enables shedding new light on fiber morphologies that may be adopted by the RecA protein. Two distinct RecA helical morphologies, the so-called "extended" and "compressed" forms, are known to play a role in homologous recombination. We investigate the variability within each form in terms of helical parameters and steric accessibility. We also address possible helical discontinuities in RecA filaments due to multiple monomer-monomer association modes. By relating local interface organization to global filament morphology, the strategies developed here to study RecA self-assembly are particularly well suited to other DNA-binding proteins and to filamentous protein assemblies in general.

  9. An integrative approach to the study of filamentous oligomeric assemblies, with application to RecA.

    Directory of Open Access Journals (Sweden)

    Benjamin Boyer

    Full Text Available Oligomeric macromolecules in the cell self-organize into a wide variety of geometrical motifs such as helices, rings or linear filaments. The recombinase proteins involved in homologous recombination present many such assembly motifs. Here, we examine in particular the polymorphic characteristics of RecA, the most studied member of the recombinase family, using an integrative approach that relates local modes of monomer/monomer association to the global architecture of their screw-type organization. In our approach, local modes of association are sampled via docking or Monte Carlo simulations. This enables shedding new light on fiber morphologies that may be adopted by the RecA protein. Two distinct RecA helical morphologies, the so-called "extended" and "compressed" forms, are known to play a role in homologous recombination. We investigate the variability within each form in terms of helical parameters and steric accessibility. We also address possible helical discontinuities in RecA filaments due to multiple monomer-monomer association modes. By relating local interface organization to global filament morphology, the strategies developed here to study RecA self-assembly are particularly well suited to other DNA-binding proteins and to filamentous protein assemblies in general.

  10. Structural studies on Mycobacterium tuberculosis RecA: Molecular plasticity and interspecies variability

    Indian Academy of Sciences (India)

    Anu V Chandran; J Rajan Prabu; Astha Nautiyal; K Neelakanteshwar Patil; K Muniyappa; M Vijayan

    2015-03-01

    Structures of crystals of Mycobacterium tuberculosis RecA, grown and analysed under different conditions, provide insights into hitherto underappreciated details of molecular structure and plasticity. In particular, they yield information on the invariant and variable features of the geometry of the P-loop, whose binding to ATP is central for all the biochemical activities of RecA. The strengths of interaction of the ligands with the P-loop reveal significant differences. This in turn affects the magnitude of the motion of the `switch’ residue, Gln195 in M. tuberculosis RecA, which triggers the transmission of ATP-mediated allosteric information to the DNA binding region. M. tuberculosis RecA is substantially rigid compared with its counterparts from M. smegmatis and E. coli, which exhibit concerted internal molecular mobility. The interspecies variability in the plasticity of the two mycobacterial proteins is particularly surprising as they have similar sequence and 3D structure. Details of the interactions of ligands with the protein, characterized in the structures reported here, could be useful for design of inhibitors against M. tuberculosis RecA.

  11. RecA Proteins from Deinococcus geothermalis and Deinococcus murrayi - Cloning, Purification and Biochemical Characterisation

    Directory of Open Access Journals (Sweden)

    Kur Józef

    2011-04-01

    Full Text Available Abstract Background Escherichia coli RecA plays a crucial role in recombinational processes, the induction of SOS responses and mutagenic lesion bypasses. It has also been demonstrated that RecA protein is indispensable when it comes to the reassembly of shattered chromosomes in γ-irradiated Deinococcus radiodurans, one of the most radiation-resistant organisms known. Moreover, some functional differences between E. coli and D. radiodurans RecA proteins have also been shown. Results In this study, recA genes from Deinococcus geothermalis and Deinococcus murrayi, bacteria that are slightly thermophilic and extremely γ-radiation resistant, were isolated, cloned and expressed in E. coli. After production and purification, the biochemical properties of DgeRecA and DmuRecA proteins were determined. Both proteins continued to exist in the solutions as heterogenous populations of oligomeric forms. The DNA binding by DgeRecA and DmuRecA proteins is stimulated by Mg2+ ions. Furthermore, both proteins bind more readily to ssDNA when ssDNA and dsDNA are in the same reaction mixture. Both proteins are slightly thermostable and were completely inactivated in 10 s at 80°C. Both proteins hydrolyze ATP and dATP in the presence of ssDNA or complementary ssDNA and dsDNA, but not in the absence of DNA or in the presence of dsDNA only, and dATP was hydrolyzed more rapidly than ATP. They were also able to promote DNA strand exchange reactions by a pathway common for other RecA proteins. However, we did not obtain DNA strand exchange products when reactions were performed on an inverse pathway, characteristic for RecA of D. radiodurans. Conclusions The characterization of DgeRecA and DmuRecA proteins made in this study indicates that the unique properties of D. radiodurans RecA are probably not common among RecA proteins from Deinococcus sp.

  12. ANTIBODIES DEFINING RAT ENDOTHELIAL-CELLS - RECA-1, A PAN-ENDOTHELIAL CELL-SPECIFIC MONOCLONAL-ANTIBODY

    NARCIS (Netherlands)

    DUIJVESTIJN, AM; VANGOOR, H; KLATTER, F; MAJOOR, GD; VANBUSSEL, E; VRIESMAN, PJCV

    1992-01-01

    We have been searching for antibodies reactive with rat endothelial cells. Two monoclonal antibodies (mAb), named RECA-1 and RECA-2 were produced and tested in immunoperoxidase staining on frozen sections of various rat tissues. Staining patterns were compared to those obtained with the mAbs OX-2, O

  13. Experimental Conditions: SE16_S10_M03_D01 [Metabolonote[Archive

    Lifescience Database Archive (English)

    Full Text Available SE16_S10_M03_D01 SE16 Effect of agricultural films for spinach leaf metabolites 2 SE16_S10... Spinacia oleracea Kurohaminstarland Leaf SE16_S10_M03 6.7mg [MassBase ID] MDLC1_35977 SE16_MS1 LC-FT

  14. Transoceanic spreading of pathogenic strains of Vibrio parahaemolyticus with distinctive genetic signatures in the recA gene.

    Directory of Open Access Journals (Sweden)

    Narjol González-Escalona

    Full Text Available Vibrio parahaemolyticus is an important human pathogen whose transmission is associated with the consumption of contaminated seafood. Consistent multilocus sequence typing for V. parahaemolyticus has shown difficulties in the amplification of the recA gene by PCR associated with a lack of amplification or a larger PCR product than expected. In one strain (090-96, Peru, 1996, the produced PCR product was determined to be composed of two recA fragments derived from different Vibrio species. To better understand this phenomenon, we sequenced the whole genome of this strain. The hybrid recA gene was found to be the result of a fragmentation of the original lineage-specific recA gene resulting from a DNA insertion of approximately 30 kb in length. This insert had a G+C content of 38.8%, lower than that of the average G+C content of V. parahaemolyticus (45.2%, and contained 19 ORFs, including a complete recA gene. This new acquired recA gene deviated 24% in sequence from the original recA and was distantly related to recA genes from bacteria of the Vibrionaceae family. The reconstruction of the original recA gene (recA3 identified the precursor as belonging to ST189, a sequence type reported previously only in Asian countries. The identification of this singular genetic feature in strains from Asia reveals new evidence for genetic connectivity between V. parahaemolyticus populations at both sides of the Pacific Ocean that, in addition to the previously described pandemic clone, supports the existence of a recurrent transoceanic spreading of pathogenic V. parahaemolyticus with the corresponding potential risk of pandemic expansion.

  15. Detection of the new cosmopolitan genus Thermoleptolyngbya (Cyanobacteria, Leptolyngbyaceae) using the 16S rRNA gene and 16S-23S ITS region.

    Science.gov (United States)

    Sciuto, Katia; Moro, Isabella

    2016-12-01

    Cyanobacteria are widespread prokaryotes that are able to live in extreme conditions such as thermal springs. Strains attributable to the genus Leptolyngbya are among the most common cyanobacteria sampled from thermal environments. Leptolyngbya is a character-poor taxon that was demonstrated to be polyphyletic based on molecular analyses. The recent joining of 16S rRNA gene phylogenies with 16S-23S ITS secondary structure analysis is a useful approach to detect new cryptic taxa and has led to the separation of new genera from Leptolyngbya and to the description of new species inside this genus and in other related groups. In this study, phylogenetic investigations based on both the 16S rRNA gene and the 16S-23S ITS region were performed alongside 16S rRNA and 16S-23S ITS secondary structure analyses on cyanobacteria of the family Leptolyngbyaceae. These analyses focused on filamentous strains sampled from thermal springs with a morphology ascribable to the genus Leptolyngbya. The phylogenetic reconstructions showed that the Leptolyngbya-like thermal strains grouped into a monophyletic lineage that was distinct from Leptolyngbya. The 16S-23S ITS secondary structure results supported the separation of this cluster. A new genus named Thermoleptolyngbya was erected to encompass these strains, and two species were described inside this new taxon: T. albertanoae and T. oregonensis.

  16. RecA bundles mediate homology pairing between distant sisters during DNA break repair

    Science.gov (United States)

    Lesterlin, Christian; Ball, Graeme; Schermelleh, Lothar; Sherratt, David J.

    2014-02-01

    DNA double-strand break (DSB) repair by homologous recombination has evolved to maintain genetic integrity in all organisms. Although many reactions that occur during homologous recombination are known, it is unclear where, when and how they occur in cells. Here, by using conventional and super-resolution microscopy, we describe the progression of DSB repair in live Escherichia coli. Specifically, we investigate whether homologous recombination can occur efficiently between distant sister loci that have segregated to opposite halves of an E. coli cell. We show that a site-specific DSB in one sister can be repaired efficiently using distant sister homology. After RecBCD processing of the DSB, RecA is recruited to the cut locus, where it nucleates into a bundle that contains many more RecA molecules than can associate with the two single-stranded DNA regions that form at the DSB. Mature bundles extend along the long axis of the cell, in the space between the bulk nucleoid and the inner membrane. Bundle formation is followed by pairing, in which the two ends of the cut locus relocate at the periphery of the nucleoid and together move rapidly towards the homology of the uncut sister. After sister locus pairing, RecA bundles disassemble and proteins that act late in homologous recombination are recruited to give viable recombinants 1-2-generation-time equivalents after formation of the initial DSB. Mutated RecA proteins that do not form bundles are defective in sister pairing and in DSB-induced repair. This work reveals an unanticipated role of RecA bundles in channelling the movement of the DNA DSB ends, thereby facilitating the long-range homology search that occurs before the strand invasion and transfer reactions.

  17. Electron microscopic visualization of the RecA protein-mediated pairing and branch migration phases of DNA strand exchange

    DEFF Research Database (Denmark)

    Register, JC; Christiansen, Gunna; Griffith, J

    1987-01-01

    examined by electron microscopy: supertwisted double-stranded (ds) DNA and linear single-stranded (ss) DNA, linear dsDNA and circular ssDNA, and linear dsDNA and colinear ssDNA. Several major observations were: (i) with RecA protein bound to the DNA, plectonemic joints were ultrastructurally...... hundreds of RecA protein monomers and several kilobases of DNA. These observations suggest that, following RecA protein-ssDNA filament formation, strand exchange proceeds by a pathway that can be divided structurally into three phases: pairing, envelopment/exchange, and release of the products....

  18. Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA.

    Science.gov (United States)

    Bell, Jason C; Plank, Jody L; Dombrowski, Christopher C; Kowalczykowski, Stephen C

    2012-11-01

    Escherichia coli RecA is the defining member of a ubiquitous class of DNA strand-exchange proteins that are essential for homologous recombination, a pathway that maintains genomic integrity by repairing broken DNA. To function, filaments of RecA must nucleate and grow on single-stranded DNA (ssDNA) in direct competition with ssDNA-binding protein (SSB), which rapidly binds and continuously sequesters ssDNA, kinetically blocking RecA assembly. This dynamic self-assembly on a DNA lattice, in competition with another protein, is unique for the RecA family compared to other filament-forming proteins such as actin and tubulin. The complexity of this process has hindered our understanding of RecA filament assembly because ensemble measurements cannot reliably distinguish between the nucleation and growth phases, despite extensive and diverse attempts. Previous single-molecule assays have measured the nucleation and growth of RecA--and its eukaryotic homologue RAD51--on naked double-stranded DNA and ssDNA; however, the template for RecA self-assembly in vivo is SSB-coated ssDNA. Using single-molecule microscopy, here we directly visualize RecA filament assembly on single molecules of SSB-coated ssDNA, simultaneously measuring nucleation and growth. We establish that a dimer of RecA is required for nucleation, followed by growth of the filament through monomer addition, consistent with the finding that nucleation, but not growth, is modulated by nucleotide and magnesium ion cofactors. Filament growth is bidirectional, albeit faster in the 5'→3' direction. Both nucleation and growth are repressed at physiological conditions, highlighting the essential role of recombination mediators in potentiating assembly in vivo. We define a two-step kinetic mechanism in which RecA nucleates on transiently exposed ssDNA during SSB sliding and/or partial dissociation (DNA unwrapping) and then the RecA filament grows. We further demonstrate that the recombination mediator protein pair

  19. Mutations in the 16S rRNA Genes of Helicobacter pylori Mediate Resistance to Tetracycline

    OpenAIRE

    Trieber, Catharine A.; Taylor, Diane E.

    2002-01-01

    Low-cost and rescue treatments for Helicobacter pylori infections involve combinations of several drugs including tetracycline. Resistance to tetracycline has recently emerged in H. pylori. The 16S rRNA gene sequences of two tetracycline-resistant clinical isolates (MIC = 64 μg/ml) were determined and compared to the consensus H. pylori 16S rRNA sequence. One isolate had four nucleotide substitutions, and the other had four substitutions and two deletions. Natural transformation with the 16S ...

  20. Genetic analysis on 16S rDNA of brucella%布鲁菌16S rDNA基因遗传分析

    Institute of Scientific and Technical Information of China (English)

    李克诚; 周邦谣; 夏菲

    2013-01-01

    目的 通过分析临床分离的布鲁菌、其他盐杆菌科细菌以及临床常见致病菌的16S rDNA基因片段的差异并构建16S rDNA系统发育树,为进一步研究布鲁菌打下基础.方法 PCR扩增临床分离株的16SrDNA并测序;从EMBL下载常见盐杆菌科细菌和临床上常见致病菌的16S rDNA序列.利用CLUSTALX和MEGA程序进行16S rDNA比对并构建系统发育树.结果 成功扩增了临床分离菌株的16S rDNA,得到测序结果,比对临床分离菌株和相关菌株后,发现了特异序列5’-ATCCCGGTCGCGGTTAGTGG-3';系统发育树表明不同种的布鲁菌间的距离非常接近;布鲁菌和醋菌属的进化距离较近,但是和其他的盐杆菌的进化距离较远.结论 在布鲁菌16S rDNA中存在特异序列5'-ATCCCGGTCGCGGTTAGTGG-3’,有可能作为探针来快速检测布鲁氏菌.%Objective To analyze and to compare the genetic characteristics of 16S rDNA gene of Brucella with other halobacteriaceae and pathogenic bacteria, and to construct phylogenetic tree to find the specific sequence. Methods 16s rDNA of Brucella isolated from clinical sample was amplified and sequenced, which was then compared with sequences of halobacteriaceae and other pathogenic bacteria downloaded from EM-BL. CLUSTALA and MEGA software were used for the comparison of the sequences and building of the phylogenetic tree. Results 16s rDNA was successfully amplified and sequenced. Meanwhile, a specific sequence of 5' -ATCCCGGTCGCGGTTAGTGG-3' was identified. Phylogenetic tree showed that the distance was very close among different species of Brucella and was also closer to acetobacter sp. . but was farther to other halobacteriaceae. Conclusions A specific sequence is present in 16S rDNA of brucella which could be used as a probe to detect Brucella.

  1. Investigation of the spectral properties of LED-based MR16s for general illumination

    Science.gov (United States)

    Brown, David F.; Nicol, David B.; Payne, Adam; Ferguson, Ian T.

    2004-10-01

    The spectral properties of commercially available LED-based and halogen MR16s were investigated. The measurements taken include TLF (Total Luminous Flux), CCT (Correlated Color Temperature), CRI (Color Rendering Index), angular variation of CCT, and luminous efficacy. The halogen MR16s were used as a baseline for comparison with LED-based MR16s. It is shown at this time that LED-based MR16s are not suitable as a direct replacement for existing alternatives due to high initial cost, low power efficiency, poor CRIs, and undesirable CCTs.

  2. Sequencing of 16S rRNA Gene: A Rapid Tool for Identification of Bacillus anthracis

    OpenAIRE

    Sacchi, Claudio T.; Whitney, Anne M.; Mayer, Leonard W.; Morey, Roger; Steigerwalt, Arnold; Boras, Ariana; Weyant, Robin S.; Popovic, Tanja

    2002-01-01

    In a bioterrorism event, a tool is needed to rapidly differentiate Bacillus anthracis from other closely related spore-forming Bacillus species. During the recent outbreak of bioterrorism-associated anthrax, we sequenced the 16S rRNA generom these species to evaluate the potential of 16S rRNA gene sequencing as a diagnostic tool. We found eight distinct 16S types among all 107 16S rRNA gene seqs fuences that differed from each other at 1 to 8 positions (0.06% to 0.5%). All 86 B. anthracis had...

  3. Use of RecA protein to enrich for homologous genes in a genomic library

    Energy Technology Data Exchange (ETDEWEB)

    Taidi-Laskowski, B.; Grumet, F.C. (Stanford Univ. School of Medicine, CA (USA)); Tyan, D. (Univ. of California, Los Angeles (USA)); Honigberg, S.M.; Radding, C.R. (Yale Univ. School of Medicine, New Haven, CT (USA))

    1988-08-25

    RecA protein-coated probe has been utilized to enrich genomic digests for desired genes in order to facilitate cloning from genomic libraries. Using a previously cloned HLA-B27 gene as the recA-coated enrichment probe, the authors obtained a mean 108x increase in the ratio of specific to nonspecific plaques in lambda libraries screened for B27 variant alleles of estimated 99% homology to the probe. Class I genes of lesser homology were less enriched. Loss of genomic DNA during the enrichment procedure can, however, restrict application of this technique whenever starting genomic DNA is very limited. Nevertheless, the impressive reduction in cloning effort and material makes recA enrichment a useful new tool for cloning homologous genes from genomic DNA.

  4. 蜡状芽胞杆菌群16S rDNA分析%Analysis of 16S rDNA in the Bacillus cereus Group

    Institute of Scientific and Technical Information of China (English)

    邹寰; 喻子牛; 孙明

    2008-01-01

    蜡状芽胞杆菌群主要包括炭疽芽胞杆菌(Bacillus anthracis)、蜡状芽胞杆菌(Bacillus cereus)、苏云金芽胞杆菌(Bacillus thuringiensis).GenBank已有这个群的8个菌株完成了全基因组序列测定.对这8株蜡状芽胞杆菌群菌株中98条16S rDNA的序列进行相互BLAST比较,发现在同一基因组内各个16S rDNA拷贝全局相似度最低为96.47%,在不同基因组间16S rDNA局部片段比对最小相似度达到99.72%,对应片段长度也有1417 bp.这点充分说明,该群的细菌完全共用同一种16S rDNA,根据16S rDNA给细菌分类的特点,它们应该属于同一个种.在亲缘关系上,枯草芽胞杆菌离蜡状芽胞杆菌群最近.

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

    Science.gov (United States)

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

    2001-02-01

    The identification of Gram-negative pathogenic and non-pathogenic bacteria commonly isolated from an orchard phylloplane may result in a time consuming and tedious process for the plant pathologist. The paper provides a simple "one-step" protocol that uses the polymerase chain reaction (PCR) to amplify intergenic spacer regions between 16S and 23S genes and a portion of 16S gene in the prokaryotic rRNA genetic loci. Amplified 16S rDNA, and restriction fragment length polymorphisms (RFLP) following EcoRI digestion produced band patterns that readily distinguished between the plant pathogen Erwinia amylovora (causal agent of fire blight in pear and apple) and the orchard epiphyte Pantoea agglomerans (formerly E. herbicola). The amplified DNA patterns of 16S-23S spacer regions may be used to differentiate E. amylovora at the intraspecies level. Isolates of E. amylovora obtained from raspberries exhibited two major fragments while those obtained from apples showed three distinct amplified DNA bands. In addition, the size of the 16S-23S spacer region differs between Pseudomonas syringae and Pseudomonas fluorescens. The RFLP pattern generated by HaeIII digestion may be used to provide a rapid and accurate identification of these two common orchard epiphytes. PMID:11166101

  6. Development of a recA Gene-Based Identification Approach for the Entire Burkholderia Genus

    OpenAIRE

    Payne, George W.; Vandamme, Peter; Morgan, Sara H.; LiPuma, John J.; Coenye, Tom; Weightman, Andrew J.; Jones, T. Hefin; Mahenthiralingam, Eshwar

    2005-01-01

    Burkholderia is an important bacterial genus containing species of ecological, biotechnological, and pathogenic interest. With their taxonomy undergoing constant revision and the phenotypic similarity of several species, correct identification of Burkholderia is difficult. A genetic scheme based on the recA gene has greatly enhanced the identification of Burkholderia cepacia complex species. However, the PCR developed for the latter approach was limited by its specificity for the complex. By ...

  7. Purification and characterization of the RecA protein from Neisseria gonorrhoeae.

    Directory of Open Access Journals (Sweden)

    Elizabeth A Stohl

    Full Text Available The strict human pathogen Neisseria gonorrhoeae is the only causative agent of the sexually transmitted infection gonorrhea. The recA gene from N. gonorrhoeae is essential for DNA repair, natural DNA transformation, and pilin antigenic variation, all processes that are important for the pathogenesis and persistence of N. gonorrhoeae in the human population. To understand the biochemical features of N. gonorrhoeae RecA (RecA(Ng, we overexpressed and purified the RecA(Ng and SSB(Ng proteins and compared their activities to those of the well-characterized E. coli RecA and SSB proteins in vitro. We observed that RecA(Ng promoted more strand exchange at early time points than RecA(Ec through DNA homologous substrates, and exhibited the highest ATPase activity of any RecA protein characterized to date. Further analysis of this robust ATPase activity revealed that RecA(Ng is more efficient at displacing SSB from ssDNA and that RecA(Ng shows higher ATPase activity during strand exchange than RecA(Ec. Using substrates created to mimic the cellular processes of DNA transformation and pilin antigenic variation we observed that RecA(Ec catalyzed more strand exchange through a 100 bp heterologous insert, but that RecA(Ng catalyzed more strand exchange through regions of microheterology. Together, these data suggest that the processes of ATP hydrolysis and DNA strand exchange may be coupled differently in RecA(Ng than in RecA(Ec. This difference may explain the unusually high ATPase activity observed for RecA(Ng with the strand exchange activity between RecA(Ng and RecA(Ec being more similar.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    OpenAIRE

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

    2015-01-01

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

  10. Factores asociados a recaídas por tuberculosis en Lima este - Perú

    Directory of Open Access Journals (Sweden)

    María Ríos Hipólito

    2002-01-01

    Full Text Available Objetivo: Determinar los factores de riesgo asociados a recaídas por tuberculosis en Lima Este - Perú, entre marzo y diciembre del 2000. Materiales y métodos: estudio caso-control. Se definió a los casos (184 como los pacientes que recibieron tratamiento con el esquema I alguna vez, egresaron como curados y volvieron a presentar otro episodio de tuberculosis BK positivo durante 1999. Los controles (368 fueron los pacientes nuevos con tuberculosis BK positivo tratados en 1998 que no recayeron. Resultados: se asociaron significativamente a las recaídas el sexo masculino, la edad mayor de 50 años, el consumo de drogas, la residencia en un área urbana, el hacinamiento, la percepción errada de la enfermedad (PEE y la desocupación, no así el contacto con un paciente tuberculoso. Luego del análisis multivariado, sólo se asociaron el área urbana, el hacinamiento, la PEE y el tratamiento irregular. Conclusiones: la residencia en un área urbana, el hacinamiento, la PEE y la irregularidad en el tratamiento son factores asociados significativamente a recaídas en pacientes con TBC pulmonar BK(+ de Lima Este, Perú.

  11. RecFOR and RecOR as distinct RecA loading pathways.

    Science.gov (United States)

    Sakai, Akiko; Cox, Michael M

    2009-01-30

    The molecular role of the RecF protein in loading RecA protein onto single-stranded DNA (ssDNA)-binding protein-coated ssDNA has been obscured by the facility with which the RecO and RecR proteins alone perform this function. We now show that RecFOR and RecOR define distinct RecA loading functions that operate optimally in different contexts. RecFOR, but not RecOR, is most effective when RecF(R) is bound near an ssDNA/double-stranded (dsDNA) junction. However, RecF(R) has no enhanced binding affinity for such a junction. RecO and RecR proteins are both required under all conditions in which the RecFOR pathway operates. The RecOR pathway is uniquely distinguished by a required interaction between RecO protein and the ssDNA binding protein C terminus. The RecOR pathway is more efficient for RecA loading onto ssDNA when no proximal dsDNA is available. A merger of new and published results leads to a new model for RecFOR function. PMID:18986990

  12. High-level synthesis of the recA protein is not sufficient for the expression of SOS functions in Escherichia coli K-12

    International Nuclear Information System (INIS)

    The influence of constitutive overproduction of the recA protein on the expression of SOS functions as measured by UV induction of mutations and prophage Λ was investigated. Constitutive high-level synthesis of the recA protein was achieved using the recombinant multicopy Co1E1 plasmid pLC 17-43, which carries the recA gene region of the E. coli chromosome. High cellular concentration of the recA protein did not result in an enhancement of either spontaneous or UV-induced mutagenesis or prophage Λ release, indicating that a high level of the recA protein is not sufficient for SOS expression. Obviously the recA protein requires, in addition to a high rate of synthesis a specific activation to trigger SOS responses. (author)

  13. 海南温泉嗜热菌的16S rDNA分析%Hainan Hot Spring Thermophiles' 16S rDNA Analysis

    Institute of Scientific and Technical Information of China (English)

    吴红萍; 孟甜; 张飞官; 陈永安; 李文芳; 王丙乾; 王锐萍

    2013-01-01

    目的:确定24株海南温泉嗜热菌菌株的分类地位.方法:Blastn分析菌株16S rDNA序列同源性;邻接法构建菌株16S rDNA序列系统发育进化树并分析菌株的进化位置;Clustax比对分析菌株的相似度和进化距离.结果:菌株LY5和LY4的16S rDNA序列与Geobacillus pallidus strain B1,partial sequence(GenBank:HM030740.1)的16SrDNA序列同源性分别为98%和97%,其他菌株的16S rDNA序列与Geobacillus subterraneus,strain R-35641(GenBank:FN428689.1)的16S rDNA序列的同源性均大于96%.Clustax比对分析表明26株菌16S rDNA序列前段(1~70bp)、中段(70bp~1420bp)、后段(1420~1484bp)的相似度分别为40%、100%和60%,进化距离分析表明菌株GT7、LY4和LY5与其他菌株进化距离较远,其余菌株之间进化距离差异不明显.综上所述,初步将24株温泉嗜热菌鉴定为土芽孢杆菌属(Geobacillus sp.).结论:16S rDNA序列分析可用于温泉嗜热菌的鉴定.

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

    Directory of Open Access Journals (Sweden)

    Maria Razzauti

    Full Text Available 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.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.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

  15. Genotypic Characterization of Bradyrhizobium Strains Nodulating Endemic Woody Legumes of the Canary Islands by PCR-Restriction Fragment Length Polymorphism Analysis of Genes Encoding 16S rRNA (16S rDNA) and 16S-23S rDNA Intergenic Spacers, Repetitive Extragenic Palindromic PCR Genomic Fingerprinting, and Partial 16S rDNA Sequencing

    OpenAIRE

    Vinuesa, Pablo; Rademaker, Jan L. W.; de Bruijn, Frans J.; Werner, Dietrich

    1998-01-01

    We present a phylogenetic analysis of nine strains of symbiotic nitrogen-fixing bacteria isolated from nodules of tagasaste (Chamaecytisus proliferus) and other endemic woody legumes of the Canary Islands, Spain. These and several reference strains were characterized genotypically at different levels of taxonomic resolution by computer-assisted analysis of 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphisms (PCR-RFLPs), 16S-23S rDNA intergenic spacer (IGS) RFLPs, and repeti...

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

    OpenAIRE

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

    2014-01-01

    In the present study, samples of rhizosphere and root nodules were collected from different areas of Pakistan to isolate plant growth promoting rhizobacteria. Identification of bacterial isolates was made by 16S rRNA gene sequence analysis and taxonomical confirmation on EzTaxon Server. The identified bacterial strains were belonged to 5 genera i.e. Ensifer, Bacillus, Pseudomona, Leclercia and Rhizobium. Phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relat...

  17. 16S rRNA Mutation Associated with Tetracycline Resistance in a Gram-Positive Bacterium

    OpenAIRE

    Ross, Jeremy I.; Eady, E Anne; Cove, Jonathan H.; Cunliffe, William J.

    1998-01-01

    A genetic basis for tetracycline resistance in cutaneous propionibacteria was suggested by comparing the nucleotide sequences of the 16S rRNA genes from 16 susceptible and 21 resistant clinical isolates and 6 laboratory-selected tetracycline-resistant mutants of a susceptible strain. Fifteen clinical isolates resistant to tetracycline were found to have cytosine instead of guanine at a position cognate with Escherichia coli 16S rRNA base 1058 in a region important for peptide chain terminatio...

  18. Comparison of two approaches for the classification of 16S rRNA gene sequences.

    Science.gov (United States)

    Chatellier, Sonia; Mugnier, Nathalie; Allard, Françoise; Bonnaud, Bertrand; Collin, Valérie; van Belkum, Alex; Veyrieras, Jean-Baptiste; Emler, Stefan

    2014-10-01

    The use of 16S rRNA gene sequences for microbial identification in clinical microbiology is accepted widely, and requires databases and algorithms. We compared a new research database containing curated 16S rRNA gene sequences in combination with the lca (lowest common ancestor) algorithm (RDB-LCA) to a commercially available 16S rDNA Centroid approach. We used 1025 bacterial isolates characterized by biochemistry, matrix-assisted laser desorption/ionization time-of-flight MS and 16S rDNA sequencing. Nearly 80 % of isolates were identified unambiguously at the species level by both classification platforms used. The remaining isolates were mostly identified correctly at the genus level due to the limited resolution of 16S rDNA sequencing. Discrepancies between both 16S rDNA platforms were due to differences in database content and the algorithm used, and could amount to up to 10.5 %. Up to 1.4 % of the analyses were found to be inconclusive. It is important to realize that despite the overall good performance of the pipelines for analysis, some inconclusive results remain that require additional in-depth analysis performed using supplementary methods.

  19. Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians

    Directory of Open Access Journals (Sweden)

    Chiari Ylenia

    2005-03-01

    Full Text Available Abstract Background Identifying species of organisms by short sequences of DNA has been in the center of ongoing discussions under the terms DNA barcoding or DNA taxonomy. A C-terminal fragment of the mitochondrial gene for cytochrome oxidase subunit I (COI has been proposed as universal marker for this purpose among animals. Results Herein we present experimental evidence that the mitochondrial 16S rRNA gene fulfills the requirements for a universal DNA barcoding marker in amphibians. In terms of universality of priming sites and identification of major vertebrate clades the studied 16S fragment is superior to COI. Amplification success was 100% for 16S in a subset of fresh and well-preserved samples of Madagascan frogs, while various combination of COI primers had lower success rates.COI priming sites showed high variability among amphibians both at the level of groups and closely related species, whereas 16S priming sites were highly conserved among vertebrates. Interspecific pairwise 16S divergences in a test group of Madagascan frogs were at a level suitable for assignment of larval stages to species (1–17%, with low degrees of pairwise haplotype divergence within populations (0–1%. Conclusion We strongly advocate the use of 16S rRNA as standard DNA barcoding marker for vertebrates to complement COI, especially if samples a priori could belong to various phylogenetically distant taxa and false negatives would constitute a major problem.

  20. The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

    Energy Technology Data Exchange (ETDEWEB)

    Galvão, C.W. [Departamento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR (Brazil); Souza, E.M. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil); Etto, R.M. [Departamento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR (Brazil); Pedrosa, F.O.; Chubatsu, L.S.; Yates, M.G. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil); Schumacher, J.; Buck, M. [Department of Life Sciences, Imperial College London, London (United Kingdom); Steffens, M.B.R. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil)

    2012-10-15

    DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX{sub Hs}) can interact with the H. seropedicae RecA protein (RecA{sub Hs}) and that RecA{sub Hs} possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX{sub Hs} inhibited 90% of the RecA{sub Hs} DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA{sub Hs}. RecA{sub Hs} ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX{sub Hs} was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX{sub Hs} protein negatively modulates the RecA{sub Hs} activities by protein-protein interactions and also by DNA-protein interactions.

  1. DNA double strand break end-processing and RecA induce RecN expression levels in Bacillus subtilis.

    Science.gov (United States)

    Cardenas, Paula P; Gándara, Carolina; Alonso, Juan C

    2014-02-01

    Bacillus subtilis cells respond to double strand breaks (DSBs) with an ordered recruitment of repair proteins to the site lesion, being RecN one of the first responders. In B. subtilis, one of the responses to DSBs is to increase RecN expression rather than modifying its turnover rate. End-processing activities and the RecA protein itself contribute to increase RecN levels after DNA DSBs. RecO is required for RecA filament formation and full SOS induction, but its absence did not significantly affect RecN expression. Neither the absence of LexA nor the phosphorylation state of RecA or SsbA significantly affect RecN expression levels. These findings identify two major mechanisms (SOS and DSB response) used to respond to DSBs, with LexA required for one of them (SOS response). The DSB response, which requires end-processing and RecA or short RecO-independent RecA filaments, highlights the importance of guarding genome stability by modulating the DNA damage responses. PMID:24373815

  2. Bacillus subtilis RecO nucleates RecA onto SsbA-coated single-stranded DNA.

    Science.gov (United States)

    Manfredi, Candela; Carrasco, Begoña; Ayora, Silvia; Alonso, Juan C

    2008-09-01

    Subsaturating amounts of Bacillus subtilis SsbA, independently of the order of addition, partially inhibit the single-stranded DNA-dependent dATPase activity of RecA. This negative effect is fully overcome when a substoichiometric amount of RecO is added. SsbA added prior to RecA does not stimulate the dATP-dependent DNA strand exchange activity; however, added after RecA it enhances the extent of strand exchange. The addition of RecO stimulates RecA-mediated joint molecule formation, although it limits the accumulation of final recombination products. Thus we suggest that RecO has a dual activity: RecO acts as a RecA mediator enabling RecA to utilize SsbA-coated single-stranded DNA as a polymerization substrate and controls RecA-mediated DNA strand exchange by limiting its extent. We herein discuss the possible mechanisms of RecO involvement in the regulation of double strand break repair and genetic transformation. PMID:18599486

  3. Separation of recombination and SOS response in Escherichia coli RecA suggests LexA interaction sites.

    Directory of Open Access Journals (Sweden)

    Anbu K Adikesavan

    2011-09-01

    Full Text Available RecA plays a key role in homologous recombination, the induction of the DNA damage response through LexA cleavage and the activity of error-prone polymerase in Escherichia coli. RecA interacts with multiple partners to achieve this pleiotropic role, but the structural location and sequence determinants involved in these multiple interactions remain mostly unknown. Here, in a first application to prokaryotes, Evolutionary Trace (ET analysis identifies clusters of evolutionarily important surface amino acids involved in RecA functions. Some of these clusters match the known ATP binding, DNA binding, and RecA-RecA homo-dimerization sites, but others are novel. Mutation analysis at these sites disrupted either recombination or LexA cleavage. This highlights distinct functional sites specific for recombination and DNA damage response induction. Finally, our analysis reveals a composite site for LexA binding and cleavage, which is formed only on the active RecA filament. These new sites can provide new drug targets to modulate one or more RecA functions, with the potential to address the problem of evolution of antibiotic resistance at its root.

  4. Repair of ultraviolet-irradiated transforming DNA in A recA mutant of Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Stuy, J.H.; Walter, R.B. (Florida State Univ., Tallahassee (USA). Dept. of Biological Science)

    1983-04-01

    Ultraviolet-irradiated transforming DNA was assayed on a wild-type strain of Haemophilus influenzae strain Rd, on an excision repair-deficient (uvr-2) mutant, on a recombination repair-deficient (recA4) mutant, and on a strain carrying both mutations. The donor DNA had a point mutation genetic marker (strAl) and a long nonhomologous plasmid-derived DNA segment inserted in the HPl prophage. The shape of the inactivation curves suggested that only recombination was responsible for the inverse square root kinetics observed with excision repair-proficient recipients.

  5. Progress in Clinical Application of 16S rRNA Gene%16S rRNA基因在临床上的应用进展

    Institute of Scientific and Technical Information of China (English)

    杨祖卿; 尚世强

    2005-01-01

    传统的细菌检测主要依靠血清学、生物化学、细菌形态学及细菌培养等方法进行分类鉴定,但前三者敏感性和特异性不高,后者费时且阳性率低.近10余年来分子生物学技术发展迅速,各种基因方法如DNA杂交、质粒图谱和16S rRNA序列分析等在临床上得到广泛应用.该文就近年来国外16S rRNA在细菌学研究及其应用的一些新进展作一综述.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

    API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method. PMID:26808495

  8. Modeling the early stage of DNA sequence recognition within RecA nucleoprotein filaments.

    Science.gov (United States)

    Saladin, Adrien; Amourda, Christopher; Poulain, Pierre; Férey, Nicolas; Baaden, Marc; Zacharias, Martin; Delalande, Olivier; Prévost, Chantal

    2010-10-01

    Homologous recombination is a fundamental process enabling the repair of double-strand breaks with a high degree of fidelity. In prokaryotes, it is carried out by RecA nucleofilaments formed on single-stranded DNA (ssDNA). These filaments incorporate genomic sequences that are homologous to the ssDNA and exchange the homologous strands. Due to the highly dynamic character of this process and its rapid propagation along the filament, the sequence recognition and strand exchange mechanism remains unknown at the structural level. The recently published structure of the RecA/DNA filament active for recombination (Chen et al., Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structure, Nature 2008, 453, 489) provides a starting point for new exploration of the system. Here, we investigate the possible geometries of association of the early encounter complex between RecA/ssDNA filament and double-stranded DNA (dsDNA). Due to the huge size of the system and its dense packing, we use a reduced representation for protein and DNA together with state-of-the-art molecular modeling methods, including systematic docking and virtual reality simulations. The results indicate that it is possible for the double-stranded DNA to access the RecA-bound ssDNA while initially retaining its Watson-Crick pairing. They emphasize the importance of RecA L2 loop mobility for both recognition and strand exchange.

  9. The specificity of the secondary DNA binding site of RecA protein defines its role in DNA strand exchange.

    OpenAIRE

    Mazin, A V; Kowalczykowski, S C

    1996-01-01

    The RecA protein-single-stranded DNA (ssDNA) filament can bind a second DNA molecule. Binding of ssDNA to this secondary site shows specificity, in that polypyrimidinic DNA binds to the RecA protein-ssDNA filament with higher affinity than polypurinic sequences. The affinity of ssDNA, which is identical in sequence to that bound in the primary site, is not always greater than that of nonhomologous DNA. Moreover, this specificity of DNA binding does not depend on the sequence of the DNA bound ...

  10. The function of the secondary DNA-binding site of RecA protein during DNA strand exchange.

    OpenAIRE

    Mazin, A V; Kowalczykowski, S C

    1998-01-01

    RecA protein features two distinct DNA-binding sites. During DNA strand exchange, the primary site binds to single-stranded DNA (ssDNA), forming the helical RecA nucleoprotein filament. The weaker secondary site binds double-stranded DNA (dsDNA) during the homology search process. Here we demonstrate that this site has a second important function. It binds the ssDNA strand that is displaced from homologous duplex DNA during DNA strand exchange, stabilizing the initial heteroduplex DNA product...

  11. Expression of the bacterial recA gene impairs genetic recombination and sporulation in a Saccharomyces cerevisiae diploid strain

    Directory of Open Access Journals (Sweden)

    Marcos Antonio de Morais Junior

    2003-01-01

    Full Text Available The Escherichia coli RecA protein (RecAp has been demonstrated to induce mutagenesis in yeast cells, although there is still little information on the role of the RecAp in yeast recombination events. We evaluated spontaneous and induced general recombination in vegetative and meiotic cells of the XS2316 strain of the yeast Saccharomyces cerevisiae bearing the recA gene. We found that RecAp decreased reciprocal recombination, gene conversion and intrachromosomal recombination and promoted an increase in error-prone processes in both vegetative and meiotic cells, while its negative effect on meiotic recombination blocked ascospore formation.

  12. Single-Molecule Imaging of DNA Pairing by RecA Reveals a 3-Dimensional Homology Search

    OpenAIRE

    Forget, Anthony L.; Kowalczykowski, Stephen C.

    2012-01-01

    DNA breaks can be repaired with high-fidelity by homologous recombination. A ubiquitous protein that is essential for this DNA template-directed repair is RecA 1 . After resection of broken DNA to produce single-stranded DNA (ssDNA), RecA assembles on this ssDNA into a filament with the unique capacity to search and find DNA sequences in double-stranded DNA (dsDNA) that are homologous to the ssDNA. This homology search is vital to recombinational DNA repair, and results in homologous pairing ...

  13. Toxicity analysis of pesticides on cyanobacterial species by 16S rDNA molecular characterization

    Directory of Open Access Journals (Sweden)

    J. I. Nirmal Kumar

    2013-06-01

    Full Text Available Damaging effects of endosulfan on native structure of DNA, evident as a result of PCR based assay such as 16S rDNA amplification and sequencing, led to formation of gaps, mismatching of base pairs and dissimilarities in entire 16S rDNA sequences of treated cultures. Endosulfan was the most fatal to Westiellopsis prolifica of 16S rDNA region at 40ppm insecticide induced series of mispairing, and other lesions amounting up to 20% dissimilarity and 7% gaps. Whereas, 16S rDNA region of Anabaena fertilissima was comparatively less influenced with 18% dissimilarity and 7% gaps in response to 12ppm endosulfan, while 16S rDNA gene of Aulosira fertilissima was the least prone to changes with 17% dissimilarity, and 5% gaps under 60ppm endosulfan stress by the end of 16 days. On the other side, impact of fungicide tebuconazole after 16 days reflected identities up to 78% and 8% gaps for 30ppm treated A. fertilissima, while 60ppm treatment instilled 79% similarities with 10% gaps in W. prolifica and 83% identities with 5% gaps of Aulosira fertilissima after 16 days.

  14. Regulated expression of the dinR and recA genes during competence development and SOS induction in Bacillus subtilis

    NARCIS (Netherlands)

    Haijema, BJ; vanSinderen, D; Winterling, K; Kooistra, J; Venema, G; Hamoen, LW

    1996-01-01

    It has been hypothesized that the dinR gene product of Bacillus subtilis acts as a repressor of the SOS regulon by binding to DNA sequences located upstream of SOS genes, including dinR and recA. Following activation as a result of DNA damage, RecA is believed to catalyse DinR-autocleavage, thus der

  15. Rare Events of Intragenus and Intraspecies Horizontal Transfer of the 16S rRNA Gene.

    Science.gov (United States)

    Tian, Ren-Mao; Cai, Lin; Zhang, Wei-Peng; Cao, Hui-Luo; Qian, Pei-Yuan

    2015-07-27

    Horizontal gene transfer (HGT) of operational genes has been widely reported in prokaryotic organisms. However, informational genes such as those involved in transcription and translation processes are very difficult to be horizontally transferred, as described by Woese's complexity hypothesis. Here, we analyzed all of the completed prokaryotic genome sequences (2,143 genomes) in the NCBI (National Center for Biotechnology Information) database, scanned for genomes with high intragenomic heterogeneity of 16S rRNA gene copies, and explored potential HGT events of ribosomal RNA genes based on the phylogeny, genomic organization, and secondary structures of the ribosomal RNA genes. Our results revealed 28 genomes with relatively high intragenomic heterogeneity of multiple 16S rRNA gene copies (lowest pairwise identity 16S rRNA gene only occurred at intragenus or intraspecies levels, which is quite different from the HGT of operational genes. Our results improve our understanding regarding the exchange of informational genes.

  16. Isolation of temperature-sensitive mutants of 16 S rRNA in Escherichia coli

    DEFF Research Database (Denmark)

    Triman, K; Becker, E; Dammel, C;

    1989-01-01

    Temperature-sensitive mutants have been isolated following hydroxylamine mutagenesis of a plasmid containing Escherichia coli rRNA genes carrying selectable markers for spectinomycin resistance (U1192 in 16 S rRNA) and erythromycin resistance (G2058 in 23 S rRNA). These antibiotic resistance...... alleles, originally identified by Morgan and co-workers, enable us to follow expression of cloned rRNA genes in vivo. Recessive mutations causing the loss of expression of the cloned 16 S rRNA gene were identified by the loss of the ability of cells to survive on media containing spectinomycin....... The mutations were localized by in vitro restriction fragment replacement followed by in vivo marker rescue and were identified by DNA sequence analysis. We report here seven single-base alterations in 16 S rRNA (A146, U153, A350, A359, A538, A1292 and U1293), five of which produce temperature...

  17. 16s rRNA的保守字和进化树重建%Conserved Words in 16s Ribosomal RNA Deduced from Evolutionary Tree Reconstruction

    Institute of Scientific and Technical Information of China (English)

    罗辽复; 贾孟文

    2002-01-01

    Evolutionary distance is defined by oligonucleotide (n-bases) frequency difference of two sequences.Phylogenetic tree is reconstructed using a set of 16S (18S) rRNA sequences and the definition of distance.The quality of trees generally improves with increasing n and reaches a plateau of best fit at n=7 or 8.So,the 7-mer or 8-mer frequencies provides a basis to describe rRNA evolution.Then,a group of 7-mers are deduced which are correlate well with evolution.Evolution-related conservative words longer than 7 bases for Bacteria and Archaea in 16S rRNA sequences have been found.They are highly conserved in nearly all species of a kingdom (or a sub-kingdom) and are located on nearly same sites of sequences. The structural meaning of these conservative words is discussed briefly.%据寡核苷(n核苷)频数差定义进化距离,由此构成16s rRNA进化树,当n=7,8时和实验资料符合很好,在寻找出全部进化相关的7-核苷的基础上,本文进一步求得了长度大于7的保守字,它们在一个界别中的诸物种中高度保守,并出现于核糖体序列的基本相同的位置上,这些保守字对于核糖体的早期进化至关重要.

  18. RecO and RecR are necessary for RecA loading in response to DNA damage and replication fork stress.

    Science.gov (United States)

    Lenhart, Justin S; Brandes, Eileen R; Schroeder, Jeremy W; Sorenson, Roderick J; Showalter, Hollis D; Simmons, Lyle A

    2014-08-01

    RecA is central to maintaining genome integrity in bacterial cells. Despite the near-ubiquitous conservation of RecA in eubacteria, the pathways that facilitate RecA loading and repair center assembly have remained poorly understood in Bacillus subtilis. Here, we show that RecA rapidly colocalizes with the DNA polymerase complex (replisome) immediately following DNA damage or damage-independent replication fork arrest. In Escherichia coli, the RecFOR and RecBCD pathways serve to load RecA and the choice between these two pathways depends on the type of damage under repair. We found in B. subtilis that the rapid localization of RecA to repair centers is strictly dependent on RecO and RecR in response to all types of damage examined, including a site-specific double-stranded break and damage-independent replication fork arrest. Furthermore, we provide evidence that, although RecF is not required for RecA repair center formation in vivo, RecF does increase the efficiency of repair center assembly, suggesting that RecF may influence the initial stages of RecA nucleation or filament extension. We further identify single-stranded DNA binding protein (SSB) as an additional component important for RecA repair center assembly. Truncation of the SSB C terminus impairs the ability of B. subtilis to form repair centers in response to damage and damage-independent fork arrest. With these results, we conclude that the SSB-dependent recruitment of RecOR to the replisome is necessary for loading and organizing RecA into repair centers in response to DNA damage and replication fork arrest. PMID:24891441

  19. Dickeya species relatedness and clade structure determined by comparison of recA sequences.

    Science.gov (United States)

    Parkinson, Neil; Stead, David; Bew, Janice; Heeney, John; Tsror Lahkim, Leah; Elphinstone, John

    2009-10-01

    Using sequences from the recA locus, we have produced a phylogeny of 188 Dickeya strains from culture collections and identified species relatedness and subspecies clade structure within the genus. Of the six recognized species, Dickeya paradisiaca, D. chrysanthemi and D. zeae were discriminated with long branch lengths. The clade containing the D. paradisiaca type strain included just one additional strain, isolated from banana in Colombia. Strains isolated from Chrysanthemum and Parthenium species made up most of the clade containing the D. chrysanthemi type strain, and the host range of this species was extended to include potato. The D. zeae clade had the largest number of sequevars and branched into two major sister clades that contained all of the Zea mays isolates, and were identified as phylotypes PI and PII. The host range was increased from six to 13 species, including potato. The recA sequence of an Australian sugar-cane strain was sufficiently distinct to rank as a new species-level branch. In contrast to these species, Dickeya dadantii, D. dianthicola and D. dieffenbachiae were distinguished with shorter branch lengths, indicating relatively closer relatedness. The recA sequence for the type strain of D. dadantii clustered separately from other strains of the species. However, sequence comparison of three additional loci revealed that the D. dadantii type strain grouped together with the six other D. dadantii strains that were sequenced. Analysis of all four loci indicated that the D. dadantii strains were most closely related to D. dieffenbachiae. Three further branches (DUC-1, -2 and -3) were associated with these three species, which all diverged from a common origin and can be considered as a species complex. The large clade containing the D. dianthicola type strain comprised 58 strains and had little sequence diversity. One sequevar accounted for the majority of these strains, which were isolated nearly exclusively from eight hosts from Europe

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

    Science.gov (United States)

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

    2014-11-11

    The effect of gemini (alkanediyl-α,ω-bis(dimethylcetylammonium bromide)) (16-s-16, s=4, 5, 6) surfactants on the interaction of ninhydrin with chromium(III) complex of glycylphenylalanine ([Cr(III)-Gly-Phe]2+) has been investigated using UV-visible spectrophotometer at different temperatures. The order of reaction with respect to [Cr(III)-Gly-Phe]2+ is unity while it is fractional with respect to ninhydrin. Whereas, the values of rate constant (kψ) increase and leveling-off regions, like conventional single chain cetyltrimethylammonium bromide (CTAB) surfactant, were observed with geminis, later produces a third region of increasing kψ at higher gemini surfactant concentrations. This unusual third-region effect of the gemini micelles is assigned to changes in their micellar morphologies. The results obtained in micellar media were treated in terms of pseudo-phase model. The values of thermodynamic parameters (Ea, ΔH# and ΔS#) and binding constants (KA and KNin) have been evaluated. PMID:24878435

  1. Non-covalent interactions between ATP and RecA DNA-repairing proteins: DFT and semiempirical calculations

    Science.gov (United States)

    Rodriguez, Jorge

    2015-03-01

    The role of Bacterial RecA in the structural maintenance of genomes and the genetic information they carry has been established. In particular, the RecA DNA-repairing protein from D. Radiodurans, a radiation-resistant bacteria, is crucial for the repair of double strand breaks (DSBs). We have performed semi-empirical free-energy calculations and QM/MM calculations to study their non-covalent interactions with ATP and ADP. Such studies provide insight into the mechanisms of ATP/ADP --> RecA energy transfer and, therefore, about specific functional uses of incoming energy for DNA repairing mechanisms. We present a detailed analysis of the non-covalent interactions which minimize the interaction Gibbs free energies leading to the most stable non-covalent binding sites. Van der Waal, hydrogen bonding and electrostatic interactions has been quantified which provides a detailed insight into the mechanisms of ATP-RecA interaction. Further, possible chemical interactions and functional roles of RecA proteins are explored based on the previously mentioned studies. Acknowledgements: Funded, in part, by DTRA award 106339 (JHR). Dr. Mark C. Palenik and Mrs. Lora Beard are gratefully acknowledged Supported in part by DTRA Award 106339.

  2. Expression of the recA gene of Pseudomonas aeruginosa PAO is inducible by DNA-damaging agents

    Energy Technology Data Exchange (ETDEWEB)

    Miller, R.V.; Kokjohn, T.A.

    1988-05-01

    Western (immunoblot) analysis using Escherichia coli anti-RecA antiserum revealed that expression of the RecA protein of Pseudomonas aeruginosa PAO is induced upon exposure of the bacterium to UV irradiation or norfloxacin, a quinolone related to nalidixic acid.

  3. The location of DNA in complexes of recA protein with double-stranded DNA. A neutron scattering study

    International Nuclear Information System (INIS)

    Purified recA protein is found as rodlike homopolymers, and it forms filamentous complexes with double-stranded DNA that are stable in the presence of ATP gamma S, a nonhydrolyzable analogue of ATP. The structure of these filaments has been described in some detail by electron microscopy. Here we confirm the mass per length of 6.5 recA/100 A in solution by small-angle neutron scattering and extend the analysis to homopolymers of recA protein, finding a mass per length of about 7 recA/100 A and a radial mass distribution (cross-sectional radius of gyration) significantly different for the two filaments. The models proposed so far for the structure of the complex have placed the DNA in the center of the filament. Here we verify this assumption using small-angle neutron scattering to locate the DNA in the complexes, exploiting the contrast variation method in D2O/H2O mixtures. Model calculations show that the natural contrast difference between DNA and protein is not sufficient to locate the DNA (which accounts for only 4.7% of the mass in the complex). When deuterated DNA is used, the contrast difference is enhanced, and model calculations and experiment then converge, indicating that the DNA is indeed near the axis of the complex

  4. Large-scale structure of RecA protein from Deinococcus radiodurance and its complexes in solution

    Science.gov (United States)

    Karelov, D. V.; Lebedev, D. V.; Suslov, A. V.; Shalguev, V. I.; Kuklin, A. I.; Islamov, A. Kh; Lauter, H.; Lanzov, V. A.; Isaev-Ivanov, V. V.

    2008-03-01

    Different conformational states of the filaments formed by RecA protein from a radiation resistant strain Deinococcus radiodurance (RecADr) in solution were investigated using small angle neutron scattering. Scattering by the protein self-polymer was consistent with a long helix model, with the pitch of the helix being lower than that in the crystal structure. Compared to those of RecA proteins from Escherichia coli and Pseudomonas aeruginosa, helical filaments of RecA from D. radiodurance exhibited a lower helical pitch and lower stability at low Mg2+ concentrations or under conditions of elevated ionic strength in the absence of ATP (adenosine triphosphate). Formation of an active filament upon binding of ATPγS and either single- or double-stranded DNA brought about a significant increase in the helix pitch and a moderate decrease in the cross-sectional gyration radius, but resulted in little change in the number of monomers per helix turn. The helix pitch value of the RecADr presynaptic complex was conservative and close to that found for other RecA proteins and their analogs.

  5. Bacterial metabarcoding by 16S rRNA gene ion torrent amplicon sequencing.

    Science.gov (United States)

    Fantini, Elio; Gianese, Giulio; Giuliano, Giovanni; Fiore, Alessia

    2015-01-01

    Ion Torrent is a next generation sequencing technology based on the detection of hydrogen ions produced during DNA chain elongation; this technology allows analyzing and characterizing genomes, genes, and species. Here, we describe an Ion Torrent procedure applied to the metagenomic analysis of 16S rRNA gene amplicons to study the bacterial diversity in food and environmental samples. PMID:25343859

  6. Novel essential gene Involved in 16S rRNA processing in Escherichia coli.

    Science.gov (United States)

    Kurata, Tatsuaki; Nakanishi, Shinobu; Hashimoto, Masayuki; Taoka, Masato; Yamazaki, Yukiko; Isobe, Toshiaki; Kato, Jun-ichi

    2015-02-27

    Biogenesis of ribosomes is a complex process mediated by many factors. While its transcription proceeds, ribosomal RNA (rRNA) folds itself into a characteristic three-dimensional structure through interaction with ribosomal proteins, during which its ends are processed. Here, we show that the essential protein YqgF, a RuvC family protein with an RNase-H-like motif, is involved in the processing of pre-16S rRNA during ribosome maturation. Indeed, pre-16S rRNA accumulated in cells of a temperature-sensitive yqgF mutant (yqgF(ts)) cultured at a non-permissive temperature. In addition, purified YqgF was shown to process the 5' end of pre-16S rRNA within 70S ribosomes in vitro. Mass spectrometry analysis of the total proteins in the yqgF(ts) mutant cells showed that the expression of genes containing multiple Shine-Dalgarno-like sequences was observed to be lower than in wild type. These results are interpreted to indicate that YqgF is involved in a novel enzymic activity necessary for the processing of pre-16S rRNA, thereby affecting elongation of translation.

  7. Effects of 16S rRNA gene mutations on tetracycline resistance in Helicobacter pylori

    NARCIS (Netherlands)

    M.M. Gerrits (Monique); M. Berning; A.H.M. van Vliet (Arnoud); E.J. Kuipers (Ernst); J.G. Kusters (Johannes)

    2003-01-01

    textabstractThe triple-base-pair 16S rDNA mutation AGA(926-928)-->TTC mediates high-level tetracycline resistance in Helicobacter pylori. In contrast, single- and double-base-pair mutations mediated only low-level tetracycline resistance and decreased growth rates in the presence o

  8. Prosthetic joint infection due to Lysobacter thermophilus diagnosed by 16S rRNA gene sequencing.

    Science.gov (United States)

    Dhawan, B; Sebastian, S; Malhotra, R; Kapil, A; Gautam, D

    2016-01-01

    We report the first case of prosthetic joint infection caused by Lysobacter thermophilus which was identified by 16S rRNA gene sequencing. Removal of prosthesis followed by antibiotic treatment resulted in good clinical outcome. This case illustrates the use of molecular diagnostics to detect uncommon organisms in suspected prosthetic infections.

  9. Bacterial metabarcoding by 16S rRNA gene ion torrent amplicon sequencing.

    Science.gov (United States)

    Fantini, Elio; Gianese, Giulio; Giuliano, Giovanni; Fiore, Alessia

    2015-01-01

    Ion Torrent is a next generation sequencing technology based on the detection of hydrogen ions produced during DNA chain elongation; this technology allows analyzing and characterizing genomes, genes, and species. Here, we describe an Ion Torrent procedure applied to the metagenomic analysis of 16S rRNA gene amplicons to study the bacterial diversity in food and environmental samples.

  10. The Characterization of Novel Tissue Microbiota Using an Optimized 16S Metagenomic Sequencing Pipeline.

    Directory of Open Access Journals (Sweden)

    Jérôme Lluch

    Full Text Available Substantial progress in high-throughput metagenomic sequencing methodologies has enabled the characterisation of bacteria from various origins (for example gut and skin. However, the recently-discovered bacterial microbiota present within animal internal tissues has remained unexplored due to technical difficulties associated with these challenging samples.We have optimized a specific 16S rDNA-targeted metagenomics sequencing (16S metabarcoding pipeline based on the Illumina MiSeq technology for the analysis of bacterial DNA in human and animal tissues. This was successfully achieved in various mouse tissues despite the high abundance of eukaryotic DNA and PCR inhibitors in these samples. We extensively tested this pipeline on mock communities, negative controls, positive controls and tissues and demonstrated the presence of novel tissue specific bacterial DNA profiles in a variety of organs (including brain, muscle, adipose tissue, liver and heart.The high throughput and excellent reproducibility of the method ensured exhaustive and precise coverage of the 16S rDNA bacterial variants present in mouse tissues. This optimized 16S metagenomic sequencing pipeline will allow the scientific community to catalogue the bacterial DNA profiles of different tissues and will provide a database to analyse host/bacterial interactions in relation to homeostasis and disease.

  11. Supramolecular hydrogel of kanamycin selectively sequesters 16S rRNA

    OpenAIRE

    Yang, Zhimou; Kuang, Yi; Li, Xinming; Zhou, Ning; Zhang, Ye; Xu, Bing

    2012-01-01

    As the first example of hydrogelator derived from aminoglycoside antibiotics, the hydrogel of kanamycin indicates that the hydrogel of aminoglycosides preserve the specific interaction with their macromolecular targets (e.g., 16S rRNA), thus illustrating a simple approach to explore and identify possible biological targets of supramolecular nanofibers/hydrogels.

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

    Science.gov (United States)

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

    2016-01-01

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

  13. 16S rRNA、16S-23S rRNA基因测序分析检测主要血流感染病原菌比较%Comparison of the role of 16S rRNA and 16S-23S rRNA gene sequence-based identification of bacteria in bloodsteam infection

    Institute of Scientific and Technical Information of China (English)

    金中淦; 葛平; 徐蓉; 陈蓉; 宣瑛; 刘学杰; 王庆忠

    2012-01-01

    目的 比较细菌16S rRNA、16S-23S rRNA基因测序分析在血流感染病原菌检测中的作用.方法 提取临床上血流感染常见的金黄色葡萄菌、表皮葡萄球菌、大肠埃希菌、粪肠球菌、肺炎链球菌、铜绿假单胞菌、阴沟肠杆菌、鲍曼不动杆菌、洛菲不动杆菌、肺炎克雷伯杆菌、化脓性链球菌、奇异变形杆菌、潘尼变形杆菌、屎肠球菌、粘质沙雷菌、宋内志贺菌、产气肠杆菌、小肠结肠炎耶尔森菌、腐生葡萄球菌基因组DNA,运用16S rRNA、16S-23S rRNA基因进行PCR扩增.扩增产物经测序后在美国国家生物技术中心( NCBI)上进行比对分析,确定菌种.结果 在所分析的19种临床血流感染常见细菌中,16S rRNA基因测序分析可将除粘质沙雷菌外的细菌鉴定到种的水平,但无法完全区分近缘种属;16S-23SrRNA成功鉴定17种细菌,除大肠埃希菌、宋内志贺菌外所有细菌均成功鉴定到单一种的水平.结论 16S-23S rRNA基因可作为血流感染细菌检测较好的分子靶标.

