16S rRNA analysis of Listeria monocytogenes and Brochothrix thermosphacta

Abstract Listeria monocytogenes and Brochothrix thermosphacta were investigated by the 16S rRNA cataloguing approach in order to determine their phylogenetic relationship. Both species are specifically, although moderately related, forming one of several sublines within the Bacillus-Lactobacillus-Streptococcus cluster of the ' Clostridium ' subbranch of Gram-positive eubacteria.     

Characterization of nitrogen-fixing Paenibacillus species by polymerase chain reaction-restriction fragment length polymorphism analysis of part of genes encoding 16S rRNA and 23S rRNA and by multilocus enzyme electrophoresis

Forty-two strains representing the eight recognized nitrogen-fixing Paenibacillus species and 12 non-identified strains were examined by restriction fragment length polymorphism (RFLP) analysis of part of 16S and 23S rRNA genes amplified by polymerase chain reaction (PCR). Eleven different 16S rDNA genotypes were obtained from the combined data of RFLP analysis with four endonucleases and they were in agreement with the established taxonomic classification. Only one group of unclassified strains (Group I) was assigned in a separate genotype, suggesting they belong to a new species. Using the 23S PCR-RFLP method only six genotypes were detected, showing that this method is less discriminative than the 16S PCR-RFLP. Using the multilocus enzyme electrophoresis (MLEE) assay, the 48 strains tested could be classified into 35 zymovars. The seven enzymatic loci tested were polymorphic and the different profiles obtained among strains allowed the grouping of strains into 10 clusters. The PCR-RFLP methods together with the MLEE assay provide a rapid tool for the characterization and the establishment of the taxonomic position of isolates belonging to this nitrogen-fixing group, which shows a great potentiality in promoting plant growth.     

拟诺卡氏菌16S rRNA,gyrB,sod和rpoB基因的系统发育分析

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。     

利用16S rRNA基因同源性分析鉴定两株明串珠菌

从酸马奶中分离出2株明串珠菌KLDS 5.0301和KLDS 5.0302,对2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,并建立明串珠菌属的系统发育树.结果表明,KLDS 5.0301的16S rRNA序列同L. garlicum的同源性百分比为100%.KLDS 5.0302的16S rRNA序列同L.mesenteroides LM2菌株的16S rRNA序列的同源性百分比为99.9%.根据系统发育树的结果,将KLDS5.0301鉴定为L.garlicum,KLDS 5.0302鉴定为L.mesenteroides.菌株KLDS 5.0301和KLDS 5.0302的16SrRNA序列已经在GeneBank申请国际序列注册号,分别为DQ239691和DQ297412.     

Bacterial,archaeal and eukaryotic diversity in Arctic sediment as revealed by 16S rRNA and 18S rRNA gene clone libraries analysis

We studied the microbial diversity in the sediment from the Kongsfjorden, Svalbard, Arctic, in the summer of 2005 based on the analysis of 16S rRNA and 18S rRNA gene clone libraries. The sequences of the cloned 16S rRNA and 18S rRNA gene inserts were used to determine the species identity or closest relatives by comparison with sequences of known species. Compared to the other samples acquired in Arctic and Antarctic, which are different from that of ours, the microbial diversity in our sediment is much higher. The bacterial sequences were grouped into 11 major lineages of the domain Bacteria: Proteobacteria (include α-, β-, γ-, δ-, and ε-Proteobacteria); Bacteroidetes; Fusobacteria; Firmicutes; Chloroflexi; Chlamydiae; Acidobacteria; Actinobacteria; Planctomycetes; Verrucomicrobiae and Lentisphaerae. Crenarchaeota were dominant in the archaeal clones containing inserts. In addition, six groups from eukaryotes including Cercozoa, Fungi, Telonema, Stramenopiles, Alveolata, and Metazoa were identified. Remarkably, the novel group Lentisphaerae was reported in Arctic sediment at the first time. Our study suggested that Arctic sediment as a unique habitat may contain substantial microbial diversity and novel species will be discovered.     

Selection of primers for optimal taxonomic classification of environmental 16S rRNA gene sequences

Microbial community profiling using 16S rRNA gene sequences requires accurate taxonomy assignments. ‘Universal'' primers target conserved sequences and amplify sequences from many taxa, but they provide variable coverage of different environments, and regions of the rRNA gene differ in taxonomic informativeness—especially when high-throughput short-read sequencing technologies (for example, 454 and Illumina) are used. We introduce a new evaluation procedure that provides an improved measure of expected taxonomic precision when classifying environmental sequence reads from a given primer. Applying this measure to thousands of combinations of primers and read lengths, simulating single-ended and paired-end sequencing, reveals that these choices greatly affect taxonomic informativeness. The most informative sequence region may differ by environment, partly due to variable coverage of different environments in reference databases. Using our Rtax method of classifying paired-end reads, we found that paired-end sequencing provides substantial benefit in some environments including human gut, but not in others. Optimal primer choice for short reads totaling 96 nt provides 82–100% of the confident genus classifications available from longer reads.     

