Presence of a novel 16S-23S rRNA gene intergenic spacer insert in Bradyrhizobium canariense strains

Seven slow-growing bacterial strains isolated from root nodules of yellow serradella (Ornithopus compressus) that originated from Asinara Island on North Western Sardinia in Italy were characterized by partial 16S rRNA gene and intergenic spacer (ITS) sequencing as well as amplified fragment length polymorphism (AFLP) genomic fingerprinting. The results indicated that the O. compressus isolates belong to the Bradyrhizobium canariense species. The analysis of ITS sequences divided the branch of B. canariense strains into two statistically separated groups (ITS clusters I and II). All the strains in ITS cluster I showed the presence of unique oligonucleotide insert TTAGAGACTTAGGTTTCTK. This insert was neither found in other described species of the family Rhizobiaceae nor in any other bacterial families and can be used as a natural and high selective genetic marker for ITS cluster I of B. canariense strains. ITS grouping of O. compressus isolates was supported by the unweighted pair group method with arithmetic averages cluster analysis of their AFLP patterns, suggesting that the strains of ITS cluster II were genetically closer to each other than to isolates from the ITS cluster I. A taxonomic importance is supposed of the revealed 19 bp ITS insert for an intraspecific division within high heterogeneous B. canariense species.     

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.     

空气环境中几种细菌检测与16S rRNA测序鉴定

采集空气环境微生物,根据菌落和革兰氏染色特征,分离5株细菌。提取分离株DNA,采用设计的16SrRNA引物扩增DNA片段;纯化PCR产物,进行测序反应并再纯化,上机读序。拼接与校对DNA序列,在NCBI网站进行Blast,鉴定细菌型别。5株菌株分别是表皮葡萄球菌、头状葡萄球菌、施氏假单胞菌、溶血葡萄球菌和大肠杆菌;其中溶血葡萄球菌是致病菌,其他4株是条件致病菌。这提示,应警惕空气环境中致病菌的感染。     

Bacterial phylogeny based on 16S and 23S rRNA sequence analysis

Abstract: Molecular phylogeny increasingly supports the understanding of organismal relationships and provides the basis for the classification of microorganisms according to their natural affiliations. Comparative sequence analysis of ribosomal RNAs or the corresponding genes currently is the most widely used approach for the reconstruction of microbial phylogeny. The highly and less conserved primary and higher order structure elements of rRNAs document the history of microbial evolution and are informative for definite phylogenetic levels. An optimal alignment of the primary structures and a careful data selection are prerequisites for reliable phylogenetic conclusions. rRNA based phylogenetic trees can be reconstructed and the significance of their topologies evaluated by applying distance, maximum parsimony and maximum likelihood methods of phylogeny inference in comparison, and by fortuitous or directed resampling of the data set. Phylogenetic trees based on almost equivalent data sets of bacterial 23S and 16S rRNAs are in good agreement and their overall topologies are supported by alternative phylogenetic markers such as elongation factors and ATPase subunits. Besides their phylogenetic information content, the differently conserved primary structure regions of rRNAs provide target sites for specific hybridization probes which have been proven to be powerful tools for the identification of microbes on the basis of their phylogenetic relationships.     

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.     

Taxonomic re-evaluation of Aphanizomenon flos-aquae NH-5 based on morphology and 16S rRNA gene sequences

The taxonomy of Aphanizomenon flos-aquae strain NH-5, a producer of cyanotoxins, was re-evaluated by comparison with six other Aphanizomenon strains using morphological characteristics and 16S rRNA gene sequences. Strain NH-5 was concluded to be improperly identified as Aph. flos-aquae based upon (1) lack of bundle formation in the trichomes, (2) location of akinetes next to heterocytes, (3) lower similarities (less than 97.5%) in the 16S rRNA gene sequences relative to Aph. flos-aquae strains, and (4) comparison within a phylogenetic tree constructed from 16S rRNA gene sequences. The Aphanizomenon strains investigated in this study are classified to four morphological groups as described by the classical taxonomy of Komárek & Kovácik (1989). This classification was supported from the phylogenetic results of 16S rRNA gene sequences. This study also discusses the generic boundaries between Aphanizomenon and Anabaena.     

