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PubMed1999

Phylogeny and diversity of Bradyrhizobium strains isolated from the root nodules of peanut (Arachis hypogaea) in Sichuan, China.

Twenty-two rhizobial strains isolated from the root nodules of two Chinese peanut cultivars (Arachis hypogaea L. Tianfu no. 3 and a local cultivar) growing at four different sites in the Sichuan province, Southwest China, were characterized by growth rate, rep-PCR, PCR-RFLP of 16S rDNA, partial sequencing of ribosomal genes, and fatty acid-methyl ester analysis (FAME), and compared with strains representing Bradyrhizobium japanicum, B. elkanii and other unclassified Bradyrhizobium sp. All peanut isolates from Sichuan were bradyrhizobia. Dendrograms constructed using the rep-PCR fingerprints grouped the strains mainly according to their geographic and cultivar origin. Based on PCR-RFLP and partial sequence analysis of 16S rDNA it appears that peanut bradyrhizobial strains from Sichuan are similar to peanut strains from Africa and Israel, and closely related to B. japonicum. In contrast, analysis of FAME data using two-dimensional principal component analysis indicated that Bradyrhizobium sp. (Arachis) were similar to, but slightly different from other bradyrhizobia. The presence and level of fatty acid 16:1 w5c was the distinguishing feature. The results of PCR-RFLP of the 16S rRNA gene, the partial sequence analysis of 16S rDNA, and FAME were in good agreement.

Arachis↗

PubMed1995

A phylogenetic analysis of micro-organisms isolated from subsurface environments.

Three methods were used to provide information on the identity and phylogenetic relatedness of 19 aerobic, chemoheterotrophic bacteria isolated from topsoil and deep subsurface sediments at a site in South Carolina. These methods were (i) analysis of selected physiological traits, (ii) restriction endonuclease analysis (REA) of genomic DNA, and (iii) analysis of 16S ribosomal RNA sequences. When the 16S rRNA sequences were compared with those for 12 standard strains, two topsoil isolates and six subsurface strains formed a tight group with the high-G+C Gram-positive bacteria and appeared to be most closely related to Arthrobacter globiformis--a coryneform-actinomycete bacterium with unusually effective survival capabilities. The rest of the subsurface isolates were scattered among the standard strains from the Proteobacteria-including the pseudomonads and Agrobacterium tumefaciens--or the low-G+C Gram-positive bacteria.

Agrobacterium tumefaciens↗

PubMed2003

Identification of Aerococcus urinae in urine samples.

To evaluate procedures for the identification of Aerococcus urinae, we examined 24 alpha-hemolytic non-enterococcal bacterial isolates from 4373 urine samples. Published procedures were compared with 16s rRNA sequencing and biochemical profiling (BBL-Crystal-GP). 16s rRNA sequencing and BBL-Crystal-GP identified the same 13 isolates as A. urinae. Published tests failed to distinguish the 13 A. urinae isolates from eight non-A. urinae isolates; several tests exhibited no discrimination. Ciprofloxacin and trimethoprim susceptibility and growth at 45 degrees C improved discrimination. For urinary isolates, standard procedures for identification of A. urinae are redundant and insufficiently discriminatory, and may need revision. BBL-Crystal-GP is an accurate alternative.

Adult↗

PubMed2002

'Candidatus mycoplasma haemodidelphidis' sp. nov., 'Candidatus mycoplasma haemolamae' sp. nov. and Mycoplasma haemocanis comb. nov., haemotrophic parasites from a naturally infected opossum (Didelphis virginiana), alpaca (Lama pacos) and dog (Canis familiaris): phylogenetic and secondary structural relatedness of their 16S rRNA genes to other mycoplasmas.

