Molecular Identification of Aeromonas and Coliform Bacteria Isolated on m Endo Media from Lake Erie Waters

Summary Bacteria from recreational waters collected from two Lake Erie beaches in Dunkirk, New York were plated onto m Endo LES media. The 16S rRNA gene was then amplified from coliform and non-coliform bacteria using the polymerase chain reaction. The PCR products were characterized by restriction fragment length polymorphism (RFLP) analysis. A total of 8 RFLP groups were identified from the analysis of 920 samples and selected PCR products from each group were sequenced. The DNA sequence analysis indicated that more than half of the bacteria identified as coliforms on the m Endo plates belonged to the genus Aeromonas from the family Aeromonadaceae. Most of the remaining coliforms were from the Enterobacteriaceae. The data indicate that m Endo agar plates allow the growth of non-coliform bacteria, especially Aeromonas species.     

Ethiopian soils harbor natural populations of rhizobia that form symbioses with common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

<p>The diversity and taxonomic relationships of 83 bean-nodulating rhizobia indigenous to Ethiopian soils were characterized by PCR-RFLP of the internally transcribed spacer (ITS) region between the 16S and 23S rRNA genes, 16S rRNA gene sequence analysis, multilocus enzyme electrophoresis (MLEE), and amplified fragment-length polymorphism. The isolates fell into 13 distinct genotypes according to PCR-RFLP analysis of the ITS region. Based on MLEE, the majority of these genotypes (70%) was genetically related to the type strain of Rhizobium leguminosarum. However, from analysis of their 16S rRNA genes, the majority was placed with Rhizobium etli. Transfer and recombination of the 16S rRNA gene from presumptively introduced R. etli to local R. leguminosarum is a possible theory to explain these contrasting results. However, it seems unlikely that bean rhizobia originating from the Americas (or Europe) extensively colonized soils of Ethiopia because Rhizobium tropici, Rhizobium gallicum, and Rhizobium giardinii were not detected and only a single ineffective isolate of R. etli that originated from a remote location was identified. Therefore, Ethiopian R. leguminosarum may have acquired the determinants for nodulation of bean from a low number of introduced bean-nodulating rhizobia that either are poor competitors for nodulation of bean or that failed to survive in the Ethiopian environment. Furthermore, it may be concluded from the genetic data presented here that the evidence for separating R. leguminosarum and R. etli into two separate species is inconclusive.     

Ribotyping of 16S and 23S rRNA genes and organization of rrn operons in members of the bacterial genera Gemmata,Planctomyces, Thermotoga,Thermus, and Verrucomicrobium

The number of organization of rrn genes of two members of the order Planctomycetales, Planctomyces limnophilus and Gemmata obscuriglobus, as well as three species from other bacterial phyla, namely Thermotoga maritima, Thermus aquaticus and Verrucomicrobium spinosum were examined by Southern blot hybridization analysis of restricted DNA with labeled 16S- and 23S rRNAs. Ribotyping analysis revealed that two species contain unlinked 16S- and 23S rRNA genes. Planctomyces limnophilus possessed two unlinked rrn genes which were separated from each other by at least 4.3 kb, and Thermus aquaticus had to unlinked 16S and 23S rRNA genes, separated from each other by at least 2.5 kb. Gemmata obscuriglobus exhibited five genes for which the organization could as yet not be determined because of the complex hybridization patterns. In the other two species, rrn genes clustered in operons. Thermotoga maritima had a single gene for each rRNA species which were separated by not more than 1.5 kb, while Verrucomicrobium spinosum had four copies of probably linked 16S and 23S rRNA genes with a maximal distance between 16S and 23S rRNA genes of 1.3 kb.     

