Phylogenetic study and identification of human pathogenic Vibrio species based on partial hsp60 gene sequences

The use of hsp60 gene sequences for phylogenetic study and identification of pathogenic marine vibrios was investigated. A 600-bp partial hsp60 gene was amplified by PCR and sequenced from 29 strains representing 15 Vibrio species within the family Vibrionaceae. Sequence comparison of the amplified partial hsp60 gene revealed 71-82% sequence identity among different Vibrio species and 96-100% sequence identity among epidemiologically distinct strains with the same species designation. This degree of discrimination allows unambiguous differentiation of all Vibrio species included in the current study from each other, as well as from Aeromonas hydrophila and Plesiomonas shigelloides, which are often misidentified as Vibrio species by conventional biochemical methods. Based on the hsp60 gene sequences, two previously unidentified shrimp isolates were found to be more closely related to Vibrio alginolyticus (93-94% sequence identity) than to Vibrio parahaemolyticus (89% sequence identity), whereas 16S rRNA gene analysis was unable to differentiate among these closely related species (95-97% sequence identity). Our results indicate that the hsp60 gene may be a useful alternative target for phylogenetic analysis and species identification of marine Vibrios to complement more conventional identification systems.     

Development and evaluation of loop-mediated isothermal amplification for rapid detection of Haemophilus parasuis

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions and has a high specificity and efficiency. We developed a LAMP assay targeting the 16S rRNA gene for rapid detection of Haemophilus parasuis. The results obtained from testing 31 H. parasuis strains and 28 other bacterial species strains showed that LAMP was as specific as, and more sensitive than, nested PCR. Fifty-five lung samples were collected from 55 healthy pigs. All the samples were negative for H. parasuis by bacterial isolation, nested PCR and LAMP, respectively. In addition, 122 lung samples were collected from 122 pigs with apparent respiratory problems. Sixty-five were positive by bacterial isolation. All the samples that were positive by bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated higher sensitivity than nested PCR, picking up 16 additional cases. The LAMP assay also gave a same result compared with the nested PCR when the two assays were used, respectively, to detect H. parasuis from samples obtained from experimentally infected pigs. We concluded that LAMP is a highly sensitive and reliable method for detection of H. parasuis infection.     

Bombiscardovia coagulans gen. nov., sp. nov., a new member of the family Bifidobacteriaceae isolated from the digestive tract of bumblebees

One hundred and eighty-seven fructose-6-phosphate phosphoketolase positive strains were isolated from the digestive tract of three different bumblebee species. Analyses of the partial 16S rRNA gene sequences of the representative strains showed only 92.8% and 92.5% similarity to Bifidobacterium coryneforme YIT 4092(T) and Bifidobacterium indicum JCM 1302(T), 92.2% similarity to Alloscardovia omnicolens CCUG 18650 and slightly reduced similarity of 91% to other members of the family Bifidobacteriaceae. On the other hand, analyses of the partial heat-shock protein 60 (hsp60) gene sequence revealed that the proposed type strain BLAPIII-AGV(T) was affiliated only to the 60 kDa chaperonin sequence of uncultured bacteria from human vagina (79-80%) and the hsp60 gene sequence of A. omnicolens CCUG 31649(T) (75.5%). The peptidoglycan type was A4α with an l-Lys-d-Asp interpeptide bridge. The polar lipids contained diphosphatidylglycerol, an unknown phospholipid, six glycolipids and two phosphoglycolipids. The major fatty acids were C(18:1), C(20:0) and C(18:0). These and other analyses indicated that the isolates represented a new genus within the family Bifidobacteriaceae. This observation was further substantiated by determination of the DNA G+C contents (46.1-47.1 mol%). Affinity of the strains to some scardovial genera (Aeriscardovia, Alloscardovia and Metascardovia) was also confirmed by their ability to grow under aerobic conditions. Besides the above mentioned differences, Bombiscardovia coagulans was found to differ from all scardovial genera in the ability to grow at temperatures as low as 5°C, which was another major phenotypically different characteristic of this new member of the family Bifidobacteriaceae. Hence, on the basis of phylogenetic analyses using partial 16S rRNA and hsp60 gene sequence data, and the temperature related phenotypic difference, we propose a novel taxa, B. coagulans gen. nov., sp. nov. (type strain=BLAPIII-AGV(T)=DSM 22924(T)=ATCC BAA-1568(T)).     