The 16S rRNA sequence of newly characterized haemotrophic bacteria in an opossum (Didelphis virginiana) and alpaca (Lama pacos) was determined. In addition, the 16S rRNA sequence of a haemotrophic parasite in the dog (Canis familiaris) was determined. Sequence alignment and evolutionary analysis as well as secondary structural similarity and signature nucleotide sequence motifs of their 16S rRNA genes, positioned these organisms in the genus Mycoplasma. The highest scoring sequence similarities were 16S rRNA genes from haemotrophic mycoplasma species (Haemobartonella and Eperythrozoon spp.). However, the lack of several higher-order structural idiosyncrasies used to define the pneumoniae group, suggests that these organisms and related haemotrophic mycoplasmas represent a new group of mycoplasmas. It is recommended that the organisms be named 'Candidatus Mycoplasma haemodidelphidis', 'Candidatus Mycoplasma haemolamae' and Mycoplasma haemocanis comb. nov., to provide some indication of the target cell and host species of these parasites, and to reflect their phylogenetic affiliation.

Animals↗

PubMed2001

Phylogenetic relationships among four species of Mullidae (Perciformes) inferred from DNA sequences of mitochondrial cytochrome b and 16S rRNA genes.

DNA sequence comparisons of two mitochondrial DNA genes were used to infer phylogenetic relationships among four species of mullids. Approximately 238bp of the mitochondrial 16S ribosomal RNA (rRNA) and 261bp of the cytochrome b (cytb) genes were sequenced from representatives of three mullid genera (Mullus, Upeneus, Pseudopeneus), present in the Mediterranean Sea. Trees were constructed using three methods: maximum likelihood (ML), neighbor joining (NJ) and parsimony (MP). The results of the analyses of these data together with published data of the same mtDNA segments of two other perciform species (Sparus aurata, Perca fluviatilis), support the previous taxonomic classification of the three genera examined, as well as the classification of the two red mullet species in the same genus.

Journal Article↗

PubMed2001

Clarification of the relationship between the members of the family Thermomonosporaceae on the basis of 16S rDNA, 16S-23S rRNA internal transcribed spacer and 23S rDNA sequences and chemotaxonomic analyses.

To resolve relationships between members of the family Thermomonosporaceae, phylogenetic analyses using three sets of nucleotide sequences from 16S rDNA, 23S rDNA and the 16S-23S internal transcribed spacer (ITS) were carried out. Nearly all species of the family were included in this study. On the basis of congruous phylogenetic results and chemotaxonomic data, the following proposals are made. First, Actinomadura libanotica, Actinomadura aurantiaca, Actinomadura glomerata and Actinomadura longicatena are transferred to the genus Actinocorallia as Actinocorallia libanotica comb. nov., Actinocorallia aurantiaca comb. nov., Actinocorallia glomerata comb. nov. and Actinocorallia longicatena comb. nov., respectively. All the species of this genus are phylogenetically coherent and of phospholipid type PII (presence of phosphatidylethanolamine), distinguishing them from other Actinomadura species that are of phospholipid type PI (absence of diagnostic phospholipids). Second, Excellospora viridilutea is transferred to the genus Actinomadura as Actinomadura viridilutea comb. nov. As a result of the proposed transfers, the family Thermomonosporaceae now contains four genera Thermomonospora, Actinomadura, Actinocorallia and Spirillospora. The genus Actinocorallia and family Thermomonosporaceae are redescribed.

Actinomycetales↗

PubMed1982

Nucleotide sequence of a Euglena gracilis chloroplast gene coding for the 16S rRNA: homologies to E. coli and Zea mays chloroplast 16S rRNA.

The nucleotide sequence of 16S rDNA from Euglena gracilis chloroplasts has been determined representing the first complete sequence of an algal chloroplast rRNA gene. The structural part of the 16S rRNA gene has 1491 nucleotides according to a comparative analysis of our sequencing results with the published 5'- and 3'-terminal "T1-oligonucleotides" from 16S rRNA from E. gracilis. Alignment with 16S rDNA from Zea mays chloroplasts and E. coli reveals 80 to 72% sequence homology, respectively. Two deletions of 9 and 23 nucleotides are found which are identical in size and position with deletions observed in 16S rDNA of maize and tobacco chloroplasts and which seem to be characteristic for all chloroplast rRNA species. We also find insertions and deletions in E. gracilis not seen in 16S rDNA of higher plant chloroplasts. The 16S rRNA sequence of E. gracilis chloroplasts can be folded by base pairing according to the general 16S rRNA secondary structure model.