Culturable Actinobacteria from the Marine Sponge Hymeniacidon perleve: Isolation and Phylogenetic Diversity by 16S rRNA gene-RFLP Analysis

A total of 106 actinobacteria associated with the marine sponge Hymeniacidon perleve collected from the Yellow Sea, China were isolated using eight different media. The number of species and genera of actinobacteria recovered from the different media varied significantly, underlining the importance of optimizing the isolation conditions. The phylogenetic diversity of the actinobacteria isolates was assessed using 16S rRNA gene amplification–restriction fragment length polymorphism (RFLP) analysis of the 106 strains with different morphologies. The RFLP fingerprinting of selected strains by HhaI-digestion of the 16S rRNA genes resulted in 11 different patterns. The HhaI-RFLP analysis gave good resolution for the identification of the actinobacteria isolates at the genus level. A phylogenetic analysis using 16S rRNA gene sequences revealed that the isolates belonged to seven genera of culturable actinobacteria including Actinoalloteichus, Micromonospora, Nocardia, Nocardiopsis, Pseudonocardia, Rhodococcus, and Streptomyces. The dominant genus was Streptomyces, which represented 74% of the isolates. Three of the strains identified are candidates for new species.     

Aeromonas piscicola sp. nov., isolated from diseased fish

Four Aeromonas strains (S1.2^T, EO-0505, TC1 and TI 1.1) isolated from moribund fish in Spain showed a restriction fragment length polymorphism (RFLP) pattern related to strains of Aeromonas salmonicida and Aeromonas bestiarum but their specific taxonomic position was unclear. Multilocus sequence analysis (MLSA) of housekeeping genes rpoD, gyrB, recA and dnaJ confirmed the allocation of these isolates to an unknown genetic lineage within the genus Aeromonas with A. salmonicida, A. bestiarum and Aeromonas popoffii as the phylogenetically nearest neighbours. Furthermore, a strain biochemically labelled as Aeromonas hydrophila (AH-3), showing a pattern of A. bestiarum based on 16S rDNA-RFLP, also clustered with the unknown genetic lineage. The genes rpoD and gyrB proved to be the best phylogenetic markers for differentiating these isolates from their neighbouring species. Useful phenotypic features for differentiating the novel species from other known Aeromonas species included their ability to hydrolyze elastin, produce acid from l-arabinose and salicin, and their inability to produce acid from lactose and use l-lactate as a sole carbon source. A polyphasic approach using phenotypic characterization, phylogenetic analysis of the 16S rRNA gene and of four housekeeping genes, as well as DNA–DNA hybridization studies and an analysis of the protein profiles by MALDI-TOF-MS, showed that these strains represented a novel species for which the name Aeromonas piscicola sp. nov. is proposed with isolate S1.2^T (=CECT 7443^T, =LMG 24783^T) as the type strain.     

Concurrent evolution of nitrogenase genes and 16S rRNA in Rhizobium species and other nitrogen fixing bacteria

It was known that nitrogenase genes and proteins are well conserved even though they are present in a large variety of phylogenetically diverse nitrogen fixing bacteria. This has lead to the speculation, among others, that nitrogen fixation (nif) genes were spread by lateral gene transfer relatively late in evolution. Here we report an attempt to test this hypothesis.We had previously established the complete nucleotide sequences of the three nitrogenase genes from Bradyrhizobium japonicum, and have now analyzed their homologies (or the amino acid sequence homologies of their gene products) with corresponding genes (and proteins) from other nitrogen fixing bacteria. There was a considerable sequence conservation which certainly reflects the strict structural requirements of the nitrogenase iron-sulfur proteins for catalytic functioning. Despite this, the sequences were divergent enough to classify them into an evolutionary scheme that was conceptually not different from the phylogenetic positions, based on 16S rRNA homology, of the species or genera harboring these genes. Only the relation of nif genes of slow-growing rhizobia (to which B. japonicum belongs) and fast-growing rhizobia was unexpectedly distant. We have, therefore, performed oligonucleotide cataloguing of their 16S rRNA, and found that there was indeed only a similarity of S _AB=0.53 between fast- and slowgrowing rhizobia.In conclusion, the results suggest that nif genes may have evolved to a large degree in a similar fashion as the bacteria which carry them. This interpretation would speak against the idea of a recent lateral distribution of nif genes among microorganisms.     