副猪嗜血杆菌aroA基因鉴定及遗传进化分析

[目的]细菌aroA基因参与芳香族氨基酸的生物合成,被成功应用于细菌分类和基因失活致弱突变菌株的构建.副猪嗜血杆菌(Hps)是感染猪出现多发性浆膜炎和关节炎的一种病原细菌,鉴定该菌aroA全基因序列将有助于鉴定遗传进化关系和突变分析.[方法]利用PCR和细菌基因组步移技术鉴定Hps的aroA基因序列,进而对不同血清型菌株该基因序列进行鉴定,并与其它革兰氏阴性细菌进行比对和遗传进化分析.[结果]自Hps血清5型基因组DNA中获得包含完整aroA基因的3.7 kb基因片段,其中aroA基因全长1314 bp,编码产物长度437 aa,分子量大小47.9 kDa,该基因上游为磷酸烯醇式丙酮酸羧化酶基因.自本试验选择的Hps不同血清型菌株中均可扩增出包含完整aroA基因的1476 bp片段,且这些不同血清型菌株间核酸序列同源性在97.7%以上.Hps血清5型aroA基因序列与巴氏杆菌科其它成员核酸序列同源性为70.6%-78.9%,与E.coli和S.typhi-murium的同源性分别为66.4%和67.2%.[结论]本试验首次对Hps的15个血清型国际参考菌株及地方分离株aroA全基因序列进行了鉴定,序列比较结果显示aroA基因在革兰氏阴性细菌中具有较高的同源性.aroA基因鉴定对构建基因失活突变菌株以研究Hps生物学特性奠定了基础.     

两株对虾幼体弧菌病病原的分离和鉴定

从患弧菌病的凡纳滨对虾（Litopenaeus vannamei）幼体中分离到两株病原菌zouA和zouB,常规形态和生理生化试验表明均为弧菌属菌种,弧菌编码鉴定系统分别鉴定为溶藻弧菌（Vibrioatginolyticus）和副溶血弧菌（V.parahaemolyticus）。副溶血弧菌R72H序列检测结果进一步证实菌株zouB为副溶血弧菌。对菌株zouA的16S rRNA基因序列分析表明该菌株与溶藻弧菌、副溶血弧菌等弧菌的相似性均高于98％,相互间不能区分;HSP60基因序列分析表明该菌株与溶藻弧菌相似性达98％以上,而与所有其它弧菌的相似性不到92％。结合表型和分子特征的鉴定结果,菌株zouA和zouB分别被鉴定为溶藻弧菌和副溶血弧菌。     

两株对虾幼体弧菌病病原的分离和鉴定

从患弧菌病的凡纳滨对虾(Litopenaeus vannamei)幼体中分离到两株病原菌zouA和zouB, 常规形态和生理生化试验表明均为弧菌属菌种, 弧菌编码鉴定系统分别鉴定为溶藻弧菌(Vibrio alginolyticus)和副溶血弧菌(V. parahaemolyticus)。副溶血弧菌R72H序列检测结果进一步证实菌株zouB为副溶血弧菌。对菌株zouA的16S rRNA基因序列分析表明该菌株与溶藻弧菌、副溶血弧菌等弧菌的相似性均高于98%, 相互间不能区分; HSP60基因序列分析表明该菌株与溶藻弧菌相似性达98%以上, 而与所有其它弧菌的相似性不到92%。结合表型和分子特征的鉴定结果, 菌株zouA和zouB分别被鉴定为溶藻弧菌和副溶血弧菌。     

Genotypic characterization of Lactic acid bacteria in gut microbiome of freshwater fish