Base Sequence↗

PubMed1993

Phylogeny of sibling species of Simulium venustum and S. verecundum (Diptera: Simuliidae) based on sequences of the mitochondrial 16S rRNA gene.

The phylogeny of four sibling species of Simulium venustum (CC, CC3, CC4, and AC(gB)) and two sibling species of S. verecundum (AA and ACD = S. rostratum) was reconstructed using nucleotide sequences of the mitochondrial large subunit rRNA gene. Separate phylogenetic analyses were performed by dividing the sequence data into: (1) helices of a computer-generated secondary structure of the rDNA; (2) loops of the computer-generated structure; (3) helices of a consensus secondary structure (deduced by comparing the computer-generated structure of black flies with the corresponding structures proposed for the fruit fly (Drosophila yakuba) and the mosquito (Aedes albopictus)); (4) loops of the consensus structure; (5) both helices and loops of the consensus secondary structure; and (6) the entire sequence regardless of secondary structure, including 11 variable sites in regions where the prediction of secondary structure was not possible. We found that different data sets led to different phylogenetic conclusions. The phylogenies based on data sets 4 and 6 were consistent with nonmolecular evidence, while the phylogenies based on other data sets were not. Our study suggests S. decorum, a morphospecies, might have shared a common ancestor with sibling species of S. venustum.

Animals↗

PubMed1996

Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis.

Sequence heterogeneities in 16S rRNA genes from individual strains of Paenibacillus polymyxa were detected by sequence-dependent separation of PCR products by temperature gradient gel electrophoresis (TGGE). A fragment of the 16S rRNA genes, comprising variable regions V6 to V8, was used as a target sequence for amplifications. PCR products from P. polymyxa (type strain) emerged as a well-defined pattern of bands in the gradient gel. Six plasmids with different inserts, individually demonstrating the migration characteristics of single bands of the pattern, were obtained by cloning the PCR products. Their sequences were analyzed as a representative sample of the total heterogeneity. An amount of 10 variant nucleotide positions in the fragment of 347 bp was observed, with all substitutions conserving the relevant secondary structures of the V6 and V8 regions in the RNA molecules. Hybridizations with specifically designed probes demonstrated different chromosomal locations of the respective rRNA genes. Amplifications of reverse-transcribed rRNA from ribosome preparations, as well as whole-cell hybridizations, revealed a predominant representation of particular sequences in ribosomes of exponentially growing laboratory cultures. Different strains of P. polymyxa showed not only remarkably differing patterns of PCR products in TGGE analysis but also discriminative whole-cell labeling with the designed oligonucleotide probes, indicating the different representation of individual sequences in active ribosomes. Our results demonstrate the usefulness of TGGE for the structural analysis of heterogeneous rRNA genes together with their expression, stress problems of the generation of meaningful data for 16S rRNA sequences and probe designs, and might have consequences for evolutionary concepts.

Bacillus↗

PubMedDate not supplied

Sequence variation of the 16S to 23S rRNA spacer region in Salmonella enterica.

The possibility for identification of Salmonella enterica serotypes by sequence analysis of the 16S to 23S rRNA internal transcribed spacer was investigated by direct sequencing of polymerase chain reaction-amplified DNA from all operons simultaneously in a collection of 25 strains of 18 different serotypes of S. enterica, and by sequencing individual cloned operons from a single strain. It was only possible to determine the first 117 bases upstream from the 23S rRNA gene by direct sequencing because of variation between the rrn operons. Comparison of sequences from this region allowed separation of only 15 out of the 18 serotypes investigated and was not specific even at the subspecies level of S. enterica. To determine the differences between internal transcribed spacers in more detail, the individual rrn operons of strain JEO 197, serotype IV 43:z4,z23:-, were cloned and sequenced. The strain contained four short internal transcribed spacer fragments of 382-384 bases in length, which were 98.4-99.7% similar to each other and three long fragments of 505 bases with 98.0-99.8% similarity. The study demonstrated a higher degree of interbacterial variation than intrabacterial variation between operons for serotypes of S. enterica.

