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.

We determined the nucleotide sequence of the entire genome of a diazotrophic endophyte, Azospirillum sp. B510. Strain B510 is an endophytic bacterium isolated from stems of rice plants (Oryza sativa cv. Nipponbare). The genome of B510 consisted of a single chromosome (3 311 395 bp) and six plasmids, designated as pAB510a (1 455 109 bp), pAB510b (723 779 bp), pAB510c (681 723 bp), pAB510d (628 837 bp), pAB510e (537 299 bp), and pAB510f (261 596 bp). The chromosome bears 2893 potential protein-encoding genes, two sets of rRNA gene clusters (rrns), and 45 tRNA genes representing 37 tRNA species. The genomes of the six plasmids contained a total of 3416 protein-encoding genes, seven sets of rrns, and 34 tRNAs representing 19 tRNA species. Eight genes for plasmid-specific tRNA species are located on either pAB510a or pAB510d. Two out of eight genomic islands are inserted in the plasmids, pAB510b and pAB510e, and one of the islands is inserted into trnfM-CAU in the rrn located on pAB510e. Genes other than the nif gene cluster that are involved in N2 fixation and are homologues of Bradyrhizobium japonicum USDA110 include fixABCX, fixNOQP, fixHIS, fixG, and fixLJK. Three putative plant hormone-related genes encoding tryptophan 2-monooxytenase (iaaM) and indole-3-acetaldehyde hydrolase (iaaH), which are involved in IAA biosynthesis, and ACC deaminase (acdS), which reduces ethylene levels, were identified. Multiple gene-clusters for tripartite ATP-independent periplasmic-transport systems and a diverse set of malic enzymes were identified, suggesting that B510 utilizes C4-dicarboxylate during its symbiotic relationship with the host plant.

We previously reported that extra-slow-growing Bradyrhizobium japonicum isolates obtained from three field sites in Japan, designated as HRS (highly reiterated sequence-possessing) strains, have high copy numbers of the insertion sequences RSalpha and RSbeta. When strain collections in the USA, Japan, Korea, Thailand and China were examined by Southern hybridization using RSalpha, RSbeta and IS1631 as probes, HRS strains were found in the Japanese, Chinese, and American collections, but not in the Korean and Thai ones. Copy number analyses of RSalpha and RSbeta, calibrated with the copy number of rrs (16S rRNA gene), indicated that the HRS stains can be divided into two major groups. Group A is comprised of members with a high copy number of RSalpha (mean+/-S.D., 139+/-27), a low number of RSbeta (mean+/-S.D., 30+/-13) sequences, and extremely slow growth rates (mean doubling time+/-S.D., 27+/-9 h). In contrast, group B is comprised of strains with a high copy number of RSbeta (mean+/-S.D., 93+/-6) and a lower number of RSalpha. These groupings of HRS strains were well correlated with phylogenetic clusters based on rrs, gyrB and serogroups (110/122 and 123/135). Growth rate of B. japonicum strains was also correlated exclusively with RSalpha copy number. The ecological, evolutionary and biotechnological implications of the findings are discussed.

In a survey of DNA fingerprints of indigenous Bradyrhizobium japonicum with the species-specific repeated sequences RSα and RSβ, 21 isolates from three field sites showed numerous RS-specific hybridization bands. The isolates were designated highly reiterated sequence-possessing (HRS) isolates, and their DNA hybridization profiles were easily distinguished from the normal patterns. Some HRS isolates from two field sites possessed extremely high numbers of RSα copies, ranging from 86 to 175 (average, 128), and showed shifts and duplications of nif- and hup-specific hybridization bands. The HRS isolates exhibited slower growth than normal isolates, although no difference in symbiotic properties was detected between the HRS and normal isolates. Nucleotide sequence analysis of 16S rRNA genes showed that HRS isolates were strains ofB. japonicum. There was no difference in the spectra of serological and hydrogenase groupings of normal and HRS isolates. Some HRS isolates possessed a tandem repeat RSα dimer that is similar to the structure of (IS30)2, which was shown to cause a burst of transpositional rearrangements in Escherichia coli. The results suggest that HRS isolates are derived from normal isolates in individual fields by genome rearrangements that may be mediated by insertion sequences such as RSα.

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Inoculation experiments with the endophytic bacterium Azospirillum sp. strain B510, an isolate from surface-sterilized stems of field-grown rice, were conducted in pots in a greenhouse, and in paddy fields in Hokkaido, Japan. B510 significantly enhanced the growth of newly generated leaves and shoot biomass under greenhouse conditions. When rice seedlings were treated with 1×10 8 CFU ml −1 , then transplanted to paddy fields, tiller numbers and seed yield significantly increased. Azospirillum sp. strain B510 is a promising bacterial inoculant for plant growth promotion and agricultural practices.

The expression of nitrogenase genes of Herbaspirillum sp. B501 associated in shoot (leaf and stem) of wild rice, Oryza officinalis, was studied by means of reverse transcription-PCR (RT-PCR) targeted at the nifH gene. RT-PCR analyses indicate that nifH transcript was detected exclusively from nitrogen-fixing cells of gfp-tagged strain B501gfp1 in both free-living and endophytic states by using a constitutive gfp gene transcript as a positive control. Transcription of nifH and nitrogen fixation in free-living cells were induced maximally at a 2% O 2 concentration and repressed in free air (21% O 2 ). nifH transcription was monitored in the endophytic cells by using total RNA extracted from B501gfp1-inoculated wild rice plants during daily light-dark cycles. The level of nifH transcription in planta varied dramatically, with a maximum during the light period. Moreover, the light radiation enhanced nifH expression even in free-living cells grown in culture. These results suggest that in planta nitrogen fixation by the endophyte shows a daily rhythm determined by the plant's light environment.

Extra-slow-growing bradyrhizobia from root nodules of field-grown soybeans harbor abundant insertion sequences (ISs) and are termed highly reiterated sequence-possessing (HRS) strains. We analyzed the genome organization of HRS strains with the focus on IS distribution and symbiosis island structure. Using pulsed-field gel electrophoresis, we consistently detected several plasmids (0.07 to 0.4 Mb) in the HRS strains (NK5, NK6, USDA135, 2281, USDA123, and T2), whereas no plasmids were detected in the non-HRS strain USDA110. The chromosomes of the six HRS strains (9.7 to 10.7 Mb) were larger than that of USDA110 (9.1 Mb). Using MiSeq sequences of 6 HRS and 17 non-HRS strains mapped to the USDA110 genome, we found that the copy numbers of ISRj1, ISRj2, ISFK1, IS1632, ISB27, ISBj8, and IS1631 were markedly higher in HRS strains. Whole-genome sequencing showed that the HRS strain NK6 had four small plasmids (136 to 212 kb) and a large chromosome (9,780 kb). Strong colinearity was found between 7.4-Mb core regions of the NK6 and USDA110 chromosomes. USDA110 symbiosis islands corresponded mainly to five small regions (S1 to S5) within two variable regions, V1 (0.8 Mb) and V2 (1.6 Mb), of the NK6 chromosome. The USDA110 nif gene cluster (nifDKENXSBZHQW-fixBCX) was split into two regions, S2 and S3, where ISRj1-mediated rearrangement occurred between nifS and nifB. ISs were also scattered in NK6 core regions, and ISRj1 insertion often disrupted some genes important for survival and environmental responses. These results suggest that HRS strains of soybean bradyrhizobia were subjected to IS-mediated symbiosis island shuffling and core genome degradation.