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.     

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。     

Phylogenetic affiliation of Aeromonas culicicola MTCC 3249(T) based on gyrB gene sequence and PCR-amplicon sequence analysis of cytolytic enterotoxin gene

We determined the gyrB gene sequences of all 17 hybridizations groups of Aeromonas. Phylogenetic trees showing the evolutionary relatedness of gyrB and 16S rRNA genes in the type strains of Aeromonas were compared. Using this approach, we determined the phylogenetic position of Aeromonas culicicola MTCC 3249(T), isolated from midgut of Culex quinquefasciatus. In the gyrB based-analysis A. culicicola MTCC 3249(T) grouped with A. veronii whereas, it grouped with A. jandaei in the 16S rRNA based tree. The number of nucleotide differences in 16S rRNA sequences was less than found with the gyrB sequence data. Most of the observed nucleotide differences in the gyrB gene were synonymous. The Cophenetic Correlation Coefficient (CCC) for gyrB sequences was 0.87 indicating this gene to be a better molecular chronometer compared to 16S rRNA for delineation of Aeromonas species. This strain was found to be positive for the cytolytic enterotoxin gene. PCR-Amplicon Sequence Analysis (PCR-ASA) of this gene showed that the isolate is affiliated to type I and is potentially pathogenic. These PCR-ASA results agreed in part with the gyrB sequence results.     

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.     

Multilocus sequence analysis reveals multiple symbiovars within Mesorhizobium species

The genus Mesorhizobium includes species nodulating several legumes, such as chickpea, which has a high agronomic importance. Chickpea rhizobia were originally described as either Mesorhizobium ciceri or M. mediterraneum. However, rhizobia able to nodulate chickpea have been shown to belong to several different species within the genus Mesorhizobium. The present study used a multilocus sequence analysis approach to infer a high resolution phylogeny of the genus Mesorhizobium and to confirm the existence of a new chickpea nodulating genospecies. The phylogenetic structure of the Mesorhizobium clade was evaluated by sequence analysis of the 16S rRNA gene, ITS region and the five core genes atpD, dnaJ, glnA, gyrB, and recA. Phylogenies obtained with the different genes are in overall good agreement and a well-supported, almost fully resolved, phylogenetic tree was obtained using the combined data. Our phylogenetic analyses of core genes sequences and their comparison with the symbiosis gene nodC, corroborate the existence of one new chickpea Mesorhizobium genospecies and one new symbiovar, M. opportunistum sv. ciceri. Furthermore, our results show that symbiovar ciceri spreads over six species of mesorhizobia. To our knowledge this study shows the most complete Mesorhizobium multilocus phylogeny to date and contributes to the understanding of how a symbiovar may be present in different species.     

The phylogenetic significance of peptidoglycan types: Molecular analysis of the genera Microbacterium and Aureobacterium based upon sequence comparison of gyrB, rpoB, recA and ppk and 16SrRNA genes

The type strains of 27 species of the genus Microbacterium, family Microbacteriaceae, were analyzed with respect to the phylogeny of the housekeeping genes coding for DNA gyrase subunit B (gyrB), RNA-polymerase subunit B (rpoB), recombinase A (recA) and polyphosphate kinase (ppk). The resulting gene trees were compared to the 16S rRNA gene phylogeny of the same species. The topology of neighbour-joining and maximum parsimony phylogenetic trees based upon nucleic acid sequences and protein sequences of housekeeping genes differed among each other and no gene tree was identical to that of the 16S rRNA gene tree. Only some species showed consistent clustering by all genes analyzed, but the majority of species branched with different neighbours in most gene trees. The failure to phylogenetically cluster type strains into two groups based upon differences in the amino acid composition of peptidoglycan on the basis of 16S rRNA gene sequence similarity, once leading to the union of the genera Microbacterium and Aureobacterium, was also seen in the analysis of recA, rpoB and gyrB gene and protein phylogenies. Analysis of the pkk gene and protein as well as of a concatenate tree, combining sequences of all five genes (total of 3.700 nucleotides), sees members of the former genus Aureobacterium and other type strains with lysine as diagnostic diamino acid to form a coherent cluster that branches within the radiation of Microbacterium species with ornithine in the peptidoglycan.     

