Detection and identification of Escherichia coli,Shigella, and Salmonella by microarrays using the gyrB gene

Commonly, 16S ribosome RNA (16S rRNA) sequence analysis has been used for identifying enteric bacteria. However, it may not always be applicable for distinguishing closely related bacteria. Therefore, we selected gyrB genes that encode the subunit B protein of DNA gyrase (a topoisomerase type II protein) as target genes. The molecular evolution rate of gyrB genes is higher than that of 16S rRNA, and gyrB genes are distributed universally among bacterial species. Microarray technology includes the methods of arraying cDNA or oligonucleotides on substrates such as glass slides while acquiring a lot of information simultaneously. Thus, it is possible to identify the enteric bacteria easily using microarray technology. We devised a simple method of rapidly identifying bacterial species through the combined use of gyrB genes and microarrays. Closely related bacteria were not identified at the species level using 16S rRNA sequence analysis, whereas they were identified at the species level based on the reaction patterns of oligonucleotides on our microarrays using gyrB genes.     

Development of PCR primers specific for the amplification and direct sequencing of gyrB genes from microbacteria, order Actinomycetales

PCR primer sets were developed for the specific amplification and sequence analyses encoding the gyrase subunit B (gyrB) of members of the family Microbacteriaceae, class Actinobacteria. The family contains species highly related by 16S rRNA gene sequence analyses. In order to test if the gene sequence analysis of gyrB is appropriate to discriminate between closely related species, we evaluate the 16S rRNA gene phylogeny of its members. As the published universal primer set for gyrB failed to amplify the responding gene of the majority of the 80 type strains of the family, three new primer sets were identified that generated fragments with a composite sequence length of about 900 nt. However, the amplification of all three fragments was successful only in 25% of the 80 type strains. In this study, the substitution frequencies in genes encoding gyrase and 16S rDNA were compared for 10 strains of nine genera. The frequency of gyrB nucleotide substitution is significantly higher than that of the 16S rDNA, and no linear correlation exists between the similarities of both molecules among members of the Microbacteriaceae. The phylogenetic analyses using the gyrB sequences provide higher resolution than using 16S rDNA sequences and seem able to discriminate between closely related species.     

Complete genome sequence of the gliding freshwater bacterium Fluviicola taffensis type strain (RW262)

Fluviicola taffensis O'Sullivan et al. 2005 belongs to the monotypic genus Fluviicola within the family Cryomorphaceae. The species is of interest because of its isolated phylogenetic location in the genome-sequenced fraction of the tree of life. Strain RW262(T) forms a monophyletic lineage with uncultivated bacteria represented in freshwater 16S rRNA gene libraries. A similar phylogenetic differentiation occurs between freshwater and marine bacteria in the family Flavobacteriaceae, a sister family to Cryomorphaceae. Most remarkable is the inability of this freshwater bacterium to grow in the presence of Na(+) ions. All other genera in the family Cryomorphaceae are from marine habitats and have an absolute requirement for Na(+) ions or natural sea water. F. taffensis is the first member of the family Cryomorphaceae with a completely sequenced and publicly available genome. The 4,633,577 bp long genome with its 4,082 protein-coding and 49 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

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。     

Climatic oscillations triggered post-Messinian speciation of Western Palearctic brown frogs (Amphibia,Ranidae)

