Isolation and Genetic Characterization of Aurantimonas and Methylobacterium Strains from Stems of Hypernodulated Soybeans

Abstract

The aims of this study were to isolate Aurantimonas and Methylobacterium strains that responded to soybean nodulation phenotypes and nitrogen fertilization rates in a previous culture-independent analysis (Ikeda et al. ISME J. 4:315-326, 2010). Two strategies were adopted for isolation from enriched bacterial cells prepared from stems of field-grown, hypernodulated soybeans: PCR-assisted isolation for Aurantimonas and selective cultivation for Methylobacterium. Thirteen of 768 isolates cultivated on Nutrient Agar medium were identified as Aurantimonas by colony PCR specific for Aurantimonas and 16S rRNA gene sequencing. Meanwhile, among 187 isolates on methanolcontaining agar media, 126 were identified by 16S rRNA gene sequences as Methylobacterium. A clustering analysis (>99% identity) of the 16S rRNA gene sequences for the combined datasets of the present and previous studies revealed 4 and 8 operational taxonomic units (OTUs) for Aurantimonas and Methylobacterium, respectively, and showed the successful isolation of target bacteria for these two groups. ERIC-and BOX-PCR showed the genomic uniformity of the target isolates. In addition, phylogenetic analyses of Aurantimonas revealed a phyllosphere-specific cluster in the genus. The isolates obtained in the present study will be useful for revealing unknown legume-microbe interactions in relation to the autoregulation of nodulation.

Impact of <i>Azospirillum</i> sp. B510 Inoculation on Rice-Associated Bacterial Communities in a Paddy Field

“…Aurantimonas has been found in above-ground parts of plants such as rice, soybean, Arabidopsis thaliana , and Jatropha curcas (1, 5, 16, 18). Their population was likely controlled in a manner similar to that of legume-rhizobia symbiosis (10).…”

Bacterial clade with the ribosomal RNA operon on a small plasmid rather than the chromosome

“…To characterize this bacterial group from a genomic viewpoint, we determined the complete genome sequence of Aureimonas sp. AU20, an isolate from the stem of a soybean plant (11). The result showed that the AU20 genome (5.2 Mb in total) contains nine circular replicons: the main chromosome (3.7 Mb) and eight other replicons (designated pAU20a to pAU20g and pAU20rrn) ( Table 1 and Fig.…”

“…Four of the strains (A. ureilytica and Aureimonas spp. AU40, D3, and N4) (11,18,19) were similar to AU20 in that their rrn sequences were assembled into small (9.4-10 kb) circular replicons that contained pPS10 repA-like genes and oriV regions similar to pAU20rrn (Fig. 1B and Fig.…”

“…Total DNA was extracted with a DNeasy Blood & Tissue Kit (QIAGEN) in accordance with the manufacturer's instructions, with slight modification (11).…”

“…Therefore, we consider it possible that the high copy number of the rrn-plasmid, compared with four rrn copies at most on the chromosome, confers a selective advantage to the Aureimonas host under changing environmental conditions. In this context, it is noteworthy that the RLC clade overlaps completely with the operational taxonomic unit whose relative abundance in soybean-associated bacterial communities fluctuates drastically in response to phenotypic and nutritional changes of the host plants (11,28). Second, split of the rrn operon from the chromosome possibly has an additional regulatory influence, for example, on the global transcription profile by redirecting RNA polymerase from the chromosome to the separate replicon.…”