Selective Phylogenetic Analysis Targeted at 16S rRNA Genes of Thermophiles and Hyperthermophiles in Deep-Subsurface Geothermal Environments

Deep-subsurface samples obtained by deep drilling are likely to be contaminated with mesophilic microorganisms in the drilling fluid, and this could affect determination of the community structure of the geothermal microflora using 16S rRNA gene clone library analysis. To eliminate possible contamination by PCR-amplified 16S rRNA genes from mesophiles, a combined thermal denaturation and enzyme digestion method, based on a strong correlation between the G+C content of the 16S rRNA gene and the optimum growth temperatures of most known prokaryotic cultures, was used prior to clone library construction. To validate this technique, hot spring fluid (76°C) and river water (14°C) were used to mimic a deep-subsurface sample contaminated with drilling fluid. After DNA extraction and PCR amplification of the 16S rRNA genes from individual samples separately, the amplified products from river water were observed to be denatured at 82°C and completely digested by exonuclease I (Exo I), while the amplified products from hot spring fluid remained intact after denaturation at 84°C and enzyme digestion with Exo I. DNAs extracted from the two samples were mixed and used as a template for amplification of the 16S rRNA genes. The amplified rRNA genes were denatured at 84°C and digested with Exo I before clone library construction. The results indicated that the 16S rRNA gene sequences from the river water were almost completely eliminated, whereas those from the hot spring fluid remained.     

Molecular Phylogenetic Analysis of Archaeal Intron-Containing Genes Coding for rRNA Obtained from a Deep-Subsurface Geothermal Water Pool

Molecular phylogenetic analysis of a naturally occurring microbial community in a deep-subsurface geothermal environment indicated that the phylogenetic diversity of the microbial population in the environment was extremely limited and that only hyperthermophilic archaeal members closely related to Pyrobaculum were present. All archaeal ribosomal DNA sequences contained intron-like sequences, some of which had open reading frames with repeated homing-endonuclease motifs. The sequence similarity analysis and the phylogenetic analysis of these homing endonucleases suggested the possible phylogenetic relationship among archaeal rRNA-encoded homing endonucleases.     

Phylogenetic Analysis of Sequences of the 12S and 16S rRNA Mitochondrial Genes in the Family Bovidae: New Evidence

The phylogeny of the family Bovidae has been inferred from our data on the 12S and 16S rRNA mtDNA gene sequences and from the results of other authors. A considerable (2460 bp) length of the analyzed fragments of these conserved genes and the use of different methods of cladogram construction allowed us to verify the systematic position of the genera Saiga, Pantholops, Procapra, and Oreamnos. Saigas were shown to be phylogenetically far closer to gazelles than black-tailed gazelles and pygmy antelopes. In general, the genetic analysis data are in agreement with the results of morphological studies.     

Phylogenetic clustering of soil microbial communities by 16S rRNA but not 16S rRNA genes

We evaluated phylogenetic clustering of bacterial and archaeal communities from redox-dynamic subtropical forest soils that were defined by 16S rRNA and rRNA gene sequences. We observed significant clustering for the RNA-based communities but not the DNA-based communities, as well as increasing clustering over time of the highly active taxa detected by only rRNA.     

Quantitative PCR Targeting 16S rRNA and Reductive Dehalogenase Genes Simultaneously Monitors Multiple Dehalococcoides Strains

The 16S rRNA gene provides insufficient information to infer the range of chloroorganic electron acceptors used by different Dehalococcoides organisms. To overcome this limitation and provide enhanced diagnostic tools for growth measurements, site assessment, and bioremediation monitoring, a quantitative real-time PCR (qPCR) approach targeting 16S rRNA genes and three Dehalococcoides reductive dehalogenase (RDase) genes with assigned function (i.e., tceA, bvcA, and vcrA) was designed and evaluated. qPCR standard curves generated for the RDase genes by use of genomic DNA from Dehalococcoides pure cultures correlated with standard curves obtained for both Bacteria- and Dehalococcoides-targeted 16S rRNA genes, suggesting that the RDase genes are useful targets for quantitative assessment of Dehalococcoides organisms. RDase gene probe/primer pairs were specific for the Dehalococcoides strains known to carry the diagnostic RDase gene sequences, and the qPCR method allowed the detection of as few as 1 to 20 and quantification of as few as 50 to 100 tceA, bvcA, or vcrA gene targets per PCR volume. The qPCR approach was applied to dechlorinating enrichment cultures, microcosms, and samples from a contaminated site. In characterized enrichment cultures where known Dehalococcoides strains were enumerated, the sum of the three RDase genes equaled the total Dehalococcoides cell numbers. In site samples and chloroethane-dechlorinating microcosms, the sum of the three RDase genes was much less than that predicted by Dehalococcoides-targeted qPCR, totaling 10 to 30% of the total Dehalococcoides cell numbers. Hence, a large number of Dehalococcoides spp. contain as-yet-unidentified RDase genes, indicating that our current understanding of the dechlorinating Dehalococcoides community is incomplete.     