  14. Oral microbiome profiles: 16S rRNA pyrosequencing and microarray assay comparison.

    Directory of Open Access Journals (Sweden)

    Jiyoung Ahn

    Full Text Available OBJECTIVES: The human oral microbiome is potentially related to diverse health conditions and high-throughput technology provides the possibility of surveying microbial community structure at high resolution. We compared two oral microbiome survey methods: broad-based microbiome identification by 16S rRNA gene sequencing and targeted characterization of microbes by custom DNA microarray. METHODS: Oral wash samples were collected from 20 individuals at Memorial Sloan-Kettering Cancer Center. 16S rRNA gene survey was performed by 454 pyrosequencing of the V3-V5 region (450 bp. Targeted identification by DNA microarray was carried out with the Human Oral Microbe Identification Microarray (HOMIM. Correlations and relative abundance were compared at phylum and genus level, between 16S rRNA sequence read ratio and HOMIM hybridization intensity. RESULTS: The major phyla, Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria were identified with high correlation by the two methods (r = 0.70∼0.86. 16S rRNA gene pyrosequencing identified 77 genera and HOMIM identified 49, with 37 genera detected by both methods; more than 98% of classified bacteria were assigned in these 37 genera. Concordance by the two assays (presence/absence and correlations were high for common genera (Streptococcus, Veillonella, Leptotrichia, Prevotella, and Haemophilus; Correlation = 0.70-0.84. CONCLUSION: Microbiome community profiles assessed by 16S rRNA pyrosequencing and HOMIM were highly correlated at the phylum level and, when comparing the more commonly detected taxa, also at the genus level. Both methods are currently suitable for high-throughput epidemiologic investigations relating identified and more common oral microbial taxa to disease risk; yet, pyrosequencing may provide a broader spectrum of taxa identification, a distinct sequence-read record, and greater detection sensitivity.

  15. Deinococcus radiodurans RecA nucleoprotein filaments characterized at the single-molecule level with optical tweezers.

    Science.gov (United States)

    Pobegalov, Georgii; Cherevatenko, Galina; Alekseev, Aleksandr; Sabantsev, Anton; Kovaleva, Oksana; Vedyaykin, Alexey; Morozova, Natalia; Baitin, Dmitrii; Khodorkovskii, Mikhail

    2015-10-23

    Deinococcus radiodurans can survive extreme doses of ionizing radiation due to the very efficient DNA repair mechanisms that are able to cope even with hundreds of double-strand breaks. RecA, the critical protein of homologous recombination in bacteria, is one of the key components of the DNA-repair system. Repair of double-strand breaks requires RecA binding to DNA and assembly of the RecA nucleoprotein helical filaments. The Escherichia coli RecA protein (EcRecA) and its interactions with DNA have been extensively studied using various approaches including single-molecule techniques, while the D. radiodurans RecA (DrRecA) remains much less characterized. However, DrRecA shows some remarkable differences from E. coli homolog. Here we combine microfluidics and single-molecule DNA manipulation with optical tweezers to follow the binding of DrRecA to long double-stranded DNA molecules and probe the mechanical properties of DrRecA nucleoprotein filaments at physiological pH. Our data provide a direct comparison of DrRecA and EcRecA binding to double-stranded DNA under identical conditions. We report a significantly faster filaments assembly as well as lower values of persistence length and contour length for DrRecA nucleoprotein filaments compared to EcRecA. Our results support the existing model of DrRecA forming more frequent and less continuous filaments relative to those of EcRecA.

  16. Molecular Interaction and Cellular Location of RecA and CheW Proteins in Salmonella enterica during SOS Response and Their Implication in Swarming

    Science.gov (United States)

    Irazoki, Oihane; Aranda, Jesús; Zimmermann, Timo; Campoy, Susana; Barbé, Jordi

    2016-01-01

    In addition to its role in DNA damage repair and recombination, the RecA protein, through its interaction with CheW, is involved in swarming motility, a form of flagella-dependent movement across surfaces. In order to better understand how SOS response modulates swarming, in this work the location of RecA and CheW proteins within the swarming cells has been studied by using super-resolution microscopy. Further, and after in silico docking studies, the specific RecA and CheW regions associated with the RecA-CheW interaction have also been confirmed by site-directed mutagenesis and immunoprecipitation techniques. Our results point out that the CheW distribution changes, from the cell poles to foci distributed in a helical pattern along the cell axis when SOS response is activated or RecA protein is overexpressed. In this situation, the CheW presents the same subcellular location as that of RecA, pointing out that the previously described RecA storage structures may be modulators of swarming motility. Data reported herein not only confirmed that the RecA-CheW pair is essential for swarming motility but it is directly involved in the CheW distribution change associated to SOS response activation. A model explaining not only the mechanism by which DNA damage modulates swarming but also how both the lack and the excess of RecA protein impair this motility is proposed. PMID:27766091

  17. Discovery and characterization of RecA protein of thermophilic bacterium Thermus thermophilus MAT72 phage Tt72 that increases specificity of a PCR-based DNA amplification.

    Science.gov (United States)

    Stefanska, Aleksandra; Kaczorowska, Anna-Karina; Plotka, Magdalena; Fridjonsson, Olafur H; Hreggvidsson, Gudmundur O; Hjorleifsdottir, Sigridur; Kristjansson, Jakob K; Dabrowski, Slawomir; Kaczorowski, Tadeusz

    2014-07-20

    The recA gene of newly discovered Thermus thermophilus MAT72 phage Tt72 (Myoviridae) was cloned and overexpressed in Escherichia coli. The 1020-bp gene codes for a 339-amino-acid polypeptide with an Mr of 38,155 which shows 38.7% positional identity to the E. coli RecA protein. When expressed in E. coli, the Tt72 recA gene did not confer the ability to complement the ultraviolet light (254nm) sensitivity of an E. coli recA mutant. Tt72 RecA protein has been purified with good yield to catalytic and electrophoretic homogeneity using a three-step chromatography procedure. Biochemical characterization indicated that the protein can pair and promote ATP-dependent strand exchange reaction resulting in formation of a heteroduplex DNA at 60°C under conditions otherwise optimal for E. coli RecA. When the Tt72 RecA protein was included in a standard PCR-based DNA amplification reaction, the specificity of the PCR assays was significantly improved by eliminating non-specific products.

  18. 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.

    Science.gov (United States)

    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

  19. A novel role for RecA under non-stress: promotion of swarming motility in Escherichia coli K-12

    Directory of Open Access Journals (Sweden)

    Blázquez Jesús

    2007-03-01

    Full Text Available Abstract Background Bacterial motility is a crucial factor in the colonization of natural environments. Escherichia coli has two flagella-driven motility types: swimming and swarming. Swimming motility consists of individual cell movement in liquid medium or soft semisolid agar, whereas swarming is a coordinated cellular behaviour leading to a collective movement on semisolid surfaces. It is known that swimming motility can be influenced by several types of environmental stress. In nature, environmentally induced DNA damage (e.g. UV irradiation is one of the most common types of stress. One of the key proteins involved in the response to DNA damage is RecA, a multifunctional protein required for maintaining genome integrity and the generation of genetic variation. Results The ability of E. coli cells to develop swarming migration on semisolid surfaces was suppressed in the absence of RecA. However, swimming motility was not affected. The swarming defect of a ΔrecA strain was fully complemented by a plasmid-borne recA gene. Although the ΔrecA cells grown on semisolidsurfaces exhibited flagellar production, they also presented impaired individual movement as well as a fully inactive collective swarming migration. Both the comparative analysis of gene expression profiles in wild-type and ΔrecA cells grown on a semisolid surface and the motility of lexA1 [Ind-] mutant cells demonstrated that the RecA effect on swarming does not require induction of the SOS response. By using a RecA-GFP fusion protein we were able to segregate the effect of RecA on swarming from its other functions. This protein fusion failed to regulate the induction of the SOS response, the recombinational DNA repair of UV-treated cells and the genetic recombination, however, it was efficient in rescuing the swarming motility defect of the ΔrecA mutant. The RecA-GFP protein retains a residual ssDNA-dependent ATPase activity but does not perform DNA strand exchange. Conclusion

  20. Sequence and secondary structure of the mitochondrial 16S ribosomal RNA gene of Ixodes scapularis.

    Science.gov (United States)

    Krakowetz, Chantel N; Chilton, Neil B

    2015-02-01

    The complete DNA sequences and secondary structure of the mitochondrial (mt) 16S ribosomal (r) RNA gene were determined for six Ixodes scapularis adults. There were 44 variable nucleotide positions in the 1252 bp sequence alignment. Most (95%) nucleotide alterations did not affect the integrity of the secondary structure of the gene because they either occurred at unpaired positions or represented compensatory changes that maintained the base pairing in helices. A large proportion (75%) of the intraspecific variation in DNA sequence occurred within Domains I, II and VI of the 16S gene. Therefore, several regions within this gene may be highly informative for studies of the population genetics and phylogeography of I. scapularis, a major vector of pathogens of humans and domestic animals in North America.

  1. A renaissance for the pioneering 16S rRNA gene

    Energy Technology Data Exchange (ETDEWEB)

    Tringe, Susannah; Hugenholtz, Philip

    2008-09-07

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

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

    Science.gov (United States)

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

    1993-01-01

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

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

    Science.gov (United States)

    Ortega, Maya

    2010-01-01

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

  4. Genomic and Haplotype Comparison of Butanol Producing Bacteria Based on 16S rDNA

    OpenAIRE

    Ekwan Nofa Wiratno; Suharjono; Agustin Krisna Wardani

    2016-01-01

    High butanol demand for transportation fuel triggers butanol production development. Exploration of butanolproducing bacteria using genomic comparison and biogeography will help to develop butanol industry. The objectives of this research were butanol production, genome comparison and haplotype analysis of butanolproducing bacteria from Ranu Pani Lake sediment using 16S rDNA sequences. The highest butanol concentrations were showed by Paenibacillus polymyxa RP 2.2 isolate (10.34 g...

  5. Effects of 16S rRNA gene mutations on tetracycline resistance in Helicobacter pylori

    OpenAIRE

    Gerrits, Monique; Berning, M.; Vliet, Arnoud; Kuipers, Ernst; Kusters, Johannes

    2003-01-01

    textabstractThe triple-base-pair 16S rDNA mutation AGA(926-928)-->TTC mediates high-level tetracycline resistance in Helicobacter pylori. In contrast, single- and double-base-pair mutations mediated only low-level tetracycline resistance and decreased growth rates in the presence of tetracycline, explaining the preference for the TTC mutation in tetracycline-resistant H. pylori isolates.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2006-02-01

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

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Phaik-Eem Lim

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

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

    OpenAIRE

    Phaik-Eem Lim; Ji Tan; I WAYAN SUANA; Praphathip Eamsobhana; Hoi Sen Yong

    2012-01-01

    The fruit fly Bactrocera caudata is a pest species of economic importance in Asia. Its larvae feed on the flowers of Cucurbitaceae such as Cucurbita moschata. To-date it is distinguished from related species based on morphological characters. Specimens of B. caudata from Peninsular Malaysia and Indonesia (Bali and Lombok) were analysed using the partial DNA sequences of cytochrome c oxidase subunit I (COI) and 16S rRNA genes. Both gene sequences revealed that B. caudata from Peninsular Malays...

  11. Binding of 16S rRNA to chloroplast 30S ribosomal proteins blotted on nitrocellulose.

    Science.gov (United States)

    Rozier, C; Mache, R

    1984-10-11

    Protein-RNA associations were studied by a method using proteins blotted on a nitrocellulose sheet. This method was assayed with Escherichia Coli 30S ribosomal components. In stringent conditions (300 mM NaCl or 20 degrees C) only 9 E. coli ribosomal proteins strongly bound to the 16S rRNA: S4, S5, S7, S9, S12, S13, S14, S19, S20. 8 of these proteins have been previously found to bind independently to the 16S rRNA. The same method was applied to determine protein-RNA interactions in spinach chloroplast 30S ribosomal subunits. A set of only 7 proteins was bound to chloroplast rRNA in stringent conditions: chloroplast S6, S10, S11, S14, S15, S17 and S22. They also bound to E. coli 16S rRNA. This set includes 4 chloroplast-synthesized proteins: S6, S11, S15 and S22. The core particles obtained after treatment by LiCl of chloroplast 30S ribosomal subunit contained 3 proteins (S6, S10 and S14) which are included in the set of 7 binding proteins. This set of proteins probably play a part in the early steps of the assembly of the chloroplast 30S ribosomal subunit.

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

    Science.gov (United States)

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

    2016-01-22

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

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

    Science.gov (United States)

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

    2016-09-01

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

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

    Science.gov (United States)

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

    2015-11-13

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

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

    Science.gov (United States)

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

    2016-02-01

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

  16. Molecular Approaches to Studying Microbial Communities: Targeting the 16S Ribosomal RNA Gene.

    Science.gov (United States)

    Fukuda, Kazumasa; Ogawa, Midori; Taniguchi, Hatsumi; Saito, Mitsumasa

    2016-09-01

    Culture-independent methods to detect microorganisms have been developed in parallel with traditional culture-based methods ever since the classification of bacteria based on 16S rRNA gene sequences was advocated in the 1970s. The development and the prevalence of culture-independent molecular technologies have provided revolutionary progress in microbial studies. The development of these technologies contributes significantly to the research of microorganisms that cannot be detected by traditional methods such as culture-dependent methods.Many molecular methods targeting the 16S rRNA gene, such as fluorescence in situ hybridization (FISH), quantitative PCR, terminal restriction fragment length polymorphism (T-RFLP), denaturing-gradient gel electrophoresis (DGGE), clone library analysis, and next-generation DNA sequencing (NGS) technologies, have been applied to various microbial studies. Notably, the advent of NGS technologies enabled a large-scale research of the bacterial community. Many recent studies using the NGS technologies have revealed that a larger number of bacteria and taxa than previously thought inhabit various parts of the human body and various places on the earth. The principles and characteristics of each molecular method are different, and each method possesses individual advantages; for example target specificity, comprehensiveness, rapidness, and cost efficiency. Therefore it is important that the methods used in studies are suitable for the objective and materials. Herein, we highlights molecular approaches targeting the 16S rRNA gene in bacterial community analysis, and focuses on the advantages and limitations of each technology. PMID:27627970

  17. Characterization of copy numbers of 16S rDNA and 16S rRNA of Candidatus Liberibacter asiaticus and the implication in detection in planta using quantitative PCR

    Directory of Open Access Journals (Sweden)

    Wang Nian

    2009-03-01

    Full Text Available Abstract Background Citrus Huanglongbing (HLB is one of the most devastating diseases on citrus and is associated with Candidatus Liberibacter spp.. The pathogens are phloem limited and have not been cultured in vitro. The current management strategy of HLB is to remove infected citrus trees and reduce psyllid populations with insecticides to prevent the spreading. This strategy requires sensitive and reliable diagnostic methods for early detection. Results We investigated the copy numbers of the 16S rDNA and 16S rRNA of the HLB pathogen and the implication of improving the diagnosis of HLB for early detection using Quantitative PCR. We compared the detection of HLB with different Quantitative PCR based methods with primers/probe targeting either 16S rDNA, beta-operon DNA, 16S rRNA, or beta-operon RNA. The 16S rDNA copy number of Ca. Liberibacter asiaticus was estimated to be three times of that of the beta-operon region, thus allowing detection of lower titer of Ca. L. asiaticus. Quantitative reverse transcriptional PCR (QRT-PCR indicated that the 16S rRNA averaged 7.83 times more than that of 16S rDNA for the same samples. Dilution analysis also indicates that QRT-PCR targeting 16S rRNA is 10 time more sensitive than QPCR targeting 16S rDNA. Thus QRT-PCR was able to increase the sensitivity of detection by targeting 16S rRNA. Conclusion Our result indicates that Candidatus Liberibacter asiaticus contains three copies of 16S rDNA. The copy number of 16S rRNA of Ca. L. asiaticus in planta averaged about 7.8 times of 16S rDNA for the same set of samples tested in this study. Detection sensitivity of HLB could be improved through the following approaches: using 16S rDNA based primers/probe in the QPCR assays; and using QRT-PCR assays targeting 16S rRNA.

  18. Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies

    KAUST Repository

    Wang, Yong

    2009-10-09

    Bacterial 16S ribosomal DNA (rDNA) amplicons have been widely used in the classification of uncultured bacteria inhabiting environmental niches. Primers targeting conservative regions of the rDNAs are used to generate amplicons of variant regions that are informative in taxonomic assignment. One problem is that the percentage coverage and application scope of the primers used in previous studies are largely unknown. In this study, conservative fragments of available rDNA sequences were first mined and then used to search for candidate primers within the fragments by measuring the coverage rate defined as the percentage of bacterial sequences containing the target. Thirty predicted primers with a high coverage rate (>90%) were identified, which were basically located in the same conservative regions as known primers in previous reports, whereas 30% of the known primers were associated with a coverage rate of <90%. The application scope of the primers was also examined by calculating the percentages of failed detections in bacterial phyla. Primers A519-539, E969- 983, E1063-1081, U515 and E517, are highly recommended because of their high coverage in almost all phyla. As expected, the three predominant phyla, Firmicutes, Gemmatimonadetes and Proteobacteria, are best covered by the predicted primers. The primers recommended in this report shall facilitate a comprehensive and reliable survey of bacterial diversity in metagenomic studies. © 2009 Wang, Qian.

  19. Overexpression of Salmonella enterica serovar Typhi recA gene confers fluoroquinolone resistance in Escherichia coli DH5α.

    Science.gov (United States)

    Yassien, M A M; Elfaky, M A

    2015-11-01

    A spontaneous fluoroquinolone-resistant mutant (STM1) was isolated from its parent Salmonella enterica serovar Typhi (S. Typhi) clinical isolate. Unlike its parent isolate, this mutant has selective resistance to fluoroquinolones without any change in its sensitivity to various other antibiotics. DNA gyrase assays revealed that the fluoroquinolone resistance phenotype of the STM1 mutant did not result from alteration of the fluoroquinolone sensitivity of the DNA gyrase isolated from it. To study the mechanism of fluoroquinolone resistance, a genomic library from the STM1 mutant was constructed in Escherichia coli DH5α and two recombinant plasmids were obtained. Only one of these plasmids (STM1-A) conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. The chromosomal insert from STM1-A, digested with EcoRI and HindIII restriction endonucleases, produced two DNA fragments and these were cloned separately into pUC19 thereby generating two new plasmids, STM1-A1 and STM1-A2. Only STM1-A1 conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. Sequence and subcloning analyses of STM1-A1 showed the presence of an intact RecA open reading frame. Unlike that of the wild-type E. coli DH5α, protein analysis of a crude STM1-A1 extract showed overexpression of a 40 kDa protein. Western blotting confirmed the 40 kDa protein band to be RecA. When a RecA PCR product was cloned into pGEM-T and introduced into E. coli DH5α, the STM1-A11 subclone retained fluoroquinolone resistance. These results suggest that overexpression of RecA causes selective fluoroquinolone resistance in E. coli DH5α. PMID:26375447

  20. Biochemical and Cellular Characterization of Helicobacter pylori RecA, a Protein with High-Level Constitutive Expression▿†

    OpenAIRE

    Orillard, Emilie; Radicella, J. Pablo; Marsin, Stéphanie

    2011-01-01

    Helicobacter pylori is a bacterial pathogen colonizing half of the world's human population. It has been implicated in a number of gastric diseases, from asymptomatic gastritis to cancer. It is characterized by an amazing genetic variability that results from high mutation rates and efficient DNA homologous recombination and transformation systems. Here, we report the characterization of H. pylori RecA (HpRecA), a protein shown to be involved in DNA repair, transformation, and mouse colonizat...

  1. Overexpression of Salmonella enterica serovar Typhi recA gene confers fluoroquinolone resistance in Escherichia coli DH5α

    Directory of Open Access Journals (Sweden)

    M.A.M. Yassien

    2015-11-01

    Full Text Available A spontaneous fluoroquinolone-resistant mutant (STM1 was isolated from its parent Salmonella enterica serovar Typhi (S. Typhi clinical isolate. Unlike its parent isolate, this mutant has selective resistance to fluoroquinolones without any change in its sensitivity to various other antibiotics. DNA gyrase assays revealed that the fluoroquinolone resistance phenotype of the STM1 mutant did not result from alteration of the fluoroquinolone sensitivity of the DNA gyrase isolated from it. To study the mechanism of fluoroquinolone resistance, a genomic library from the STM1 mutant was constructed in Escherichia coli DH5α and two recombinant plasmids were obtained. Only one of these plasmids (STM1-A conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. The chromosomal insert from STM1-A, digested with EcoRI and HindIII restriction endonucleases, produced two DNA fragments and these were cloned separately into pUC19 thereby generating two new plasmids, STM1-A1 and STM1-A2. Only STM1-A1 conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. Sequence and subcloning analyses of STM1-A1 showed the presence of an intact RecA open reading frame. Unlike that of the wild-type E. coli DH5α, protein analysis of a crude STM1-A1 extract showed overexpression of a 40 kDa protein. Western blotting confirmed the 40 kDa protein band to be RecA. When a RecA PCR product was cloned into pGEM-T and introduced into E. coli DH5α, the STM1-A11 subclone retained fluoroquinolone resistance. These results suggest that overexpression of RecA causes selective fluoroquinolone resistance in E. coli DH5α.

  2. recA and catalase in H sub 2 O sub 2 -mediated toxicity in Neisseria gonorrhoeae

    Energy Technology Data Exchange (ETDEWEB)

    Hassett, D.J.; Charniga, L.; Cohen, M.S. (Univ. of North Carolina, Chapel Hill (USA))

    1990-12-01

    Neisseria gonorrhoeae cells defective in the biosynthesis of the recA gene product are no more sensitive to hydrogen peroxide than wild-type cells. Although gonococci possess nearly 100-fold-greater catalase levels than Escherichia coli, they are more susceptible to hydrogen peroxide than this organism. The natural niche of gonococci undoubtedly results in exposure to oxidant stress; however, they do not demonstrate particularly efficient antioxidant defense systems.

  3. DNA breakage, repair and lethality after 125I decay in rec+ and recA strains of Escherichia coli

    International Nuclear Information System (INIS)

    Iodine-125 decays by electron capture and is known to cause extensive molecular fragmentation via the Auger effect. 125I was incorporated into the DNA of exponentially-growing E. coli K12 AB2487, a recA mutant, and E. coli K12 AB2497, the corresponding rec+ strain as 5-iododeoxyuridine (IUdR), an analogue of thymidine. Radioactive bacteria were stored at -1960C, and samples were periodically assayed for loss of viability and for the induction of double-strand breaks (DSBs) in DNA. Each 125I decay in the DNA of either strain induced one DSB, i.e. proportional to (DSB) = 1.0. For the recA strain, proportional to (lethal) = 0.9 and for the rec+ strain, 0.4. Assays for biological repair of DSBs, involving incubation of thawed samples in growth-medium at 370C before the extraction of DNA, demonstrated significant repair of 125I-induced DSBs by rec+ cells but none by recA cells. For small numbers of decays, there was approximately a 1 : 1 correlation, for either strain, between lethal decays and post-incubation residual DSBs. Comparison with data for larger numbers of decays indicated that a typical rec+ cell can repair no more than three to four DSBs per completed genome (2.5 x 109 daltons). (author)

  4. RecA Binding to a Single Double-Stranded DNA Molecule: A Possible Role of DNA Conformational Fluctuations

    Science.gov (United States)

    Leger, J. F.; Robert, J.; Bourdieu, L.; Chatenay, D.; Marko, J. F.

    1998-10-01

    Most genetic regulatory mechanisms involve protein-DNA interactions. In these processes, the classical Watson-Crick DNA structure sometimes is distorted severely, which in turn enables the precise recognition of the specific sites by the protein. Despite its key importance, very little is known about such deformation processes. To address this general question, we have studied a model system, namely, RecA binding to double-stranded DNA. Results from micromanipulation experiments indicate that RecA binds strongly to stretched DNA; based on this observation, we propose that spontaneous thermal stretching fluctuations may play a role in the binding of RecA to DNA. This has fundamental implications for the protein-DNA binding mechanism, which must therefore rely in part on a combination of flexibility and thermal fluctuations of the DNA structure. We also show that this mechanism is sequence sensitive. Theoretical simulations support this interpretation of our experimental results, and it is argued that this is of broad relevance to DNA-protein interactions.

  5. Microvariation artifacts introduced by PCR and cloning of closely related 16S rRNA gene sequences

    NARCIS (Netherlands)

    Speksnijder, A.G.C.L.; Kowalchuk, G.A.; Jong, S. de; Kline, E.; Stephen, J.R.; Laanbroek, H.J.

    2001-01-01

    A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a m

  6. Microvariation Artifacts Introduced by PCR and Cloning of Closely Related 16S rRNA Gene Sequences

    NARCIS (Netherlands)

    Speksnijder, A.G.C.L.; Kowalchuk, G.A.; Jong, de S.; Kline, E.; Stephen, J.R.; Laanbroek, H.J.

    2001-01-01

    A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a m

  7. A comparison of rpoB and 16S rRNA as markers in pyrosequencing studies of bacterial diversity

    NARCIS (Netherlands)

    Vos, M.; Quince, C.; Pijl, A.S.; De Hollander, M.; Kowalchuk, G.A.

    2012-01-01

    Background The 16S rRNA gene is the gold standard in molecular surveys of bacterial and archaeal diversity, but it has the disadvantages that it is often multiple-copy, has little resolution below the species level and cannot be readily interpreted in an evolutionary framework. We compared the 16S r

  8. Characterization of the Pseudomonas aeruginosa recA gene: the Les- phenotype

    International Nuclear Information System (INIS)

    The Les- phenotype (lysogeny establishment deficient) is a pleiotropic effect of the lesB908 mutation of Pseudomonas aeruginosa PAO. lesB908-containing strains are also (i) deficient in general recombination, (ii) sensitive to UV irradiation, and (iii) deficient in UV-stimulated induction of prophages. The P. aeruginosa recA-containing plasmid pKML3001 complemented each of these pleiotropic characteristics of the lesB908 mutation, supporting the hypothesis that lesB908 is an allele of the P. aeruginosa recA gene. The phenotypic effects of the lesB908 mutation may be best explained by the hypothesis that the lesB908 gene product is altered in such a way that it has lost synaptase activity but possesses intrinsic protease activity in the absence of DNA damage. The Les- phenotype is a result of the rapid destruction of newly synthesized phage repressor, resulting in lytic growth of the infecting virus. This hypothesis is consistent with the observations that increasing the number of copies of the phage repressor gene by increasing the multiplicity of infection (i.e., average number of phage genomes per cell) or by introducing the cloned phage repressor gene into a lesB908 mutant will also suppress the Les- phenotype in a phage-specific fashion

  9. Genomic and Haplotype Comparison of Butanol Producing Bacteria Based on 16S rDNA

    Directory of Open Access Journals (Sweden)

    Ekwan Nofa Wiratno

    2016-04-01

    Full Text Available High butanol demand for transportation fuel triggers butanol production development. Exploration of butanolproducing bacteria using genomic comparison and biogeography will help to develop butanol industry. The objectives of this research were butanol production, genome comparison and haplotype analysis of butanolproducing bacteria from Ranu Pani Lake sediment using 16S rDNA sequences. The highest butanol concentrations were showed by Paenibacillus polymyxa RP 2.2 isolate (10.34 g.L-1, followed by Bacillus methylotrophicus RP 3.2 and B. methylotrophicus RP 7.2 isolate (10.11 g.L-1 and 9.63 g.L-1 respectively. Paenibacillus polymyxa RP 2.2 showed similarity in nucleotide composition (ATGC with B. methylotrophicus RP 3.2, B. methylotrophicus RP 7.2, P. polymyxa CR1, Bacillus amyloliquefaciens NELB-12, and Paenibacillus polymyxa WR-2. Clostridium acetobutylicum ATCC 824 showed similarity in nucleotide composition (ATGC with Clostridium saccharoperbutylacetonicum N1-4, and Clostridium saccharobutylicum Ox29. The lowest G+C content was C. saccharobutylicum Ox29 (51.35%, and the highest was B. methylotrophicus RP 7.2 (55.33%. Conserved region of 16S rDNA (1044 bp were consisted of 17 conserved sequences. The number of Parsimony Informative Site (PIS was 319 spot and single tone was 48 spot. We found in this study that all of butanolproducing bacterial DNA sequences have clustered to 8 haplotypes. Based on the origin of sample, there were three haplotype groups. Bacteria from group A were could produce butanol 8.9-10.34 g.L-1, group B 9.2-14.2 g.L-1 and group C was could produce butanol 0.47 g.L-1. The haplotype analysis of bacteria based on 16S rDNA sequences in this study could predict capability of butanol production.

  10. CORE: a phylogenetically-curated 16S rDNA database of the core oral microbiome.

    Directory of Open Access Journals (Sweden)

    Ann L Griffen

    Full Text Available Comparing bacterial 16S rDNA sequences to GenBank and other large public databases via BLAST often provides results of little use for identification and taxonomic assignment of the organisms of interest. The human microbiome, and in particular the oral microbiome, includes many taxa, and accurate identification of sequence data is essential for studies of these communities. For this purpose, a phylogenetically curated 16S rDNA database of the core oral microbiome, CORE, was developed. The goal was to include a comprehensive and minimally redundant representation of the bacteria that regularly reside in the human oral cavity with computationally robust classification at the level of species and genus. Clades of cultivated and uncultivated taxa were formed based on sequence analyses using multiple criteria, including maximum-likelihood-based topology and bootstrap support, genetic distance, and previous naming. A number of classification inconsistencies for previously named species, especially at the level of genus, were resolved. The performance of the CORE database for identifying clinical sequences was compared to that of three publicly available databases, GenBank nr/nt, RDP and HOMD, using a set of sequencing reads that had not been used in creation of the database. CORE offered improved performance compared to other public databases for identification of human oral bacterial 16S sequences by a number of criteria. In addition, the CORE database and phylogenetic tree provide a framework for measures of community divergence, and the focused size of the database offers advantages of efficiency for BLAST searching of large datasets. The CORE database is available as a searchable interface and for download at http://microbiome.osu.edu.

  11. Clinical identification of bacteria in human chronic wound infections: culturing vs. 16S ribosomal DNA sequencing

    Directory of Open Access Journals (Sweden)

    Rhoads Daniel D

    2012-11-01

    Full Text Available Abstract Background Chronic wounds affect millions of people and cost billions of dollars in the United States each year. These wounds harbor polymicrobial biofilm communities, which can be difficult to elucidate using culturing methods. Clinical molecular microbiological methods are increasingly being employed to investigate the microbiota of chronic infections, including wounds, as part of standard patient care. However, molecular testing is more sensitive than culturing, which results in markedly different results being reported to clinicians. This study compares the results of aerobic culturing and molecular testing (culture-free 16S ribosomal DNA sequencing, and it examines the relative abundance score that is generated by the molecular test and the usefulness of the relative abundance score in predicting the likelihood that the same organism would be detected by culture. Methods Parallel samples from 51 chronic wounds were studied using aerobic culturing and 16S DNA sequencing for the identification of bacteria. Results One hundred forty-five (145 unique genera were identified using molecular methods, and 68 of these genera were aerotolerant. Fourteen (14 unique genera were identified using aerobic culture methods. One-third (31/92 of the cultures were determined to be Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis with higher relative abundance scores were more likely to be detected by culture as demonstrated with regression modeling. Conclusion Discordance between molecular and culture testing is often observed. However, culture-free 16S ribosomal DNA sequencing and its relative abundance score can provide clinicians with insight into which bacteria are most abundant in a sample and which are most likely to be detected by culture.

  12. Globicatella sanguinis bacteraemia identified by partial 16S rRNA gene sequencing

    DEFF Research Database (Denmark)

    Abdul-Redha, Rawaa Jalil; Balslew, Ulla; Christensen, Jens Jørgen;

    2007-01-01

    Globicatella sanguinis is a gram-positive coccus, resembling non-haemolytic streptococci. The organism has been isolated infrequently from normally sterile sites of humans. Three isolates obtained by blood culture could not be identified by Rapid 32 ID Strep, but partial sequencing of the 16S r......RNA gene revealed the identity of the isolated bacteria, and supplementary biochemical tests confirmed the species identification. The cases histories illustrate the dilemma of finding relevant, newly recognized, opportunistic pathogens and the identification achievement (s) that can be obtained by using...

  13. Molecular phylogeny of silk-producing insects based on 16S ribosomal RNA and cytochrome oxidase subunit I genes

    Indian Academy of Sciences (India)

    B. Mahendran; S. K. Ghosh; S. C. Kundu

    2006-04-01

    We have examined the molecular-phylogenetic relationships between nonmulberry and mulberry silkwormspecies that belong to the families Saturniidae, Bombycidae and Lasiocampidae using 16S ribosomal RNA (16S rRNA) and cytochrome oxidase subunit I (coxI) gene sequences. Aligned nucleotide sequences of 16S rRNA and coxI from 14 silk-producing species were used for construction of phylogenetic trees by maximum likelihood and maximum parsimony methods. The tree topology on the basis of 16S rRNA supports monophyly for members of Saturniidae and Bombycidae. Weighted parsimony analysis weighted towards transversions relative to transitions (ts, tv4) for coxI resulted in more robust bootstrap support over unweighted parsimony and favours the 16S rRNA tree topology. Combined analysis reflected clear biogeographic pattern, and agrees with morphological and cytological data.

  14. 16S rRNA甲基化介导的氨基糖苷类耐药%Resistance mechanism against aminoglycosides mediated by 16S rRNA methylation

    Institute of Scientific and Technical Information of China (English)

    张晓文

    2012-01-01

    Aminoglycosid.es have been used for the treatment of a broad range of life -threatening Gram-positive and Grarrmeg-ative bacterial infections. These agents bind to the A site of the 16S rRNA of the bacterial 30S ribosomal subunit and subsequently block its growth through interference with its protein synthesis . 16S rRNA methylation is capable of conferring an extraordinarily high level of resistance against most of the clinically important aminoglycosides . Previous research has shown that this phenomenon is media -ted by some 16S rRNA methylase. Because of the clinical importance of these enzymes , further global dissemination of 16S rRNA methylase genes among pathogenic bacilli will be a cause of great concern in the near future . This article presents an overview on the action mechanism, origin, classification and genetic environment of 16S rRNA methylase.%氨基糖苷类抗生素在治疗革兰阳性和阴性细菌引起的感染中起着重要的作用,可通过与细菌30S核糖体亚基的16S rRNA的A位点结合而阻碍蛋白质的合成.16S rRNA甲基化作用可导致细菌对氨基糖苷类药物高水平耐药,大量研究显示这一现象是由一类16S rRNA甲基化酶所介导的.由于16S rRNA甲基化酶在临床上的重要性,为引起医务人员的重视,文中将从此类酶的作用机制、起源、分类以及基因环境等方面作一综述.

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

    Science.gov (United States)

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

    2016-01-01

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

  16. GENE 16S RRNA SEQUENCE PHYLOGENETIC ANALYSIS OF LYSINE PRODUCERS STRAINS

    Directory of Open Access Journals (Sweden)

    G. S. Andriiash

    2014-12-01

    Full Text Available The phylogenetic relationships of strainsproducers of essential amino acids of aspartate family Brevibacterium sp. UCM Ac-674 (Brevibacterium sp. 90, Brevibacterium sp. IMV Ac-5004 (Brevibacterium sp. 90H, Brevibacterium sp. UCM Ac-675 (Brevibacterium sp. E531, mutant strain Brevibacterium sp. IMV B-7447 from the «Collections strains and lines of plants for food and agricultural biotechnology SO “Institute for Food Biotechnology and Genomics” of National Academy of Sciences of Ukraine were investigated. The affiliation strain Brevibacterium sp. IMV B-7447 to the genus Brevibacterium within the sequences of the genes based on 16S rRNA was confirmed. The dendogram of phylogenetic relationships of studied strains and related strains Brevibacterium from database GenBank was constructed. It was shown that by the criterion of homology gene sequences based on 16S rRNA the investigated strains-producers belong to three phylogenetic groups. It was established that the mutant strain Brevibacterium sp. ІMV B-7447 has no analogues in the database GenBank.

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

    Directory of Open Access Journals (Sweden)

    Muhammad Naveed

    2014-09-01

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

  18. Sequence analysis of mitochondrial 16S ribosomal RNA gene fragment from seven mosquito species

    Indian Academy of Sciences (India)

    Yogesh S Shouche; Milind S Patole

    2000-12-01

    Mosquitoes are vectors for the transmission of many human pathogens that include viruses, nematodes and protozoa. For the understanding of their vectorial capacity, identification of disease carrying and refractory strains is essential. Recently, molecular taxonomic techniques have been utilized for this purpose. Sequence analysis of the mitochondrial 16S rRNA gene has been used for molecular taxonomy in many insects. In this paper, we have analysed a 450 bp hypervariable region of the mitochondrial 16S rRNA gene in three major genera of mosquitoes, Aedes, Anopheles and Culex. The sequence was found to be unusually A + T rich and in substitutions the rate of transversions was higher than the transition rate. A phylogenetic tree was constructed with these sequences. An interesting feature of the sequences was a stretch of Ts that distinguished between Aedes and Culex on the one hand, and Anopheles on the other. This is the first report of mitochondrial rRNA sequences from these medically important genera of mosquitoes.

  19. Molecular phylogeny of western Atlantic Farfantepenaeus and Litopenaeus shrimp based on mitochondrial 16S partial sequences.

    Science.gov (United States)

    Maggioni, R; Rogers, A D; Maclean, N; D'Incao, F

    2001-01-01

    Partial sequences for the 16S rRNA mitochondrial gene were obtained from 10 penaeid shrimp species: Farfantepenaeus paulensis, F. brasiliensis, F. subtilis, F. duorarum, F. aztecus, Litopenaeus schmitti, L. setiferus, and Xiphopenaeus kroyeri from the western Atlantic and L. vannamei and L. stylirostris from the eastern Pacific. Sequences were also obtained from an undescribed morphotype of pink shrimp (morphotype II) usually identified as F. subtilis. The phylogeny resulting from the 16S partial sequences showed that these species form two well-supported monophyletic clades consistent with the two genera proposed in a recent systematic review of the suborder Dendrobranchiata. This contrasted with conclusions drawn from recent molecular phylogenetic work on penaeid shrimps based on partial sequences of the mitochondrial COI region that failed to support recent revisions of the Dendrobranchiata based on morphological analysis. Consistent differences observed in the sequences for morphotype II, coupled with previous allozyme data, support the conclusion that this is a previously undescribed species of Farfantepenaeus. PMID:11161743

  20. Phylogenetic analyses of some genera in Oedipodidae (Orthoptera: Acridoidea) based on 16S mitochondrial partialgene sequences

    Institute of Scientific and Technical Information of China (English)

    Xiang-Chu Yin; Xin-Jiang Li; Wen-Qiang Wang; Hong Yin; Cheng-Quan Cao; Bao-Hua Ye; Zhan Yin

    2008-01-01

    Based on the 16S mitochondrial partial gene sequences of 29 genera, containing 26 from Oedipodidae and one each from Tanaoceridae, Pyrgomorphidae and Tetrigidae (as outgroups), the homologus sequences were compared and phylogenetic analyses were performed. A phylogenetic tree was inferred by neighbor-joining (N J). The results of sequences compared show that: (i) in a total of 574 bp of Oedipodidae, the number of substituted nucleotides was 265 bp and the average percentages ofT, C, A and G were 38.3%,11.4%, 31.8% and 18.5%, respectively, and the content of A+T (70.1%) was distinctly richer than that of C+G (29.9%); and (ii) the average nucleotide divergence of 16S rDNA sequences among genera of Oedipodidae were 9.0%, among families of Acridoidea were 17.0%, and between superfamilies (Tetrigoidea and Acridoidea) were 23.9%, respectively. The phylogenetic tree indicated: (i) the Oedipodidae was a monophyletic group, which suggested that the taxonomic status of this family was confLrrned; (ii) the genus Heteropternis separated from the other Oedipodids first and had another unique sound-producing structure in morphology, which is the type-genus of subfamily Heteropterninae; and (iii) the relative intergeneric relationship within the same continent was closer than that of different continents, and between the Eurasian genera and the African genera, was closer than that between Eurasians and Americans.

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

    Science.gov (United States)

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

    2014-01-01

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

  2. Phylogenetic analysis of 16S mitochondrial DNA data in sloths and anteaters

    Directory of Open Access Journals (Sweden)

    Maria Claudene Barros

    2003-01-01

    Full Text Available We sequenced part of the 16S rRNA mitochondrial gene in 17 extant taxa of Pilosa (sloths and anteaters and used these sequences along with GenBank sequences of both extant and extinct sloths to perform phylogenetic analysis based on parsimony, maximum-likelihood and Bayesian methods. By increasing the taxa density for anteaters and sloths we were able to clarify some points of the Pilosa phylogenetic tree. Our mitochondrial 16S results show Bradypodidae as a monophyletic and robustly supported clade in all the analysis. However, the Pleistocene fossil Mylodon darwinii does not group significantly to either Bradypodidae or Megalonychidae which indicates that trichotomy best represents the relationship between the families Mylodontidae, Bradypodidae and Megalonychidae. Divergence times also allowed us to discuss the taxonomic status of Cyclopes and the three species of three-toed sloths, Bradypus tridactylus, Bradypus variegatus and Bradypus torquatus. In the Bradypodidae the split between Bradypus torquatus and the proto-Bradypus tridactylus / B. variegatus was estimated as about 7.7 million years ago (MYA, while in the Myrmecophagidae the first offshoot was Cyclopes at about 31.8 MYA followed by the split between Myrmecophaga and Tamandua at 12.9 MYA. We estimate the split between sloths and anteaters to have occurred at about 37 MYA.

  3. Sequencing of an atypical Brucella strain 16S rDNA%1株非典型布鲁杆菌的16S rDNA序列分析

    Institute of Scientific and Technical Information of China (English)

    刘志国; 王妙; 刘日宏; 崔步云

    2015-01-01

    目的 分析非典型布鲁杆菌的16S rDNA序列,评价16S rDNA序列测定方法鉴定布鲁杆菌的可行性.方法 用常规方法对l株非典型菌株进行初步鉴定;提取菌株的DNA,双向测定16S rDNA全序列,在NCBI对该序列进行Blast比对,用DNAMAN软件进行菌株的16S rDNA序列一致性比对;同时,从GenBank下载与布鲁杆菌有血清学交叉反应菌株的16S rDNA全序列,用MEGA 6.0构建16S rDNA系统发生树.结果 常规鉴定显示,试验菌株为非典型布鲁杆菌.试验菌株的16S rDNA序列与布鲁杆菌基因的相似性为99%,与牛种布鲁杆菌544A、羊种布鲁杆菌16M的16S rDNA序列的一致性为96.99%.系统发生树显示,试验菌株为布鲁杆菌,且与牛种布鲁杆菌544A、羊种布鲁杆菌16M有较为密切的亲缘关系.结论 试验菌株为非典型布鲁杆菌,16S rDNA序列测定是一种简便、快速、准确的非典型布鲁杆菌鉴定方法.%Objective To sequence an atypical Brucella strain 16S rDNA,and to evaluate the feasibility of 16S rDNA sequencing method for identification of Brucella.Methods Preliminary identification of atypical strains was carried out with conventional method.Strain DNA was extracted,and 16S rDNA complete sequence was bidirectional sequenced,and Blast in NCBI and DNAMAN software were used for comparison of the sequence identities of the 16S rDNA.Moreover,16S rDNA complete sequence of the stains those were known to cross-react serologically with Brucella was downloaded from GenBank,MEGA 6.0 was used to construct the phylogenetic tree.Results The conventional identification results revealed that it was an atypical Brucella,the gene similarity between the sequences of the test strain 16S rDNA and Brucella was 99%,between 16S rDNA sequence of Brucella abortus 544A and Brucella melitensis 16M was 96.99%.Phylogenetic tree revealed that the test stain was a Brucella,and closely related to Brucella abortus 544A and Brucella melitensis 16M.Conclusion The

  4. Enhanced DNA binding affinity of RecA protein from Deinococcus radiodurans.

    Science.gov (United States)

    Warfel, Jaycob D; LiCata, Vince J

    2015-07-01

    Deinococcus radiodurans (Dr) has a significantly more robust DNA repair response than Escherichia coli (Ec), which helps it survive extremely high doses of ionizing radiation and prolonged periods of desiccation. DrRecA protein plays an essential part in this DNA repair capability. In this study we directly compare the binding of DrRecA and EcRecA to the same set of short, defined single (ss) and double stranded (ds) DNA oligomers. In the absence of cofactors (ATPγS or ADP), DrRecA binds to dsDNA oligomers more than 20 fold tighter than EcRecA, and binds ssDNA up to 9 fold tighter. Binding to dsDNA oligomers in the absence of cofactor presumably predominantly monitors DNA end binding, and thus suggests a significantly higher affinity of DrRecA for ds breaks. Upon addition of ATPγS, this species-specific affinity difference is nearly abolished, as ATPγS significantly decreases the affinity of DrRecA for DNA. Other findings include that: (1) both proteins exhibit a dependence of binding affinity on the length of the ssDNA oligomer, but not the dsDNA oligomer; (2) the salt dependence of binding is modest for both species of RecA, and (3) in the absence of DNA, DrRecA produces significantly shorter and/or fewer free-filaments in solution than does EcRecA. The results suggest intrinsic biothermodynamic properties of DrRecA contribute directly to the more robust DNA repair capabilities of D. radiodurans.

  5. 福建华溪蟹线粒体DNA COI和16S rRNA基因序列的遗传多样性%Genetic diversity on mitochondrial DNA COI and 16S rRNA of Sinopotamon fukienense

    Institute of Scientific and Technical Information of China (English)

    石林波; 张小燕; 汪雁; 王云龙; 周宪民; 邹节新

    2013-01-01

    目的 探讨福建华溪蟹(Sinopotamonfukienense)的遗传多样性.方法 采用PCR结合DNA测序技术,测定S.fukienense的线粒体COI和16S rRNA基因序列的组成.经比对获得639 bp长度的COI基因序列和526 bp的16S rRNA基因序列,以对比分析S.fukienense的遗传多样性.结果 S.fukienense基于COI基因核苷酸多样性(Pi)为0.048 4,高于其基于16S rRNA基因核苷酸多样性(Pi)为0.021 6.同时,S.fukienense基于COI基因单倍型间的平均遗传距离(P)为0.048,大于其基于16S rRNA基因单倍型间的平均遗传距离(P)0.026.结论 COI序列在分析S.fukienense遗传异变时的作用更优于16S rRNA基因序列.%The aim of this study was to explore the genetic diversity of the freshwater crab S.fukienense based on the gene sequence CO I and 16S rRNA.The genes of CO I and 16S rRNA were amplified with PCR and subsequently sequenced.The base composition and sequence variation of them were analyed with Mega version 4.0 and DnaSP version 4.10.We obtained 639 bp nucleotide sequence of gene COI and 526 bp nucleotide sequence of gene 16S rRNA.The nucleotide diversity based on gene COI (Pi=0.048 4) was higher than that based on 16S rRNA (Pi=0.021 6).At the same time,the average genetic distance of haplotypes based on gene COI (P=0.048) was larger than that based on 16S rRNA (P=0.026).Results suggest that the COI sequence is better than the 16S rRNA sequence in the analysis of genetic mutation for S.fukienense.

  6. 16S rRNA基因测序技术在肝脓疡细菌鉴定中的作用%Usefulness of 16S rRNA Gene Sequencing for Identification of Bacteria from Pyogenic Liver Abscess

    Institute of Scientific and Technical Information of China (English)

    宋伦圭

    2014-01-01

    目的:评价16S核糖体RNA (rRNA)基因测序在肝脓疡中细菌鉴定中的应用价值。方法2012年1月-2013年12月间共20例肝脓疡行经皮置管引流的患者,分别行脓液培养,血培养和16S rRNA基因测序。利用454 GS Junior System对脓液基因组DNA行PCR和16S rRNA基因测序。脓液培养,血液培养和16S rRNA 基因测序结果进行分别评价。结果脓液和血液培养阳性的患者分别是9例(45%)和4例(20%)。16S rRNA基因测序细菌鉴定率为90%,明显高于传统的培养方法。结论16S rRNA基因测序方法较传统的培养方法能更准确和有效对肝脓疡进行细菌鉴定。%Background/Aims To evaluate the usefulness of 16S ribosomal RNA (rRNA) gene sequencing for an accurate and better identification of bacteria from pyogenic liver abscess (PLA). Methodology 20 patients with PLA were included who underwent percutaneous catheter drainage, abscess culture, blood culture and 16S rRNA gene sequencing for isolates from January 2012 to December 2013. Genomic DNAs of abscess fluids were subjected to PCR and sequencing of 16S rRNA gene by on a 454 GS Junior System. The results were evaluated between abscess cultures, blood and 16S rRNA gene sequencing for isolates. Results Abscess and blood cultures were positive in 9 (45%) and 4 (20%) patients, respectively. The 16S rRNA gene sequencing showed with 90% identification of bacteria a significantly greater identification than conventional cultured methods. Conclusion This study showed a greater usefulness of 16S rRNA gene sequencing than conventional cultured methods for accurate and better identification of bacteria from PLA.

  7. Identiifcation of pathogenic microorganism by sequencing 16S rRNA gene%16S rRNA基因序列分析法鉴定病原细菌

    Institute of Scientific and Technical Information of China (English)

    朱飞舟; 陈利玉; 陈汉春

    2013-01-01

    目的:运用16S rRNA 基因序列分析法鉴定14种细菌,为该方法的临床应用奠定基础。方法:提取细菌DNA,采用通用引物PCR扩增16S rRNA 基因片段并测序。将测序结果用Blastn 在线软件在Nucleotide 数据库中进行序列同源性比对,根据序列同源性鉴定病原细菌。结果:12种细菌可以鉴定到“种”,2种细菌可以鉴定到“属”。结论:16S rRNA 基因序列分析是一种有效的病原细菌鉴定方法。%Objective: To identify 14 bacteria by sequencing the 16S rRNA gene and establish the basis for clinical application in the future. Methods: DNA samples of the 14 bacteria were extracted. The 16S rRNA genes were amplified by PCR and sequenced with common primers. The sequences of the 16S rRNA genes were aligned by online software Blastn in nucleotide database. The bacteria were identified according to the homology of their 16S rRNA genes. Results: Twelve bacteria were classified to species, the other 2 bacteria were classified to genus. Conclusion: 16S rRNA gene sequence analysis is useful in identifying pathogenic bacteria.

  8. Diversity of 16S rDNA and environmental factor influencing microorganisms in Malan ice core

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The research on extrempholic microorganisms in glacial low-temperature environment receives more attention than ever before. Due to the successive chronological records in ice core, it is important to initiate microbiological studies on ice core samples. 23 samples from one ice core, drilled from central Qinghai-Tibetan Plateau, were analyzed. The number of total microorganisms and culturable microorganisms in different layers showed that it related with the content of dust in ice. It is suggested that the distribution of microorganisms in ice depends on the transportation of materials during glacier development. The bacteria diversity in Malan Glacier was analyzed by 16S rDNA sequencing methods, which showed that many sequences were similar to known psychrophilic bacteria.

  9. Multi-site-specific 16S rRNA methyltransferase RsmF from Thermus thermophilus

    DEFF Research Database (Denmark)

    Demirci, Hasan; Larsen, Line H G; Hansen, Trine;

    2010-01-01

    Cells devote a significant effort toward the production of multiple modified nucleotides in rRNAs, which fine tune the ribosome function. Here, we report that two methyltransferases, RsmB and RsmF, are responsible for all four 5-methylcytidine (m(5)C) modifications in 16S rRNA of Thermus...... thermophilus. Like Escherichia coli RsmB, T. thermophilus RsmB produces m(5)C967. In contrast to E. coli RsmF, which introduces a single m(5)C1407 modification, T. thermophilus RsmF modifies three positions, generating m(5)C1400 and m(5)C1404 in addition to m(5)C1407. These three residues are clustered near...

  10. Genetic Diversity in Populations of Sepiella maindroni Using 16S rRNA Gene Sequence Analysis

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Part of the 16S rRNA gene is amplified with PCR and sequenced for 5 populations of common Chinese cuttlefish Sepiella maindroni: three from the South China Sea, one from East China Sea and one from Japan. The result shows that a total of 5 nucleotide positions are found to have gaps or insertions of base pairs among these individuals, and 13 positions are examined to be variable in all the sequences, which range from 494 to 509 base pairs. All of the individuals are grouped into 7 haplotypes (h1-h7). No marked genetic difference is observed among those populations. All of the individuals from Nagasaki belong to h1 and the h3 haplotype is found only in the coastal waters of China. AG transition in Nucleotide 255 is suggested to be taken as a kind of genetic marker to identify the populations distributed in East-South China Sea and the Nagasaki waters of Japan.

  11. discussion on validity of rana maoershanensis based on partial sequence of 16s rrna gene

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    rana maoershanensis found in mt.maoershan in guangxi,china was reported as a new species in 2007,but there was no molecular data for this frog.the partial sequences (543 bp) of 16s rrna gene from 12 specimens of 3 brown frog species (rana hanluica,r.maoershanensis and r.chensinensis) were analyzed with 17 specimens of 9 species from genbank.the nucleotide sequence divergence between r.maoershanensis and the other brown frog species were 4.5%-6.5%,with 22-30 nucleotide substitutions at this locus.the phylogenetic relationships based on mp,ml,and bayesian inference indicate that the brown frogs from southern china were diverged into three groups (clades a,b and c).r.maoershanensis was clustered together a well-supported subclade (b-l).it is suggested that r.maoershanensis is a valid species.

  12. Preliminary study on mitochondrial 16S rRNA gene sequences and phylogeny of flatfishes (Pleuronectiformes)

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    A 605 bp section of mitochondrial 16S rRNA gene from Paralichthys olivaceus, Pseudorhombus cinnamomeus, Psetta maxima and Kareius bicoloratus, which represent 3 families of Order Pleuronectiformes was amplified by PCR and sequenced to show the molecular systematics of Pleuronectiformes for comparison with related gene sequences of other 6 flatfish downloaded from GenBank. Phylogenetic analysis based on genetic distance from related gene sequences of 10 flatfish showed that this method was ideal to explore the relationship between species, genera and families. Phylogenetic trees set-up is based on neighbor-joining, maximum parsimony and maximum likelihood methods that accords to the general rule of Pleuronectiformes evolution. But they also resulted in some confusion. Unlike data from morphological characters, P. olivaceus clustered with K.bicoloratus, but P. cinnamomeus did not cluster with P. olivaceus, which is worth further studying.

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

    Science.gov (United States)

    Dorsch, M; Lane, D; Stackebrandt, E

    1992-01-01

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

  14. 16S rRNA amplicon sequencing dataset for conventionalized and conventionally raised zebrafish larvae.

    Science.gov (United States)

    Davis, Daniel J; Bryda, Elizabeth C; Gillespie, Catherine H; Ericsson, Aaron C

    2016-09-01

    Data presented here contains metagenomic analysis regarding the sequential conventionalization of germ-free zebrafish embryos. Zebrafish embryos that underwent a germ-free sterilization process immediately after fertilization were promptly exposed to and raised to larval stage in conventional fish water. At 6 days postfertilization (dpf), these "conventionalized" larvae were compared to zebrafish larvae that were raised in conventional fish water never undergoing the initial sterilization process. Bacterial 16S rRNA amplicon sequencing was performed on DNA isolated from homogenates of the larvae revealing distinct microbiota variations between the two groups. The dataset described here is also related to the research article entitled "Microbial modulation of behavior and stress responses in zebrafish larvae" (Davis et al., 2016) [1]. PMID:27508247

  15. Extensive 16S rRNA gene sequence diversity in Campylobacter hyointestinalis strains: taxonomic and applied implications

    DEFF Research Database (Denmark)

    Harrington, C.S.; On, Stephen L.W.

    1999-01-01

    Phylogenetic relationships of Campylobacter hyointestinalis subspecies were examined by means of 16S rRNA gene sequencing. Sequence similarities among C. hyointestinalis subsp. lawsonii strains exceeded 99.0 %, but values among C. hyointestinalis subsp. hyointestinalis strains ranged from 96...... of the genus Campylobacter, emphasizing the need for multiple strain analysis when using 16S rRNA gene sequence comparisons for taxonomic investigations....

  16. Species identification and profiling of complex microbial communities using shotgun Illumina sequencing of 16S rRNA amplicon sequences.

    OpenAIRE

    Ong, SH; Kukkillaya, VU; Wilm, A; Lay, C; Ho, EX; Low, L; Hibberd, ML; Nagarajan, N.

    2013-01-01

    The high throughput and cost-effectiveness afforded by short-read sequencing technologies, in principle, enable researchers to perform 16S rRNA profiling of complex microbial communities at unprecedented depth and resolution. Existing Illumina sequencing protocols are, however, limited by the fraction of the 16S rRNA gene that is interrogated and therefore limit the resolution and quality of the profiling. To address this, we present the design of a novel protocol for shotgun Illumina sequenc...

  17. Comparative evolution of the recA gene of surface and deep subsurface microorganisms (an evolutionary clock of intermediate rate). Final report

    Energy Technology Data Exchange (ETDEWEB)

    Miller, R.V.

    1998-04-01

    Because of the ability of the recA protein product to maintain both DNA integrity and increase genetic diversity, this gene may be essential to the survival of microorganisms following the damaging effects of numerous environmental stresses such as exposure to solar UV radiation, exposure to gamma radiation, starvation, and changing environments. While the various activities and amino-acid sequence of recA have been highly conserved among the eubacteria and archaea, little is known as to whether a strict structure-function relationship has been conserved. In other words, are the same regions of this highly plastic, functionally heterogeneous protein involved in the same catalytic capacities throughout the bacterial kingdom? While it is reasonable to assume that this type of conservation has also occurred, we felt it necessary to test the assumption by demonstrating that mutations in different genera of bacteria which eliminate similar functions (i.e., lead to similar phenotypes) are caused by changes in the amino-acid sequence in the same regions of their recA proteins. Therefore, we located the changes in nucleotide sequence in two recA mutants of P. aeruginosa which displayed mutant phenotypes in recombination and UV resistance. Our assumption was that if structure-function relationships held, these mutations would be found in areas already identified as essential for the function of the E. coli recA protein.

  18. Experimental study of 16S rRNA gene sequence analysis for identification of atypical bacteria%16S rRNA基因序列分析鉴定非典型细菌的实验研究

    Institute of Scientific and Technical Information of China (English)

    沈亚娟; 夏云

    2013-01-01

    目的 建立以16S rRNA基因序列分析为基础的细菌鉴定方法,并初步将其应用于临床常规细菌的鉴定.方法 选择临床微生物实验室不能准确鉴定的细菌,以16S rRNA为靶序列,在两端保守区设计引物,PCR反应扩增目的 片段,测序后与数据库中已知细菌的16S rRNA序列进行序列比对.结果 13株菌中,有11株与数据库中的已知16S rRNA序列相似性达99.0%以上,成功鉴定到种的水平.结论 16S rRNA基因序列分析的方法可快速、准确地鉴定不典型菌株,可作为细菌常规鉴定的补充方法.%Objective To establish an approach for bacteria identification based on 16S rRNA gene sequence analysis ,and apply it to clinical routine bacteria identification initially .Methods Bacteria which can not be accurately identified in clinical microbiologi -cal laboratory were selected .16S rRNA served as target sequence ,primers were designed to match the conserved region at both ends and target fragments were amplified by means of PCR reaction .After sequencing ,their sequences were compared with 16S rRNA sequence of known bacteria in the database .Results Among 13 strains ,11 strains showed sequence similarity of 99 .0% with 16S rRNA sequence of known bacteria in the database ,and were successfully identified to the species level .Conclusion 16S rRNA gene sequence analysis can be applied to identify atypical bacteria quickly and accurately ,and serve as a supplementary method of routine bacteria identification .

  19. 16S rRNA is Exchangeable in Escherichia coli%外源16S rRNA在大肠杆菌中的可替换性研究

    Institute of Scientific and Technical Information of China (English)

    李晓丹; 杨瑾; 曹慧; 崔中利

    2007-01-01

    16S rRNA对于构建功能性核糖体是十分重要的,人们普遍将其作为原核生物进化中保守的系统发育标志.为了进一步研究16S rRNA的进化,本文将Escherichia coli中最后一个拷贝的16S rRNA基因替换为Bacillus subtilis的16S rRNA 基因,得到了菌株SQ110BSX.菌株SQ110BSX的代时与出发菌株SQ110基本一致,但是SQ110BSX表现出冷敏感性,而且rRNA/蛋白比值为SQ110的148%.实验结果表明,菌株SQ110BSX中的核糖体效率明显下降.由于E.coli和B.subtilis在遗传距离上较远,两者的可替换性证明了16S rRNA的高度保守性.%16S rRNA plays an important role in the functional construction of ribosomes and is considered to be a conserved phylogenic marker of prokaryotic evolution.To elucidate the evolution of 16S rRNA, we succeeded in replacing the 16S rRNA gene of an E.coli strain with all rrn operons deleted with the 16S rRNA gene of Bacillus subtilis.We found that manipulated strain SQ110BSX showed similar characteristics with the starting E.coli strain SQ110 in terms of generation time.Strain SQ110BSX was cold sensitive, and the rRNA/protein ratio of it increased to 148% of the starting strain.These results indicate that ribosome efficiency has decreased.In view of the phylogenic distance between E.coli and B.subtilis, 16S rRNA was demonstrated to have a highly exchangeable property between these two species of different evolution status.