DNA extraction for 16S rRNA gene analysis to determine genetic diversity in deep sediment communities

A protocol was devised which permitted the extraction of DNA from deep marine sediments up to 503 m below the sea floor. These sediments have been laid down over the last 3 million years. 16S rRNA gene sequences were amplified from the DNA by the polymerase chain reaction. The details of the successful extraction and polymerase chain reaction methodology varied between samples from different depths. This emphasizes the attention to detail required to allow the diversity of bacteria in these deep sediments to be studied.     

Application of recA and rpoB sequence analysis on phylogeny and molecular identification of Geobacillus species

Aims:  Some Geobacillus species have highly similar 16S rRNA gene sequences, making 16S rDNA sequence analysis-based identification problematic. To overcome this limitation, recA and rpoB sequence analysis was evaluated as an alternative for distinguishing Geobacillus species.
Methods and Results:  The phylogram of 16S rRNA gene sequences inferred from the neighbour-joining method showed that nine clusters of Geobacillus species were characterized with bootstrap values >90%. The recA and rpoB sequences of 10 reference strains in clusters V, VIb and VIc were amplified and sequenced using consensus primers. Alignment of recA sequences in clusters V, VIb and VIc revealed three types of recA genes, consistent with the putative amino acid sequences and in vivo recA splicing analysis. The phylogram constructed from rpoB sequences showed more divergence than that constructed from 16S rRNA gene sequences.
Conclusions:  recA and rpoB sequence analysis differentiated closely-related Geobacillus species and provided direct evidence for reclassifying some species dubiously categorized as Geobacilli . Additionally, this study revealed three types of recA genes in the different Geobacillus species.
Significance and Impact of the Study:  This study highlights the advantage of recA and rpoB sequence analysis to supplement 16S rRNA gene sequence analysis for efficient and convenient determination of Geobacillus species.     

纳豆芽胞杆菌16S rRNA基因的克隆及其系统进化分析

纳豆芽胞杆菌是从豆豉中分离出的一种具有益生功能的芽胞杆菌。该研究从纳豆芽胞杆菌提取基因组DNA,以芽胞杆菌16S rRNA基因的通用引物,用PCR方法成功扩增出纳豆芽胞杆菌的部分16S rRNA基因,所克隆序列长1 435 bp,G＋C含量为55%,该序列已被GeneBank收录,其编号为AY864812。BLAST分析结果显示,AY864812与GeneBank中收录的枯草芽胞杆菌16S rRNA基因同源性最高,其中与AY601722的同源性为100%.用Clustalx 1.8对相关序列进行系统进化分析,结果显示纳豆芽胞杆菌与枯草芽胞杆菌在进化关系上的地位最近,从分子水平上证实了纳豆芽胞杆菌是枯草杆菌的1个亚种。     

嗜盐菌HBCC-2的16S rRNA基因测序分析及其培养特性

从连云港台南盐场海盐生产区中分离纯化到一株嗜盐古菌HBCC-2,该菌株经PCR扩增后,测定其16S rRNA基因序列,采用BLAST软件对基因库中基因序列进行同源性比较,选取其相似性序列,采用Clustalx1.8和MEGA3.1软件对其16S rDNA序列进行了系统发育分析研究,结果表明HBCC-2菌株与菌株Halorubrum sp.GSL5.48的相似性达99%,结合其形态观察及生理生化反应特性,初步确定该菌株属于嗜盐红菌属(Halorubrum),菌株HBCC-2的16S rDNA序列已登陆到GenBank,其序列号为EF687739.通过比较不同NaCl浓度、pH和培养温度对该菌株生长的影响情况,研究了该菌株的生长特性,结果表明NaCl浓度为4mol/L、温度为35℃和pH为7.0的培养条件下其生长最佳.     

Evaluation of different partial 16S rRNA gene sequence regions for phylogenetic analysis of microbiomes

Operational taxonomic units (OTUs) are conventionally defined at a phylogenetic distance (0.03—species, 0.05—genus, 0.10—family) based on full-length 16S rRNA gene sequences. However, partial sequences (700 bp or shorter) have been used in most studies. This discord may affect analysis of diversity and species richness because sequence divergence is not distributed evenly along the 16S rRNA gene. In this study, we compared a set each of bacterial and archaeal 16S rRNA gene sequences of nearly full length with multiple sets of different partial 16S rRNA gene sequences derived therefrom (approximately 440-700 bp), at conventional and alternative distance levels. Our objective was to identify partial sequence region(s) and distance level(s) that allow more accurate phylogenetic analysis of partial 16S rRNA genes. Our results showed that no partial sequence region could estimate OTU richness or define OTUs as reliably as nearly full-length genes. However, the V1-V4 regions can provide more accurate estimates than others. For analysis of archaea, we recommend the V1-V3 and the V4-V7 regions and clustering of species-level OTUs at 0.03 and 0.02 distances, respectively. For analysis of bacteria, the V1-V3 and the V1-V4 regions should be targeted, with species-level OTUs being clustered at 0.04 distance in both cases.     

