Hisayuki Mitsui

Publications of Hisayuki Mitsui

• Complete Genome Sequence of Bradyrhizobium sp. S23321: Insights into Symbiosis Evolution in Soil Oligotrophs.

  Authors: Takashi Okubo, Takahiro Tsukui, Hiroko Maita, Shinobu Okamoto, Kenshiro Oshima, Takatomo Fujisawa, Akihiro Saito, Hiroyuki Futamata, Reiko Hattori, Yumi Shimomura [......] Yuko Takada Hoshino, Hideki Hirakawa, Hisayuki Mitsui, Kimihiro Terasawa, Manabu Itakura, Shusei Sato, Wakako Ikeda-Ohtsubo, Natsuko Sakakura, Eli Kaminuma, Kiwamu Minamisawa

  Microbes and environments / JSME. 03/2012;

  Bradyrhizobium sp. S23321 is an oligotrophic bacterium isolated from paddy field soil. Although S23321 is phylogenetically close to Bradyrhizobium japonicum USDA110, a legume symbiont, it is unableBradyrhizobium sp. S23321 is an oligotrophic bacterium isolated from paddy field soil. Although S23321 is phylogenetically close to Bradyrhizobium japonicum USDA110, a legume symbiont, it is unable to induce root nodules in siratro, a legume often used for testing Nod factor-dependent nodulation. The genome of S23321 is a single circular chromosome, 7,231,841 bp in length, with an average GC content of 64.3%. The genome contains 6,898 potential protein-encoding genes, one set of rRNA genes, and 45 tRNA genes. Comparison of the genome structure between S23321 and USDA110 showed strong colinearity; however, the symbiosis islands present in USDA110 were absent in S23321, whose genome lacked a chaperonin gene cluster (groELS3) for symbiosis regulation found in USDA110. A comparison of sequences around the tRNA-Val gene strongly suggested that S23321 contains an ancestral-type genome that precedes the acquisition of a symbiosis island by horizontal gene transfer. Although S23321 contains a nif (nitrogen fixation) gene cluster, the organization, homology, and phylogeny of the genes in this cluster were more similar to those of photosynthetic bradyrhizobia ORS278 and BTAi1 than to those on the symbiosis island of USDA110. In addition, we found genes encoding a complete photosynthetic system, many ABC transporters for amino acids and oligopeptides, two types (polar and lateral) of flagella, multiple respiratory chains, and a system for lignin monomer catabolism in the S23321 genome. These features suggest that S23321 is able to adapt to a wide range of environments, probably including low-nutrient conditions, with multiple survival strategies in soil and rhizosphere.

• Nitrate-dependent N₂O emission from intact soybean nodules via denitrification by Bradyrhizobium japonicum bacteroids.

  Authors: Junta Hirayama, Shima Eda, Hisayuki Mitsui, Kiwamu Minamisawa

  Applied and environmental microbiology. 12/2011; 77(24):8787-90.

  In the presence of nitrate, N₂O emission increased markedly from soybean roots inoculated with nosZ mutant of Bradyrhizobium japonicum, but not from soybean roots inoculated with a napA nosZ doubleIn the presence of nitrate, N₂O emission increased markedly from soybean roots inoculated with nosZ mutant of Bradyrhizobium japonicum, but not from soybean roots inoculated with a napA nosZ double mutant, indicating that B. japonicum bacteroids in soybean nodules are able to convert the exogenously supplied nitrate into N₂O via a denitrification pathway.

• The genotype of the calcium/calmodulin-dependent protein kinase gene (CCaMK) determines bacterial community diversity in rice roots under paddy and upland field conditions.

  Authors: Seishi Ikeda, Takashi Okubo, Naoya Takeda, Mari Banba, Kazuhiro Sasaki, Haruko Imaizumi-Anraku, Shinsuke Fujihara, Yoshinari Ohwaki, Kenshiro Ohshima, Yoshimichi Fukuta, Shima Eda, Hisayuki Mitsui, Masahira Hattori, Tadashi Sato, Takuro Shinano, Kiwamu Minamisawa

  Applied and environmental microbiology. 07/2011; 77(13):4399-405.