Base Sequence↗

PubMed1998

Phylogenetic characterization of the bacterium-like organism associated with marginal chlorosis of strawberry and proposition of a Candidatus taxon for the organism, 'Candidatus phlomobacter fragariae'.

Marginal chlorosis is a new disease of strawberry which was first seen in France in 1988. A phloem-restricted bacterium-like organism was found associated with the disease. Even though the organism could not be cultured and resembles in this way most other phloem-restricted pathogens, characterization was achieved from the sequence of its PCR-generated 16S rDNA, and comparison with other organisms. From these studies, the strawberry agent was found to be a new bacterium within group 3 of the gamma subclass of Proteobacteria, a group of Gram-negative bacteria including, in particular, insect symbionts or parasites as well as enterobacteria. Its closest relative, Arsenophonus nasoniae, is the causal agent of the son-killer trait in wasps. The two bacteria share 92% 16S rDNA sequence identity. We propose a Candidatus taxon for the marginal-chlorosis-associated bacterium, 'Candidatus Phlomobacter fragariae'.

Fruit↗

PubMed2000

Acholeplasma vituli sp. nov., from bovine serum and cell cultures.

Organisms isolated from commercial foetal bovine serum and from cell culture lines containing such serum supplements were found to consist of non-helical, non-motile, pleomorphic coccoid forms. One strain (FC 097-2T) cultivated directly from foetal bovine serum was selected for characterization. In ultrastructural examination, individual round cells lacked cell wall structures and cells varied in size, with a mean diameter of about 700 nm. However, variable numbers of cells were filterable through membranes of 300 nm. Optimum growth occurred between 30 and 37 degrees C. The organism fermented glucose, fructose and mannose, but did not hydrolyse arginine. The strain was insensitive to 500 U penicillin ml(-1) and was capable of growing in the absence of serum or cholesterol. The organism was serologically distinct from all 13 currently described species in the genus Acholeplasma and from other sterol-requiring species in the genus Mycoplasma, using growth inhibition, immunoperoxidase and immunofluorescence tests. Strain FC 097-2T was found to have a DNA G+C composition between 37.6 +/- 1 mol% and 38.3 +/- 1 mol%. The genome size was determined to be 2095 kbp. The 16S rDNA sequence of strain FC 097-2T was compared to 16S rDNA sequences of other mollicutes in nucleotide databases. No deposited sequence was found to be identical; the closest relatives were several members of the genus Acholeplasma. On the basis of these findings and other similarities to acholeplasmas in morphology and growth, the absence of a sterol requirement for growth, and similar genomic characteristics, the organism was assigned to the genus Acholeplasma. Strain FC 097-2T is designated the type strain (ATCC 700667T) of a new species, Acholeplasma vituli.

Acholeplasma↗

PubMed2003

In most Bradyrhizobium groups sequence comparison of 16S-23S rDNA internal transcribed spacer regions corroborates DNA-DNA hybridizations.

In an extension of a previous small-scale test to assess the use of 16S-23S rDNA internal transcribed spacer (ITS) sequences for rapid grouping of bradyrhizobia, we have sequenced the ITS region of 32 isolates of Bradyrhizobium that had previously been studied using AFLP and DNA-DNA hybridizations. We also included representatives of Afipia and Rhodopseudomonas. Our results indicate that ITS sequences are very diverse among bradyrhizobia. Nevertheless, for most of the bradyrhizobia, the grouping of ITS sequences was in line with AFLP results and DNA-DNA hybridization data. Strains that have at least 95.5% ITS sequence similarity belong to the same genospecies, i.e. they have more than 60% DNA-DNA hybridization values. The ITS sequences can therefore provide a relatively fast way to guide strain identification and aid selection of the reference groups that should be included in DNA-DNA hybridization experiments for precise genotypic identification. The Bradyrhizobium strains isolated from Aeschynomene species showed a much larger diversity in ITS sequences than other bradyrhizobia, possibly as a result of lateral exchange. The above ITS sequence similarity criterion for genospecies therefore does not apply to them, but they can easily be distinguished from other Bradyrhizobium genospecies because they have a distinct tRNA(ala) gene.