The present study focused on identification and genotypic characterization of Lactic acid bacteria (LAB) in the intestine of freshwater fish. 76 strains of LAB were isolated and identified by 16S rRNA gene sequences and hsp60 gene sequences as different strains of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus fermentum, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus brevis, Lactobacillus reuteri, Lactobacillus salivarius, Pediococcus pentosaceus, Pediococcus acidilactici, Weissella paramesenteroides, Weissella cibaria, Enterococcus faecium, and Enterococcus durans. The hsp60 gene showed a higher level of sequence variation among the isolates examined, with lower interspecies sequence similarity providing more resolutions at the species level than the 16S rRNA gene. Phylogenetic tree derived from hsp60 gene sequences with higher bootstrap values at the nodal branches was more consistent as compared to phylogenetic tree constructed from 16S rRNA gene sequences. Closely related species L. plantarum and L. pentosus as well as species L. delbrueckii subsp. bulgaricus and L. fermentum were segregated in different cluster in hsp60 phylogenetic tree whereas such a distribution was not apparent in 16S rRNA phylogenetic tree. In silico restriction analysis revealed a high level of polymorphism within hsp60 gene sequences. Restriction pattern with enzymes AgsI and MseI in hsp60 gene sequences allowed differentiation of all the species including closely related species L. plantarum and L. pentosus, E. faecium and E. durans. In general, hsp60 gene with higher evolutionary divergence proved to be a better phylogenetic marker for the group LAB.     

Lactobacillus Strain Diversity Based on Partial hsp60 Gene Sequences and Design of PCR-Restriction Fragment Length Polymorphism Assays for Species Identification and Differentiation

A phylogenetic tree showing diversities among 116 partial (499-bp) Lactobacillus hsp60 (groEL, encoding a 60-kDa heat shock protein) nucleotide sequences was obtained and compared to those previously described for 16S rRNA and tuf gene sequences. The topology of the tree produced in this study showed a Lactobacillus species distribution similar, but not identical, to those previously reported. However, according to the most recent systematic studies, a clear differentiation of 43 single-species clusters was detected/identified among the sequences analyzed. The slightly higher variability of the hsp60 nucleotide sequences than of the 16S rRNA sequences offers better opportunities to design or develop molecular assays allowing identification and differentiation of either distant or very closely related Lactobacillus species. Therefore, our results suggest that hsp60 can be considered an excellent molecular marker for inferring the taxonomy and phylogeny of members of the genus Lactobacillus and that the chosen primers can be used in a simple PCR procedure allowing the direct sequencing of the hsp60 fragments. Moreover, in this study we performed a computer-aided restriction endonuclease analysis of all 499-bp hsp60 partial sequences and we showed that the PCR-restriction fragment length polymorphism (RFLP) patterns obtainable by using both endonucleases AluI and TacI (in separate reactions) can allow identification and differentiation of all 43 Lactobacillus species considered, with the exception of the pair L. plantarum/L. pentosus. However, the latter species can be differentiated by further analysis with Sau3AI or MseI. The hsp60 PCR-RFLP approach was efficiently applied to identify and to differentiate a total of 110 wild Lactobacillus strains (including closely related species, such as L. casei and L. rhamnosus or L. plantarum and L. pentosus) isolated from cheese and dry-fermented sausages.     

The dnaJ gene as a novel phylogenetic marker for identification of Vibrio species

The utility of the dnaJ gene for identifying Vibrio species was investigated by analyzing dnaJ sequences of 57 type strains and 22 clinical strains and comparing sequence homologies with those of the 16S rDNA gene and other housekeeping genes (recA, rpoA, hsp60). Among the 57 Vibrio species, the mean sequence similarity of the dnaJ gene (77.9%) was significantly less than that of the 16S rDNA gene (97.2%), indicating a high discriminatory power of the dnaJ gene. Most Vibrio species were, therefore, differentiated well by dnaJ sequence analysis. Compared to other housekeeping genes, the dnaJ gene showed better resolution than recA or rpoA for differentiating Vibrio coralliilyticus from Vibrio neptunius and Vibrio harveyi from Vibrio rotiferianus. Among the clinical strains, all 22 human pathogenic strains, including an atypical strain, were correctly identified by the dnaJ sequence. Our findings suggest that analysis of the dnaJ gene sequence can be used as a new tool for the identification of Vibrio species.     