  20. 16S rRNA partial gene sequencing for the differentiation and molecular subtyping of Listeria species.

    Science.gov (United States)

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

    2013-12-01

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

  1. FastGroupII: A web-based bioinformatics platform for analyses of large 16S rDNA libraries

    Directory of Open Access Journals (Sweden)

    McNairnie Pat

    2006-02-01

    Full Text Available Abstract Background High-throughput sequencing makes it possible to rapidly obtain thousands of 16S rDNA sequences from environmental samples. Bioinformatic tools for the analyses of large 16S rDNA sequence databases are needed to comprehensively describe and compare these datasets. Results FastGroupII is a web-based bioinformatics platform to dereplicate large 16S rDNA libraries. FastGroupII provides users with the option of four different dereplication methods, performs rarefaction analysis, and automatically calculates the Shannon-Wiener Index and Chao1. FastGroupII was tested on a set of 16S rDNA sequences from coral-associated Bacteria. The different grouping algorithms produced similar, but not identical, results. This suggests that 16S rDNA datasets need to be analyzed in multiple ways when being used for community ecology studies. Conclusion FastGroupII is an effective bioinformatics tool for the trimming and dereplication of 16S rDNA sequences. Several standard diversity indices are calculated, and the raw sequences are prepared for downstream analyses.

  2. Phylogenetic analysis of the Listeria monocytogenes based on sequencing of 16S rRNA and hlyA genes.

    Science.gov (United States)

    Soni, Dharmendra Kumar; Dubey, Suresh Kumar

    2014-12-01

    The discrimination between Listeria monocytogenes and Listeria species has been detected. The 16S rRNA and hlyA were PCR amplified with set of oligonucleotide primers with flank 1,500 and 456 bp fragments, respectively. Based on the differences in 16S rRNA and hlyA genes, a total 80 isolates from different environmental, food and clinical samples confirmed it to be L. monocytogenes. The 16S rRNA sequence similarity suggested that the isolates were similar to the previously reported ones from different habitats by others. The phylogenetic interrelationships of the genus Listeria were investigated by sequencing of 16S rRNA and hlyA gene. The 16S rRNA sequence indicated that genus Listeria is comprised of following closely related but distinct lines of descent, one is the L. monocytogenes species group (including L. innocua, L. ivanovii, L. seeligeri and L. welshimeri) and other, the species L. grayi, L. rocourtiae and L. fleischmannii. The phylogenetic tree based on hlyA gene sequence clearly differentiates between the L. monocytogenes, L. ivanovii and L. seeligeri. In the present study, we identified 80 isolates of L. monocytogenes originating from different clinical, food and environmental samples based on 16S rRNA and hlyA gene sequence similarity.

  3. Avances en el tratamiento de los pacientes con Leucemia Promielocítica Aguda en Recaída

    Directory of Open Access Journals (Sweden)

    A. Sanz Miguel

    2006-04-01

    Full Text Available Hasta la demostración de la actividad sobresaliente del trióxido de arsénico (ATO en recaídas de pacientes con leucemia promielocítica (LPA, el tratamiento de rescate en esta enfermedad consistía en la readministración de ácido holo-trans retinóico (ATRA y quimioterapia para inducir la remisión, generalmente conteniendo citarabina a altas dosis, seguido de consolidación y/o trasplante de progenitores hematopoyéticos (TPH.

  4. Avances en el tratamiento de los pacientes con Leucemia Promielocítica Aguda en Recaída

    OpenAIRE

    A. Sanz Miguel

    2006-01-01

    Hasta la demostración de la actividad sobresaliente del trióxido de arsénico (ATO) en recaídas de pacientes con leucemia promielocítica (LPA), el tratamiento de rescate en esta enfermedad consistía en la readministración de ácido holo-trans retinóico (ATRA) y quimioterapia para inducir la remisión, generalmente conteniendo citarabina a altas dosis, seguido de consolidación y/o trasplante de progenitores hematopoyéticos (TPH).

  5. Identification of Pheretima aspergillum by CO Ⅰ and 16S rRNA with DNA Molecular Marker Methods%基于COⅠ与16S rRNA基因对广地龙的DNA分子鉴定研究

    Institute of Scientific and Technical Information of China (English)

    韦健红; 李薇; 吴文如; 喻良文

    2012-01-01

    OBJECTIVE: To establish a rapid, accurate and standardized DNA molecular marker method for the identification of Pheretima aspergillum. METHODS: The CO Ⅰ and 16S rRNA gene sequences of P. aspergillum from 5 different populations were determined using CodonCode Aligner sequence assembly. In addition, the CO Ⅰ and 16S rRNA gene sequences of P. aspergillum were downloaded from GenBank, and intraspecific and interspecific K2P genetic distance between P. aspergillum and counterfeit were calculated with MEGA 4.1 to construct NJ and MP phylogenetic tree. RESULTS: The variation sites and information sites of CO Ⅰ were higher than those of 16S rRNA. There were no insertions and deletions of CO Ⅰ , and there were 4 insertions and deletions of 16S rRNA. The interspecific genetic distances of CO Ⅰ and 16S rRNA sequences were significantly greater than intraspecific ones, and CO Ⅰ and 16S rRNA gene can be identified from the other earthworm species. CONCLUSION: The CO I and 16S rRNA gene can provide reference for the identification of the animal Chinese medicine at the molecular level, and accumulate information for DNA barcode of animal Chinese medicine.%目的:建立一种快速、准确和标准化的广地龙DNA分子标记鉴别方法.方法:测定了5个不同居群广地龙的线粒体细胞色素酶亚单位(CO)Ⅰ和16S rRNA基因序列,采用CodonCode Aligner进行序列拼接,通过下载GenBank地龙原动物的CO Ⅰ与16S rRNA序列,采用MEGA 4.1计算广地龙及其伪品地龙的种内、种间的K2P遗传距离,并基于K2P模型构建NJ和MP树.结果:COⅠ变异位点、信息位点均高于16S rRNA,CO Ⅰ基因无插入和缺失,16S rRNA存在4个插入和缺失.CO Ⅰ和16S rRNA序列种间遗传距离均明显大于种内,CO Ⅰ和16S rRNA基因均能将广地龙从其他地龙或蚯蚓物种鉴别开来.结论:获得的广地龙CO Ⅰ和16S rRNA序列可为动物性中药材地龙的分子水平鉴定提供参考,为动物性中药材DNA条

  6. 16S rRNA in identification and typing of Clostridium botulinum%16S rRNA在肉毒梭菌分型鉴定中的应用

    Institute of Scientific and Technical Information of China (English)

    卢卫嘉; 毛晓燕; 熊颖; 张超; 王建锋

    2011-01-01

    Objective To establish a rapid, reliable method for identifying and typing of Clostridium botulinum using 16S rDNA sequencing and phylogenetic tree construction. Methods Clostridium genome templates were extracted from 9 strains, including LCL063, 830110, LC175, LCL155, 66418, N153, 61082, ALASKA, and IWANAIs 16S rRNA genes were amplifed by PCR with the 16S rRNA specific primers, and then the PCR products were cloned to pGEM -T Easy vector and sequenced. Finally, the sequence of 16S rDNA was analyzed by Clustal and Mega program; the phylogenetic trees were constructed by Neighor joining and Maximum parsimony. Results It was found that LCL063, 830110, and LCL175 were BONT/E producing Clostridium butyricums; IWANAI and ALASKA were Clostridium botulinum type E. The results of the present method were consistent with those of the conventional method. Conclusion 16S rRNA sequencing combined with phylogenetic tree analysis is a rapid and accurate method in Clostridium botulinum identification, and the method may serve as a criterion for bacterial typing with the completion of ribosomal RNA data bank.%目的 建立一种快速、可靠的对肉毒梭菌进行分型鉴定的手段.方法 以从LCL063、830110、LC175、LCL155、66418、N153、61082、ALASKA、IWANAI共9株梭菌中提取的基因组DNA为模板,利用16S rRNA特异性引物分别进行PCR扩增并进行T-A克隆转化、测序.通过Clustal和Mega软件分析16S rDNA序列,以NJ法和MP法构建进化树,分析其种属特异性.结果 16S rRNA分型结果可判断出LCL063、830110、LCL175为产E型毒素的酪酸梭菌.IWANAI与ALASKA株为E型肉毒梭菌.与传统分型鉴定得到的结果一致.结论 16S rRNA在肉毒梭菌分型鉴定中具有快速、准确的优势,随着核糖体库的不断完善,有望成为细菌分型鉴定的标准依据.

  7. RFLP Analysis of 16S rRNA Genes of Bacterial Community in Water Sample of a Petroleum Reservoir%油藏水样细菌群落16S rRNA基因的RFLP分析

    Institute of Scientific and Technical Information of China (English)

    陈平; 李辉; 牟伯中

    2005-01-01

    通过16S rRNA基因的限制性酶切片段长度多态性分析(RFLP)方法,考察了油藏水样中细菌群落及多样性.从水样中分离纯化微生物总DNA,选择性扩增细菌16S rRNA基因,并构建16S rDNA克隆文库.337 个16S rDNA克隆片段分别用限制性内切酶HinfⅠ和HaeⅢ酶切分析,得到 74 个操作分类单元 (OTUs) ,其中数量最多的 4 个OTUs共占克隆子总数的73.6%,另外 70 个OTUs的丰度均处于较低水平,有 57 个 OTUs 仅含有 1 个克隆子.结果表明,运用RFLP方法分析16S rDNA克隆片段能够有效评估油藏水样中的细菌群落和多样性.

  8. Cloning and Sequencing of Phytoplasma 16S rRNA Gene From Infected Mulberry in China%桑黄化型萎缩病病原体16S rRNA基因的序列分析

    Institute of Scientific and Technical Information of China (English)

    夏志松; 難波成任

    2004-01-01

    用PCR法克隆了中国桑黄化型萎缩病病原植原体的16S rRNA基因,并进行了序列分析.结果表明:克隆的基因大小为1 372 bp,与日本桑萎缩病病原植原体16S rRNA的同源性高达99.85%,只存在个别碱基的突变.Blastj检索结果显示与中国桑黄化型萎缩病病原体16S rRNA的基因同源性高达99%的有来源于玉米、洋葱、土豆等50多种其它植物的植原体16S rRNA基因,表明植物萎缩病病原体16S rRNA基因具高度保守性.

  9. Application of 16S rRNA Gene Library Method in Bacterium Flora Analysis of Infected Wound%16S rRNA基因文库方法在感染标本菌群分析中的应用

    Institute of Scientific and Technical Information of China (English)

    常玉梅; 刘海燕

    2013-01-01

    目的 探讨16S rRNA基因文库方法对感染标本菌群的鉴定效果.方法 采集70例创伤患者的感染伤口脓液或渗出液标本,使用通用引物扩增标本中所有细菌的16S rRNA基因片段,构建16S rRNA基因文库,挑取阳性克隆测序,分析感染细菌的数量与种类.结果 从150个克隆子中检测7种不同酶切类型的克隆,鉴定出7类微生物,其中屎肠球菌种类最多,占88%,坚强肠球菌比例占2%,另5类序列与非可培养细菌类群的16S rRNA基因序列相似性较高,均占2%.结论 感染标本中屎肠球菌含量丰富,应用16S rRNA基因文库的方法可有效分离到感染标本中的非可培养菌.

  10. Technologically important extremophile 16S rRNA sequence Shannon entropy and fractal property comparison with long term dormant microbes

    Science.gov (United States)

    Holden, Todd; Gadura, N.; Dehipawala, S.; Cheung, E.; Tuffour, M.; Schneider, P.; Tremberger, G., Jr.; Lieberman, D.; Cheung, T.

    2011-10-01

    Technologically important extremophiles including oil eating microbes, uranium and rocket fuel perchlorate reduction microbes, electron producing microbes and electrode electrons feeding microbes were compared in terms of their 16S rRNA sequences, a standard targeted sequence in comparative phylogeny studies. Microbes that were reported to have survived a prolonged dormant duration were also studied. Examples included the recently discovered microbe that survives after 34,000 years in a salty environment while feeding off organic compounds from other trapped dead microbes. Shannon entropy of the 16S rRNA nucleotide composition and fractal dimension of the nucleotide sequence in terms of its atomic number fluctuation analyses suggest a selected range for these extremophiles as compared to other microbes; consistent with the experience of relatively mild evolutionary pressure. However, most of the microbes that have been reported to survive in prolonged dormant duration carry sequences with fractal dimension between 1.995 and 2.005 (N = 10 out of 13). Similar results are observed for halophiles, red-shifted chlorophyll and radiation resistant microbes. The results suggest that prolonged dormant duration, in analogous to high salty or radiation environment, would select high fractal 16S rRNA sequences. Path analysis in structural equation modeling supports a causal relation between entropy and fractal dimension for the studied 16S rRNA sequences (N = 7). Candidate choices for high fractal 16S rRNA microbes could offer protection for prolonged spaceflights. BioBrick gene network manipulation could include extremophile 16S rRNA sequences in synthetic biology and shed more light on exobiology and future colonization in shielded spaceflights. Whether the high fractal 16S rRNA sequences contain an asteroidlike extra-terrestrial source could be speculative but interesting.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2015-03-01

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

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

    Directory of Open Access Journals (Sweden)

    Nathan D. Olson

    2015-03-01

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

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

    Science.gov (United States)

    Schwenk, Dagmar; Nohynek, Liisa; Rischer, Heiko

    2014-06-01

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

  15. 16S rRNA-based detection of oral pathogens in coronary atherosclerotic plaque

    Directory of Open Access Journals (Sweden)

    Mahendra Jaideep

    2010-01-01

    Full Text Available Background: Atherosclerosis develops as a response of the vessel wall to injury. Chronic bacterial infections have been associated with an increased risk for atherosclerosis and coronary artery disease. The ability of oral pathogens to colonize in coronary atheromatous plaque is well known. Aim: The aim of this study was to detect the presence of Treponema denticola, Porphyromonas gingivalis and Campylobacter rectus in the subgingival and atherosclerotic plaques of patients with coronary artery disease. Materials and Methods: Fifty-one patients in the age group of 40-80 years with coronary artery disease were selected for the study. DNA was extracted from the plaque samples. The specific primers for T. denticola, C. rectus and P. gingivalis were used to amplify a part of the 16S rRNA gene by polymerase chain reaction. Statistical Analysis Used: Chi-square analysis, correlation coefficient and prevalence percentage of the microorganisms were carried out for the analysis. Results: Of the 51 patients, T. denticola, C. rectus and P. gingivalis were detected in 49.01%, 21.51% and 45.10% of the atherosclerotic plaque samples. Conclusions: Our study revealed the presence of bacterial DNA of the oral pathogenic microorganisms in coronary atherosclerotic plaques. The presence of the bacterial DNA in the coronary atherosclerotic plaques in significant proportion may suggest the possible relationship between periodontal bacterial infection and genesis of coronary atherosclerosis.

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

    Science.gov (United States)

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

    2013-12-27

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

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

    Science.gov (United States)

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

    1997-01-01

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

  18. Tratamiento de esquizofrénicos crónicos: uso de bajas dosis de neurolépticos y recaídas.

    Directory of Open Access Journals (Sweden)

    Pilar Moreno Raymundo

    1996-01-01

    Full Text Available Hemos estudiado 109 pacientes esquizofrénicos atendidos durante un año en el CSM de Getafe. De sus historias clínicas hemos CSM recogido la dosis de mantenimiento con neurolépticos, datos sociodemográficos y las recaídas en el último año. La dosis de mantenimiento con neurolépticos mayoritariamente empleada se cuentra dentro del rango considerado como dosis baja por diferentes autores. El uso de estas bajas dosis no determina un mayor número de recaídas.

  19. Adicción a los opiáceros : mecanismos neurobiológicos implicados en la recaída

    OpenAIRE

    Ribeiro do Couto, Bruno Rodolfo

    2005-01-01

    RESUMEN El objetivo general de la presente Tesis Doctoral fue el de estudiar los mecanismos neurobiológicos subyacentes al fenómeno de recaída, analizando qué neurotransmisores y qué sistemas de neurotransmisión pueden estar involucrados en esta conducta. Mediante el condicionamiento de preferencia de lugar desarrollamos un modelo de recaída utilizando como sujetos ratones macho de la cepa OF1. Nuestra hipótesis fue que la administración de morfina inducirá una preferencia p...

  20. 16S rRNA基因检测对新生儿败血症诊断价值的Meta分析%Meta-analysis of value of 16S rRNA gene detection in diagnosis of neonatal sepsis

    Institute of Scientific and Technical Information of China (English)

    王远明; 杜惠容; 陈恒; 张辉

    2012-01-01

    目的 16S rRNA 基因检测是诊断新生儿败血症的方法之一,但是尚无研究综合评价其诊断价值,本研究拟系统评价16S rRNA基因检测在新生儿败血症中的诊断价值.方法 在Cochrane图书馆、Medline、Embase、Science Direct、中国期刊全文数据库、万方、维普等数据库中查找利用16S rRNA基因检测诊断新生儿败血症的文献.QUADAS工具评价纳入文献的质量,Meta-Disc 1.4 软件检验异质性并根据其结果选择相应效应模型计算纳入研究的合并敏感性、特异性等指标,绘制汇总受试者工作特征(SROC)曲线,综合评价16S rRNA基因检测的诊断价值.结果共有8篇文献共计2 543例新生儿纳入本次研究,Meta分析显示16S rRNA基因检测对新生儿败血症的合并诊断价值分别为:敏感度为0.93,特异度为0.97,阳性似然比为25.12,阴性似然比为0.08,诊断比值比为323.40.SROC曲线下面积为0.99.结论 16S rRNA基因检测中对新生儿败血症具有较高的诊断价值,可作为诊断新生儿败血症重要工具.%Objective 16S Rrna gene detection is one of the methods for the diagnosis of neonatal sepsis, but there is not any study evaluating its diagnostic value comprehensively. So this study aims to evaluate the diagnostic value of 16S Rrna gene detection in neonatal sepsis comprehensively. Methods Literature on diagnosis of neonatal sepsis using the 16S Rrna gene detection was searched in Cochrane Library, Medline, Embase, Science Direct, CNKI, Wanfang, VIP and other databases. QUADAS tool was used to evaluate the quality of literature; Meta-Disc 1.4 software was used to test heterogeneity, based on which the relevant effect model was selected to calculate the combined sensitivity, specificity and other indicators of the literature, the summary receiver operating characteristic (SROC) curve was drawn and the diagnostic value of 16S Rrna gene detection was evaluated comprehensively. Results A total of 8 pieces of

  1. Identification of clinical uncommon bacteria by sequence analysis of 16S rRNA gene%基于16S rRNA序列鉴定临床不常见细菌

    Institute of Scientific and Technical Information of China (English)

    吴智刚; 李小蓝; 吴奎海

    2013-01-01

    目的 以16S rRNA基因为靶序列,建立核糖体测序方法鉴定临床不常见细菌.方法 利用通用引物聚合酶链反应(PCR)扩增细菌16S rRNA基因,对PCR产物进行测序,在GenBank中对测序结果进行Blastn分析.结果 10株临床常见细菌测序结果与表型鉴定符合,检测出4株临床不常见细菌.结论 16S rRNA基因测序可以作为鉴定临床不常见细菌的重要方法之一.

  2. The effects of lexA101, recB21, recF143 and uvrD3 mutations on liquid-holding recovery in ultraviolet-irradiated Escherichia coli K12 recA56

    International Nuclear Information System (INIS)

    Using an Escherichia coli K12 recA strain, we have tested the effects of incorporating additional mutations affecting deoxyribonucleic acid (DNA) repair on ultraviolet-radiation sensitivity and in the expression of liquid-holding recovery (LHR). (This laboratory had previously shown that a mutation at uvrA, uvrB or uvrC blocked LHR in a recA strain). In the recA56 background, an additional lexA101 mutation had no effect on UV-radiation sensitivity or LHR. The addition of a recB21 mutation to recA56 did not alter UV-radiation sensitivity, but greatly increased the rate of LHR. The recB gene product (exonuclease V) appears to act as a competitive inhibitor both of excision repair and of photoreactivation under liquid-holding (LH) conditions. The uvrD3 mutation increased the radiation sensitivity of a recA strain, and almost completely blocked LHR. The recA uvrD strain showed more DNA degradation and DNA double-strand breaks during LH than did the recA strain. Rhe recF143 mutation increased both UV-radiation sensitivity and LHR in a recA strain, suggesting that the recF gene product may also function in recA-independent pathways of DNA repair. (orig.)

  3. [Strategy of selecting 16S rRNA hypervariable regions for metagenome-phylogenetic marker genes based analysis].

    Science.gov (United States)

    Zhang, Jun-yi; Zhu, Bing-chuan; Xu, Chao; Ding, Xiao; Li, Jun-feng; Zhang, Xue-gong; Lu, Zu-hong

    2015-11-01

    The advent of next generation sequencing technology enables parallel analysis of the whole microbial community from multiple samples. Particularly, sequencing 16S rRNA hypervariable tags has become the most efficient and cost-effective method for assessing microbial diversity. Due to its short read length of the 2nd-generation sequencing methods that cannot cover the full 16S rRNA genomic region, specific hypervariable regions or V-regions must be selected to act as the proxy. Over the past decade, selection of V-regions has not been consistent in assessing microbial diversity. Here we evaluated the current strategies of selecting 16S rRNA hypervariable regions for surveying microbial diversity. The environmental condition was considered as one of the important factors for selection of 16S rRNA hypervariable regions. We suggested that a pilot study to test different V-regions is required in bacterial diversity studies based on 16S rRNA genes.

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

    Science.gov (United States)

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

    2016-06-01

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

  5. Differentiation Between Bacillus thuringiensis and Bacillus cereus by 16S rDNA-PCR and ERIC-PCR

    Institute of Scientific and Technical Information of China (English)

    LI Haitao; LIU Dongming; GAO Jiguo

    2011-01-01

    16S rDNA and ERIC (Enterobacteia Repetitive Intergenic Consensus Sequences) based on PCR method were tested for the effectiveness of the differentiation of B. thuringiensis and B. cereus. 16S rDNA-PCR primers were designed based on the sequence difference in variable regions of B. cereus 16S rDNA and B. thuringiensis 16S rDNA, 16S rDNA-PCR showed no obvious difference between B. cereus and B. thuringiensis. The only difference was that one 1600-bp amplificon could be obtained from all the three B. Cereus strains, and none amplificon from any B. thuringiensis strains. ERIC was optimized based on previous reports. The genonlic DNA was used for the template of ER1C-PCR, and the following DNA fingerprints were analyzed by the agarose gel electrophoresis. The results showed that DNA fingerprint of three B. thuringiensis strains had a unique amplicon less than 100-bp, while DNA fingerprint of three B. cereus" strains had none. Moreover, DNA fingerprint of B. cereus showed a 700-bp amplicon, but didn't have any DNA fingerprints ofB. thuringiensis genome. Therefore, ERIC-PCR technique should be able to be used for the differentiation of B. thuringiensis and B. cereus.

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

    Science.gov (United States)

    Allen, Julie M; Burleigh, J Gordon; Light, Jessica E; Reed, David L

    2016-01-01

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

  7. Sequence analysis of 16S rDNA derived from a high yield lipase strain%一株高产脂肪酶产生菌16S rDNA的序列分析

    Institute of Scientific and Technical Information of China (English)

    孙新城; 马淑玲; 张玲丽; 张浩; 景建洲

    2012-01-01

    [目的]对分离获得的高产脂肪酶菌株进行鉴定,为其改造和更好利用奠定基础.[方法]对从食堂下水道中分离获得的一株高效产脂肪酶细菌(JLΠ-4)进行培养,提取其基因组DNA.设计16S rDNA通用引物,扩增16S rDNA基因片段,并连接到pUC19-T载体上,转化大肠杆菌DH5X,经PCR鉴定的阳性克隆摇菌培养后测序.[结果]提取获得较高质量的基因组DNA,扩增获得新分离菌株16S rDNA基因片段,长度为1528 bp,BLAST相似性比对分析结果表明,其与伯克霍尔德氏菌16S rDNA序列相似性达97%,是一株与伯克霍尔德氏菌最近的革兰氏阴性菌.[结论]初步将高产脂肪酶细菌JTΠ-4鉴定为唐菖蒲伯克霍尔德菌.%[Objective]The present study was conducted to identify the separated high yield lipase strain to provide theoretical research references and to enhance the high yield lipase strain's transformation and application. [Method]A strain of bacterium with high yield lipase JLIT-4 was isolated from the sewage of a canteen, then its genomic DNA was extracted. The gene fragments of 16S rDNA were amplified using 16S rDNA universal primers and connected to pUC19-T vector; the fragments are then transformed into E. Coli DH5X. The positive clones identified by the PCR method were cultured and sequenced. [Result]Quality genome DNA was successfully extracted. The 16S rDNA gene fragments of newly isolated strain were amplified with the length of 1528 bp. According to comparison analysis of BLAST, 16S rDNA sequence similarity between the strains and Burkholderia(DQ355168) were 97%, so the lipase producing strains were identified as gram-negative bacteria that were most similar to the structures of Burkholderia. [ Conclusion ]The high-yield lipase JLΠ-4 was primarily identified as Burkholderia gladioli.

  8. Advance in application of 16S rRNA gene in bacteriology%16S rRNA基因序列分析技术在细菌分类中应用的研究进展

    Institute of Scientific and Technical Information of China (English)

    杨霞; 陈陆; 王川庆

    2008-01-01

    由于16S rRNA基因序列的保守性和存在的普遍性,应用16S rRNA作为分子指标已逐渐成为微生物检测和分类鉴定的一种强有力工具.文章就该基因的特征、研究方法、检测方法及临床应用与研究的新进展等作以简要综述,同时对存在的问题进行了探讨.

  9. Investigating bacterial populations in styrene-degrading biofilters by 16S rDNA tag pyrosequencing.

    Science.gov (United States)

    Portune, Kevin J; Pérez, M Carmen; Álvarez-Hornos, F Javier; Gabaldón, Carmen

    2015-01-01

    Microbial biofilms are essential components in the elimination of pollutants within biofilters, yet still little is known regarding the complex relationships between microbial community structure and biodegradation function within these engineered ecosystems. To further explore this relationship, 16S rDNA tag pyrosequencing was applied to samples taken at four time points from a styrene-degrading biofilter undergoing variable operating conditions. Changes in microbial structure were observed between different stages of biofilter operation, and the level of styrene concentration was revealed to be a critical factor affecting these changes. Bacterial genera Azoarcus and Pseudomonas were among the dominant classified genera in the biofilter. Canonical correspondence analysis (CCA) and correlation analysis revealed that the genera Brevundimonas, Hydrogenophaga, and Achromobacter may play important roles in styrene degradation under increasing styrene concentrations. No significant correlations (P > 0.05) could be detected between biofilter operational/functional parameters and biodiversity measurements, although biological heterogeneity within biofilms and/or technical variability within pyrosequencing may have considerably affected these results. Percentages of selected bacterial taxonomic groups detected by fluorescence in situ hybridization (FISH) were compared to results from pyrosequencing in order to assess the effectiveness and limitations of each method for identifying each microbial taxon. Comparison of results revealed discrepancies between the two methods in the detected percentages of numerous taxonomic groups. Biases and technical limitations of both FISH and pyrosequencing, such as the binding of FISH probes to non-target microbial groups and lack of classification of sequences for defined taxonomic groups from pyrosequencing, may partially explain some differences between the two methods.

  10. 16S rRNA gene survey of microbial communities in Winogradsky columns.

    Directory of Open Access Journals (Sweden)

    Ethan A Rundell

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

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

    Science.gov (United States)

    Ellegaard, Kirsten M.; Engel, Philipp

    2016-01-01

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

  12. 16S rRNA gene sequencing as a tool to study microbial populations in foods and process environments

    DEFF Research Database (Denmark)

    Buschhardt, Tasja; Hansen, Tina Beck; Bahl, Martin Iain;

    2015-01-01

    reference. Results: Taxonomic assignments and abundances of sequences in the total community and in the Enterobacteriaceae subpopulation were affected by the 16S rRNA gene variable region, DNA extraction methods, and polymerases chosen. However, community compositions were very reproducible when the same...... methods were used. Conclusions: Altogether, we have shown that conclusions from population studies based on 16S rRNA gene sequencing need to be made with caution. Overcoming the constraints, we believe that population studies can give new research possibilities for e.g. interaction studies, identification......Introduction: Methodological constraints during culturing and biochemical testing have left the true microbiological diversity of foods and process environments unexplored. Culture-independent molecular methods, such as 16S rRNA gene sequencing, may provide deeper insight into microbial communities...

  13. Phylogeny of the cuttlefishes (Mollusca:Cephalopoda) based on mitochondrial COI and 16S rRNA gene sequence data

    Institute of Scientific and Technical Information of China (English)

    LIN Xiangzhi; ZHENG Xiaodong; XIAO Shu; WANG Rucai

    2004-01-01

    To clarify cuttlefish phylogeny, mitochondrial cytochrome c oxidase subunit I (COI) gene and partial 16S rRNA gene are sequenced for 13 cephalopod species. Phylogenetic trees are constructed, with the neighbor-joining method.Coleoids are divided into two main lineages, Decabrachia and Octobrachia. The monophyly of the order Sepioidea,which includes the families Sepiidae, Sepiolidae and Idiosepiidae, is not supported. From the two families of Sepioidea examined, the Sepiolidae are polyphyletic and are excluded from the order. On the basis of 16S rRNA and amino acid of COI gene sequences data, the two genera (Sepiella and Sepia) from the Sepiidae can be distinguished, but do not have a visible boundary using COI gene sequences. The reason is explained. This suggests that the 16S rDNA of cephalopods is a precious tool to analyze taxonomic relationships at the genus level, and COI gene is fitter at a higher taxonomic level (i.e., family).

  14. Effect of mutations in the A site of 16 S rRNA on aminoglycoside antibiotic-ribosome interaction

    DEFF Research Database (Denmark)

    Recht, M I; Douthwaite, S; Dahlquist, K D;

    1999-01-01

    Decoding of genetic information occurs upon interaction of an mRNA codon-tRNA anticodon complex with the small subunit of the ribosome. The ribosomal decoding region is associated with highly conserved sequences near the 3' end of 16 S rRNA. The decoding process is perturbed by the aminoglycoside...... of universally conserved nucleotides at 1406 to 1408 and 1494 to 1495 in the decoding region of plasmid-encoded bacterial 16 S rRNA. Phenotypic changes range from the benign effect of U1406-->A or A1408-->G substitutions, to the highly deleterious 1406G and 1495 mutations that assemble into 30 S subunits...... but are defective in forming functional ribosomes. Changes in the local conformation of the decoding region caused by these mutations were identified by chemical probing of isolated 30 S subunits. Ribosomes containing 16 S rRNA with mutations at positions 1408, 1407+1494, or 1495 had reduced affinity...

  15. RecJ nuclease is required for SOS induction after introduction of a double-strand break in a RecA loading deficient recB mutant of Escherichia coli.

    Science.gov (United States)

    Vlasić, Ignacija; Ivancić-Baće, Ivana; Imesek, Mirna; Mihaljević, Boris; Brcić-Kostić, Krunoslav

    2008-09-01

    The SOS response is an important mechanism which allows Escherichia coli cells to maintain genome integrity. Two key proteins in SOS regulation are LexA (repressor) and RecA (coprotease). The signal for SOS induction is generated at the level of a RecA filament. Depending on the type of DNA damage, a RecA filament is produced by specific activities (helicase, nuclease and RecA loading) of either RecBCD, RecF or a hybrid recombination pathway. It was recently demonstrated that RecA loading activity is essential for the induction of the SOS response after UV-irradiation. In this paper we studied the genetic requirements for SOS induction after introduction of a double-strand break (DSB) by the I-SceI endonuclease in a RecA loading deficient recB mutant (recB1080). We monitored SOS induction by assaying beta-galactosidase activity and compared induction of the response between strains having one or more inactivated mechanisms of RecA loading and their derivatives. We found that simultaneous inactivation of both RecA loading functions (in recB1080 recO double mutant) partially impairs SOS induction after introduction of a DSB. However, we found that the RecJ nuclease is essential for SOS induction after the introduction of a DSB in the recB1080 mutant. This result indicates that RecJ is needed to prepare ssDNA for subsequent loading of RecA protein. It implies that an additional type of RecA loading could exist in the cell. PMID:18445487

  16. Application of the Sequences Analysis of the 16S rRNA Gene and ITS of 16S-23S rRNA to the Systematic Study of the Genus Arthrospira and Spirulina%16S rRNA基因与16S-23S rRNA转录单元内间隔区序列分析及其在节旋藻和螺旋藻分类鉴定中的应用

    Institute of Scientific and Technical Information of China (English)

    茅云翔; 杨官品; 张宝红; 张学成

    2001-01-01

    测定了节旋藻属3个品系和螺旋藻属1个品系的全长16S rRNA基因和16S-23S rRNA转录单元内间隔区序列(ITS),分析了已知的节旋藻、螺旋藻和相关品系的相应序列的同源性,构建了系统发生树,并评价了这两段DNA序列在节旋藻、螺旋藻种属分类和种质鉴定中的意义.结果表明:(1)16S rRNA基因序列和ITS序列均可用于节旋藻属和螺旋藻属的属间分类,以两序列为基础的系统学分析结果一致;(2)ITS序列变异程度高于16S rDNA序列,适用于节旋藻和螺旋藻属内品系或种质鉴定;(3)节旋藻属可明确界定,16S rRNA基因序列相似性大于98%,ITS序列相似性大于88%;(4)螺旋藻属某些品系间16S rDNA序列和ITS序列相似性较低,与不同属间的序列相似性程度为同一水平.

  17. Defining reference sequences for Nocardia species by similarity and clustering analyses of 16S rRNA gene sequence data.

    Directory of Open Access Journals (Sweden)

    Manal Helal

    Full Text Available BACKGROUND: The intra- and inter-species genetic diversity of bacteria and the absence of 'reference', or the most representative, sequences of individual species present a significant challenge for sequence-based identification. The aims of this study were to determine the utility, and compare the performance of several clustering and classification algorithms to identify the species of 364 sequences of 16S rRNA gene with a defined species in GenBank, and 110 sequences of 16S rRNA gene with no defined species, all within the genus Nocardia. METHODS: A total of 364 16S rRNA gene sequences of Nocardia species were studied. In addition, 110 16S rRNA gene sequences assigned only to the Nocardia genus level at the time of submission to GenBank were used for machine learning classification experiments. Different clustering algorithms were compared with a novel algorithm or the linear mapping (LM of the distance matrix. Principal Components Analysis was used for the dimensionality reduction and visualization. RESULTS: The LM algorithm achieved the highest performance and classified the set of 364 16S rRNA sequences into 80 clusters, the majority of which (83.52% corresponded with the original species. The most representative 16S rRNA sequences for individual Nocardia species have been identified as 'centroids' in respective clusters from which the distances to all other sequences were minimized; 110 16S rRNA gene sequences with identifications recorded only at the genus level were classified using machine learning methods. Simple kNN machine learning demonstrated the highest performance and classified Nocardia species sequences with an accuracy of 92.7% and a mean frequency of 0.578. CONCLUSION: The identification of centroids of 16S rRNA gene sequence clusters using novel distance matrix clustering enables the identification of the most representative sequences for each individual species of Nocardia and allows the quantitation of inter- and intra

  18. Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome

    Institute of Scientific and Technical Information of China (English)

    Carlos; W; Nossa; William; E; Oberdorf; Jφrn; A; Aas; Bruce; J; Paster; Todd; Z; DeSantis; Eoin; L; Brodie; Daniel; Malamud; Michael; A; Poles

    2010-01-01

    AIM:To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome.METHODS:A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral,esophageal,and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species.Candidate primers evaluated were from the European rRNA database.To assess the effect of sequence length on accuracy of classifica...

  19. Expanded versions of the 16S and 23S ribosomal RNA mutation databases (16SMDBexp and 23SMDBexp)

    OpenAIRE

    Triman, K L; Peister, A; Goel, R A

    1998-01-01

    Expanded versions of the Ribosomal RNA Mutation Databases provide lists of mutated positions in 16S and 16S-like ribosomal RNA (16SMDBexp) and 23S and 23S-like ribosomal RNA (23SMDBexp) and the identity of each alteration. Alterations from organisms other than Escherichia coli are reported at positions according to the E.coli numbering system. Information provided for each mutation includes: (i) a brief description of the phenotype(s) associated with each mutation, (ii) whether a mutant pheno...

  20. Analysis of the genotypes among different strains of common Mycobacteria based on 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer sequences%常见分枝杆菌种内不同株之间16S rRNA基因和16S-23SrRNA ITS序列分析结果的比较

    Institute of Scientific and Technical Information of China (English)

    黄至澄; 徐黔宁; 闫李侠; 陈保文; 王国治

    2011-01-01

    目的 针对常见分枝杆菌不同株对其基因序列进行分析,比较分析结果.方法 利用16S rRNA Gene和16S-23S rRNAITS(转录间隔区序列)分析法分别对97株共7种DSMZ/ATCC引进的常见分枝杆菌进行种内不同株之间基因差异性分析,对比两种分型结果的异同.结果 16S rRNA基因可将13株草分枝杆菌分为3个型别,18株偶发分枝杆菌分为6个型别,17株耻垢分枝杆菌分为4个型别,8株戈登分枝杆菌分为3个型别,9株龟分枝杆菌龟亚种分为3个型别,15株堪萨斯分枝杆菌分为2个型别,17株产鼻疽分枝杆菌分为1个型别;而16S-23S rRNA ITS可依次将上述分枝杆菌分为3个、15个、7个、3个、4个、3个、5个型别.结论 16S rRNA G ene分析和16S-23S rRNA ITS分析均是分枝杆菌基因型分析的可靠方法,此外,16S-23SrRNA ITS的种内多态性高于16S rRNA Gene.

  1. Flow Cytometry-Assisted Cloning of Specific Sequence Motifs from Complex 16S rRNA Gene Libraries

    DEFF Research Database (Denmark)

    Nielsen, Jeppe Lund; Schramm, Andreas; Bernhard, Anne E.;

    2004-01-01

      FLOW CYTOMETRY-ASSISTED CLONING OF SPECIFIC SEQUENCE MOTIFS FROM COMPLEX 16S RRNA GENE LIBRARIES Jeppe L. Nielsen,1 Andreas Schramm,1,2 Anne E. Bernhard,1 Gerrit J. van den Engh,3 and David A. Stahl1* Department of Civil and Environmental Engineering, University of Washington,1 and Institute......-assisted cloning, was used to recover sequences affiliated with a unique lineage within the Bacteroidetes not abundant in a clone library of environmental 16S rRNA genes.  ...

  2. Escherichia coli Vertebral Osteomyelitis Diagnosed According to Broad-range 16S rRNA Gene Polymerase Chain Reaction (PCR).

    Science.gov (United States)

    Shibata, Satoshi; Tanizaki, Ryutaro; Watanabe, Koji; Makabe, Kenta; Shoda, Naoki; Kutsuna, Satoshi; Nagamatsu, Maki; Oka, Shinichi; Ohmagari, Norio

    2015-01-01

    Identifying the causative agent of pyogenic osteomyelitis is often challenging, especially when antibiotics are administered before a biopsy. We herein present a case of osteomyelitis in the cervical vertebrae presenting with progressive paralytic symptoms, in which we successfully identified Escherichia coli from a biopsy specimen using broad-range 16S rRNA gene polymerase chain reaction (PCR) even though sensitive antibiotics had been used for more than 50 days before the biopsy. Broad-range 16S rRNA gene PCR is a useful diagnostic method, especially when prebiopsy antibiotics are unavoidably used for a clinically unstable state.

  3. Rapid detection of the mutations of neisseria gonorrhoeae 16S rRNA gene using DNA chip%DNA芯片快速检测淋球菌16S rRNA基因突变

    Institute of Scientific and Technical Information of China (English)

    周文明; 杨森; 赵建龙; 曹慧敏; 张学军

    2012-01-01

    目的 探讨新研制的DNA芯片应用于快速检测淋球菌16S rRNA 基因突变的临床应用价值.方法 根据淋球菌16SrRNA 基因的序列信息设计探针并制作DNA芯片,PCR扩增并荧光标记包含16S rRNA 基因突变的目的 DNA片段,与芯片杂交,同时以测序法为对照.结果 87份泌尿生殖道拭子均可被DNA芯片检测,同时检测发现有1株淋球菌16S rRNA基因突变(2709 C→T),与测序结果 一致.结论 DNA芯片检测淋球菌16SrRNA基因突变具有快速、高特异性和高灵敏度,可以应用于对大观霉素的耐药性临床检测.%To develop a new method, DNA chip, for rapid detection the mutations of neisseria gonorrhoeae 16S rRNA gene. Methods Probe were design according to the sequence of neisseria gonorrhoeae 16S rRNA genes, then DNA chip was made. DNA fragment which contains 16S rRNA gene mutation was amplified by PCR technique, labeled with cy5 fluorescence, and then hybridized with DNA chip. Results of DNA sequencing were used as the control. Results All of urogenital swab specimens were detected by DNA chip. We found one specimen which had mutation in gene 16S rRNA( 2709 C->T), which was consistency between the DNA chip and DNA sequencing. Conclusion DNA chip is a rapid technique with high sensitivity and specificity for the detection of mutations of neisseria gonorrhoeae 16SrRNA gene. This method can be used for detection of spectinomycin resistance in clinical practice.

  4. 16S/18S ribosomal DNA clone library analysis of rumen microbial diversity

    International Nuclear Information System (INIS)

    The rumen contains a complex ecosystem where billions of bacteria, archaea, protozoa and fungi reside. This diverse microbiota is well adapted to live in the rumen and play an important role in the digestion of feed and nutrient supply to the host in the form of microbial protein and volatile fatty acids. It is estimated that the rumen microbial population consists of about 106 protozoa/ml, 103-107 fungi/ml, 1010 bacteria/ml, and 109 methanogens/ml. To better understand the complex relationships in the rumen, it is necessary to gain an insight into the diversity of the rumen microbes and how the quantity and composition of rumen micro-organisms are altered by a number of different host factors such as age, genetics and diet. In the past, the diversity of micro-organisms from the digestive tracts of domesticated ruminants has been identified by classical microbiological techniques. However, given the fastidious growth requirements of rumen micro-organisms, it is reasonable to concede that the culture-dependent methods may select against some species, or taxonomic groups, leading researchers to underestimate the microbial diversity that is actually present in the rumen. In fact, it has been speculated that 90% of micro-organisms in nature have escaped traditional cultivation methods. Therefore, a major challenge in microbial ecology has been to assess the diversity and structure of natural microbial communities. The field of molecular biology has advanced with many innovative technological breakthroughs. The ability to extract and to isolate high-molecular weight DNA from rumen digesta, PCR amplify genes from specific microbial groups and obtain gene sequence data is now a routine event. The small subunit ribosomal RNA (SSU-rRNA) gene, called 16S in prokaryotes and 18S in eukaryotes, is the most widely used molecular marker to presumptively identify morphologically indistinguishable species, to infer their phylogenetic relationships, and to elucidate microbial

  5. Identification of Bacillus strains isolated from Yacai by 16S rRNA gene sequencing%利用16S rRNA序列鉴定分离自芽菜中的芽孢杆菌

    Institute of Scientific and Technical Information of China (English)

    张良; 吴华昌; 邓静; 李萍萍; 肖辰; 沈芳

    2012-01-01

    从宜宾芽菜中分离优势菌群,选取4株芽孢杆菌,分别为B1、B2、B3、B4.对4株菌的16S rRNA基因经PCR扩增测序,将测序结果同该属内菌株的16S rRNA序列作多序列比较,并建立芽孢杆菌属的系统发育树.结合细菌形态学生理生化特性鉴定结果,结果表明菌株B1、B3为枯草芽孢杆菌,菌株B2为解淀粉芽孢杆菌,菌株B4为乙酰微小杆菌.%Four dominant Bacillus strains named Bl, B2, B3 and B4 were isolated from Yacai (a kind of Yibin pickles in China). The 16S rRNA genes of these strains were amplified in vitro and sequenced. Then a phylogenetic tree was constructed by multiple alignments of their sequences with other 16S rRNA gene sequences of Bacillus. According to 16S rRNA gene analysis combined with morphological, physiological and biochemical characteristics, B1 and B3 were identified as Bacillus subtilis, B2 was Bacillus amyloliquefaciens and B4 was Exiguobacterium acetylicum.

  6. Thinking beside the box: Should we care about the non-coding strand of the 16S rRNA gene?

    Science.gov (United States)

    Garcia-Mazcorro, Jose F; Barcenas-Walls, Jose R

    2016-08-01

    The 16S rRNA gene (16S rDNA) codes for RNA that plays a fundamental role during translation in the ribosome and is used extensively as a marker gene to establish relationships among bacteria. However, the complementary non-coding 16S rDNA (nc16S rDNA) has been ignored. An idea emerged in the course of analyzing bacterial 16S rDNA sequences in search for nucleotide composition and substitution patterns: Does the nc16S rDNA code? If so, what does it code for? More importantly: Does 16S rDNA evolution reflect its own evolution or the evolution of its counterpart nc16S rDNA? The objective of this minireview is to discuss these thoughts. nc strands often encode small RNAs (sRNAs), ancient components of gene regulation. nc16S rDNA sequences from different bacterial groups were used to search for possible matches in the Bacterial Small Regulatory RNA Database. Intriguingly, the sequence of one published sRNA obtained from Legionella pneumophila (GenBank: AE0173541) showed high non-random similarity with nc16S rDNA corresponding in part to the V5 region especially from Legionella and relatives. While the target(s) of this sRNA is unclear at the moment, its mere existence might open up a new chapter in the use of the 16S rDNA to study relationships among bacteria.

  7. Thinking beside the box: Should we care about the non-coding strand of the 16S rRNA gene?

    Science.gov (United States)

    Garcia-Mazcorro, Jose F; Barcenas-Walls, Jose R

    2016-08-01

    The 16S rRNA gene (16S rDNA) codes for RNA that plays a fundamental role during translation in the ribosome and is used extensively as a marker gene to establish relationships among bacteria. However, the complementary non-coding 16S rDNA (nc16S rDNA) has been ignored. An idea emerged in the course of analyzing bacterial 16S rDNA sequences in search for nucleotide composition and substitution patterns: Does the nc16S rDNA code? If so, what does it code for? More importantly: Does 16S rDNA evolution reflect its own evolution or the evolution of its counterpart nc16S rDNA? The objective of this minireview is to discuss these thoughts. nc strands often encode small RNAs (sRNAs), ancient components of gene regulation. nc16S rDNA sequences from different bacterial groups were used to search for possible matches in the Bacterial Small Regulatory RNA Database. Intriguingly, the sequence of one published sRNA obtained from Legionella pneumophila (GenBank: AE0173541) showed high non-random similarity with nc16S rDNA corresponding in part to the V5 region especially from Legionella and relatives. While the target(s) of this sRNA is unclear at the moment, its mere existence might open up a new chapter in the use of the 16S rDNA to study relationships among bacteria. PMID:27412167

  8. ASSESSMENT OF FECAL POLLUTION SOURCES IN PLUM CREEK WATERSHED USING BACTEROIDETES 16S RDNA-BASED ASSAYS

    Science.gov (United States)

    Recently, 16S rDNA Bacteroidetes-targeted PCR assays were developed to discriminate between ruminant and human fecal pollution. These assays are rapid and relatively inexpensive but have been used in a limited number of studies. In this study, we evaluated the efficacy o...

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

    Science.gov (United States)

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

  10. Unexpected Diagnosis of Cerebral Toxoplasmosis by 16S and D2 Large-Subunit Ribosomal DNA PCR and Sequencing

    DEFF Research Database (Denmark)

    Kruse, Alexandra Yasmin Collin; Kvich, Lasse Andersson; Eickhardt-Dalbøge, Steffen Robert;

    2015-01-01

    The protozoan parasite Toxoplasma gondii causes severe opportunistic infections. Here, we report an unexpected diagnosis of cerebral toxoplasmosis. T. gondii was diagnosed by 16S and D2 large-subunit (LSU) ribosomal DNA (rDNA) sequencing of a cerebral biopsy specimen and confirmed by T. gondii...

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

    Science.gov (United States)

    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...

  12. A Web-Hosted R Workflow to Simplify and Automate the Analysis of 16S NGS Data

    Science.gov (United States)

    Next-Generation Sequencing (NGS) produces large data sets that include tens-of-thousands of sequence reads per sample. For analysis of bacterial diversity, 16S NGS sequences are typically analyzed in a workflow that containing best-of-breed bioinformatics packages that may levera...

  13. Group-specific 16S rRNA-targeted oligonucleotide probes to identify thermophilic bacteria in marine hydrothermal vents

    NARCIS (Netherlands)

    Harmsen, HJM; Prieur, D; Jeanthon, C

    1997-01-01

    Four 16S rRNA-targeted oligonucleotide probes were designed for the detection of thermophilic members of the domain Bacteria known to thrive in marine hydrothermal systems, We developed and characterized probes encompassing most of the thermophilic members of the genus Bacillus, most species of the

  14. Microbial community structure of Arctic multiyear sea ice and surface seawater by 454 sequencing of the 16S RNA gene

    DEFF Research Database (Denmark)

    Bowman, Jeff S.; Rasmussen, Simon; Blom, Nikolaj;

    2011-01-01

    community in MYI at two sites near the geographic North Pole using parallel tag sequencing of the 16S rRNA gene. Although the composition of the MYI microbial community has been characterized by previous studies, microbial community structure has not been. Although richness was lower in MYI than...

  15. Phylogenetic analysis of Thai oyster (Ostreidae) based on partial sequences of the mitochondrial 16S rDNA gene

    DEFF Research Database (Denmark)

    Bussarawit, Somchai; Gravlund, Peter; Glenner, Henrik;

    2006-01-01

    Ten oyster species of the family Ostreidae (Subfamilies Crassostreinae and Lophinae) from Thailand were studied using morphological data and mitochondrial 16S rDNA gene sequences. Additional sequence data from five specimens of Ostreidae and one specimen of Tridacna gigas were downloaded from Gen...

  16. Improved taxonomic assignment of human intestinal 16S rRNA sequences by a dedicated reference database

    NARCIS (Netherlands)

    Ritari, Jarmo; Salojärvi, Jarkko; Lahti, Leo; Vos, de Willem M.

    2015-01-01

    Background: Current sequencing technology enables taxonomic profiling of microbial ecosystems at high resolution and depth by using the 16S rRNA gene as a phylogenetic marker. Taxonomic assignation of newly acquired data is based on sequence comparisons with comprehensive reference databases to f

  17. 16S rRNA gene sequencing in routine identification of anaerobic bacteria isolated from blood cultures

    DEFF Research Database (Denmark)

    Justesen, Ulrik Stenz; Skov, Marianne Nielsine; Knudsen, Elisa;

    2010-01-01

    A comparison between conventional identification and 16S rRNA gene sequencing of anaerobic bacteria isolated from blood cultures in a routine setting was performed (n = 127). With sequencing, 89% were identified to the species level, versus 52% with conventional identification. The times...

  18. First report of neonatal bacteremia caused by "Haemophilus quentini" diagnosed by 16S rRNA gene sequencing, Italy.

    Science.gov (United States)

    Giufrè, Maria; Cardines, Rita; Degl'Innocenti, Roberto; Cerquetti, Marina

    2015-10-01

    We report the first case of neonatal bacteremia caused by a "Haemophilus quentini" isolate in Italy. The isolate was differentiated from H. influenzae by 16S rRNA sequencing and was characterized by comparison with the wild-type "H. quentini" CCUG 36167. Both isolates carried substitutions in penicillin-binding protein 3 but were susceptible to aminopenicillins.

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

    NARCIS (Netherlands)

    S.A. Boers (Stefan A.); J.P. Hays (John P.); R. Jansen (Ruud)

    2015-01-01

    textabstract16S 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 limitat

  20. Direct 16S rRNA gene sequencing of polymicrobial culture-negative samples with analysis of mixed chromatograms

    DEFF Research Database (Denmark)

    Hartmeyer, Gitte N; Justesen, Ulrik S

    2010-01-01

    Two cases involving polymicrobial culture-negative samples were investigated by 16S rRNA gene sequencing, with analysis of mixed chromatograms. Fusobacterium necrophorum, Prevotella intermedia and Streptococcus constellatus were identified from pleural fluid in a patient with Lemierre's syndrome...

  1. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and standard protocol for genomovar assignment.

    Science.gov (United States)

    LaFrentz, B R; Waldbieser, G C; Welch, T J; Shoemaker, C A

    2014-07-01

    Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare, and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing of this important fish pathogen. Interpretation of restriction patterns can be difficult due to the lack of a formal description of the expected number and sizes of DNA fragments generated for each of the described genomovars. In this study, partial 16S rRNA gene sequences (ca. 1250-bp fragment) from isolates representing each described genomovar and isolates generating unique restriction patterns were cloned and sequenced. The results demonstrated that some isolates contained up to three different 16S rRNA genes whose sequences generate different RFLP patterns due to intragenomic heterogeneity within HaeIII restriction sites. The occurrence of HaeIII restriction sites within the portion of the 16S rRNA gene used for typing the F. columnare isolates and intragenomic heterogeneity within these sites explained the restriction patterns observed following RFLP analyses. This research provides a standard protocol for typing isolates of F. columnare by RFLP and a formal description of the expected restriction patterns for the previously described genomovars I, II, II-B and III. Additionally, we describe a new genomovar, I/II.

  2. Apples with Oranges: Comparing the GS-FLX vs Ion Torrent Platforms for 16S Metagenomics Studies

    OpenAIRE

    Paech, Sam; McDermott, Stewart; Men, Artem; McGrath, Ken

    2012-01-01

    Decreasing costs and faster turn-around times mean that next-generation platforms are more readily accessible to metagenomics researchers. We compared the suitability of the GS-FLX (XLR70) and Ion Torrent (314 with 100bp chemistry) to generate data sets that are suitable for 16S-based taxonomic metagenomics.

  3. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and standard protocol for genomovar assignment

    Science.gov (United States)

    Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing this important fish pathogen. Interpretation of restriction patterns can be difficult due to th...

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

    Science.gov (United States)

    Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing this important fish pathogen. Interpretation of restriction patterns can be difficult due to th...

  5. Species identification and profiling of complex microbial communities using shotgun Illumina sequencing of 16S rRNA amplicon sequences.

    Directory of Open Access Journals (Sweden)

    Swee Hoe Ong

    Full Text Available The high throughput and cost-effectiveness afforded by short-read sequencing technologies, in principle, enable researchers to perform 16S rRNA profiling of complex microbial communities at unprecedented depth and resolution. Existing Illumina sequencing protocols are, however, limited by the fraction of the 16S rRNA gene that is interrogated and therefore limit the resolution and quality of the profiling. To address this, we present the design of a novel protocol for shotgun Illumina sequencing of the bacterial 16S rRNA gene, optimized to amplify more than 90% of sequences in the Greengenes database and with the ability to distinguish nearly twice as many species-level OTUs compared to existing protocols. Using several in silico and experimental datasets, we demonstrate that despite the presence of multiple variable and conserved regions, the resulting shotgun sequences can be used to accurately quantify the constituents of complex microbial communities. The reconstruction of a significant fraction of the 16S rRNA gene also enabled high precision (>90% in species-level identification thereby opening up potential application of this approach for clinical microbial characterization.

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

    Science.gov (United States)

    Pratihar, Suman; Bhattacharya, Manojit; Deuti, Kaushik

    2016-07-01

    Genus Duttaphrynus (Amphibia: Anura: Bufonidae) is endemic to southwestern and southern China and throughout southern Asia. Duttaphrynus phylogeny was also under debate for many years. 12S and 16S rDNAs help us to elucidate Duttaphrynus phylogeny. PMID:26155970

  7. TaxCollector: Modifying Current 16S rRNA Databases for the Rapid Classification at Six Taxonomic Levels

    Directory of Open Access Journals (Sweden)

    Eric W. Triplett

    2010-07-01

    Full Text Available The high level of conservation of 16S ribosomal RNA gene (16S rRNA in all Prokaryotes makes this gene an ideal tool for the rapid identification and classification of these microorganisms. Databases such as the Ribosomal Database Project II (RDP-II and the Greengenes Project offer access to sets of ribosomal RNA sequence databases useful in identification of microbes in a culture-independent analysis of microbial communities. However, these databases do not contain all of the taxonomic levels attached to the published names of the bacterial and archaeal sequences. TaxCollector is a set of scripts developed in Python language that attaches taxonomic information to all 16S rRNA sequences in the RDP-II and Greengenes databases. These modified databases are referred to as TaxCollector databases, which when used in conjunction with BLAST allow for rapid classification of sequences from any environmental or clinical source at six different taxonomic levels, from domain to species. The TaxCollector database prepared from the RDP-II database is an important component of a new 16S rRNA pipeline called PANGEA. The usefulness of TaxCollector databases is demonstrated with two very different datasets obtained using samples from a clinical setting and an agricultural soil. The six TaxCollector scripts are freely available on http://taxcollector.sourceforge.net and on http://www.microgator.org.

  8. Bacterial diversity in the Uranium mill-tailings Gittersee as estimated via a 16S rDNA approach

    International Nuclear Information System (INIS)

    Bacterial diversity in a soil sample collected from uranium mill-tailings called Gittersee and situated near the city of Dresden, Germany, was analysed by using a culture-independent 16S rDNA approach exploiting PCR amplification primers 7F and 1513R. The results were compared with those obtained earlier analysing the same sample by using another primer pair, namely 43F-1404R. The two 16S rDNA approaches demonstrated that Proteobacteria were the most predominant group in the sample, followed by Cytophaga/Flavobacterium/ Bacteroidesand by Gram positive bacteria with low and also with high G+C content too. A large number of 16S rDNA sequences from two libraries were identical or almost identical. However, the ratio between the bacterial groups represented in them significantly differed. 7F-1513R primer set retrieved in addition to the above mentioned sequences, also 16S rRNA of green non-sulphur bacteria and representatives of the AD1 and the OP11 divisions. The latter indicates that the 7F-1513R primer set seems to be more reliable in analyses of bacterial diversity. (authors)

  9. Bacterial diversity in Philippine fermented mustard (burong mustasa) as revealed by 16S rRNA gene analysis.

    Science.gov (United States)

    Larcia, L L H; Estacio, R C; Dalmacio, L M M

    2011-12-01

    Previous studies on the bacterial profile of burong mustasa, a traditional Philippine fermented food, had been conducted using culture-dependent techniques. Since these methods may underestimate the total microbiota of a sample, a culture-independent study was done to determine the bacterial diversity in burong mustasa through molecular biology techniques. Bacterial DNA was isolated from fermented mustard samples at different stages of fermentation. The isolated genomic DNA was amplified by PCR using specific primers for the 16S ribosomal RNA gene (16S rDNA). The 1.5 kb amplicons obtained were subjected to nested PCR using primers for the internal variable region of the 16S rDNA. The 585 bp nested PCR amplicons were then subjected to denaturing gradient gel electrophoresis (DGGE) to separate the different bacteria present in each sample. Distinct and unique bands in the DGGE profile were excised, reamplified, purified and sequenced for bacterial identification. Molecular cloning of the 1.5 kb 16S rDNA was also performed using the pGEM-T Easy Vector System. The cloned gene was sequenced for bacterial identification. The identified microbiota in burong mustasa at different stages of fermentation include lactic acid bacteria and several uncultured bacteria (initial up to the final stages); acetic acid bacteria (middle stage); and Streptobacillus and Fusobacterium species (initial stage). The potential probiotic bacteria found in burong mustasa are Weissella and Lactobacillus. PMID:22146686

  10. Comparison of gull-specific assays targeting 16S rRNA gene of Catellicoccus marimammalium and Streptococcus spp.

    Science.gov (United States)

    Gulls have been implicated as a source of fecal contamination in inland and coastal waters. Only one gull-specific assay is currently available (i.e., gull2 qPCR assay). This assay is based on the 16S rRNA gene of Catellicocclls marimammalium and has showed a high level of host-s...

  11. Rare-Earth Calcium Oxyborate Piezoelectric Crystals ReCa4O(BO33: Growth and Piezoelectric Characterizations

    Directory of Open Access Journals (Sweden)

    Fapeng Yu

    2014-07-01

    Full Text Available Rare-earth calcium oxyborate crystals, ReCa4O(BO33 (ReCOB, Re = Er, Y, Gd, Sm, Nd, Pr, and La , are potential piezoelectric materials for ultrahigh temperature sensor applications, due to their high electrical resistivity at elevated temperature, high piezoelectric sensitivity and temperature stability. In this paper, different techniques for ReCOB single-crystal growth are introduced, including the Bridgman and Czochralski pulling methods. Crystal orientations and the relationships between the crystallographic and physical axes of the monoclinic ReCOB crystals are discussed. The procedures for dielectric, elastic, electromechanical and piezoelectric property characterization, taking advantage of the impedance method, are presented. In addition, the maximum piezoelectric coefficients for different piezoelectric vibration modes are explored, and the optimized crystal cuts free of piezoelectric cross-talk are obtained by rotation calculations.