Bacterial Typing Techniques↗

PubMed2003

Elevation of three subspecies of Pectobacterium carotovorum to species level: Pectobacterium atrosepticum sp. nov., Pectobacterium betavasculorum sp. nov. and Pectobacterium wasabiae sp. nov.

A collection of 42 strains belonging to the five subspecies of Pectobacterium carotovorum (subspecies atrosepticum, betavasculorum, carotovorum, odoriferum and wasabiae) and 11 reference and type strains of biovars of Pectobacterium chrysanthemi, Pectobacterium cacticidum and Brenneria paradisiaca were studied by DNA-DNA hybridization, numerical taxonomy of 120 phenotypic characteristics, serology and new phylogenetic analysis of previously reported sequences from a database of aligned 16S rDNA sequences. The P. carotovorum subspecies formed a clade according to neighbour-joining methods, but they formed two paraphyletic clusters according to maximum-likelihood and maximum-parsimony. However, phylogenetic analysis of 16S rDNA sequences alone is not sufficient to justify generic differentiation and therefore, it is proposed to retain the P. carotovorum subspecies in the genus Pectobacterium. The strains of P. carotovorum were distributed in four genomospecies: genomospecies 1, harbouring all strains of subsp. atrosepticum, genomospecies 2, including the strains of subsp. betavasculorum isolated from sugar beet, sunflower, potato, hyacinth and artichoke, genomospecies 3, clustering all strains of subsp. wasabiae isolated from wasabi in Japan, and genomospecies 4, gathering together strains of subsp. carotovorum and strains of subsp. odoriferum. Four strains of P. carotovorum subsp. carotovorum remained unclustered. Biochemical criteria, deduced from a numerical taxonomy study of phenotypic characteristics and serological reactions, allowed discrimination of strains belonging to the four genomospecies. Thus, it is proposed that three genomospecies be elevated to species level as Pectobacterium atrosepticum sp. nov. (type strain CFBP 1526T =LMG 2386T =NCPPB 549T =ICMP 1526T), Pectobacterium betavasculorum sp. nov. (type strain CFBP 2122T =LMG 2464T =NCPPB 2795T =ICMP 4226T) and Pectobacterium wasabiae sp. nov. (type strain CFBP 3304T =LMG 8404T =NCPPB 3701T =ICMP 9121T). Only two subspecies are maintained within P. carotovorum, subsp. carotovorum (type strain CFBP 2046T =LMG 2404T =NCPPB 312T =ICMP 5702T) and subsp. odoriferum (type strain CFBP 1878T =LMG 5863T =NCPPB 3839T = ICMP 11553T), for which discriminating tests are available.

Enterobacteriaceae↗

PubMed2003

Physiological and molecular characterization of anaerobic benzene-degrading mixed cultures.

Nine distinct anaerobic benzene-degrading cultures were enriched from sediment samples from four different sites. These cultures used nitrate, sulphate or CO2 as electron acceptors. The shortest doubling times were observed in nitrate-reducing cultures, although cell yield was lowest in these cultures. The highest substrate concentration utilized and maximum absolute rates of benzene degraded (in micro M day-1) were observed in methanogenic cultures. The microbial compositions of a methanogenic and nitrate-reducing culture were determined from a clone library of 16S rRNA genes. Five Bacterial 16S rRNA sequences, one of which resembled a clone previously found in a sulphate-reducing, benzene-degrading culture and four Archaeal 16S rRNA sequences were identified in a methanogenic culture. Four Bacterial and no Archaeal 16S rRNA sequences were identified in a nitrate-reducing culture. The relative abundance of the four nitrate-reducing putative species was determined by slot blot hybridization. Two green sulphur bacteria together formed 52% of the clone library, but were found to be less than 4% of the culture by slot blot analysis. One of the cloned 16S rRNA gene sequences comprised 70% of the culture and was phylogenetically 93% similar to both Azoarcus and Dechloromonas species, which have been shown to degrade aromatic compounds, including benzene, under nitrate-reducing conditions.

