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.     

Arcobacter bivalviorum sp. nov. and Arcobacter venerupis sp. nov., new species isolated from shellfish

A group of ten Arcobacter isolates (Gram negative, slightly curved motile rods, oxidase positive) was recovered from mussels (nine) and from clams (one). These isolates could not be assigned to any known species using the molecular identification methods specific for this genus (16S rDNA-RFLP and m-PCR). The aim of this study is to establish the taxonomic position of these isolates. The 16S rRNA gene sequence similarity of mussel strain F4(T) to the type strains of all other Arcobacter species ranged from 91.1% to 94.8%. The species most similar to the clams' strain F67-11(T) were Arcobacter defluvii (CECT 7697(T), 97.1%) and Arcobacter ellisii (CECT 7837(T), 97.0%). On the basis of phylogenetic analyses with 16S rRNA, rpoB, gyrB and hsp60 genes, the mussel and clam strains formed two different, new lineages within the genus Arcobacter. These data, together with their different phenotypic characteristics and MALDI-TOF mass spectra, revealed that these strains represent two new species, for which the names Arcobacter bivalviorum (type strain F4(T)=CECT 7835(T)=LMG 26154(T)) and Arcobacter venerupis (type strain F67-11(T)=CECT 7836(T)=LMG 26156(T)) are proposed.     

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.     

干酪乳杆菌的近缘种及亚种部分看家基因的系统发育分析

【背景】16S rRNA基因序列分析已广泛应用于细菌的分类鉴定,但是存在一定局限性,而使用看家基因作为分子标记在近缘种及亚种间的系统发育分析中具有其独特的优势。【目的】研究16S rRNA、uvr C (核酸外切酶ABC,C亚基)和mur E (UDP-N-乙酰胞壁酰三肽合酶)基因序列对干酪乳杆菌的近缘种及亚种的区分能力。【方法】采用分离自传统发酵乳中的6株干酪乳杆菌为研究对象,选取uvr C和mur E基因片段,通过PCR扩增、测序,结合已公布的干酪乳杆菌的近缘种或亚种的相应序列计算遗传距离、构建系统发育树,并与16S rRNA基因序列分析技术进行比较。【结果】研究发现Lactobacilluscasei及相近种间的uvr C、mur E和联合基因(uvr C-mur E)构建的系统发育树拓扑结构与16S rRNA基因结果基本一致,区别在于相似性的不同,其分别为79.00%-99.16%、89.08%-99.20%、76.56%-99.69%和99.58%-100%。基于16S rRNA基因不能区分干酪乳杆菌的近缘种及亚种,而看家基因uvr C和mur E基因序列能够很好地区分干酪乳杆菌的近缘种及亚种,并且将uvr C和mur E基因串联使用后,试验菌株与参考菌株的分类关系更加清晰。【结论】联合基因(uvr C-mur E)可作为16SrRNA基因的辅助工具用于干酪乳杆菌的近缘种及亚种的快速准确鉴定。     

Members of the Cytophaga–Flavobacterium–Bacteroides phylum as intracellular bacteria of acanthamoebae: proposal of 'Candidatus Amoebophilus asiaticus'

Three Gram-negative, rod-shaped bacteria that were found intracellularly in two environmental and one clinical Acanthamoeba sp. isolates were analysed. Two endocytobionts showing a parasitic behaviour were propagated successfully outside their amoebal host cells and were identified subsequently by comparative 16S rRNA sequence analysis as being most closely affiliated with Flavobacterium succinicans (99% 16S rRNA sequence similarity) or Flavobacterium johnsoniae (98% 16S rRNA sequence similarity). One endocytobiont could neither be cultivated outside its original Acanthamoeba host ( Acanthamoeba sp. TUMSJ-321) nor transferred into other amoebae. Electron microscopy revealed that the amoebal trophozoites and cysts were almost completely filled with cells of this endosymbiont which are surrounded by a host-derived membrane. According to 16S rRNA sequence analysis, this endosymbiont could also be assigned to the Cytophaga – Flavobacterium – Bacteroides (CFB) phylum, but was not closely affiliated to any recognized species within this phylogenetic group (less than 82% 16S rRNA sequence similarity). Identity and intracellular localization of this endosymbiont were confirmed by application of a specific fluorescently labelled 16S rRNA-targeted probe. Based on these findings, we propose classification of this obligate Acanthamoeba endosymbiont as ' Candidatus Amoebophilus asiaticus'. Comparative 18S rRNA sequence analysis of the host of ' Candidatus Amoebophilus asiaticus' revealed its membership with Acanthamoeba 18S rDNA sequence type T4 that comprises the majority of all Acanthamoeba isolates.     