一株氯氰菊酯降解菌16SrDNA，gyrB和GyrB的系统发育分析

从农药厂污水处理池中分离得到一株氯氰菊酯降解菌,在30℃,pH7．0的条件下,无机盐培养基中100mg／L的氯氰菊酯,经过7．5天,能够降解大约52．3％,外加碳源能够明显提高其降解性能。生理生化实验结合16SrDNA,册诏和GyrB的系统发育分析,将其归为Gordonia菌属。在16SrDNA水平上,其与G．amicalis DSM44461和G．hydrophobica DSM44015^T的相似值最高,为98．1％;而在gyrB和GyrB水平上,其与G.hydrophobica JCM10086的相似值最高,分别为86．8％和91,1％。通过对所构建的系统发育树进行评估,表明16SrDNA序列适用于将分离菌株鉴定到属的水平上,而gyrB和GyrB更适用于在属内种的水平上进行系统发育的分析。     

Use of the dnaJ gene for the detection and identification of all Legionella pneumophila serogroups and description of the primers used to detect 16S rDNA gene sequences of major members of the genus Legionella

We sequenced about 930 bp of the dnaJ gene from 15 Legionella pneumophila serogroups and some other members of the genus Legionella. As L. pneumophila 16S rDNA sequences could not discriminate between all subspecies and serogroups, we assessed the use of dnaJ gene sequences to differentiate between Legionella subspecies as well as between L. pneumophila serogroups. A phylogenetic analysis revealed that dnaJ gene sequences were more variable between the L. pneumophila serogroups than mip gene and 16S rDNA sequences. By studying 61 strains from 41 species of the genus Legionella, as well as other genera, we established a PCR method that could amplify 285 bp of dnaJ gene from all L. pneumophila serogroups. This primer set was more sensitive than mip gene primers and was able to detect 0.25 ng of purified DNA. We also describe the 16S rDNA primers that were used to detect most Legionella genus members.     

16S rRNA及rpoC1基因用于螺旋藻、节旋藻系统发育的比较北大核心CSCD

【目的】利用16S rRNA和rpoC1基因分子标记研究螺旋藻、节旋藻的系统发育关系,并对其区分能力进行比较。【方法】以84株螺旋藻、节旋藻为研究对象,对其进行16S rRNA、rpoC1基因序列的扩增、测序及分析,并对构建的系统发育树进行对比。【结果】rpoC1基因序列保守位点所占比例49.7%、平均G+C百分含量47.7%和序列相似度76%–100%明显低于16S rRNA基因序列的79.4%、55.6%和91%–100%,其变异程度高于16S rRNA基因;基于16S rRNA、rpoC1基因构建的系统发育NJ树拓扑结构基本一致,84株实验藻株分为2个属3个类群,其中仅F-351、F-904-2、F-1070和TJBC14-1藻株为螺旋藻,其余均为节旋藻;虽然2个基因都不能区分形态种和地理种,但rpoC1基因NJ树的置信度(100%)高于16S rRNA基因(99%),属内分群效果也明显优于16S rRNA基因。【结论】支持了螺旋藻、节旋藻为两个不同属的结论,且在属内分类时rpoC1基因比16S rRNA基因具有更高的区分度。     

On molecular taxonomy: what is in a name?