新疆艾比湖嗜盐菌的细菌视紫红质和16S rRNA基因序列的研究

为了研究分析嗜盐古生菌物种与细菌视紫红质(BR)蛋白基因资源,从40份土壤、湖水及淤泥样品中分离出148株嗜盐菌,对其中6株菌采用聚合酶链式反应(PCR)方法对其编码螺旋C至螺旋G的蛋白基因片段和16SrRNA基因进行了扩增,并测定了基因的核苷酸序列。与已报道的相应片段进行对比,ABDH10,ABDH1I和ABDH40中的螺旋C至螺旋G的蛋白与其他菌株差异显著。基于16SrRNA序列的同源性比较以及系统发育学研究表明,ABDH10和ABDH40是Natronorubrum属下的新成员和Natrinema属下的新成员,ABDH40的16SrRNA序列已登录到GenBank,其序列号为AY989910。ABDH11中的螺旋C至螺旋G的蛋白与其他菌株差异显著。     

Questionable 16S ribosomal RNA gene annotations are frequent in completed microbial genomes

According to recent reports, many ribosomal RNA gene annotations are still questionable, and the use of inappropriate tools for annotation has been blamed. However, we believe that the abundant 16S rRNA partial sequence in the databases, mainly created by culture-independent PCR methods, is another main cause of the ambiguous annotations of 16S rRNA. To examine the current status of 16S rRNA gene annotations in complete microbial genomes, we used as a criterion the conserved anti-SD sequence, located at the 3′ end of the 16S rRNA gene, which is commonly overlooked by culture-independent PCR methods. In our large survey, 859 16S rRNA gene sequences from 252 different species of the microbial complete genomes were inspected. 67 species (234 genes) were detected with ambiguous annotations. The common anti-SD sequence and other conserved 16S rRNA sequence features could be detected in the downstream-intergenic regions for almost every questionable sequence, indicating that many of the 16S rRNA genes were annotated incorrectly. Furthermore, we found that more than 91.5% of the 93,716 sequences of the available 16S rRNA in the main databases are partial sequences. We also performed BLAST analysis for every questionable rRNA sequence, and most of the best hits in the analysis were rRNA partial sequences. This result indicates that partial sequences are prevalent in the databases, and that these sequences have significantly affected the accuracy of microbial genomic annotation. We suggest that the annotation of 16S rRNA genes in newly complete microbial genomes must be done in more detail, and that revision of questionable rRNA annotations should commence as soon as possible.     

Mycobacterium species identification--a new approach via dnaJ gene sequencing

The availability of the dnaJ1 gene for identifying Mycobacterium species was examined by analyzing the complete dnaJ1 sequences (approximately 1200 bp) of 56 species (54 of them were type strains) and comparing sequence homologies with those of the 16S rRNA gene and other housekeeping genes (rpoB, hsp65). Among the 56 Mycobacterium species, the mean sequence similarity of the dnaJ1 gene (80.4%) was significantly less than that of the 16S rRNA, rpoB and hsp65 genes (96.6%, 91.3% and 91.1%, respectively), indicating a high discriminatory power of the dnaJ1 gene. Seventy-one clinical isolates were correctly clustered to the corresponding type strains, showing isolates belonging to the same species. In order to propose a method for strain identification, we identified an area with a high degree of polymorphism, bordered by conserved sequences, that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (approximately 350 bp) allows accurate species identification and may be used as a new tool for the identification of Mycobacterium species.     

Multiphasic approach reveals genetic diversity of environmental and patient isolates of Mycobacterium mucogenicum and Mycobacterium phocaicum associated with an outbreak of bacteremias at a Texas hospital