Speciation of two sympatric coastal fish species, Girella punctata and Girella leonina (Perciformes, Kyphosidae)

Girella punctata and Girella leonina are sympatric sister species showing extensive distributional overlap in shallow rocky reefs in the Pacific Ocean south of the Japanese Islands. Differences between the two species in external morphological characters, such as number of pored lateral line scales, colour of opercular flap and shape of caudal fin, are congruent with genetic divergence. Nucleotide identity between the two species in the 3.3 kbp region of partial mitochondrial DNA containing the D-loop region, in 12S and 16S ribosomal RNA (rRNA) and transfer RNA genes is 95%. To estimate divergence time, Bayesian analysis was conducted using a dataset comprising concatenated nucleotide sequences from the two rRNA genes of three girellid and nine other fish species. Using the Elopomorpha – Clupeocephala split (265 million years ago (mya)) as a calibration point, divergence between G. punctata and G. leonina is estimated as having occurred 6.0±1.4 mya. Speciation is suggested to have been caused by geographical isolation associated with formation of the Japanese Islands, which resulted in disjunction of Girella habitat.     

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.     

Arcobacter ellisii sp. nov., isolated from mussels

As part of a study carried out for detecting Arcobacter spp. in shellfish, three mussel isolates that were Gram-negative slightly curved rods, non-spore forming, showed a new 16S rDNA-RFLP pattern with a specific identification method for the species of this genus. Sequences of the 16S rRNA gene and those of the housekeeping genes rpoB, gyrB and hsp60 provided evidence that these mussel strains belonged to an unknown genetic lineage within the genus Arcobacter. The similarity between the 16S rRNA gene sequence of the representative strain (F79-6^T) and type strains of the other Arcobacter species ranged between 94.1% with A. halophilus and 99.1% with the recently proposed species A. defluvii (CECT 7697^T). DDH results between strain F79-6^T and the type strain of the latter species were below 70% (53 ± 3.0%). Phenotypic characteristics together with MALDITOF mass spectra differentiated the new mussel strains from all other Arcobacter species. All the results indicate that these strains represent a new species, for which the name Arcobacter ellisii sp. nov. with the type strain F79-6^T (=CECT 7837^T = LMG 26155^T) is proposed.     

基于16S rRNA和rpoB基因的分子系统发育分析在铜绿假单胞菌鉴定中的应用

为对比16S rRNA和rpo B基因分子系统发育分析与传统表型分类法对铜绿假单胞菌的鉴定,评估16S rRNA和rpo B基因序列分析在铜绿假单胞菌鉴定中的应用,用表型分类方法对临床自动微生物鉴定系统鉴定为铜绿假单胞菌的23株分离株进行再鉴定,PCR扩增23株分离株16S rRNA和rpo B基因片段,并测序进行系统发育分析。结果表明,表型再鉴定结果与自动微生物鉴定系统鉴定结果一致。基于两个基因的系统发育分析均显示分离株p22与不动杆菌属序列聚为一枝,其余22株分离株与铜绿假单胞菌序列聚为一枝。因此p22应鉴定为不动杆菌,16S rRNA和rpo B基因序列分析均能准确鉴定铜绿假单胞菌并能较好建立假单胞菌属内种间关系。     

Phylogenetic Studies of <Emphasis Type="Italic">Gordonia</Emphasis> Species Based on <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">secA1</Emphasis> Gene Analyses

Phylogenetic relations within the genus Gordonia were analyzed using partial gyrB and secA1 gene sequences of 23 type species in comparison with those of 16S rRNA gene. The gyrB and secA1 phylogenies showed agreement with that constructed using 16S rRNA gene sequences. The degrees of divergence of the gyrB and secA1 genes were approximately 3.4 and 1.7 times greater, respectively, than that of 16S rRNA gene. The gyrB gene showed more discriminatory power than either the secA1 or 16S rRNA gene, facilitating clear differentiation of any two Gordonia species using gyrB gene analysis. Our data indicate that gyrB and secA1 gene sequences are useful as markers for phylogenetic study and identification at the species level of the genus Gordonia.     