一株耐热嗜盐菌的分离及16SrRNA基因序列分析

目的利用盐固体分离培养基,从西藏自治区澜沧江边康宁镇一个47℃的盐井样品中分离纯化到一株耐热嗜盐菌菌株YJ0232。方法通过形态观察、生理生化特性和16srRNA基因序列分析,鉴定嗜盐菌菌株YJ0232分类学地位。结果菌株YJ0232初步鉴定为中度嗜盐菌,属于盐单胞菌属（Halomonassp．）菌株。其16SrRNA基因序列已被GenBank数据库收录,序列号为EU029645。结论本研究对澜沧江高盐环境微生物资源进行了初步探索研究。可为今后研究同类极端环境中新的物种资源以及微生物多样性提供参考。     

Molecular identification of pufferfish species using PCR amplification and restriction analysis of a segment of the 16S rRNA gene

This study amplified the mitochondrial 16S rRNA gene using polymerase chain reaction (PCR) with a template of total DNA from muscle tissues of nine pufferfish species collected from the coastal area of Okinawa Islands in Japan: Pleuranacanthus sceleratus, Triodon macropterus, Chelonodon patoca, Sphoeroides pachygaster, Arothron hispidus, A. stellatus, A. manilensis, A. mappa, and A. nigropunctatus. Then nucleotide sequence encoding a partial region of the 16S rRNA gene was compared among species. The sequenced fragment was also used to select restriction enzymes, yielding species-specific restriction fragment length polymorphisms (RFLP). The sequence of the segment of the 16S rRNA gene consisted of about 615 nucleotides and showed interspecies variations in the targeted region. After calculation of corresponding RFLP-patterns of nine species investigated with suitable restriction enzymes, three restriction enzymes – BanII, DdeI, and NlaIII – were found to be sufficient for identification of all nine species. Successful testing of this methodology in frozen and heated food samples suggests its utility for pufferfish species authentication in food products.     

16S rRNA gene-based analysis of mucosa-associated bacterial community and phylogeny in the chicken gastrointestinal tracts: from crops to ceca

Mucosa-associated microbiota from different regions of the gastrointestinal (GI) tract of adult broilers was studied by analysis of 16S rRNA gene sequences. The microbiota mainly comprised Gram-positive bacteria along the GI tract. Fifty-one operational taxonomic units (OTUs) (from 98 clones) were detected in the ceca, as compared with 13 OTUs (from 49 clones) in the crops, 11 OTUs (from 51 clones) in the gizzard, 14 OTUs (from 52 clones) in the duodenum, 12 OTUs (from 50 clones) in the jejunum and nine OTUs (from 50 clones) in the ileum. Ceca were dominantly occupied by clostridia-related sequences (40%) with other abundant sequences being related to Faecalibacterium prausnitzii (14%), Escherichia coli (11%), lactobacilli (7%) and Ruminococcus (6%). Lactobacilli were predominant in the upper GI tract and had the highest diversity in the crop. Both Lactobacillus aviarius and Lactobacillus salivarius were the predominant species among lactobacilli. Candidatus division Arthromitus was also abundant in the jejunum and ileum.     

Application of rpoB sequence similarity analysis, REP-PCR and BOX-PCR for the differentiation of species within the genus Geobacillus

Aim:  To investigate the applicability of rpoB gene, which encodes the β subunit of RNA polymerase, to be used as an alternative to 16S rRNA for sequence similarity analysis in the thermophilic genus Geobacillus. Rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (REP‐ and BOX‐polymerase chain reaction) were also used. Methods and Results:  rpoB DNA (458 bp) were amplified from 21 Geobacillus‐ and Bacillus type strains, producing different BOX‐ and REP‐PCR profiles, in addition to 11 thermophilic isolates of Geobacillus and Bacillus species from a Santorini volcano habitat. The sequences and the phylogenetic tree of rpoB were compared with those obtained from 16S rRNA gene analysis. The results demonstrated between 90–100% (16S rRNA) and 74–100% (rpoB) similarity among examined bacteria. Conclusion:  BOX‐ and REP‐PCR can be applied for molecular typing within Geobacillus genus. rpoB sequence similarity analysis permits a more accurate discrimination of the species within the Geobacillus genus than the more commonly used 16S rRNA. Significance and Impact of the Study:  The obtained results suggested that rpoB sequence similarity analysis is a powerful tool for discrimination between species within the ecologically and industrially important strains of Geobacillus genus.     

Partial rpoB gene sequencing for identification of Leptospira species

The usual target for sequence-based identification of Leptospira species is the 16S rRNA gene. However, because the 16S rRNA gene is not polymorphic enough, it is necessary to sequence a 1500 bp segment of this gene for accurate identification. Based on the alignment of previously determined rpoB of three Leptospira strains, we designed and tested a primer pair that enabled us to amplify and sequence a 600 bp segment of Leptospira rpoB. This segment was species-specific for the 16 species tested, but was unable to separate Leptospira interrogans serovars accurately. For the 11 L. interrogans serovars tested, only seven genotypes could be determined. We thus think that analysis of partial rpoB may be useful as an initial screening test for the identification of a new isolate of Leptospira and detection or identification of Leptospira in clinical or environmental samples, but not for serovar determination.