  The effects of the Oryza sativa calcium/calmodulin-dependent protein kinase OsCCaMK genotype (dominant homozygous [D], heterozygous [H], recessive homozygous [R]) on rice root-associated bacteria,The effects of the Oryza sativa calcium/calmodulin-dependent protein kinase OsCCaMK genotype (dominant homozygous [D], heterozygous [H], recessive homozygous [R]) on rice root-associated bacteria, including endophytes and epiphytes, were examined by using a Tos17 rice mutant line under paddy and upland field conditions. Roots were sampled at the flowering stage and were subjected to clone library analyses. The relative abundance of Alphaproteobacteria was noticeably decreased in R plants under both paddy and upland conditions (0.8% and 3.0%, respectively) relative to those in D plants (10.3% and 17.4%, respectively). Population shifts of the Sphingomonadales and Rhizobiales were mainly responsible for this low abundance in R plants. The abundance of Anaerolineae (Chloroflexi) and Clostridia (Firmicutes) was increased in R plants under paddy conditions. The abundance of a subpopulation of Actinobacteria (Saccharothrix spp. and unclassified Actinosynnemataceae) was increased in R plants under upland conditions. Principal coordinate analysis revealed unidirectional community shifts in relation to OsCCaMK gene dosage under both conditions. In addition, shoot length, tiller number, and plant weight decreased as the OsCCaMK gene dosage decreased under upland conditions. These results suggest significant impacts of OsCCaMK on both the diversity of root-associated bacteria and rice plant growth under both paddy and upland field conditions.

• Isolation and genetic characterization of Aurantimonas and Methylobacterium strains from stems of hypernodulated soybeans.

  Authors: Mizue Anda, Seishi Ikeda, Shima Eda, Takashi Okubo, Shusei Sato, Satoshi Tabata, Hisayuki Mitsui, Kiwamu Minamisawa

  Microbes and environments / JSME. 06/2011; 26(2):172-80.

  The aims of this study were to isolate Aurantimonas and Methylobacterium strains that responded to soybean nodulation phenotypes and nitrogen fertilization rates in a previous culture-independentThe aims of this study were to isolate Aurantimonas and Methylobacterium strains that responded to soybean nodulation phenotypes and nitrogen fertilization rates in a previous culture-independent analysis (Ikeda et al. ISME J. 4:315-326, 2010). Two strategies were adopted for isolation from enriched bacterial cells prepared from stems of field-grown, hypernodulated soybeans: PCR-assisted isolation for Aurantimonas and selective cultivation for Methylobacterium. Thirteen of 768 isolates cultivated on Nutrient Agar medium were identified as Aurantimonas by colony PCR specific for Aurantimonas and 16S rRNA gene sequencing. Meanwhile, among 187 isolates on methanol-containing agar media, 126 were identified by 16S rRNA gene sequences as Methylobacterium. A clustering analysis (>99% identity) of the 16S rRNA gene sequences for the combined datasets of the present and previous studies revealed 4 and 8 operational taxonomic units (OTUs) for Aurantimonas and Methylobacterium, respectively, and showed the successful isolation of target bacteria for these two groups. ERIC- and BOX-PCR showed the genomic uniformity of the target isolates. In addition, phylogenetic analyses of Aurantimonas revealed a phyllosphere-specific cluster in the genus. The isolates obtained in the present study will be useful for revealing unknown legume-microbe interactions in relation to the autoregulation of nodulation.

• Identification of Mesorhizobium loti genes relevant to symbiosis by using signature-tagged mutants.

  Authors: Naoganchaolu Borjigin, Keisuke Furukawa, Yoshikazu Shimoda, Satoshi Tabata, Shusei Sato, Shima Eda, Kiwamu Minamisawa, Hisayuki Mitsui

  Microbes and environments / JSME. 06/2011; 26(2):165-71.

  Signature-tagged mutagenesis was applied to Mesorhizobium loti, a nitrogen-fixing root-nodule symbiont of the leguminous plant Lotus japonicus. We arranged 1,887 non-redundant mutant strains of M.Signature-tagged mutagenesis was applied to Mesorhizobium loti, a nitrogen-fixing root-nodule symbiont of the leguminous plant Lotus japonicus. We arranged 1,887 non-redundant mutant strains of M. loti into 75 sets, each consisting of 24 to 26 strains with a different tag in each strain. These sets were each inoculated en masse onto L. japonicus plants. Comparative analysis of total DNA extracted from inoculants and resulting nodules based on quantitative PCR led to the selection of 69 strains as being reduced in relative abundance during nodulation. Plant assays conducted with individual strains confirmed that 3 were defective in nodulation (Nod(-)) and that 10 were Nod(+) but defective in nitrogen fixation (Fix(-)); in each case, the symbiosis deficiency could be attributed to the transposon insertion carried by that strain. Although the remaining 56 strains were Fix(+), 33 of them showed significantly reduced competitiveness during nodulation. Among the mutants we identified are known genes that are diverse in predicted function as well as some genes of unknown function, which demonstrates the validity of this screening procedure for functional genomics in rhizobia.