银色裂腹鱼线粒体基因组序列特征与系统进化分析

银色裂腹鱼(Schizothorax argentatus)在我国仅分布于新疆地区的伊犁河流域,是我国裂腹鱼类中珍稀濒危品种之一,具有较高的科研和经济价值。本研究采用高通量测序技术获得了银色裂腹鱼长度为16580 bp的线粒体基因组全序列,其基因组成和排列顺序均与典型的脊椎动物相似,共有13个蛋白质编码基因、22个tRNA基因、2个rRNA基因和1个非编码区(D-loop)。碱基组成分别为A(30.25%)、G(17.28%)、C(27.20%)和T(25.27%),呈现明显的AT偏好性和反G偏倚。tRNA基因中仅tRNA-Ser(GCU)因缺少二氢尿嘧啶茎而无法形成典型的三叶草结构。ND6基因的AT-skew和GC-skew值波动最大,揭示该基因经历的选择和突变压力可能与其他基因不同。银色裂腹鱼线粒体控制区包含了3个不同的结构域:终止序列区(ETAS)、中央保守区(CSB-F、CSB-E、CSB-D和CSB-B)和保守序列区(CSB1、CSB2和CSB3),且在CSB3下游约50 bp处识别到鲤形目(Cypriniformes)鱼类中普遍存在的保守序列片段TT(AT)nGTG。基于28种裂腹鱼属鱼类线粒体基因组全序列构建的系统发育关系表明银色裂腹鱼分化时间较早,与其他类群亲缘关系较远,这可能与其所生活的水域地理位置和水文环境有密切关系。     

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。     

Diversity and geographical distribution of rhizobia associated with <Emphasis Type="Italic">Lespedeza</Emphasis> spp. in temperate and subtropical regions of China

Eighty-eight root-nodule isolates from Lespedeza spp. grown in temperate and subtropical regions of China were characterized by a polyphasic approach. Nine clusters were defined in numerical taxonomy and SDS-PAGE analysis of whole cell proteins. Based upon further characterizations of amplified 16S rDNA restriction analysis (ARDRA), PCR-based restriction fragment length polymorphism of ribosomal IGS, 16S rDNA sequence analysis and DNA-DNA hybridization, these isolates were identified as Bradyrhizobium japonicum, B. elkanii, B. yuanmingense, Mesorhizobium amorphae, M. huakuii, Sinorhizobium meliloti and three genomic species related to B. yuanmingense, Rhizobium gallicum and R. tropici. The Bradyrhizobium species and R. tropici-related rhizobia were mainly isolated from the subtropical region and the species of Mesorhizobium, S. meliloti and R. gallicum-related species were all isolated from the temperate region. Phylogenetic analyses of nifH and nodC indicated that the symbiotic genes of distinct rhizobial species associated with Lespedeza spp. might have different origins and there was no evidence for lateral gene transfer of symbiotic genes. The results obtained in the present study and in a previous report demonstrated that Lespedeza spp. are nodulated by rhizobia with diverse genomic backgrounds and these Lespedeza-nodulating rhizobia were not specific to the host species, but specific to their geographic origins. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. GenBank sequence accession numbers: The GenBank accession numbers were EF61095 through EF061114 and EF051240 for acquired 16S rDNA sequences; EF153395 through EF153402 for nifH sequences; and EF153403 through EF153410 for nodC sequences.     

Proposal to assign Aeromonas diversa sp. nov. as a novel species designation for Aeromonas group 501

The Aeromonas group 501, also named Aeromonas sp. HG13, is taxonomically close to A. schubertii. Results obtained in previous studies, including DNA–DNA hybridization and DNA fingerprinting, suggest that Aeromonas group 501 could constitute a different Aeromonas species. In this work we have performed a polyphasic study with the two strains comprising the Aeromonas sp. HG13 in order to propose a formal species name. They could be differentiated from A. schubertii by the indole and lysine decarboxylase tests and the utilization of l-lactate. Phenotypically, both strains were also easily separated from the other Aeromonas species. Sequence analysis of the 16S rRNA gene showed high sequence similarities (>97%) between Aeromonas group 501 and all Aeromonas species. Nevertheless, sequence divergences of cpn60, dnaJ, gyrB and rpoD genes were higher than the intraspecific threshold values established for each gene (3.5%, 3.3%, 2.3% and 2.6%, respectively), while sequence divergences between strains CDC 2478-85^T and CDC 2555-87 were low (0.6–1.1%). The DNA G+C content of the type strain was 62.2 mol%. Phenotypic and genotypic evidence strongly suggests that the Aeromonas group 501 is a novel species of the genus Aeromonas, for which the name Aeromonas diversa sp. nov. is proposed. The type strain is CDC 2478-85^T (=CECT 4254^T=ATCC 43946^T=LMG 17321^T).     

