Partial 16S Ribosomal DNA Sequencing Research Articles

Molecular evidence for the presence of novel actinomycete lineages in a temperate forest soil

PCR primers were designed to selectively recover partial (∼1100 bp) actinomycete 16S ribosomal DNA sequences from a temperate forest soil. A gene library was made and colony PCR was used to identify clones containing inserts. Unique clones were identified and partial or complete insert sequences were determined for 53 clones. Phylogenetic analyses revealed that 46 (87%) of the clones sampled contained 16S rDNA sequences which fell within the actinomycete radiation. The largest group of 34 sequences formed two closely related monophyletic groups in the 16S rRNA tree, which in turn formed a weakly supported sister group with the sequence fromActinomadura madurae. Four novel 16S rDNA lineages were detected inMycobacterium, one inPropionibacterium and one inCorynebacterium. Three novel sequences weakly grouped withSporichthya polymorpha. Two sequences formed an isolated lineage not closely related to any of the reference actinomycetes. Our results lend strong support to the hypothesis that cultured (and sequenced) actinomycetes do not adequately describe the diversity of this group in the environment.

Identification of grass-associated and toluene-degrading diazotrophs, Azoarcus spp., by analyses of partial 16S ribosomal DNA sequences

The genus Azoarcus includes nitrogen-fixing, grass-associated strains as well as denitrying toluene degraders. In order to identify and group members of the genus Azoarcus, phylogenetic analysis based on partial sequences of 16S rRNA genes (16S rDNAs) is proposed. 16S rRNA-targeted PCR using specific primers to exclude amplification in the majority of other members of the beta subclass of the class Proteobacteria was combined with direct sequencing of the PCR products. Tree inference from comparisons of 446-bp rDNA fragments yielded similar results for the three known Azoarcus spp. sequences and for analysis of the complete 16S rDNA sequence. These three species formed a phylogenetically coherent group with representatives of two other Azoarcus species which were subjected to 16S rRNA sequencing in this study. This group was related to Rhodocyclus purpureus and Thauera selenatis. New isolates and also sequences of so far uncultured bacteria from roots of Kallar grass were assigned to the genus Azoarcus as well. Also, strains degrading monoaromatic hydrocarbons anaerobically in the presence of nitrate clustered within this genus, albeit not with grass-associated isolates. All representative members of the five species harboring rhizospheric bacteria were able to form N2O from nitrate and showed anaerobic growth on malic acid with nitrate but not on toluene. In order to visualize different Azoarcus spp. by whole-cell in situ hybridizations, we generated 16S rRNA-targeted, fluorescent probes by in vitro transcription directly from PCR products which spanned the variable region V2. Hybridization was species specific for Azoarcus communis and Azoarcus indigens.(ABSTRACT TRUNCATED AT 250 WORDS)

Genomic Heterogeneity among French Rhizobium Strains Isolated from Phaseolus vulgaris L.

Levels of DNA relatedness between strains isolated from root nodules of Phaseolus vulgaris and reference strains of different Rhizobium species were determined by performing DNA-DNA hybridization experiments (S1 nuclease method). The nine strains examined were members of three genomic groups previously delineated by a restriction fragment length polymorphism analysis among strains isolated from P. vulgaris at different sites in France. In agreement with the results of the restriction fragment length polymorphism analysis, three genomic species were found. We confirmed that one of these species corresponded to Rhizobium leguminosarum since the strain examined was 100% related to the type strain of this species. The other two species were new genomic species which were less than 21% related to reference strains belonging to other Rhizobium species, including Rhizobium etli and Rhizobium tropici, and were 18% related to each other. As determined by an analysis of partial 16S ribosomal DNA sequences, each of the genomic species was found to belong to a lineage independent from the lineages of previously described Rhizobium species. Nevertheless, they were included in the group formed by the fast-growing Rhizobium species. Both genomic species 1 and genomic species 2 contained a majority of strains which were capable of nodulating both P. vulgaris and Leucaena leucocephala, like R. tropici. However, they also contained strains with a nodulation phenotype restricted to P. vulgaris, like R. leguminosarum bv. phaseoli and R. etli bv. phaseoli. Our data are the first evidence that in Europe species other than R. leguminosarum nodulate P. vulgaris.