Anaerobiosis↗

PubMed1997

Development of oligonucleotide primers from the 16S-23S rRNA intergenic sequences for identifying different dairy and probiotic lactic acid bacteria by PCR.

Spacer regions between the 16S and 23S rRNA genes of different dairy and probiotic lactic acid bacteria were amplified by the polymerase chain reaction (PCR) with conserved primers and the nucleotide sequences of these spacer regions were determined. Amplification/oligonucleotide primer pairs were designed for Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus helveticus and Streptococcus thermophilus based on the differences in the nucleotide sequences of the 16S-23S rRNA spacer regions. Also a primer pair identifying both Lb. paracasei and Lb. rhamnosus was designed. In addition to conventional PCR in a heat block a rapid PCR method in an Air Thermocycler (ATC) with glass capillaries was used.

Base Sequence↗

PubMed2000

[A Carnobacterium-like organism isolated form a patient with multiple bacterial synergistic gangrene].

An atypical lactic-acid producing gram positive rod Y6 strain was studied in this report, which was isolated form clinic sample of a patient with multiple bacterial synergistic gangrene, and could not be identified by routine method. A 1.5 kb of 16S rDNA of Y6 strain was synthesized and sequenced. Comparative 16S rDNA sequence analyses revealed that stain Y6 is most closely related to the genus of Carnobacterium. The overall similarity value between Y6 strain and Carnobacterium species are 93% to 97%. The signature nucleotides in 16S rDNA primary sequence of strain Y6 and that of genus of Carnobacterium were identical. The biological features of Y6 strain are very similar to that of Carnobacterium, such as lactic acid as main end product of in PYG broth, no gas produced form fermentation of glucose, catalase negative, no motility. Data suggested that Y6 strain is very similar to the genus of Carnobacterium, of which no strain has been isolated form clinical sample so far. Based on the results obtained, we names Y6 strain as Carnobacterium-like organism.

Base Sequence↗

PubMed1999

Relationships of bradyrhizobia from the legumes Apios americana and Desmodium glutinosum.

Multilocus enzyme electrophoresis, partial 23S rRNA sequences, and nearly full-length 16S rRNA sequences all indicated high genetic similarity among root-nodule bacteria associated with Apios americana, Desmodium glutinosum, and Amphicarpaea bracteata, three common herbaceous legumes whose native geographic ranges in eastern North America overlap extensively. A total of 19 distinct multilocus genotypes (electrophoretic types [ETs]) were found among the 35 A. americana and 33 D. glutinosum isolates analyzed. Twelve of these ETs (representing 78% of all isolates) were either identical to ETs previously observed in A. bracteata populations, or differed at only one locus. Within both 23S and 16S rRNA genes, several isolates from A. americana and D. glutinosum were either identical to A. bracteata isolates or showed only single nucleotide differences. Growth rates and nitrogenase activities of A. bracteata plants inoculated with isolates from D. glutinosum were equivalent to levels found with native A. bracteata bacterial isolates, but none of the three A. americana isolates tested had high symbiotic effectiveness on A. bracteata. Phylogenetic analysis of both 23S and 16S rRNA sequences indicated that both A. americana and D. glutinosum harbored rare bacterial genotypes similar to Bradyrhizobium japonicum USDA 110. However, the predominant root nodule bacteria on both legumes were closely related to Bradyrhizobium elkanii.

Bradyrhizobium↗