Gene sequences of small portions of the genome are often used for premature detailed taxonomic changes, neglecting polyphasic taxonomy, which should also consider phenotypical characteristics. Three examples are given: (i) Recently, members of the genera Eperythrozoon and Haemobartonella have been moved, correctly so, from the Rickettsiales to the Mycoplasmatales, but were assigned to the genus Mycoplasma, mostly on the basis of 16S rRNA sequence analysis. Not only is the 16S rRNA sequence similarity between 'classical' Mycoplasma and these species of Eperythrozoon and Haemobartonella less than that between some other well-recognised bacterial genera, but their biological differences amply justify their classification in different genera of the Mycoplasmatales. Furthermore, the move creates considerable confusion, as it necessitates new names for some species, with more confusion likely to come when the 16S rRNA sequences of the type species of Eperythrozoon, a name which has priority over Mycoplasma, will be analysed. (ii) In the Rickettsiales, members of the genera Anaplasma, Ehrlichia, Cowdria, Neorickettsia and Wolhbachia are so closely related phylogenetically on the basis of 16S rRNA sequences, and for some also of groESL operon sequences, that they have recently been fused, correctly so, into one family, the Anaplasmataceae, while the tribes Ehrlichieae and Wolbachieae have been abolished. Sequence diversity within the 'classical' genus Ehrlichia has led to classifying E. phagocytophila (including E. equi and the agent of human granulocytic ehrlichiosis), E. platys and E. bovis in the genus Anaplasma, while others have been retained in Ehrlichia, which also includes Cowdria ruminantium. E. sennetsu and E. risticii have been transferred to the genus Neorickettsia. 16S rRNA and GroEL sequences of 'classical' Anaplasma and some members of 'classical' Ehrlichia do show a close relationship, but differences in citrate synthase gene sequences, the GC content of this gene, and sequences of the gene encoding the beta-subunit of RNA polymerase, not to speak of the phenotypical differences, do not justify the fusion into one genus. Because of the phylogenetical diversity in Ehrlichia it is recommended that a new genus name be created for the E. phagocytophila genogroup (and E. platys and E. bovis). (iii) One of the conclusions of studies on the phylogeny of ticks of the subfamilies Rhipicephalinae and Hyalomminae, based on nucleotide sequences from 12S rRNA, cytochrome c oxidase I, the internal transcribed spacer 2, 18S rRNA, as well as morphological characters, is that Boophilus should be considered as a subgenus of Rhipicephalus. While Boophilus and Rhipicephalus are undoubtedly close, the obviously important morphological and biological differences between the genera Rhipicephalus and Boophilus are thus overruled by similarities in the sequences of a number of genes and this leads to considerable confusion. Polyphasic taxonomy amply justifies maintaining Boophilus as a separate genus, phylogenetically near to Rhipicephalus. This note is a plea for a cautious and balanced approach to taxonomy, taking into account molecular genotypical information, as far as is possible from different genes, as well as phenotypical characteristics.     

The comparative amino acid sequences, substrate specificities and gene or cDNA nucleotide sequences of some prokaryote and eukaryote amidinotransferases: implications for evolution

The amino acid sequences of the amidinotransferases and the nucleotide sequences of their genes or cDNA from four Streptomyces species (seven genes) and from the kidneys of rat, pig, human and human pancreas were compared. The overall amino acid and nucleotide sequences of the prokaryotes and eukaryotes were very similar and further, three regions were identified that were highly identical. Evidence is presented that there is virtually zero chance that the overall and high identity regions of the amino acid sequence similarities and the overall nucleotide sequence similarities between Streptomyces and mammals represent random match. Both rat and lamprey amidinotransferases were able to use inosamine phosphate, the amidine group acceptor of Streptomyces. We have concluded that the structure and function of the amidinotransferases and their genes has been highly conserved through evolution from prokaryotes to eukaryotes. The evolution has occurred with: (1) a high degree of retention of nucleotide and amino acid sequences; (2) a high degree of retention of the primitive Streptomyces guanine+cytosine (G+C) third codon position composition in certain high identity regions of the eukaryote cDNA; (3) a decrease in the specificities for the amidine group acceptors; and (4) most of the mutations silent in the regions suggested to code for active sites in the enzymes.     