  12. Genotyping of bacteria belonging to the former Erwinia genus by PCR-RFLP analysis of a recA gene fragment.

    Science.gov (United States)

    Waleron, Małgorzata; Waleron, Krzysztof; Podhajska, Anna J; Lojkowska, Ewa

    2002-02-01

    Genotypic characterization, based on the analysis of restriction fragment length polymorphism of the recA gene fragment PCR product (recA PCR-RFLP), was performed on members of the former Erwinia genus. PCR primers deduced from published recA gene sequences of Erwinia carotovora allowed the amplification of an approximately 730 bp DNA fragment from each of the 19 Erwinia species tested. Amplified recA fragments were compared using RFLP analysis with four endonucleases (AluI, HinfI, TasI and Tru1I), allowing the detection of characteristic patterns of RFLP products for most of the Erwinia species. Between one and three specific RFLP groups were identified among most of the species tested (Erwinia amylovora, Erwinia ananas, Erwinia cacticida, Erwinia cypripedii, Erwinia herbicola, Erwinia mallotivora, Erwinia milletiae, Erwinia nigrifluens, Erwinia persicina, Erwinia psidii, Erwinia quercina, Erwinia rhapontici, Erwinia rubrifaciens, Erwinia salicis, Erwinia stewartii, Erwinia tracheiphila, Erwinia uredovora, Erwinia carotovora subsp. atroseptica, Erwinia carotovora subsp. betavasculorum, Erwinia carotovora subsp. odorifera and Erwinia carotovora subsp. wasabiae). However, in two cases, Erwinia chrysanthemi and Erwinia carotovora subsp. carotovora, 15 and 18 specific RFLP groups were detected, respectively. The variability of genetic patterns within these bacteria could be explained in terms of their geographic origin and/or wide host-range. The results indicated that PCR-RFLP analysis of the recA gene fragment is a useful tool for identification of species and subspecies belonging to the former Erwinia genus, as well as for differentiation of strains within E. carotovora subsp. carotovora and E. chrysanthemi. PMID:11832521

  13. Crystal structure of THEP1 from the hyperthermophile Aquifex aeolicus: a variation of the RecA fold

    Directory of Open Access Journals (Sweden)

    Wittinghofer Alfred

    2005-03-01

    Full Text Available Abstract Background aaTHEP1, the gene product of aq_1292 from Aquifex aeolicus, shows sequence homology to proteins from most thermophiles, hyperthermophiles, and higher organisms such as man, mouse, and fly. In contrast, there are almost no homologous proteins in mesophilic unicellular microorganisms. aaTHEP1 is a thermophilic enzyme exhibiting both ATPase and GTPase activity in vitro. Although annotated as a nucleotide kinase, such an activity could not be confirmed for aaTHEP1 experimentally and the in vivo function of aaTHEP1 is still unknown. Results Here we report the crystal structure of selenomethionine substituted nucleotide-free aaTHEP1 at 1.4 Å resolution using a multiple anomalous dispersion phasing protocol. The protein is composed of a single domain that belongs to the family of 3-layer (α/β/α-structures consisting of nine central strands flanked by six helices. The closest structural homologue as determined by DALI is the RecA family. In contrast to the latter proteins, aaTHEP1 possesses an extension of the β-sheet consisting of four additional β-strands. Conclusion We conclude that the structure of aaTHEP1 represents a variation of the RecA fold. Although the catalytic function of aaTHEP1 remains unclear, structural details indicate that it does not belong to the group of GTPases, kinases or adenosyltransferases. A mainly positive electrostatic surface indicates that aaTHEP1 might be a DNA/RNA modifying enzyme. The resolved structure of aaTHEP1 can serve as paradigm for the complete THEP1 family.

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

    Directory of Open Access Journals (Sweden)

    Xiao Li Shi

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

  15. The Identification of Discriminating Patterns from 16S rRNA Gene to Generate Signature for Bacillus Genus.

    Science.gov (United States)

    More, Ravi P; Purohit, Hemant J

    2016-08-01

    The 16S ribosomal RNA (16S rRNA) gene has been widely used for the taxonomic classification of bacteria. A molecular signature is a set of nucleotide patterns, which constitute a regular expression that is specific to each particular taxon. Our main goal was to identify discriminating nucleotide patterns in 16S rRNA gene and then to generate signatures for taxonomic classification. To demonstrate our approach, we used the phylum Firmicutes as a model using representative taxa Bacilli (class), Bacillales (order), Bacillaceae (family), and Bacillus (genus), according to their dominance at each hierarchical taxonomic level. We applied combined composite vector and multiple sequence alignment approaches to generate gene-specific signatures. Further, we mapped all the patterns into the different hypervariable regions of 16S rRNA gene and confirmed the most appropriate distinguishing region as V3-V4 for targeted taxa. We also examined the evolution in discriminating patterns of signatures across taxonomic levels. We assessed the comparative classification accuracy of signatures with other methods (i.e., RDP Classifier, KNN, and SINA). Results revealed that the signatures for taxa Bacilli, Bacillales, Bacillaceae, and Bacillus could correctly classify isolate sequences with sensitivity of 0.99, 0.97, 0.94, and 0.89, respectively, and specificity close to 0.99. We developed signature-based software DNA Barcode Identification (DNA BarID) for taxonomic classification that is available at website http://www.neeri.res.in/DNA_BarID.htm . This pattern-based study provides a deeper understanding of taxon-specific discriminating patterns in 16S rRNA gene with respect to taxonomic classification. PMID:27104769

  16. 16S-23S rRNA Gene Intergenic Spacer Region Variability Helps Resolve Closely Related Sphingomonads.

    Science.gov (United States)

    Tokajian, Sima; Issa, Nahla; Salloum, Tamara; Ibrahim, Joe; Farah, Maya

    2016-01-01

    Sphingomonads comprise a physiologically versatile group many of which appear to be adapted to oligotrophic environments, but several also had features in their genomes indicative of host associations. In this study, the extent variability of the 16S-23S rDNA intergenic spacer (ITS) sequences of 14 ATCC reference sphingomonad strains and 23 isolates recovered from drinking water was investigated through PCR amplification and sequencing. Sequencing analysis of the 16S-23S rRNA gene ITS region revealed that the ITS sizes for all studied isolates varied between 415 and 849 bp, while their G+C content was 42.2-57.9 mol%. Five distinct ITS types were identified: ITS(none) (without tRNA genes), ITS(Ala(TGC)), ITS(Ala(TGC)+Ile(GAT)), ITS(Ile(GAT)+Ala(TGC)), and ITS (Ile(GAT)+Pseudo). All of the identified tRNA(Ala(TGC)) molecules consisted of 73 bases, and all of the tRNA(Ile(GAT)) molecules consisted of 74 bases. We also detected striking variability in the size of the ITS region among the various examined isolates. Highest variability was detected within the ITS-2. The importance of this study is that this is the first comparison of the 16S-23S rDNA ITS sequence similarities and tRNA genes from sphingomonads. Collectively the data obtained in this study revealed the heterogeneity and extent of variability within the ITS region compared to the 16S rRNA gene within closely related isolates. Sequence and length polymorphisms within the ITS region along with the ITS types (tRNA-containing or lacking and the type of tRNA) and ITS-2 size and sequence similarities allowed us to overcome the limitation we previously encountered in resolving closely related isolates based on the 16S rRNA gene sequence.

  17. The Identification of Discriminating Patterns from 16S rRNA Gene to Generate Signature for Bacillus Genus.

    Science.gov (United States)

    More, Ravi P; Purohit, Hemant J

    2016-08-01

    The 16S ribosomal RNA (16S rRNA) gene has been widely used for the taxonomic classification of bacteria. A molecular signature is a set of nucleotide patterns, which constitute a regular expression that is specific to each particular taxon. Our main goal was to identify discriminating nucleotide patterns in 16S rRNA gene and then to generate signatures for taxonomic classification. To demonstrate our approach, we used the phylum Firmicutes as a model using representative taxa Bacilli (class), Bacillales (order), Bacillaceae (family), and Bacillus (genus), according to their dominance at each hierarchical taxonomic level. We applied combined composite vector and multiple sequence alignment approaches to generate gene-specific signatures. Further, we mapped all the patterns into the different hypervariable regions of 16S rRNA gene and confirmed the most appropriate distinguishing region as V3-V4 for targeted taxa. We also examined the evolution in discriminating patterns of signatures across taxonomic levels. We assessed the comparative classification accuracy of signatures with other methods (i.e., RDP Classifier, KNN, and SINA). Results revealed that the signatures for taxa Bacilli, Bacillales, Bacillaceae, and Bacillus could correctly classify isolate sequences with sensitivity of 0.99, 0.97, 0.94, and 0.89, respectively, and specificity close to 0.99. We developed signature-based software DNA Barcode Identification (DNA BarID) for taxonomic classification that is available at website http://www.neeri.res.in/DNA_BarID.htm . This pattern-based study provides a deeper understanding of taxon-specific discriminating patterns in 16S rRNA gene with respect to taxonomic classification.

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

    Science.gov (United States)

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

    2011-04-28

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

  19. 16S rRNA terminal restriction fragment length polymorphism for the characterization of the nasopharyngeal microbiota.

    Directory of Open Access Journals (Sweden)

    Silvio D Brugger

    Full Text Available A novel non-culture based 16S rRNA Terminal Restriction Fragment Length Polymorphism (T-RFLP method using the restriction enzymes Tsp509I and Hpy166II was developed for the characterization of the nasopharyngeal microbiota and validated using recently published 454 pyrosequencing data. 16S rRNA gene T-RFLP for 153 clinical nasopharyngeal samples from infants with acute otitis media (AOM revealed 5 Tsp509I and 6 Hpy166II terminal fragments (TFs with a prevalence of >10%. Cloning and sequencing identified all TFs with a prevalence >6% allowing a sufficient description of bacterial community changes for the most important bacterial taxa. The conjugated 7-valent pneumococcal polysaccharide vaccine (PCV-7 and prior antibiotic exposure had significant effects on the bacterial composition in an additive main effects and multiplicative interaction model (AMMI in concordance with the 16S rRNA 454 pyrosequencing data. In addition, the presented T-RFLP method is able to discriminate S. pneumoniae from other members of the Mitis group of streptococci, which therefore allows the identification of one of the most important human respiratory tract pathogens. This is usually not achieved by current high throughput sequencing protocols. In conclusion, the presented 16S rRNA gene T-RFLP method is a highly robust, easy to handle and a cheap alternative to the computationally demanding next-generation sequencing analysis. In case a lot of nasopharyngeal samples have to be characterized, it is suggested to first perform 16S rRNA T-RFLP and only use next generation sequencing if the T-RFLP nasopharyngeal patterns differ or show unknown TFs.

  20. Molecular phylogeny of three Moina species based on 16S rDNA gene sequences%三种裸腹溞的16S rDNA序列分析与系统分类探讨

    Institute of Scientific and Technical Information of China (English)

    徐敏; 邓道贵; 张海军; 王文平; 晁秋杰

    2015-01-01

    为了检验已记录的3种裸腹溞(发头裸腹溞(Moina irrasa)、短型裸腹溞(Moina brachiata)、微型裸腹溞(Moina micrura))的系统分类,用试剂盒法分别提取3种裸腹溞的基因组DNA.利用特异性引物,通过PCR扩增了3种裸腹溞的16S rDNA部分序列,并与来自GenBank中每个种类相似度较高的裸腹溞属序列进行分析.结果表明,3种裸腹溞的平均种间相似度为88.7%,碱基中A+T含量均明显高于G+C含量.本研究的发头裸腹溞的16S rDNA序列与GenBank所下载的多刺裸腹溞(Moina macrocopa)的16S rDNA序列相似度为99%,遗传距离(K2P双参考模型)为0.5%,属种内范围;两个地区的短型裸腹溞测得的16S rDNA序列与GenBank下载的欧洲短型裸腹溞的16S rDNA序列序列相似度相对较低(88%~90%),遗传距离较大(13.2% ~ 13.5%左右),已达到属内种间分化水平.基于16S rDNA构建的NJ树和贝叶斯树也支持以上结论.结果表明,本研究的发头裸腹溞可能为多刺裸腹溞,本研究用的短型裸腹溞与GenBank下载的欧洲短型裸腹溞已经达到种间分化的标准.由于缺乏物种形态资料和其他分子标记的对比,3种裸腹溞的分类地位还需进行更深入的探讨.

  1. 16S rRNA甲基化酶在产KPC酶肺炎克雷伯菌中的分布%The distribution of 16S rRNA methylase genes in KPC-producing Klebsiella pneumoniae strains

    Institute of Scientific and Technical Information of China (English)

    陆理英; 张伟丽; 杨青; 周华; 俞云松

    2009-01-01

    目的 了解产肺炎克雷伯菌碳青霉烯酶-2型(KPC-2)肺炎克雷伯菌中16S rRNA甲基化酶基因的分布.方法 收集37株产KPC-2肺炎克雷伯菌,使用琼脂稀释法测定其对阿米卡星、庆大霉素和奈替米星的最小抑菌浓度(MIC),PCR扩增6种16S rRNA甲基化酶基因:armA、rmtA、rmtB、rmtC、rmtD和npmA.结果 产KPC-2肺炎克雷伯菌对阿米卡星、庆大霉素和奈替米星的耐药率均为97.3%(MIC50≥1024μg/mL),其中8株检出arms基因,25株检出rmtB基因,同时检出armA和rmtB基因的有4株,未检测到rmtA、rmtC、rmtD和npmA阳性菌株.16S rRNA甲基化酶基因总阳性率为78.4%(29/37).结论 16S rRNA甲基化酶基因armA和rmtB在产KPC-2肺炎克雷伯菌中广泛分布.%Objective To investigate the distribution of 16S rRNA methylase genes in Klebsiella pneumoniae strains producing Klebsiella pneumoniae ealbapenenase type 2(KPC-2).Methods A total of 37 Klebsiella pneumoniae isolates producing KPC-2 were collected.The minimal inhibitory concentrations (MICs)of these strains to amikacin,gentamyein and netilmicin were determinated by agal dilution method.Six 16S rRNA methylase genes(armA,rmtA,rmtB,rmtC,rmtD and npmA)were detected by PCR.Results The resistant rates to amikacin,gentamycin and netilmicin were 97.3%(MIC50≥1024μg/mL).Among those resistant strains,8 were armr/A positive,25 were rmtB positive,4 were both armA and rmtB positive.and no other 16S rRNA methylase genes were found.The total positive rate of 16S rRNA methylase genes was 78.4%(29/37).Conclusion 16S rRNA methylase genes armA and rmtB ale prevalent in Klebsiella pneumoniae strains producing KPC-2.

  2. 病犬大肠杆菌16S rRNA甲基化酶基因检测%Molecular Detection of 16S rRNA Methylase Genes Among Escherichia coli Strains Isolated from Diseased Dogs

    Institute of Scientific and Technical Information of China (English)

    陈玉霞; 李德喜; 杜向党; 潘玉善; 刘建华; 苑丽; 张素梅

    2011-01-01

    为了解郑州市发病犬大肠杆菌对氨基糖苷类药物高度耐药的16S rRNA甲基化酶流行情况,主要研究测定了分离自河南郑州市宠物医院发病犬的123株大肠杆菌对氨基糖苷类代表药物阿米卡星的敏感性;分别设计6种16S rRNA甲基化酶基因特异性引物,对耐药分离株进行16S rRNA甲基化酶基因PCR扩增检测.检测结果显示,在发病犬大肠杆菌中仅检测到armA和rmtB,其检出率分别为3.25%(4/123)和38.2%(47/123).其中,有3.25%(4/123)的大肠杆菌可同时检测到armA和rmtB.这些结果提示,此地区发病犬大肠杆菌16S rRNA甲基化酶基因以rmtB为主.病犬细菌一旦携带这些耐药基因,可导致对氨基糖苷类药物高度耐药,应引起重视.%In order to investigate the epidemiology of 16S rRNA methylases which mediated the high level resistance to aminoglycosides among Escherichia coli strains isolated from diseased dogs in zhengzhou city of He' nan Province. 123 E. coli strains were isolated from clinic samples in diseased dogs. Antibacterial susceptibility determinations were performed and six types of 16S rRNA methylase genes were detected by PCR. Of six types of 16S rRNA methylase genes, only armA and rmtB were detected and the positive rates among these E. coli strains were 3.25% (4/123)and 38.2% (47/123), respectively. The concurrence rate of armA and rmtB genes in E. coli strains in diseased dogs was 3.25%. These results suggested 16S rRNA methylases among E. coli strains isolated from diseased dogs were prevalent in Zhengzhou City of He' nan Province, with rmtB dominant. One the pathogens in dogs carried these resistant genes, which could result in the high level resistance to aminoglycosides. So, this serious situation should cause concern.

  3. The study of 16S rRNA methylase genes in Enterobacter cloacae%庆大霉素耐药阴沟肠杆菌16S rRNA甲基化酶基因的研究

    Institute of Scientific and Technical Information of China (English)

    李玉珍; 吴燕峰; 李红玉; 杨银梅

    2011-01-01

    目的 了解庆大霉素耐药阴沟肠杆菌中16S rRNA甲基化酶基因的分布情况.方法 筛选临床分离的庆大霉素耐药的阴沟肠杆菌30株.采用聚合酶链反应(PCR)方法扩增16S rRNA甲基化酶五种相关耐药基因armA、rmtA、rmtB、rmtC和rmtD,PCR产物进行电泳分析和测序,抗菌药物的药物敏感性试验采用纸片扩散法进行,Whonet 5.4软件进行数据分析.结果 30株庆大霉素耐药阴沟肠杆菌中16S rRNA甲基化酶基因的检出率为56.7%(17/30),其中30.0%(9/30)检出armA基因、26.7%(8/30)检出rmtB基因,未检出rmtA、rmtC和rmtD基因.30株庆大霉素耐药阴沟肠杆菌对妥布霉素、阿米卡星耐药率分别为93.3%,83.3%;对其他抗菌药物除亚胺培南较敏感外,其余抗菌药物耐药率大于60.0%.结论 庆大霉素耐药阴沟肠杆菌中16S rRNA甲基化酶基因主要是armA和rmtB基因,碳青霉烯类对该菌有较好体外抗菌活性.%Objective To investigate the distribution of 16S rRNA methylase genes in gentamicin-resistant Enterobacter cloacae.Method 30 strains of clinically isolated Enterobacter cloacae were collected.Five 16S rRNA methylase genes including armA, rmtA, rmtB, rmtC and rmtD, were detected by polymerase chain reaction (PCR).The PCR products were confirmed by electrophoresis analysis and sequencing.The Antimicrobial Susceptibility Testing was conducted by disk diffusion method.The data were analyzed by Whonet 5.4 software.Result The relevance ratio of 16S rRNA methylase genes were 56.7% (17/30) including armA genes 30.0% (9/30) and rmtB genes 26.7% (8/30).None of the isolates carried rmtA, rmtC and rmtD genes.The resistence rate of Enterobacter cloacae to amikacin and tobramycin were 93.3% and 83.3% respectively.While being sensitive to imipenem, the strains were resistant to many other antimicrobial drugs with the resistance rate over 60.0%.Conclusion 16S rRNA methylase genes in Enterobacter cloacae are mainly armA and rmtB genes

  4. Factores de riesgo que influyen en la recaída de consumo de drogas lícitas e ilícitas en adolescentes atendidos en el Instituto sobre Alcoholismo y Farmacodependencia

    OpenAIRE

    Tatiana Blanco-Álvarez; Silvia Jiménez-Morales

    2015-01-01

    El objetivo de la presente investigación fue determinar los factores de riesgo asociados con las recaídas en el consumo de drogas en adolescentes que han recibido tratamiento en el IAFA. Metodología: Estudio cuantitativo, con alcance correlacional y transversal. Instrumentos: Entrevista de Recaídas para Usuarios de Conductas Adictivas, Inventario de Situaciones Precipitantes de Recaídas, Escala de Control Atencional, Inventario de Habilidades de Afrontamiento, Escala de Depresión, Ansiedad y ...

  5. Bacterial diversity of soil under eucalyptus assessed by 16S rDNA sequencing analysis Diversidade bacteriana de solo sob eucaliptos obtida por seqüenciamento do 16S rDNA

    OpenAIRE

    Érico Leandro da Silveira; Rodrigo Matheus Pereira; Denilson César Scaquitto; Eliamar Aparecida Nascimbém Pedrinho; Silvana Pómpeia Val-Moraes; Ester Wickert; Lúcia Maria Carareto-Alves; Eliana Gertrudes Macedo Lemos

    2006-01-01

    Studies on the impact of Eucalyptus spp. on Brazilian soils have focused on soil chemical properties and isolating interesting microbial organisms. Few studies have focused on microbial diversity and ecology in Brazil due to limited coverage of traditional cultivation and isolation methods. Molecular microbial ecology methods based on PCR amplified 16S rDNA have enriched the knowledge of soils microbial biodiversity. The objective of this work was to compare and estimate the bacterial diversi...

  6. The use of 16s rDNA methods in soil microbial ecology Uso de métodos 16S rDNA em ecologia microbiana do solo

    OpenAIRE

    Andrew Macrae

    2000-01-01

    New and exciting molecular methods, many using the 16S small sub-unit ribosomal nucleic acid molecule, are opening the microbial "black box" in soil. These studies have added much to our knowledge of microbial diversity in soils, and are beginning to advance our understanding of the relationship between this diversity and its function in soil processes. Over the next few years, the knowledge gained from molecular studies will, we hope, lead to improvements in sustainable land management and s...

  7. 我国重要帘蛤科(Veneridae)贝类的16S rRNA序列系统学分析%MOLECULAR PHYLOGENY OF VENERIDAE (MOLLUSCA, BIVALVIA) BASED ON 16S rRNA SEQUENCES

    Institute of Scientific and Technical Information of China (English)

    赵婷; 吴琪; 潘宝平

    2013-01-01

    本文对我国隶属于帘蛤科(Veneridae)10个亚科、17个属、20种贝类的16S rRNA基因片段进行了系统学分析,上述动物的16S rRNA片段长度在438-648bp之间,利用PAUP软件包在对序列比对基础上构建了邻接系统树(NJ)和最大拟然系统树(ML).16S rRNA数据显示,我国帘蛤科贝类由三个主要分支组成,美女蛤亚科中的加夫蛤属(Gafrarium)可能是一个单型属,该属与美女蛤属合并为加夫蛤属比较恰当.帘蛤亚科与雪蛤亚科应属于不连续的分类单元.另外,青蛤亚科与仙女蛤亚科均应作为独立的亚科存在.本文的研究结论与修订后的帘蛤科形态分类观点一致.

  8. Application of bacterial 16S rDNA amplification and sequencing in the classification and identification of bacteria%16S rDNA扩增及测序在细菌鉴定与分类中的应用

    Institute of Scientific and Technical Information of China (English)

    朱诗应; 戚中田

    2013-01-01

    Bacterial 16S rDNA amplification and sequencing is a new tool which has been widely used to identify bacterial species and perform taxonomic studies . The application of this technology for identification of uncultivable bacteria , differentiating species with high DNA sequence similarity and discovering novel bacterial genus and species are introduced in this paper . Future perspective of the method in clinical microbiology laboratories is also discussed .%16S rDNA扩增及测序技术在细菌的鉴定与分类研究中发挥着越来越重要的作用.本文就16S rDNA结构、可变区和保守区部分序列或全序列在临床上细菌鉴定和新细菌识别等方面的研究进展进行综述,并对其在临床实验室中的应用进行展望.

  9. Advances and Applications on Methodology of 16S rRNA Sequencing in Gut Microbiota Analysis%16S rRNA测序技术在肠道微生物中的应用研究进展

    Institute of Scientific and Technical Information of China (English)

    李东萍; 郭明璋; 许文涛

    2015-01-01

    16S rRNA sequencing is one of the high-throughput-sequencing-based methods used in gut microbiota analysis. Almost all the bacterial species in gut microbiota can be quantified through 16S rRNA sequencing, which has made this method into the mainstream. Two issues are very important in the application of 16S rRNA sequencing:sequencing strategy and bioinformatic analysis. In this review, three aspects of the sequencing strategy, including sequencing platform, sequencing region, and data size were discussed. While on bioinformatic analysis, the advance in sequences cluster and annotation, microbiota structure analysis, key taxa screening and functional analysis were reviewed here.%16S rRNA测序是高通量测序依赖的肠道微生物研究方法之一,该方法可以对肠道微生物中的所有菌种进行精确定量,因此正逐渐成为研究肠道微生物菌种丰度变化的主流。肠道微生物16S rRNA测序的应用过程中有两个问题至关重要,一是如何根据需要选择测序方案;二是面对高通量测序得到的海量数据,如何进行生物信息学分析,以得到具有生物学意义的结果。从测序平台、测序片段、测序数据量的选择3个方面讨论了如何选择测序方案,并从序列聚类与注释、群落结构分析、关键分类单位的筛选与功能分析等方面对目前常用的生物信息学分析手段进行综述。

  10. 食品中弓形菌16S rRNA特异性扩增检测方法的建立%Development of a specific amplification method of Arcobacter 16S rRNA gene in foods

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    针对弓形菌16SrRNA基因合成1对引物,通过对聚合酶链式反应(PCR)扩增条件的优化,建立了检测弓形菌的PCR方法。3株弓形菌标准菌株PCR产物测序结果与NCBI上公布的弓形菌16S rRNA基因序列进行比对,比对结果表明3株弓形菌测序结果与NCBI上公布的弓形菌16S rRNA基因序列同源性均在99%以上。3株弓形菌标准菌株均特异性地扩增出了长度为1202bp的片段,其他19株不同种类的菌株均无扩增产物出现。55份食品样品用Johnson-Murano肉汤增菌后用此法进行检测,其中6份样品为弓形菌阳性,阳性率为10.9%。上述实验结果表明,方法特异性强、操作简便,节省了检测时间,可用于食品中弓形菌的快速检测。%One pair of primers were used to amplify 16S rRNA sequence of Arcobacter,and a PCR method for detection of Arcobacter was developed with optimization of PCR amplification conditions.PCR products of 3 different Arcobacter type strains were sequenced and compared with 16S rRNA genes of Arcobacter published on NCBI.It's proved that the homology between the PCR products and 16S rRNA genes of Arcobacter published were all above 99%.3 different type strains of Arcobacter produced a 1202 bp amplified band,while all of the other 19 different bacteria didn't produced any band.55 food samples were detected whether Arcobacter present or not by the PCR method following enrichment in Johnson-Murano broth,and 6 samples were detected positive for this microorganism,namely with the positive ratio of 10.9%.The result suggested that the method developed was specific,easy to operate and timesaving,and could be used for rapid detection of Arcobacter in foods.

  11. 16S rDNA在婴幼儿配方乳粉阪崎肠杆菌鉴定中的应用%Application of 16S rDNA in Enterobacter Sakazakii Identiifcation for Infant Formula Milk Powder

    Institute of Scientific and Technical Information of China (English)

    林玉宙

    2015-01-01

    选取近几年从婴幼儿配方乳粉生产过程环境及产品中收集、经国标GB/T 4789.40-2010检验和API20E鉴定为阪崎肠杆菌阳性的菌株16株,利用MicroSEQ ID微生物鉴定系统进行16S rDNA基因测序分析,构建系统发育树,鉴定种属。结果显示,这16份经API20E鉴定为阪崎肠杆菌阳性的菌株,经16S rDNA基因测序证实,其中2份为梨形肠杆菌,1份为克氏柠檬酸杆菌,其余13份样品均为阪崎肠杆菌,且与数据库中标准菌株的同源性达到99.5%以上。16S rDNA基因测序方法在婴幼儿配方乳粉企业进行阪崎肠杆菌鉴定和溯源方面具有广阔的前景。%Sixteen Enterobacter sakazaki strains col ected from the environment and products during infant formula milk powder production process and identiifed to be positive by GB/T 4789.40-2010 as wel as API20E were analyzed by 16S rDNA gene se-quencing using MicroSEQ ID microbial identiifcation system,and the system development tree was built to identify the species. The results showed that two of 16 Enterobacter sakazaki strains were Enterobacter pyrinus,and one was Citrobacter koseri,and the other thirteen were Enterobacter sakazaki and the homology with standard Enterobacter sakazaki strain in the database was more than 99.5%. 16S rDNA gene sequencing method has the broad prospect in the identiifcation and traceability of Enterobacter saka-zaki for infant formula enterprises.

  12. Isolation and 16S DNA characterization of soil microorganisms from tropical soils capable of utilizing the herbicides hexazinone and tebuthiuron.

    Science.gov (United States)

    Mostafa, Fadwa I Y; Helling, Charles S

    2003-11-01

    Six non-fermentative bacteria were isolated from Colombian (South America) and Hawaiian (USA) soils after enrichment with minimal medium supplemented with two herbicides, hexazinone (Hex) and tebuthiuron (Teb). Microscopic examination and physiological tests were followed by partial 16S DNA sequence analysis, using the first 527 bp of the 16S rRNA gene for bacterial identification. The isolated microorganisms (and in brackets, the herbicide that each degraded) were identified as: from Colombia. Methylobacterium organophilum [Teb], Paenibacillus pabuli [Teb], and Micrmbacterium foliorum [Hex]; and from Hawaii, Methylobacterium radiotolerans [Teb], Paenibacillus illinoisensis [Hex], and Rhodococcus equi [Hex]. The findings further explain how these herbicides, which have potential for illicit coca (Erythroxylum sp.) control, dissipate following their application to tropical soils. PMID:14649709

  13. Phylogenetic relationship of 16 Oedipodidae species (Insecta: Orthoptera) based on the 16S rRNA gene sequences

    Institute of Scientific and Technical Information of China (English)

    HUI-MENG LU; YUAN HUANG

    2006-01-01

    The sequences of the mitochondrial 16S rRNA gene of 16 Oedipodidae species were amplified and sequenced. All sequences were aligned and analyzed and the phyloge netic relationships were inferred. The properties of 16S gene in Oedipodidae showed typical patterns of many insects such as a high A+T content and variable distance-dependent transition/transversion ratios. The 0.2 weight for sites of loops may be advisable for phylogeny reconstruction using the maximum parsimony method. The phylogenetic analysis results do not support the current subfamily classification systems of Oedipodidae. Bryodemellinae and Bryodeminae are closely related and should be merged as one subfamily. The status of Oedipodinae and Locustinae is also problematic.

  14. Phylogenetic relationships of five species of Dorippinae (Crustacea, Decapoda) revealed by 16S rDNA sequence analysis

    Institute of Scientific and Technical Information of China (English)

    FANYu; LIXinzheng; SONGLinsheng; CAIZhonghua

    2004-01-01

    A molecular phylogeny is presented for the subfamily Dorippinae (including 9 individuals, representing 5 species and 4 genera), based on the sequence data from 16S rRNA gene. Two-cluster test between lineages in these phylogenetic trees has been performed. On the basis of rate constancy, the rate of nucleotide substitutions of 16S rDNA sequence data is estimated as 0.27% per million years. The analysis strongly supports the recognition of the Dorippinae as a monophyletic subfamily. Phylogenetic tree indicates that the subfamily Dorippinae is divided into two main clades, and genus Dorippe appears basal in the subfamily, diverging from other species 36.6 Ma ago. It is also clear that the Heikea is closely related to the genus Neodorippe. The divergence time between them is 15.8 Ma.

  15. Identification of bacteria associated with underground parts of Crocus sativus by 16S rRNA gene targeted metagenomic approach.

    Science.gov (United States)

    Ambardar, Sheetal; Sangwan, Naseer; Manjula, A; Rajendhran, J; Gunasekaran, P; Lal, Rup; Vakhlu, Jyoti

    2014-10-01

    Saffron (Crocus sativus L), an autumn-flowering perennial sterile plant, reproduces vegetatively by underground corms. Saffron has biannual corm-root cycle that makes it an interesting candidate to study microbial dynamics in its rhizosphere and cormosphere (area under influence of corm). Culture independent 16S rRNA gene metagenomic study of rhizosphere and cormosphere of Saffron during flowering stage revealed presence of 22 genera but none of the genus was common in all the three samples. Bulk soil bacterial community was represented by 13 genera with Acidobacteria being dominant. In rhizosphere, out of eight different genera identified, Pseudomonas was the most dominant genus. Cormosphere bacteria comprised of six different genera, dominated by the genus Pantoea. This study revealed that the bacterial composition of all the three samples is significantly different (P rhizosphere, cormosphere and bulk soil of Saffron, using cultivation independent 16S rRNA gene targeted metagenomic approach. PMID:24989343

  16. Salinity inhibits post transcriptional processing of chloroplast 16S rRNA in shoot cultures of jojoba (Simmondsia chinesis).

    Science.gov (United States)

    Mizrahi-Aviv, Ela; Mills, David; Benzioni, Aliza; Bar-Zvi, Dudy

    2005-03-01

    Chloroplast metabolism is rapidly affected by salt stress. Photosynthesis is one of the first processes known to be affected by salinity. Here, we report that salinity inhibits chloroplast post-transcriptional RNA processing. A differentially expressed 680-bp cDNA, containing the 3' sequence of 16S rRNA, transcribed intergenic spacer, exon 1 and intron of tRNA(Ile), was isolated by differential display reverse transcriptase PCR from salt-grown jojoba (Simmondsia chinesis) shoot cultures. Northern blot analysis indicated that although most rRNA appears to be fully processed, partially processed chloroplast 16S rRNA accumulates in salt-grown cultures. Thus, salinity appears to decrease the processing of the rrn transcript. The possible effect of this decreased processing on physiological processes is, as yet, unknown.

  17. Vertical stratification of microbial communities in the Red Sea revealed by 16S rDNA pyrosequencing

    OpenAIRE

    Qian, Pei-Yuan; Wang, Yong; Lee, On On; Lau, Stanley C K; Yang, Jiangke; Lafi, Feras F.; Al-Suwailem, Abdulaziz; Wong, Tim YH

    2010-01-01

    The ecosystems of the Red Sea are among the least-explored microbial habitats in the marine environment. In this study, we investigated the microbial communities in the water column overlying the Atlantis II Deep and Discovery Deep in the Red Sea. Taxonomic classification of pyrosequencing reads of the 16S rRNA gene amplicons showed vertical stratification of microbial diversity from the surface water to 1500 m below the surface. Significant differences in both bacterial and archaeal diversit...

  18. Determination of 16S rRNA Sequences of Enterococci and Application to Species Identification of Nonmotile Enterococcus gallinarum Isolates

    OpenAIRE

    Patel, Robin; Piper, Kerryl E.; Rouse, Mark S; Steckelberg, James M.; Uhl, Jim R.; Kohner, Peggy; Hopkins, Marlene K.; Cockerill, Franklin R.; Kline, Bruce C.

    1998-01-01

    The 16S rRNA sequences of enterococcal species E. faecium, E. faecalis, E. gallinarum, E. casseliflavus/flavescens, E. dispar, E. pseudoavium, E. sulfureus, E. malodoratus, E. raffinosus, E. cecorum, E. hirae, E. saccharolyticus, E. seriolicida, E. mundtii, E. avium, E. durans, E. columbae, and E. solitarius are presented herein. These data were utilized to confirm the species identification of two nonmotile E. gallinarum isolates which had been previously phenotypically identified as E. faec...

  19. Family- and Genus-Level 16S rRNA-Targeted Oligonucleotide Probes for Ecological Studies of Methanotrophic Bacteria

    OpenAIRE

    J. Gulledge; Ahmad, A; Steudler, P. A.; Pomerantz, W. J.; Cavanaugh, Colleen Marie

    2001-01-01

    Methanotrophic bacteria play a major role in the global carbon cycle, degrade xenobiotic pollutants, and have the potential for a variety of biotechnological applications. To facilitate ecological studies of these important organisms, we developed a suite of oligonucleotide probes for quantitative analysis of methanotroph-specific 16S rRNA from environmental samples. Two probes target methanotrophs in the family Methylocystaceae (type II methanotrophs) as a group. No oligonucleotide signature...

  20. The structure of the archaebacterial ribosomal protein S7 and its possible interaction with 16S rRNA.

    Science.gov (United States)

    Hosaka, H; Yao, M; Kimura, M; Tanaka, I

    2001-11-01

    Ribosomal protein S7 is one of the ubiquitous components of the small subunit of the ribosome. It is a 16S rRNA-binding protein positioned close to the exit of the tRNA, and it plays a role in initiating assembly of the head of the 30S subunit. Previous structural analyses of eubacterial S7 have shown that it has a stable alpha-helix core and a flexible beta-arm. Unlike these eubacterial proteins, archaebacterial or eukaryotic S7 has an N-terminal extension of approximately 60 residues. The crystal structure of S7 from archaebacterium Pyrococcus horikoshii (PhoS7) has been determined at 2.1 A resolution. The final model of PhoS7 consists of six major alpha-helices, a short 3(10)-helix and two beta-stands. The major part (residues 18-45) of the N-terminal extension of PhoS7 reinforces the alpha-helical core by well-extended hydrophobic interactions, while the other part (residues 46-63) is not visible in the crystal and is possibly fixed only by interacting with 16S rRNA. These differences in the N-terminal extension as well as in the insertion (between alpha1 and alpha2) of the archaebacterial S7 structure from eubacterial S7 are such that they do not necessitate a major change in the structure of the currently available eubacterial 16S rRNA. Some of the inserted chains might pass through gaps formed by helices of the 16S rRNA.

  1. Short-read assembly of full-length 16S amplicons reveals bacterial diversity in subsurface sediments.

    Directory of Open Access Journals (Sweden)

    Christopher S Miller

    Full Text Available In microbial ecology, a fundamental question relates to how community diversity and composition change in response to perturbation. Most studies have had limited ability to deeply sample community structure (e.g. Sanger-sequenced 16S rRNA libraries, or have had limited taxonomic resolution (e.g. studies based on 16S rRNA hypervariable region sequencing. Here, we combine the higher taxonomic resolution of near-full-length 16S rRNA gene amplicons with the economics and sensitivity of short-read sequencing to assay the abundance and identity of organisms that represent as little as 0.01% of sediment bacterial communities. We used a new version of EMIRGE optimized for large data size to reconstruct near-full-length 16S rRNA genes from amplicons sheared and sequenced with Illumina technology. The approach allowed us to differentiate the community composition among samples acquired before perturbation, after acetate amendment shifted the predominant metabolism to iron reduction, and once sulfate reduction began. Results were highly reproducible across technical replicates, and identified specific taxa that responded to the perturbation. All samples contain very high alpha diversity and abundant organisms from phyla without cultivated representatives. Surprisingly, at the time points measured, there was no strong loss of evenness, despite the selective pressure of acetate amendment and change in the terminal electron accepting process. However, community membership was altered significantly. The method allows for sensitive, accurate profiling of the "long tail" of low abundance organisms that exist in many microbial communities, and can resolve population dynamics in response to environmental change.

  2. 16s rRNA Identification of Pediococcus spp. from Broiler and Studies of Adherence Ability on Immobilized Mucus

    OpenAIRE

    Ema Damayanti; Lies Mira Yusiati; Achmad Dinoto

    2015-01-01

    The objectives of this research were to study taxonomical status of lactic acid bacteria (LAB) isolated from broiler and adherence ability on mucus in vitro. Molecular analysis was performed by analyzing 16S rRNA gene using universal primer. The adherence assay on mucus was carried out using microplate method with total plate count (TPC), absorbance (A550) and confirmed by scanning electron microscopy (SEM). The results of this studies revealed that three of LAB isolates have closed relation ...

  3. Analysis of 16S rRNA amplicon sequencing options on the Roche/454 next-generation titanium sequencing platform.

    Directory of Open Access Journals (Sweden)

    Hideyuki Tamaki

    Full Text Available BACKGROUND: 16S rRNA gene pyrosequencing approach has revolutionized studies in microbial ecology. While primer selection and short read length can affect the resulting microbial community profile, little is known about the influence of pyrosequencing methods on the sequencing throughput and the outcome of microbial community analyses. The aim of this study is to compare differences in output, ease, and cost among three different amplicon pyrosequencing methods for the Roche/454 Titanium platform METHODOLOGY/PRINCIPAL FINDINGS: The following three pyrosequencing methods for 16S rRNA genes were selected in this study: Method-1 (standard method is the recommended method for bi-directional sequencing using the LIB-A kit; Method-2 is a new option designed in this study for unidirectional sequencing with the LIB-A kit; and Method-3 uses the LIB-L kit for unidirectional sequencing. In our comparison among these three methods using 10 different environmental samples, Method-2 and Method-3 produced 1.5-1.6 times more useable reads than the standard method (Method-1, after quality-based trimming, and did not compromise the outcome of microbial community analyses. Specifically, Method-3 is the most cost-effective unidirectional amplicon sequencing method as it provided the most reads and required the least effort in consumables management. CONCLUSIONS: Our findings clearly demonstrated that alternative pyrosequencing methods for 16S rRNA genes could drastically affect sequencing output (e.g. number of reads before and after trimming but have little effect on the outcomes of microbial community analysis. This finding is important for both researchers and sequencing facilities utilizing 16S rRNA gene pyrosequencing for microbial ecological studies.

  4. Assessing hog lagoon waste contamination in the Cape Fear Watershed using Bacteroidetes 16S rRNA gene pyrosequencing.

    Science.gov (United States)

    Arfken, Ann M; Song, Bongkeun; Mallin, Michael A

    2015-09-01

    Hog lagoons can be major sources of waste and nutrient contamination to watersheds adjacent to pig farms. Fecal source tracking methods targeting Bacteroidetes 16S rRNA genes in pig fecal matter may underestimate or fail to detect hog lagoon contamination in riverine environments. In order to detect hog lagoon wastewater contamination in the Cape Fear Watershed, where a large number of hog farms are present, we conducted pyrosequencing analyses of Bacteroidetes 16S rRNA genes in hog lagoon waste and identified new hog lagoon-specific marker sequences. Additional pyrosequencing analyses of Bacteroidetes 16S rRNA genes were conducted with surface water samples collected at 4 sites during 5 months in the Cape Fear Watershed. Using an operational taxonomic unit (OTU) identity cutoff value of 97 %, these newly identified hog lagoon markers were found in 3 of the river samples, while only 1 sample contained the pig fecal marker. In the sample containing the pig fecal marker, there was a relatively high percentage (14.1 %) of the hog lagoon markers and a low pig fecal marker relative abundance of 0.4 % in the Bacteroidetes 16S rRNA gene sequences. This suggests that hog lagoon contamination must be somewhat significant in order for pig fecal markers to be detected, and low levels of hog lagoon contamination cannot be detected targeting only pig-specific fecal markers. Thus, new hog lagoon markers have a better detection capacity for lagoon waste contamination, and in conjunction with a pig fecal marker, provide a more comprehensive and accurate detection of hog lagoon waste contamination in susceptible watersheds.

  5. Asaia bogorensis peritonitis identified by 16S ribosomal RNA sequence analysis in a patient receiving peritoneal dialysis.

    Science.gov (United States)

    Snyder, Richard W; Ruhe, Jorg; Kobrin, Sidney; Wasserstein, Alan; Doline, Christa; Nachamkin, Irving; Lipschutz, Joshua H

    2004-08-01

    Here the authors report a case of refractory peritonitis leading to multiple hospitalizations and the loss of peritoneal dialysis access in a patient on automated peritoneal dialysis, caused by Asaia bogorensis, a bacterium not previously described as a human pathogen. This organism was identified by sequence analysis of the 16S ribosomal RNA gene. Unusual microbial agents may cause peritonitis, and molecular microbiological techniques are important tools for identifying these agents.

  6. Cilantro microbiome before and after nonselective pre-enrichment for Salmonella using 16S rRNA and metagenomic sequencing

    OpenAIRE

    Jarvis, Karen G.; White, James R.; Grim, Christopher J.; Ewing, Laura; Ottesen, Andrea R; Beaubrun, Junia Jean-Gilles; Pettengill, James B; Brown, Eric; Hanes, Darcy E.

    2015-01-01

    Background Salmonella enterica is a common cause of foodborne gastroenteritis in the United States and is associated with outbreaks in fresh produce such as cilantro. Salmonella culture-based detection methods are complex and time consuming, and improvments to increase detection sensitivity will benefit consumers. In this study, we used 16S rRNA sequencing to determine the microbiome of cilantro. We also investigated changes to the microbial community prior to and after a 24-hour nonselective...

  7. Predictive microbiology combined with metagenomic analysis targeted on the 16S rDNA : A new approach for food quality

    OpenAIRE

    Delhalle, Laurent; Ellouze, Mariem; Taminiau, Bernard; Korsak Koulagenko, Nicolas; Nezer, Carine

    2013-01-01

    OBJECTIVES The food spoilage process is mainly caused by alteration micro-organisms and classical culture-based methods have therefore been used to assess the microbiological quality of food. These techniques are simple to implement but may not be relevant to understand the modifications of the microbial ecology which occur in the food product in response to different changes in the environmental conditions. Metagenomic analysis targeted on 16S ribosomal DNA can bring about a solution to t...

  8. Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing

    OpenAIRE

    Diaz, Patricia I.; Abusleme, Loreto; Hong, Bo-Young; Amanda K. Dupuy; Linda D Strausbaugh

    2014-01-01

    Background and objective: The advent of next-generation sequencing has significantly facilitated characterization of the oral microbiome. Despite great efforts in streamlining the processes of sequencing and data curation, upstream steps required for amplicon library generation could still influence 16S rRNA gene-based microbial profiles. Among upstream processes, DNA extraction is a critical step that could represent a great source of bias. Accounting for bias introduced by extraction proced...

  9. Unique 16S rRNA sequences of Eurythenes gryllus (Crustacea: Amphipoda: Lysianassidae) from the Gulf of Mexico abyssal plain

    OpenAIRE

    Elva Escobar-Briones; Eduardo Nájera-Hillman; Fernando Álvarez

    2010-01-01

    Amphipods of the species Eurythenes gryllus were collected at 2 locations on the abyssal plain (~3 400 m) of the Gulf of Mexico in order to test whether or not these scavenger amphipods are isolated in this peripheral sea or show connectivity by their predominant swimming behavior, moving horizontally along the abyssal water masses in the region. Partial sequences of the mitochondrial 16S rRNA gene from 2 individuals of E. gryllus were determined and showed small differences when compared to ...

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

    OpenAIRE

    Mu Peng; Xiaoxue Zi; Qiuyu Wang

    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. Acidobacte...

  11. Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing.

    Science.gov (United States)

    Shin, Jongoh; Lee, Sooin; Go, Min-Jeong; Lee, Sang Yup; Kim, Sun Chang; Lee, Chul-Ho; Cho, Byung-Kwan

    2016-01-01

    Demands for faster and more accurate methods to analyze microbial communities from natural and clinical samples have been increasing in the medical and healthcare industry. Recent advances in next-generation sequencing technologies have facilitated the elucidation of the microbial community composition with higher accuracy and greater throughput than was previously achievable; however, the short sequencing reads often limit the microbial composition analysis at the species level due to the high similarity of 16S rRNA amplicon sequences. To overcome this limitation, we used the nanopore sequencing platform to sequence full-length 16S rRNA amplicon libraries prepared from the mouse gut microbiota. A comparison of the nanopore and short-read sequencing data showed that there were no significant differences in major taxonomic units (89%) except one phylotype and three taxonomic units. Moreover, both sequencing data were highly similar at all taxonomic resolutions except the species level. At the species level, nanopore sequencing allowed identification of more species than short-read sequencing, facilitating the accurate classification of the bacterial community composition. Therefore, this method of full-length 16S rRNA amplicon sequencing will be useful for rapid, accurate and efficient detection of microbial diversity in various biological and clinical samples. PMID:27411898

  12. Diversity of Archaea in Icelandic hot springs based on 16S rRNA and chaperonin genes.

    Science.gov (United States)

    Mirete, Salvador; de Figueras, Carolina G; González-Pastor, Jose E

    2011-07-01

    The diversity of archaeal communities growing in four hot springs (65-90 °C, pH 6.5) was assessed with 16S rRNA gene primers specific for the domain Archaea. Overall, mainly uncultured members of the Desulfurococcales, the Thermoproteales and the Korarchaeota, were identified. Based on this diversity, a set of chaperonin heat-shock protein (Hsp60) gene sequences from different archaeal species were aligned to design two degenerate primer sets for the amplification of the chaperonin gene: Ths and Kor (which can also detect the korarchaeotal chaperonin gene from one of the samples). A phylogenetic tree was constructed using the chaperonin sequences retrieved and other sequences from cultured representatives. The Alpha and Beta paralogs of the chaperonin gene were observed within the main clades and orthologs among them. Cultivated representatives from these clades were assigned to either paralog in the chaperonin tree. Uncultured representatives observed in the 16S rRNA gene analysis were found to be related to the Desulfurococcales. The topologies of the 16S rRNA gene and chaperonin phylogenetic trees were compared, and similar phylogenetic relationships were observed. Our results suggest that the chaperonin Hsp60 gene may be used as a phylogenetic marker for the clades found in this extreme environment.

  13. Studies on the ability of partially iodinated 16S RNA to participate in 30S ribosome assembly.

    Science.gov (United States)

    Schendel, P L; Craven, G R

    1976-11-01

    Deproteinated 16S RNA was iodinated at pH 5.0 in an aqueous solution containing TlCl3 plus KI for 1-5 hours at 42 degrees C. Under these conditions 33 moles of iodine are incorporated per mole of RNA. As judged by sucrose gradient sedimentation, the iodinated RNA does not exhibit any large alteration in conformation as compared to unmodified 16S. The iodinated RNA was examined for its ability to reconstitute with total 30S proteins. Sedimentation velocity analysis reveals that the reconstituted subunit has a sedimentation constant of approximately 20S. In addition, protein analysis of particles reconstituted with 16S RNA iodinated for 5 hours indicates that proteins S2, S10, S13, S14, S15, S17, S18, S19, and S21 are no longer able to participate in the 30S assembly process and that proteins S6, S16 and S20 are present in reduced amounts. The ramifications of these results concerning protein-RNA and RNA-RNA interactions occurring in ribosome assembly are discussed.

  14. Identification of forensically important beetles (Coleoptera: Histeridae) in China based on 16S rRNA and Cyt b.

    Science.gov (United States)

    Su, R N; Guo, Y D; Xie, D; Peng, Y L; Cai, J F; Hua, F; Sheng, L H

    2013-09-01

    Exact identification of an insect sample is usually the first essential step in a forensic entomological analysis. However, the morphological similarity of beetles in the level of species usually poses a challenge for forensic scientists within their routine work. As a supplementary to traditional morphological method, molecular genetics identification turns out to be simple and time-saving. A molecular identification method involving a 288-bp segment of the 16S ribosomal RNA (16S rRNA) gene and a 334-bp segment of the cytochrome b (Cyt b) gene from 23 histerid beetles specimens, collected from 7 locations in 6 Chinese provinces, was evaluated. The 16S rRNA and Cyt b genes are sequenced to examine the ability of the region, resolve species identities and enrich the local databases. The monophyletic branches of the phylogenetic tree showed the potential of the markers in identifying beetles within families. Combined analysis is a more accurate approach for species identication than independent analysis.

  15. Complete ecological isolation and cryptic diversity in Polynucleobacter bacteria not resolved by 16S rRNA gene sequences.

    Science.gov (United States)

    Hahn, Martin W; Jezberová, Jitka; Koll, Ulrike; Saueressig-Beck, Tanja; Schmidt, Johanna

    2016-07-01

    Transplantation experiments and genome comparisons were used to determine if lineages of planktonic Polynucleobacter almost indistinguishable by their 16S ribosomal RNA (rRNA) sequences differ distinctively in their ecophysiological and genomic traits. The results of three transplantation experiments differing in complexity of biotic interactions revealed complete ecological isolation between some of the lineages. This pattern fits well to the previously detected environmental distribution of lineages along chemical gradients, as well as to differences in gene content putatively providing adaptation to chemically distinct habitats. Patterns of distribution of iron transporter genes across 209 Polynucleobacter strains obtained from freshwater systems and representing a broad pH spectrum further emphasize differences in habitat-specific adaptations. Genome comparisons of six strains sharing ⩾99% 16S rRNA similarities suggested that each strain represents a distinct species. Comparison of sequence diversity among genomes with sequence diversity among 240 cultivated Polynucleobacter strains indicated a large cryptic species complex not resolvable by 16S rRNA sequences. The revealed ecological isolation and cryptic diversity in Polynucleobacter bacteria is crucial in the interpretation of diversity studies on freshwater bacterioplankton based on ribosomal sequences.

  16. Clinical Fusobacterium mortiferum Isolates Cluster with Undifferentiated Clostridium rectum Species Based on 16S rRNA Gene Phylogenetic Analysis.

    Science.gov (United States)

    Lee, Yangsoon; Eun, Chang Soo; Han, Dong Soo

    2016-05-01

    The most commonly encountered clinical Fusobacterium species are F. nucleatum and F. necrophorum; other Fusobacteria, such as F. mortiferum and F. varium, have occasionally been isolated from human specimens. Clostridium rectum is a gram-positive species characterized as a straight bacillus with oval sub-terminal spores. The close 16S rRNA gene sequence relationship of C. rectum with the genus Fusobacterium is unexpected given their very different phenotypic characteristics. Between 2014 and 2015, a total of 19 Fusobacterium isolates were recovered from the colonic tissue of 10 patients at a university hospital. All isolates were identified based on 16S rRNA gene sequencing. The phylogenetic relationship among these isolates was estimated using the neighbor-joining method and the Molecular Evolutionary Genetic Analysis (MEGA) version 6. Based on phylogenetic analysis, the F. mortiferum isolates clustered into two groups - F. mortiferum DSM 19809 (group I) and F. mortiferum ATCC 25557 (group II) - even though they are of the same species. Furthermore, the F. mortiferum DSM 19809 (group I) showed a close phylogenetic relationship with C. rectum, even though C. rectum is classified as a gram-positive spore-producing bacillus. C. rectum is clearly unrelated to the genus Clostridium as it shows highest 16S rRNA gene sequence similarity with species from the genus Fusobacterium Therefore, additional methods such as Gram staining and other biochemical methods should be performed for Fusobacterium identification.

  17. Molecular Diagnosis of Periprosthetic Joint Infection by Quantitative RT-PCR of Bacterial 16S Ribosomal RNA

    Directory of Open Access Journals (Sweden)

    Mel S. Lee

    2013-01-01

    Full Text Available The diagnosis of periprosthetic joint infection is sometimes straightforward with purulent discharge from the fistula tract communicating to the joint prosthesis. However it is often difficult to differentiate septic from aseptic loosening of prosthesis because of the high culture-negative rates in conventional microbiologic culture. This study used quantitative reverse transcription polymerase chain reaction (RT-qPCR to amplify bacterial 16S ribosomal RNA in vitro and in 11 clinical samples. The in vitro analysis demonstrated that the RT-qPCR method was highly sensitive with the detection limit of bacterial 16S rRNA being 0.148 pg/μl. Clinical specimens were analyzed using the same protocol. The RT-qPCR was positive for bacterial detection in 8 culture-positive cases (including aerobic, anaerobic, and mycobacteria and 2 culture-negative cases. It was negative in one case that the final diagnosis was confirmed without infection. The molecular diagnosis of bacterial infection using RT-qPCR to detect bacterial 16S rRNA around a prosthesis correlated well with the clinical findings. Based on the promising clinical results, we were attempting to differentiate bacterial species or drug-resistant strains by using species-specific primers and to detect the persistence of bacteria during the interim period before the second stage reimplantation in a larger scale of clinical subjects.

  18. New screening software shows that most recent large 16S rRNA gene clone libraries contain chimeras.

    Science.gov (United States)

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

    2006-09-01

    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/.

  19. A comparison of rpoB and 16S rRNA as markers in pyrosequencing studies of bacterial diversity.

    Directory of Open Access Journals (Sweden)

    Michiel Vos

    Full Text Available BACKGROUND: The 16S rRNA gene is the gold standard in molecular surveys of bacterial and archaeal diversity, but it has the disadvantages that it is often multiple-copy, has little resolution below the species level and cannot be readily interpreted in an evolutionary framework. We compared the 16S rRNA marker with the single-copy, protein-coding rpoB marker by amplifying and sequencing both from a single soil sample. Because the higher genetic resolution of the rpoB gene prohibits its use as a universal marker, we employed consensus-degenerate primers targeting the Proteobacteria. METHODOLOGY/PRINCIPAL FINDINGS: Pyrosequencing can be problematic because of the poor resolution of homopolymer runs. As these erroneous runs disrupt the reading frame of protein-coding sequences, removal of sequences containing nonsense mutations was found to be a valuable filter in addition to flowgram-based denoising. Although both markers gave similar estimates of total diversity, the rpoB marker revealed more species, requiring an order of magnitude fewer reads to obtain 90% of the true diversity. The application of population genetic methods was demonstrated on a particularly abundant sequence cluster. CONCLUSIONS/SIGNIFICANCE: The rpoB marker can be a complement to the 16S rRNA marker for high throughput microbial diversity studies focusing on specific taxonomic groups. Additional error filtering is possible and tests for recombination or selection can be employed.

  20. Metagenomic Analysis of Slovak Bryndza Cheese Using Next-Generation 16S rDNA Amplicon Sequencing

    Directory of Open Access Journals (Sweden)

    Planý Matej

    2016-06-01

    Full Text Available Knowledge about diversity and taxonomic structure of the microbial population present in traditional fermented foods plays a key role in starter culture selection, safety improvement and quality enhancement of the end product. Aim of this study was to investigate microbial consortia composition in Slovak bryndza cheese. For this purpose, we used culture-independent approach based on 16S rDNA amplicon sequencing using next generation sequencing platform. Results obtained by the analysis of three commercial (produced on industrial scale in winter season and one traditional (artisanal, most valued, produced in May Slovak bryndza cheese sample were compared. A diverse prokaryotic microflora composed mostly of the genera Lactococcus, Streptococcus, Lactobacillus, and Enterococcus was identified. Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris were the dominant taxons in all tested samples. Second most abundant species, detected in all bryndza cheeses, were Lactococcus fujiensis and Lactococcus taiwanensis, independently by two different approaches, using different reference 16S rRNA genes databases (Greengenes and NCBI respectively. They have been detected in bryndza cheese samples in substantial amount for the first time. The narrowest microbial diversity was observed in a sample made with a starter culture from pasteurised milk. Metagenomic analysis by high-throughput sequencing using 16S rRNA genes seems to be a powerful tool for studying the structure of the microbial population in cheeses.

  1. DNA sequencing reveals limited heterogeneity in the 16S rRNA gene from the rrnB operon among five Mycoplasma hominis isolates

    DEFF Research Database (Denmark)

    Mygind, T; Birkelund, Svend; Christiansen, Gunna

    1998-01-01

    To investigate the intraspecies heterogeneity within the 16S rRNA gene of Mycoplasma hominis, five isolates with diverse antigenic profiles, variable/identical P120 hypervariable domains, and different 16S rRNA gene RFLP patterns were analysed. The 16S rRNA gene from the rrnB operon was amplified...... by PCR and the PCR products were sequenced. Three isolates had identical 16S rRNA sequences and two isolates had sequences that differed from the others by only one nucleotide....

  2. Research progress in exogenous 16S rRNA methylase%外源性16srRNA甲基化酶研究进展

    Institute of Scientific and Technical Information of China (English)

    费秋萍; 张顺; 蔡挺; 胡珊珊; 吴春丽

    2016-01-01

    外源性16S rRNA甲基化酶能介导细菌对多种氨基糖苷类抗菌药物高水平耐药,目前已发现10种外源性16S rRNA甲基化酶;该综述分析了外源性16S rRNA甲基化酶与两类内源性16S rRNA甲基化酶的联系,发现外源性16S rRNA甲基化酶可阻碍特定的内源性16S rRNA甲基化酶的甲基化作用,影响细菌的生长及对抗菌药物的敏感性,对基因环境的分析表明外源性16S rRNA甲基化酶基因常与其他耐药基因位于同一可移动基因元件上,耐药机制复杂,应继续监测外源性16S rRNA甲基化酶的流行情况,深入探讨其耐药机制的形成,以期早日改善临床致病菌耐药情况。%Exogenous 16S rRNA methylase can mediate a mechanism of bacteria with high level resistance to many aminoglycosides ,and ten kinds of exogenous 16S rRNA methylase have already been reported .This essay has an‐alyzed the association of exogenous 16S rRNA methylase with two kinds of endogenous 16S rRNA methylase .The result showed that exogenous 16S rRNA methylase could defect the growth of strains and change the strain's sus‐ceptibility to antibiotics by hindering the methylation of specific endogenous 16S rRNA methylase .The analysis of genetic environment suggested that exogenous 16S rRNA methylase produced a complex resistance mechanism as their encoding genes are mostly locating on transferable plasmids with other resistance determinants .It required us to have a further monitoring of the exogenous 16S rRNA methylase and seek for the resistance mechanism to mini‐mize the occurrence of drug resistance .

  3. Loop-mediated isothermal amplification assay for 16S rRNA methylase genes in Gram-negative bacteria.

    Science.gov (United States)

    Nagasawa, Mitsuaki; Kaku, Mitsuo; Kamachi, Kazunari; Shibayama, Keigo; Arakawa, Yoshichika; Yamaguchi, Keizo; Ishii, Yoshikazu

    2014-10-01

    Using the loop-mediated isothermal amplification (LAMP) method, we developed a rapid assay for detection of 16S rRNA methylase genes (rmtA, rmtB, and armA), and investigated 16S rRNA methylase-producing strains among clinical isolates. Primer Explorer V3 software was used to design the LAMP primers. LAMP primers were prepared for each gene, including two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB). Detection was performed with the Loopamp DNA amplification kit. For all three genes (rmtA, rmtB, and armA), 10(2) copies/tube could be detected with a reaction time of 60 min. When nine bacterial species (65 strains saved in National Institute of Infectious Diseases) were tested, which had been confirmed to possess rmtA, rmtB, or armA by PCR and DNA sequencing, the genes were detected correctly in these bacteria with no false negative or false positive results. Among 8447 clinical isolates isolated at 36 medical institutions, the LAMP method was conducted for 191 strains that were resistant to aminoglycosides based on the results of antimicrobial susceptibility tests. Eight strains were found to produce 16S rRNA methylase (0.09%), with rmtB being identified in three strains (0.06%) of 4929 isolates of Enterobacteriaceae, rmtA in three strains (0.10%) of 3284 isolates of Pseudomonas aeruginosa, and armA in two strains (0.85%) of 234 isolates of Acinetobacter spp. At present, the incidence of strains possessing 16S rRNA methylase genes is very low in Japan. However, when Gram-negative bacteria showing high resistance to aminoglycosides are isolated by clinical laboratories, it seems very important to investigate the status of 16S rRNA methylase gene-harboring bacilli and monitor their trends among Japanese clinical settings.

