Identification of Comamonas species using 16S rRNA gene sequence

A bacterial strain Bz02 was isolated from a water sample collected from river Gomti at the Indian city of Lucknow. We characterized the strain using 16S rRNA sequence. Phylogenetic analysis showed that the strain formed a monophyletic clade with members of the genus Comamonas. The closest phylogenetic relative was Comamonas testosteroni with 95% 16S rRNA gene sequence similarity. It is proposed that the identified strain Bz02 be assigned as the type strain of a species of the genus Comamonas (Comamonas sp Bz02) based on 16S rRNA gene sequence search in Ribosomal Database Project, small subunit rRNA and large subunit rRNA databases together with the phylogenetic tree analysis. The sequence is deposted in GenBank with the accession number {"type":"entrez-nucleotide","attrs":{"text":"FJ211417","term_id":"209164434"}}FJ211417.     

DNA sequence of the 16S rRNA/23S rRNA intercistronic spacer of two rDNA operons of the archaebacterium Methanococcus vannielii

The DNA sequence of the spacer (plus flanking) regions separating the 16S rRNA and 23S rRNA genes of two presumptive rDNA operons of the archaebacterium Methanococcus vannielii was determined. The spacers are 156 and 242 base pairs in size and they share a sequence homology of 49 base pairs following the 3' terminus of the 16S rRNA gene and of about 60 base pairs preceding the 5' end of the 23S rRNA gene. The 242 base pair spacer, in addition contains a sequence which can be transcribed into tRNAAla, whereas no tRNA-like secondary structure can be delineated from the 156 base pair spacer region. Almost complete sequence homology was detected between the end of the 16S rRNA gene and the 3' termini of either Escherichia coli or Halobacterium halobium 16S rRNA, whereas the putative 5' terminal 23S rRNA sequence shared partial homology with E. coli 23S rRNA and eukaryotic 5.8S rRNA.     

菌种1137116S rRNA序列分析及鉴定

通过PCR方法扩增菌种11371的16S rRNA基因并测序,将序列提交GenBank(登录号:DQ531606),并与其他链霉菌属种进行比较,通过DNAStar软件得到菌种16S rRNA基因序列进化树。同时采用插片法、显微镜观察等方法对株菌11371进行形态特征、培养特征、生理生化特征鉴定。结果表明,11371的16S rRNA序列与其他链霉菌具有一定的同源性,结合生理、生化指标鉴定结果,进一步确定菌种为不吸水链霉菌一株新亚种(Streptomyces ahygroscopicus subsp.wuzhouensis n.sub-sp.),菌株11371 16S rRNA序列为GenBank中首例Streptomyces ahygroscopicus的16S rRNA序列。     

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。     

一株番茄青枯菌拮抗菌的鉴定及抗病效果初探

从形态、生理生化、16S rDNA3个方面确定了番茄青枯菌拮抗菌株3-1-16的分类地位。光学显微镜下观察到菌体为杆状细胞,革兰氏染色均匀,并可见菌体染成蓝紫色。透射电镜进一步观察到细胞内有许多颗粒状物质,无伴胞晶体。Biolog鉴定,3-1-16与巨大芽孢杆菌(Bacillus megaterium)具有最高相似率为98%。16S rRNA分析,3-1-16与巨大芽孢杆菌MO31同源性最高为99.4%。聚类分析显示3-1-16与3株巨大芽孢杆菌聚成一支,支持度为100%。生理生化特征及培养特征测定结果表明,菌株3-1-16鉴定为巨大芽孢杆菌(Bacillus megaterium)。盆栽试验表明该菌株对番茄青枯病防病效果达到81.3%。     

Analysis of DNA encoding 23S rRNA and 16S–23S rRNA intergenic spacer regions from Plesiomonas shigelloides

Amplification of the gene encoding 23S rRNA of Plesiomonas shigelloides by polymerase chain reaction (PCR), with primers complementary to conserved regions of 16S and the 3' end of 23S rRNA genes, resulted in a DNA fragment of approximately 3 kb. This fragment was cloned in Escherichia coli and its nucleotide sequence determined. The region encoding 23S rRNA shows high homology with the published sequences of 23S rRNA from other members of the gamma division of Proteobacteria. The sequence of the intergenic spacer region, between the 16S and 23S rRNA genes, was determined in a further two clones. In one the sequence of a single tRNA(Glu) was found which was absent from the other two. This variation in sequence suggests that the different clones may be derived from different ribosomal RNA operons.     