Rapid Identification of <Emphasis Type="Italic">Lactobacillus</Emphasis><Emphasis Type="Italic">acidophilus</Emphasis> by Restriction Analysis of the 16S–23S rRNA Intergenic Spacer Region and Flanking 23S rRNA Gene

A rapid molecular approach was developed for the initial identification of Lactobacillus acidophilus strains which are difficult to identify using a single biochemical test. The 16S–23S rRNA intergenic spacer regions and flanking 23S rRNA genes of 19 strains of lactobacilli were amplified and the nucleotide sequences and restriction site polymorphisms were analyzed. AluI was the most useful of the restriction enzymes analyzed and produced reproducible digestion profiles in the L. helveticus, L. plantarum, and L. casei groups, as well as in L. acidophilus. This restriction fragment length polymorphism method may be useful for the identification of L. acidophilus strains in dairy products.     

Ribosomal RNA genes in species of theCynareae tribe (Compositae). I.

Summary By means of Southern blot hybridization, the structure of the ribosomal DNA in six species of theCynareae tribe has been analyzed. Artichoke and wild artichoke possess only one type of ribosomal genes 13 kb long;Onopordum acanthium has at least two types of rDNA repeats 9.9 kb and 10.3 kb long andO. illyricum has a third gene type 9.7 kb long; inGalactites tomentosa there are at least four ribosomal gene types of 10.9, 11.6, 11.5, and 10kb;Carduus nutans possesses two ribosomal gene types of the same length of 12.5 kb that vary in the nucleotide sequence of the external spacer. The rRNA genes of all the species studied have an identical restriction mapping in the 18 S and 25 S regions, differences in length and/or nucleotide sequence are present in the external spacer.     

Ribosomal protein S12 as a site for streptomycin resistance in Nicotiana chloroplasts

Summary A streptomycin resistant Nicotiana plastome mutant, X/str ^R6, was subjected to molecular analysis. In this mutant, a single nucleotide transition, C » T, in the chloroplast gene for ribosomal protein S12 alters codon 90 from proline to serine while the nucleotide sequence of the chloroplast 16 S rRNA gene is identical to that of the wild type. Mutant X/str ^R6 thus differs from several previously reported streptomycin resistant chloroplast mutants which are altered in the gene for 16 S rRNA.     

Evolutionary history of an MHC gene in two leporid species: characterisation of <Emphasis Type="Italic">Mhc-DQA</Emphasis> in the European brown hare and comparison with the European rabbit

We surveyed the genetic diversity of the expressed major histocompatibility complex class II DQA locus in natural populations of European brown hares, Lepus europaeus, from Austria and Belgium (267 individuals in total). Based on cDNA sequences, we designed hare-specific primers to amplify the highly variable second exon of the DQA gene. Using cloning–sequencing methodology and capillary electrophoresis single-strand conformation polymorphism, we found ten alleles of the DQA exon 2 locus across these two European regions, of which eight are described for the first time. To search for signals of selection and recombination in the evolution of the DQA gene within the leporids, we augmented our sample with orthologous DQA alleles from the European rabbit, Oryctolagus cuniculus, in order to carry out a species level, species pairwise comparison. We found evidence of recombination in the history of the DQA sequences in leporids with some recombinant alleles bridging the species divide. In both species, selection on peptide binding site codons can be detected, though stronger for the rabbit. This result suggests that there may be a differential selection pressure in the deeper evolutionary history of these two species due to differences in several demographic and ecological traits likely subjecting them to differential selection by parasites. Finally, evolutionary relationships show a widespread and statistically significant intermingling of alleles from the two species. The many macroparasites shared between hares and rabbits may explain this pattern of trans-species polymorphism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequence data reported in this paper have been submitted to Genbank and have been assigned the accession numbers FJ225335–FJ225346.