Rapid discrimination and classification of the <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> group based on a partial <Emphasis Type="Italic">dnaK</Emphasis> sequence and DNA fingerprinting techniques

The Lactobacillus plantarum group comprises five very closely related species. Some species of this group are considered to be probiotic and widely applied in the food industry. In this study, we compared the use of two different molecular markers, the 16S rRNA and dnaK gene, for discriminating phylogenetic relationships amongst L. plantarum strains using sequencing and DNA fingerprinting. The average sequence similarity for the dnaK gene (89.2%) among five type strains was significantly less than that for the 16S rRNA (99.4%). This result demonstrates that the dnaK gene sequence provided higher resolution than the 16S rRNA and suggests that the dnaK could be used as an additional phylogenetic marker for L. plantarum. Species-specific profiles of the Lactobacillus strains were obtained with RAPD and RFLP methods. Our data indicate that phylogenetic relationships between these strains are easily resolved using sequencing of the dnaK gene or DNA fingerprinting assays.     

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.     

POLYPHASIC STUDY OF ANTARCTIC CYANOBACTERIAL STRAINS1

We isolated 59 strains of cyanobacteria from the benthic microbial mats of 23 Antarctic lakes, from five locations in two regions, in order to characterize their morphological and genotypic diversity. On the basis of their morphology, the cyanobacteria were assigned to 12 species that included four Antarctic endemic taxa. Sequences of the ribosomal RNA gene were determined for 56 strains. In general, the strains closely related at the 16S rRNA gene level belonged to the same morphospecies. Nevertheless, divergences were observed concerning the diversity in terms of species richness, novelty, and geographical distribution. For the 56 strains, 21 operational taxonomic units (OTUs, defined as groups of partial 16S rRNA gene sequences with more than 97.5% similarity) were found, including nine novel and three exclusively Antarctic OTUs. Sequences of Petalonema cf. involvens and Chondrocystis sp. were determined for the first time. The internally transcribed spacer (ITS) between the 16S and the 23S rRNA genes was sequenced for 33 strains, and similar groupings were observed with the 16S rRNA gene and the ITS, even when the strains were derived from different lakes and regions. In addition, 48 strains were screened for antimicrobial and cytotoxic activities, and 17 strains were bioactive against the gram‐positive Staphylococcus aureus, or the fungi Aspergillus fumigatus and Cryptococcus neoformans. The bioactivities were not in coincidence with the phylogenetic relationships, but rather were specific to certain strains.     

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。     

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.     

Gene sequence heterogeneity of Corallococcus coralloides strains isolated from geographically diverse locations

Thirty-three strains classified as Corallococcus coralloides isolated from mostly soil samples in 14 countries of four continents, were subjected to phylogenetic analyses. Based on 16S rDNA analyses the strains form a highly related cluster, sharing above 98.7% sequence similarity. Four groups were recognized within this cluster, only one of which, containing two strains from St. Lucia, Lower Antilles, was exclusively defined by strains from the same sample. The other groups contained members from different countries, even continents. The largest group embraced the type strains of C. coralloides DSM 2259(T) and Corallococcus exiguus 14696(T) which were almost indistinguishable in their 16S rRNA gene sequence. Corallococcus macrosporus DSM 14697(T) grouped outside the C. coralloides cluster, showing a higher relationship to a member of Myxococcus. The topology of the tree generated on the basis of the partial gyrase B (gyrB) gene sequence supports the rRNA gene tree, though some differences in the order of branching were observed. As judged by the binary similarity values the higher resolution power of gyrB sequences was confirmed. From a taxonomic standpoint, the size of myxospores is not a valuable taxonomic criterion, as small- and medium-sized myxospores are members of the same group. If the species status of C. coralloides and C. exiguus is verified by other methods (e.g. DNA-DNA hybridisation, RiboTyping), the genus Corallococcus may embrace a broad range of yet-to-be described novel species. The presence of strains within the same sample displaying higher relatedness to strains from other locations points towards an intensive dispersal of myxospores across continents.     