  4. Development of an Analysis Pipeline Characterizing Multiple Hypervariable Regions of 16S rRNA Using Mock Samples.

    Directory of Open Access Journals (Sweden)

    Jennifer J Barb

    Full Text Available There is much speculation on which hypervariable region provides the highest bacterial specificity in 16S rRNA sequencing. The optimum solution to prevent bias and to obtain a comprehensive view of complex bacterial communities would be to sequence the entire 16S rRNA gene; however, this is not possible with second generation standard library design and short-read next-generation sequencing technology.This paper examines a new process using seven hypervariable or V regions of the 16S rRNA (six amplicons: V2, V3, V4, V6-7, V8, and V9 processed simultaneously on the Ion Torrent Personal Genome Machine (Life Technologies, Grand Island, NY. Four mock samples were amplified using the 16S Ion Metagenomics Kit™ (Life Technologies and their sequencing data is subjected to a novel analytical pipeline.Results are presented at family and genus level. The Kullback-Leibler divergence (DKL, a measure of the departure of the computed from the nominal bacterial distribution in the mock samples, was used to infer which region performed best at the family and genus levels. Three different hypervariable regions, V2, V4, and V6-7, produced the lowest divergence compared to the known mock sample. The V9 region gave the highest (worst average DKL while the V4 gave the lowest (best average DKL. In addition to having a high DKL, the V9 region in both the forward and reverse directions performed the worst finding only 17% and 53% of the known family level and 12% and 47% of the genus level bacteria, while results from the forward and reverse V4 region identified all 17 family level bacteria.The results of our analysis have shown that our sequencing methods using 6 hypervariable regions of the 16S rRNA and subsequent analysis is valid. This method also allowed for the assessment of how well each of the variable regions might perform simultaneously. Our findings will provide the basis for future work intended to assess microbial abundance at different time points

  5. Evaluación conductual en prevención de recaídas en la adicción a las drogas : estado actual y aplicaciones clínicas

    OpenAIRE

    Secades Villa, Roberto

    1997-01-01

    La alta tasa de recaídas continúa siendo uno de los problemas más importantes en el tratamiento de la adicción a las drogas. Este trabajo proporciona una revisión de las principales estrategias de evaluación de los procesos involucrados en la recaída. Se proponen cinco tipos de estrategias: autorregistros, descripción de recaídas pasadas, fantasías de recaída, observación directa y autoinformes. Se pone especial énfasis en la utilidad clínica de los procedimientos y en su implicación para pla...

  6. Bacterial 16S rDNA sequence analysis of Siberian tiger faecal flora%东北虎粪细菌区系的16S rRNA基因序列分析

    Institute of Scientific and Technical Information of China (English)

    图雅; 朱伟云; 陆承平

    2005-01-01

    为研究东北虎粪微生物区系建立了东北虎粪细菌的16S rDNA文库.通过EcoRⅠ和HindⅢ分别对阳性克隆进行酶切分析,从东北虎的16S rDNA文库中分别获得了15个具有酶切差异的克隆.BLAST分析结果显示,在15个克隆中,10个克隆与梭菌属成员有97%以上的同源性,其中有6个序列与诺维梭菌A型(Clostridium novyi type A)有99%的同源性,为诺维梭菌A型;4个序列与猪粪细菌RT-18B(Swine manure bacterium RT-18B)有97%的同源性,为消化链球菌属(Peptostreptococcus)成员.其它序列与GenBank中登录的序列同源性低于97%,为5种未培养细菌,其中4种16S rRNA基因序列分别与Clostridium pascui 、破伤风梭菌E88(Clostridium tetani E88)、梭菌(Clostridium sp.)14505及产气荚膜梭菌(Clostridiumperfringens)有94%~95%的相似性.第5种与肉杆菌(Carnobacterium sp.)R-7279株有94%的同源性.

  7. 16S rDNA测序快速鉴定废水生物处理系统目标细菌%Use of 16S rDNA sequencing for quickly determining target bacteria in biological wastewater treatment system

    Institute of Scientific and Technical Information of China (English)

    李永峰; 任南琪; 杨传平; 陈瑛; 郑国香; 胡立杰

    2005-01-01

    在废水生物处理系统中建立分子生物学技术快速鉴定有关微生物,具有十分重要的意义.以发酵法生物制氢系统的活性污泥分离培养的厌氧发酵细菌为研究对象,采用16S rDNA碱基测序分子生物学技术,通过生物信息学数据库NCBI将DNA序列输入、比对,可以直接鉴定从废水生物处理系统中分离培养的细菌进行种属科的系统发育学地位的判定,从而简化了细菌鉴定程序.通过16S rDNA测序技术,进行了分离产氢细菌的分子生物学鉴定,发现生物制氢厌氧活性污泥中所分离的细菌可能存在的菌属有:Lactobacillus,Clostridium,Klebsiella,Propionibacterium和可能新发现的1个新属Biohydrogenbacterium等5个菌属的菌种,其中新属Biohydrogenbacterium的菌种都以利用碳水化合物生产氢气为其主要特征.16S rDNA测序技术为废水生物处理系统的细菌学研究提供了方便、准确和经济的技术基础.

  8. Bacterial diversity of soil under eucalyptus assessed by 16S rDNA sequencing analysis Diversidade bacteriana de solo sob eucaliptos obtida por seqüenciamento do 16S rDNA

    Directory of Open Access Journals (Sweden)

    Érico Leandro da Silveira

    2006-10-01

    Full Text Available Studies on the impact of Eucalyptus spp. on Brazilian soils have focused on soil chemical properties and isolating interesting microbial organisms. Few studies have focused on microbial diversity and ecology in Brazil due to limited coverage of traditional cultivation and isolation methods. Molecular microbial ecology methods based on PCR amplified 16S rDNA have enriched the knowledge of soils microbial biodiversity. The objective of this work was to compare and estimate the bacterial diversity of sympatric communities within soils from two areas, a native forest (NFA and an eucalyptus arboretum (EAA. PCR primers, whose target soil metagenomic 16S rDNA were used to amplify soil DNA, were cloned using pGEM-T and sequenced to determine bacterial diversity. From the NFA soil 134 clones were analyzed, while 116 clones were analyzed from the EAA soil samples. The sequences were compared with those online at the GenBank. Phylogenetic analyses revealed differences between the soil types and high diversity in both communities. Soil from the Eucalyptus spp. arboretum was found to have a greater bacterial diversity than the soil investigated from the native forest area.Estudos sobre impacto do Eucalyptus spp. em solos brasileiros têm focalizado propriedades químicas do solo e isolamento de microrganismos de interesse. No Brasil há pouco enfoque em ecologia e diversidade microbiana, devido às limitações dos métodos tradicionais de cultivo e isolamento. A utilização de métodos moleculares no estudo da ecologia microbiana baseados na amplificação por PCR do 16S rDNA têm enriquecido o conhecimento da biodiversidade microbiana dos solos. O objetivo deste trabalho foi comparar e estimar a diversidade bacteriana de comunidades simpátricas em solos de duas áreas: uma floresta nativa (NFA e outra adjacente com arboreto de eucaliptos (EAA. Oligonucleotídeos iniciadores foram utilizados para amplificar o 16S rDNA metagenômico do solo, o qual foi

  9. 基于16S rDNA序列和RFLP分析的病鳗分离菌株鉴定%Identification of Bacteria Isolated from Diseased Eels Using 16S rDNA PCR / RFLP Analysis

    Institute of Scientific and Technical Information of China (English)

    郭松林; 关瑞章; 冯建军; 杨求华

    2012-01-01

    Using 16S rDNA gene sequences and restriction fragment length polymorphism (RFLP) a- nalysis, the study initially identified 30 strains of bacteria isolated from European eel (Anguilla anguilla) , Japanese eel (Anguilla japonica) and American eel (Anguilla rostrata) according to the homology compari- son with bacterial 16S rDNA gene sequence which had been submitted into the C, eneBank database. The re- sults showed that the 30 bacteria could be broadly divided into 7 genera, including Aeromonas sp. , Pseudo- monas sp. , P/es/omonas sp. , Klebsh:lla sp. , Citrobacter sp. , Acinetobacter sp. and Enterobacter sp. Re- sults of the study indicated some of the isolates could be suspected as pathogenic strains which caused diseases to eels.%结合16S rDNA基因序列和限制性片段长度多态性(RFLP)的分析方法,通过与GenBank库中已递交的细菌16S rDNA基因序列进行同源性比较,对分离自发病鳗鲡(欧洲鳗鲡,日本鳗鲡和美洲鳗鲡)的30株细菌进行初步鉴定和分类.结果表明,这些细菌可大致分为气单胞菌属(Aeromonas sp.)、假单胞菌属(Pseudomonas sp.)、邻单胞菌属(Plesiomonas sp.)、克雷伯氏菌属(Klebsiella sp.)、柠檬酸杆菌属(Citrobacter sp.)、不动杆菌属(Acinetobacter sp.)和肠杆菌属(Enterobacter sp.)等7个菌属.分析认为,部分分离菌株可能是引起鳗鲡病害的疑似致病性菌株.

  10. 霍乱弧菌毒力基因检测与16S rRNA基因分型研究%Toxigene detection and 16S rRNA gene typing of Vibrio cholerae

    Institute of Scientific and Technical Information of China (English)

    张政; 朱水荣

    2006-01-01

    目的:了解浙江省霍乱弧菌毒力基因携带情况和16S rRNA基因型状况,为霍乱防治提供科学依据.方法:利用16S rRNA基因探针分析了浙江省不同时期分离的88株霍乱弧菌经Bgl Ⅰ消化的16S rRNA基因限制性酶谱,以多重PCR法对140株霍乱弧菌进行6种毒力相关基因(ctxA、rtxA、Ace、TcpA、Cri、Zot)检测分析.结果:发现各菌株的杂交片断范围为2~12 Kb,每个菌株有6~9条杂交带不等.88株霍乱菌株可分为9个16S rRNA基因型(ribotype,RT),其中埃尔托型霍乱弧菌(el tor vibrio cholerae,EVC)可分为6个RT,O139群霍乱弧菌(vibrio cholerae O139,VC O139)分为3个RT;所有VC O139菌株均携带3种以上毒力基因,86.3%的菌株携带全部6种毒力基因,所有EVC流行株均携带2种以上毒力基因,79.1%的菌株携带全部6种毒力基因.结论:认为霍乱流行菌株基因型的变迁可能是引起新一次流行的原因.结合毒素基因检测结果,我们认为VC O139与EVC在遗传特征上有相近的特点,但又有所区别.

  11. 奶牛子宫内膜炎致病菌的16S rRNA序列鉴定%Identification of the pathogenic bacteria from endometritis cow by 16S rRNA sequence analysis

    Institute of Scientific and Technical Information of China (English)

    张洪波; 杨宏军; 何洪彬; 杨少华; 王长法; 高运东; 仲跻峰; 葛利江

    2009-01-01

    [目的]对奶牛产后子宫内膜炎致病菌进行16S rRNA序列鉴定.[方法]从产后子宫内膜炎患牛子宫分泌物中分离致病菌,通过细菌培养、纯化、分离、革兰氏染色和生化试验进行初步鉴定.选取代表性菌株11株,利用细菌通用引物,通过PCR方法,对其16S rRNA基因的核苷酸序列进行扩增,将扩增产物与pMD19-T载体连接构建克隆载体,经PCR和双酶切鉴定正确后测序,测序结果与GenBank中已注册菌株的16S rRNA基因序列进行比对.[结果]共分离到致病菌株60株,有代表性的11株细菌归类为:SD01为琼氏不动杆菌,SD02为粪肠球菌,SD03为金黄色葡萄球菌,SD04为中间葡萄球菌,SD05为溶血葡萄球菌,SD06为鲁菲不动杆菌,SD07为无乳链球菌,SD08为芽孢杆菌,SD09为枯草芽孢杆菌,SD10为大肠杆菌,SD11为假单胞杆菌.[结论]通过国际公认的16S rRNA序列鉴定技术,准确鉴定出引起奶牛子宫内膜炎的致病菌种类,为临床治疗该病提供了理论依据.

  12. How long should older people take antidepressants to prevent relapse? Por quanto tempo os idosos devem tomar antidepressivos para evitar recaídas?

    Directory of Open Access Journals (Sweden)

    Jon Spear

    2002-04-01

    Full Text Available Patients with depressive disorder have a high risk of relapse after recovery from a depressive episode. Can the relapse of depressive disorder be prevented or delayed for older adults? This paper reviews the evidence from randomised clinical trials and open label trials of the effectiveness of maintenance antidepressant therapy for older adults with depressive disorder. It also examines the evidence for the effectiveness of psychosocial and psychotherapeutic interventions. The paper concludes with recommendations for clinical practice and future research.Pacientes com transtorno depressivo apresentam alto risco de recorrência e recaída. É possível prevenir a recaída ou a recorrência do episódio depressivo ou retardá-lo em fases tardias da vida? Este artigo revisa ensaios clínicos aleatorizados e não-aleatorizados com o objetivo de estabelecer se o tratamento antidepressivo de manutenção reduz o risco de recaída e recorrência de depressão em idosos. O artigo também examina a evidência atualmente disponível sobre a eficácia das intervenções psicossociais e psicoterapêuticas. O artigo conclui com recomendações para a prática clínica e pesquisas futuras.

  13. DNA compaction in the early part of the SOS response is dependent on RecN and RecA.

    Science.gov (United States)

    Odsbu, Ingvild; Skarstad, Kirsten

    2014-05-01

    The nucleoids of undamaged Escherichia coli cells have a characteristic shape and number, which is dependent on the growth medium. Upon induction of the SOS response by a low dose of UV irradiation an extensive reorganization of the nucleoids occurred. Two distinct phases were observed by fluorescence microscopy. First, the nucleoids were found to change shape and fuse into compact structures at midcell. The compaction of the nucleoids lasted for 10-20 min and was followed by a phase where the DNA was dispersed throughout the cells. This second phase lasted for ~1 h. The compaction was found to be dependent on the recombination proteins RecA, RecO and RecR as well as the SOS-inducible, SMC (structural maintenance of chromosomes)-like protein RecN. RecN protein is produced in high amounts during the first part of the SOS response. It is possible that the RecN-mediated 'compact DNA' stage at the beginning of the SOS response serves to stabilize damaged DNA prior to recombination and repair.

  14. DNA compaction in the early part of the SOS response is dependent on RecN and RecA.

    Science.gov (United States)

    Odsbu, Ingvild; Skarstad, Kirsten

    2014-05-01

    The nucleoids of undamaged Escherichia coli cells have a characteristic shape and number, which is dependent on the growth medium. Upon induction of the SOS response by a low dose of UV irradiation an extensive reorganization of the nucleoids occurred. Two distinct phases were observed by fluorescence microscopy. First, the nucleoids were found to change shape and fuse into compact structures at midcell. The compaction of the nucleoids lasted for 10-20 min and was followed by a phase where the DNA was dispersed throughout the cells. This second phase lasted for ~1 h. The compaction was found to be dependent on the recombination proteins RecA, RecO and RecR as well as the SOS-inducible, SMC (structural maintenance of chromosomes)-like protein RecN. RecN protein is produced in high amounts during the first part of the SOS response. It is possible that the RecN-mediated 'compact DNA' stage at the beginning of the SOS response serves to stabilize damaged DNA prior to recombination and repair. PMID:24615185

  15. The discovery of error-prone DNA polymerase V and its unique regulation by RecA and ATP.

    Science.gov (United States)

    Goodman, Myron F

    2014-09-26

    My career pathway has taken a circuitous route, beginning with a Ph.D. degree in electrical engineering from The Johns Hopkins University, followed by five postdoctoral years in biology at Hopkins and culminating in a faculty position in biological sciences at the University of Southern California. My startup package in 1973 consisted of $2,500, not to be spent all at once, plus an ancient Packard scintillation counter that had a series of rapidly flashing light bulbs to indicate a radioactive readout in counts/minute. My research pathway has been similarly circuitous. The discovery of Escherichia coli DNA polymerase V (pol V) began with an attempt to identify the mutagenic DNA polymerase responsible for copying damaged DNA as part of the well known SOS regulon. Although we succeeded in identifying a DNA polymerase, one that was induced as part of the SOS response, we actually rediscovered DNA polymerase II, albeit in a new role. A decade later, we discovered a new polymerase, pol V, whose activity turned out to be regulated by bound molecules of RecA protein and ATP. This Reflections article describes our research trajectory, includes a review of key features of DNA damage-induced SOS mutagenesis leading us to pol V, and reflects on some of the principal researchers who have made indispensable contributions to our efforts.

  16. Bacterial community structure in High-Arctic snow and freshwater as revealed by pyrosequencing of 16S rRNA genes and cultivation

    DEFF Research Database (Denmark)

    Møller, Annette K.; Søborg, Ditte A.; Abu Al-Soud, Waleed;

    2013-01-01

    The bacterial community structures in High-Arctic snow over sea ice and an ice-covered freshwater lake were examined by pyrosequencing of 16S rRNA genes and 16S rRNA gene sequencing of cultivated isolates. Both the pyrosequence and cultivation data indicated that the phylogenetic composition...

  17. Evidence of Multiple Treponema Phylotypes Involved in Bovine Digital Dermatitis as Shown by 16S rRNA Gene Analysis and Fluorescence In Situ Hybridization

    DEFF Research Database (Denmark)

    Klitgaard, Kirstine; Boye, Mette; Capion, Nynne;

    2008-01-01

    of the bacteria involved in DD lesions of cattle by using culture-independent molecular methods. Ten different phylotypes of Treponema were identified either by 16S rRNA gene sequencing of bacteria from DD lesions or by fluorescence in situ hybridization (FISH) analysis using phylotype-specific 16S r...

  18. Comparison of COBAS AMPLICOR Neissefia gonorrhoeae PCR, including confirmation with N-gonorrhoeae-specific 16S rRNA PCR, with traditional culture

    NARCIS (Netherlands)

    Luijt, DS; Bos, PAJ; van Zwet, AA; Vader, PCV; Schirm, J

    2005-01-01

    A total of 3,023 clinical specimens were tested for Neisseria gonorrhoeae by using COBAS AMPLICOR (CA) PCR and confirmation of positives by N. gonorrhoeae-specific 16S rRNA PCR. The sensitivity of CA plus 16S rRNA PCR was 98.8%, compared to 68.2% for culture. Confirmation of CA positives increased t

  19. Comparison of COBAS AMPLICOR Neisseria gonorrhoeae PCR, including confirmation with N. gonorrhoeae-specific 16S rRNA PCR, with traditional culture

    NARCIS (Netherlands)

    Luijt, D.S.; Bos, P.A.; van Zwet, A.A.; Voorst-Vader, P.C.; Schirm, J.

    2005-01-01

    : A total of 3,023 clinical specimens were tested for Neisseria gonorrhoeae by using COBAS AMPLICOR (CA) PCR and confirmation of positives by N. gonorrhoeae-specific 16S rRNA PCR. The sensitivity of CA plus 16S rRNA PCR was 98.8%, compared to 68.2% for culture. Confirmation of CA positives increased

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

    Science.gov (United States)

    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...

  1. Identification of signature and primers specific to genus Pseudomonas using mismatched patterns of 16S rDNA sequences

    Directory of Open Access Journals (Sweden)

    Kapley A

    2003-05-01

    Full Text Available Abstract Background Pseudomonas, a soil bacterium, has been observed as a dominant genus that survives in different habitats with wide hostile conditions. We had a basic assumption that the species level variation in 16S rDNA sequences of a bacterial genus is mainly due to substitutions rather than insertion or deletion of bases. Keeping this in view, the aim was to identify a region of 16S rDNA sequence and within that focus on substitution prone stretches indicating species level variation and to derive patterns from these stretches that are specific to the genus. Results Repeating elements that are highly conserved across different species of Pseudomonas were considered as guiding markers to locate a region within the 16S gene. Four repeating patterns showing more than 80% consistency across fifty different species of Pseudomonas were identified. The sub-sequences between the repeating patterns yielded a continuous region of 495 bases. The sub-sequences after alignment and using Shanon's entropy measure yielded a consensus pattern. A stretch of 24 base positions in this region, showing maximum variations across the sampled sequences was focused for possible genus specific patterns. Nine patterns in this stretch showed nearly 70% specificity to the target genus. These patterns were further used to obtain a signature that is highly specific to Pseudomonas. The signature region was used to design PCR primers, which yielded a PCR product of 150 bp whose specificity was validated through a sample experiment. Conclusions The developed approach was successfully applied to genus Pseudomonas. It could be tried in other bacterial genera to obtain respective signature patterns and thereby PCR primers, for their rapid tracking in the environmental samples.

  2. Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system.

    Science.gov (United States)

    Schloss, Patrick D; Jenior, Matthew L; Koumpouras, Charles C; Westcott, Sarah L; Highlander, Sarah K

    2016-01-01

    Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina's MiSeq, have allowed researchers to obtain millions of high quality but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3-V5, V1-V3, V1-V5, V1-V6, and V1-V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1-V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina's MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.

  3. Flow Cytometric and 16S Sequencing Methodologies for Monitoring the Physiological Status of the Microbiome in Powdered Infant Formula Production

    Science.gov (United States)

    Anvarian, Amir H. P.; Cao, Yu; Srikumar, Shabarinath; Fanning, Séamus; Jordan, Kieran

    2016-01-01

    The aim of this study was to develop appropriate protocols for flow cytometric (FCM) and 16S rDNA sequencing investigation of the microbiome in a powdered infant formula (PIF) production facility. Twenty swabs were collected from each of the three care zones of a PIF production facility and used for preparing composite samples. For FCM studies, the swabs were washed in 200 mL phosphate buffer saline (PBS). The cells were harvested by three-step centrifugation followed by a single stage filtration. Cells were dispersed in fresh PBS and analyzed with a flow cytometer for membrane integrity, metabolic activity, respiratory activity and Gram characteristics of the microbiome using various fluorophores. The samples were also plated on agar plates to determine the number of culturable cells. For 16S rDNA sequencing studies, the cells were harvested by centrifugation only. Genomic DNA was extracted using a chloroform-based method and used for 16S rDNA sequencing studies. Compared to the dry low and high care zones, the wet medium care zone contained a greater number of viable, culturable, and metabolically active cells. Viable but non-culturable cells were also detected in dry-care zones. In total, 243 genera were detected in the facility of which 42 were found in all three care zones. The greatest diversity in the microbiome was observed in low care. The genera present in low, medium and high care were mostly associated with soil, water, and humans, respectively. The most prevalent genera in low, medium and high care were Pseudomonas, Acinetobacter, and Streptococcus, respectively. The integration of FCM and metagenomic data provided further information on the density of different species in the facility. PMID:27446009

  4. Lyme disease caused by Borrelia burgdorferi with two homeologous 16S rRNA genes: a case report

    Directory of Open Access Journals (Sweden)

    Lee SH

    2016-04-01

    Full Text Available Sin Hang Lee,1,21Pathology Department, Milford Hospital, Milford, CT, USA; 2Milford Molecular Diagnostics, Milford, CT, USA Abstract: Lyme disease (LD, the most common tick-borne disease in North America, is believed to be caused exclusively by Borrelia burgdorferi sensu stricto and is usually diagnosed by clinical evaluation and serologic assays. As reported previously in a peer-reviewed article, a 13-year-old boy living in the Northeast of the USA was initially diagnosed with LD based on evaluation of his clinical presentations and on serologic test results. The patient was treated with a course of oral doxycycline for 28 days, and the symptoms resolved. A year later, the boy developed a series of unusual symptoms and did not attend school for 1 year. A LD specialist reviewed the case and found the serologic test band patterns nondiagnostic of LD. The boy was admitted to a psychiatric hospital. After discharge from the psychiatric hospital, a polymerase chain reaction test performed in a winter month when the boy was 16 years old showed a low density of B. burgdorferi sensu lato in the blood of the patient, confirmed by partial 16S rRNA (ribosomal RNA gene sequencing. Subsequent DNA sequencing analysis presented in this report demonstrated that the spirochete isolate was a novel strain of B. burgdorferi with two homeologous 16S rRNA genes, which has never been reported in the world literature. This case report shows that direct DNA sequencing is a valuable tool for reliable molecular diagnosis of Lyme and related borrelioses, as well as for studies of the diversity of the causative agents of LD because LD patients infected by a rare or novel borrelial variant may produce an antibody pattern that can be different from the pattern characteristic of an infection caused by a typical B. burgdorferi sensu stricto strain. Keywords: Lyme disease, Borrelia burgdorferi, homeologous 16S rRNA genes, DNA sequencing

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

    Directory of Open Access Journals (Sweden)

    Chia Sing eChan

    2015-03-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-03-17

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

  7. 鸡卡氏杆菌的分子鉴定及其16S rRNA基因序列分析%Molecular Identification of Gallibacterium from Hen and Analysis of Its 16S rRNA Gene

    Institute of Scientific and Technical Information of China (English)

    皇甫和平; 付仁一; 郭伦涛; 王川庆; 陈陆; 杨霞; 刘红英; 彭志锋; 郑鹿平; 徐雪; 刘慧敏

    2009-01-01

    Two strains of Gallibacterium, isolated from one laying hen flock in Zhengzhou City of Henan Province, were identified by the morphological observation, genus-specific PCR, and analysis of 16S rRNA gene, which was used to generate the phylogenetic tree, with the 21 members of the 12 genera belonging to Pasteurellaceae to analyze the homology. Two strains were named Yu-ZZ-HL-I-SLG and Yu-ZZ-HL-II-GZ. The comparative result of the 16S rDNA sequence shows that the 2 isolated strains are identical in sequence; the highest identity (99.9%) was observed between the isolated strain and one of the strains of Gallibacterium anatis (AF228002), the homologies between the isolated strain and 3 strains of gallibacterium accessed in NCBI (AF228016, Gallibacterium genomosp.1, AF228017, Gallibacterium genomosp.2, AF228018, Gallibacterium genomosp.1) were above 97.1%, higher than that of the isolated strain and the other strains of the other 11 genera which were between 90.7%-93.2%. It can be seen from the phylogenetic tree that the 2 isolated strains and the other 4 strain of gallibacterium fell into the same branch, furthermore the 2 isolated strains and the strain of Gallibacterium anatis locate in an internal branch, indicating that the 2 isolated strains belong to Gallibacterium anatis.

  8. 16S rRNA Gene Sequence Analysis and Fermentation Products Identification of Amycolatopsis sp.FJNU1011%Amycolatopsis sp. FJNU1011的16S rRNA基因序列分析和发酵产物鉴定

    Institute of Scientific and Technical Information of China (English)

    李力; 刘小玲; 黄连琴; 黄建忠

    2013-01-01

    从土壤中分离1株具有抗革兰氏阳性菌和抗肿瘤活性的放线菌,其16S rRNA的序列聚类分析结果表明该菌可能是拟无枝酸菌属中的一个新种,定命为Amycolatopsis sp.FJNU1011.通过液相色谱质谱联用仪对其发酵提取物进行分析,发现不仅含有5种抗肿瘤作用kigamicins的组分,并且含有新的kigamicins结构类似物.%A strain isolated from soil has the inhibitory activities against G + bacterial and tumor. The sequence analysis of 16S rRNA gene showed that the strain is a species of amycolatop sis, named Amycolatopsis sp. FJNU1011. With the analysis by HPLC-MS, Amycolatopsis sp. FJNU1011 can produce not only kigamicins, but also one new uncharacterized analogue of kigam icins.

  9. 16s rDNA文库法分析番石榴果实蝇共生菌组成%Composition of symbiotic bacteria associated with Bactrocera correcta analyzed by 16s rDNA libraries

    Institute of Scientific and Technical Information of China (English)

    戴阳; 李志红; 柳丽君; 吴佳教; 邓裕亮

    2012-01-01

    [Objective] The composition of symbiotic bacteria associated with Guava fruit fly, Bacirocera correcta (Bezzi), was investigated to provide the basis for further study on physiological function of the symbionts and co-evolution between bacteria and the host. [Method] 16s rDNA libraries of laboratory population for B. correcta were constructed, and BLAST analysis of bacterial composition and abundance was carried out in this study. [Result] The dominant symbionts in B. correcta male adults were Pseudomonas aeruginosa and Lactococcus lactis, while symbiotic bacteria in female adults were Enterobacter bacteria. [Conclusion] Various bacteria are present in the Guava fruit fly, B. correcta.%[目的]探索分析番石榴果实蝇[ Bactrocera correcta (Bezzi)]的共生菌组成,为进一步探索共生菌的生理功能以及与宿主的协同进化关系奠定基础.[方法]构建番石榴果实蝇室内种群共生菌的16s rDNA文库,BLAST分析其共生菌的组成以及丰度.[结果]室内雄虫共生菌主要为Pseudomonas aeruginosa和Lactococcus lactis,雌虫共生菌主要为Enterobacter属的细菌.[结论]本研究结果证明番石榴果实蝇存在多种共生菌.

  10. Research on the PCR Amplication to 16S rRNA Gene of Oil Microorganisms%石油微生物16S rRNA基因PCR扩增研究

    Institute of Scientific and Technical Information of China (English)

    卞立红; 曲丽娜; 汪洋; 张虹; 黄永红

    2010-01-01

    掌握石油微生物的16S rRNA基因保守区的扩增方法是先进分子水平鉴定微生物种类一种有效的实验方法.通过研究利用分子生物技术提高对石油微生物的了解,进行未来对石油的开采,摸索出石油微生物的DNA提取方法,16S rRNA基因的PCR扩增反应条件的研究进而初步鉴定菌种类型.本文是以大庆采油三厂分离纯化的石油微生物为实验材料,摸索合适的石油微生物基因组DNA的提取方法及合适的PCR扩增条件和反应体系,在本实验室后续开展石油微生物在分子水平方面的实践指导中有重要意义.

  11. Sampling of intestinal microbiota and targeted amplification of bacterial 16S rRNA genes for microbial ecologic analysis.

    Science.gov (United States)

    Tong, Maomeng; Jacobs, Jonathan P; McHardy, Ian H; Braun, Jonathan

    2014-11-03

    Dysbiosis of host-associated commensal microbiota is emerging as an important factor in risk and phenotype of immunologic, metabolic, and behavioral diseases. Accurate analysis of microbial composition and functional state in humans or mice requires appropriate collection and pre-processing of biospecimens. Methods to sample luminal and mucosal microbiota from human or mouse intestines and to profile microbial phylogenetic composition using 16S rRNA sequencing are presented here. Data generated using the methods in this unit can be used for downstream quantitative analysis of microbial ecology.

  12. Tetracycline-Resistant Clinical Helicobacter pylori Isolates with and without Mutations in 16S rRNA-Encoding Genes

    OpenAIRE

    Wu, Jeng Yih; Kim, Jae J.; Reddy, Rita; W.M. Wang; David Y Graham; Kwon, Dong H.

    2005-01-01

    Tetracycline-resistant Helicobacter pylori strains have been increasingly reported worldwide. However, only a small number of tetracycline-resistant strains have been studied with regard to possible mechanisms of resistance and those studies have focused on mutations in the tetracycline binding sites of 16S rRNA-encoding genes. We here report studies of 41 tetracycline-resistant H. pylori strains (tetracycline MICs, 4 to 32 μg/ml) from North America (n = 12) and from East Asia (n = 29). DNA s...

  13. Terminal restriction fragment length polymorphism (T-RFLP) profiling of bacterial 16S rRNA genes.

    Science.gov (United States)

    Osborne, Catherine A

    2014-01-01

    T-RFLP profiling is a very effective method for comparing many samples in an environmental microbiology study, because fingerprints of microbial diversity can be generated in a sensitive, reproducible, and cost-effective manner. This protocol describes the steps required to generate T-RFLP profiles of the dominant members of a bacterial community, by PCR amplification of the bacterial 16S rRNA genes and three restriction endonuclease digests to generate three different profiles for each sample. The generation of multiple profiles per sample provides enough information to confidently differentiate rich environmental bacterial communities.

  14. 16S and 23S plastid rDNA phylogenies of Prototheca species and their auxanographic phenotypes1

    Science.gov (United States)

    Ewing, Aren; Brubaker, Shane; Somanchi, Aravind; Yu, Esther; Rudenko, George; Reyes, Nina; Espina, Karen; Grossman, Arthur; Franklin, Scott

    2014-01-01

    Because algae have become more accepted as sources of human nutrition, phylogenetic analysis can help resolve the taxonomy of taxa that have not been well studied. This can help establish algal evolutionary relationships. Here, we compare Auxenochlorella protothecoides and 23 strains of Prototheca based on their complete 16S and partial 23S plastid rDNA sequences along with nutrient utilization (auxanographic) profiles. These data demonstrate that some of the species groupings are not in agreement with the molecular phylogenetic analyses and that auxanographic profiles are poor predictors of phylogenetic relationships. PMID:25937672

  15. Plastid 16s rDNA sequence analysis of Phaeocystis glogosa from South China Sea%南海球形棕囊藻质体16S rDNA序列分析

    Institute of Scientific and Technical Information of China (English)

    马奔; 章群; 司从利; 黄小彧

    2011-01-01

    测定了采自香港2株球形棕囊藻和湛江1株等鞭金藻质体16S rDNA基因序列,结合GenBank中下载的32条同源序列,分析序列特征并构建分子系统树.发现600 bp序列中有可变位点42个,简约信息位点33个,可变位点和简约信息位点分别为全序列的7.0%和5.5%.A+T的含量(52.5%)略大于C+G含量(47.5%),与已报道的棕囊藻属叶绿体和线粒体基因相似;在分子系统树上,同属藻种聚类在一起,属间遗传距离分别为0.025 5~0.050 3.棕囊藻属属内遗传距离0-0.005 0,不同藻种相混杂,并未独立成支;质体16s rDNA部分序列虽不能区分棕囊藻属内不同藻种,但能鉴定出GenBank中27个环境样品为棕囊藻属藻株,从而能确定其地理分布和相对丰度,对棕囊藻赤潮的预测预报具有重要意义.质体16srDNA在其他赤潮藻类物种鉴定方面的应用需要进一步研究.%Partial sequences of plastid 16S rDNA in the two Strains of Phaeocystis globosa isolated from Hong Kong and one Isochrysis strain isolated from Zhangjiang in South China Sea were determined,combined with 32 homologous sequences downloaded from GenBank, the sequence information were analyzed and phylogenetic tree were reconstructed. 42 variable sites and 33 parsimony-informative sites were found in the aligned 600 bp sequences, 7.0% and 5.5% sites were variable and parsimony-informative. The contents of (A+TX52.5%) were a little more than those of (G+C)(47.5%), which were similar to those plastid and mitochondrial genes of Phaeocystis..In the phylogenetic tree, algae strains of the same genus were clustered, and genetic distances among genus were between 0.025 5-0.050 3. Genetic distances within the genus Phaeocystis were less than 0.005 0, and different species were intertwined without isolated clade for each species. Although partial sequences of plastid 16S rDNA could not differentiate different species in the genus Phaeocystis, which may due to the shortage of

  16. Prophage induction and differential RecA and UmuDAb transcriptome regulation in the DNA damage responses of Acinetobacter baumannii and Acinetobacter baylyi.

    Directory of Open Access Journals (Sweden)

    Janelle M Hare

    Full Text Available The SOS response to DNA damage that induces up to 10% of the prokaryotic genome requires RecA action to relieve LexA transcriptional repression. In Acinetobacter species, which lack LexA, the error-prone polymerase accessory UmuDAb is instead required for ddrR induction after DNA damage, suggesting it might be a LexA analog. RNA-Seq experiments defined the DNA damage transcriptome (mitomycin C-induced of wild type, recA and umuDAb mutant strains of both A. baylyi ADP1 and A. baumannii ATCC 17978. Of the typical SOS response genes, few were differentially regulated in these species; many were repressed or absent. A striking 38.4% of all ADP1 genes, and 11.4% of all 17978 genes, were repressed under these conditions. In A. baylyi ADP1, 66 genes (2.0% of the genome, including a CRISPR/Cas system, were DNA damage-induced, and belonged to four regulons defined by differential use of recA and umuDAb. In A. baumannii ATCC 17978, however, induction of 99% of the 152 mitomycin C-induced genes depended on recA, and only 28 of these genes required umuDAb for their induction. 90% of the induced A. baumannii genes were clustered in three prophage regions, and bacteriophage particles were observed after mitomycin C treatment. These prophages encoded esvI, esvK1, and esvK2, ethanol-stimulated virulence genes previously identified in a Caenorhabditis elegans model, as well as error-prone polymerase alleles. The induction of all 17978 error-prone polymerase alleles, whether prophage-encoded or not, was recA dependent, but only these DNA polymerase V-related genes were de-repressed in the umuDAb mutant in the absence of DNA damage. These results suggest that both species possess a robust and complex DNA damage response involving both recA-dependent and recA-independent regulons, and further demonstrates that although umuDAb has a specialized role in repressing error-prone polymerases, additional regulators likely participate in these species' transcriptional

  17. 16S rRNA gene phylogenesis of culturable predominant bacteria from diseased Apostichopus japonicus (Holothuroidea, Echinodermata)

    Science.gov (United States)

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

    2009-06-01

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

  18. Comparison of 16S rDNA-PCR Amplification and Culture of Cerebrospinal Fluid for Diagnosis of Bacterial Meningitis

    Directory of Open Access Journals (Sweden)

    Farshad Foroughi

    2010-12-01

    Full Text Available Objective:Early and accurate diagnosis of bacterial meningitis is of critical concern. Optimum and rapid laboratory facilities are not routinely available for detecting the etiologic agents of meningitis. The objective of this study was to compare polymerase chain reaction (PCR assay with culture for detection of bacteria in central nervous system (CNS samples from patients suspected to have meningitis. Methods: One-hundred CSF samples were obtained and divided into two parts. One part of samples was used for standard bacterial culture and gram staining. The remaining was used for DNA extraction. PCR assay was performed with universal primers for 16S rDNA gene of bacteria. Performance characteristics of the test were determined. Findings:The PCR method was able to detect bacteria in all 36 culture-positive and in 38 of 64 culture-negative cases showing sensitivity and specificity of 100% and 40.6% respectively. Positive predictive value was 48.6% and negative predictive value 100%, however, Kappa coefficient showed the correlation of the 2 methods to be at 0.33. Conclusion:There are advantages and disadvantages in performance characteristics of the conventional CSF culture and universal CSF 16S rDNA PCR. Therefore, it is recommended to use both methods in clinical practice, particularly in suspicious contaminated samples, with presumable presence of fastidious or slow growing bacteria because of antibiotic consumption.

  19. Phylogenetic relationships of true butterflies (Lepidoptera: Papilionoidea) inferred from COI, 16S rRNA and EF-1α sequences.

    Science.gov (United States)

    Kim, Man Il; Wan, Xinlong; Kim, Min Jee; Jeong, Heon Cheon; Ahn, Neung-Ho; Kim, Ki-Gyoung; Han, Yeon Soo; Kim, Iksoo

    2010-11-01

    The molecular phylogenetic relationships among true butterfly families (superfamily Papilionoidea) have been a matter of substantial controversy; this debate has led to several competing hypotheses. Two of the most compelling of those hypotheses involve the relationships of (Nymphalidae + Lycaenidae) + (Pieridae + Papilionidae) and (((Nymphalidae + Lycaenidae) + Pieridae) + Papilionidae). In this study, approximately 3,500 nucleotide sequences from cytochrome oxidase subunit I (COI), 16S ribosomal RNA (16S rRNA), and elongation factor-1 alpha (EF-1α) were sequenced from 83 species belonging to four true butterfly families, along with those of three outgroup species belonging to three lepidopteran superfamilies. These sequences were subjected to phylogenetic reconstruction via Bayesian Inference (BI), Maximum Likelihood (ML), and Maximum Parsimony (MP) algorithms. The monophyletic Pieridae and monophyletic Papilionidae evidenced good recovery in all analyses, but in some analyses, the monophylies of the Lycaenidae and Nymphalidae were hampered by the inclusion of single species of the lycaenid subfamily Miletinae and the nymphalid subfamily Danainae. Excluding those singletons, all phylogenetic analyses among the four true butterfly families clearly identified the Nymphalidae as the sister to the Lycaenidae and identified this group as a sister to the Pieridae, with the Papilionidae identified as the most basal linage to the true butterfly, thus supporting the hypothesis: (Papilionidae + (Pieridae + (Nymphalidae + Lycaenidae))).

  20. Phylogeny of fireflies (Coleoptera: Lampyridae) inferred from mitochondrial 16S ribosomal DNA, with references to morphological and ethological traits

    Institute of Scientific and Technical Information of China (English)

    LI Xueyan; YANG Shuang; XIE Meng; LIANG Xingcai

    2006-01-01

    We sequenced partial mitochondrial 16S ribosomal DNA (16S rDNA) of 18 firefly species from Southwest of China.Combined with homologous sequences previously reported, phylogenetic trees including Japanese, Korean and Chinese species were reconstructed by neighbor-joining, maximum parsimony and Bayesian methods. All reconstructions agree on most nodes of the trees. Monophyly of Lampyridae is not supported because Rhagophthalmus ohbai in Rhagophthalmidae is included within it. Lamprigera, a genus placed unreliably in Lampyrinae, shows a close relationship to Amydetinae. Monophyly of Luciolinae is not supported because Pristolycus sagulatus (Lampyrinae) is included within it. In the Luciolinae, monophyly of Curtos and Hotaria is well established, respectively, but both morphological and molecular data continue to indicate that Luciola is not monophyletic and its subdivision is indeed necessary. Within the Lampyrinae, both Pyrocoelia and Diaphanes are not monophyletic, but monophyly of Pyrocoelia + Diaphanes is well supported. Phylogeny of Diaphanes is discussed for the first time. Generic placement of a newly discovered species (Diaphanes pectinealis Li et Liang)sharing some characters of Pyrocoelia and Diaphanes challenges the delimitation of these two similar genera. With references to the firefly mating systems, we suggest that more emphases should be placed on those sexually selected characters such as antennal structure in taxonomy of Lampyridae.

  1. 16s rRNA Identification of Pediococcus spp. from Broiler and Studies of Adherence Ability on Immobilized Mucus

    Directory of Open Access Journals (Sweden)

    Ema Damayanti

    2015-11-01

    Full Text Available The objectives of this research were to study taxonomical status of lactic acid bacteria (LAB isolated from broiler and adherence ability on mucus in vitro. Molecular analysis was performed by analyzing 16S rRNA gene using universal primer. The adherence assay on mucus was carried out using microplate method with total plate count (TPC, absorbance (A550 and confirmed by scanning electron microscopy (SEM. The results of this studies revealed that three of LAB isolates have closed relation to Pediococcus acidilactici (99.9% species.Three isolates of P. acidilactici have adherence ability on broiler mucus higher than that on porcine mucin with an adherence percentage of 55.5% versus 50.8% and absorbance A550 of 0.061 versus 0.051, respectively. The highest adherence ability showed by P. acidilactici R02 with adherence percentage was 59.3% and absorbance A550 = 0.068. Adherence on mucus were affected by the addition of 3 g/l of gastric juice and 0.3% (b/v of bile salt. Adherence analysis using SEM also showed that the adherence on broiler mucus was higher than the adherence on porcine mucin. Altogether this adherence studies, suggest that three isolates of P. acidilactici LAB were capable of colonizing host intestinal mucus in vitro as important property to be promising probiotic bacteria for broiler.Key words : adherence, broiler, Pediococcus, mucus, 16S rRNA

  2. Native Valve Endocarditis due to Corynebacterium striatum confirmed by 16S Ribosomal RNA Sequencing: A Case Report and Literature Review

    Science.gov (United States)

    2016-01-01

    Corynebacterium species are non-fermentous Gram-positive bacilli that are normal flora of human skin and mucous membranes and are commonly isolated in clinical specimens. Non-diphtheriae Corynebacterium are regarded as contaminants when found in blood culture. Currently, Corynebacterium striatum is considered one of the emerging nosocomial agents implicated in endocarditis and serious infections. We report a case of native-valve infective endocarditis caused by C. striatum, which was misidentified by automated identification system but identified accurately by 16S ribosomal RNA sequencing, in a 55-year-old male patient. The patient had two mobile vegetations on his mitral valve, both of which had high embolic risk. Through surgical valve replacement and an antibiotic regimen, the patient recovered completely. In unusual clinical scenarios, C. striatum should not be simply dismissed as a contaminant when isolated from clinical specimens. The possibility of C. striatum infection should be considered even in an immunocompetent patient, and we suggest a genotypic assay, such as 16S rRNA sequencing, to confirm species identity. PMID:27659439

  3. Arrested development of the myxozoan parasite, Myxobolus cerebralis, in certain populations of mitochondrial 16S lineage III Tubifex tubifex

    Science.gov (United States)

    Baxa, D.V.; Kelley, G.O.; Mukkatira, K.S.; Beauchamp, K.A.; Rasmussen, C.; Hedrick, R.P.

    2008-01-01

    Laboratory populations of Tubifex tubifex from mitochondrial (mt)16S ribosomal DNA (rDNA) lineage III were generated from single cocoons of adult worms releasing the triactinomyxon stages (TAMs) of the myxozoan parasite, Myxobolus cerebralis. Subsequent worm populations from these cocoons, referred to as clonal lines, were tested for susceptibility to infection with the myxospore stages of M. cerebralis. Development and release of TAMs occurred in five clonal lines, while four clonal lines showed immature parasitic forms that were not expelled from the worm (non-TAM producers). Oligochaetes from TAM- and non-TAM-producing clonal lines were confirmed as lineage III based on mt16S rDNA and internal transcribed spacer region 1 (ITS1) sequences, but these genes did not differentiate these phenotypes. In contrast, random amplified polymorphic DNA analyses of genomic DNA demonstrated unique banding patterns that distinguished the phenotypes. Cohabitation of parasite-exposed TAM- and non-TAM-producing phenotypes showed an overall decrease in expected TAM production compared to the same exposure dose of the TAM-producing phenotype without cohabitation. These studies suggest that differences in susceptibility to parasite infection can occur in genetically similar T. tubifex populations, and their coexistence may affect overall M. cerebralis production, a factor that may influence the severity of whirling disease in wild trout populations. ?? 2007 Springer-Verlag.

  4. Analysis of the cystic fibrosis lung microbiota via serial Illumina sequencing of bacterial 16S rRNA hypervariable regions.

    Directory of Open Access Journals (Sweden)

    Heather Maughan

    Full Text Available The characterization of bacterial communities using DNA sequencing has revolutionized our ability to study microbes in nature and discover the ways in which microbial communities affect ecosystem functioning and human health. Here we describe Serial Illumina Sequencing (SI-Seq: a method for deep sequencing of the bacterial 16S rRNA gene using next-generation sequencing technology. SI-Seq serially sequences portions of the V5, V6 and V7 hypervariable regions from barcoded 16S rRNA amplicons using an Illumina short-read genome analyzer. SI-Seq obtains taxonomic resolution similar to 454 pyrosequencing for a fraction of the cost, and can produce hundreds of thousands of reads per sample even with very high multiplexing. We validated SI-Seq using single species and mock community controls, and via a comparison to cystic fibrosis lung microbiota sequenced using 454 FLX Titanium. Our control runs show that SI-Seq has a dynamic range of at least five orders of magnitude, can classify >96% of sequences to the genus level, and performs just as well as 454 and paired-end Illumina methods in estimation of standard microbial ecology diversity measurements. We illustrate the utility of SI-Seq in a pilot sample of central airway secretion samples from cystic fibrosis patients.

  5. A grammar-based distance metric enables fast and accurate clustering of large sets of 16S sequences

    Directory of Open Access Journals (Sweden)

    Benson Andrew K

    2010-12-01

    Full Text Available Abstract Background We propose a sequence clustering algorithm and compare the partition quality and execution time of the proposed algorithm with those of a popular existing algorithm. The proposed clustering algorithm uses a grammar-based distance metric to determine partitioning for a set of biological sequences. The algorithm performs clustering in which new sequences are compared with cluster-representative sequences to determine membership. If comparison fails to identify a suitable cluster, a new cluster is created. Results The performance of the proposed algorithm is validated via comparison to the popular DNA/RNA sequence clustering approach, CD-HIT-EST, and to the recently developed algorithm, UCLUST, using two different sets of 16S rDNA sequences from 2,255 genera. The proposed algorithm maintains a comparable CPU execution time with that of CD-HIT-EST which is much slower than UCLUST, and has successfully generated clusters with higher statistical accuracy than both CD-HIT-EST and UCLUST. The validation results are especially striking for large datasets. Conclusions We introduce a fast and accurate clustering algorithm that relies on a grammar-based sequence distance. Its statistical clustering quality is validated by clustering large datasets containing 16S rDNA sequences.

  6. Molecular and functional diversity of PGPR fluorescent Pseudomonads based on 16S rDNA-RFLP and RAPD markers.

    Science.gov (United States)

    Singh, Bhim Pratap

    2015-09-01

    The genetic and functional diversity of plant growth promoting rhizobacterial (PGPR) fluorescent pseudomonads associated with chickpea (Cicer arietinum L.) rhizosphere was analyzed. In total, 34 isolates along with two reference isolates were screened for various plant growth promoting traits (phosphorous solubilization, ACC deaminase, HCN, IAA and siderophore productions) and antagonist activity against four fungal phytopathogens and three bacterial pathogens. Most of the isolates, that showed PGPR activity, also showed antagonistic activity against all the three fungal pathogens. The genetic relationship was assessed by using random amplification of polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (16S rDNA-RFLP). Relationship between both the markers was analyzed based on mantel test and a negative correlation was observed. The study concluded that PGPR traits appeared to be strain specific rather than specific to any phylogenetic group. The study also reported that 16S rDNA based profiling differentiated PGPR fluorescent Pseudomonas on the basis of location rather than biological trait. RAPD profiling could be useful to differentiate among the closely related isolates. The genetic and functional diversity of fluorescent pseudomonads, associated with the chickpea rhizosphere, has useful ecological role and potential utilization in sustainable agriculture.

  7. IDENTIFICATION OF Staphylococcus sp. STRAINS ISOLATED FROM POSITIVE WIDAL BLOOD BASED ON 16S rRNA GENE SEQUENCES

    Directory of Open Access Journals (Sweden)

    Sri Darmawati

    2015-12-01

    Full Text Available The purpose of this study is to identify 8 strains of Staphylococcus genus members isolated from positive Widal blood (4 strains of Staphylococcus saprophyticus, 1 strain of Staphylococcus warneri, 2 strains of Staphylococcus hominis, 1 strain of Staphylococcus xylosus and 1 strain of Staphylococcus capitis based on 16S rRNA gene sequences. The methods used in this study are conducting PCR of 16S rRNA gene, cloning genes using T-Vector pMD20 which is transformed to E. coli DH5α, sequencing. The results show that four strains (BA 47.4, BA 19.2, KD 29.5 and TG 09.1 are identified as Stap. Saprophyticus strains of Stap. saprophyticus members of ATCC 15305T (99.01-100% similarity. The strain of TG 01.3 is identified as Stap. Warneri strain of TG 01.3 of Stap. Warneri members of ATCC 27836T (99.74-99.93% similarity, 2strains (KT 29.2 and KD 35.1 are identified as of Stap. hominis members of DSM 20328T (99.4-99.67% similarity. The strain of KT 30.5 is not identified

  8. Detection of novel organisms associated with salpingitis, by use of 16S rDNA polymerase chain reaction.

    Science.gov (United States)

    Hebb, Jennifer K; Cohen, Craig R; Astete, Sabina G; Bukusi, Elizabeth A; Totten, Patricia A

    2004-12-15

    Although Chlamydia trachomatis and Neisseria gonorrhoeae are established causes of salpingitis, the majority of cases have no known etiology. We used broad-range 16S rDNA polymerase chain reaction to identify novel, possibly uncultivable, bacteria associated with salpingitis and identified bacterial 16S sequences in Fallopian-tube specimens from 11 (24%) of 45 consecutive women with laparoscopically confirmed acute salpingitis (the case patients) and from 0 of 44 women seeking tubal ligations (the control subjects) at Kenyatta National Hospital, Nairobi, Kenya. Bacterial phylotypes most closely related to Leptotrichia spp. were detected as the sole phylotypes in 1, and mixed with other bacterial phylotypes in 2, specimens. Novel bacterial phylotypes and those associated with bacterial vaginosis, including Atopobium vaginae, were identified in 3 specimens. N. gonorrhoeae and Streptococcus pyogenes were identified in 2 and 1 specimens, respectively. The finding of novel phylotypes associated with salpingitis has important implications for the etiology, pathogenesis, and treatment of this important reproductive-tract disease syndrome. PMID:15551209

  9. Identification of Thiobacillus ferrooxidans strains based on restriction fragment length polymorphism analysis of 16S rDNA.

    Science.gov (United States)

    Kamimura, K; Wakai, S; Sugio, T

    2001-01-01

    The 16S rDNA sequences from ten strains of Thiobacillus ferrooxidans were amplified by PCR. The products were compared by performing restriction fragment length polymorphism (RFLP) analysis with restriction endonucleases Alu I, Hap II, Hha I, and Hae III. The RFLP patterns revealed that T. ferrooxidans could be distinguished from other iron- or sulphur-oxidizing bacteria such as T. thiooxidans NB1-3, T. caldus GO-1, Leptospirillum ferrooxidans and the marine iron-oxidizing bacterium strain KU2-11. The RFLP patterns obtained with Alu I, Hap II, and Hae III were the same for nine strains of T. ferrooxidans except for strain ATCC 13661. The RFLP patterns for strains NASF-1 and ATCC 13661 with Hha I were distinct from those for other T. ferrooxidans strains. The 16S rDNA sequence of T. ferrooxidans NASF-1 possessed an additional restriction site for Hha I. These results show that iron-oxidizing bacteria isolated from natural environments were rapidly identified as T. ferrooxidans by the method combining RFLP analysis with physiological analysis. PMID:11414499

  10. Identification of bacteria associated with underground parts of Crocus sativus by 16S rRNA gene targeted metagenomic approach.

    Science.gov (United States)

    Ambardar, Sheetal; Sangwan, Naseer; Manjula, A; Rajendhran, J; Gunasekaran, P; Lal, Rup; Vakhlu, Jyoti

    2014-10-01

    Saffron (Crocus sativus L), an autumn-flowering perennial sterile plant, reproduces vegetatively by underground corms. Saffron has biannual corm-root cycle that makes it an interesting candidate to study microbial dynamics in its rhizosphere and cormosphere (area under influence of corm). Culture independent 16S rRNA gene metagenomic study of rhizosphere and cormosphere of Saffron during flowering stage revealed presence of 22 genera but none of the genus was common in all the three samples. Bulk soil bacterial community was represented by 13 genera with Acidobacteria being dominant. In rhizosphere, out of eight different genera identified, Pseudomonas was the most dominant genus. Cormosphere bacteria comprised of six different genera, dominated by the genus Pantoea. This study revealed that the bacterial composition of all the three samples is significantly different (P < 0.05) from each other. This is the first report on the identification of bacteria associated with rhizosphere, cormosphere and bulk soil of Saffron, using cultivation independent 16S rRNA gene targeted metagenomic approach.

  11. 16S rRNA Gene Phylogenesis of Culturable Predominant Bacteria from Diseased Apostichopus japonicus(Holothuroidea, Echinodermata)

    Institute of Scientific and Technical Information of China (English)

    MA Haiyan; JIANG Guoliang; WU Zhiqiang; WANG Xin

    2009-01-01

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

  12. Application of 16S rRNA metagenomics to analyze bacterial communities at a respiratory care centre in Taiwan.

    Science.gov (United States)

    Tang, Chuan Yi; Yiu, Siu-Ming; Kuo, Han-Yueh; Tan, Te-Sheng; Liao, Ki-Hok; Liu, Chih-Chin; Hon, Wing-Kai; Liou, Ming-Li

    2015-03-01

    In this study, we applied a 16S ribosomal RNA (rRNA) metagenomics approach to survey inanimate hospital environments (IHEs) in a respiratory care center (RCC). A total of 16 samples, including 9 from medical devices and 7 from workstations, were analyzed. Besides, clinical isolates were retrospectively analyzed during the sampling period in the RCC. A high amount of microbial diversity was detected, with an average of 1,836 phylotypes per sample. In addition to Acinetobacter, more than 60 % of the bacterial communities present among the top 25 abundant genera were dominated by skin-associated bacteria. Differences in bacterial profiles were restricted to individual samples. Furthermore, compliance with hand hygiene guidelines may be unsatisfactory among hospital staff according to a principal coordinate analysis that indicated clustering of bacterial communities between devices and workstations for most of the sampling sites. Compared to the high incidence of clinical isolates in the RCC, only Staphylococcus and Acinetobacter were highly abundant in the IHEs. Despite Acinetobacter was the most abundant genus present in IHEs of the RCC, potential pathogens, e.g., Acinetobacter baumannii, might remain susceptible to carbapenem. This study is the first in Taiwan to demonstrate a high diversity of human-associated bacteria in the RCC via 16S rRNA metagenomics, which allows for new assessment of potential health risks in RCCs, aids in the evaluation of existing sanitation protocols, and furthers our understanding of the development of healthcare-associated infections.

  13. Rapid identification and classification of bacteria by 16S rDNA restriction fragment melting curve analyses (RFMCA).

    Science.gov (United States)

    Rudi, Knut; Kleiberg, Gro H; Heiberg, Ragnhild; Rosnes, Jan T

    2007-08-01

    The aim of this work was to evaluate restriction fragment melting curve analyses (RFMCA) as a novel approach for rapid classification of bacteria during food production. RFMCA was evaluated for bacteria isolated from sous vide food products, and raw materials used for sous vide production. We identified four major bacterial groups in the material analysed (cluster I-Streptococcus, cluster II-Carnobacterium/Bacillus, cluster III-Staphylococcus and cluster IV-Actinomycetales). The accuracy of RFMCA was evaluated by comparison with 16S rDNA sequencing. The strains satisfying the RFMCA quality filtering criteria (73%, n=57), with both 16S rDNA sequence information and RFMCA data (n=45) gave identical group assignments with the two methods. RFMCA enabled rapid and accurate classification of bacteria that is database compatible. Potential application of RFMCA in the food or pharmaceutical industry will include development of classification models for the bacteria expected in a given product, and then to build an RFMCA database as a part of the product quality control. PMID:17367680

  14. 16S rRNA is a better choice than COI for DNA barcoding hydrozoans in the coastal waters of China

    Institute of Scientific and Technical Information of China (English)

    ZHENG Lianming; HE Jinru; LIN Yuanshao; CAO Wenqing; ZHANG Wenjing

    2014-01-01

    Identification of hydrozoan species is challenging, even for taxonomic experts, due to the scarcity of distinct morphological characters and phenotypic plasticity. DNA barcoding provides an efficient method for spe-cies identification, however, the choice between mitochondrial cytochrome c oxidase subunit I (COI) and large subunit ribosomal RNA gene (16S) as a standard barcode for hydrozoans is subject to debate. Herein, we directly compared the barcode potential of COI and 16S in hydrozoans using 339 sequences from 47 pelagic hydrozoan species. Analysis of Kimura 2-parameter genetic distances (K2P) documented the mean intraspecific/interspecific variation for COI and 16S to be 0.004/0.204 and 0.003/0.223, respectively. An obvi-ous“barcoding gap”was detected for all species in both markers and all individuals of a species clustered together in both the COI and 16S trees. These results suggested that the species within the studied taxa can be efficiently and accurately identified by COI and 16S. Furthermore, our results confirmed that 16S was a better phylogenetic marker for hydrozoans at the genus level, and in some cases at the family level. Con-sidering the resolution and effectiveness for barcoding and phylogenetic analyses of Hydrozoa, we strongly recommend 16S as the standard barcode for hydrozoans.

  15. Species identification of meat products based on mtDNA 16S rRNA gene sequencing%应用mtDNA16S rRNA基因测序鉴定肉产品种属

    Institute of Scientific and Technical Information of China (English)

    艾斯卡尔·买买提; 马合木提·哈力克; 日沙来提·吐尔地; 古丽茹合萨·艾海提

    2011-01-01

    Objective Applied mtDNA 16S rRNA gene sequencing method performed species identification of meat products. Methods 15 unknown animal muscle samples were given for inspection by related authorities, pork, beef and lamb were bought and used as a control. Extract DNA by conventional phenol-chloroform method, amplified mtDNA 16S rRNA gene by using the general primer and the specific primers respectively, products were examined by 1. 5% agarose gel electrophoresis. Amplified sequences were sequenced by Shanghai Bio-Engineering Company and then compared the identities through sequencing and BLAST aligning. Results All PCR products from 15 received samples amplified with general primer were showed positive band; neither one of them had positive band while used the sheep specific primer; while used specific primer of pig and bovine 14 and 1 of testing samples had amplified positive band respectively. The identity of them with pork and beef were 96% ~ 100% respectively while sequenced and BLAST aligned. Conclusion Among the 15 unknown meat products 14were pork and one was beef.%目的 应用mtDNA 16S rRNA基因测序方法进行肉产品种属鉴定.方法 15份有关部门送检的未知动物肌肉样本,3份市购猪、牛和羊的肌肉为对照样本.常规酚-氯仿法提取模板DNA,分别用通用引物和特异性引物扩增mtDNA的16S rRNA基因片段,产物用1.5%琼脂糖电泳检测,扩增的基因片段由上海生物工程公司进行序列测定及基因库同源性匹配查询.结果 经通用引物扩增15份未知样本,均检测出阳性条带;用羊特异性引物扩增,无未知样本检出阳性条带;用猪及牛特异性引物扩增,分别有14份和1份样本检出阳性条带,经测序及同源性查询,分别与猪和牛的同源性为96%~100%.结论 送检未知肉产品样本中14份是猪肉,1份是牛肉.