三角帆蚌五个野生群体线粒体DNA 16S rRNA遗传特性

对中国主要淡水湖泊(鄱阳湖、洞庭湖、太湖、洪泽湖及巢湖)三角帆蚌5个野生群体的线粒体DNA 16S rRNA基因片段进行了扩增和测序,得到473bp的碱基序列,没有发现插入/缺失突变的核苷酸位点。检测到了32个多态性核苷酸位点,共7种单倍型。鄱阳湖群体的222(C→G)和325(A→G)位点,太湖群体的233(A→G)位点,巢湖群体的40(A→G)、138(A→T)和294(C→T)位点,洪泽湖群体的241(A→C)位点的变异可以作为区分群体分子遗传标记位点。洞庭湖群体未发现特异位点。在5个群体间鄱阳湖群体多态性位点、核苷酸多态性、单倍型多态性和单倍型数量4个指标都最高,表明鄱阳湖群体具有最为丰富的遗传结构,遗传变异最大,可作为三角帆蚌选育的基础群体。     

从线粒体基因探讨中国大头蛙群的分类及其属内地位

Partial sequences of the mitochondrial 12S rRNA and 16S rRNA gene were determined for 8 populations of three species of Chinese Limnonectes, and aligned with the published sequences of Limnonectes from other parts of the world. When Nanorana parker, Paa boulengeri, Fejervarya limnocharis and Hoplobatrachus rugulosus was used as outgroup taxa (Accession Nos. AY158705, AY313685, AF206111, AF206491, AY322311). The sequences of the 12S rRNA and 16S rRNA genes totaled 950 nueleotide positions with gaps including 510 variable sites. We reconstructed phylogenetie trees using Clustal X 1.8, Mega 2.1 and PHYLIP 3.5e software, and using the maximum parsimony and maximum likelihood methods, respectively. Our analyses suggest that these fanged frogs from China are another monophyletie group in addition to the four monophyletie groups identified by previous studies. The Chinese Limnonectes were grouped into three elades (BCL 55% ). The first elade contains one species (BCL 100% ), from a population of Limnoneetes fragilis from Hainan Province. The second contains four individuals (BCL 100% ), i. e. two populations of Limnonectes kuhlii from Yunnan Province. The third contains one species (BCL 100% ), i. e. five populations of Limnonectes fujianensis from Fujian Province and 1 from Taiwan Province. The resulted phylogenetie trees indicate L. fragilis is basal to L. kuhlii L. fujianensis 。     

Bacterial diversity in two coastal lagoons deduced from 16S rDNA PCR amplification and partial sequencing

Abstract: Amplification and sequence analysis of the 16S rRNA genes from DNA samples extracted directly from the environment allows the study of microbial diversity in natural ecosystems without the need for cultivation. In this study this methodology has been applied to two coastal lagoons. Activity and numbers of heterotrophic bacteria have indicated that, as expected, Prévost lagoon (located on the French Mediterranean coast) is more eutrophic than that of the Arcachon Bay (French Atlantic coast). Analysis of partial 16S rRNA gene sequences revealed that, in both environments, a relatively large number of clones related to Cytophaga/Flexibacter/Bacteroides as well as to α-Proteobacteria were found. One hundred percent similarity with the sequences of the data bases were not found for any of the more than a hundred clones studied, in fact for most clones maximum similarity was below 95% for the approx. 200 bases sequenced. Similarity was not higher with any of the sequences found for the 14 isolates (pure cultures) obtained from the same samples. Redundancy, i.e. number of identical sequences, was higher in the samples from Arcachon. In addition, sequences related to representatives of ten major phylogenetic branches of Bacteria were obtained from Prévost lagoon; however only five branches were represented by the data from Arcachon. These findings indicated a higher bacterial phylogenetic diversity in the Prévost lagoon.     

16S rRNA gene sequencing on a benchtop sequencer: accuracy for identification of clinically important bacteria

Aims

Test the choice of 16S rRNA gene amplicon and data analysis method on the accuracy of identification of clinically important bacteria utilizing a benchtop sequencer.

Methods and Results

Nine 16S rRNA amplicons were tested on an Ion Torrent PGM to identify 41 strains of clinical importance. The V1–V2 region identified 40 of 41 isolates to the species level. Three data analysis methods were tested, finding that the Ribosomal Database Project's SequenceMatch outperformed BLAST and the Ion Reporter Metagenomics analysis pipeline. Lastly, 16S rRNA gene sequencing mixtures of four species through a six log range of dilution showed species were identifiable even when present as 0·1% of the mixture.

Conclusions

Sequencing the V1–V2 16S rRNA gene region, made possible by the increased read length Ion Torrent PGM sequencer's 400 base pair chemistry, may be a better choice over other commonly used regions for identifying clinically important bacteria. In addition, the SequenceMatch algorithm, freely available from the Ribosomal Database Project, is a good choice for matching filtered reads to organisms. Lastly, 16S rRNA gene sequencing's sensitivity to the presence of a bacterial species at 0·1% of a mixture suggests it has sufficient sensitivity for samples in which important bacteria may be rare.

Significance and Impact of the Study

We have validated 16S rRNA gene sequencing on a benchtop sequencer including simple mixtures of organisms; however, our results highlight deficits for clinical application in place of current identification methods.     