Characterization of the genus Bifidobacterium by automated ribotyping and 16S rRNA gene sequences

In order to characterize the genus Bifidobacterium, ribopatterns and approximately 500 bp (Escherichia coli positions 27 to 520) of 16S rRNA gene sequences of 28 type strains and 64 reference strains of the genus Bifidobacterium were determined. Ribopatterns obtained from Bifidobacterium strains were divided into nine clusters (clusters I-IX) with a similarity of 60%. Cluster V, containing 17 species, was further subdivided into 22 subclusters with a similarity of 90%. In the genus Bifidobacterium, four groups were shown according to Miyake et al.: (i) the Bifidobacterium longum infantis-longum-suis type group, (ii) the B. catenulatum-pseudocatenulatum group, (iii) the B. gallinarum-saeculare-pullorum group, and (iv) the B. coryneforme-indicum group, which showed higher than 97% similarity of the 16S rRNA gene sequences in each group. Using ribotyping analysis, unique ribopatterns were obtained from these species, and they could be separated by cluster analysis. Ribopatterns of six B. adolescentis strains were separated into different clusters, and also showed diversity in 16S rRNA gene sequences. B. adolescentis consisted of heterogeneous strains. The nine strains of B. pseudolongum subsp. pseudolongum were divided into five subclusters. Each type strain of B. pseudolongum subsp. pseudolongum and B. pseudolongum subsp. globosum and two intermediate groups, which were suggested by Yaeshima et al., consisted of individual clusters. B. animalis subsp. animalis and B. animalis subsp. lactis could not be separated by ribotyping using Eco RI. We conclude that ribotyping is able to provide another characteristic of Bifidobacterium strains in addition to 16S rRNA gene sequence phylogenetic analysis, and this information suggests that ribotyping analysis is a useful tool for the characterization of Bifidobacterium species in combination with other techniques for taxonomic characterization.     

A pseudogene cluster in the leader region of the Euglena chloroplast 16S-23S rRNA genes.

The nucleotide sequence of a region (leader region) preceding the 5'-end of 16S-23S rRNA gene region of Euglena gracilis chloroplast DNA was compared with the homologous sequences that code for the 16S-23S rRNA operons of Euglena and E. coli. The leader region shows close homology in sequence to the 16S-23S rRNA gene region of Euglena (Orozco et al. (1980) J. Biol.Chem. 255, 10997-11003) as well as to the rrnD operon of E. coli, suggesting that it was derived from the 16S-23S rRNA gene region by gene duplication. It was shown that the leader region had accumulated nucleotide substitutions at an extremely rapid rate in its entirety, similar to the rate of tRNAIle pseudogene identified in the leader region. In addition, the leader region shows an unique base content which is quite distinct from those of 16S-23S rRNA gene regions of Euglena and E. coli, but again is similar to that of the tRNAIle pseudogene. The above two results strongly suggest that the leader region contains a pseudogene cluster which was derived from a gene cluster coding for the functional 16S-23S rRNA operon possibly by imperfect duplication during evolution of Euglena chloroplast DNA.     

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。     

Genetic diversity of Acacia tortilis ssp. raddiana rhizobia in Tunisia assessed by 16S and 16S-23S rDNA genes analysis

AIMS: In order to understand the genetic diversity of Acacia tortilis ssp. raddiana-rhizobia in Tunisia, isolates from nine geographical locations were obtained and analysed. METHODS AND RESULTS: Characterization using restriction fragment length polymorphism analysis (RFLP) of PCR-amplified 16S rRNA gene and the intergenic spacer (IGS) between the 16S and 23S rRNA genes was undertaken. Symbiotic efficiency of the strains was also estimated. Analysis of the 16S rRNA by PCR-RFLP showed that the isolates were phylogenetically related to Ensifer ssp., Rhizobium tropicii-IIA, and Rhizobium tumefaciens species. Analysis of 16S-23S spacer by PCR-RFLP showed a high diversity of these rhizobia and revealed eleven additional groups, which indicates that these strains are genetically very diverse. Full 16S rRNA gene-sequencing showed that the majority of strains form a new subdivion inside the genera Ensifer, with Ensifer meliloti being its nearest neighbour. Nodulation test performed on the plant host demonstrated differences in the infectivity among the strains. CONCLUSION: Rhizobial populations that nodulate specifically and efficiently Acacia tortilis ssp. raddiana in representative soils of Tunisia is dominated by E. meliloti-like genomospecies. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides the first clear characterization and symbiotic efficiency data of rhizobia strains nodulating A. tortilis in Tunisia.     