Between March and May 2006, a Texas hospital identified five Mycobacterium mucogenicum bloodstream infections among hospitalized oncology patients using fluorescence high-performance liquid chromatography analysis of mycolic acids. Isolates from blood cultures were compared to 16 isolates from environmental sites or water associated with this ward. These isolates were further characterized by hsp65, 16S rRNA, and rpoB gene sequencing, hsp65 PCR restriction analysis, and molecular typing methods, including repetitive element PCR, random amplified polymorphic DNA PCR, and pulsed-field gel electrophoresis (PFGE) of large restriction fragments. Three of five patient isolates were confirmed as M. mucogenicum and were in a single cluster as determined by all identification and typing methods. The remaining two patient isolates were identified as different strains of Mycobacterium phocaicum by rpoB sequence analysis. One of these matched an environmental isolate from a swab of a hand shower in the patient's room, while none of the clinical isolates of M. mucogenicum matched environmental strains. Among the other 15 environmental isolates, 11 were identified as M. mucogenicum and 4 as M. phocaicum strains, all of which were unrelated by typing methods. Although the 16S rRNA gene sequences matched for all 14 M. mucogenicum isolates, there were two each of the hsp65 and rpoB sequevars, seven PCR typing patterns, and 12 PFGE patterns. Among the seven M. phocaicum isolates were three 16S rRNA sequevars, two hsp65 sequevars, two rpoB sequevars, six PCR typing patterns, and six PFGE patterns. This outbreak represents the first case of catheter-associated bacteremia caused by M. phocaicum and the first report of clinical isolates from a U.S. hospital. The investigation highlights important differences in the available typing methods for mycobacteria and demonstrates the genetic diversity of these organisms even within narrow confines of time and space.     

Genetic diversity of Datisca cannabina-compatible Frankia strains as determined by sequence analysis of the PCR-amplified 16S rRNA gene.

The presence of Frankia strains in soil samples collected from northern areas of Pakistan was detected by inoculating Coriaria nepalensis and Datisca cannabina plants. The abundance of compatible Frankia strains in some areas was indicated by profuse nodulation of the host plants, whereas soil samples from other localities failed to result in nodulation. An oligonucleotide probe (COR/DAT) directed against the 16S rRNA gene of the endophytes of Coriaria and Datisca spp. that did not cross-react with the RNA gene of Frankia strains isolated from other hosts was developed. Genetic diversity among Frankia strains nodulating D. cannabina was determined by sequence analysis of the partial 16S rRNA gene amplified from nodules induced by soil samples from different localities by PCR. Four types of Frankia sequences and one non-Frankia sequence were detected by hybridization with a Frankia genus probe and the COR/DAT probe as well as by sequence analysis of the cloned PCR products.     

Culturable aerobic bacteria from the upstream region of a karst water rivulet

The composition of 681 aerobic and heterotrophic strains that were isolated on two different media was assessed at four sampling points along a ~300 m stretch of a karst water rivulet. Based on partial sequence analysis of 16S rRNA genes, members of 35 genera were identified; however, only a few species dominated as their representatives were repeatedly isolated at different sampling sites. Determination of the phylum affiliation showed that the isolates included members of Bacteriodetes (especially the genus Flavobacterium) and Proteobacteria (mainly Pseudomonas and Stenotrophomonas). MALDI-TOF analysis and/or similarities of partial sequences of flavobacterial strains resulted in the generation of almost complete 16S rRNA gene sequences for 100 isolates, about 60 of which may represent novel phylospecies. The number as well as the intra-phylum distribution of the isolates changed with distance from the discharge site. While phylogenetically restricted at the spring, diversity increased at downstream sampling sites.     

The genomic heterogeneity among Mycobacterium terrae complex displayed by sequencing of 16S rRNA and hsp 65 genes

The species identification within Mycobacterium terrae complex has been known to be very difficult. In this study, the genomic diversity of M. terrae complex with eighteen clinical isolates, which were initially identified as M. terrae complex by phenotypic method, was investigated, including that of three type strains (M. terrae, M. nonchromogenicum, and M. triviale ). 16S rRNA and 65-kDa heat shock protein (hsp 65) gene sequences of mycobacteria were determined and aligned with eleven other references for the comparison using similarity search against the GenBank and Ribosomal Database Project II (RDP) databases. 16S rRNA and hsp 65 genes of M. terrae complex showed genomic heterogeneity. Amongst the eighteen clinical isolates, nine were identified as M. nonchromogenicum, eight as M. terrae, one as M. mucogenicum with the molecular characteristic of rapid growth. M. nonchromogenicum could be subdivided into three subgroups, while M. terrae could be subdivided into two subgroups using a 5 bp criterion (>1% difference). Seven isolates in two subgroups of M. nonchromogenicum were Mycobacterium sp. strain MCRO 6, which was closely related to M. nonchromogenicum. The hsp 65 gene could not differentiate one M. nonchromogenicum from M. avium or one M. terrae from M. intracellulare. The nucleotide sequence analysis of 16S rRNA and hsp 65 genes was shown to be useful in identifying the M. terrae complex, but hsp 65 was less discriminating than 16S rRNA.     