• Involvement of the smeAB multidrug efflux pump in resistance to plant antimicrobials and contribution to nodulation competitiveness in Sinorhizobium meliloti.

  Authors: Shima Eda, Hisayuki Mitsui, Kiwamu Minamisawa

  Applied and environmental microbiology. 03/2011; 77(9):2855-62.

  The contributions of multicomponent-type multidrug efflux pumps to antimicrobial resistance and nodulation ability in Sinorhizobium meliloti were comprehensively analyzed. Computational searchesThe contributions of multicomponent-type multidrug efflux pumps to antimicrobial resistance and nodulation ability in Sinorhizobium meliloti were comprehensively analyzed. Computational searches identified genes in the S. meliloti strain 1021 genome encoding 1 pump from the ATP-binding cassette family, 3 pumps from the major facilitator superfamily, and 10 pumps from the resistance-nodulation-cell division family, and subsequently, these genes were deleted either individually or simultaneously. Antimicrobial susceptibility tests demonstrated that deletion of the smeAB pump genes resulted in increased susceptibility to a range of antibiotics, dyes, detergents, and plant-derived compounds and, further, that specific deletion of the smeCD or smeEF genes in a ΔsmeAB background caused a further increase in susceptibility to certain antibiotics. Competitive nodulation experiments revealed that the smeAB mutant was defective in competing with the wild-type strain for nodulation. The introduction of a plasmid carrying smeAB into the smeAB mutant restored antimicrobial resistance and nodulation competitiveness. These findings suggest that the SmeAB pump, which is a major multidrug efflux system of S. meliloti, plays an important role in nodulation competitiveness by mediating resistance toward antimicrobial compounds produced by the host plant.

• Autoregulation of nodulation interferes with impacts of nitrogen fertilization levels on the leaf-associated bacterial community in soybeans.

  Authors: Seishi Ikeda, Mizue Anda, Shoko Inaba, Shima Eda, Shusei Sato, Kazuhiro Sasaki, Satoshi Tabata, Hisayuki Mitsui, Tadashi Sato, Takuro Shinano, Kiwamu Minamisawa

  Applied and environmental microbiology. 01/2011; 77(6):1973-80.

  The diversities leaf-associated bacteria on nonnodulated (Nod(-)), wild-type nodulated (Nod(+)), and hypernodulated (Nod(++)) soybeans were evaluated by clone library analyses of the 16S rRNA gene.The diversities leaf-associated bacteria on nonnodulated (Nod(-)), wild-type nodulated (Nod(+)), and hypernodulated (Nod(++)) soybeans were evaluated by clone library analyses of the 16S rRNA gene. To analyze the impact of nitrogen fertilization on the bacterial leaf community, soybeans were treated with standard nitrogen (SN) (15 kg N ha(-1)) or heavy nitrogen (HN) (615 kg N ha(-1)) fertilization. Under SN fertilization, the relative abundance of Alphaproteobacteria was significantly higher in Nod(-) and Nod(++) soybeans (82% to 96%) than in Nod(+) soybeans (54%). The community structure of leaf-associated bacteria in Nod(+) soybeans was almost unaffected by the levels of nitrogen fertilization. However, differences were visible in Nod(-) and Nod(++) soybeans. HN fertilization drastically decreased the relative abundance of Alphaproteobacteria in Nod(-) and Nod(++) soybeans (46% to 76%) and, conversely, increased those of Gammaproteobacteria and Firmicutes in these mutant soybeans. In the Alphaproteobacteria, cluster analyses identified two operational taxonomic units (OTUs) (Aurantimonas sp. and Methylobacterium sp.) that were especially sensitive to nodulation phenotypes under SN fertilization and to nitrogen fertilization levels. Arbuscular mycorrhizal infection was not observed on the root tissues examined, presumably due to the rotation of paddy and upland fields. These results suggest that a subpopulation of leaf-associated bacteria in wild-type Nod(+) soybeans is controlled in similar ways through the systemic regulation of autoregulation of nodulation, which interferes with the impacts of N levels on the bacterial community of soybean leaves.

• Community- and genome-based views of plant-associated bacteria: plant-bacterial interactions in soybean and rice.

  Authors: Seishi Ikeda, Takashi Okubo, Mizue Anda, Hideo Nakashita, Michiko Yasuda, Shusei Sato, Takakazu Kaneko, Satoshi Tabata, Shima Eda, Ayumi Momiyama, Kimihiro Terasawa, Hisayuki Mitsui, Kiwamu Minamisawa

  Plant & cell physiology. 09/2010; 51(9):1398-410.

  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 plantsDiverse 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.

• Temperature-dependent expression of type III secretion system genes and its regulation in Bradyrhizobium japonicum.