大黄鱼16SrRNA和18SrRNA基因的测定与序列分析

利用多对引物,扩增并测定出大黄鱼16SrRNA基因和18SrRNA基因的部分序列,其长度分别为1202bp和1275bp,16SrRNA基因序列的GC含量为46．12％,18SrRNA基因的Gc含量为53．oo％。将大黄鱼16SrRNA基因序列与GenBank中15种硬骨鱼类的同源序列结合,同时将其18SrRNA基因序列与GenBank中9种脊索动物的同源序列相结合,运用软件获得各自序列间差异百分比,转换和颠换数值等信息。基于这两种基因序列,利用NJ法和BI法,分别构建16种硬骨鱼类和10种脊索动物的分子系统树。18SrRNA构建的系统树包括三大支,一支为哺乳类、鸟类和爬行类共6个物种,一支为两栖类的1个物种,另一支为2种硬骨鱼类。16SrRNA构建的系统树显示大黄鱼所在的石首鱼科与鲈科和盖刺鱼科亲缘关系较近。此外还讨论了这两个基因的序列特征。     

Molecular Analysis of Deep-Sea Hydrothermal Vent Aerobic Methanotrophs by Targeting Genes of 16S rRNA and Particulate Methane Monooxygenase

Molecular diversity of deep-sea hydrothermal vent aerobic methanotrophs was studied using both 16S ribosomalDNA and pmoA encoding the subunit A of particulate methane monooxygenase (pMOA). Hydrothermal vent plume and chimney samples were collected from back-arc vent at Mid-Okinawa Trough (MOT), Japan, and the Trans-Atlantic Geotraverse (TAG) site along Mid-Atlantic Ridge, respectively. The target genes were amplified by polymerase chain reaction from the bulk DNA using specific primers and cloned. Fifty clones from each clone library were directly sequenced. The 16S rDNA sequences were grouped into 3 operational taxonomic units (OTUs), 2 from MOT and 1 from TAG. Two OTUs (1 MOT and 1 TAG) were located within the branch of type I methanotrophic ?-Proteobacteria. Another MOT OTU formed a unique phylogenetic lineage related to type I methanotrophs. Direct sequencing of 50 clones each from the MOT and TAG samples yielded 17 and 4 operational pmoA units (OPUs), respectively. The phylogenetic tree based on the pMOA amino acid sequences deduced from OPUs formed diverse phylogenetic lineages within the branch of type I methanotrophs, except for the OPU MOT-pmoA-8 related to type X methanotrophs. The deduced pMOA topologies were similar to those of all known pMOA, which may suggest that the pmoA gene is conserved through evolution. Neither the 16S rDNA nor pmoA molecular analysis could detect type II methanotrophs, which suggests the absence of type II methanotrophs in the collected vent samples.     

Rhizobial 16S rRNA and dnaK Genes: Mosaicism and the Uncertain Phylogenetic Placement of Rhizobium galegae

The phylogenetic relatedness among 12 agriculturally important species in the order Rhizobiales was estimated by comparative 16S rRNA and dnaK sequence analyses. Two groups of related species were identified by neighbor-joining and maximum-parsimony analysis. One group consisted of Mesorhizobium loti and Mesorhizobium ciceri, and the other group consisted of Agrobacterium rhizogenes, Rhizobium tropici, Rhizobium etli, and Rhizobium leguminosarum. Although bootstrap support for the placement of the remaining six species varied, A. tumefaciens, Agrobacterium rubi, and Agrobacterium vitis were consistently associated in the same subcluster. The three other species included Rhizobium galegae, Sinorhizobium meliloti, and Brucella ovis. Among these, the placement of R. galegae was the least consistent, in that it was placed flanking the A. rhizogenes-Rhizobium cluster in the dnaK nucleotide sequence trees, while it was placed with the other three Agrobacterium species in the 16S rRNA and the DnaK amino acid trees. In an effort to explain the inconsistent placement of R. galegae, we examined polymorphic site distribution patterns among the various species. Localized runs of nucleotide sequence similarity were evident between R. galegae and certain other species, suggesting that the R. galegae genes are chimeric. These results provide a tenable explanation for the weak statistical support often associated with the phylogenetic placement of R. galegae, and they also illustrate a potential pitfall in the use of partial sequences for species identification.     