Comparison of 16S rRNA sequencing with conventional and commercial phenotypic techniques for identification of enterococci from the marine environment

AIMS: To compare accuracy of genus and species level identification of presumptive enterococci isolates from the marine environment using conventional biochemical testing, four commercial identification systems and 16S rRNA sequence analysis. METHODS AND RESULTS: Ninety-seven environmental bacterial isolates identified as presumptive enterococci on mEI media were tested using conventional and Enterococcus genus screen biochemical tests, four commercial testing systems and 16S rRNA sequencing. Conventional and Enterococcus genus screen biochemical testing, 16S rRNA sequencing and two commercial test systems achieved an accuracy of > or = 94% for Enterococcus genus confirmation. Conventional biochemical testing and 16S rRNA sequencing achieved an accuracy of > or = 90% for species level identification. CONCLUSIONS: For confirmation of Enterococcus genus from mEI media, conventional or genus screen biochemical testing, 16S rRNA sequencing and the four commercial systems were correct 79-100% of the time. For speciation to an accuracy of 90% or better, either conventional biochemical testing or 16S rRNA sequencing is required. SIGNIFICANCE AND IMPACT OF THE STUDY: Accurate identification of presumptive environmental Enterococcus isolates to genus and species level is an integral part of laboratory quality assurance and further characterization of Enterococcus species from pollution incidents. This investigation determines the ability of six different methods to correctly identify environmental isolates.     

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.     

Detection of Laribacter hongkongensis using species-specific duplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA intergenic spacer region (ISR)

Aims: For the rapid detection of Laribacter hongkongensis, which is associated with human community‐acquired gastroenteritis and traveller’s diarrhoea, we developed a duplex species‐specific PCR assay. Methods and Results: Full‐length of the 16S–23S rRNA intergenic spacer region (ISR) sequences of 52 L. hongkongensis isolates were obtained by PCR‐based sequencing. Two species‐specific primer pairs targeting 16S rRNA gene and ISR were designed for duplex PCR detection of L. hongkongensis. The L. hongkongensis species‐specific duplex PCR assay showed 100% specificity, and the minimum detectable level was 2·1 × 10^?2 ng μl^?1 genomic DNA which corresponds to 5000 CFU ml^?1. Conclusions: The high specificity and sensitivity of the assay make it suitable for rapid detection of L. hongkongensis. Significance and Impact of the Study: This species‐specific duplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify L. hongkongensis and may have applications in both clinical and environmental microbiology.     

Phylogenetic analysis of the family Rhizobiaceae and related bacteria by sequencing of 16S rRNA gene using PCR and DNA sequencer

Abstract The 16S rRNA gene sequences of 19 strains covering 97% of the molecules were determined for the members of the family Rhizobiaceae and related bacteria by PCR and DNA sequencer. The three biovars of Agrobacterium were located separately, whereas Agrobacterium rubi clustered with A. tumefaciens . Phylogenetic locations for the species of the genera Rhizobium, Sinorhizobium, Agrobacterium, Phylobacterium, Mycoplana (M. dimorpha), Ochrobactrum, Brucella and Rochalimaea (a rickettsia) were intermingled with each other with the similarity values higher than 92%. The family Rhizobiaceae should be redefined including the above-mentioned genera despite the ability for plant association and nitrogen fixation. Bradyrhizobium japonicum and Mycoplana bullata were far remote from the other species and should be excluded from this family.     

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。     

鼠三毛滴虫新疆灰仓鼠分离株形态学观察及其16SrRNA基因分析

目的：对从新疆实验动物研究中心饲养的封闭群灰仓鼠体内分离到的1株鞭毛虫进行形态学鉴定及基因鉴定。方法取灰仓鼠回盲部内容物进行直接涂片和常规姬姆萨染色后镜检观察,提取虫体总DNA,PCR扩增该鞭毛虫的16S rRNA基因,测序后与国外已报道的鞭毛虫进行核酸同源性分析,并应用MEGA5.22软件绘制系统发育进化树。结果形态学观察表明分离到的鞭毛虫为鼠三毛滴虫。测序后核酸同源性分析表明鼠三毛滴虫新疆灰仓鼠分离株16S rRNA序列与国外已报道的三毛滴虫高度同源。系统发育进化树表明鼠三毛滴虫新疆灰仓鼠分离株序列与已报道的鼠三毛滴虫16S rRNA（序列号AY886846.1）位于同一进化分支,与其他相关三毛滴虫亲缘关系较远。结论形态学鉴定和16 S rRNA基因分析表明,此次从新疆实验动物研究中心饲养的封闭群灰仓鼠体内分离到的鞭毛虫为鼠三毛滴虫。