  Authors: Min Wei, Keisuke Takeshima, Tadashi Yokoyama, Kiwamu Minamisawa, Hisayuki Mitsui, Manabu Itakura, Takakazu Kaneko, Satoshi Tabata, Kazuhiko Saeki, Hirofumi Omori, Shigeyuki Tajima, Toshiki Uchiumi, Mikiko Abe, Satoshi Ishii, Takuji Ohwada

  Molecular plant-microbe interactions : MPMI. 05/2010; 23(5):628-37.

  The genome-wide expression profiles of Bradyrhizobium japonicum in response to soybean (Glycine max (L.) Merr.) seed extract (SSE) and genistein were monitored with time at a low temperature (15The genome-wide expression profiles of Bradyrhizobium japonicum in response to soybean (Glycine max (L.) Merr.) seed extract (SSE) and genistein were monitored with time at a low temperature (15 degrees C). A comparison with the expression profiles of the B. japonicum genome previously captured at the common growth temperature (30 degrees C) revealed that the expression of SSE preferentially induced genomic loci, including a large gene cluster encoding the type III secretion system (T3SS), were considerably delayed at 15 degrees C, whereas most nodulation (nod) gene loci, including nodD1 and nodW, were rapidly and strongly induced by both SSE and genistein. Induction of the T3SS genes was progressively activated upon the elevation of temperature to 30 degrees C and positively responded to culture population density. In addition, genes nolA and nodD2 were dramatically induced by SSE, concomitantly with the expression of T3SS genes. However, the deletion mutation of nodD2 but not nolA led to elimination of the T3SS genes expression. These results indicate that the expression of the T3SS gene cluster is tightly regulated with integration of environmental cues such as temperature and that NodD2 may be involved in its efficient induction in B. japonicum.

• Thiosulfate-dependent chemolithoautotrophic growth of Bradyrhizobium japonicum.

  Authors: Sachiko Masuda, Shima Eda, Seishi Ikeda, Hisayuki Mitsui, Kiwamu Minamisawa

  Applied and environmental microbiology. 02/2010; 76(8):2402-9.

  Thiosulfate-oxidizing sox gene homologues were found at four loci (I, II, III, and IV) on the genome of Bradyrhizobium japonicum USDA110, a symbiotic nitrogen-fixing bacterium in soil. In fact, B.Thiosulfate-oxidizing sox gene homologues were found at four loci (I, II, III, and IV) on the genome of Bradyrhizobium japonicum USDA110, a symbiotic nitrogen-fixing bacterium in soil. In fact, B. japonicum USDA110 can oxidize thiosulfate and grow under a chemolithotrophic condition. The deletion mutation of the soxY(1) gene at the sox locus I, homologous to the sulfur-oxidizing (Sox) system in Alphaproteobacteria, left B. japonicum unable to oxidize thiosulfate and grow under chemolithotrophic conditions, whereas the deletion mutation of the soxY(2) gene at sox locus II, homologous to the Sox system in green sulfur bacteria, produced phenotypes similar to those of wild-type USDA110. Thiosulfate-dependent O(2) respiration was observed only in USDA110 and the soxY(2) mutant and not in the soxY(1) mutant. In the cells, 1 mol of thiosulfate was stoichiometrically converted to approximately 2 mol of sulfate and consumed approximately 2 mol of O(2). B. japonicum USDA110 showed (14)CO(2) fixation under chemolithotrophic growth conditions. The CO(2) fixation of resting cells was significantly dependent on thiosulfate addition. These results show that USDA110 is able to grow chemolithoautotrophically using thiosulfate as an electron donor, oxygen as an electron acceptor, and carbon dioxide as a carbon source, which likely depends on sox locus I including the soxY(1) gene on USDA110 genome. Thiosulfate oxidation capability is frequently found in members of the Bradyrhizobiaceae, which phylogenetic analysis showed to be associated with the presence of sox locus I homologues, including the soxY(1) gene of B. japonicum USDA110.

• 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.

• The cbbL gene is required for thiosulfate-dependent autotrophic growth of Bradyrhizobium japonicum.

  Authors: Sachiko Masuda, Shima Eda, Chiaki Sugawara, Hisayuki Mitsui, Kiwamu Minamisawa

  Microbes and environments / JSME. 01/2010; 25(3):220-3.