Oscillating glacial cycles over the past 2.4 million years are proposed to have had a major impact on the diversity of contemporary species communities. We used mitochondrial and nuclear DNA sequence data to infer phylogenetic relationships within Western Palearctic brown frogs and to test the influence of Pliocene and Pleistocene climatic changes on their evolution. We sequenced 1976bp of the mitochondrial genes 16S rRNA and cytochrome b and of the nuclear rhodopsin gene for all current species and subspecies. Based on an established allozyme clock for Western Palearctic water frogs and substitution rate constancy among water frogs and brown frogs, we calibrated a molecular clock for 1425bp of the 16S and rhodopsin genes. We applied this clock to date speciation events among brown frogs. Western Palearctic brown frogs underwent a basal post-Messinian radiation about 4 million years ago (mya) into five major clades: three monotypic lineages (Rana dalmatina, Rana latastei, Rana graeca), an Anatolian lineage, and a lineage comprising Rana italica, Rana arvalis, and all Iberian taxa. Polytypic lineages radiated further in concordance with the onset of climatic oscillations ca. 3.2, 2.0, and 1.0-0.6 mya, respectively. The dated fossil record corroborates our paleobiogeographic scenario. We conclude that drastic climatic changes followed by successive temperature oscillations "trapped" most brown frog species in their southern European glacial refugia with enough time to speciate. Substantial dispersal was only possible during extensive interglacial periods of a constant subtropical climate.     

Complete genome sequence of strain s85, a novel member of the family Flavobacteriaceae

An agar-degrading marine bacterium identified as a novel member of the family Flavobacteriaceae (strain S85) was isolated from seawater in Micronesia. The sequenced strain S85 genome is composed of 3,384,629 bp in a circular chromosome, which includes 2,883 complete open reading frames.     

The phylogenetic relationships of cyanobacteria inferred from 16S rRNA,gyrB, rpoC1 and rpoD1 gene sequences

Phylogenetic analysis of cyanobacteria was carried out using the small subunit rRNA (16S rRNA), DNA gyrase subunit B (gyrB), DNA-dependent RNA polymerase gamma subunit (rpoC1) and a principal sigma factor of E. coli sigma(70) type for DNA-dependent RNA polymerase (rpoD1) gene sequences of 24 strains which contained 5 subgroups of cyanobacteria-3 strains of the Chroococcales, 5 strains of the Pluerocapsales, 7 strains of the Oscillatoriales, 7 strains of the Nostocales and 2 strains of the Stigonematales. Degenerated PCR primers of gyrB, rpoC1 and rpoD1 genes were designed using consensus amino acid sequences registered in GenBank. The phylogenetic positions of cyanobacteria were resolved through phylogenetic analysis based on 16S rDNA, gyrB, rpoC1 and rpoD1 gene sequences. Phylogenies of gyrB, rpoC1 and rpoD1 support 16S rRNA-based classification of cyanobacteria. Interestingly, phylogenies from amino acid sequences deduced from gyrB and combined amino acid sequences deduced from rpoC1 and rpoD1 genes strongly support that of 16S rRNA, but the branching pattens of the trees based on 16S rDNA, GyrB, rpoC1, rpoD1 and combined amino acid sequences deduced from rpoC1 and rpoD1 were not congruent. In this study, we showed the correlation among phylogenetic relationships of 16S rDNA, gyrB, rpoC1 and rpoD1 genes. The phylogenetic trees based on the sequences of 16S rDNA, GyrB, rpoC1, rpoD1 and the combined amino acid sequences deduced from rpoC1 and rpoD1 showed that the lateral gene transfer of rRNA might be suspected for Synechocystis sp. PCC 6803.     

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。     

Genetic analysis of housekeeping genes of members of the genus Acholeplasma: phylogeny and complementary molecular markers to the 16S rRNA gene