Discrimination of Bacillus anthracis and closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microarray

Analysis of 16S rRNA sequences is a commonly used method for the identification and discrimination of microorganisms. However, the high similarity of 16S and 23S rRNA sequences of Bacillus cereus group organisms (up to 99-100%) and repeatedly failed attempts to develop molecular typing systems that would use DNA sequences to discriminate between species within this group have resulted in several suggestions to consider B. cereus and B. thuringiensis, or these two species together with B. anthracis, as one species. Recently, we divided the B. cereus group into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, based on 16S rRNA, 23S rRNA and gyrB gene sequences and identified subgroup-specific makers in each of these three genes. Here we for the first time demonstrated discrimination of these seven subgroups, including subgroup Anthracis, with a 3D gel element microarray of oligonucleotide probes targeting 16S and 23S rRNA markers. This is the first microarray enabled identification of B. anthracis and discrimination of these seven subgroups in pure cell cultures and in environmental samples using rRNA sequences. The microarray bearing perfect match/mismatch (p/mm) probe pairs was specific enough to discriminate single nucleotide polymorphisms (SNPs) and was able to identify targeted organisms in 5min. We also demonstrated the ability of the microarray to determine subgroup affiliations for B. cereus group isolates without rRNA sequencing. Correlation of these seven subgroups with groupings based on multilocus sequence typing (MLST), fluorescent amplified fragment length polymorphism analysis (AFLP) and multilocus enzyme electrophoresis (MME) analysis of a wide spectrum of different genes, and the demonstration of subgroup-specific differences in toxin profiles, psychrotolerance, and the ability to harbor some plasmids, suggest that these seven subgroups are not based solely on neutral genomic polymorphisms, but instead reflect differences in both the genotypes and phenotypes of the B. cereus group organisms.     

Taxonomic study of "tufted mitior" strains of streptococci (Streptococcus sanguinis biotype 11); recognition of a new genospecies

The taxonomic position of tufted strains of streptococci, phenotypically resembling Streptococcus mitis and previously referred to as 'tufted mitior' was investigated. By 16S rRNA sequence analysis, it was clear that the "tufted mitior" strains belonged to the mitis group of species within the genus Streptococcus. It was confirmed that these strains were taxonomically independent at the species level, sharing less than 43%, DNA-DNA similarity with all established species of the mitis group. However biochemical test data obtained, using three commercial identification kits (Rapid ID32 Strep, STREPTOGRAM, and Biolog GP-plate) together with in-house biochemical tests employing 4-MUF-linked fluorogenic substrates did not reveal sufficient differential tests with which to identify the "tufted mitior" strains unequivocally. From these data, we conclude that these "tufted mitior" strains represent a new taxon within the mitis group of the genus Streptococcus, and propose that they should be considered as a genospecies until differential phenotypic characteristics are found for their identification.     

Sequence and phylogenetic analyses of novel glucosyltransferase genes of mutans streptococci isolated from pig oral cavity

Nucleotide sequences of water-insoluble glucan-producing glucosyltransferase (gtf) genes of new mutans streptococci isolated from pig oral cavity, Streptococcus orisuis JCM14035, and of Streptococcus criceti HS-6 were determined. The gtf gene of S. orisuis JCM14035 consisted of a 4,401 bp ORF encoding for a 1,466 amino acids, and was revealed to belong to the gtfI group. The percent homology of amino acid sequence of the GTF-I from S. orisuis and S. criceti are 95.0%, however, this score ranges from 77.0% to 78.0% when compared to Streptococcus sobrinus 6715. The deduced N-terminal amino acid sequence was considered responsible for the secretion of GTF-I in S. orisuis JCM14035 and S. criceti HS-6 with high similarity to known GTF proteins from other streptococci. In addition, two other conserved regions, i.e., N-terminal putative catalytic-site and C-terminal glucan binding domain, were also found in GTF-Is of S. orisuis JCM14035 and S. criceti HS-6. Phylogenetic analysis suggested that S. orisuis JCM14035 and S. criceti HS-6, closely related to each other, resemble S. sobrinus and S. downei based on the amino acid sequences of the GTFs.     

