Nitrogen-fixing bacteria were isolated from the stems of wild and cultivated rice on a modified Rennie medium. Based on 16S ribosomal DNA (rDNA) sequences, the diazotrophic isolates were phylogenetically close to four genera: Herbaspirillum, Ideonella, Enterobacter, and Azospirillum. Phenotypic properties and signature sequences of 16S rDNA indicated that three isolates (B65, B501, and B512) belong to the Herbaspirillum genus. To examine whether Herbaspirillum sp. strain B501 isolated from wild rice, Oryza officinalis, endophytically colonizes rice plants, the gfp gene encoding green fluorescent protein (GFP) was introduced into the bacteria. Observations by fluorescence stereomicroscopy showed that the GFP-tagged bacteria colonized shoots and seeds of aseptically grown seedlings of the original wild rice after inoculation of the seeds. Conversely, for cultivated rice Oryza sativa, no GFP fluorescence was observed for shoots and only weak signals were observed for seeds. Observations by fluorescence and electron microscopy revealed that Herbaspirillum sp. strain B501 colonized mainly intercellular spaces in the leaves of wild rice. Colony counts of surface-sterilized rice seedlings inoculated with the GFP-tagged bacteria indicated significantly more bacterial populations inside the original wild rice than in cultivated rice varieties. Moreover, after bacterial inoculation, in planta nitrogen fixation in young seedlings of wild rice, O. officinalis, was detected by the acetylene reduction and 15 N 2 gas incorporation assays. Therefore, we conclude that Herbaspirillum sp. strain B501 is a diazotrophic endophyte compatible with wild rice, particularly O. officinalis.

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.Through the symbiotic nitrogen fixation process, bacteria belonging to the family Rhizobiaceae convert atmospheric dinitrogen (N 2 ) to ammonia (NH 3 ), which can be effectively used by host legume plants. The establishment of a rhizobiumlegume symbiosis requires induction of new developmental programs in the partners. The symbiotic interaction begins with signal exchanges of flavonoids and Nod factors (lipochitooligosaccharides) between the two partners (6). In legume nodules, microaerobic environments trigger the rhizobial expression of nitrogen-fixing genes, such as nif and fix, via an oxygen-sensing system (13). However, the establishment of nitrogen-fixing symbiosis probably requires more complex steps triggered by reciprocal signal exchanges that lead to the organogenesis of nodules, differentiation of microsymbionts, and efficacy of nodulation (27). In addition to this symbiotic lifestyle, rhizobia survive in soils with many environment stresses, such as nutrient starvation.Lotus japonicus is a promising model legume for studying molecular interactions between symbiosis partners (20). Schauser et al. (40) first identified the plant regulatory gene nin, which is responsible for the nodule organogenesis program, in this legume. Recently, the receptor-like kinase genes have...

bWe report the complete genome sequence of Acidovorax sp. strain KKS102, a polychlorinated-biphenyl-degrading strain isolated from a soil sample in Tokyo. The genome contains a single circular 5,196,935-bp chromosome and no plasmids.

Sinorhizobium meliloti is a root-nodulating, nitrogen-fixing bacterium. An S. meliloti strain that is mutant for the rpoH(1) gene, which encodes a sigma(32)-like protein, elicits the formation of ineffective nodules on the host plant alfalfa. We characterized the rpoH(1) mutant for phenotypes related to symbiosis. Alfalfa nodules formed by the rpoH(1) mutant exhibited greatly reduced levels of acetylene reduction activity compared to the wild-type nodules. Whereas intracellular colonization by rhizobia was observed in a zone just below the apical meristem, we found ultrastructural abnormalities and signs of degeneration of bacteroids within many host cells in the proximally adjacent zone. In the proximal part of the nodule, only a few nodule cells contained bacteroids. In contrast, the rpoH(1) mutant showed normal induction of nitrogen fixation gene expression in microaerobic culture. These results suggest that the rpoH(1) mutation causes early senescence of bacteroids during the endosymbiotic process, but does not affect the invasion process or the synthesis of the nitrogenase machinery. The rpoH(1) mutant exhibited increased sensitivity to various agents and to acid pH, suggesting that RpoH(1) is required to protect the bacterial cell against environmental stresses encountered within the host. Since RpoH(1) was previously reported to be required for the synthesis of some heat shock proteins (Hsps), we examined the transcription of several genes for Hsp homologs. We found that transcription of groESL(5), lon, and clpB after heat shock was RpoH(1)-dependent, and conserved nucleotide sequences were found in the -35 and -10 regions upstream of the transcription start sites of these genes. Although groESL(5) expression is almost completely dependent on RpoH(1), we found that a groESL(5) mutant strain is still capable of normal symbiotic nitrogen fixation on alfalfa.

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