The partial nucleotide sequences of the rpoB and gyrB genes as well as the complete sequence of the 16S-23S rRNA intergenic transcribed spacer (ITS) were determined for all known Acholeplasma species. The same genes of Mesoplasma and Entomoplasma species were also sequenced and used to infer phylogenetic relationships among the species within the orders Entomoplasmatales and Acholeplasmatales. The comparison of the ITS, rpoB, and gyrB phylogenetic trees with the 16S rRNA phylogenetic tree revealed a similar branch topology suggesting that the ITS, rpoB, and gyrB could be useful complementary phylogenetic markers for investigation of evolutionary relationships among Acholeplasma species. Thus, the multilocus phylogenetic analysis of Acholeplasma multilocale sequence data (ATCC 49900 (T) = PN525 (NCTC 11723)) strongly indicated that this organism is most closely related to the genera Mesoplasma and Entomoplasma (family Entomoplasmataceae) and form the branch with Mesoplasma seiffertii, Mesoplasma syrphidae, and Mesoplasma photuris. The closest genetic relatedness of this species to the order Entomoplasmatales was additionally supported by the finding that A. multilocale uses UGA as the tryptophan codon in its gyrB and gyrA sequences. Use of the UGA codon for encoding tryptophan was previously reported as a unique genetic feature of Entomoplasmatales and Mycoplasmatales but not of Acholeplasmatales. These data, as well as previously published data on metabolic features of A. multilocale, leads to the proposal to reclassify A. multilocale as a member of the family Entomoplasmataceae.     

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.     

Microheterogeneity in 16S ribosomal DNA-defined bacterial populations from a stratified planktonic environment is related to temporal changes and to ecological adaptations

Temporal changes of the bacterioplankton from a meromictic lake (Lake Vilar, Banyoles, Spain) were analyzed with four culture-independent techniques: epifluorescence microscopy, PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting, fluorescence in situ whole-cell hybridization and flow cytometry sorting. Microscopically, blooms of one cyanobacterium (Synechococcus sp.-like), one green sulfur bacterium (Chlorobium phaeobacteroides-like), and one purple sulfur bacterium (Thiocystis minor-like) were observed at different depths and times. DGGE retrieved these populations and, additionally, populations related to the Cytophaga-Flavobacterium-Bacteroides phylum as predominant community members. The analyses of partial 16S ribosomal DNA sequences from the DGGE fingerprints (550 bp analyzed) revealed higher genetic diversity than expected from microscopic observation for most of these groups. Thus, the sequences of two Synechococcus spp. (both had a similarity of 97% to Synechococcus sp. strain PCC6307 in 16S rRNA), two Thiocystis spp. (similarities to Thiocystis minor of 93 and 94%, respectively), and three Cytophaga spp. (similarities to Cytophaga fermentans of 88 and 89% and to Cytophaga sp. of 93%, respectively) were obtained. The two populations of Synechococcus exhibited different pigment compositions and temporal distributions and their 16S rRNA sequences were 97.3% similar. The two Thiocystis populations differed neither in pigment composition nor in morphology, but their 16S rRNA sequences were only 92.3% similar and they also showed different distributions over time. Finally, two of the Cytophaga spp. showed 96.2% similarity between the 16S rRNA sequences, but one of them was found to be mostly attached to particles and only in winter. Thus, the identity of the main populations changed over time, but the function of the microbial guilds was maintained. Our data showed that temporal shifts in the identity of the predominant population is a new explanation for the environmental 16S rRNA microdiversity retrieved from microbial assemblages and support the hypothesis that clusters of closely related 16S rRNA environmental sequences may actually represent numerous closely related, yet ecologically distinct, populations.     

Draft Genome Sequence of Strain P7-3-5, a New Flavobacteriaceae Bacterium Isolated from Intertidal Sand

The Flavobacteriaceae bacterium strain P7-3-5 was isolated from intertidal sand of the Yellow Sea, China. Analysis of the 16S rRNA gene sequences showed that strain P7-3-5 formed a distinct phylogenetic lineage within the family Flavobacteriaceae. The genome of strain P7-3-5 was sequenced to facilitate the physiological, ecological, and evolutionary studies of the bacteria within the family Flavobacteriaceae.     