Phylogenetic relationships of Oriental torrent frogs in the genus Amolops and its allies (Amphibia, Anura, Ranidae)

We investigated the phylogenetic relationships among 20 species of Oriental torrent frogs in the genus Amolops and its allies from China and Southeast Asia based on 1346-bp sequences of the mitochondrial 12S and 16S rRNA genes. Oriental species of the tribe Ranini form a monophyletic group containing 11 clades (Rana temporaria + Pseudoamolops, R. chalconota, four clades of Amolops, Meristogenys, three clades of Huia species, and Staurois) for which the phylogenetic relationships are unresolved. The genus Amolops consists of southern Chinese, southwestern Chinese, Thai, and Vietnamese-Malaysian lineages, but their relationships are also unresolved. The separation of southern and southwestern lineages within China conforms to previous morphological and karyological results. Species of Huia do not form a monophyletic group, whereas those of Meristogenys are monophyletic. Because P. sauteri is a sister species of R. temporaria, distinct generic status of Pseudoamolops is unwarranted.     

Diversity and evolution of 5S rRNA gene family organization in Pythium

The 5S rRNA gene family organization among 87 species and varieties of Pythium was investigated to assess evolutionary stability of the two patterns detected and to determine which pattern is likely the ancestral state in the genus. Species with filamentous sporangia (Groups A-C according to the ITS phylogenetic tree for Pythium) had 5S genes linked to the rDNA repeat that were predominantly coded for on the DNA strand opposite to the one with the other rRNA genes (‘inverted’ orientation). A small group of species with contiguous sporangia (Group D) is related to Groups A-C but had unlinked 5S genes. The main group of species with spherical zoosporangia (Groups E-J) generally had unlinked 5S genes in tandem arrays. The six species in Group K, although they also have spherical sporangia, had linked genes on the same strand as the other rRNA genes ‘non-inverted’ and most of them had pairs of tandem 5S genes. The evolutionary stability of 5S sequence organization was compared with the stability of morphological characters as interpreted from a phylogeny based on ITS sequence analysis. Features of 5S sequence organization were found to be just as consistent within groups as were the morphological characters. To determine the ancestral type of 5S family organization, a survey of Phytophthora strains was conducted to supply an outgroup reference. The most parsimonious interpretation of the data in this survey yielded the tentative conclusion that the linked condition of the 5S sequences was ancestral.     

Comparative phylogenetic analyses of Halomonas variabilis and related organisms based on 16S rRNA, gyrB and ectBC gene sequences

Halomonas variabilis and phylogenetically related organisms were isolated from various habitats such as Antarctic terrain and saline ponds, deep-sea sediment, deep-sea waters affected by hydrothermal plumes, and hydrothermal vent fluids. Ten strains were selected for physiological and phylogenetic characterization in detail. All of those strains were found to be piezotolerant and psychrotolerant, as well as euryhaline halophilic or halotolerant. Their stress tolerance may facilitate their wide occurrence, even in so-called extreme environments. The 16S rDNA-based phylogenetic relationship was complemented by analyses of the DNA gyrase subunit B gene (gyrB) and genes involved in the synthesis of the major compatible solute, ectoine: diaminobutyric acid aminotransferase gene (ectB) and ectoine synthase gene (ectC). The phylogenetic relationships of H. variabilis and related organisms were very similar in terms of 16S rDNA, gyrB, and ectB. The ectC-based tree was inconsistent with the other phylogenetic trees. For that reason, ectC was inferred to derive from horizontal transfer.     

Identification and differentiation of Lactobacillus, Streptococcus and Bifidobacterium species in fermented milk products with bifidobacteria

The aim of this study was to identify and discriminate bacteria contained in commercial fermented milks with bifidobacteria by the use of amplified ribosomal DNA restriction analysis (ARDRA) and randomly amplified polymorphic DNA (RAPD) techniques. ARDRA of the 16S rDNA gene and RAPD were performed on 13 Lactobacillus strains, 13 Streptococcus and 13 Bifidobacterium strains isolated from commercial fermented milk. Lactobacillus delbrueckii, Streptococcus thermophilus and Bifidobacterium animalis isolates were identified by genus- and species-PCR and also, they were differentiated at genus and species level by ARDRA using MwoI restriction enzyme. The ARDRA technique allowed for the discrimination among these three related genus with the use of only one restriction enzyme, since distinctive profiles were obtained for each genus. Therefore it can be a simple, rapid and useful method for routine identification. Also, RAPD technique allowed the discrimination of all bacteria contained in dairy products, at genus- and strain-level by the performance of one PCR reaction.