Selective and Sensitive Method for PCR Amplification of Escherichia coli 16S rRNA Genes in Soil

A set of PCR primers targeting 16S rRNA gene sequences was designed, and PCR parameters were optimized to develop a robust and reliable protocol for selective amplification of Escherichia coli 16S rRNA genes. The method was capable of discriminating E. coli from other enteric bacteria, including its closest relative, Shigella. Selective amplification of E. coli occurred only when the annealing temperature in the PCR was elevated to 72°C, which is 10°C higher than the optimum for the primers. Sensitivity was retained by modifying the length of steps in the PCR, by increasing the number of cycles, and most importantly by optimizing the MgCl₂ concentration. The PCR protocol developed can be completed in less then 2 h and, by using Southern hybridization, has a detection limit of ca. 10 genomic equivalents per reaction. The method was demonstrated to be effective for detecting E. coli DNA in heterogeneous DNA samples, such as those extracted from soil.     

Investigation of the Microbial Ecology of Intertidal Hot Springs by Using Diversity Analysis of 16S rRNA and Chitinase Genes

The microbial diversity of intertidal hot springs on the seashore of northwest Iceland was examined by combining directed in situ enrichments, artificial support colonization, and mat sampling. Analysis of 16S rRNA genes revealed the presence of clones related to both marine and terrestrial, thermophilic, mesophilic, and psychrophilic microorganisms scattered among 11 bacterial divisions. No archaea were found. The species composition of the enrichments was affected by the length of the hot periods experienced at low tide and was very different from those found in the biomass. A total of 36 chitinase genes were detected by molecular screening of the samples with degenerate primers for glycoside hydrolase family 18. The chitinase gene diversity was at least twofold higher in the enrichment samples than in the controls, indicating that a much higher diversity of hydrolytic genes can be accessed with this approach.     

Phylogenetic Analysis of Pasteuria penetrans by 16S rRNA Gene Cloning and Sequencing

Pasteuria penetrans is an endospore-forming bacterial parasite of Meloidogyne spp. This organism is among the most promising agents for the biological control of root-knot nematodes. In order to establish the phylogenetic position of this species relative to other endospore-forming bacteria, the 16S ribosomal genes from two isolates of P. penetrans, P-20, which preferentially infects M. arenaria race 1, and P-100, which preferentially infects M. incognita and M. javanica, were PCR-amplified from a purified endospore extraction. Universal primers for the 16S rRNA gene were used to amplify DNA which was cloned, and a nucleotide sequence was obtained for 92% of the gene (1,390 base pairs) encoding the 16S rDNA from each isolate. Comparison of both isolates showed identical sequences that were compared to 16S rDNA sequences of 30 other endospore-forming bacteria obtained from GenBank. Parsimony analyses indicated that P. penetrans is a species within a clade that includes Alicyclobacillus acidocaldarius, A. cycloheptanicus, Sulfobacillus sp., Bacillus tusciae, B. schlegelii, and P. ramosa. Its closest neighbor is P. ramosa, a parasite of Daphnia spp. (water fleas). This study provided a genomic basis for the relationship of species assigned to the genus Pasteuria, and for comparison of species that are parasites of different phytopathogenic nematodes.     

Phylogenetic studies of the rRNA group II pseudomonads based on 16S rRNA gene sequences

Nearly complete sequences of 16S rRNA genes were determined for eight bacterial strains representing five species of the rRNA homology group II pseudomonads that are members of the beta subclass of the class Proteobacteria. Comparative analysis with published sequence data indicated that Pseudomonas andropogonis, Ps. caryophylli, Ps. gladioli pv. gladioli and Ps. cepacia aggregate in one coherent cluster at 94·2% sequence similarity; Ps. solanacearum and Ps. pickettii shared 95·3% and 92·8% similarity with Alcaligenes eutrophus in another cluster. Both clusters joined at 87·8% similarity, which is similar to that for genera in this subclass of Proteobacteria. Based on this study and on comparison with other works we suggest that these species are separated from authentic pseudomonads and constitute a new genus or possibly two related genera accommodating Ps. andropogonis, Ps. caryophylli, Ps. gladioli, Ps. cepacia, and Ps. solanacearum, Ps. pickettii and A. eutrophus, respectively. Four strains of Ps. solanacearum representing Biovars 1, 2, 3 and 4 were subdivided into two clusters at 99·1% sequence similarity, in agreement with other published phenotypic and genotypic studies. The two clusters may be potentially regarded as subspecies.     