Bacterial diversity based on 16S rRNA and gyrB genes at Yinshan mine, China

The diversity of bacterial communities at three sites impacted by acid mine drainage (AMD) from the Yinshan Mine in China was studied using comparative sequence analysis of two molecular markers, the 16S rRNA and gyrB genes. The phylogenetic analyses retrieved sequences from six classes of bacteria, Nitrospira, Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Acidobacteria, and Actinobacteria, as well as sequences related to the plastid of the cyanobacterium Cyanidium acidocaldarium and also some unknown bacteria. The results of phylogenetic analyses based on gyrB and 16S rRNA were compared. This confirmed that gyrB gene analysis may be a useful tool, in addition to the comparative sequence analysis of the 16S rRNA gene, for the analysis of microbial community compositions. Moreover, the Mantel test showed that the geochemical characteristics, especially the pH value and the concentration of iron, strongly influenced the composition of the microbial communities.     

大头蛙和脆皮大头蛙线粒体3个基因的测定及两栖类亲缘关系研究

现存的两栖类系统发生关系一直存在争议,特别是3个目间的亲缘关系。本文设计了5对引物,扩增和测定了大头蛙和脆皮大头蛙线粒体12S和16S rRNA基因和Cytb基因的全序列。在对所测序列进行分析的同时,基于3个基因全序列的相加数据,运用MEGA 3.1和PHYLIP 3.64软件中的NJ法、MP法和ML法,对两爬类17个物种,以鱼类非洲肺鱼为外群,重建出3个树形完全一致的分子系统树。研究结果显示:现存两栖类中无尾目和有尾目为姐妹群关系,并推断有尾目内小鲵科和隐鳃鲵科亲缘关系较近。此外,在研究两栖类系统发生关系方面,作者分析前人研究中产生两种不同观点的可能原因,同时总结了在此类研究中产生偏差的几种影响因素。     

Pukyongia salina gen. nov., sp. nov., a novel genus in the family Flavobacteriaceae

A Gram-negative aerobic bacterium, designated RR4-38T, was isolated from a biofilter in a seawater recirculating aqua-culture system (RAS) in Busan, South Korea. The bacteria were irregular, short, rod-shaped, non-motile, oxidase-positive, and catalase-negative. Growth of the strain RR4-38T was observed at 15–35·C (optimum, 25–30·C), pH 5.5–9.5 (optimum, pH 8.0), and in the presence of 0–5% (w/v) NaCl (optimum, 3%). Phylogenetic analysis based on the 16S rRNA gene sequences showed that the strain RR4-38T formed a distinct lineage with close genera Ulvibacter (≤ 95.01% 16S rRNA gene sequence similarity), Aureitalea (94.74%), Aureisphaera (≤ 93.27%), and Jejudonia (93.07%) that all belong to the family Flavobacteriaceae. Whole-genome sequence comparison revealed that the ANI (average nucleotide identity) and digital DDH (DNA-DNA hybridization) values between strain RR4-38T and the two closest strains, Ulvibacter antarcticus DSM 23424T and Aureitalea marina S1-66T, were 68.96–69.88% and 17.4–19%, respectively. The genome analysis revealed that the strain might be involved in biodegradation of organic debris produced by farmed fish in aquaculture systems. The predominant respiratory quinone was menaquinone MK-6 and the major cellular fatty acids were iso-C15:0 (26.5%), iso-C17:0 3-OH (16.4%), iso-C15:1 G (15%), and iso-C16:0 3-OH (9.6%). The major cellular polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, unidentified aminolipids, and glycolipids. Based on phenotypic, chemotaxonomic, and phylogenetic features, strain RR4-38t represents a novel genus and species in the family Flavobacteriaceae, for which the name Pukyongia salina gen. nov., sp. nov. is proposed. The type strain is RR4-38T (= KCTC 52651T = DSM 108068T).     

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.     