  16. Studies on the Application of 16s rDNA for Rapid Detection of Streptoccocus Group C%利用16S rDNA快速鉴定C群链球菌的研究

    Institute of Scientific and Technical Information of China (English)

    王伟伟; 陈义平; 张喜悦; 赵永刚; 刘春菊; 王志亮

    2007-01-01

    本试验应用PCR方法,根据16S rDNA两端的保守序列,用prmer5.0软件设计引物扩增待检细菌,然后对扩增产物DNA片段进行测序、序列分析,经与GenBank中的已知序列进行同源性比较后,发现该未知菌属于链球菌C群.该方法比传统的细菌鉴定方法省时省力,并且能将细菌定型到群,结果准确、快速、重复性好.

  17. Dynamic changes of intestinal 16S rDNA metagenome in 5 infants%动态监测婴儿肠道16S rDNA宏基因组5例报告

    Institute of Scientific and Technical Information of China (English)

    马丽亚; 张敏; 王辉林; 陈睿; 黄艳; 梁小琴; 卢光进

    2014-01-01

    目的:探讨婴儿肠道16S rDNA宏基因组的动态变化。方法收集5例健康婴儿生后3 d、1个月、6个月、1岁时粪便样本共17份,提取细菌总DNA,采用新一代高通量测序技术对16S rDNA的V6高变区测序并进行物种分类、丰度及多样性分析。结果17份样品共产生原始测序数据为2190.66 Mbp,Operational Taxonomic Units(OTU)数量36~308;优势菌门包括变形菌门、厚壁菌门、拟杆菌门和放线菌门;不同婴儿间肠道菌群分布有差异;科水平>1%的物种生后3 d时2~4种,1个月、6个月时增至最多7种,1岁时达10种;4个时间点的npShannon和Simpson指数均值分别为1.117、1.460、2.088、2.50和0.443、0.408、0.229、0.143。结论婴儿粪便中含丰富细菌基因组;细菌物种丰度及分类存在个体差异;细菌多样性随日龄而增加,婴儿肠道菌群结构出生后逐渐趋向复杂和多样。%Objective To investigate the dynamic changes of intestinal 16S rDNA metagenome in healthy infants. Methods Seventeen fecal samples were collected at ages of 3 days, 1 month, 6 months and 1 year in 5 infants. Total bacterial DNAs were extracted and submitted high throughout sequencing on the V6 viable region of 16S rDNA. Tags and Operational Taxonomic Units (OTU) were then obtained and analysis of taxonomy, abundance and alpha diversity were performed. Results In total 2 190.66 Mbp raw data in 17 samples were produced. The OTU numbers ranged from 36 to 308. The dominate phylum included Proteobacteria, Firmicutes and Bacteroidetes and Actinobacteria. The bacterial families>1%increased from only 2-4 per sample on day 3 to 7 at 1 or 6 months, 10 at 12 months. The average npShannon and Simpson index on day 3, at 1 month, 6 months and 1 year were 1.117, 1.460, 2.088, 2.50 and 0.443, 0.408, 0.229, 0.143 respectively. Conclusions Infants’ intestines harbor abounding bacterial genomes. Distinct individual differences exist in infants in terms of

  18. Diversity of Ulva spp.(Enteromorpha spp.) epiphytic bacteria based on 16S rDNA sequences%基于16S rDNA分析的浒苔外生细菌多样性研究

    Institute of Scientific and Technical Information of China (English)

    周新倩; 杨锐; 吴小凯; 黄艳花

    2016-01-01

    为研究浙江近海浒苔Ulva spp.(Enteromorpha spp.)外生细菌多样性,采用传统的形态学和16S rDNA测序分析的方法,从舟山朱家尖、宁海国华电厂和奉化南沙3个地区分离到可培养的浒苔外生细菌及其周围海水细菌65株.根据细菌菌落特征和革兰氏染色结果等将分离到的细菌分为26种表型.16S rDNA序列测序比对发现:菌株与不动杆菌属(Acinetobacter)、交替单胞菌属(Alteromonas)、弧菌属(Vibrio)、假交替单胞菌属(Pseudoalteromonas)、微小杆菌属(Exiguobacterium)、芽孢杆菌属(Bacillus)、赤细菌属(Erythrobacter)、微球菌属(Micrococcus)、假单胞菌属(Pseudomonas)、深海杆菌属(Idiomarina)、Phaeobacter、Roseivirgaj和Silicibacter等23个属相应菌株具有较高同源性.对不同地区浒苔外生细菌进行了Shannon-Wiener多样性指数分析.研究表明:(1)宁海国华电厂的浒苔外生细菌多样性最丰富,多样性指数为93.98%;(2)浒苔外生细菌与其生活地区密切相关,其群落组成具有地域差异,其优势类群也不尽相同,但均归属于变形菌门.

  19. Physiological and Biochemical Characteristics of Thermophilic Bacteria and Its 16S rDNA Sequence Analysis%高温菌的生理生化及其16S rDNA序列分析

    Institute of Scientific and Technical Information of China (English)

    李华芝; 李秀艳; 韩波波

    2010-01-01

    利用高温菌耐热嗜热的特性和有机物好氧分解的基本原理,从厨余垃圾处理系统中分离筛选到6株在65℃能产生淀粉酶、脂肪酶、蛋白质酶及纤维素酶的高温高效菌种,并对其生理生化和16S rDNA进行了分析.结果表明:筛选出的6株高温菌,经鉴定都有芽孢,并具有兼氧性;对其进行16S rDNA区段序列测定显示,6株高温菌属于Geobacillus thermoglucosidasius菌种中的不同亚种;分离筛选出的高温菌经过2h的迟缓期后很快进入对数生长期,且持续时间较长,生长速度尤以HB6最快;6株高温菌株全部具有淀粉、纤维素、蛋白质降解活性,5株有脂肪降解活性.可见高温菌具有更高的有机物降解活性和更快的代谢速率.可提高有机物质的降解速率,缩短工艺运行时间,有利于工业化生产,在厨余资源化利用上具有广泛的应用前景.

  20. Evolutionary Relationships among Three Belocera Species Inferred from 16 S rDNA Sequences%三种簇角飞虱属昆虫16 S rDNA序列及系统进化研究

    Institute of Scientific and Technical Information of China (English)

    侯晓晖; 陈祥盛

    2012-01-01

    通过PCR扩增获得了飞虱科3属5种昆虫的16 S rDNA序列,对DNA序列进行测定,分析了其序列组成及变异.采用Mega 4.0软件,利用邻接法(NJ)和最大简约法(MP)进行5种飞虱的系统发育分析,结果显示构建的MP和NJ分子系统树拓扑结构一致,3个簇角飞虱属物种中华簇角飞虱(Belocera.sinensis)、褐额簇角飞虱(B.fuscifrons)和爬竹簇角飞虱(B.ampelocalamus)单独聚为一支,与偏角飞虱属海南偏角飞虱(Neobelocera hainanensis)的亲缘关系较近,与短头飞虱属显脊短头飞虱(Epeurysa distincta)的亲缘关系较远,分子生物学分析结果与形态学研究结果一致.%The 16 S Rdna sequences of 5 plant hoppers belonging to 3 genus in the family of Delphacidae were obtained by PCR and then sequenced. The sequence composition and mutation were analyzed. Molecular phylogenetic trees were constructed by maximum parsimony method (MP) and neighbor-joining method (NJ) using Mega 4.0 software. The result showed that the topologies of MP tree and NJ tree constructed were the same. Belocera Sinensis, B. Juscifrons and B. Ampelocalamus were clustered into one group, which had closer relationship to Neobelocera hainanensis while further distance from Epeurysa dis-tincta. These results were identical with those of morphology research.

  1. Identification of Corn Bacterial Wilt Pathogen by Nested-PCR Based on 16S rDNA%16S nested-PCR技术检测玉米细菌性枯萎病菌

    Institute of Scientific and Technical Information of China (English)

    吴琼; 陈枝楠; 范怀忠; 金显忠

    2005-01-01

    玉米细菌性枯萎病是玉米上的重要种传病害,病原菌为Pantoea stewartii subsp.stewartii.本研究设计了16S通用引物,扩增该病菌及其近似种的16S rDNA,通过序列测定和分析,针对该病菌设计了特异性引物,采用nested-PCR技术,能够准确地区别该病菌及其近似种,检测的灵敏度在DNA水平上达到10-3pg级,检测活菌则达到2 cfu.检测人工污染的玉米种子时,不受种子提取液中其它物质的干扰,灵敏度依然达到2 cfu.%Pantoea stewartii subsp. stewartii is the seed-borne causative agent of bacterial wilt and leaf blight of sweet corn and maize. In this study, a specific 16S rDNA-based nested-PCR (nPCR) was developed to identify the bacterium from a range of closely related bacteria. This PCR assay was proved to be highly sensitive with the detection limit of 10 -3pg genomic DNA or 2 colony forming units (cfu). Moreover, this PCR assay successfully detected the target bacteria as few as 2 cfu when tested with artificially contaminated seeds, and the sensitivity was not affected by seed compounds.

  2. Isolation and 16s rdna sequence analysis of bacteria from dieback affected mango orchards in southern pakistan

    International Nuclear Information System (INIS)

    A broad range of microorganisms are involved in various mango plant diseases such as fungi, algae and bacteria. In order to study the role of bacteria in mango dieback, a survey of infected mango plants in southern Pakistan was carried out. A number of bacterial isolates were obtained from healthy looking and infected mango trees, and their characterization was undertaken by colony PCR and subsequent sequence analysis of 16S rDNA. These analyses revealed the presence of various genera including Acinetobacter, Bacillus, Burkholderia, Cronobacter, Curtobacterium, Enterobacter, Erwinia, Exiguobacterium, Halotelea, Lysinibacillus, Micrococcus, Microbacterium, Pantoea, Pseudomonas, Salmonella and Staphylococcus. It is noteworthy that several members of these genera have been reported as plant pathogens. The present study provided baseline information regarding the phytopathogenic bacteria associated with mango trees in southern Pakistan. (author)

  3. Detection of Borrelia-specific 16S rRNA sequence in total RNA extracted from Ixodes ricinus ticks

    Directory of Open Access Journals (Sweden)

    Ž. Radulović

    2010-08-01

    Full Text Available A reverse transcriptase - polymerase chain reaction based assay for Borrelia species detection in ticks was developed. The method was based on amplification of 552 nucleotide bases long sequence of 16S rRNA, targeted by Borrelia specific primers. In the present study, total RNA extracted from Ixodes ricinus ticks was used as template. The results showed higher sensitivity for Borrelia detection as compared to standard dark-field microscopy. Method specificity was confirmed by cloning and sequencing of obtained 552 base pairs long amplicons. Phylogenetic analysis of obtained sequences showed that they belong to B. lusitaniae and B. afzelii genospecies. RT-PCR based method presented in this paper could be very useful as a screening test for detecting pathogen presence, especially when in investigations is required extraction of total RNA from ticks.

  4. 产凝集素乳杆菌的筛选及PCR-16S rDNA鉴定

    Institute of Scientific and Technical Information of China (English)

    徐进; 赵声兰; 陈朝银

    2008-01-01

    目的从泡菜汁中分离产凝集素乳杆菌并鉴定。方法通过凝集实验筛选产凝集素的乳杆菌,用聚合酶链式反应(PCR)扩增16S rDNA可变区基因鉴定产凝集素乳杆菌。结果此菌株呈革兰阳性,短杆状,产乳酸。通过同源性比较,此菌株与Lactobacillus plantarum strain p215的同源性为99%。结论分离得到了产凝集素的菌株,鉴定表明此菌株为植物乳杆菌。

  5. Legionella Bozemanae, a New Cause of Septic Arthritis diagnosed by 16S PCR followed by specific culture

    DEFF Research Database (Denmark)

    Just, Søren Andreas; Bonde Knudsen, John; Skov, Marianne Nielsine;

    This is the first report ever to demonstrate that L. Bozemanae can colonize synovial joints leading to infectious arthritis. L. Bozemanae is a rare Legionella species, earlier described as a cause of cavitating lung infections with up to 40% mortality (2). L. Bozemanae is missed by standard cultu...... cultures, however, the case was diagnosed by combining 16S PCR and sequencing followed by culture under specific conditions, a method which may help in the diagnosis of septic arthritis caused by unusual pathogens not detected by standard culture.......This is the first report ever to demonstrate that L. Bozemanae can colonize synovial joints leading to infectious arthritis. L. Bozemanae is a rare Legionella species, earlier described as a cause of cavitating lung infections with up to 40% mortality (2). L. Bozemanae is missed by standard...

  6. Description of the male, redescription of the female and 16S rDNA sequence of Ixodes aulacodi (Ixodidae).

    Science.gov (United States)

    Chiţimia-Dobler, Lidia; D'Amico, Gianluca; Yao, Patrick Kouassi; Kalmár, Zsuzsa; Gherman, Călin Mircea; Mihalca, Andrei Daniel; Estrada-Peña, Agustin

    2016-04-01

    Ixodes (Afrixodes) aulacodiArthur, 1956 is a poorly known species that has been recorded predominantly in the wet countries of western and central Africa, mainly associated to the greater cane rat Thryonomys swinderianus (Temmink). We herein redescribe the female, describe the male (ascribed to the species from specimens found in copula) and provide the 16S rDNA sequence. We also provide complete illustrations of the adults based on specimens found on greater cane rats in Ivory Coast. Ixodes aulacodi is included in the group of species of the subgenus Afrixodes that have horseshoe shaped anal groove, and which lack auriculae and cornua. The female is easily separated when compared with other species because of a unique combination of characters: All the coxae have internal spurs, coxa II has two external spurs, syncoxae are absent, and trochanters I-III have one spur each. The male has a notched hypostome and lacks syncoxae, auriculae and cornua. PMID:26803353

  7. Protocols for 16S rDNA Array Analyses of Microbial Communities by Sequence-Specific Labeling of DNA Probes

    Directory of Open Access Journals (Sweden)

    Knut Rudi

    2003-01-01

    Full Text Available Analyses of complex microbial communities are becoming increasingly important. Bottlenecks in these analyses, however, are the tools to actually describe the biodiversity. Novel protocols for DNA array-based analyses of microbial communities are presented. In these protocols, the specificity obtained by sequence-specific labeling of DNA probes is combined with the possibility of detecting several different probes simultaneously by DNA array hybridization. The gene encoding 16S ribosomal RNA was chosen as the target in these analyses. This gene contains both universally conserved regions and regions with relatively high variability. The universally conserved regions are used for PCR amplification primers, while the variable regions are used for the specific probes. Protocols are presented for DNA purification, probe construction, probe labeling, and DNA array hybridizations.

  8. Phylogeny and divergence time estimation of cheilostome bryozoans based on mitochodrial 16S rRNA sequences

    Institute of Scientific and Technical Information of China (English)

    HAO Jiasheng; LI Chunxiang; SUN Xiaoyan; YANG Qun

    2005-01-01

    The mitochondrial 16S rDNA sequences of 40 species of cheilostome bryozoans including those of 24 species newly determined were used to reconstruct the phylogenetic tree using neighboring-joining and maximum-parsimony methods. By applying molecular clock technique on the basis of the appropriate phylogeny and the fossil record, the divergence times of the two main cheilostome groups, Anasca and Ascophora sensu stricto, were estimated. The results show that the molecular phylogeny of the higher taxonomic groups (superfamilies and higher taxa) of cheilostome bryozoans is mostly in conflict with the morphology-based phylogenetic trees; the divergence of the extant groups of Anasca and those of Ascophora sensu stricto is estimated to have happened about 263 Ma (Permian Guadalupian Epoch) and 183 Ma (Early Jurassic), respectively.

  9. Reducing the effects of PCR amplification and sequencing artifacts on 16S rRNA-based studies.

    Directory of Open Access Journals (Sweden)

    Patrick D Schloss

    Full Text Available The advent of next generation sequencing has coincided with a growth in interest in using these approaches to better understand the role of the structure and function of the microbial communities in human, animal, and environmental health. Yet, use of next generation sequencing to perform 16S rRNA gene sequence surveys has resulted in considerable controversy surrounding the effects of sequencing errors on downstream analyses. We analyzed 2.7×10(6 reads distributed among 90 identical mock community samples, which were collections of genomic DNA from 21 different species with known 16S rRNA gene sequences; we observed an average error rate of 0.0060. To improve this error rate, we evaluated numerous methods of identifying bad sequence reads, identifying regions within reads of poor quality, and correcting base calls and were able to reduce the overall error rate to 0.0002. Implementation of the PyroNoise algorithm provided the best combination of error rate, sequence length, and number of sequences. Perhaps more problematic than sequencing errors was the presence of chimeras generated during PCR. Because we knew the true sequences within the mock community and the chimeras they could form, we identified 8% of the raw sequence reads as chimeric. After quality filtering the raw sequences and using the Uchime chimera detection program, the overall chimera rate decreased to 1%. The chimeras that could not be detected were largely responsible for the identification of spurious operational taxonomic units (OTUs and genus-level phylotypes. The number of spurious OTUs and phylotypes increased with sequencing effort indicating that comparison of communities should be made using an equal number of sequences. Finally, we applied our improved quality-filtering pipeline to several benchmarking studies and observed that even with our stringent data curation pipeline, biases in the data generation pipeline and batch effects were observed that could potentially

  10. Carbonate formation by anaerobic oxidation of methane: Evidence from lipid biomarker and fossil 16S rDNA

    Science.gov (United States)

    Stadnitskaia, A.; Nadezhkin, D.; Abbas, B.; Blinova, V.; Ivanov, M. K.; Sinninghe Damsté, J. S.

    2008-04-01

    Carbonate chimneys and other carbonate structures occur widespread in the Gulf of Cadiz and probably reflect the presence of cold seeps and associated release of methane in the geological past, possibly in the Early Pleistocene, but it is unclear under what conditions and by which processes these carbonates were formed. We studied a fossil methane-related carbonate crust collected from the Kidd mud volcano in the gulf. Concentrations of microbial lipids, their stable carbon isotope composition, sequences of fossil 16S rRNA genes of anaerobic methanotrophic archaea in combination with mineralogical and carbon and oxygen isotopic composition of carbonate were obtained for seven different horizons of the crust. This combination of organic and inorganic geochemical techniques with molecular ecological methods gave a consistent view on processes resulting in the formation of the crust and indicated that it took place in two phases and in a downward direction. Archaeal lipid biomarkers and fossil 16S rRNA gene sequence data revealed the dominance of archaeal ANME-2 group and elevated methane partial pressures during the formation of the top part of the crust. The lower part of the carbonate was likely formed in an environment with reduced methane fluxes as revealed by the dominance of fossil remains of ANME-1 archaea. The combination of these methods can be used as an effective tool to reconstruct in unprecedented detail the palaeo-biogeochemical processes resulting in the formation of carbonate fabrics. This interdisciplinary strategy may also be applied for other fossil methane-derived carbonates, generating new concepts and knowledge about past methane-related carbonate systems.

  11. Lyme disease caused by Borrelia burgdorferi with two homeologous 16S rRNA genes: a case report.

    Science.gov (United States)

    Lee, Sin Hang

    2016-01-01

    Lyme disease (LD), the most common tick-borne disease in North America, is believed to be caused exclusively by Borrelia burgdorferi sensu stricto and is usually diagnosed by clinical evaluation and serologic assays. As reported previously in a peer-reviewed article, a 13-year-old boy living in the Northeast of the USA was initially diagnosed with LD based on evaluation of his clinical presentations and on serologic test results. The patient was treated with a course of oral doxycycline for 28 days, and the symptoms resolved. A year later, the boy developed a series of unusual symptoms and did not attend school for 1 year. A LD specialist reviewed the case and found the serologic test band patterns nondiagnostic of LD. The boy was admitted to a psychiatric hospital. After discharge from the psychiatric hospital, a polymerase chain reaction test performed in a winter month when the boy was 16 years old showed a low density of B. burgdorferi sensu lato in the blood of the patient, confirmed by partial 16S rRNA (ribosomal RNA) gene sequencing. Subsequent DNA sequencing analysis presented in this report demonstrated that the spirochete isolate was a novel strain of B. burgdorferi with two homeologous 16S rRNA genes, which has never been reported in the world literature. This case report shows that direct DNA sequencing is a valuable tool for reliable molecular diagnosis of Lyme and related borrelioses, as well as for studies of the diversity of the causative agents of LD because LD patients infected by a rare or novel borrelial variant may produce an antibody pattern that can be different from the pattern characteristic of an infection caused by a typical B. burgdorferi sensu stricto strain.

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

    Directory of Open Access Journals (Sweden)

    Keys Christine E

    2010-03-01

    Full Text Available Abstract 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

  13. Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing

    Directory of Open Access Journals (Sweden)

    Loreto Abusleme

    2014-04-01

    Full Text Available Background and objective: The advent of next-generation sequencing has significantly facilitated characterization of the oral microbiome. Despite great efforts in streamlining the processes of sequencing and data curation, upstream steps required for amplicon library generation could still influence 16S rRNA gene-based microbial profiles. Among upstream processes, DNA extraction is a critical step that could represent a great source of bias. Accounting for bias introduced by extraction procedures is important when comparing studies that use different methods. Identifying the method that best portrays communities is also desirable. Accordingly, the aim of this study was to evaluate bias introduced by different DNA extraction procedures on oral microbiome profiles. Design: Four DNA extraction methods were tested on mock communities consisting of seven representative oral bacteria. Additionally, supragingival plaque samples were collected from seven individuals and divided equally to test two commonly used DNA extraction procedures. Amplicon libraries of the 16S rRNA gene were generated and sequenced via 454-pyrosequencing. Results: Evaluation of mock communities revealed that DNA yield and bacterial species representation varied with DNA extraction methods. Despite producing the lowest yield of DNA, a method that included bead beating was the only protocol capable of detecting all seven species in the mock community. Comparison of the performance of two commonly used methods (crude lysis and a chemical/enzymatic lysis+column-based DNA isolation on plaque samples showed no effect of extraction protocols on taxa prevalence but global community structure and relative abundance of individual taxa were affected. At the phylum level, the latter method improved the recovery of Actinobacteria, Bacteroidetes, and Spirochaetes over crude lysis. Conclusion: DNA extraction distorts microbial profiles in simulated and clinical oral samples, reinforcing the

  14. Lyme disease caused by Borrelia burgdorferi with two homeologous 16S rRNA genes: a case report

    Science.gov (United States)

    Lee, Sin Hang

    2016-01-01

    Lyme disease (LD), the most common tick-borne disease in North America, is believed to be caused exclusively by Borrelia burgdorferi sensu stricto and is usually diagnosed by clinical evaluation and serologic assays. As reported previously in a peer-reviewed article, a 13-year-old boy living in the Northeast of the USA was initially diagnosed with LD based on evaluation of his clinical presentations and on serologic test results. The patient was treated with a course of oral doxycycline for 28 days, and the symptoms resolved. A year later, the boy developed a series of unusual symptoms and did not attend school for 1 year. A LD specialist reviewed the case and found the serologic test band patterns nondiagnostic of LD. The boy was admitted to a psychiatric hospital. After discharge from the psychiatric hospital, a polymerase chain reaction test performed in a winter month when the boy was 16 years old showed a low density of B. burgdorferi sensu lato in the blood of the patient, confirmed by partial 16S rRNA (ribosomal RNA) gene sequencing. Subsequent DNA sequencing analysis presented in this report demonstrated that the spirochete isolate was a novel strain of B. burgdorferi with two homeologous 16S rRNA genes, which has never been reported in the world literature. This case report shows that direct DNA sequencing is a valuable tool for reliable molecular diagnosis of Lyme and related borrelioses, as well as for studies of the diversity of the causative agents of LD because LD patients infected by a rare or novel borrelial variant may produce an antibody pattern that can be different from the pattern characteristic of an infection caused by a typical B. burgdorferi sensu stricto strain. PMID:27186082

  15. Evaluation of PCR-generated chimeras, mutations, and heteroduplexes with 16S rRNA gene-based cloning.

    Science.gov (United States)

    Qiu, X; Wu, L; Huang, H; McDonel, P E; Palumbo, A V; Tiedje, J M; Zhou, J

    2001-02-01

    To evaluate PCR-generated artifacts (i.e., chimeras, mutations, and heteroduplexes) with the 16S ribosomal DNA (rDNA)-based cloning approach, a model community of four species was constructed from alpha, beta, and gamma subdivisions of the division Proteobacteria as well as gram-positive bacterium, all of which could be distinguished by HhaI restriction digestion patterns. The overall PCR artifacts were significantly different among the three Taq DNA polymerases examined: 20% for Z-Taq, with the highest processitivity; 15% for LA-Taq, with the highest fidelity and intermediate processitivity; and 7% for the conventionally used DNA polymerase, AmpliTaq. In contrast to the theoretical prediction, the frequency of chimeras for both Z-Taq (8.7%) and LA-Taq (6.2%) was higher than that for AmpliTaq (2.5%). The frequencies of chimeras and of heteroduplexes for Z-Taq were almost three times higher than those of AmpliTaq. The total PCR artifacts increased as PCR cycles and template concentrations increased and decreased as elongation time increased. Generally the frequency of chimeras was lower than that of mutations but higher than that of heteroduplexes. The total PCR artifacts as well as the frequency of heteroduplexes increased as the species diversity increased. PCR artifacts were significantly reduced by using AmpliTaq and fewer PCR cycles (fewer than 20 cycles), and the heteroduplexes could be effectively removed from PCR products prior to cloning by polyacrylamide gel purification or T7 endonuclease I digestion. Based upon these results, an optimal approach is proposed to minimize PCR artifacts in 16S rDNA-based microbial community studies.

  16. 16S rRNA gene pyrosequencing reveals bacterial dysbiosis in the duodenum of dogs with idiopathic inflammatory bowel disease.

    Directory of Open Access Journals (Sweden)

    Jan S Suchodolski

    Full Text Available 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.

  17. Differentiation of Acidithiobacillus ferrooxidans and A. thiooxidans strains based on 16S-23S rDNA spacer polymorphism analysis.

    Science.gov (United States)

    Bergamo, Rogério F; Novo, Maria Teresa M; Veríssimo, Ricardo V; Paulino, Luciana C; Stoppe, Nancy C; Sato, Maria Inês Z; Manfio, Gilson P; Prado, Paulo Inácio; Garcia, Oswaldo; Ottoboni, Laura M M

    2004-09-01

    Restriction fragment length polymorphism (RFLP) and sequence analyses of the PCR-amplified 16S-23S rDNA intergenic spacer (ITS) were used for differentiating Acidithiobacillus thiooxidans strains from other related acidithiobacilli, including A. ferrooxidans and A. caldus. RFLP fingerprints obtained with AluI, DdeI, HaeIII, HinfI and MspI enabled the differentiation of all Acidithiobacillus reference strains into species groups. The A. thiooxidans strains investigated (metal mine isolates) yielded identical RFLP patterns to the A. thiooxidans type strain (ATCC 19377(T)), except for strain DAMS, which had a distinct pattern for all enzymes tested. Fourteen A. ferrooxidans mine strains were assigned to 3 RFLP groups, the majority of which were grouped with A. ferrooxidans ATCC 23270(T). The spacer region of one representative strain from each of the RFLP groups obtained was subjected to sequence analysis, in addition to eleven additional A. thiooxidans strains isolated from sediment and water samples, and A. caldus DSM 8584(T). The tRNA(IIe) and tRNA(Ala) genes, present in all strains analyzed, showed high sequence similarity. Phylogenetic analysis of the ITS sequences differentiated all three Acidithiobacillus species. Inter- and infraspecific genetic variations detected were mainly due to the size and sequence polymorphism of the ITS3 region. Mantel tests showed no significant correlation between ITS sequence similarity and the geographical origin of strains. The results showed that the 16S-23S rDNA spacer region is a useful target for the development of molecular-based methods aimed at the detection, rapid differentiation and identification of acidithiobacilli.

  18. 16S partial gene mitochondrial DNA and internal transcribed spacers ribosomal DNA as differential markers of Trichuris discolor populations.

    Science.gov (United States)

    Callejón, R; Halajian, A; de Rojas, M; Marrugal, A; Guevara, D; Cutillas, C

    2012-05-25

    Comparative morphological, biometrical and molecular studies of Trichuris discolor isolated from Bos taurus from Spain and Iran was carried out. Furthermore, Trichuris ovis isolated from B. taurus and Capra hircus from Spain has been, molecularly, analyzed. Morphological studies revealed clear differences between T. ovis and T. discolor isolated from B. taurus but differences were not observed between populations of T. discolor isolated from different geographical regions. Nevertheless, the molecular studies based on the amplification and sequencing of the internal transcribed spacers 1 and 2 ribosomal DNA and 16S partial gene mitochondrial DNA showed clear differences between both populations of T. discolor from Spain and Iran suggesting two cryptic species. Phylogenetic studies corroborated these data. Thus, phylogenetic trees based on ITS1, ITS2 and 16S partial gene sequences showed that individuals of T. discolor from B. taurus from Iran clustered together and separated, with high bootstrap values, of T. discolor isolated from B. taurus from Spain, while populations of T. ovis from B. taurus and C. hircus from Spain clustered together but separated with high bootstrap values of both populations of T. discolor. Furthermore, a comparative phylogenetic study has been carried out with the ITS1and ITS2 sequences of Trichuris species from different hosts. Three clades were observed: the first clustered all the species of Trichuris parasitizing herbivores (T. discolor, T. ovis, Trichuris leporis and Trichuris skrjabini), the second clustered all the species of Trichuris parasitizing omnivores (Trichuris trichiura and Trichuris suis) and finally, the third clustered species of Trichuris parasitizing carnivores (Trichuris muris, Trichuris arvicolae and Trichuris vulpis).

  19. Phylogenetic relationships within the family Halomonadaceae based on comparative 23S and 16S rRNA gene sequence analysis.

    Science.gov (United States)

    de la Haba, Rafael R; Arahal, David R; Márquez, M Carmen; Ventosa, Antonio

    2010-04-01

    A phylogenetic study of the family Halomonadaceae was carried out based on complete 16S rRNA and 23S rRNA gene sequences. Several 16S rRNA genes of type strains were resequenced, and 28 new sequences of the 23S rRNA gene were obtained. Currently, the family includes nine genera (Carnimonas, Chromohalobacter, Cobetia, Halomonas, Halotalea, Kushneria, Modicisalibacter, Salinicola and Zymobacter). These genera are phylogenetically coherent except Halomonas, which is polyphyletic. This genus comprises two clearly distinguished clusters: group 1 includes Halomonas elongata (the type species) and the species Halomonas eurihalina, H. caseinilytica, H. halmophila, H. sabkhae, H. almeriensis, H. halophila, H. salina, H. organivorans, H. koreensis, H. maura and H. nitroreducens. Group 2 comprises the species Halomonas aquamarina, H. meridiana, H. axialensis, H. magadiensis, H. hydrothermalis, H. alkaliphila, H. venusta, H. boliviensis, H. neptunia, H. variabilis, H. sulfidaeris, H. subterranea, H. janggokensis, H. gomseomensis, H. arcis and H. subglaciescola. Halomonas salaria forms a cluster with Chromohalobacter salarius and the recently described genus Salinicola, and their taxonomic affiliation requires further study. More than 20 Halomonas species are phylogenetically not within the core constituted by the Halomonas sensu stricto cluster (group 1) or group 2 and, since their positions on the different phylogenetic trees are not stable, they cannot be recognized as additional groups either. In general, there is excellent agreement between the phylogenies based on the two rRNA gene sequences, but the 23S rRNA gene showed higher resolution in the differentiation of species of the family Halomonadaceae.

  20. RecET driven chromosomal gene targeting to generate a RecA deficient Escherichia coli strain for Cre mediated production of minicircle DNA

    Directory of Open Access Journals (Sweden)

    Coutelle Charles

    2006-03-01

    Full Text Available Abstract Background Minicircle DNA is the non-replicating product of intramolecular site-specific recombination within a bacterial minicircle producer plasmid. Minicircle DNA can be engineered to contain predominantly human sequences which have a low content of CpG dinucleotides and thus reduced immunotoxicity for humans, whilst the immunogenic bacterial origin and antibiotic resistance marker gene sequences are entirely removed by site-specific recombination. This property makes minicircle DNA an excellent vector for non-viral gene therapy. Large-scale production of minicircle DNA requires a bacterial strain expressing tightly controlled site-specific recombinase, such as Cre recombinase. As recombinant plasmids tend to be more stable in RecA-deficient strains, we aimed to construct a recA- bacterial strain for generation of minicircle vector DNA with less chance of unwanted deletions. Results We describe here the construction of the RecA-deficient minicircle DNA producer Escherichia coli HB101Cre with a chromosomally located Cre recombinase gene under the tight control of the araC regulon. The Cre gene expression cassette was inserted into the chromosomal lacZ gene by creating transient homologous recombination proficiency in the recA- strain HB101 using plasmid-born recET genes and homology-mediated chromosomal "pop-in, pop-out" of the plasmid pBAD75Cre containing the Cre gene and a temperature sensitive replication origin. Favourably for the Cre gene placement, at the "pop-out" step, the observed frequency of RecET-led recombination between the proximal regions of homology was 10 times higher than between the distal regions. Using the minicircle producing plasmid pFIXluc containing mutant loxP66 and loxP71 sites, we isolated pure minicircle DNA from the obtained recA- producer strain HB101Cre. The minicircle DNA preparation consisted of monomeric and, unexpectedly, also multimeric minicircle DNA forms, all containing the hybrid loxP66

  1. 16S rRNA-targeted probes for specific detection of Thermoanaerobacterium spp., Thermoanaerobacterium thermosaccharolyticum, and Caldicellulosiruptor spp. by fluorescent in situ hybridization in biohydrogen producing systems

    DEFF Research Database (Denmark)

    O-Thong, Sompong; Prasertsan, P.; Karakashev, Dimitar Borisov;

    2008-01-01

    16S rRNA gene targeted oligonucleotide probes for specific detection of genera Thermoanaerobacterium (Tbm1282), Caldicellulosiruptor (Ccs432), and specie Thermoanaerobacterium thermosaccharolyticum (Tbmthsacc184) were designed and used to monitor the spatial distribution of hydrogen producing bac...

  2. Cyanobacterial ecotypes in different optical microenvironments of a 68 C hot spring mat community revealed by 16S-23S rRNA internal transcribed spacer region variation

    DEFF Research Database (Denmark)

    Ferris, Mike J.; Kühl, Michael; Wieland, Andrea;

    2003-01-01

    distinct populations over the vertical interval. We were unable to identify patterns in genetic variation in Synechococcus 16S rRNA sequences that correlate with different vertically distributed populations. However, patterns of variation at the internal transcribed spacer locus separating 16S and 23S r......RNA genes suggested the existence of closely related but genetically distinct populations corresponding to different functional populations occurring at different depths....

  3. Systematic use of universal 16S rRNA gene polymerase chain reaction (PCR) and sequencing for processing pleural effusions improves conventional culture techniques.

    Science.gov (United States)

    Insa, Rosario; Marín, Mercedes; Martín, Adoración; Martín-Rabadán, Pablo; Alcalá, Luís; Cercenado, Emilia; Calatayud, Laura; Liñares, Josefina; Bouza, Emilio

    2012-03-01

    Conventional culture of pleural fluid samples frequently provides false-negative results. Universal polymerase chain reaction (PCR) of the 16S ribosomal ribonucleic acid (rRNA) gene (16S PCR) has proven useful in the diagnosis of various bacterial infections. We conducted a prospective study to assess the value of 16S PCR in the etiologic diagnosis of pleural effusion. All pleural fluid samples received for culture were also studied using 16S PCR. Positive samples were sequenced for identification. Clinical records and conventional culture results were analyzed to classify pleural fluid samples as infected or not infected. We studied 723 samples. We excluded 188 samples because they were obtained from a long-term chest tube, there was a diagnosis of mycobacterial infection, or there were insufficient data to classify the episode. Finally, 535 pleural fluid samples were analyzed. According to our criteria, 82 (15.3%) were infected and 453 (84.7%) were not infected. In the infected samples, 16S PCR was positive in 67 samples (81.7%) while conventional culture was positive in 45 (54.9%). There were 4 false positives with 16S PCR (0.9%) and 12 with culture (2.6%). The values for the etiologic diagnosis of bacterial pleural effusion of conventional culture compared with 16S PCR were as follows: sensitivity, 54.9%/81.7%; specificity, 97.4%/99.1%; positive predictive value, 76.3%/94.4%; negative predictive value, 92.6%/96.8%; and accuracy, 90.8%/96.5%.When compared with conventional culture, 16S PCR plus sequencing substantially improves the etiologic diagnosis of infectious pleural effusion. In our opinion, this technique should be added to the routine diagnostic armamentarium of clinical microbiology laboratories.

  4. Soil Acidobacterial 16S rRNA Gene Sequences Reveal Subgroup Level Differences between Savanna-Like Cerrado and Atlantic Forest Brazilian Biomes

    OpenAIRE

    Catão, Elisa C. P.; Lopes, Fabyano A. C.; Janaína F. Araújo; Alinne P. de Castro; Barreto, Cristine C.; Mercedes M.C. Bustamante; Betania F. Quirino; Krüger, Ricardo H.

    2014-01-01

    16S rRNA sequences from the phylum Acidobacteria have been commonly reported from soil microbial communities, including those from the Brazilian Savanna (Cerrado) and the Atlantic Forest biomes, two biomes that present contrasting characteristics of soil and vegetation. Using 16S rRNA sequences, the present work aimed to study acidobacterial diversity and distribution in soils of Cerrado savanna and two Atlantic forest sites. PCA and phylogenetic reconstruction showed that the acidobacterial ...

  5. Evaluation of Partial 16S Ribosomal DNA Sequencing for Identification of Nocardia Species by Using the MicroSeq 500 System with an Expanded Database

    OpenAIRE

    Cloud, Joann L.; Patricia S Conville; Croft, Ann; Harmsen, Dag; Witebsky, Frank G.; Carroll, Karen C.

    2004-01-01

    Identification of clinically significant nocardiae to the species level is important in patient diagnosis and treatment. A study was performed to evaluate Nocardia species identification obtained by partial 16S ribosomal DNA (rDNA) sequencing by the MicroSeq 500 system with an expanded database. The expanded portion of the database was developed from partial 5′ 16S rDNA sequences derived from 28 reference strains (from the American Type Culture Collection and the Japanese Collection of Microo...

  6. Characterization by 16S rRNA gene analysis and in situ hybridization of bacteria living in the hindgut of a deposit-feeding echinoid (Echinodermata)

    OpenAIRE

    da Silva, S.G.; Gillan, D. C.; Dubilier, N.; De Ridder, C.

    2006-01-01

    The hindgut caecum of the deposit-feeding echinoid Echinocardium cordatum harbours a symbiotic bacterial microflora, organized into layered mats around detrital particles owing to the proliferation of filamentous bacteria. The bacterial community was analysed using 16S rRNA gene analysis and fluorescence in situ hybridization. The purpose was to characterize its biodiversity and to identify its predominant members. The majority of the 16S sequences belong to the delta -Proteobacteria (61.5%),...

  7. Causas de recaída e de busca por tratamento referidas por dependentes químicos em uma unidade de reabilitação

    OpenAIRE

    Flávia Regina Mendes Carvalho; Tatiana Brusamarello; Andréa Noeremberg Guimarães; Marcio Roberto Paes; Mariluci Alves Maftum

    2011-01-01

    Objetivos: Identificar as causas de recaída e de busca por tratamento pelos dependentes químicos.Metodologia: Pesquisa qualitativa, exploratória, realizada em Unidade de Reabilitação de Adictos de um hospital psiquiátrico do Paraná, Brasil. Os dados foram coletados nos meses de maio a junho de 2010, através de entrevista semiestruturada com 12 dependentes químicos. Posteriormente, foram analisados e organizados em categorias temáticas.Resultados: Da análise, emergiram cinco categorias: o meio...

  8. The approaches to mathematical modeling of recA, umuD genes expression in bacteria Escherichia coli after UV-irradiation

    International Nuclear Information System (INIS)

    The modern data of recA, umuD genes expression of the system of SOS-repair at classical object of radiation genetic researches - bacteria Escherichia coli, after ultraviolet irradiation are presented. Essentially a new method of analysis of SOS-genes expression is considered. It was shown that using this method it is possible to determine the character of induction of some SOS-genes more precisely. The possible approach to the mathematical description of SOS-response of cells by construction of the system of the differential equations is presented

  9. 16S rDNA在乳酸菌菌种鉴定及聚类分析上的应用%Application of 16S rDNA fingerprint on the strain identification and traceability isolated from yogurts

    Institute of Scientific and Technical Information of China (English)

    杨柳; 杨捷琳; 谌鸿超; 曹敏仪; 潘良文

    2013-01-01

    Microecological products are paid more and more attention.Most commercial microecological products on local market are the yogurts which contain lactic acid bacteria.The common technology of strain identification are the physiological and biochemical test or the application of PCR with some defects such as time-consuming or need specialized person.We used Riboprinter to complete the subspecies level of identification of 16 lactobacillus bacterias isolated from 11 yogurt samples based on the separation and purification of the bacterias.Not only chose the optimization of the culture conditions,but also established the "Traceability File" used for the quality research.What's more,the use of the 16s rDNA fingerprint analysis and real time PCR helped to confirm the verification of the results from the colony identification.%近年来,微生态制剂日益受到市场关注.市售微生态食品以益生菌酸奶或乳酸饮料为主,其中活的乳酸菌含量及菌株特性是决定微生态制剂质量的关键.目前常用的菌种鉴定技术包括生理生化方法或PCR方法,前者工作量大周期长,后者需要专业人员操作,且难以区分死活细菌.本文选取了11种市售的乳酸菌产品,采用MC、MRL、TPY等乳酸菌专用培养基进行乳酸菌计数及菌种分离,进一步用荧光定量PCR方法验证了产品中常规培养法难以分离的双歧杆菌,在此基础上,采用Riboprinter基因指纹图谱分析仪,对分离自11种酸奶样品的16株乳酸菌菌进行了核酸水平的鉴定,建立了16S rDNA聚类分析分子图谱,方便并简化对微生态产品的质量进行“物证相符”的考证,初步分析了目前市场上市售酸奶所用菌种的种属相关性.

  10. Diagnóstico de Mycoplasma genitalium por amplificación de los genes MgPa y ARN ribosomal 16S Diagnosis of Mycoplasma genitalium by MgPa and rRNA 16S gene amplification

    Directory of Open Access Journals (Sweden)

    Carmen Fernández-Molina

    2008-10-01

    Full Text Available OBJETIVO: El microorganismo Mycoplasma genitalium se ha relacionado con la uretritis no gonocócica (UNG. La técnica de PCR se ha convertido en el principal método de detección de este patógeno. En consecuencia, debe aplicarse un método de diagnóstico mediante la amplificación de fragmentos de ADN por la técnica PCR. MATERIAL Y MÉTODOS: Se seleccionaron los cebadores MGF-MGR y MgPaF-MgPaR, complementarios de los genes de ARNr 16S y MgPa de M. genitalium, respectivamente. Se efectuaron ensayos de especificidad y sensibilidad y se estudiaron muestras clínicas. RESULTADOS: La PCR con cada grupo de cebadores utilizado fue específica sólo para M. genitalium y la sensibilidad fue mayor con el grupo de cebadores MGF-MGR. En el estudio de 34 muestras clínicas, 18.5% fue positivo a M. genitalium y se encontró un mayor número de muestras positivas al utilizar los cebadores MgPaF-MgPaR. CONCLUSIONES: Debe aplicarse en la práctica clínica el diagnóstico de M. genitalium mediante la amplificación del ADN por PCR en los pacientes con UNG.OBJECTIVE: Mycoplasma genitalium has been associated with nongonococcal urethritis (NGU. Diagnosis by PCR has become the primary detection method for this organism. Thus, diagnosis by DNA amplification using the PCR technique should be utilized. MATERIAL AND METHODS: GMF/GMR and MgpF/MgpR primer pairs, complementary to the M. genitalium 16S rRNA and MgPa genes, respectively, were selected. Specificity and sensibility assays were conducted and clinical samples were studied. RESULTS: The PCR with each primer pair was specific only for M. genitalium, and the sensibility was higher with the GMF/GMR primers. In the study of 34 clinical samples, 18,5% were positive for M. genitalium, with more positive samples when the MgpF/MgpR primers were used. CONCLUSIONS: DNA amplification by PCR should be applied in clinical practice to the diagnosis of M. genitalium in patients with NGU should using.

  11. 家兔胚胎胃肠道细菌分离及16S rRNA基因序列分析%Isolation and 16S rRNA gene sequence analysis of gastrointestinal bacteria in the rabbit embryos

    Institute of Scientific and Technical Information of China (English)

    郭海勇; 单晓枫; 吴静; 孙文怡; 于超; 王好; 钱爱东

    2012-01-01

    为研究家兔胚胎胃肠道细菌菌群的种类,对常规饲养、正常妊娠的家兔胚胎后期胃肠道细菌进行分离,基于分离细菌的生理生化特性、16S rRNA基因序列的同源性比较及系统发育分析,初步确定家兔胚胎后期胃肠道中存在的微生物茵群有微球菌属(Micrococcus)、葡萄球菌属(Staphylococcus)、棒杆菌属(Corynebacterium)以及蜡样芽孢杆菌(Bacillus cereus)等细菌,其序列同源性均为97%~99%.分离细菌均为G+细菌,家兔胚胎胃肠道中细菌的平均数量达到0.8×102 cfu/g~1.2×102 cfu/g,本研究为哺乳动物胚胎时期存在于胃肠道中细菌的深入研究奠定了基础.%In order to study the variety of gastrointestinal bacteria in the rabbit embryos, the bacteria of gastrointestinal tract in the later embryonic period were isolated from the conventional feeding and normal pregnant rabbits. Based on the bacterial physiological and biochemical properties, homology and phylogenetic analysis of 16S rRNA gene sequence, four isolates were identified as the genera Micrococcus, Staphylococcus, Corynebacterium and Bacillus cereus, all of which have 97% to 99% similarity. The bacteria were the gram-positive bacterium and the average number of bacteria was 0.8xl02 cfu/g to 1.2X102 cfu/g in gastrointestinal tract of the rabbit embryo in the later embryonic period. This study would lay a foundation for further studies on bacteria of gastrointestinal tract from the mammalian embryo stage.

  12. Phylogenetic Analysis of Veneridae (Mollusca: Bivalvia) based on Mitochondrial 16S rDNA%基于16S rDNA序列的帘蛤科贝类分子系统发育研究

    Institute of Scientific and Technical Information of China (English)

    程汉良; 周旻纯; 陈冬勤; 彭永兴; 董志国; 易乐飞; 孟学平; 申欣

    2012-01-01

    对21种帘蛤科贝类线粒体16S rRNA基因片段的核苷酸序列进行了分析,以探讨这一序列在种质鉴定、分子系统发育研究中的应用价值.试验结果表明,所有物种扩增片段长度504~642 bp,具有明显的长度多态性,序列A+T含量59.2%~69.0%,明显高于GC含量.物种间共有变异位点447个,其中简约信息位点351个.以16S rDNA片段序列为标记,以中国蛤蜊做外群,构建了帘蛤科贝类的系统发生树,拓扑结构显示,帘蛤科贝类形成3个明显类群,缀锦蛤亚科的13个种形成一个单系群,其结点自展值为93%.第二类群由雪蛤亚科、帘蛤亚科和镜蛤亚科的种类组成,结点自展值为87%.第三类群由仙女蛤亚科、青蛤亚科、楔形蛤亚科和文蛤亚科的种类组成,结点自展值为100%.结合拓扑结构分析和序列比对分析,本研究支持将短文蛤和丽文蛤订为文蛤的同物异名的观点,建议将丽文蛤和短文蛤订为文蛤的地理亚种;支持将薄片镜蛤和Dosinia angulosa订为2个独立种的观点;宜将波纹巴非蛤和织锦巴非蛤订为2个独立种.

  13. Methanosarcina acetivorans 16S rRNA and transcription factor nucleotide fluctuation with implications in exobiology and pathology

    Science.gov (United States)

    Holden, Todd; Tremberger, G., Jr.; Cheung, E.; Subramaniam, R.; Sullivan, R.; Schneider, P.; Flamholz, A.; Marchese, P.; Hiciano, O.; Yao, H.; Lieberman, D.; Cheung, T.

    2008-08-01

    Cultures of the methane-producing archaea Methanosarcina, have recently been isolated from Alaskan sediments. It has been proposed that methanogens are strong candidates for exobiological life in extreme conditions. The spatial environmental gradients, such as those associated with the polygons on Mars' surface, could have been produced by past methanogenesis activity. The 16S rRNA gene has been used routinely to classify phenotypes. Using the fractal dimension of nucleotide fluctuation, a comparative study of the 16S rRNA nucleotide fluctuation in Methanosarcina acetivorans C2A, Deinococcus radiodurans, and E. coli was conducted. The results suggest that Methanosarcina acetivorans has the lowest fractal dimension, consistent with its ancestral position in evolution. Variation in fluctuation complexity was also detected in the transcription factors. The transcription factor B (TFB) was found to have a higher fractal dimension as compared to transcription factor E (TFE), consistent with the fact that a single TFB in Methanosarcina acetivorans can code three different TATA box proteins. The average nucleotide pair-wise free energy of the DNA repair genes was found to be highest for Methanosarcina acetivorans, suggesting a relatively weak bonding, which is consistent with its low prevalence in pathology. Multitasking capacity comparison of type-I and type-II topoisomerases has been shown to correlate with fractal dimension using the methicillin-resistant strain MRSA 252. The analysis suggests that gene adaptation in a changing chemical environment can be measured in terms of bioinformatics. Given that the radiation resistant Deinococcus radiodurans is a strong candidate for an extraterrestrial origin and that the cold temperature Psychrobacter cryohalolentis K5 can function in Siberian permafrost, the fractal dimension comparison in this study suggests that a chemical resistant methanogen could exist in extremely cold conditions (such as that which existed on early

  14. 布鲁氏菌和幽门螺杆菌等6种病原菌16S rRNA和cagA等基因的克隆与鉴定%Cloning and identification of 16S rRNA and cagA genes from six pathogenic bacteria including Brucella and Helicobacter pylori

    Institute of Scientific and Technical Information of China (English)

    高正琴; 岳秉飞; 贺争鸣

    2010-01-01

    目的 克隆并鉴定布鲁氏菌的16S rRNA、支原体的16S rRNA、泰泽氏菌的16S rRNA、空肠弯曲菌的flaA、肝螺杆菌的flaB和幽门螺杆菌的cagA基因,为建立实时荧光定量PCR检测方法作准备.方法 设计、合成布鲁氏菌的16S rRNA、支原体的16S rRNA、泰泽氏菌的16S rRNA、空肠弯曲菌flaA、肝螺杆菌flaB和幽门螺杆菌的cagA基因的特异性引物;PCR扩增、克隆16S rRNA、flaA、flaB和cagA基因;对重组质粒进行酶切鉴定、PCR鉴定和测序分析.结果 布鲁氏菌的16S rRNA、支原体的16S rRNA、泰泽氏菌的16S rRNA、空肠弯曲杆菌的flaA、肝螺杆菌的flaB和幽门螺杆菌的cagA基因PCR扩增产物分别在612 bp、600 bp、922 bp、637 bp、1 545 bp和1 168 bp处出现特异性目的 条带,结果均与预期扩增的目的 片段大小一致.将PCR产物分别与pMD18-T载体连接并转化感受态细菌大肠埃希菌JM109.对重组质粒pT-BC16S rRNA、pT-MP16S rRNA、pT-CP16S rRNA、pT-CJflaA、pT-HHflaB和pT-HPcagA分别进行酶切鉴定及PCR鉴定,结果均可见特异性目的 条带.测序结果显示,布鲁氏菌的16S rRNA、支原体的16S rRNA、泰泽氏菌的16S rRNA、空肠弯曲菌的flaA、肝螺杆菌的flaB和幽门螺杆菌的cagA基因序列与GenBank中细菌相应序列同源性高达100 %,说明上述6种致病菌的16S rRNA、flaA、flaB和cagA基因已成功克隆.结论 成功克隆了布鲁氏菌16S rRNA、支原体的16S rRNA、泰泽氏菌的16S rRNA、空肠弯曲菌的flaA、肝螺杆菌的flaB和幽门螺杆菌的cagA基因,为建立实时荧光定量PCR检测方法奠定了基础.

  15. Bacillus subtilis RecA and its accessory factors, RecF, RecO, RecR and RecX, are required for spore resistance to DNA double-strand break.

    Science.gov (United States)

    Vlašić, Ignacija; Mertens, Ramona; Seco, Elena M; Carrasco, Begoña; Ayora, Silvia; Reitz, Günther; Commichau, Fabian M; Alonso, Juan C; Moeller, Ralf

    2014-02-01

    Bacillus subtilis RecA is important for spore resistance to DNA damage, even though spores contain a single non-replicating genome. We report that inactivation of RecA or its accessory factors, RecF, RecO, RecR and RecX, drastically reduce survival of mature dormant spores to ultrahigh vacuum desiccation and ionizing radiation that induce single strand (ss) DNA nicks and double-strand breaks (DSBs). The presence of non-cleavable LexA renders spores less sensitive to DSBs, and spores impaired in DSB recognition or end-processing show sensitivities to X-rays similar to wild-type. In vitro RecA cannot compete with SsbA for nucleation onto ssDNA in the presence of ATP. RecO is sufficient, at least in vitro, to overcome SsbA inhibition and stimulate RecA polymerization on SsbA-coated ssDNA. In the presence of SsbA, RecA slightly affects DNA replication in vitro, but addition of RecO facilitates RecA-mediated inhibition of DNA synthesis. We propose that repairing of the DNA lesions generates a replication stress to germinating spores, and the RecA·ssDNA filament might act by preventing potentially dangerous forms of DNA repair occurring during replication. RecA might stabilize a stalled fork or prevent or promote dissolution of reversed forks rather than its cleavage that should require end-processing. PMID:24285298

  16. Discordant 16S and 23S rRNA gene phylogenies for the genus Helicobacter: implications for phylogenetic inference and systematics.

    Science.gov (United States)

    Dewhirst, Floyd E; Shen, Zeli; Scimeca, Michael S; Stokes, Lauren N; Boumenna, Tahani; Chen, Tsute; Paster, Bruce J; Fox, James G

    2005-09-01

    Analysis of 16S rRNA gene sequences has become the primary method for determining prokaryotic phylogeny. Phylogeny is currently the basis for prokaryotic systematics. Therefore, the validity of 16S rRNA gene-based phylogenetic analyses is of fundamental importance for prokaryotic systematics. Discrepancies between 16S rRNA gene analyses and DNA-DNA hybridization and phenotypic analyses have been noted in the genus Helicobacter. To clarify these discrepancies, we sequenced the 23S rRNA genes for 55 helicobacter strains representing 41 taxa (>2,700 bases per sequence). Phylogenetic-tree construction using neighbor-joining, parsimony, and maximum likelihood methods for 23S rRNA gene sequence data yielded stable trees which were consistent with other phenotypic and genotypic methods. The 16S rRNA gene sequence-derived trees were discordant with the 23S rRNA gene trees and other data. Discrepant 16S rRNA gene sequence data for the helicobacters are consistent with the horizontal transfer of 16S rRNA gene fragments and the creation of mosaic molecules with loss of phylogenetic information. These results suggest that taxonomic decisions must be supported by other phylogenetically informative macromolecules, such as the 23S rRNA gene, when 16S rRNA gene-derived phylogeny is discordant with other credible phenotypic and genotypic methods. This study found Wolinella succinogenes to branch with the unsheathed-flagellum cluster of helicobacters by 23S rRNA gene analyses and whole-genome comparisons. This study also found intervening sequences (IVSs) in the 23S rRNA genes of strains of 12 Helicobacter species. IVSs were found in helices 10, 25, and 45, as well as between helices 31' and 27'. Simultaneous insertion of IVSs at three sites was found in H. mesocricetorum. PMID:16109952

  17. First microbiota assessments of children's paddling pool waters evaluated using 16S rRNA gene-based metagenome analysis.

    Science.gov (United States)

    Sawabe, Toko; Suda, Wataru; Ohshima, Kenshiro; Hattori, Masahira; Sawabe, Tomoo

    2016-01-01

    Insufficient chloric sterilization of children's paddling pool waters increases the risk of diarrheal illness. Therefore, we investigated the microbiota changes after children use pools. First, we applied 16S rRNA gene-based metagenome analysis to understand the dynamics of microbiota in pool water, especially with respect to the bio-contamination by potential pathogens. Proteobacteria were major taxa detected in every pool water sample after children spent time in the pool. In more detail, Gammaproteobacteria comprised the dominant class, which was followed by Betaproteobacteria. Five phyla, Bacteroidetes, Firmicutes, Actinobacteria and Deinococcus-Thermus phyla were minor groups. The pool water microbiota are likely to be a consortium of intestinal and skin microbiota from humans. Interestingly, the ratio of Gammaproteobacteria and Betaproteobacteria differed according to the age of the children who used the pool, which means the pool water was additionally contaminated by soil microbiota as a result of the children's behavior. Furthermore, potential pathogens, such as Campylobacter spp., Comamonas testosteroni and Burkholderia pseudomallei, were also found. Considering the standard plate counts, the abundances of these human pathogens are unlikely to be a sufficiently infectious dose. We suggest the importance of sanitary measures in paddling pool waters to reduce bio-contamination from both humans and the environment.

  18. Structural insights of microbial community of Deulajhari (India hot spring using 16s-rRNA based metagenomic sequencing

    Directory of Open Access Journals (Sweden)

    Archana Singh

    2016-03-01

    Full Text Available Insights about the distribution of the microbial community prove to be the major goal of understanding microbial ecology which remains to be fully deciphered. Hot springs being hub for the thermophilic microbiota attract the attention of the microbiologists. Deulajhari hot spring cluster is located in the Angul district of Odisha. Covered within a wooded area, Deulajhari hot spring is also fed by the plant litter resulting in a relatively high amount of total organic content (TOC. For the first time, Illumina sequencing based biodiversity analysis of microbial composition is studied through amplicon metagenome sequencing of 16s rRNA targeting V3‐V4 region using metagenomic DNA from the hot spring sediment. Over 28 phyla were detected through the amplicon metagenome sequencing of which the most dominating phyla at the existing physiochemical parameters like; temperature 69 °C, pH 8.09, electroconductivity 0.025 dSm−1 and total organic carbon 0.356%, were Proteobacteria (88.12%, Bacteriodetes (10.76%, Firmicutes (0.35%, Spirochetes (0.18% and chloroflexi (0.11%. Approximately 713 species were observed at the above physiochemical parameters. The analysis of the metagenome provides the quantitative insights into microbial populations based on the sequence data in Deulajhari hot spring. Metagenome sequence is deposited to SRA database which is available at NCBI with accession no. SRX1459736.

  19. A ribonucleoprotein fragment of the 30 S ribosome of E. coli containing two contiguous domains of the 16 S RNA.

    Science.gov (United States)

    Spitnik-Elson, P; Elson, D; Avital, S; Abramowitz, R

    1982-08-11

    Ribonucleoprotein fragments of the 30 S ribosome of E. coli have been prepared by limited ribonuclease digestion and mild heating of the ribosome in a constant ionic environment. One such fragment has been described previously. A second electrophoretically homogeneous fragment has now been isolated and its RNA and protein moieties have been characterized. It contains the 5' half of the 16 S RNA, encompassing domains I and II except for the extreme 5' terminus and several small gaps. Seven proteins are present: S4, S5, S6, S8, S12, S15 and S20. The RNA binding sites of five of these proteins are known, and all are RNA sequences that are present in the fragment. Published neutron scattering and immuno-electron microscopic data indicate that six of the proteins are clustered together in a cross sectional slice through the center of the subunit. After deproteinization, the RNA moiety gives two bands in gel electrophoresis, one containing domains I and II and the other, essentially only domain II. The former, although larger, migrates faster in gel electrophoresis, indicating that RNA domains I and II interact with each other in such a way as to become more compact than domain II by itself.

  20. Bacterial community composition of anthropogenic biochar and Amazonian anthrosols assessed by 16S rRNA gene 454 pyrosequencing.

    Science.gov (United States)

    Taketani, Rodrigo Gouvêa; Lima, Amanda Barbosa; da Conceição Jesus, Ederson; Teixeira, Wenceslau Geraldes; Tiedje, James M; Tsai, Siu Mui

    2013-08-01

    Biochar (BC) is a common minor constituent of soils and is usually derived from the burning of wood materials. In the case of Amazonian dark earth (ADE) soils, the increased amount of this material is believed to be due to anthropogenic action by ancient indigenous populations. In this study, we use 16S rRNA gene pyrosequencing to assess the bacterial diversity observed in the BC found in ADEs as well as in the dark earth itself and the adjacent Acrisol. Samples were taken from two sites, one cultivated with manioc and one with secondary forest cover. Analyses revealed that the community structure found in each sample had unique features. At a coarse phylogenetic resolution, the most abundant phyla in all sequence libraries were Actinobacteria, Acidobacteria, Verrucomicrobia and Proteobacteria that were present in similar relative abundance across all samples. However, the class composition varied between them highlighting the difference between the Acrisol and the remaining samples. This result was also corroborated by the comparison of the OTU composition (at 97 % identity). Also, soil coverage has shown an effect over the community structure observed in all samples. This pattern was found to be significant through unweighted UniFrac as well as P tests. These results indicate that, although the ADEs are found in patches within the Acrisols, the contrasting characteristics found between them led to the development of significantly different communities. PMID:23743632

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

    Directory of Open Access Journals (Sweden)

    Mu Peng

    2015-09-01

    Full Text Available 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.