基于16S rRNA部分序列中国蝙蝠科蝙蝠系统发生分析

蝙蝠科蝙蝠遍布全世界,是蝙蝠中种类最多的一个科.尽管从形态学、胚胎学和分子生物学等方面认为蝙蝠科内长翼蝠亚科应该提升到科、鼠耳蝠属应该提升到亚科的分类地位,但是其科内的系统关系长期以来一直处于争议之中.本文对蝙蝠科11种38个标本线粒体16S rRNA部分序列进行了测序,并结合以前报道的13种(属于7科13属)蝙蝠的线粒体16S rRNA部分序列构建了系统树,结果表明:长翼蝠亚科可以提升到科的分类地位、鼠耳蝠属提升到亚科的分类地位,这与前人报道的结果一致;相对于由鼠耳蝠亚科、彩蝠亚科和管鼻蝠亚科构成的分支,蝙蝠亚科和Antrozoinae是一个并系群.蝙蝠亚科内的亲缘关系也进行了进一步的讨论.     

Phylogenetic analysis of the genus Aquaspirillum based on 16S rRNA gene sequences

Phylogenetic analysis of 15 species of the genus Aquaspirillum based on 16S rRNA gene (rDNA) sequences indicated that the genus Aquaspirillum is phylogenetically heterogeneous and the species could be divided into four groups as follows: Aquaspirillum serpens, the type species of this genus, A. dispar and A. putridiconchylium are situated in the family Neisseriaceae; members of the second group, A. gracile, A. delicatum, A. anulus, A. giesbergeri, A. sinuosum, A. metamorphum and A. psychrophilum, are included in the family Comamonadaceae; the two members of the third group, A. arcticum and A. autotrophicum, are included in the family Oxalobacteriaceae; and members of the fourth group, A. polymorphum, A. peregrinum, and A. itersonii, are included in the alpha-subdivision of Proteobacteria. Thus, phylogenetic studies indicated that all the species excepting A. serpens, the type species, should be transferred to distinct genera.     

Gene Analysis of Trehalose-producing Enzymes from Hyperthermophilic Archaea in Sulfolobales

The genes encoding new trehalose-producing enzymes from S. acidocaldarius ATCC33909 were cloned to analyze the distribution of these genes in Sulfolobales. Comparison of the amino acid sequences with S. solfataricus KM1 showed approximately 50% similarity. Southern analysis suggests that homologues of the trehalose-producing enzyme genes exist widely in Sulfolobales and strains in Sulfolobales were classified into three kinds of genotypes.     

Phylogenetic analysis of the genus Microbacterium based on 16S rRNA gene sequences

Abstract 16S rRNA gene (rDNA) studies of the six species of the genus Microbacterium, M. lacticum, M. laevaniformans, M. dextranolyticum, M. imperiale, M. arborescens and M. aurum , were performed and the primary structures were compared with those of 29 representative actinobacteria and related organisms. Phylogenetic analysis indicated that six species of the genus Microbacterium and representative four species of the genus Aureobacterium appear to be phylogenetically coherent as was suggested by Rainey et al., although the peptidoglycan types of these two genera are different (peptidoglycan type B1 or B2). Thus, the phylogenetical analyses revealed that members of actinobacteria with group B-peptidoglycan do not cluster according to their peptidoglycan types, but form compact cluster different from actinobacteria or actinomycetes with group A-peptidoglycan.     

Phylogenetic analysis based evolutionary study of 16S rRNA in known Pseudomonas sp

Molecular evolution analysis of 16S rRNA sequences of native Pseudomonas strains and different fluorescent pseudomonads wereconducted on the basis of Molecular Evolutionary Genetics Analysis version 5.2 (MEGA5.2). Topological evaluations showcommon origin for native strains with other known strains with available sequences at GenBank database. Phylogenetic affiliationof different Pseudomonas sp based on 16S rRNA gene shows that molecular divergence contributes to the genetic diversity ofPseudomonas sp. Result indicate direct dynamic interactions with the rhizospheric pathogenic microbial community. The selectionpressure acting on 16S rRNA gene was related to the nucleotide diversity of Pseudomonas sp in soil rhizosphere community amongdifferent agricultural crops. Besides, nucleotide diversity among the whole population was very low and tajima test statistic value(D) was also slightly positive (Tajima׳s test statistics D value 0.351). This data indicated increasing trends of infection of soil-bornepathogens under gangetic-alluvial regions of West Bengal due to high degree of nucleotide diversity with decreased population ofplant growth promoting rhizobacteria like fluorescent Pseudomonads in soil.