Yasuyuki Kawaharada

Publications of Yasuyuki Kawaharada

• Complete genomic structure of the cultivated rice endophyte Azospirillum sp. B510.

  Authors: Takakazu Kaneko, Kiwamu Minamisawa, Tsuyoshi Isawa, Hiroki Nakatsukasa, Hisayuki Mitsui, Yasuyuki Kawaharada, Yasukazu Nakamura, Akiko Watanabe, Kumiko Kawashima, Akiko Ono [......] Chika Takahashi, Chiharu Minami, Tsunakazu Fujishiro, Mitsuyo Kohara, Midori Katoh, Naomi Nakazaki, Shinobu Nakayama, Manabu Yamada, Satoshi Tabata, Shusei Sato

  DNA research : an international journal for rapid publication of reports on genes and genomes. 02/2010; 17(1):37-50.

  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 sativaWe 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 N(2) 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 C(4)-dicarboxylate during its symbiotic relationship with the host plant.

• Identification of the Mesorhizobium loti gene responsible for glycerophosphorylation of periplasmic cyclic beta-1,2-glucans.

  Authors: Yasuyuki Kawaharada, Hiromasa Kiyota, Shima Eda, Kiwamu Minamisawa, Hisayuki Mitsui

  FEMS microbiology letters. 11/2009;

  Periplasmic cyclic beta-1,2-glucans play a crucial role in symbiosis as well as in hypo-osmotic adaptation for rhizobia. These glucans are modified in many species by anionic substituents such asPeriplasmic cyclic beta-1,2-glucans play a crucial role in symbiosis as well as in hypo-osmotic adaptation for rhizobia. These glucans are modified in many species by anionic substituents such as glycerophosphoryl and succinyl ones, but their role remains to be examined. In this work, the cgmA homolog is shown to be responsible for glycerophosphorylation of cyclic beta-1,2-glucans in Mesorhizobium loti. The mutation in cgmA converted most anionic glucans into neutral ones, leaving a small amount of succinylated ones. An additional mutation in opgC, which encodes a succinyltransferase homolog, abolished the residual succinyl substituents in the cgmA-mutant background. The double mutant in cgmA and opgC did not show any significant phenotypic differences from the wild type during both vegetative growth and symbiosis. It is concluded that the anionic substituents make a minor contribution, if any, to the effectiveness of cyclic beta-1,2-glucans in M. loti.

• Aerobic vanillate degradation and C1 metabolism in Bradyrhizobium japonicum.

  Authors: Nirinya Sudtachat, Naofumi Ito, Manabu Itakura, Sachiko Masuda, Shima Eda, Hisayuki Mitsui, Yasuyuki Kawaharada, Kiwamu Minamisawa

  Applied and environmental microbiology. 07/2009;

  Bradyrhizobium japonicum, a symbiotic nitrogen-fixing soil bacterium, has multiple gene copies for aromatic degradation on the genome, and is able to use low concentrations of vanillate, aBradyrhizobium japonicum, a symbiotic nitrogen-fixing soil bacterium, has multiple gene copies for aromatic degradation on the genome, and is able to use low concentrations of vanillate, a methoxylated lignin monomer, as an energy source. A transcriptome analysis indicated that one set of vanA1B, pcaG1H1, and genes for C1 catabolism are upregulated in B. japonicum USDA110 cells grown in vanillate [Ito et al. 2006. Microbes Environ. 21:240-250x]. To examine the functions of these genes in vanillate degradation, we tested cell growth and substrate consumption of vanA1B, pcaG1H1, and mxaF mutants of USDA110. The vanA1B and pcaG1H1 mutants were unable to grow in minimal media containing 1 mM vanillate and protocatechuate, respectively, although wild-type USDA110 was able to grow in both media, indicating that the upregulated copies of vanA1B and pcaG1H1 are exclusively responsible for vanillate degradation. Mutating mxaF eliminated expression of gfa and flhA, which contribute to glutathione-dependent C1 metabolism. The mxaF mutant had markedly lower cell growth in medium containing vanillate than the wild-type strain. In the presence of protocatechuate, there was no difference in cell growth between the mxaF mutant and the wild-type strain. These results suggest that the C1 pathway genes are required for efficient vanillate catabolism. In addition, wild-type USDA110 oxidized methanol, whereas the mxaF mutant did not, suggesting that the metabolic capability of the C1 pathway in B. japonicum extends to methanol oxidation. The mxaF mutant showed normal nodulation and N2 fixation phenotypes with soybeans, which was not similar to symbiotic phenotypes of methylotrophic rhizobia.

• Structural characterization of neutral and anionic glucans from Mesorhizobium loti.

  Authors: Yasuyuki Kawaharada, Hiromasa Kiyota, Shima Eda, Kiwamu Minamisawa, Hisayuki Mitsui

  Carbohydrate research. 10/2008; 343(14):2422-7.

  The periplasmic glucans of Mesorhizobium loti were isolated and separated into fractions according to their acidity. NMR spectroscopy confirmed their backbone structure to be a cyclicThe periplasmic glucans of Mesorhizobium loti were isolated and separated into fractions according to their acidity. NMR spectroscopy confirmed their backbone structure to be a cyclic beta-(1-->2)-d-glucan as in the case of other rhizobia, and revealed no non-glycosidic substituents in the neutral fraction, and glycerophosphoryl and succinyl residues as major and minor substituents, respectively, in the anionic fractions. MALDI-TOF mass spectrometry showed that the anionic glucans contain one, two, or three such substituents per molecule according to their acidity, and, in contrast, that all the anionic subfractions have a similar size distribution to that of the neutral glucans, where molecules composed of 20-24 glucosyl residues are predominant. These results clarify the periplasmic glucan composition in terms of charge-to-mass ratios in M. loti cells.

• A Mesorhizobium loti mutant with reduced glucan content shows defective invasion of its host plant Lotus japonicus.

  Authors: Yasuyuki Kawaharada, Shima Eda, Kiwamu Minamisawa, Hisayuki Mitsui

  Microbiology (Reading, England). 01/2008; 153(Pt 12):3983-93.

  Random transposon mutagenesis led to the isolation of a novel Mesorhizobium loti mutant that is defective in nitrogen fixation during symbiosis with Lotus japonicus. The mutated locus, designatedRandom transposon mutagenesis led to the isolation of a novel Mesorhizobium loti mutant that is defective in nitrogen fixation during symbiosis with Lotus japonicus. The mutated locus, designated cep, encodes a putative cell-envelope protein displaying no significant sequence similarity to proteins with known functions. This mutant elicits the formation of nodule-like bumps and root-hair curling, but not the elongation of infection threads, on L. japonicus roots. This is reminiscent of the phenotypes of rhizobial mutants impaired in cyclic beta-glucan biosynthesis. The cep mutant exhibits partially reduced content of cell-associated glucans and intermediate deficiency of motility under hypo-osmotic conditions as compared to a glucan-deficient mutant. Second-site pseudorevertants of the cep mutant were isolated by selecting for restoration of symbiotic nitrogen fixation. A subset of pseudorevertants restored both symbiotic capability and glucan content to levels comparable to that of the wild-type. These results suggest that the Cep product acts on a successful symbiosis by affecting cell-associated glucan content.