Isolation and characterization of 23 carbofuran-degrading bacteria from soils from distant geographical areas

The aim of this work was to isolate, identify and type carbofuran-degrading bacteria from two geographically distant soils. Restriction Fragment Length Polymorphism (RFLP) patterns of the 16S rRNA gene and partial 16S rRNA sequence analysis were used to classify the 23 isolates obtained. Nine of them showed high similarity to Pseudomonas strains, seven showed similarity to the Flexibacter/Cytophaga/Bacteroides group and the remainder showed similarity to other bacterial genera. Isolates within the same group were sub-typed by comparing partial 16S rRNA sequences and SDS-PAGE analysis of their total protein profiles. Many of the UK isolates showed similarity to the Pseudomonas genera, while most of the Greek isolates showed similarity to the Flexibacter/Cytophaga/Bacteroides group. Only two Chrysobacterium strains isolated from both the UK and Greek soils were identical.     

Variations in the 16S-23S rRNA internal transcribed spacer of fibrolytic Butyrivibrio isolates from the reindeer rumen

Strains of Butyrivibrio are principal cellulytic bacteria in the rumen of the High Arctic Svalbard reindeer ( Rangifer tarandus platyrhynchus ). According to phylogenetic analysis based on 16S rRNA gene sequencing, Butyrivibrio can be divided into three subgroups within the Clostridia class of the phylum Firmicutes, but the current phenotypic and genotypic differentiation within the family Lachnospiraceae is insufficient. This current study describes the sequence diversity of the 16S-23S rRNA intergenic transcribed spacer (ITS) region of Butyrivibrio isolates from reindeer. A total of 17 different ITS sequences with sizes between 449 and 784 nt were obtained. Genes encoding tRNA(Ile) and tRNA(Ala) were identified in four of the sequences. Phylogenetic neighbor-joining trees were constructed based on the ITS sequence and compared with a phylogenetic neighbor-joining tree based on 16S rRNA gene sequences previously obtained for the same isolates. These comparisons indicated a better differentiation between strains in the ITS sequence than the 16S rRNA gene based tree. Through this study, a better means for identifying and tracking fibrolytic and potentially probiotic Butyrivibrio strains in reindeer and other ruminants has been provided.     

Pseudoscardovia suis gen. nov., sp. nov., a new member of the family Bifidobacteriaceae isolated from the digestive tract of wild pigs (Sus scrofa)

Seventeen fructose-6-phosphate phosphoketolase-positive bacterial strains were isolated from the digestive tract of wild pigs (Sus scrofa). Most of them were identified as Bifidobacterium boum according to sequences of 16S rRNA gene. Two strains isolated from the small intestine content had unusual morphology of cells in comparison with bifidobacteria. Cells growing in liquid anaerobic media were regular shaped rods arranged mostly in pairs. These isolates showed relatively low 16S rRNA gene sequence similarities (maximum identity of 94%) to members of the family Bifidobacteriaceae. Nevertheless, phylogenetic analyses of 16S rRNA, hsp60 and xfp gene sequences revealed that these strains are more related to recently described Neoscardovia, Aeriscardovia and other scardovial genera, than to Bifidobacterium species. Partial gene sequences of other phylogenetic markers showed low (65.8–89.5%) similarities to genome sequences of bifidobacteria and Gardnerella vaginalis. The major fatty acids detected in cells of the representative strain DPTE4^T were C_16:0, C_18:1, C_14:0. The peptidoglycan type of the DPTE4^T strain was A3β l-Orn(l-Lys)-l-Ser(l-Ala)-l-Ala₂. Polar lipid analysis revealed two phosphoglycolipids and phospholipids, a glycolipid and diphosphatidylglycerol. The results of phylogenetic, genotypic and phenotypic analyses support the proposal of a novel taxa, Pseudoscardovia suis gen. nov., sp. nov. (type strain = DPTE4^T = DSM 24744^T = CCM 7942^T).     

Discordant 16S and 23S rRNA gene phylogenies for the genus Helicobacter: implications for phylogenetic inference and systematics

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.