Noriyuki Nukui

Expression Islands Clustered on the Symbiosis Island of the Mesorhizobium loti Genome

Rhizobia are symbiotic nitrogen-fixing soil bacteria that are associated with host legumes. The establishment of rhizobial symbiosis requires signal exchanges between partners in microaerobic environments that result in mutualism for the two partners. We developed a macroarray for Mesorhizobium loti MAFF303099, a microsymbiont of the model legume Lotus japonicus, and monitored the transcriptional dynamics of the bacterium during symbiosis, microaerobiosis, and starvation. Global transcriptional profiling demonstrated that the clusters of genes within the symbiosis island (611 kb), a transmissible region distinct from other chromosomal regions, are collectively expressed during symbiosis, whereas genes outside the island are downregulated. This finding implies that the huge symbiosis island functions as clustered expression islands to support symbiotic nitrogen fixation. Interestingly, most transposase genes on the symbiosis island were highly upregulated in bacteroids, as were nif, fix, fdx, and rpoN. The genome region containing the fixNOPQ genes outside the symbiosis island was markedly upregulated as another expression island under both microaerobic and symbiotic conditions. The symbiosis profiling data suggested that there was activation of amino acid metabolism, as well as nif-fix gene expression. In contrast, genes for cell wall synthesis, cell division, DNA replication, and flagella were strongly repressed in differentiated bacteroids. A highly upregulated gene in bacteroids, mlr5932 (encoding 1-aminocyclopropane-1-carboxylate deaminase), was disrupted and was confirmed to be involved in nodulation enhancement, indicating that disruption of highly expressed genes is a useful strategy for exploring novel gene functions in symbiosis.

Rhizobitoxine production by Bradyrhizobium elkanii enhances nodulation and competitiveness on Macroptilium atropurpureum

ABSTRACT Application of 1-aminoocyclopropane-1-carboxylic acid, an ethylene precursor, decreased nodulation of Macroptilium atropurpureum by Bradyrhizobium elkanii. B. elkaniiproduces rhizobitoxine, an ethylene synthesis inhibitor. Elimination of rhizobitoxine production in B. elkanii increased ethylene evolution and decreased nodulation and competitiveness on M. atropurpureum. These results suggest that rhizobitoxine enhances nodulation and competitiveness of B. elkanii on M. atropurpureum.

Expression of the 1-aminocyclopropane-1-carboxylic acid deaminase gene requires symbiotic nitrogen-fixing regulator gene nifA2 in Mesorhizobium loti MAFF303099.

ABSTRACT Many soil bacteria contain 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which degrades ACC, a precursor of the phytohormone ethylene. In order to examine the regulation of the acdS gene encoding ACC deaminase in Mesorhizobium loti MAFF303099 during symbiosis with the host legume Lotus japonicus, we introduced the β-glucuronidase (GUS) gene into acdS so that GUS was expressed under control of the acdS promoter, and we also generated disruption mutants with mutations in a nitrogen fixation regulator gene, nifA. The histochemical GUS assay showed that there was exclusive expression of acdS in mature root nodules. Two homologous nifA genes, mll5857 and mll5837, were found in the symbiosis island of M. loti and were designated nifA1 and nifA2, respectively. Quantitative reverse transcription-PCR demonstrated that nifA2 disruption resulted in considerably diminished expression of acdS, nifH, and nifA1 in bacteroid cells. In contrast, nifA1 disruption slightly enhanced expression of the acdS transcripts and suppressed nifH to some extent. These results indicate that the acdS gene and other symbiotic genes are positively regulated by the NifA2 protein, but not by the NifA1 protein, in M. loti. The mode of gene expression suggests that M. loti acdS participates in the establishment and/or maintenance of mature nodules by interfering with the production of ethylene, which induces negative regulation of nodulation.