The Evolutionary History of the Genus Acanthamoeba and the Identification of Eight New 18S rRNA Gene Sequence Types

ABSTRACT The 18S rRNA gene ( Rns ) phylogeny of Acanthamoeba is being investigated as a basis for improvements in the nomenclature and taxonomy of the genus. We previously analyzed Rns sequences from 18 isolates from morphological groups 2 and 3 and found that they fell into four distinct evolutionary lineages we called sequence types T1-T4. Here, we analyzed sequences from 53 isolates representing 16 species and including 35 new strains. Eight additional lineages (sequence types T5-T12) were identified. Four of the 12 sequence types included strains from more than one nominal species. Thus, sequence types could be equated with species in some cases or with complexes of closely related species in others. The largest complex, sequence type T4, which contained six closely related nominal species, included 24 of 25 keratitis isolates. Rns sequence variation was insufficient for full phylogenetic resolution of branching orders within this complex, but the mixing of species observed at terminal nodes confirmed that traditional classification of isolates has been inconsistent. One solution to this problem would be to equate sequence types and single species. Alternatively, additional molecular information will be required to reliably differentiate species within the complexes. Three sequence types of morphological group 1 species represented the earliest divergence in the history of the genus and, based on their genetic distinctiveness, are candidates for reclassification as one or more novel genera.     

Phylogenetic diversity of culturable bacteria from Antarctic sandy intertidal sediments

The diversity of culturable bacteria associated with sandy intertidal sediments from the coastal regions of the Chinese Antarctic Zhongshan Station on the Larsemann Hills (Princess Elizabeth Land, East Antarctica) was investigated. A total of 65 aerobic heterotrophic bacterial strains were isolated at 4°C. Microscopy and 16S rRNA gene sequence analysis indicated that the isolates were dominated by Gram-negative bacteria, while only 16 Gram-positive strains were isolated. The bacterial isolates fell in five phylogenetic groups: Alpha- and Gammaproteobacteria, Bacteroidetes, Actinobacteria and Firmicutes. Based on phylogenetic trees, all the 65 isolates were sorted into 29 main clusters, corresponding to at least 29 different genera. Based on sequence analysis (<98% sequence similarity), the Antarctic isolates belonged to at least 37 different bacterial species, and 14 of the 37 bacterial species (37.8%) represented potentially novel taxa. These results indicated a high culturable diversity within the bacterial community of the Antarctic sandy intertidal sediments.     

Aeromonas tecta sp. nov., isolated from clinical and environmental sources

Five Aeromonas strains, isolated from both clinical and environmental sources and characterized by a polyphasic approach, including phylogenetic analysis derived from gyrB, rpoD, and 16S rRNA gene sequencing, as well as DNA-DNA hybridization, extensive biochemical and antibiotic susceptibility tests, were recognized as members of an unknown, or undescribed, Aeromonas species. These "Aeromonas eucrenophila-like" strains were closely related to the species A. eucrenophila and Aeromonas encheleia, but they were negative for indole and acid from glycerol tests. Therefore, based on the results of the phylogenetic analyses and DNA-DNA pairing data of these strains, a novel species of the genus Aeromonas is described, for which the name Aeromonas tecta is proposed with isolate F518(T) (CECT7082(T), DSM17300(T), MDC91(T)) as the type strain.     

Flavobacterium oncorhynchi sp. nov., a new species isolated from rainbow trout (Oncorhynchus mykiss)

Eighteen isolates of a Gram-negative, catalase and oxidase-positive, rod-shaped bacterium, recovered from diseased rainbow trout (Oncorhynchus mykiss), were characterized, using a polyphasic taxonomic approach. Studies based on comparative 16S rRNA gene sequence analysis showed that that the eighteen new isolates shared 99.2-100% sequence similarities. Phylogenetic analysis revealed that isolates from trout belonged to the genus Flavobacterium, showing the highest sequence similarities to F. chungangense (98.6%), F. frigidimaris (98.1%), F. hercynium (97.9%) and F. aquidurense (97.8%). DNA-DNA reassociation values between the trout isolates (exemplified by strain 631-08(T)) and five type strains of the most closely related Flavobacterium species exhibited less than 27% similarity. The G+C content of the genomic DNA was 33.0 mol%. The major respiratory quinone was observed to be menaquinone 6 (MK-6) and iso-C(15:0), C(15:0) and C(16:1) ω7c the predominant fatty acids. The polar lipid profile of strain 631-08(T) consisted of phosphatidylethanolamine, unknown aminolipids AL1 and AL3, lipids L1, L2, L3 and L4 and phospholipid PL1. The novel isolates were differentiated from related Flavobacterium species by physiological and biochemical tests. On the basis of the evidence from this polyphasic study, it is proposed that the isolates from rainbow trout be classified as a new species of the genus Flavobacterium, Flavobacterium oncorhynchi sp. nov. The type strain is 631-08(T) (= CECT 7678(T) = CCUG 59446(T)).     