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

    Science.gov (United States)

    Peng, Mu; Zi, Xiaoxue; Wang, Qiuyu

    2015-09-24

    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.

  3. First microbiota assessments of children's paddling pool waters evaluated using 16S rRNA gene-based metagenome analysis.

    Science.gov (United States)

    Sawabe, Toko; Suda, Wataru; Ohshima, Kenshiro; Hattori, Masahira; Sawabe, Tomoo

    2016-01-01

    Insufficient chloric sterilization of children's paddling pool waters increases the risk of diarrheal illness. Therefore, we investigated the microbiota changes after children use pools. First, we applied 16S rRNA gene-based metagenome analysis to understand the dynamics of microbiota in pool water, especially with respect to the bio-contamination by potential pathogens. Proteobacteria were major taxa detected in every pool water sample after children spent time in the pool. In more detail, Gammaproteobacteria comprised the dominant class, which was followed by Betaproteobacteria. Five phyla, Bacteroidetes, Firmicutes, Actinobacteria and Deinococcus-Thermus phyla were minor groups. The pool water microbiota are likely to be a consortium of intestinal and skin microbiota from humans. Interestingly, the ratio of Gammaproteobacteria and Betaproteobacteria differed according to the age of the children who used the pool, which means the pool water was additionally contaminated by soil microbiota as a result of the children's behavior. Furthermore, potential pathogens, such as Campylobacter spp., Comamonas testosteroni and Burkholderia pseudomallei, were also found. Considering the standard plate counts, the abundances of these human pathogens are unlikely to be a sufficiently infectious dose. We suggest the importance of sanitary measures in paddling pool waters to reduce bio-contamination from both humans and the environment. PMID:26671497

  4. The phylogeny of native and exotic scallops cultured in China based on 16S rDNA sequences

    Institute of Scientific and Technical Information of China (English)

    LIU Baozhong; DONG Bo; XIANG Jianhai; WANG Zaizhao

    2007-01-01

    Scallops of the Family Pectinidae are a valuable resource in marine industry of the world.Understanding the phylogeny of the family is important for the development of the industry. In this study,partial 16S mitochondrial rDNA genes were obtained from 8 scallop species that are commonly cultured indigenous and transplanted species in China. Phylogenetic relationships of Pectinidae were analyzed based on the 8 sequences and other 5 published ones in GenBank, representing 9 genera of the family. The molecular phylogeny trees were constructed using 3 methods with software PHYLIP. The results showe that total 13 species of scallops clustered in 4 clades. Pecten maximus joins P. jacobaeus then Amusium pleuronectes in cluster, indicating close relationship of genus Amusium with Pecten in evolution. P. yessoensis is close to Chlamysfarreri and C. islandica. No enough material was available to single out genus Patinopecten as an independent monophyletic subfamily. The position ofAdamussium colbecki indicates that it is far from genus Pecten but near to genus Chlamys in evolution.

  5. Characterization of Lactobacillus from Algerian goat's milk based on phenotypic, 16S rDNA sequencing and their technological properties

    Directory of Open Access Journals (Sweden)

    Ahmed Marroki

    2011-03-01

    Full Text Available Nineteen strains of Lactobacillus isolated from goat's milk from farms in north-west of Algeria were characterized. Isolates were identified by phenotypic, physiological and genotypic methods and some of their important technological properties were studied. Phenotypic characterization was carried out by studying physiological, morphological characteristics and carbohydrate fermentation patterns using API 50 CHL system. Isolates were also characterized by partial 16S rDNA sequencing. Results obtained with phenotypic methods were correlated with the genotypic characterization and 13 isolates were identified as L. plantarum, two isolates as L. rhamnosus and one isolate as L. fermentum. Three isolates identified as L. plantarum by phenotypic characterization were found to be L. pentosus by the genotypic method. A large diversity in technological properties (acid production in skim milk, exopolysaccharide production, aminopeptidase activity, antibacterial activity and antibiotic susceptibility was observed. Based on these results, two strains of L. plantarum (LbMS16 and LbMS21 and one strain of L. rhamnosus (LbMF25 have been tentatively selected for use as starter cultures in the manufacture of artisanal fermented dairy products in Algeria.

  6. Analysis of the chronic wound microbiota of 2,963 patients by 16S rDNA pyrosequencing.

    Science.gov (United States)

    Wolcott, Randall D; Hanson, John D; Rees, Eric J; Koenig, Lawrence D; Phillips, Caleb D; Wolcott, Richard A; Cox, Stephen B; White, Jennifer S

    2016-01-01

    The extent to which microorganisms impair wound healing is an ongoing controversy in the management of chronic wounds. Because the high diversity and extreme variability of the microbiota between individual chronic wounds lead to inconsistent findings in small cohort studies, evaluation of a large number of chronic wounds using identical sequencing and bioinformatics methods is necessary for clinicians to be able to select appropriate empiric therapies. In this study, we utilized 16S rDNA pyrosequencing to analyze the composition of the bacterial communities present in samples obtained from patients with chronic diabetic foot ulcers (N = 910), venous leg ulcers (N = 916), decubitus ulcers (N = 767), and nonhealing surgical wounds (N = 370). The wound samples contained a high proportion of Staphylococcus and Pseudomonas species in 63 and 25% of all wounds, respectively; however, a high prevalence of anaerobic bacteria and bacteria traditionally considered commensalistic was also observed. Our results suggest that neither patient demographics nor wound type influenced the bacterial composition of the chronic wound microbiome. Collectively, these findings indicate that empiric antibiotic selection need not be based on nor altered for wound type. Furthermore, the results provide a much clearer understanding of chronic wound microbiota in general; clinical application of this new knowledge over time may help in its translation to improved wound healing outcomes. PMID:26463872

  7. Vertical stratification of microbial communities in the Red Sea revealed by 16S rDNA pyrosequencing

    KAUST Repository

    Qian, Peiyuan

    2010-07-29

    The ecosystems of the Red Sea are among the least-explored microbial habitats in the marine environment. In this study, we investigated the microbial communities in the water column overlying the Atlantis II Deep and Discovery Deep in the Red Sea. Taxonomic classification of pyrosequencing reads of the 16S rRNA gene amplicons showed vertical stratification of microbial diversity from the surface water to 1500 m below the surface. Significant differences in both bacterial and archaeal diversity were observed in the upper (2 and 50 m) and deeper layers (200 and 1500 m). There were no obvious differences in community structure at the same depth for the two sampling stations. The bacterial community in the upper layer was dominated by Cyanobacteria whereas the deeper layer harbored a large proportion of Proteobacteria. Among Archaea, Euryarchaeota, especially Halobacteriales, were dominant in the upper layer but diminished drastically in the deeper layer where Desulfurococcales belonging to Crenarchaeota became the dominant group. The results of our study indicate that the microbial communities sampled in this study are different from those identified in water column in other parts of the world. The depth-wise compositional variation in the microbial communities is attributable to their adaptations to the various environments in the Red Sea. © 2011 International Society for Microbial Ecology All rights reserved.

  8. Microbial diversity analysis of former salterns in southern Taiwan by 16S rRNA-based methods.

    Science.gov (United States)

    Wang, Chung-Yi; Ng, Chang-Chai; Chen, Tseng-Wei; Wu, Sz-Jie; Shyu, Yuan-Tay

    2007-12-01

    The microbiota diversity of the former salterns in southern Taiwan was investigated by denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH). Soil samples from three salterns were analyzed using DGGE and 16S rRNA from 502 colonies representing 5 archaea and 18 bacteria taxonomic groups. Each representative taxonomic group was further identified, whereas 8.7% of clones were unclassified microorganisms. Chromohalobacter, Halomonas and Virgibacillus are dominant in the Biemen saltern, Chiguensis saltern and Szutsau saltern, respectively. During FISH analysis, several taxonomic-specific probes were used. The DAPI-stained-cell count in the Szutsao saltern had a higher number of microorganisms (4.58 x 10(7) cell/cm(3)) than the other salterns. Archaea occupied 2.7-6.6% whereas bacteria accounted for 37.2-52.9% of total microbial population at the three sites. Among these three sampling sites, the Szutsao saltern had the highest diversity in halophilic microbial composition, as indicated by DGGE and FISH. PMID:18072239

  9. Sequencing of 16S rRNA reveals a distinct salivary microbiome signature in Behçet's disease.

    Science.gov (United States)

    Coit, Patrick; Mumcu, Gonca; Ture-Ozdemir, Filiz; Unal, Ali Ugur; Alpar, Ugur; Bostanci, Nagihan; Ergun, Tulin; Direskeneli, Haner; Sawalha, Amr H

    2016-08-01

    Behçet's disease (BD) is characterized by recurrent oro-genital ulcers, mucocutaneous lesions, and serious organ involvement. We investigated the salivary microbiome in BD using high-throughput sequencing of the 16S rRNA V4 region. Stimulated saliva samples were collected from 31 BD patients and 15 healthy controls, and in 9 BD patients, a second saliva sample was collected following dental and periodontal treatment. Sequence analysis identified a total of 908 operational taxonomic units (OTUs) present across all samples. Patients had a microbial community structure that is significantly less diverse than healthy controls. The most overabundant species in BD was Haemophilus parainfluenzae, while the most depleted included Alloprevotella rava and species in the genus Leptotrichia. Periodontal treatment improved oral health indices in BD but had no short-term effect on bacterial community structure. Neither the BD-associated genetic risk locus within the HLA-B/MICA region nor being on immunosuppressive medications explained the differences between patients and controls. PMID:27283393

  10. Comparison of Bacteroides-Prevotella 16S rRNA genetic markers for fecal samples from different animal species

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    Peng, Mu; Zi, Xiaoxue; Wang, Qiuyu

    2015-10-01

    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

  12. Molecular phylogenetics of genus Mytilus based on the 16S rRNA sequences%基于16S rRNA序列初步探讨贻贝属的系统发育

    Institute of Scientific and Technical Information of China (English)

    沈玉帮; 李家乐; 牟月军

    2009-01-01

    通过比较贻贝属5个物种包括中国的两种贻贝的线粒体16S rRNA基因部分序列,来初步确定它们的系统发育关系和了解中国沿海两种贻贝的遗传多样性情况.以Perna viridis为外群,采用NJ法和MP法构建分子系统树.系统发育分析表明,5种贻贝(Mytilus californianus, M. corcuscus, M. galloprovincialis, M. edulis, M. trossulus)在系统树上依次进行分叉,呈放射状.M. californianus最为原始,M. corcuscus次之.每一个贻贝物种都形成单系.其中,M. edulis和M. trossulus是非常相似的,M. corcuscus和M. californianus的亲缘关系近.同时发现,我国沿海分布的紫贻贝(M. galloprovincialis)和厚壳贻贝(M. corcuscus)的遗传多样性都较高,但厚壳贻贝的遗传多样性要低于紫贻贝,可能是由于厚壳贻贝过度被渔民开采等导致厚壳贻贝群体大小降低的缘故.这里系统发育分析为将来进行物种进化、迁移和育种方面的比较研究提供理论基础.

  13. Design of Vibrio 16S rRNA Gene Specific Primers and Their Application in the Analysis of Seawater Vibrio Community

    Institute of Scientific and Technical Information of China (English)

    LIU Yong; YANG Guanpin; WANG Hualei; CHEN Jixiang; SHI Xianming; ZOU Guiwei; WEI Qiwei; SUN Xiuqin

    2006-01-01

    The pathogenic species of genus Vibrio cause vibriosis, one of the most prevalent diseases of maricultured animals and seafood consumers. Monitoring their kinetics in the chain of seafood production, processing and consumption is of great importance for food and mariculture safety. In order to enrich Vibrio-representing 16S ribosomal RNA gene (rDNA) fragments and identify these bacteria further real-timely and synchronously among bacterial flora in the chain, a pair of primers that selectively amplify Vibrio 16S rDNA fragments were designed with their specificities and coverage testified in the analysis of seawater Vibrio community. The specificities and coverage of two primers, VF169 and VR744, were determined theoretically among bacterial 16S rDNAs available in GenBank by using BLAST program and practically by amplifying Vibrio 16S rDNA fragments from seawater DNA. More than 88.3% of sequences in GenBank, which showed identical matches with VR744, belong to Vibrio genus. A total of 33 clones were randomly selected and sequenced. All of the sequences showed their highest similarities to and clustered around those of diverse known Vibrio species. The primers designed are capable of retrieving a wide range of Vibrio 16S rDNA fragments specifically among bacterial flora in seawater, the most important natural environment of seafood cultivation.

  14. Phylogenetic analysis of Pomacea canaliculata isolates collected from rice fields in different origins of China by combined mitochondrial 12S and 16S genes.

    Science.gov (United States)

    Li, Xiao-Yan; Bian, Qing-Qing; Zhao, Guang-Hui

    2015-02-01

    To study the genetic relationships of Pomacea canaliculata collected from rice fields in China, the mitochondrial (mt) 12S and 16S of 9 P. canaliculata isolates from 5 southern provinces in China were sequenced and analyzed. The intra-specific sequence variations of P. canaliculata were 0-1.1% for 12S and 0--0.6% for 16S, while the inter-specific variations among common Pomacea species in mt 12S and 16S were 3.0-11.7% and 2.3-10.1%, respectively. Phylogenetic analysis based on combined sequences of mt 12S and 16S revealed complex genetic structure of P. canaliculata in China. Two phylogenetic groups of P. canaliculata were indicated in China with one group sistered to P. canaliculata isolates from USA, and two groups were even found in the same province. The phylogenetic relationships of Pomacea spp. also could be effectively inferred by combined sequences of mt 12S and 16S. These findings provided basic information for further study of population genetics and diffusion pattern of P. canaliculata in China as well as in the world.

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

    Science.gov (United States)

    Myer, Phillip R; Kim, MinSeok; Freetly, Harvey C; Smith, Timothy P L

    2016-08-01

    Next generation sequencing technologies have vastly changed the approach of sequencing of the 16S rRNA gene for studies in microbial ecology. Three distinct technologies are available for large-scale 16S sequencing. All three are subject to biases introduced by sequencing error rates, amplification primer selection, and read length, which can affect the apparent microbial community. In this study, we compared short read 16S rRNA variable regions, V1-V3, with that of near-full length 16S regions, V1-V8, using highly diverse steer rumen microbial communities, in order to examine the impact of technology selection on phylogenetic profiles. Short paired-end reads from the Illumina MiSeq platform were used to generate V1-V3 sequence, while long "circular consensus" reads from the Pacific Biosciences RSII instrument were used to generate V1-V8 data. The two platforms revealed similar microbial operational taxonomic units (OTUs), as well as similar species richness, Good's coverage, and Shannon diversity metrics. However, the V1-V8 amplified ruminal community resulted in significant increases in several orders of taxa, such as phyla Proteobacteria and Verrucomicrobia (P niche-specific database to use in analyzing data from shorter read technologies when budgetary constraints preclude use of near-full length 16S sequencing. PMID:27282101

  16. Combining flow cytometry and 16S rRNA gene pyrosequencing: A promising approach for drinking water monitoring and characterization

    KAUST Repository

    Prest, Emmanuelle I E C

    2014-10-01

    The combination of flow cytometry (FCM) and 16S rRNA gene pyrosequencing data was investigated for the purpose of monitoring and characterizing microbial changes in drinking water distribution systems. High frequency sampling (5min intervals for 1h) was performed at the outlet of a treatment plant and at one location in the full-scale distribution network. In total, 52 bulk water samples were analysed with FCM, pyrosequencing and conventional methods (adenosine-triphosphate, ATP; heterotrophic plate count, HPC). FCM and pyrosequencing results individually showed that changes in the microbial community occurred in the water distribution system, which was not detected with conventional monitoring. FCM data showed an increase in the total bacterial cell concentrations (from 345±15×103 to 425±35×103cellsmL-1) and in the percentage of intact bacterial cells (from 39±3.5% to 53±4.4%) during water distribution. This shift was also observed in the FCM fluorescence fingerprints, which are characteristic of each water sample. A similar shift was detected in the microbial community composition as characterized with pyrosequencing, showing that FCM and genetic fingerprints are congruent. FCM and pyrosequencing data were subsequently combined for the calculation of cell concentration changes for each bacterial phylum. The results revealed an increase in cell concentrations of specific bacterial phyla (e.g., Proteobacteria), along with a decrease in other phyla (e.g., Actinobacteria), which could not be concluded from the two methods individually. The combination of FCM and pyrosequencing methods is a promising approach for future drinking water quality monitoring and for advanced studies on drinking water distribution pipeline ecology. © 2014 Elsevier Ltd.

  17. Combining flow cytometry and 16S rRNA gene pyrosequencing: a promising approach for drinking water monitoring and characterization.

    Science.gov (United States)

    Prest, E I; El-Chakhtoura, J; Hammes, F; Saikaly, P E; van Loosdrecht, M C M; Vrouwenvelder, J S

    2014-10-15

    The combination of flow cytometry (FCM) and 16S rRNA gene pyrosequencing data was investigated for the purpose of monitoring and characterizing microbial changes in drinking water distribution systems. High frequency sampling (5 min intervals for 1 h) was performed at the outlet of a treatment plant and at one location in the full-scale distribution network. In total, 52 bulk water samples were analysed with FCM, pyrosequencing and conventional methods (adenosine-triphosphate, ATP; heterotrophic plate count, HPC). FCM and pyrosequencing results individually showed that changes in the microbial community occurred in the water distribution system, which was not detected with conventional monitoring. FCM data showed an increase in the total bacterial cell concentrations (from 345 ± 15 × 10(3) to 425 ± 35 × 10(3) cells mL(-1)) and in the percentage of intact bacterial cells (from 39 ± 3.5% to 53 ± 4.4%) during water distribution. This shift was also observed in the FCM fluorescence fingerprints, which are characteristic of each water sample. A similar shift was detected in the microbial community composition as characterized with pyrosequencing, showing that FCM and genetic fingerprints are congruent. FCM and pyrosequencing data were subsequently combined for the calculation of cell concentration changes for each bacterial phylum. The results revealed an increase in cell concentrations of specific bacterial phyla (e.g., Proteobacteria), along with a decrease in other phyla (e.g., Actinobacteria), which could not be concluded from the two methods individually. The combination of FCM and pyrosequencing methods is a promising approach for future drinking water quality monitoring and for advanced studies on drinking water distribution pipeline ecology.

  18. Bacterial diversity and community structure of supragingival plaques in adults with dental health or caries revealed by 16S pyrosequencing

    Directory of Open Access Journals (Sweden)

    Cuicui Xiao

    2016-07-01

    Full Text Available Dental caries has a polymicrobial etiology within the complex oral microbial ecosystem. However, the overall diversity and structure of supragingival plaque microbiota in adult dental health and caries are not well understood. Here, 160 supragingival plaque samples from patients with dental health and different severities of dental caries were collected for bacterial genomic DNA extraction, pyrosequencing by amplification of the 16S rDNA V1–V3 hypervariable regions, and bioinformatic analysis. High-quality sequences (2,261,700 clustered into 10,365 operational taxonomic units (OTUs; 97% identity, representing 453 independent species belonging to 122 genera, 66 families, 34 orders, 21 classes, and 12 phyla. All groups shared 7522 OTUs, indicating the presence of a core plaque microbiome. Smooth rarefaction curves were suggestive of plaque microbial diversity. α diversity analysis showed that healthy plaque microbial diversity exceeded that of dental caries, with the diversity decreasing gradually with the severity of caries. The dominant phyla of plaque microbiota included Bacteroidetes, Actinobacteria, Proteobacteria, Firmicutes, Fusobacteria, and TM7. The dominant genera included Capnocytophaga, Prevotella, Actinomyces, Corynebacterium, Neisseria, Streptococcus, Rothia, and Leptotrichia. β diversity analysis showed that the plaque microbial community structure was similar in all groups and that group members were relatively constant, only showing differences in abundance. Analysis of composition differences identified 10 health-related and 21 caries-related genera. Key genera (27 that potentially contributed to plaque microbiota distributions between groups were identified. Finally, co-occurrence network analysis and function prediction were performed. Treatment strategies directed toward modulating microbial interactions and their functional output should be further developed.

  19. Culture dependent bacteria in commercial fishes: Qualitative assessment and molecular identification using 16S rRNA gene sequencing

    KAUST Repository

    Alikunhi, Nabeel M.

    2016-05-27

    Fish contaminations have been extensively investigated in Saudi coasts, but studies pertaining to bacterial pathogens are meager. We conducted qualitative assessment and molecular identification of culture dependent bacteria in 13 fish species collected from three fishing sites and a local fish market in Jeddah, Saudi Arabia. The bacterial counts of gills, skin, gut and muscle were examined on agar plates of Macconkey’s (Mac), Eosin methylene blue (EMB) and Thiosulfate Citrate Bile Salts (TCBS) culture media. Bacterial counts exhibited interspecific, locational and behavioral differences. Mugil cephalus exhibited higher counts on TCBS (all body-parts), Mac (gills, muscle and gut) and EMB (gills and muscle). Samples of Area I were with higher counts, concurrent to seawater and sediment samples, revealing the influence of residing environment on fish contamination. Among feeding habits, detritivorous fish harbored higher bacterial counts, while carnivorous group accounted for lesser counts. Counts were higher in skin of fish obtained from market compared to field samples, revealing market as a major source of contamination. Bacterial counts of skin were positively correlated with other body-parts indicating influence of surface bacterial biota in overall quality of fish. Hence, hygienic practices and proper storage facilities in the Jeddah fish market is recommended to prevent adverse effect of food-borne illness in consumers. Rahnella aquatilis (Enterobacteriaceae) and Photobacterium damselae (Vibrionaceae) were among the dominant species identified from fish muscle samples using Sanger sequencing of 16S rRNA. This bacterial species are established human pathogens capable of causing foodborne illness with severe antibiotic resistance. Opportunistic pathogens such as Hafnia sp. (Enterobacteriaceae) and Pseudomonas stutzeri (Pseudomonadaceae) were also identified from fish muscle. These findings indicate bacterial contamination risk in commonly consumed fish of

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

    Science.gov (United States)

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

    2016-04-01

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

  1. Rapid 16S rRNA next-generation sequencing of polymicrobial clinical samples for diagnosis of complex bacterial infections.

    Directory of Open Access Journals (Sweden)

    Stephen J Salipante

    Full Text Available 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.

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

    Science.gov (United States)

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

    2016-04-01

    Bacterial diversity in a seawater recirculating aquaculture system (RAS) was investigated using 16S rRNA amplicon sequencing to understand the roles of bacterial communities in the system. The RAS was operated at nine different combinations of temperature (15°C, 20°C, and 25°C) and salinity (20‰, 25‰, and 32.5‰). Samples were collected from five or six RAS tanks (biofilters) for each condition. Fifty samples were analyzed. Proteobacteria and Bacteroidetes were most common (sum of both phyla: 67.2% to 99.4%) and were inversely proportional to each other. Bacteria that were present at an average of ≥ 1% included Actinobacteria (2.9%) Planctomycetes (2.0%), Nitrospirae (1.5%), and Acidobacteria (1.0%); they were preferentially present in packed bed biofilters, mesh biofilters, and maturation biofilters. The three biofilters showed higher diversity than other RAS tanks (aerated biofilters, floating bed biofilters, and fish tanks) from phylum to operational taxonomic unit (OTU) level. Samples were clustered into several groups based on the bacterial communities. Major taxonomic groups related to family Rhodobacteraceae and Flavobacteriaceae were distributed widely in the samples. Several taxonomic groups like [Saprospiraceae], Cytophagaceae, Octadecabacter, and Marivita showed a cluster-oriented distribution. Phaeobacter and Sediminicola-related reads were detected frequently and abundantly at low temperature. Nitrifying bacteria were detected frequently and abundantly in the three biofilters. Phylogenetic analysis of the nitrifying bacteria showed several similar OTUs were observed widely through the biofilters. The diverse bacterial communities and the minor taxonomic groups, except for Proteobacteria and Bacteroidetes, seemed to play important roles and seemed necessary for nitrifying activity in the RAS, especially in packed bed biofilters, mesh biofilters, and maturation biofilters. PMID:27033205

  3. Evidence that E. coli ribosomal protein S13 has two separable functional domains involved in 16S RNA recognition and protein S19 binding.

    Science.gov (United States)

    Schwarzbauer, J; Craven, G R

    1985-09-25

    We have found that E. coli ribosomal protein S13 recognizes multiple sites on 16S RNA. However, when protein S19 is included with a mixture of proteins S4, S7, S8, S16/S17 and S20, the S13 binds to the complex with measurably greater strength and with a stoichiometry of 1.5 copies per particle. This suggests that the protein may have two functional domains. We have tested this idea by cleaving the protein into two polypeptides. It was found that one of the fragments, composed of amino acid residues 84-117, retained the capacity to bind 16S RNA at multiple sites. Protein S19 had no affect on the strength or stoichiometry of the binding of this fragment. These data suggest that S13 has a C-terminal domain primarily responsible for RNA recognition and possibly that the N-terminal region is important for association with protein S19.

  4. Identification of the forensically important beetles Nicrophorus japonicus, Ptomascopus plagiatus and Silpha carinata (Coleoptera: Silphidae) based on 16S rRNA gene in China.

    Science.gov (United States)

    Tang, Z C; Guo, Y D; Zhang, X W; Shi, J; Yang, K T; Li, X L; Chen, Y Q; Cai, J F

    2012-09-01

    Sarcophagous beetles play an important role in estimating postmortem interval time (PMI) in the later stages decomposition of carcasses. However, the morphological similarity of beetles usually poses a challenge for forensic scientists within their routine work. As a supplementary to traditional morphological method, molecular genetics identification is simple and time-saving. A molecular identification method involving a 288-bp segment of the 16S ribosomal RNA (16S rRNA) gene from 15 beetles of Silphidae (Coleoptera), collected from 5 locations in 4 Chinese provinces, was evaluated. Phenogram analysis of the sequenced segments by the unweighted pairgroup method analysis (UPGMA) method showed that all specimens were properly assigned into four species with strong similarity, which indicated the possibility of separation congeneric species with the short 16S rRNA fragment. These results will be instrumental for implementation of the Chinese database of forensically relevant beetles.

  5. Fastidious Gram-Negatives: Identification by the Vitek 2 Neisseria-Haemophilus Card and by Partial 16S rRNA Gene Sequencing Analysis

    DEFF Research Database (Denmark)

    Wolff Sönksen, Ute; Christensen, Jens Jørgen; Nielsen, Lisbeth;

    2010-01-01

    Taxonomy and identification of fastidious Gram negatives are evolving and challenging. We compared identifications achieved with the Vitek 2 Neisseria-Haemophilus (NH) card and partial 16S rRNA gene sequence (526 bp stretch) analysis with identifications obtained with extensive phenotypic...... 16S rRNA gene sequence analysis results: For 76 strains phenotypic and sequencing identifications were identical, for 23 strains the sequencing identifications were either probable or possible, and for one strain only the genus was confirmed. Thus, the Vitek 2 NH system identifies most...... of the commonly occurring species included in the database. Some strains of rarely occurring species and strains of non-database species closely related to database species cause problems. Partial 16S rRNA gene sequence analysis performs well, but does not always suffice, additional phenotypical characterization...

  6. Combined analyses of the ITS loci and the corresponding 16S rRNA genes reveal high micro- and macrodiversity of SAR11 populations in the Red Sea.

    Directory of Open Access Journals (Sweden)

    David Kamanda Ngugi

    Full Text Available Bacteria belonging to the SAR11 clade are among the most abundant prokaryotes in the pelagic zone of the ocean. 16S rRNA gene-based analyses indicate that they constitute up to 60% of the bacterioplankton community in the surface waters of the Red Sea. This extremely oligotrophic water body is further characterized by an epipelagic zone, which has a temperature above 24 °C throughout the year, and a remarkable uniform temperature (~22 °C and salinity (~41 psu from the mixed layer (~200 m to the bottom at over 2000 m depth. Despite these conditions that set it apart from other marine environments, the microbiology of this ecosystem is still vastly understudied. Prompted by the limited phylogenetic resolution of the 16S rRNA gene, we extended our previous study by sequencing the internal transcribed spacer (ITS region of SAR11 in different depths of the Red Sea's water column together with the respective 16S fragment. The overall diversity captured by the ITS loci was ten times higher than that of the corresponding 16S rRNA genes. Moreover, species estimates based on the ITS showed a highly diverse population of SAR11 in the mixed layer that became diminished in deep isothermal waters, which was in contrast to results of the related 16S rRNA genes. While the 16S rRNA gene-based sequences clustered into three phylogenetic subgroups, the related ITS fragments fell into several phylotypes that showed clear depth-dependent shifts in relative abundances. Blast-based analyses not only documented the observed vertical partitioning and universal co-occurrence of specific phylotypes in five other distinct oceanic provinces, but also highlighted the influence of ecosystem-specific traits (e.g., temperature, nutrient availability, and concentration of dissolved oxygen on the population dynamics of this ubiquitous marine bacterium.

  7. Interaction of ribosomal proteins S5, S6, S11, S12, S18 and S21 with 16 S rRNA.

    Science.gov (United States)

    Stern, S; Powers, T; Changchien, L M; Noller, H F

    1988-06-20

    We have examined the effects of assembly of ribosomal proteins S5, S6, S11, S12, S18 and S21 on the reactivities of residues in 16 S rRNA towards chemical probes. The results show that S6, S18 and S11 interact with the 690-720 and 790 loop regions of 16 S rRNA in a highly co-operative manner, that is consistent with the previously defined assembly map relationships among these proteins. The results also indicate that these proteins, one of which (S18) has previously been implicated as a component of the ribosomal P-site, interact with residues near some of the recently defined P-site (class II tRNA protection) nucleotides in 16 S rRNA. In addition, assembly of protein S12 has been found to result in the protection of residues in both the 530 stem/loop and the 900 stem regions; the latter group is closely juxtaposed to a segment of 16 S rRNA recently shown to be protected from chemical probes by streptomycin. Interestingly, both S5 and S12 appear to protect, to differing degrees, a well-defined set of residues in the 900 stem/loop and 5'-terminal regions. These observations are discussed in terms of the effects of S5 and S12 on streptomycin binding, and in terms of the class III tRNA protection found in the 900 stem of 16 S rRNA. Altogether these results show that many of the small subunit proteins, which have previously been shown to be functionally important, appear to be associated with functionally implicated segments of 16 S rRNA.

  8. 16S rRNA gene sequencing is a non-culture method of defining the specific bacterial etiology of ventilator-associated pneumonia.

    Science.gov (United States)

    Xia, Li-Ping; Bian, Long-Yan; Xu, Min; Liu, Ying; Tang, Ai-Ling; Ye, Wen-Qin

    2015-01-01

    Ventilator-associated pneumonia (VAP) is an acquired respiratory tract infection following tracheal intubation. The most common hospital-acquired infection among patients with acute respiratory failure, VAP is associated with a mortality rate of 20-30%. The standard bacterial culture method for identifying the etiology of VAP is not specific, timely, or accurate in identifying the bacterial pathogens. This study used 16S rRNA gene metagenomic sequencing to identify and quantify the pathogenic bacteria in lower respiratory tract and oropharyngeal samples of 55 VAP patients. Sequencing of the 16S rRNA gene has served as a valuable tool in bacterial identification, particularly when other biochemical, molecular, or phenotypic identification techniques fail. In this study, 16S rRNA gene sequencing was performed in parallel with the standard bacterial culture method to identify and quantify bacteria present in the collected patient samples. Sequence analysis showed the colonization of multidrug-resistant strains in VAP secretions. Further, this method identified Prevotella, Proteus, Aquabacter, and Sphingomonas bacterial genera that were not detected by the standard bacterial culture method. Seven categories of bacteria, Streptococcus, Neisseria, Corynebacterium, Acinetobacter, Staphylococcus, Pseudomonas and Klebsiella, were detectable by both 16S rRNA gene sequencing and standard bacterial culture methods. Further, 16S rRNA gene sequencing had a significantly higher sensitivity in detecting Streptococcus and Pseudomonas when compared to standard bacterial culture. Together, these data present 16S rRNA gene sequencing as a novel VAP diagnosis tool that will further enable pathogen-specific treatment of VAP.

  9. Combined analyses of the ITS loci and the corresponding 16S rRNA genes reveal high micro- and macrodiversity of SAR11 populations in the Red Sea.

    KAUST Repository

    Ngugi, David Kamanda

    2012-11-20

    Bacteria belonging to the SAR11 clade are among the most abundant prokaryotes in the pelagic zone of the ocean. 16S rRNA gene-based analyses indicate that they constitute up to 60% of the bacterioplankton community in the surface waters of the Red Sea. This extremely oligotrophic water body is further characterized by an epipelagic zone, which has a temperature above 24 °C throughout the year, and a remarkable uniform temperature (~22 °C) and salinity (~41 psu) from the mixed layer (~200 m) to the bottom at over 2000 m depth. Despite these conditions that set it apart from other marine environments, the microbiology of this ecosystem is still vastly understudied. Prompted by the limited phylogenetic resolution of the 16S rRNA gene, we extended our previous study by sequencing the internal transcribed spacer (ITS) region of SAR11 in different depths of the Red Sea\\'s water column together with the respective 16S fragment. The overall diversity captured by the ITS loci was ten times higher than that of the corresponding 16S rRNA genes. Moreover, species estimates based on the ITS showed a highly diverse population of SAR11 in the mixed layer that became diminished in deep isothermal waters, which was in contrast to results of the related 16S rRNA genes. While the 16S rRNA gene-based sequences clustered into three phylogenetic subgroups, the related ITS fragments fell into several phylotypes that showed clear depth-dependent shifts in relative abundances. Blast-based analyses not only documented the observed vertical partitioning and universal co-occurrence of specific phylotypes in five other distinct oceanic provinces, but also highlighted the influence of ecosystem-specific traits (e.g., temperature, nutrient availability, and concentration of dissolved oxygen) on the population dynamics of this ubiquitous marine bacterium.

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

    Science.gov (United States)

    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.

  11. Composition of fecal microbiota of laboratory mice derived from Japanese commercial breeders using 16S rRNA gene clone libraries

    Science.gov (United States)

    NOZU, Ryoko; UENO, Masami; HAYASHIMOTO, Nobuhito

    2016-01-01

    The fecal microbiota of six mice derived from three Japanese commercial breeders was analyzed by using 16S rRNA gene clone libraries to construct a database for analyzing the gut microbiota of laboratory mice. The 566 clones were obtained from the clone libraries generated from the fecal DNA samples derived from BALB/c, C57BL/6N, DBA/2 and ICR mice. Among these 566 clones, there were 446 unique 16S rRNA gene sequences. When grouped at the 98% similarity level, the 446 unique sequences consisted of 103 Clostridiales, 43 Bacteroidales, 5 Lactobacillus and 3 Erysipelotricaceae, as well as sequences from 11 other phyla. PMID:26902692

  12. Diversity, Dynamics and Activity of Bacterial Communities during Production of an Artisanal Sicilian Cheese as Evaluated by 16S rRNA Analysis

    OpenAIRE

    Randazzo, C.L.; Torriani, S.; Akkermans, A.D.L.; Vos, de, R.; Vaughan, E E

    2002-01-01

    The diversity and dynamics of the microbial communities during the manufacturing of Ragusano cheese, an artisanal cheese produced in Sicily (Italy), were investigated by a combination of classical and culture-independent approaches. The latter included PCR, reverse transcriptase-PCR (RT-PCR), and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes (rDNA). Bacterial and Lactobacillus group-specific primers were used to amplify the V6 to V8 and V1 to V3 regions of the 16S rRNA gene...

  13. Thermoelectric behaviour of SmS and Smsub(0.84)Gdsub(0.16)S at high pressures and temperatures

    International Nuclear Information System (INIS)

    The 4f → 5d electronic phase transition in SmS has been studied using thermoelectric power as a probe. The variation of thermopower with pressure in Smsub(0.84)Gdsub(0.16)S and in the high pressure phase of SmS is anomalous, characterized by a rather large pressure coefficient. The temperature coefficient of thermopower in Smsub(0.84)Gdsub(0.16)S is large and negative at low pressures leading to a change of sign at higher temperatures. The anomalies can be understood on the basis of the ICF model. (author)

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

    OpenAIRE

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

    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 pollu...

  15. Interconversion of active and inactive 30 S ribosomal subunits is accompanied by a conformational change in the decoding region of 16 S rRNA

    DEFF Research Database (Denmark)

    Moazed, D; Van Stolk, B J; Douthwaite, S;

    1986-01-01

    Zamir, Elson and their co-workers have shown that 30 S ribosomal subunits are reversibly inactivated by depletion of monovalent or divalent cations. We have re-investigated the conformation of 16 S rRNA in the active and inactive forms of the 30 S subunit, using a strategy that is designed......' regions of 16 S rRNA. The inactive form also shows significantly decreased reactivity at positions 1533 to 1538 (the Shine-Dalgarno region), in agreement with earlier findings. The principal changes in reactivity involve the universally conserved nucleotides G926, C1395, A1398 and G1401. The three purines...

  16. Estimation of Pig Fecal Contamination in a River Catchment by Real-Time PCR Using Two Pig-Specific Bacteroidales 16S rRNA Genetic Markers▿

    OpenAIRE

    Mieszkin, Sophie; Furet, Jean-Pierre; Corthier, Gérard; Gourmelon, Michèle

    2009-01-01

    The microbiological quality of coastal or river water can be affected by fecal contamination from human or animal sources. To discriminate pig fecal pollution from other pollution, a library-independent microbial source tracking method targeting Bacteroidales host-specific 16S rRNA gene markers by real-time PCR was designed. Two pig-specific Bacteroidales markers (Pig-1-Bac and Pig-2-Bac) were designed using 16S rRNA gene Bacteroidales clone libraries from pig feces and slurry. For these two ...

  17. Diversity of Free-Living and Attached Bacteria in Offshore Western Mediterranean Waters as Depicted by Analysis of Genes Encoding 16S rRNA

    OpenAIRE

    Acinas, Silvia G; Antón, Josefa; Rodríguez-Valera, Francisco

    1999-01-01

    In a previous study (S. G. Acinas, F. Rodríguez-Valera, and C. Pedrós-Alió, FEMS Microbiol. Ecol. 24:27–40, 1997), community fingerprinting by 16S rDNA restriction analysis applied to Mediterranean offshore waters showed that the free-living pelagic bacterial community was very different from the bacterial cells aggregated or attached to particles of more than about 8 μm. Here we have studied both assemblages at three depths (5, 50, and 400 m) by cloning and sequencing the 16S rDNA obtained f...

  18. Comparative Analysis of 16S rRNA and amoA Genes from Archaea Selected with Organic and Inorganic Amendments in Enrichment Culture

    OpenAIRE

    Xu, Mouzhong; Schnorr, Jon; Keibler, Brandon; Holly M Simon

    2012-01-01

    We took advantage of a plant-root enrichment culture system to characterize mesophilic soil archaea selected through the use of organic and inorganic amendments. Comparative analysis of 16S rRNA and amoA genes indicated that specific archaeal clades were selected under different conditions. Three amoA sequence clades were identified, while for a fourth group, identified by 16S rRNA gene analysis alone and referred to as the “root” clade, we detected no corresponding amoA gene. The amoA-contai...

  19. Quantification of Hyphomicrobium Populations in Activated Sludge from an Industrial Wastewater Treatment System as Determined by 16S rRNA Analysis

    OpenAIRE

    Layton, A C; Karanth, P. N.; Lajoie, C. A.; Meyers, A J; Gregory, I. R.; Stapleton, R. D.; Taylor, D E; Sayler, G. S.

    2000-01-01

    The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homolog...

  20. Factores de riesgo que influyen en la recaída de consumo de drogas lícitas e ilícitas en adolescentes atendidos en el Instituto sobre Alcoholismo y Farmacodependencia

    Directory of Open Access Journals (Sweden)

    Tatiana Blanco-Álvarez

    2015-12-01

    Full Text Available El objetivo de la presente investigación fue determinar los factores de riesgo asociados con las recaídas en el consumo de drogas en adolescentes que han recibido tratamiento en el IAFA. Metodología: Estudio cuantitativo, con alcance correlacional y transversal. Instrumentos: Entrevista de Recaídas para Usuarios de Conductas Adictivas, Inventario de Situaciones Precipitantes de Recaídas, Escala de Control Atencional, Inventario de Habilidades de Afrontamiento, Escala de Depresión, Ansiedad y Estrés, Escala Multidimensional de Apoyo Social Recibido, Escala de Dificultades en la Regulación Emocional, y Cuestionario Breve de Confianza Situacional. Muestra: 107 adolescentes que reciben atención ambulatoria en el Centro de Menores del Instituto de Alcoholismo y Farmacodependencia. Conclusiones: las variables intrapersonales como regulación emocional (conductas dirigidas a metas, control atencional (atención focalizada, estados emocionales negativos y búsqueda de sensaciones positivas son factores de riesgo determinantes en las recaídas de consumo de drogas.

  1. Species-level core oral bacteriome identified by 16S rRNA pyrosequencing in a healthy young Arab population

    Directory of Open Access Journals (Sweden)

    Nezar Noor Al-hebshi

    2016-05-01

    Full Text Available Background: Reports on the composition of oral bacteriome in Arabs are lacking. In addition, the majority of previous studies on other ethnic groups have been limited by low-resolution taxonomic assignment of next-generation sequencing reads. Furthermore, there has been a conflict about the existence of a ‘core’ bacteriome. Objective: The objective of this study was to characterize the healthy core oral bacteriome in a young Arab population at the species level. Methods: Oral rinse DNA samples obtained from 12 stringently selected healthy young subjects of Arab origin were pyrosequenced (454's FLX chemistry for the bacterial 16S V1–V3 hypervariable region at an average depth of 11,500 reads. High-quality, non-chimeric reads ≥380 bp were classified to the species level using the recently described, prioritized, multistage assignment algorithm. A core bacteriome was defined as taxa present in at least 11 samples. The Chao2, abundance-based coverage estimator (ACE, and Shannon indices were computed to assess species richness and diversity. Results: Overall, 557 species-level taxa (211±42 per subject were identified, representing 122 genera and 13 phyla. The core bacteriome comprised 55 species-level taxa belonging to 30 genera and 7 phyla, namely Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, Fusobacteria, Saccharibacteria, and SR1. The core species constituted between 67 and 87% of the individual bacteriomes. However, the abundances differed by up to three orders of magnitude among the study subjects. On average, Streptococcus mitis, Rothia mucilaginosa, Haemophilus parainfluenzae, Neisseria flavescence/subflava group, Prevotella melaninogenica, and Veillonella parvula group were the most abundant. Streptococcus sp. C300, a taxon never reported in the oral cavity, was identified as a core species. Species richness was estimated at 586 (Chao2 and 614 (ACE species, whereas diversity (Shannon index averaged at 3.99. Conclusions

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

    Science.gov (United States)

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

    2006-12-01

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

  3. RecFOR proteins target RecA protein to a DNA gap with either DNA or RNA at the 5' terminus: implication for repair of stalled replication forks.

    Science.gov (United States)

    Morimatsu, Katsumi; Wu, Yun; Kowalczykowski, Stephen C

    2012-10-12

    The repair of single-stranded gaps in duplex DNA by homologous recombination requires the proteins of the RecF pathway. The assembly of RecA protein onto gapped DNA (gDNA) that is complexed with the single-stranded DNA-binding protein is accelerated by the RecF, RecO, and RecR (RecFOR) proteins. Here, we show the RecFOR proteins specifically target RecA protein to gDNA even in the presence of a thousand-fold excess of single-stranded DNA (ssDNA). The binding constant of RecF protein, in the presence of the RecOR proteins, to the junction of ssDNA and dsDNA within a gap is 1-2 nm, suggesting that a few RecF molecules in the cell are sufficient to recognize gDNA. We also found that the nucleation of a RecA filament on gDNA in the presence of the RecFOR proteins occurs at a faster rate than filament elongation, resulting in a RecA nucleoprotein filament on ssDNA for 1000-2000 nucleotides downstream (5' → 3') of the junction with duplex DNA. Thus, RecA loading by RecFOR is localized to a region close to a junction. RecFOR proteins also recognize RNA at the 5'-end of an RNA-DNA junction within an ssDNA gap, which is compatible with their role in the repair of lagging strand gaps at stalled replication forks. PMID:22902627

  4. DETECTION OF EHRLICHIA SP. ISOLATED FROM THE TICK BOOPHILUS MICROPLUS OF TIBET BY SEQUENCE ANALYSIS OF 16S rRNA GENES%用16S rRNA基因序列分析从西藏的微小牛蜱中检出的埃立克体

    Institute of Scientific and Technical Information of China (English)

    温博海; 蹇锐; 张有植

    2003-01-01

    用埃立克体属特异性套式PCR和DNA序列测定从西藏微小牛蜱检出边缘无形体和埃立克体;用半套式PCR扩得该埃立克体的16S rRNA基因,并测定了它的序列;将该埃立克体的16S rRNA基因序列与其它埃立克体的16S rRNA基因序列进行对比分析,结果证明它的16S rRNA基因与埃立克体属的犬埃立克体群16S rRNA基因相似水平最高(97%~98%);用16S rRNA基因序列做系统发育分析,结果显示该采自西藏的微小牛蜱的埃立克体可能是与查菲埃立克体密切相关的一种新埃立克体.

  5. 应用16S rRNA基因序列分析快速鉴定临床标本中的革兰氏阳性杆菌%Quick identification of Gram-positive bacilli in clinical specimens using 16S rRNA gene sequence analysis

    Institute of Scientific and Technical Information of China (English)

    姚纲; 胡红焱; 梁慧; 张健鹏

    2014-01-01

    目的 探索一种快速鉴定临床标本中革兰氏阳性杆菌的方法.方法 利用PCR技术扩增待检菌株的16S rRNA基因序列,通过分析待检菌株的16 S rRNA基因序列对其进行鉴定.结果 5株待检菌株的16S rRNA基因序列均成功扩增,其中4株的16S rRNA基因序列与基因库中已注册的核酸序列相似率达99.9%以上,将其鉴定到种的水平,1株的16S rRNA基因序列与基因库中雷弗森菌属的核酸序列相似率为97.09%,将其鉴定为雷弗森菌属.结论 应用16S rRNA基因序列分析可快速、准确地鉴定临床标本中的革兰氏阳性杆菌.

  6. Fastidious Gram-Negatives: Identification by the Vitek 2 Neisseria-Haemophilus Card and by Partial 16S rRNA Gene Sequencing Analysis

    DEFF Research Database (Denmark)

    Wolff Sönksen, Ute; Christensen, Jens Jørgen; Nielsen, Lisbeth;

    2010-01-01

    16S rRNA gene sequence analysis results: For 76 strains phenotypic and sequencing identifications were identical, for 23 strains the sequencing identifications were either probable or possible, and for one strain only the genus was confirmed. Thus, the Vitek 2 NH system identifies most...

  7. Escherichia coli cafA gene encodes a novel RNase, designated as RNase G, involved in processing of the 5' end of 16S rRNA.

    Science.gov (United States)

    Wachi, M; Umitsuki, G; Shimizu, M; Takada, A; Nagai, K

    1999-06-01

    We found that the Escherichia coli cafA::cat mutant accumulated a precursor of 16S rRNA. This precursor migrated to the same position with 16.3S precursor found in the BUMMER strain that is known to be deficient in the 5' end processing of 16S rRNA. Accumulation of 16. 3S rRNA in the BUMMER mutant was complemented by introduction of a plasmid carrying the cafA gene. The mutant type cafA gene cloned from the BUMMER strain had a 11-bp deletion in its coding region. A small amount of the mature 16S rRNA was still formed in the cafA::cat mutant. This residual activity was found to be due to RNase E encoded by the rne/ams gene by rifampicin-chase experiments of the cafA::cat ams1 double mutant. These results indicated that the cafA gene encodes a novel RNase responsible for processing of the 5' end of 16S rRNA. PMID:10362534

  8. Identification of Novel RNA-Protein Contact in Complex of Ribosomal Protein S7 and 3'-Terminal Fragment of 16S rRNA in E. coli.

    Science.gov (United States)

    Golovin, A V; Khayrullina, G A; Kraal, B; Kopylov, Capital A Cyrillic М

    2012-10-01

    For prokaryotes in vitro, 16S rRNA and 20 ribosomal proteins are capable of hierarchical self- assembly yielding a 30S ribosomal subunit. The self-assembly is initiated by interactions between 16S rRNA and three key ribosomal proteins: S4, S8, and S7. These proteins also have a regulatory function in the translation of their polycistronic operons recognizing a specific region of mRNA. Therefore, studying the RNA-protein interactions within binary complexes is obligatory for understanding ribosome biogenesis. The non-conventional RNA-protein contact within the binary complex of recombinant ribosomal protein S7 and its 16S rRNA binding site (236 nucleotides) was identified. UV-induced RNA-protein cross-links revealed that S7 cross-links to nucleotide U1321 of 16S rRNA. The careful consideration of the published RNA- protein cross-links for protein S7 within the 30S subunit and their correlation with the X-ray data for the 30S subunit have been performed. The RNA - protein cross-link within the binary complex identified in this study is not the same as the previously found cross-links for a subunit both in a solution, and in acrystal. The structure of the binary RNA-protein complex formed at the initial steps of self-assembly of the small subunit appears to be rearranged during the formation of the final structure of the subunit.

  9. Identification of bacterial species associated with the sheep scab mite (Psoroptes ovis) by using amplified genes coding for 16S rRNA.

    Science.gov (United States)

    Hogg, J C; Lehane, M J

    1999-09-01

    This was the first molecular study of the bacterial flora of the sheep scab mite (Psoroptes ovis). A sequence analysis of genes coding for 16S rRNA revealed that Serratia marcescens and bacteria closely related to Staphylococcus intermedius or Staphylococcus chromogens and Alloiococcus otitidis were present. These bacteria were associated with skin lesions, dermatitis, and otitis media caused by P. ovis.

  10. Identification of Bacterial Species Associated with the Sheep Scab Mite (Psoroptes ovis) by Using Amplified Genes Coding for 16S rRNA

    OpenAIRE

    Hogg, J. C.; Lehane, M.J.

    1999-01-01

    This was the first molecular study of the bacterial flora of the sheep scab mite (Psoroptes ovis). A sequence analysis of genes coding for 16S rRNA revealed that Serratia marcescens and bacteria closely related to Staphylococcus intermedius or Staphylococcus chromogens and Alloiococcus otitidis were present. These bacteria were associated with skin lesions, dermatitis, and otitis media caused by P. ovis.

  11. Sequence and phylogenetic analyses of the chloroplast 16S rRNA, tufA, and rbcL genes from Bryopsis hypnoides

    Institute of Scientific and Technical Information of China (English)

    L(U) Fang; WANG Guangce

    2011-01-01

    Using shotgun sequencing data,the complete sequences of chloroplast 16S rRNA and tufA genes were acquired from native specimens of Bryopsis hypnoides (Qingdao,China).There are two group Ⅰ introns in the 16S rRNA gene,which is structurally similar to that of Caulerpa sertularioides (Bryopsidales,Chlorophyta).The chloroplast-encoded tufA gene sequence is 1230 bp long,very AT-rich (61.5%),and is similar to previously published 16S rRNA sequences of bryopsidinean algae.Phylogenetic analyses based on chloroplast 16S rRNA and tufA gene sequence data support previous hypotheses that the Bryopsidineae,Halimedineae,and Ostreobidineae are three distinct lineages.These results also confirmed the exclusion of Avrainvillea from the family Udoteaceae.Phylogenetic analyses inferred that the genus Bryopsis as sister to Derbesia; however,this clade lacked robust nodal support.Moreover,the phylogenetic tree inferred from rbcL GenBank sequences,combined with the geographical distributions of Bryopsis species,identified a strongly supportive clade for three differently distributed Asian Bryopsis species.The preliminary results suggesting that these organisms are of distinct regional endemism.

  12. Optimal eukaryotic 18S and universal 16S/18S ribosomal RNA primers and their application in a study of symbiosis.

    Science.gov (United States)

    Wang, Yong; Tian, Ren Mao; Gao, Zhao Ming; Bougouffa, Salim; Qian, Pei-Yuan

    2014-01-01

    Eukaryotic 18S ribosomal RNA (rRNA) gene primers that feature a wide coverage are critical in detecting the composition of eukaryotic microscopic organisms in ecosystems. Here, we predicted 18S rRNA primers based on consecutive conserved sites and evaluated their coverage efficiency and scope of application to different eukaryotic groups. After evaluation, eight of them were considered as qualified 18S primers based on coverage rate. Next, we examined common conserved regions in prokaryotic 16S and eukaryotic 18S rRNA sequences to design 16S/18S universal primers. Three 16S/18S candidate primers, U515, U1390 and U1492, were then considered to be suitable for simultaneous amplification of the rRNA sequences in three domains. Eukaryotic 18S and prokaryotic 16S rRNA genes in a sponge were amplified simultaneously using universal primers U515 and U1390, and the subsequent sorting of pyrosequenced reads revealed some distinctive communities in different parts of the sample. The real difference in biodiversity between prokaryotic and eukaryotic symbionts could be discerned as the dissimilarity between OTUs was increased from 0.005 to 0.1. A network of the communities in external and internal parts of the sponge illustrated the co-variation of some unique microbes in certain parts of the sponge, suggesting that the universal primers are useful in simultaneous detection of prokaryotic and eukaryotic microbial communities.

  13. Optimal eukaryotic 18S and universal 16S/18S ribosomal RNA primers and their application in a study of symbiosis.

    Directory of Open Access Journals (Sweden)

    Yong Wang

    Full Text Available Eukaryotic 18S ribosomal RNA (rRNA gene primers that feature a wide coverage are critical in detecting the composition of eukaryotic microscopic organisms in ecosystems. Here, we predicted 18S rRNA primers based on consecutive conserved sites and evaluated their coverage efficiency and scope of application to different eukaryotic groups. After evaluation, eight of them were considered as qualified 18S primers based on coverage rate. Next, we examined common conserved regions in prokaryotic 16S and eukaryotic 18S rRNA sequences to design 16S/18S universal primers. Three 16S/18S candidate primers, U515, U1390 and U1492, were then considered to be suitable for simultaneous amplification of the rRNA sequences in three domains. Eukaryotic 18S and prokaryotic 16S rRNA genes in a sponge were amplified simultaneously using universal primers U515 and U1390, and the subsequent sorting of pyrosequenced reads revealed some distinctive communities in different parts of the sample. The real difference in biodiversity between prokaryotic and eukaryotic symbionts could be discerned as the dissimilarity between OTUs was increased from 0.005 to 0.1. A network of the communities in external and internal parts of the sponge illustrated the co-variation of some unique microbes in certain parts of the sponge, suggesting that the universal primers are useful in simultaneous detection of prokaryotic and eukaryotic microbial communities.

  14. Seasonal dynamics of anammox bacteria in estuarial sediment of the Mai Po Nature Reserve revealed by analyzing the 16S rRNA and hydrazine oxidoreductase (hzo) genes.

    Science.gov (United States)

    Li, Meng; Cao, Huiluo; Hong, Yi-Guo; Gu, Ji-Dong

    2011-01-01

    The community and population dynamics of anammox bacteria in summer (wet) and winter (dry) seasons in estuarial mudflat sediment of the Mai Po Nature Reserve were investigated by 16S rRNA and hydrazine oxidoreductase (hzo) genes. 16S rRNA phylogenetic diversity showed that sequences related to 'Kuenenia' anammox bacteria were presented in summer but not winter while 'Scalindua' anammox bacteria occurred in both seasons and could be divided into six different clusters. Compared to the 16S rRNA genes, the hzo genes revealed a relatively uniform seasonal diversity, with sequences relating to 'Scalindua', 'Anammoxoglobus', and planctomycete KSU-1 found in both seasons. The seasonal specific bacterial groups and diversity based on the 16S rRNA and hzo genes indicated strong seasonal community structures in estuary sediment of this site. Furthermore, the higher abundance of hzo genes in summer than winter indicates clear seasonal population dynamics. Combining the physicochemical characteristics of estuary sediment in the two seasons and their correlations with anammox bacteria community structure, we proposed the strong seasonal dynamics in estuary sediment of Mai Po to be due to the anthropogenic and terrestrial inputs, especially in summer, which brings in freshwater anammox bacteria, such as 'Kuenenia', interacting with the coastal marine anammox bacteria 'Scalindua'.

  15. Identification of a novel, invasive, not-yet-cultivated Treponema sp in the large intestine of pigs by PCR amplification of the 16S rRNA gene

    DEFF Research Database (Denmark)

    Mølbak, Lars; Schou, Kirstine Klitgaard; Jensen, Tim Kåre;

    2006-01-01

    Laser capture microdissection in combination with fluorescent in situ hybridization was used to identify an unknown species of spirochetes from the pig colonic mucosa. The 16S rRNA gene was PCR amplified, and the closest related type strain was Treponema bryantii(T) (90.1%). The spirochete, here...

  16. Investigating the etiology of bovine digital dermatitis by a combination of 16S rRNA gene analysis and fluorescence in situ hybridization

    DEFF Research Database (Denmark)

    Schou, Kirstine Klitgaard; Rasmussen, Marianne; Capion, Nynne;

    of the disease. Here, a PCR-based approach targeting the 16S rRNA gene along with fluorescence in situ hybridization was used to determine the prevalence and diversity of 17 Treponema phylotypes in 85 digital dermatitis lesions from six Danish dairy herds as well as additional biopsies of healthy skin...

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

    Science.gov (United States)

    Atherly, Todd; Ziemer, Cherie J

    2014-04-01

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

  18. Expression of gyrB and 16S ribosomal RNA genes as indicators of growth and physiological activities of Legionella pneumophila.

    Science.gov (United States)

    Okuno, Toshihiro; Tani, Katsuji; Yamaguchi, Nobuyasu; Nasu, Masao

    2015-01-01

    To determine whether the DNA gyrase (gyrB) and 16S ribosomal RNA (16S rRNA) genes can be used as indicators of the biological activities of Legionella pneumophila, the expression levels were estimated. The ratio of mRNA/DNA in gyrB was 0.7 in mid log phase and decreased drastically after the log phase. For 16S rRNA, the ratio was highest in mid log phase (7.0×10(3)), and the value that was about 10% of that in the log phase was maintained for six days. The rRNA may be vital in the resting or active but nonculturable cells that are not growing but physiologically active. The expression levels of gyrB mRNA and 16S rRNA can be used as indicators of the growth activity and the physiological activity of L. pneumophila, respectively. Therefore, by measurement of these indicators, we can evaluate the activities of Legionella cells in various environments.

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

    Science.gov (United States)

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

    2015-08-25

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

  20. Endodontic bacteria from primary and persistent endodontic lesions in Chinese patients as identified by cloning and 16S ribosomal DNA gene sequencing

    Institute of Scientific and Technical Information of China (English)

    LI Xin; ZHU Xiao-fei; ZHANG Cheng-fei; Peter Cathro; CJ Seneviratne; SHEN Song

    2013-01-01

    Background Few literatures pertain to the 16S ribosomal DNA (16S rDNA) analysis of bacteria contributing to primary and persistent endodontic lesions,with no information available for the Chinese population.As such,we investigated endodontic bacteria associated with primary and persistent endodontic lesions in adult Chinese patients living in Beijing,China using 16S rDNA gene sequencing techniques.Methods Endodontic microbial samples were obtained from fourteen adult Chinese patients and subjected to DNA extraction.Pllymerase chain reaction (PCR) products were cloned and 100 clones from each generated library were randomly selected.Purified plasmid DNA with 16S rDNA gene inserts was sequenced,and the sequences were searched against GenBank databases using the BLASTN algorithm.Only significant identification with the highest-scored BLAST result and 99% minimum similarity was considered for phylotyping.Results More than 150 taxa were obtained.Primary endodontic infection was mainly associated with Burkholderia cepacia,Actinomyces,Aranicola spp.and Streptococcus sanguinis,whilst Burkholderia cepacia was predominant in the persistent endodontic infections.Conclusion There is a difference in the species profile associated with endodontic infections of Chinese patients living in Beiiing in comoarison to other geographical or ethnic reports.

  1. Differentiation of Shewanella putrefaciens and Shewanella alga on the basis of whole-cell protein profiles, ribotyping, phenotypic characterization, and 16S rRNA gene sequence analysis

    DEFF Research Database (Denmark)

    Vogel, Birte Fonnesbech; Jørgensen, K.; Christensen, H.;

    1997-01-01

    by 16S rRNA gene sequence analysis. It is concluded that the isolates must be considered two different species, S. alga and S. putrefaciens, and that most mesophilic isolates formerly identified as S. putrefaciens belong to S. alga. The ecological role and potential pathogenicity of S. alga can...

  2. True microbiota involved in chronic lung infection of cystic fibrosis patients found by culturing and 16S rRNA gene analysis

    DEFF Research Database (Denmark)

    Rudkjøbing, Vibeke Børsholt; Thomsen, Trine R; Alhede, Morten;

    2011-01-01

    Patients suffering from cystic fibrosis (CF) develop chronic lung infection. In this study, we investigated the microorganisms present in transplanted CF lungs (n = 5) by standard culturing and 16S rRNA gene analysis. A correspondence between culturing and the molecular methods was observed...

  3. Decoupled distance-decay patterns between dsrA and 16S rRNA genes among salt marsh sulfate-reducing bacteria.

    Science.gov (United States)

    Angermeyer, Angus; Crosby, Sarah C; Huber, Julie A

    2016-01-01

    In many habitats, microorganisms exhibit significant distance-decay patterns as determined by analysis of the 16S rRNA gene and various other genetic elements. However, there have been few studies that examine how the similarities of both taxonomic and functional genes co-vary over geographic distance within a group of ecologically related microbes. Here, we determined the biogeographic patterns of the functional dissimilatory sulfite reductase gene (dsrA) and the 16S rRNA gene in sulfate-reducing bacterial communities of US East Coast salt marsh sediments. Distance-decay, ordination and statistical analyses revealed that the distribution of 16S rRNA genes is strongly influenced by geographic distance and environmental factors, whereas the dsrA gene is not. Together, our results indicate that 16S rRNA genes are likely dispersal limited and under environmental selection, whereas dsrA genes appear randomly distributed and not selected for by any expected environmental variables. Selection, drift, dispersal and mutation are all factors that may help explain the decoupled biogeographic patterns for the two genes. These data suggest that both the taxonomic and functional elements of microbial communities should be considered in future studies of microbial biogeography to aid in our understanding of the diversity, distribution and function of microorganisms in the environment.