Chryseobacterium miricola sp. nov., a novel species isolated from condensation water of space station Mir

Classification of strain W3-B1, which was isolated from condensation water in the Russian space laboratory Mir, was investigated by a polyphasic taxonomic approach. Cells of strain W3-B1 were nonmotile, asporogenous, gram-negative slender rods with rounded ends. 16S rRNA gene sequence analysis indicated that organism should be placed in the genus Chryseobacterium. This organism contains menaquinone MK-6 as the predominent isoprenoid quinone and 3-OH iso 17:0 (40%), iso 15:0 (33%) as the major fatty acids. Phylogenetically, the nearest relative of strain W3-B1 is Chryseobacterium meningosepticum with sequence similarity of 98.4%, but DNA-DNA hybridization resulted in similarity values of only 52.3%. The G+C mol% is 34.6 mol%. Based upon results obtained by morphological, biochemical, chemotaxonomic, and molecular methods, strain W3-B1 was clearly distinguishable from other Chryseobacterium species. For these reasons, a novel species of family Flavobacteriaceae is proposed; strain W3-B1(T) (= GTC 862(T) = JCM 11413(T) = DSM 14571(T)) is the type strain.     

Detection and diversity assessment of Xylella fastidiosa in field-collected plant and insect samples by using 16S rRNA and gyrB sequences

The causal agent of diseases in many economically important plants is attributed to the xylem-limited bacterium Xylella fastidiosa. The detection of this plant pathogen has been hampered due to its difficult isolation and slow growth on plates. Nearly complete nucleotide sequences of the 16S rRNA gene and partial sequences of the gyrB gene were determined for 18 strains of X. fastidiosa isolated from different plant hosts. A phylogenetic analysis, based on gyrB, grouped strains in three clusters; grape-isolated strains formed one cluster, citrus-coffee strains formed another cluster, and a third cluster resulted from all other strains. Primer pairs designed for the 16S rRNA and gyrB genes were extensively searched in databases to verify their in silico specificity. Primer pairs were certified with 30 target and 36 nontarget pure cultures of microorganisms, confirming 100% specificity. A multiplex PCR protocol was developed and its sensitivity tested. Sequencing of PCR products confirmed the validity of the multiplex PCR. Xylella fastidiosa was detected in field-collected plants, disease vector insects, and nonsymptomatic but infected plants. Specific detection of X. fastidiosa may facilitate the understanding of its ecological significance and prevention of spread of the disease.     

Sequencing and analysis of the internal transcribed spacers (ITSs) and coding regions in the EcoR I fragment of the ribosomal DNA of the Japanese pond frog Rana nigromaculata

The rDNA of eukaryotic organisms is transcribed as the 40S-45S rRNA precursor, and this precursor contains the following segments: 5' - ETS - 18S rRNA - ITS 1 - 5.8S rRNA - ITS 2 - 28S rRNA - 3'. In amphibians, the nucleotide sequences of the rRNA precursor have been completely determined in only two species of Xenopus. In the other amphibian species investigated so far, only the short nucleotide sequences of some rDNA fragments have been reported. We obtained a genomic clone containing the rDNA precursor from the Japanese pond frog Rana nigromaculata and analyzed its nucleotide sequence. The cloned genomic fragment was 4,806 bp long and included the 3'-terminus of 18S rRNA, ITS 1, 5.8S rRNA, ITS 2, and a long portion of 28S rRNA. A comparison of nucleotide sequences among Rana, the two species of Xenopus, and human revealed the following: (1) The 3'-terminus of 18S rRNA and the complete 5.8S rRNA were highly conserved among these four taxa. (2) The regions corresponding to the stem and loop of the secondary structure in 28S rRNA were conserved between Xenopus and Rana, but the rate of substitutions in the loop was higher than that in the stem. Many of the human loop regions had large insertions not seen in amphibians. (3) Two ITS regions had highly diverged sequences that made it difficult to compare the sequences not only between human and frogs, but also between Xenopus and Rana. (4) The short tracts in the ITS regions were strictly conserved between the two Xenopus species, and there was a corresponding sequence for Rana. Our data on the nucleotide sequence of the rRNA precursor from the Japanese pond frog Rana nigromaculata were used to examine the potential usefulness of the rRNA genes and ITS regions for evolutionary studies on frogs, because the rRNA precursor contains both highly conserved regions and rapidly evolving regions.