Stenotrophomonas interspecies differentiation and identification by gyrB sequence analysis

Stenotrophomonas species are found commonly in environmental and clinical samples; Stenotrophomonas maltophilia is an important opportunistic pathogen of humans. Traditional phenotyping protocols, as well as genotyping by 16S rRNA gene sequence analysis, do not reliably distinguish the species of Stenotrophomonas. Sequence analyses of two targeted PCR-amplified regions of the gyrB gene, which encodes the β-subunit of DNA gyrase, enabled resolution and identification of these species. Most type strains of the different species of Stenotrophomonas exhibited more than 7% dissimilarity in the gyrB gene sequences. Among these, strains identified as the same species exhibited sequence dissimilarities up to 4.6% and 5.9% for the two regions, respectively. Strains identified as S. maltophilia, with 16S rRNA gene sequence similarities > 99.0%, were grouped within a 'S. maltophilia complex'; these organisms exhibited gyrB similarities as low as 93%. Many of these strains possessed genomic DNA similarities with the type strain of S. maltophilia CCUG 5866(T) below 70%. These data, including gyrB sequence comparisons, indicate that strains identified as S. maltophilia may comprise distinct, new species.     

Aerobic anoxygenic photosynthesis in Roseobacter clade bacteria from diverse marine habitats

The marine Roseobacter clade comprises several genera of marine bacteria related to the uncultured SAR83 cluster, the second most abundant marine picoplankton lineage. Cultivated representatives of this clade are physiologically heterogeneous, and only some have the capability for aerobic anoxygenic photosynthesis, a process of potentially great ecological importance in the world's oceans. In an attempt to correlate phylogeny with ecology, we investigated the diversity of Roseobacter clade strains from various marine habitats (water samples, biofilms, laminariae, diatoms, and dinoflagellate cultures) by using the 16S rRNA gene as a phylogenetic marker gene. The potential for aerobic anoxygenic photosynthesis was determined on the genetic level by PCR amplification and sequencing of the pufLM genes of the bacterial photosynthesis reaction center and on the physiological level by detection of bacteriochlorophyll (Bchl) a. A collection of ca. 1,000 marine isolates was screened for members of the marine Roseobacter clade by 16S rRNA gene-directed multiplex PCR and sequencing. The 42 Roseobacter clade isolates found tended to form habitat-specific subclusters. The pufLM genes were detected in two groups of strains from dinoflagellate cultures but in none of the other Roseobacter clade isolates. Strains within the first group (the DFL-12 cluster) also synthesized Bchl a. Strains within the second group (the DFL-35 cluster) formed a new species of Roseovarius and did not produce Bchl a under the conditions investigated here, thus demonstrating the importance of genetic methods for screening of cultivation-dependent metabolic traits. The pufL genes of the dinoflagellate isolates were phylogenetically closely related to pufL genes from Betaproteobacteria, confirming similar previous observations which have been interpreted as indications of gene transfer events.     

Isolation and molecular characterization of genetically diverse antagonistic, diazotrophic red-pigmented vibrios from different mangrove rhizospheres

Genetic diversity of red-pigmented vibrios from different mangrove rhizospheres ( Porteresia coarctata, Avicennia marina and Rhizophora mucronata ) collected from Pichavaram mangrove, India was investigated. Twenty red–pink pigmented strains were isolated, 16S rRNA gene analyses indicted that these isolates belong to the genus Vibrio and were phylogenetically closely related to the type strains of Vibrio rhizosphaerae and Vibrio ruber . The rep-PCR analysis using GTG₅ and BOX primers had similar groupings, and segregated these pigmented Vibrio isolates including two type strains into seven unique genotypic groups (REP groups A1–A7). The rhizosphere of P. coarctata harbors highly genetically diverse groups of red-pigmented vibrios compared to other plants. Multilocus sequence analysis using four genetic loci ( pyrH, recA, rpoA , 16S rRNA) clearly supported the hypothesis that strains MSSRF38 (REP group A5) and MSSRF39 (REP group A6) could represent new Vibrio species. Biological functions of these vibrios were also determined and it was found that all these isolates have antagonistic activity against phytopathogens, and isolates belonging to REP groups A5 and A6 were positive for nifH gene by PCR. In conclusion, this study for the first time demonstrates the occurrence of genetically diverse groups of antagonistic, diazotrophic red-pigmented vibrios from different mangrove plants and suggests a new ecological role for vibrios as heterotrophic plant associated rhizobacteria.