  4. [Identification of Hydrocarbon-Oxidizing Dietzia Bacteria from Petroleum Reservoirs Based on Phenotypic Properties and Analysis of the 16S rRNA and gyrB Genes].

    Science.gov (United States)

    Nazina, T N; Shumkova, E S; Sokolova, D Sh; Babich, T L; Zhurina, M V; Xue, Yan-Fen; Osipov, G A; Poltaraus, A B; Tourova, T P

    2015-01-01

    The taxonomic position of hydrocarbon-oxidizing bacterial strains 263 and 32d isolated from formation water of the Daqing petroleum reservoir (PRC) was determined by polyphasic taxonomy techniques, including analysis of the 16S rRNA and the gyrB genes. The major chemotaxonomic characteristics of both strains, including the IV type cell wall, composition of cell wall fatty acids, mycolic acids, and menaquinones, agreed with those typical of Dietzia strains. The DNA G+C content of strains 263 and 32d were 67.8 and 67.6 mol%, respectively. Phylogenetic analysis of the 16S rRNA gene of strain 32d revealed 99.7% similarity to the gene of D. maris, making it possible to identify strain 32d as belonging to this species. The 16S rRNA gene sequence of strain 263 exhibited 99.7 and 99.9% similarity to those of D. natronolimnaea and D. cercidiphylli YIM65002(T), respectively. Analysis of the gyrB genes of the subterranean isolates and of a number of Dietzia type strains confirmed classiffication of strain 32d as a D. maris strain and of strain 263, as a D. natronolimnaea strain. A conclusion was made concerning higher resolving power of phylogenetic analysis of the gyrB gene compared to the 16S rRNA gene analysis in the case of determination of the species position of Dietzia isolates.

  5. Coamplification of eukaryotic DNA with 16S rRNA gene-based PCR primers: possible consequences for population fingerprinting of complex microbial communities.

    Science.gov (United States)

    Huys, Geert; Vanhoutte, Tom; Joossens, Marie; Mahious, Amal S; De Brandt, Evie; Vermeire, Severine; Swings, Jean

    2008-06-01

    The main aim of this study was to evaluate the specificity of three commonly used 16S rRNA gene-based polymerase chain reaction (PCR) primer sets for bacterial community analysis of samples contaminated with eukaryotic DNA. The specificity of primer sets targeting the V3, V3-V5, and V6-V8 hypervariable regions of the 16S rRNA gene was investigated in silico and by community fingerprinting of human and fish intestinal samples. Both in silico and PCR-based analysis revealed cross-reactivity of the V3 and V3-V5 primers with the 18S rRNA gene of human and sturgeon. The consequences of this primer anomaly were illustrated by denaturing gradient gel electrophoresis (DGGE) profiling of microbial communities in human feces and mixed gut of Siberian sturgeon. DGGE profiling indicated that the cross-reactivity of 16S rRNA gene primers with nontarget eukaryotic DNA might lead to an overestimation of bacterial biodiversity. This study has confirmed previous sporadic indications in literature indicating that several commonly applied 16S rRNA gene primer sets lack specificity toward bacteria in the presence of eukaryotic DNA. The phenomenon of cross-reactivity is a potential source of systematic error in all biodiversity studies where no subsequent analysis of individual community amplicons by cloning and sequencing is performed.

  6. Self-organizing maps: A tool to ascertain taxonomic relatedness based on features derived from 16S rDNA sequence

    Indian Academy of Sciences (India)

    D V Raje; H J Purohit; Y P Badhe; S S Tambe; B D Kulkarni

    2010-12-01

    Exploitation of microbial wealth, of which almost 95% or more is still unexplored, is a growing need. The taxonomic placements of a new isolate based on phenotypic characteristics are now being supported by information preserved in the 16S rRNA gene. However, the analysis of 16S rDNA sequences retrieved from metagenome, by the available bioinformatics tools, is subject to limitations. In this study, the occurrences of nucleotide features in 16S rDNA sequences have been used to ascertain the taxonomic placement of organisms. The tetra- and penta-nucleotide features were extracted from the training data set of the 16S rDNA sequence, and was subjected to an artificial neural network (ANN) based tool known as self-organizing map (SOM), which helped in visualization of unsupervised classification. For selection of significant features, principal component analysis (PCA) or curvilinear component analysis (CCA) was applied. The SOM along with these techniques could discriminate the sample sequences with more than 90% accuracy, highlighting the relevance of features. To ascertain the confidence level in the developed classification approach, the test data set was specifically evaluated for Thiobacillus, with Acidiphilium, Paracocus and Starkeya, which are taxonomically reassigned. The evaluation proved the excellent generalization capability of the developed tool. The topology of genera in SOM supported the conventional chemo-biochemical classification reported in the Bergey manual.

  7. Characterization of microbial communities found in the human vagina by analysis of terminal restriction fragment length polymorphisms of 16S rRNA genes

    NARCIS (Netherlands)

    Coolen, MJL; Post, E; Davis, CC; Forney, LJ

    2005-01-01

    To define and monitor the structure of microbial communities found in the human vagina, a cultivation-independent approach based on analyses of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes was developed and validated. Sixteen bacterial strains commonly found in the h

  8. Phylogeny of some mycoplasmas from ruminants based on 16S rRNA sequences and definition of a new cluster within the hominis group.

    Science.gov (United States)

    Pettersson, B; Uhlén, M; Johansson, K E

    1996-10-01

    Almost complete (> 96%) 16S rRNA sequences from nine ruminant mycoplasmas have been determined by solid-phase DNA sequencing. Polymorphisms were found in four of the 16S rRNA sequences, which indicated the existence of two different rRNA operons. Seven polymorphisms were found in Mycoplasma agalatiae, three were found in Mycoplasma bovis, one was found in Mycoplasma alkalescens, and one was found in Mycoplasma bovirhinis. The sequence data were used for construction of phylogenetic trees. All but one of the ruminant mycoplasmas sequenced in this work clustered in the hominis group. A close relationship was found between M. agalactiae and M. bovis, with a 99% nucleotide similarity between their 16S rRNA sequences. They were also found to be members of the Mycoplasma lipophilum cluster of the hominis group. Furthermore, the 16S rRNA comparisons showed that Mycoplasma alkalescens and Mycoplasma canadense are closely related (> 98.5%), and these species were found to cluster in the Mycoplasma hominis cluster of the hominis group. Interestingly, M. bovirhinis grouped in a new phylogenetic cluster of the hominis group. The new cluster, which was supported by bootstrap percentage values, signature nucleotide analysis, and higher-order structural elements, was named the Mycoplasma synoviae cluster. Mycoplasma bovoculi, Mycoplasma conjunctivae, and Mycoplasma ovipneumoniae clustered in the Mycoplasma neurolyticum cluster of the hominis group. Mycoplasma alvi clustered with Mycoplasma pirum in the M. pneumoniae cluster of the pneumoniae group.

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

    Directory of Open Access Journals (Sweden)

    Sarah M. Boomer

    2009-12-01

    Full Text Available 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.

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

    Directory of Open Access Journals (Sweden)

    Bryan E. Dutton

    2002-12-01

    Full Text Available 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.

  11. 16S ribosomal RNA-based methods to monitor changes in the hindgut bacterial community of piglets after oral administration of Lactobacillus sobrius S1

    NARCIS (Netherlands)

    Su, Y.; Yao, W.; Perez-Gutierrez, O.N.; Smidt, H.; Zhu, W.Y.

    2008-01-01

    16S ribosomal RNA (rRNA) gene based PCR/denaturing gradient gel electrophoresis (DGGE) and real-time PCR were used to monitor the changes in the composition of microbiota in the hindgut of piglets after oral administration of Lactobacillus sobrius S1. Six litters of neonatal piglets were divided ran

  12. Bacterial diversity in a soil sample from Uranium mining waste pile as estimated via a culture-independent 16S rDNA approach

    International Nuclear Information System (INIS)

    Bacterial diversity was studied in a soil sample collected from a uranium mining waste pile situated near the town of Johanngeorgenstadt, Germany. As estimated by ICP-MS analysis the studied sample was highly contaminated with Fe, Al, Mn, Zn, As, Pb and U. The 16S rDNA retrieval, applied in this study, demonstrated that more than the half of the clones of the constructed 16S rDNA library were represented by individual RFLP profiles. This indicates that the composition of the bacterial community in the sample was very complex. However, several 16S rDNA RFLP groups were found to be predominant and they were subjected to a sequence analysis. The most predominant group, which represented about 13% of the clones of the 16S rDNA library, was affiliated with the Holophaga/Acidobacterium phylum. Significant was also the number of the proteobacterial sequences which were distributed in one predominant α-proteobacterial cluster representing 11% of the total number of clones and in two equal-sized β- and γ-proteobacterial clusters representing each 6% of the clones. Two smaller groups representing both 2% of the clones were affiliated with Nitrospira and with the novel division WS3. Three of the analysed sequences were evaluated as a novel, not yet described lineage and one as a putative chimera. (authors)

  13. Identification of the RsmG methyltransferase target as 16S rRNA nucleotide G527 and characterization of Bacillus subtilis rsmG mutants

    DEFF Research Database (Denmark)

    Nishimura, Kenji; Johansen, Shanna K; Inaoka, Takashi;

    2007-01-01

    The methyltransferase RsmG methylates the N7 position of nucleotide G535 in 16S rRNA of Bacillus subtilis (corresponding to G527 in Escherichia coli). Disruption of rsmG resulted in low-level resistance to streptomycin. A growth competition assay revealed that there are no differences in fitness...

  14. High Prevalence of Metallo-β-Lactamase and 16S rRNA Methylase Coproduction among Imipenem-Resistant Pseudomonas aeruginosa Isolates in Brazil▿

    OpenAIRE

    Doi, Yohei; Ghilardi, Angela C. R.; Adams, Jennifer; de Oliveira Garcia, Doroti; Paterson, David L.

    2007-01-01

    Rates of metallo-β-lactamase and 16S rRNA methylase production were investigated in 51 imipenem-resistant Pseudomonas aeruginosa clinical isolates collected from hospitals in São Paulo, Brazil. Of them, 57% and 75% produced SPM-1 and RmtD, respectively. Of note, 51% produced both enzymes, suggesting that their coproduction is already common in this geographic area.

  15. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development - Poster

    Science.gov (United States)

    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 ...

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

    Science.gov (United States)

    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 ...

  17. Investigation of Microbial Diversity in Geothermal Hot Springs in Unkeshwar, India, Based on 16S rRNA Amplicon Metagenome Sequencing.

    Science.gov (United States)

    Mehetre, Gajanan T; Paranjpe, Aditi; Dastager, Syed G; Dharne, Mahesh S

    2016-02-25

    Microbial diversity in geothermal waters of the Unkeshwar hot springs in Maharashtra, India, was studied using 16S rRNA amplicon metagenomic sequencing. Taxonomic analysis revealed the presence of Bacteroidetes, Proteobacteria, Cyanobacteria, Actinobacteria, Archeae, and OD1 phyla. Metabolic function prediction analysis indicated a battery of biological information systems indicating rich and novel microbial diversity, with potential biotechnological applications in this niche.

  18. Quantitative Selective PCR of 16S Ribosomal DNA Correlates Well with Selective Agar Plating in Describing Population Dynamics of Indigenous Pseudomonas spp. in Soil Hot Spots

    OpenAIRE

    Johnsen, Kaare; Enger, Øivind; Jacobsen, Carsten S.; Thirup, Laila; Torsvik, Vigdis

    1999-01-01

    We used a quantitative PCR method targeting 16S ribosomal DNA using competitive PCR for specific detection of indigenous Pseudomonas DNA in soil hot spots. The amount of Pseudomonas DNA corresponded to the number of culturable Pseudomonas bacteria on Gould’s S1 agar. This represents the first use of PCR for quantification of indigenous bacteria in more than one sample of soil.

  19. Comparison of Gull Feces-specific Assays Targeting the 16S rRNA Gene of Catellicoccus Marimammalium and Streptococcus spp.

    Science.gov (United States)

    Two novel gull-specific qPCR assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR-green-based assay targeting Streptococcus spp. (i.e., gull3) and a TaqMan qPCR assay targeting Catellicoccus marimammalium (i.e., gull4). The main objectives ...

  20. Prevalence of 16S rRNA methylase, modifying enzyme, and extended-spectrum beta-lactamase genes among Acinetobacter baumannii isolates.

    Science.gov (United States)

    Liu, Zhenru; Ling, Baodong; Zhou, Liming

    2015-08-01

    Multidrug-resistant Acinetobacter baumannii has become a worldwide problem, and methylation of 16S rRNA has recently emerged as a new mechanism of resistance to aminoglycosides, which is mediated by a newly recognized group of 16S rRNA methylases. 16S rRNA methylase confers a high-level resistance to all 4,6-substituted deoxystreptamine aminoglycosides that are currently used in clinical practice. Some of the A. baumannii isolates have been found to coproduce extended-spectrum beta-lactamases (ESBLs), contributing to their multidrug resistance. The aim of this study was to detect the determinants of the 16S rRNA methylase genes armA, rmtA, rmtB, rmtC, rmtD, rmtE, and npmA, the modifying enzyme genes aac(6')-Ib, ant(3″)-Ia, aph(3')-I, and the extended-spectrum beta-lactamase genes bla(TEM), bla(SHV), and bla(CTX-M-3) among A. baumannii isolates in northeastern Sichuan, China. Minimum inhibitory concentrations (MICs) of 21 different antimicrobial agents against the A. baumannii isolates were determined. The clinical isolates showed a high level of resistance (MIC≧256 μg/ml) to aminoglycosides, which ranged from 50·1 to 83·8%. The resistances to meropenem and imipenem, two of the beta-lactam antibiotics and the most active antibiotics against A. baumannii, were 9·1 and 8·2%, respectively. Among 60 amikacin-resistant isolates, only the 16S rRNA methylase gene armA was found to be prevalent (66·7%), but the other 16S rRNA methylase genes rmtA, rmtB, rmtC, rmtD, rmtE, and npmA were not detected. The prevalences of the modifying enzyme genes aac (6')-Ib, ant (3″)-Ia, and aph (3')-I were 51·7, 81·7, and 58·3%, respectively, which are different from a previous study in which the occurrences of these genes were 3, 64, and 72%, respectively. Among the 40 isolates that were armA-positive, the prevalences of bla(TEM), bla(SHV), and bla(CTX-M-3) genes were detected for the first time in China, and their occurrences were 45, 65, and 52·5%, respectively. In all, A

  1. Rapid identification of Nesseria Meningitidis by PCR 16S rRNA gene polymerase chain reaction%建立16S rRNA基因聚合酶链反应方法快速检测脑膜炎奈瑟菌

    Institute of Scientific and Technical Information of China (English)

    牛桓彩; 田国忠

    2011-01-01

    Objective To evaluate the efficiency of 16S rRNA gene polymerase chain reaction (16S rRNA PCR) to detect Nesseria Meningitidis (N. meningitidis) in various clinical/non-clinical samples. Methods The primers-specific were designed to amplify 16S rRNA genes of N. meningitides by PCR assay according to the analysis of conserved and variable regions in bacterial 16S rRNA genes Nesseria. And compared it to crgA-PCR assay for identification of N. meningitides from the literature. Results The positive predictive value of 16S rRNA PCR and crgA-PCR assays for detecting N. meningitides both was 100% (68/68). The negative predictive value of 16S rRNA PCR and crgA-PCR assays for detecting N. meningitides was 100% (49/49) and 46.94% (23/49) respectively. Youden Index of 16S rRNA PCR and crgA-PCR assays for detecting N. meningitides was 1.00 and 0. 47 respectively. The detection rate of N. rneningitides in 188 pharyngeal swab specimens which were collected from health children was investigated by crgA-PCR and 16S rRNA PCR assays. The detection rates of 16S rRNA PCR and crgA-PCR assays was 18. 62% (35/188) and 15.96% (30/188)respectively. The consistency of two PCR assays was 7.98% (15/188). Conclusion The 16S rRNA PCR assay could be considered as a rapid, reliable and feasible method for detecting N. meningitides in pure strains and pharyngeal swab specimens than that of crgA-PCR assay.%目的 建立以16S rRNA基因为靶基因的聚合酶链反应(PCR)方法.用于快速检测脑膜炎奈瑟菌.方法 根据GenBank公布的奈瑟菌属16S rRNA基因序列,使用DNAstar软件设计引物,应用PCR方法特异检测脑膜炎奈瑟菌,对部分菌株结合16S rRNA基因测序和APIRNH生化鉴定系统以验证该方法的准确性,同时与文献报道的检测脑膜炎奈瑟菌crgA-PCR方法进行比较.结果 16S rRNA-PCR与crgA-PCR方法检测脑膜炎奈瑟菌阳性预测值皆为100%,阴性预测值分别为100%和46.94%,假阳性率分别为0%和36.73%,

  2. Identification and characterization of Salmonella typhi isolates from Southwest Sumba District, East Nusa Tenggara based on 16S rRNA gene sequences

    Directory of Open Access Journals (Sweden)

    CHARIS AMARANTINI

    2011-01-01

    Full Text Available Amarantini C, Sembiring L, Kushadiwijaya H, Asmara W (2011 Identification and characterization of Salmonella typhi isolates from Southwest Sumba District, East Nusa Tenggara based on 16S rRNA gene sequences. Biodiversitas 12: 1-6. The incidence rate of typhoid fever in the Southwest Sumba District, East Nusa Tenggara was approximately about 725/100,000. In spite of such rate, there was not much known-yet about the molecular epidemiology of the disease. Thus, having accurate data and a strong discriminatory ability was crucial to scrutinize the molecular epidemiology of S. typhi with a molecular phylogenetic approach based on 16S rRNA gene sequences. Sixteen isolates representative of S. typhi from different geographical regions in Southwest Sumba District along with the reference strain S. typhi NCTC 786 had been identified and characterised based on 16S rRNA gene sequences using PCR amplification and sequencing. The 16S rRNA sequences data were aligned with the corresponding available S. typhi sequences retrieved from the NCBI database by using CLUSTAL X software. Phylogenetic trees were generated with PHYLIP software package. Molecular phylogenetic analysis indicated that all the isolates belong to S. typhi species were suggested by their relativity with the type strain of S. typhi ATCC19430T. It was also found that the isolates which belong to S. typhi species formed several different centers of diversity within the 16S rRNA gene tree. Each clade consisted of the strains from different geographical places in the District. Thus, to conclude the inquiry, there was evident inter-geographical spread of the strains and it tended to spread further into more remote areas in the District.

  3. Community analysis of chronic wound bacteria using 16S rRNA gene-based pyrosequencing: impact of diabetes and antibiotics on chronic wound microbiota.

    Directory of Open Access Journals (Sweden)

    Lance B Price

    Full Text Available BACKGROUND: Bacterial colonization is hypothesized to play a pathogenic role in the non-healing state of chronic wounds. We characterized wound bacteria from a cohort of chronic wound patients using a 16S rRNA gene-based pyrosequencing approach and assessed the impact of diabetes and antibiotics on chronic wound microbiota. METHODOLOGY/PRINCIPAL FINDINGS: We prospectively enrolled 24 patients at a referral wound center in Baltimore, MD; sampled patients' wounds by curette; cultured samples under aerobic and anaerobic conditions; and pyrosequenced the 16S rRNA V3 hypervariable region. The 16S rRNA gene-based analyses revealed an average of 10 different bacterial families in wounds--approximately 4 times more than estimated by culture-based analyses. Fastidious anaerobic bacteria belonging to the Clostridiales family XI were among the most prevalent bacteria identified exclusively by 16S rRNA gene-based analyses. Community-scale analyses showed that wound microbiota from antibiotic treated patients were significantly different from untreated patients (p = 0.007 and were characterized by increased Pseudomonadaceae abundance. These analyses also revealed that antibiotic use was associated with decreased Streptococcaceae among diabetics and that Streptococcaceae was more abundant among diabetics as compared to non-diabetics. CONCLUSIONS/SIGNIFICANCE: The 16S rRNA gene-based analyses revealed complex bacterial communities including anaerobic bacteria that may play causative roles in the non-healing state of some chronic wounds. Our data suggest that antimicrobial therapy alters community structure--reducing some bacteria while selecting for others.

  4. First report on the bacterial diversity in the distal gut of dholes (Cuon alpinus) by using 16S rRNA gene sequences analysis.

    Science.gov (United States)

    Chen, Lei; Zhang, Honghai; Liu, Guangshuai; Sha, Weilai

    2016-05-01

    The aim of this study was to investigate the bacterial community in the distal gut of dholes (Cuon alpinus) based on the analysis of bacterial 16S rRNA gene sequences. Fecal samples were collected from five healthy unrelated dholes captured from Qilian Mountain in Gansu province of China. The diversity of the fecal bacteria community was investigated by constructing a polymerase chain reaction (PCR)-amplified 16S rRNA gene clone library. Bacterial 16S rRNA gene was amplified by using universal bacterial primers 27F and 1492R. A total of 275 chimera-free near full length 16S rRNA gene sequences were collected, and 78 non-redundant bacteria phylotypes (operational taxonomical units, OTUs) were identified according to the 97 % sequence similarity. Forty-two OTUs (53.8 %) showed less than 98 % sequence similarity to 16S rRNA gene sequences reported previously. Phylogenetic analysis demonstrated that dhole bacterial community comprised five different phyla, with the majority of sequences being classified within the phylum Bacteroidetes (64.7 %), followed by Firmicutes (29.8 %), Fusobacteria (4.7 %),Proteobacteria (0.4 %), and Actinobacteria (0.4 %). The only order Bacteroidales in phylum Bacteroidetes was the most abundant bacterial group in the intestinal bacterial community of dholes. Firmicutes and Bacteroidetes were the two most diverse bacterial phyla with 46.2 and 44.9 % of OTUs contained, respectively. Bacteroidales and Clostridiales were the two most diverse bacterial orders that contained 44.9 and 39.7 % of OTUs, respectively.

  5. Pyrosequencing of mcrA and archaeal 16S rRNA genes reveals diversity and substrate preferences of methanogen communities in anaerobic digesters.

    Science.gov (United States)

    Wilkins, David; Lu, Xiao-Ying; Shen, Zhiyong; Chen, Jiapeng; Lee, Patrick K H

    2015-01-01

    Methanogenic archaea play a key role in biogas-producing anaerobic digestion and yet remain poorly taxonomically characterized. This is in part due to the limitations of low-throughput Sanger sequencing of a single (16S rRNA) gene, which in the past may have undersampled methanogen diversity. In this study, archaeal communities from three sludge digesters in Hong Kong and one wastewater digester in China were examined using high-throughput pyrosequencing of the methyl coenzyme M reductase (mcrA) and 16S rRNA genes. Methanobacteriales, Methanomicrobiales, and Methanosarcinales were detected in each digester, indicating that both hydrogenotrophic and acetoclastic methanogenesis was occurring. Two sludge digesters had similar community structures, likely due to their similar design and feedstock. Taxonomic classification of the mcrA genes suggested that these digesters were dominated by acetoclastic methanogens, particularly Methanosarcinales, while the other digesters were dominated by hydrogenotrophic Methanomicrobiales. The proposed euryarchaeotal order Methanomassiliicoccales and the uncultured WSA2 group were detected with the 16S rRNA gene, and potential mcrA genes for these groups were identified. 16S rRNA gene sequencing also recovered several crenarchaeotal groups potentially involved in the initial anaerobic digestion processes. Overall, the two genes produced different taxonomic profiles for the digesters, while greater methanogen richness was detected using the mcrA gene, supporting the use of this functional gene as a complement to the 16S rRNA gene to better assess methanogen diversity. A significant positive correlation was detected between methane production and the abundance of mcrA transcripts in digesters treating sludge and wastewater samples, supporting the mcrA gene as a biomarker for methane yield.

  6. Microbial Contaminants of Cord Blood Units Identified by 16S rRNA Sequencing and by API Test System, and Antibiotic Sensitivity Profiling.

    Science.gov (United States)

    França, Luís; Simões, Catarina; Taborda, Marco; Diogo, Catarina; da Costa, Milton S

    2015-01-01

    Over a period of ten months a total of 5618 cord blood units (CBU) were screened for microbial contamination under routine conditions. The antibiotic resistance profile for all isolates was also examined using ATB strips. The detection rate for culture positive units was 7.5%, corresponding to 422 samples.16S rRNA sequence analysis and identification with API test system were used to identify the culturable aerobic, microaerophilic and anaerobic bacteria from CBUs. From these samples we recovered 485 isolates (84 operational taxonomic units, OTUs) assigned to the classes Bacteroidia, Actinobacteria, Clostridia, Bacilli, Betaproteobacteria and primarily to the Gammaproteobacteria. Sixty-nine OTUs, corresponding to 447 isolates, showed 16S rRNA sequence similarities above 99.0% with known cultured bacteria. However, 14 OTUs had 16S rRNA sequence similarities between 95 and 99% in support of genus level identification and one OTU with 16S rRNA sequence similarity of 90.3% supporting a family level identification only. The phenotypic identification formed 29 OTUs that could be identified to the species level and 9 OTUs that could be identified to the genus level by API test system. We failed to obtain identification for 14 OTUs, while 32 OTUs comprised organisms producing mixed identifications. Forty-two OTUs covered species not included in the API system databases. The API test system Rapid ID 32 Strep and Rapid ID 32 E showed the highest proportion of identifications to the species level, the lowest ratio of unidentified results and the highest agreement to the results of 16S rRNA assignments. Isolates affiliated to the Bacilli and Bacteroidia showed the highest antibiotic multi-resistance indices and microorganisms of the Clostridia displayed the most antibiotic sensitive phenotypes. PMID:26512991

  7. Characterization of Mycobacterium leprae Genotypes in China--Identification of a New Polymorphism C251T in the 16S rRNA Gene.

    Science.gov (United States)

    Yuan, Youhua; Wen, Yan; You, Yuangang; Xing, Yan; Li, Huanying; Weng, Xiaoman; Wu, Nan; Liu, Shuang; Zhang, Shanshan; Zhang, Wenhong; Zhang, Ying

    2015-01-01

    Leprosy continues to be prevalent in some mountainous regions of China, and genotypes of leprosy strains endemic to the country are not known. Mycobacterium lepromatosis is a new species that was discovered in Mexico in 2008, and it remains unclear whether this species exists in China. Here, we conducted PCR- restriction fragment length polymorphism (RFLP) analysis to classify genotypes of 85 DNA samples collected from patients from 18 different provinces. All 171 DNA samples from skin biopsies of leprosy patients were tested for the presence of Mycobacterium leprae and Mycobacterium lepromatosis by amplifying the 16S rRNA gene using nested PCR, followed by DNA sequencing. The new species M. lepromatosis was not found among the 171 specimens from leprosy patients in 22 provinces in China. However, we found three SNP genotypes among 85 leprosy patients. A mutation at C251T in the 16S rRNA gene was found in 76% of the strains. We also found that the strains that showed the 16S rRNA C251T mutation belonged to SNP type 3, whereas strains without the point mutation belonged to SNP type 1. The SNP type 3 leprosy strains were observed in patients from both the inner and coastal regions of China, but the SNP type 1 strains were focused only in the coastal region. This indicated that the SNP type 3 leprosy strains were more prevalent than the SNP type 1 strains in China. In addition, the 16S rRNA gene sequence mutation at C251T also indicated a difference in the geographical distribution of the strains. To our knowledge, this is the first report of a new polymorphism in 16S rRNA gene in M. leprae in China. Our findings shed light on the prevalent genotypes and provide insight about leprosy transmission that are important for leprosy control in China. PMID:26196543

  8. Characterization of Mycobacterium leprae Genotypes in China--Identification of a New Polymorphism C251T in the 16S rRNA Gene.

    Directory of Open Access Journals (Sweden)

    Youhua Yuan

    Full Text Available Leprosy continues to be prevalent in some mountainous regions of China, and genotypes of leprosy strains endemic to the country are not known. Mycobacterium lepromatosis is a new species that was discovered in Mexico in 2008, and it remains unclear whether this species exists in China. Here, we conducted PCR- restriction fragment length polymorphism (RFLP analysis to classify genotypes of 85 DNA samples collected from patients from 18 different provinces. All 171 DNA samples from skin biopsies of leprosy patients were tested for the presence of Mycobacterium leprae and Mycobacterium lepromatosis by amplifying the 16S rRNA gene using nested PCR, followed by DNA sequencing. The new species M. lepromatosis was not found among the 171 specimens from leprosy patients in 22 provinces in China. However, we found three SNP genotypes among 85 leprosy patients. A mutation at C251T in the 16S rRNA gene was found in 76% of the strains. We also found that the strains that showed the 16S rRNA C251T mutation belonged to SNP type 3, whereas strains without the point mutation belonged to SNP type 1. The SNP type 3 leprosy strains were observed in patients from both the inner and coastal regions of China, but the SNP type 1 strains were focused only in the coastal region. This indicated that the SNP type 3 leprosy strains were more prevalent than the SNP type 1 strains in China. In addition, the 16S rRNA gene sequence mutation at C251T also indicated a difference in the geographical distribution of the strains. To our knowledge, this is the first report of a new polymorphism in 16S rRNA gene in M. leprae in China. Our findings shed light on the prevalent genotypes and provide insight about leprosy transmission that are important for leprosy control in China.

  9. Analysis of 16S rRNA gene sequences and circulating cell-free DNA from plasma of chronic fatigue syndrome and non-fatigued subjects

    Directory of Open Access Journals (Sweden)

    Unger Elizabeth R

    2002-12-01

    Full Text Available Abstract Background The association of an infectious agent with chronic fatigue syndrome (CFS has been difficult and is further complicated by the lack of a known lesion or diseased tissue. Cell-free plasma DNA could serve as a sentinel of infection and disease occurring throughout the body. This type of systemic sample coupled with broad-range amplification of bacterial sequences was used to determine whether a bacterial pathogen was associated with CFS. Plasma DNA from 34 CFS and 55 non-fatigued subjects was assessed to determine plasma DNA concentration and the presence of bacterial 16S ribosomal DNA (rDNA sequences. Results DNA was isolated from 81 (91% of 89 plasma samples. The 55 non-fatigued subjects had higher plasma DNA concentrations than those with CFS (average 151 versus 91 ng and more CFS subjects (6/34, 18% had no detectable plasma DNA than non-fatigued subjects (2/55, 4%, but these differences were not significant. Bacterial sequences were detected in 23 (26% of 89. Only 4 (14% CFS subjects had 16S rDNA sequences amplified from plasma compared with 17 (32% of the non-fatigued (P = 0.03. All but 1 of the 23 16S rDNA amplicon-positive subjects had five or more unique sequences present. Conclusions CFS subjects had slightly lower concentrations or no detectable plasma DNA than non-fatigued subjects. There was a diverse array of 16S rDNA sequences in plasma DNA from both CFS and non-fatigued subjects. There were no unique, previously uncharacterized or predominant 16S rDNA sequences in either CFS or non-fatigued subjects.

  10. PCR-Independent Detection of Bacterial Species-Specific 16S rRNA at 10 fM by a Pore-Blockage Sensor

    Science.gov (United States)

    Esfandiari, Leyla; Wang, Siqing; Wang, Siqi; Banda, Anisha; Lorenzini, Michael; Kocharyan, Gayane; Monbouquette, Harold G.; Schmidt, Jacob J.

    2016-01-01

    A PCR-free, optics-free device is used for the detection of Escherichia coli (E. coli) 16S rRNA at 10 fM, which corresponds to ~100–1000 colony forming units/mL (CFU/mL) depending on cellular rRNA levels. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is sought for the detection of pathogenic microbes in food, water and body fluids. Since 16S rRNA sequences are species specific and are present at high copy number in viable cells, these nucleic acids offer an attractive target for microbial pathogen detection schemes. Here, target 16S rRNA of E. coli at 10 fM concentration was detected against a total RNA background using a conceptually simple approach based on electromechanical signal transduction, whereby a step change reduction in ionic current through a pore indicates blockage by an electrophoretically mobilized bead-peptide nucleic acid probe conjugate hybridized to target nucleic acid. We investigated the concentration detection limit for bacterial species-specific 16S rRNA at 1 pM to 1 fM and found a limit of detection of 10 fM for our device, which is consistent with our previous finding with single-stranded DNA of similar length. In addition, no false positive responses were obtained with control RNA and no false negatives with target 16S rRNA present down to the limit of detection (LOD) of 10 fM. Thus, this detection scheme shows promise for integration into portable, low-cost systems for rapid detection of pathogenic microbes in food, water and body fluids. PMID:27455337

  11. Microbial Contaminants of Cord Blood Units Identified by 16S rRNA Sequencing and by API Test System, and Antibiotic Sensitivity Profiling

    Science.gov (United States)

    França, Luís; Simões, Catarina; Taborda, Marco; Diogo, Catarina; da Costa, Milton S.

    2015-01-01

    Over a period of ten months a total of 5618 cord blood units (CBU) were screened for microbial contamination under routine conditions. The antibiotic resistance profile for all isolates was also examined using ATB strips. The detection rate for culture positive units was 7.5%, corresponding to 422 samples.16S rRNA sequence analysis and identification with API test system were used to identify the culturable aerobic, microaerophilic and anaerobic bacteria from CBUs. From these samples we recovered 485 isolates (84 operational taxonomic units, OTUs) assigned to the classes Bacteroidia, Actinobacteria, Clostridia, Bacilli, Betaproteobacteria and primarily to the Gammaproteobacteria. Sixty-nine OTUs, corresponding to 447 isolates, showed 16S rRNA sequence similarities above 99.0% with known cultured bacteria. However, 14 OTUs had 16S rRNA sequence similarities between 95 and 99% in support of genus level identification and one OTU with 16S rRNA sequence similarity of 90.3% supporting a family level identification only. The phenotypic identification formed 29 OTUs that could be identified to the species level and 9 OTUs that could be identified to the genus level by API test system. We failed to obtain identification for 14 OTUs, while 32 OTUs comprised organisms producing mixed identifications. Forty-two OTUs covered species not included in the API system databases. The API test system Rapid ID 32 Strep and Rapid ID 32 E showed the highest proportion of identifications to the species level, the lowest ratio of unidentified results and the highest agreement to the results of 16S rRNA assignments. Isolates affiliated to the Bacilli and Bacteroidia showed the highest antibiotic multi-resistance indices and microorganisms of the Clostridia displayed the most antibiotic sensitive phenotypes. PMID:26512991

  12. Phylogenetic and characteristic analysis of 16S rDNA and rpoB gene sequence of Klebsiella%克雷伯菌16S rDNA和rpoB基因系统进化及序列特征分析

    Institute of Scientific and Technical Information of China (English)

    郭晓琳; 王多春; 阚飙; 张燕敏; 张怡; 左颖; 魏来; 高燕

    2009-01-01

    目的 比较分析克雷伯菌属的种之间16S rDNA和rpoB系统进化发育关系和序列进化特征.方法 选取经生化鉴定的克雷伯菌18株,提取菌株染色体作为模板,分别使用16S rDNA和rpoB通用引物扩增并测序16S rDNA和rpoB序列.与GenBank中目前已公布的8种克雷伯属菌株16S rDNA和rpoB序列各8条、除克雷伯菌外其他肠道菌株的16S rDNA和rpoB序列各9条一起,共计各35条16SrDNA和rpoB序列,在MEGA 4.0中建立进化树并进行分群分析,使用DNAStar/MegAlign程序比较分析8种克雷伯菌的种间16S rDNA和rpoB序列变异碱基位点,并做分歧度分析.结果 在所有受试的16SrDNA和rpoB的各35条序列中,16S rDNA和rpoB进化发育树都将克雷伯菌区分为3个群:分离的18株克雷伯菌中,15株肺炎亚种与GenBank中除产酸克雷伯菌和运动克雷伯菌外的其余6种克雷伯菌聚为一群(Ⅰ),其余3株克雷伯菌(FX246、FX280和FX286),经生化鉴定为产酸克雷伯菌,与GenBank中产酸克雷伯菌(DQ835530)聚为一群(Ⅱ);而GenBank中的运动克雷伯菌单独聚为一群(Ⅲ);进一步的分析,在rpoB进化发育树中,无沦足Ⅰ群和Ⅱ群,还足Ⅰ群内的两个亚群,rpoB进化发育树的结点处自引导值都明显高于16S rDNA进化发育树;而且rpoB对产酸克雷伯菌的分群优于16S rDNA.对克雷伯菌的序列分析发现,16S rDNA序列存在41个碱基变异位点,有4个易变区,rpoB序列存在63个碱基变异位点,有1个易变区;克雷伯菌16S rDNA和rpoB的相似性分别为95.9%~100%和90.2%~100%.进一步的克雷伯菌种间序列分歧值分析,rpoB分歧值(0~10.6)高于16S rDNA(0~4.0).结论 克雷伯菌rpoB比16S rDNA具有更高的分歧度,在克雷伯菌属内种的分子分类和鉴定中,rpoB比16S rDNA基因更具优越性.%Objective To compare and analyze the phylogenetic tree and sequence variant characteristics of Klebsiella species between 16S rDNA and rpoB. Methods Eighteen isolates

  13. Quantification of 16S gene and its relation with the CO2 emission and soil properties in areas under management of sugarcane (Saccharum spp.)

    Science.gov (United States)

    Moitinho, Mara Regina; da Silva Bicalho, Elton; De Bortoli Teixeira, Daniel; La Scala, Newton, Jr.

    2015-04-01

    A diversity of microorganisms has an essential role in the recycling of soil chemical elements, controlling, for example, the dynamics of carbon de)ion and stabilization, and consequently the patterns of soil CO2 emission. In this sense, the objectives of this study were: (i) to estimate and compare the genetic diversity of microorganisms in soils under different sugarcane (Saccharum spp.) managements using molecular techniques based on metagenomic studies, and (ii) investigate the relationship of soil CO2 emission (FCO2) with microbiological results and soil chemical and physical properties in the evaluated managements. This study was conducted in agricultural areas located in southern Brazil, in which the following sugarcane managements were used: green and burned residues management, a sugarcane area under reform, and a native forest (used as a reference of the original soil condition). FCO2, soil temperature, and soil moisture were measured over 10 days, and at the end of the measurements soil samples were taken in order to determine the physical and chemical soil properties. The determination of the diversity of soil microorganisms was carried out by means of molecular techniques based on 16S rRNA gene sequencing. The highest mean value for FCO2 (3.25 μmol m-2s-1) was observed in the sugarcane area under reform, and the lowest values (1.85 and 1.27 μmol m-2s-1) were observed respectively in the green residue management and native forest areas. This same pattern was also observed when the 16S gene was quantified. In this case, the largest number of copies of this gene was found in the sugarcane area under reform (4.3x1010 copies of 16S rRNA gene per gram of dry soil), and its smallest number of copies was found in the green residues management area (1.7x1010 copies of 16S rRNA gene per gram of dry soil). The largest number of copies of the 16S gene associated to the highest values of FCO2, both observed in the sugarcane area under reform, could be related to

  14. Guidelines for interpretation of 16S rRNA gene sequence-based results for identification of medically important aerobic Gram-positive bacteria.

    Science.gov (United States)

    Woo, Patrick C Y; Teng, Jade L L; Wu, Jeff K L; Leung, Fion P S; Tse, Herman; Fung, Ami M Y; Lau, Susanna K P; Yuen, Kwok-Yung

    2009-08-01

    This study is believed to be the first to provide guidelines for facilitating interpretation of results based on full and 527 bp 16S rRNA gene sequencing and MicroSeq databases used for identifying medically important aerobic Gram-positive bacteria. Overall, full and 527 bp 16S rRNA gene sequencing can identify 24 and 40 % of medically important Gram-positive cocci (GPC), and 21 and 34 % of medically important Gram-positive rods (GPR) confidently to the species level, whereas the full-MicroSeq and 500-MicroSeq databases can identify 15 and 34 % of medically important GPC and 14 and 25 % of medically important GPR confidently to the species level. Among staphylococci, streptococci, enterococci, mycobacteria, corynebacteria, nocardia and members of Bacillus and related taxa (Paenibacillus, Brevibacillus, Geobacillus and Virgibacillus), the methods and databases are least useful for identification of staphylococci and nocardia. Only 0-2 and 2-13 % of staphylococci, and 0 and 0-10 % of nocardia, can be confidently and doubtfully identified, respectively. However, these methods and databases are most useful for identification of Bacillus and related taxa, with 36-56 and 11-14 % of Bacillus and related taxa confidently and doubtfully identified, respectively. A total of 15 medically important GPC and 18 medically important GPR that should be confidently identified by full 16S rRNA gene sequencing are not included in the full-MicroSeq database. A total of 9 medically important GPC and 21 medically important GPR that should be confidently identified by 527 bp 16S rRNA gene sequencing are not included in the 500-MicroSeq database. 16S rRNA gene sequence results of Gram-positive bacteria should be interpreted with basic phenotypic tests results. Additional biochemical tests or sequencing of additional gene loci are often required for definitive identification. To improve the usefulness of the MicroSeq databases, bacterial species that can be confidently identified by 16S r

  15. Phylogenetic analysis of Nocardiopsis species based on 16S rRNA,gyrB, sod and rpoB gene sequences%拟诺卡氏菌16S rRNA,gyrB,sod和rpoB基因的系统发育分析

    Institute of Scientific and Technical Information of China (English)

    杨玲玲; 职晓阳; 李文均

    2007-01-01

    为了更好地了解拟诺卡氏菌属(Nocardiopsis)各物种间的系统发育关系,该属现有有效描述种的gyrB,sod和rpoB基因的部分序列被测定,结合16S rRNA基因,对拟诺卡氏菌属进行了系统发育重建.研究发现拟诺卡氏菌属gyrB,sod和rpoB基因的平均相似性分别为87.7%、87.3%和94.1%,而16S rRNA基因的平均相似性则达到96.65%,3个看家基因均比16S rRNA具有更高的分歧度.比较基于不同基因的系统树发现,由gyrB基因得到的系统树拓扑结构与16S rRNA得到的结构在亚群上基本一致.因此,gyrB基因在拟诺卡氏菌属的系统分类上比16S rRNA基因更具优越性.

  16. CLONING AND SEQUENCING OF 16S RRNA GENE OF PHYTOPLASMA CWB1 STRAIN ASSOCIATED WITH CACTUS WITCHES'BROOM%仙人掌丛枝病植原体CWB1株系16S rRNA基因克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    蔡红; 李凡; 孔宝华; 陈海如

    2001-01-01

    对表现丛枝症状的仙人掌植株总DNA进行植原体16S rRNA基因PCR扩增,得到一条约1.5kb的特异片段,表明植株中有植原体存在,将此植原体株系命名为CWB1.把此特异片段与pGEM-T Easy载体连接并转化到大肠杆菌JM109感受态细胞中,通过PCR鉴定、限制性内切酶(Eco RI)酶切分析及核苷酸序列分析,均表明克隆成功.序列分析结果显示,此株系的16S rRNA基因全长1489个碱基,与属于植原体16S rⅡ-C亚组的Faba bean phyllody植原体同源率最高,为99.7%.通过16S rRNA基因核酸序列同源性比较,认为该株系属于16S rⅡ-C亚组,基本确定了其分类地位.

  17. 鸭疫里氏杆菌广西分离株16S rRNA基因序列的测定和系统进化分析%Cloning sequence and system evolvement analysis of 16S rRNA genes of Riemerella anatipestifer Guangxi isolates

    Institute of Scientific and Technical Information of China (English)

    陈泽祥; 潘艳; 禤雄标; 谢永平; 许力干; 郑列丰

    2007-01-01

    选取3株不同血清型的鸭疫里氏杆菌广西分离株GXRA01、GXRA07和GXRA09,利用细菌通用引物,通过PCR方法成功扩增出16S rRNA的部分基因片段,通过克隆、序列测定获得3个分离株16S rRNA基因的核苷酸序列,并与GenBank中注册的一些菌株的16S rRNA基因序列进行比较、系统进化关系分析发现:3株RA分属两个基因群,I群(GXRA01)与AY871818和AY871819 的16S rRNA 序列的同源性达99.9%,Ⅱ群(GXRA07、GXRA09)与GenBank中16S rRNA序列的同源性达99.3%~99.6%,GXRA07与GXRA09之间的同源性为100%,表明这3株菌株应为鸭疫里氏杆菌.

  18. Two genetic clusters in swine hemoplasmas revealed by analyses of the 16S rRNA and RNase P RNA genes.

    Science.gov (United States)

    Watanabe, Yusaku; Fujihara, Masatoshi; Obara, Hisato; Nagai, Kazuya; Harasawa, Ryô

    2011-12-01

    Only two hemoplasma species, Eperythrozoon parvum and Mycoplasma suis, have been recognized in pigs. Here we demonstrate the genetic variations among six hemoplasma strains detected from pigs, by analyzing the 16S rRNA and RNase P RNA (rnpB) genes, and propose a novel hemoplasma taxon that has not been described previously. Phylogenetic trees based on the nucleotide sequence of the 16S rRNA gene indicated that these six hemoplasmas were divided into two clusters representing M. suis and a novel taxon. We further examined the primary and secondary structures of the nucleotide sequences of the rnpB gene of the novel taxon, and found it distinct from that of M. suis. In conclusion, we unveiled a genetic cluster distinct from M. suis, suggesting a new swine hemoplasma species or E. parvum. Our findings also suggest that this novel cluster should be included in the genus Mycoplasma.

  19. Characterization of bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, as determined by 16S rDNA analysis.

    Science.gov (United States)

    Escalante, Adelfo; Rodríguez, María Elena; Martínez, Alfredo; López-Munguía, Agustín; Bolívar, Francisco; Gosset, Guillermo

    2004-06-15

    The bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, was studied in 16S rDNA clone libraries from three pulque samples. Sequenced clones identified as Lactobacillus acidophilus, Lactobacillus strain ASF360, L. kefir, L. acetotolerans, L. hilgardii, L. plantarum, Leuconostoc pseudomesenteroides, Microbacterium arborescens, Flavobacterium johnsoniae, Acetobacter pomorium, Gluconobacter oxydans, and Hafnia alvei, were detected for the first time in pulque. Identity of 16S rDNA sequenced clones showed that bacterial diversity present among pulque samples is dominated by Lactobacillus species (80.97%). Seventy-eight clones exhibited less than 95% of relatedness to NCBI database sequences, which may indicate the presence of new species in pulque samples.

  20. Phylogeny and Taxonomy of Archaea: A Comparison of the Whole-Genome-Based CVTree Approach with 16S rRNA Sequence Analysis

    Directory of Open Access Journals (Sweden)

    Guanghong Zuo

    2015-03-01

    Full Text Available A tripartite comparison of Archaea phylogeny and taxonomy at and above the rank order is reported: (1 the whole-genome-based and alignment-free CVTree using 179 genomes; (2 the 16S rRNA analysis exemplified by the All-Species Living Tree with 366 archaeal sequences; and (3 the Second Edition of Bergey’s Manual of Systematic Bacteriology complemented by some current literature. A high degree of agreement is reached at these ranks. From the newly proposed archaeal phyla, Korarchaeota, Thaumarchaeota, Nanoarchaeota and Aigarchaeota, to the recent suggestion to divide the class Halobacteria into three orders, all gain substantial support from CVTree. In addition, the CVTree helped to determine the taxonomic position of some newly sequenced genomes without proper lineage information. A few discrepancies between the CVTree and the 16S rRNA approaches call for further investigation.

  1. Phylogenetic systematics of Barn Owl (Tyto alba (Scopoli, 1769 complex inferred from mitochondrial rDNA (16S rRNA taxonomic implication

    Directory of Open Access Journals (Sweden)

    Mansour Aliabadian

    2012-09-01

    Full Text Available The Barn owl, Tyto alba (Scopoli, 1769, occurs worldwide and shows a considerable amount of morphological and geographical variations, leading to the recognition of many subspecies throughout the world. Yet, no comprehensive study has not been done on this species. Data from mitochondrial gene (16S Ribosomal RNA (16S with 569 bp length were analyzed for 41 individuals around the world. Maximum likelihood (ML, maximum parsimony (MP and Bayesian analysis showed two distinct clades including alba clad (old world and furcata clad (new world. The amount of genetic variation within each of these clades ranged from 0.5-1.7 but variation between clades was 3.7. This data may suggest that Barn owls of the Old World may be a separate species from those of the New World.

  2. 16S rRNA Gene Sequence-Based Identification of Bacteria in Automatically Incubated Blood Culture Materials from Tropical Sub-Saharan Africa.

    Directory of Open Access Journals (Sweden)

    Hagen Frickmann

    Full Text Available The quality of microbiological diagnostic procedures depends on pre-analytic conditions. We compared the results of 16S rRNA gene PCR and sequencing from automatically incubated blood culture materials from tropical Ghana with the results of cultural growth after automated incubation.Real-time 16S rRNA gene PCR and subsequent sequencing were applied to 1500 retained blood culture samples of Ghanaian patients admitted to a hospital with an unknown febrile illness after enrichment by automated culture.Out of all 1500 samples, 191 were culture-positive and 98 isolates were considered etiologically relevant. Out of the 191 culture-positive samples, 16S rRNA gene PCR and sequencing led to concordant results in 65 cases at species level and an additional 62 cases at genus level. PCR was positive in further 360 out of 1309 culture-negative samples, sequencing results of which suggested etiologically relevant pathogen detections in 62 instances, detections of uncertain relevance in 50 instances, and DNA contamination due to sample preparation in 248 instances. In two instances, PCR failed to detect contaminants from the skin flora that were culturally detectable. Pre-analytical errors caused many Enterobacteriaceae to be missed by culture.Potentially correctable pre-analytical conditions and not the fastidious nature of the bacteria caused most of the discrepancies. Although 16S rRNA gene PCR and sequencing in addition to culture led to an increase in detections of presumably etiologically relevant blood culture pathogens, the application of this procedure to samples from the tropics was hampered by a high contamination rate. Careful interpretation of diagnostic results is required.

  3. 16S rRNA sequences of Bartonella bacilliformis and cat scratch disease bacillus reveal phylogenetic relationships with the alpha-2 subgroup of the class Proteobacteria.

    OpenAIRE

    O'Connor, S P; Dorsch, M; Steigerwalt, A G; Brenner, D J; Stackebrandt, E

    1991-01-01

    The primary structures of 16S rRNAs of Bartonella bacilliformis, an isolate of the cat scratch disease (CSD) bacillus, and a strain phenotypically similar to the CSD bacillus were determined by reverse transcriptase sequencing. These microorganisms were found to be members of the alpha-2 subgroup of the class Proteobacteria. The sequence from B. bacilliformis was most closely related to the rRNA of Rochalimaea quintana (91.7% homology), the etiologic agent of trench fever. The sequence from t...

  4. Impact of Fishmeal Replacement in Diets for Gilthead Sea Bream (Sparus aurata) on the Gastrointestinal Microbiota Determined by Pyrosequencing the 16S rRNA Gene

    OpenAIRE

    Estruch, Guillem; COLLADO AMORES, MARÍA CARMEN; Peñaranda, D.S.; Tomas-Vidal, A.; Jover Cerda, Miguel; Pérez-Martínez, G; Martínez-Llorens, Silvia

    2015-01-01

    [EN] Recent studies have demonstrated the impact of diet on microbiota composition, but the essential need for the optimization of production rates and costs forces farms and aquaculture production to carry out continuous dietary tests. In order to understand the effect of total fishmeal replacement by vegetable-based feed in the sea bream (Sparus aurata), the microbial composition of the stomach, foregut, midgut and hindgut was analysed using high-throughput 16S rDNA sequ...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2015-07-17

    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 × 10(3) to 2.28 × 10(6) and 4.17 × 10(2) to 1.99 × 10(5), 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.

  8. Metagenomic analysis targeted on the 16S ribosomal DNA to study the quality of meat : a example with raw minced beef meat

    OpenAIRE

    Delhalle, Laurent; Taminiau, Bernard; Nezer, Carine; Daube, Georges

    2013-01-01

    Introduction: Steak tartare is a popular meat dish in Belgium and some other European countries. This meat preparations due to their raw nature, is highly sensitive to bacterial spoilage. A better understanding of the bacterial content of this product will thus be insightful to control the risk of spoilage. Metagenomics targeted on the 16S ribosomal DNA has appeared as a powerful tool to study bacterial composition of food samples. The aim of this study is to identify the bacterial populatio...

  9. Mutation detection analysis of a region of 16S-like ribosomal RNA gene of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii

    OpenAIRE

    Khairnar Krishna; Parija Subhash

    2008-01-01

    Abstract Background The level of intra-species genetic variation in Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii populations in a localized geographic area, like Puducherry, India, remains unknown. Methods In the present study the existence of genetic variation in the nested multiplex polymerase chain reaction (NM-PCR) amplified region of the 16S-like ribosomal RNA genes of E. histolytica, E. dispar and E. moshkovskii was investigated by riboprinting and single strand con...

  10. Development of Amplified 16S Ribosomal DNA Restriction Analysis for Identification of Actinomyces Species and Comparison with Pyrolysis-Mass Spectrometry and Conventional Biochemical Tests

    OpenAIRE

    Hall, Val; O’Neill, G. L.; Magee, J T; Duerden, B I

    1999-01-01

    Identification of Actinomyces spp. by conventional phenotypic methods is notoriously difficult and unreliable. Recently, the application of chemotaxonomic and molecular methods has clarified the taxonomy of the group and has led to the recognition of several new species. A practical and discriminatory identification method is now needed for routine identification of clinical isolates. Amplified 16S ribosomal DNA restriction analysis (ARDRA) was applied to reference strains (n = 27) and clinic...

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

    OpenAIRE

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

    2015-01-01

    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...

  12. Use of 16S-23S rRNA Intergenic Spacer Region PCR and Repetitive Extragenic Palindromic PCR Analyses of Escherichia coli Isolates To Identify Nonpoint Fecal Sources

    OpenAIRE

    Seurinck, Sylvie; Verstraete, Willy; Siciliano, Steven D.

    2003-01-01

    Despite efforts to minimize fecal input into waterways, this kind of pollution continues to be a problem due to an inability to reliably identify nonpoint sources. Our objective was to find candidate source-specific Escherichia coli fingerprints as potential genotypic markers for raw sewage, horses, dogs, gulls, and cows. We evaluated 16S-23S rRNA intergenic spacer region (ISR)-PCR and repetitive extragenic palindromic (rep)-PCR analyses of E. coli isolates as tools to identify nonpoint fecal...

  13. A PCR Assay To Discriminate Human and Ruminant Feces on the Basis of Host Differences in Bacteroides-Prevotella Genes Encoding 16S rRNA

    OpenAIRE

    Bernhard, Anne E.; Field, Katharine G.

    2000-01-01

    Our purpose was to develop a rapid, inexpensive method of diagnosing the source of fecal pollution in water. In previous research, we identified Bacteroides-Prevotella ribosomal DNA (rDNA) PCR markers based on analysis. These markers length heterogeneity PCR and terminal restriction fragment length polymorphism distinguish cow from human feces. Here, we recovered 16S rDNA clones from natural waters that were close phylogenetic relatives of the markers. From the sequence data, we designed spec...

  14. Identification of Nonpoint Sources of Fecal Pollution in Coastal Waters by Using Host-Specific 16S Ribosomal DNA Genetic Markers from Fecal Anaerobes

    OpenAIRE

    Bernhard, Anne E.; Field, Katharine G.

    2000-01-01

    We describe a new PCR-based method for distinguishing human and cow fecal contamination in coastal waters without culturing indicator organisms, and we show that the method can be used to track bacterial marker sequences in complex environments. We identified two human-specific genetic markers and five cow-specific genetic markers in fecal samples by amplifying 16S ribosomal DNA (rDNA) fragments from members of the genus Bifidobacterium and the Bacteroides-Prevotella group and performing leng...

  15. Phylogenetic Relationship of Duttaphrynus melanostictus From India and China as Revealed from the Study of 12S and 16S mtDNA Genes

    OpenAIRE

    Sanjib Kr. Das; Debojyoti Dutta

    2013-01-01

    In the present study, the phylogenetic relationship of Duttaphrynus melanostictus from West Bengal, India with other members of the Bufonidiae group was undertaken using partial mitochondrial DNA (mtDNA) genes. Mitochondria were isolated from the liver of Duttaphrynus melanostictus by a non-conventional method of membrane filtration. The technique allows trapping of mitochondria on cellulose acetate membrane followed by mtDNA isolation. 12S ribosomal RNA and 16S ribosomal RNA was sequenced wi...

  16. Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16S rRNA-targeted probes by colony hybridization and PCR.

    OpenAIRE

    Kaufmann, P; Pfefferkorn, A; Teuber, M; Meile, L

    1997-01-01

    A Bifidobacterium genus-specific target sequence in the V9 variable region of the 16S rRNA has been elaborated and was used to develop a hybridization probe. The specificity of this probe, named lm3 (5'-CGGGTGCTI*CCCACTTTCATG-3'), was used to identify all known type strains and distinguish them from other bacteria. All of the 30 type strains of Bifidobacterium which are available at the German culture collection Deutsche Sammlung von Mikroorganismen und Zellkulturen, 6 commercially available ...

  17. Identification to the species level of Lactobacillus isolated in probiotic prospecting studies of human, animal or food origin by 16S-23S rRNA restriction profiling

    OpenAIRE

    Neumann Elisabeth; Teixeira Santuza MR; Horta Maria F; Mota Rodrigo M; Moreira João; Nicoli Jacques R.; Nunes Álvaro C

    2005-01-01

    Abstract Background The accurate identification of Lactobacillus and other co-isolated bacteria during microbial ecological studies of ecosystems such as the human or animal intestinal tracts and food products is a hard task by phenotypic methods requiring additional tests such as protein and/or lipids profiling. Results Bacteria isolated in different probiotic prospecting studies, using de Man, Rogosa and Sharpe medium (MRS), were typed at species level by PCR amplification of 16S-23S rRNA i...

  18. Microbial Contaminants of Cord Blood Units Identified by 16S rRNA Sequencing and by API Test System, and Antibiotic Sensitivity Profiling

    OpenAIRE

    França, Luís; Simões, Catarina; Taborda, Marco; Diogo, Catarina; da Costa, Milton S.

    2015-01-01

    Over a period of ten months a total of 5618 cord blood units (CBU) were screened for microbial contamination under routine conditions. The antibiotic resistance profile for all isolates was also examined using ATB strips. The detection rate for culture positive units was 7.5%, corresponding to 422 samples.16S rRNA sequence analysis and identification with API test system were used to identify the culturable aerobic, microaerophilic and anaerobic bacteria from CBUs. From these samples we recov...

  19. Effect of single base changes and the absence of modified bases in 16S RNA on the reconstitution and function of Escherichia coli 30S ribosomes

    International Nuclear Information System (INIS)

    The gene coding for E. coli 16S rRNA was placed in pUC19 under the control of the strong class III T7 promoter, phi 10, by ligation of the 1490 bp BclI/BstEII fragment of the rrnB operon with appropriate synthetic oligodeoxynucleotides. Such constructs allowed efficient in vitro synthesis of full-length transcripts (up to 900 mol RNA/mol template) free of modified bases. The synthetic RNA could be assembled into 30S subunits upon addition of E. coli 30S ribosomal proteins. The particles co-sedimented with authentic 30S particles and were electron microscopically indistinguishable from them. Upon addition of 50S subunits, codon-dependent P-site binding of tRNA and codon-dependent polypeptide synthesis were >80% of 30S reconstituted from natural 16S RNA and >50% of isolated 30S. UV-induced crosslinking of P-site bound AcVal-tRNA to residue C1400 was preserved. Changing C1400 to A had little effect on reconstitution, P-site binding, or polypeptide synthesis. However, the substitution of C1499 by G markedly inhibited assembly. The effect on P-site binding and polypeptide synthesis is under study. These results show (1) none of the modified bases of 16S RNA are essential for protein synthesis, (2) substitution of A for C1400 has little functional effect, and (3) position 1400 may be important for ribosome assembly

  20. Evaluation of PCR primer selectivity and phylogenetic specificity by using amplification of 16S rRNA genes from betaproteobacterial ammonia-oxidizing bacteria in environmental samples.

    Science.gov (United States)

    Junier, Pilar; Kim, Ok-Sun; Hadas, Ora; Imhoff, Johannes F; Witzel, Karl-Paul

    2008-08-01

    The effect of primer specificity for studying the diversity of ammonia-oxidizing betaproteobacteria (betaAOB) was evaluated. betaAOB represent a group of phylogenetically related organisms for which the 16S rRNA gene approach is especially suitable. We used experimental comparisons of primer performance with water samples, together with an in silico analysis of published sequences and a literature review of clone libraries made with four specific PCR primers for the betaAOB 16S rRNA gene. With four aquatic samples, the primers NitA/NitB produced the highest frequency of ammonia-oxidizing-bacterium-like sequences compared to clone libraries with products amplified with the primer combinations betaAMOf/betaAMOr, betaAMOf/Nso1255g, and NitA/Nso1225g. Both the experimental examination of ammonia-oxidizing-bacterium-specific 16S rRNA gene primers and the literature search showed that neither specificity nor sensitivity of primer combinations can be evaluated reliably only by sequence comparison. Apparently, the combination of sequence comparison and experimental data is the best approach to detect possible biases of PCR primers. Although this study focused on betaAOB, the results presented here more generally exemplify the importance of primer selection and potential primer bias when analyzing microbial communities in environmental samples.