A one-step reaction for the rapid identification of Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti using oligonucleotide primers designed from the 16S-23S rRNA intergenic sequences

Aims:  Species-specific primers targeting the 16S–23S ribosomal DNA (rDNA) intergenic spacer region (ISR) were designed to rapidly discriminate between Lactobacillus mindensis , Lactobacillus panis , Lactobacillus paralimentarius , Lactobacillus pontis and Lactobacillus frumenti species recently isolated from French sourdough.
Methods and Results:  The 16S–23S ISRs were amplified using primers 16S/p2 and 23S/p7, which anneal to positions 1388–1406 of the 16S rRNA gene and to positions 207–189 of the 23S rRNA gene respectively, Escherichia coli numbering (GenBank accession number V00331 ). Clone libraries of the resulting amplicons were constructed using a pCR2·1 TA cloning kit and sequenced. Species-specific primers were designed based on the sequences obtained and were used to amplify the 16S–23S ISR in the Lactobacillus species considered. For all of them, two PCR amplicons, designated as small ISR (S-ISR) and large ISR (L-ISR), were obtained. The L-ISR is composed of the corresponding S-ISR, interrupted by a sequence containing tRNA^Ile and tRNA^Ala genes. Based on these sequences, species-specific primers were designed and proved to identify accurately the species considered among 30 reference Lactobacillus species tested.
Conclusions:  Designed species-specific primers enable a rapid and accurate identification of L. mindensis , L. paralimentarius , L. panis , L. pontis and L. frumenti species among other lactobacilli.
Significance and Impact of the Study:  The proposed method provides a powerful and convenient means of rapidly identifying some sourdough lactobacilli, which could be of help in large starter culture surveys.     

Variation in the 16S-23S rRNA intergenic spacer regions in Vibrio parahaemolyticus strains are due to indels nearby their tRNAGlu

Vibrio parahaemolyticus contains 11 rRNA operons each including one of six 16S-23S rRNA gene intergenic spacer classes differing in size and nucleotide sequence. Some of the spacer classes may differ between isolates. We observed that the differences in the spacers between isolates are generally in two spacer classes present in single copies in the genome, one class containing tRNA(Ala) and tRNA(Glu) and the other tRNA(Glu) exclusively. Moreover, these differences are due to indels located nearby their tRNA(Glu). Comparison of the nucleotide sequence between spacer classes suggests that intragenomic nonreciprocal recombination causes the size variations observed in the spacer regions of V. parahaemolyticus strains.     

基于线粒体16S rRNA基因对中国狼蛛科(Lycosidae)系统发育研究

将自测的中国狼蛛科Lycosidae4亚科6属26种和从GenBank中检索到的北美2种豹蛛的mtD-NA-16S rRNA序列进行比较;以漏斗蛛科1种蜘蛛作为外群,对碱基序列的组成和遗传距离进行了分析,采用Bayesian方法和最大简约法(MP)构建分子系统树。研究结果表明:16SrRNA基因的部分序列为340bp到360bp,A T含量平均为75%,存在较强的A T含量偏向性;序列共有157个碱基存在变异,其中79个简约信息位点。狼蛛科各属间的遗传距离介于0.026￣0.200之间。2种建树方法均表明:科内的属及属内的种优先聚在一起;水狼蛛属相对马蛛属是狼蛛科中较为原始的类群,分化较早;獾蛛属作为1个单系群与熊蛛属合为1个并系,属于狼蛛亚科。狼蛛科6属间的分子系统关系为(Pirata(Hippasa(Trochsa Arctosa(Pardosa Wadicosa))))。     

新疆罗布泊周边地区极端环境嗜盐菌的研究

为了研究分析新疆罗布泊周边地区pH值5-6的盐湖嗜盐古菌资源。从湖中分离筛选出一批嗜盐古菌,对其进行了生理生化特性研究,发现其中6株菌的生理特性和产酶特性比较特殊,并采用PCR方法扩增出其16SrRNA基因（16S rDNA）,并测定了基因的核苷酸序列。基于16S rDNA序列的同源性比较以及16S rDNA序列的系统发育学研究表明,菌株B20-RDX是盐盒菌属Haloarchaeon属中新种成员,GenBank登录号为FJ561285,该菌株为革兰氏阴性菌,最适盐浓度25%,最适pH 8.0,能产过氧化氢酶、淀粉酶,对四环素有抗性,能利用精氨酸和丁二酸盐。迄今为止,国内极少有关罗布泊周边地区极端环境微生物研究的报道,该研究可为今后研究同类极端环境中新的物种资源开发应用以及微生物多样性研究提供素材和参考。     

