Hisayuki Mitsui

Endophytic Colonization and In Planta Nitrogen Fixation by a Herbaspirillum sp. Isolated from Wild Rice Species

ABSTRACT 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, andAzospirillum. 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 15N2gas incorporation assays. Therefore, we conclude thatHerbaspirillum sp. strain B501 is a diazotrophic endophyte compatible with wild rice, particularly O. officinalis.

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.

Complete Genomic Structure of the Cultivated Rice Endophyte Azospirillum sp. B510

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.

Isolation and Characterization of Endophytic Bacteria from Wild and Traditionally Cultivated Rice Varieties

Abstract Endophytic bacteria were isolated from surface-sterilized stems, seeds, and leaf sheaths of wild and traditionally cultivated rice varieties. Phylogenetic analyses based on 16S rDNA revealed a wide divergence among the isolates. However, the most frequently isolated groups were Methylobacterium sp. in the α-subdivision of Proteobacteria, and Curtobacterium sp. in the high G+ C Gram-positive group. Various phenotypic traits that are expected to be involved in the persistence and functions of the bacteria were analyzed: Most of the isolates from rice excreted pectinase, were motile, and showed an osmotic resistance to 0.6 M sucrose. These traits may be involved in endophytic characteristics in rice. About 50% of the isolates showed a cellulase activity. A few isolates fixed nitrogen, produced indole-3-acetic acid, and formed capsules. These activities were partially correlated with the phylogenetic group.

Complete Genome Sequence of Acidovorax sp. Strain KKS102, a Polychlorinated-Biphenyl Degrader

We 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.

Community- and Genome-Based Views of Plant-Associated Bacteria: Plant–Bacterial Interactions in Soybean and Rice

Diverse microorganisms are living as endophytes in plant tissues and as epiphytes on plant surfaces in nature. Questions about driving forces shaping the microbial community associated with plants remain unanswered. Because legumes developed systems to attain endosymbioses with rhizobia as well as mycorrhizae during their evolution, the above questions can be addressed using legume mutants relevant to genes for symbiosis. Analytical methods for the microbial community have recently been advanced by enrichment procedures of plant-associated microbes and culture-independent analyses targeting the small subunit of rRNA in microbial ecology. In this review, we first deal with interdisciplinary works on the global diversity of bacteria associated with field-grown soybeans with different nodulation genotypes and nitrogen application. A subpopulation of Proteobacteria in aerial parts of soybean shoots was likely to be regulated through both the autoregulation system for plant-rhizobium symbiosis and the nitrogen signaling pathway, suggesting that legumes accommodate a taxonomically characteristic microbial community through unknown plant-microbe communications. In addition to the community views, we then show multiphasic analysis of a beneficial rice endophyte for comparative bacterial genomics and plant responses. The significance and perspectives of community- and genome-based approaches are discussed to achieve a better understanding of plant-microbe interactions.

Development of a bacterial cell enrichment method and its application to the community analysis in soybean stems.

A method was developed for enriching bacterial cells from soybean stems which was recalcitrant for a culture-independent analysis of bacterial community due to the interference with plant DNA. Stem homogenates were fractionated by a series of differential centrifugations followed by a Nycodenz density gradient centrifugation. The efficiency of bacterial cell enrichment was assessed by ribosomal intergenic spacer analysis (RISA). The intensity and the number of bacterial amplicons of RISA were markedly increased in the DNA extracted from the enriched bacterial cells compared to that in the DNA directly extracted from soybean stems. The phylogenetic diversity of the enriched bacterial cells was evaluated by analyzing a clone library of 16S rRNA gene in comparison with those of the culturable fractions of the enriched and non-enriched stem-associated bacteria, endophytic bacteria, and epiphytic bacteria. The results indicated that the method was able to enrich both endophytic and epiphytic bacteria from soybean stems, and was useful to assess the bacterial diversity based on a 16S rRNA gene clone library. When the sequence data from all clones (1,332 sequences) were combined, 72 operational taxonomic units were affiliated with Proteobacteria (Alpha-, Beta-, and Gammaproteobacteria), Actinobacteria, Firmicutes, and Bacteroidetes, which also provided the most comprehensive set of data on the bacterial diversity in the aerial parts of soybeans.

Incubation time and media requirements of culturable bacteria from different phylogenetic groups

Previously, time courses of the colony formation process of soil bacteria on agar plates were simulated by colony-forming curves (CFCs). Bacteria were isolated from the colonies that appeared during an incubation period corresponding to each component curve of the CFC, respectively, and they were placed into groups I, II, III or IV, which were based on the order of colonization, so that their physiological characteristics could be studied (Gorlach, K. et al. (1994) J. Gen. Appl. Microbiol. 40, 507–517). We report here on the results of phylogenetic analysis of the isolates from each CFC group. A wide variety of eubacteria were included in the collection. Strains closely related to Arthrobacter were those from CFC groups I or II, groups of faster growers, and strains in Proteobacteria α-subdivision were those from groups III or IV, which grew more slowly. In contrast, some phylogenetic groups, such as Firmicutes low G+C, Cytophaga/Flexibacter/Bacteroides and Proteobacteria β-divisions exhibited heterogeneity regarding CFC grouping. As for their requirement for media, isolates belonging to the Cytophaga/Flexibacter/Bacteroides division, the Proteobacteria α-subdivision, β-subdivision and the Firmicutes high G+C division, except for Arthrobacter-related strains, were oligotrophic bacteria. In contrast, those belonging to the Firmicutes low G+C division, the Proteobacteria γ-subdivision and the A. globiformis-related group in the Firmicutes high G+C division were not oligotrophic.

DNA sequence and mutational analysis of rhizobitoxine biosynthesis genes in Bradyrhizobium elkanii

ABSTRACT We cloned and sequenced a cluster of genes involved in the biosynthesis of rhizobitoxine, a nodulation enhancer produced by Bradyrhizobium elkanii. The nucleotide sequence of the cloned 28.4-kb DNA region encompassing rtxAshowed that several open reading frames (ORFs) were located downstream of rtxA. A large-deletion mutant of B. elkanii, USDA94Δrtx::Ω1, which lacks rtxA, ORF1 (rtxC), ORF2, and ORF3, did not produce rhizobitoxine, dihydrorhizobitoxine, or serinol. The broad-host-range cosmid pLAFR1, which contains rtxAand these ORFs, complemented rhizobitoxine production in USDA94Δrtx::Ω1. Further complementation experiments involving cosmid derivatives obtained by random mutagenesis with a kanamycin cassette revealed that at least rtxAand rtxC are necessary for rhizobitoxine production. Insertional mutagenesis of the N-terminal and C-terminal regions ofrtxA indicated that rtxA is responsible for two crucial steps, serinol formation and dihydrorhizobitoxine biosynthesis. An insertional mutant of rtxC produced serinol and dihydrorhizobitoxine but no rhizobitoxine. Moreover, thertxC product was highly homologous to the fatty acid desaturase of Pseudomonas syringae and included the copper-binding signature and eight histidine residues conserved in membrane-bound desaturase. This result suggested thatrtxC encodes dihydrorhizobitoxine desaturase for the final step of rhizobitoxine production. In light of results from DNA sequence comparison, gene disruption experiments, and dihydrorhizobitoxine production from various substrates, we discuss the biosynthetic pathway of rhizobitoxine and its evolutionary significance in bradyrhizobia.

Expression of the nifH Gene of a Herbaspirillum Endophyte in Wild Rice Species: Daily Rhythm during the Light-Dark Cycle

ABSTRACT 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% O2 concentration and repressed in free air (21% O2). 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 plants light environment.