日本囊对虾4个地理群体线粒体16S rRNA序列及遗传结构分析

对我国东南沿海日本囊对虾(Marsupenaeus japonicus)的4个地理群体广东群体(GD)、台湾群体(TW)、福建群体(FJ)和浙江群体(ZJ)的线粒体16S rRNA基因片段进行PCR扩增,对产物进行测序后分析。经比对获得470bp的核苷酸分析序列,发现了16个变异位点,得到了10种单倍型。广东、台湾、福建和浙江群体的核苷酸多样性依次分别为0.0008、0.0010、0.0051、0.0015,各群体均存在独有的单倍型和共有单倍型。群体遗传距离分析表明各群体间保持着一定的遗传差异,其中福建群体与其他群体之间存在着较远的遗传距离并保持了较高的遗传多样性。另外,利用其423bp的16S rRNA同源序列探讨了对虾科6个属共12种对虾的系统进化关系,囊对虾属与沟对虾属亲缘关系较近聚为一支,其他4个属的10种对虾聚为一支。     

Molecular cloning and comparative sequence analyses of rRNA operons in Streptomyces nodosus ATCC 14899.

The genome of Streptomyces nodosus contains six ribosomal RNA (rRNA) operons. Four of the rRNA operons; rrnB, rrnD, rrnE and rrnF were cloned. We have completely sequenced all four operons, including a region 750 base pairs (bp) upstream of the 16S rRNA gene. The three rRNA genes present in each operon were closely linked in the order 16S-23S-5S. A sequence comparison of the four operons showed more than 99% sequence similarity between the corresponding 16S and 23S rRNA genes, and more than 97% similarity between 5S rRNA genes. The sequence differences observed between 23S rRNA genes appeared to be localized in two specific regions. Substantial sequence differences were found in the region upstream of the 16S rRNA gene as well as in the internal transcribed spacers. No tRNA gene was found in the 16S-23S spacer regions.     

Suitability of partial 16S ribosomal RNA gene sequence analysis for the identification of dangerous bacterial pathogens

AIMS: In a bioterrorism event a rapid tool is needed to identify relevant dangerous bacteria. The aim of the study was to assess the usefulness of partial 16S rRNA gene sequence analysis and the suitability of diverse databases for identifying dangerous bacterial pathogens. METHODS AND RESULTS: For rapid identification purposes a 500-bp fragment of the 16S rRNA gene of 28 isolates comprising Bacillus anthracis, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Yersinia pestis, and eight genus-related and unrelated control strains was amplified and sequenced. The obtained sequence data were submitted to three public and two commercial sequence databases for species identification. The most frequent reason for incorrect identification was the lack of the respective 16S rRNA gene sequences in the database. CONCLUSIONS: Sequence analysis of a 500-bp 16S rDNA fragment allows the rapid identification of dangerous bacterial species. However, for discrimination of closely related species sequencing of the entire 16S rRNA gene, additional sequencing of the 23S rRNA gene or sequencing of the 16S-23S rRNA intergenic spacer is essential. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides comprehensive information on the suitability of partial 16S rDNA analysis and diverse databases for rapid and accurate identification of dangerous bacterial pathogens.     

Sequence Analysis and Comparison of 16S rRNA, 23S rRNA and 16S/23S Intergenic Spacer Region of Greening Bacterium Associated with Yellowing Disease (Huanglongbing) of Kinnow Mandarin

High incidence (up to 40%) of symptoms of yellowing and yellow mottling was observed in 5–8 years old orchards of kinnow mandarin {Citrus reticulate Balanco (‘King’ × ‘Willow mandarin’)} in the Punjab state of India during a survey in January 2007. These symptoms are often confused with nutrient deficiency and other stress related disorders. However, a greening bacterium has been attributed to cause the disease. The disease was graft transmissible and sequencing of 16S rRNA, 16S/23S intergenic spacer region and 23S rRNA of the greening bacterium associated with yellowing disease in kinnow mandarin confirmed it to be Candidatus Liberibacter asiaticus (‘Ca L. asiaticus’) showing maximum identity of 95.9% with ‘Ca L. asiaticus’ from USA and Brazil in 16S rRNA. The study indicates definite association of ‘Ca L. asiaticus’ with yellowing/chlorotic mottling symptoms of greening disease of kinnow mandarin in Punjab state of India.