  Bradyrhizobium japonicum is a facultative chemolithoautotroph capable of using thiosulfate and H(2) as an electron donor and CO(2) as a carbon source. In B. japonicum USDA110, the mutant of cbbL geneBradyrhizobium japonicum is a facultative chemolithoautotroph capable of using thiosulfate and H(2) as an electron donor and CO(2) as a carbon source. In B. japonicum USDA110, the mutant of cbbL gene encoding a large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was unable to grow using thiosulfate and H(2) as an electron donor. The cbbL deletion mutant was able to grow and oxidize thiosulfate in the presence of succinate. These results showed that the major route of CO(2) fixation for thiosulfate-dependent chemoautotrophic growth is the Calvin-Benson-Bassham cycle involving RuBisCO in B. japonicum.

• 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.

• Community shifts of soybean stem-associated bacteria responding to different nodulation phenotypes and N levels.

  Authors: Seishi Ikeda, Takashi Okubo, Takakazu Kaneko, Shoko Inaba, Tomiya Maekawa, Shima Eda, Shusei Sato, Satoshi Tabata, Hisayuki Mitsui, Kiwamu Minamisawa

  The ISME journal. 11/2009;

  The diversity of stem-associated bacteria of non-nodulated (Nod(-)), wild-type nodulated (Nod(+)) and hypernodulated (Nod(++)) soybeans were evaluated by clone library analyses of the 16S ribosomalThe diversity of stem-associated bacteria of non-nodulated (Nod(-)), wild-type nodulated (Nod(+)) and hypernodulated (Nod(++)) soybeans were evaluated by clone library analyses of the 16S ribosomal RNA gene. Soybeans were dressed with standard nitrogen (SN) fertilization (15 kg N ha(-1)) and heavy nitrogen (HN) fertilization (615 kg N ha(-1)). The relative abundance of Alphaproteobacteria in Nod(+) soybeans (66%) was smaller than that in Nod(-) and Nod(++) soybeans (75-76%) under SN fertilization, whereas that of Gammaproteobacteria showed the opposite pattern (23% in Nod(+) and 12-16% in Nod(-) and Nod(++) soybeans). Principal coordinate analysis showed that the bacterial communities of Nod(-) and Nod(++) soybeans were more similar to each other than to that of Nod(+) soybeans under SN fertilization. HN fertilization increased the relative abundance of Gammaproteobacteria in all nodulation phenotypes (33-57%) and caused drastic shifts of the bacterial community. The clustering analyses identified a subset of operational taxonomic units (OTUs) at the species level in Alpha- and Gammaproteobacteria responding to both the nodulation phenotypes and nitrogen fertilization levels. Meanwhile, the abundance of Betaproteobacteria was relatively constant in all libraries constructed under these environmental conditions. The relative abundances of two OTUs in Alphaproteobacteria (Aurantimonas sp. and Methylobacterium sp.) were especially sensitive to nodulation phenotype and were drastically decreased under HN fertilization. These results suggested that a subpopulation of proteobacteria in soybeans is controlled in a similar manner through both the regulation systems of plant-rhizobia symbiosis and the nitrogen signaling pathway in plants.The ISME Journal advance online publication, 19 November 2009; doi:10.1038/ismej.2009.119.

• Development of a Bacterial Cell Enrichment Method and its Application to the Community Analysis in Soybean Stems.

  Authors: Seishi Ikeda, Takakazu Kaneko, Takashi Okubo, Lynn Rallos, Shima Eda, Hisayuki Mitsui, Shusei Sato, Yasukazu Nakamura, Satoshi Tabata, Kiwamu Minamisawa

  Microbial ecology. 09/2009;

  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. StemA 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.

• 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.

• Nitrous oxide emission and microbial community in the rhizosphere of nodulated soybeans during the late growth period.

  Authors: Shoko Inaba, Kenji Tanabe, Shima Eda, Seishi Ikeda, Atsushi Higashitani, Hisayuki Mitsui, Kiwamu Minamisawa

  Microbes and environments / JSME. 01/2009; 24(1):64-7.

  We examined N(2)O emissions from the rhizosphere of field-grown soybeans during the late growth stage (99-117 days after sowing). Marked emissions were detected from the nodulated root systems ofWe examined N(2)O emissions from the rhizosphere of field-grown soybeans during the late growth stage (99-117 days after sowing). Marked emissions were detected from the nodulated root systems of field-grown soybeans, whereas a non-nodulating soybean mutant showed no emission. Degraded nodules exclusively generated the N(2)O. A culture-independent analysis of microbial communities showed Bradyrhizobium sp., Acidvorax facilis, Salmonella enterica, Xanthomonas sp., Enterobacter cloacae, Pseudomonas putida, Fusarium sp., nematodes, and other protozoans to be more abundant in the degraded nodules, suggesting that some of these organisms participate in the N(2)O emission process in the soybean rhizosphere.

• Nodulation-dependent communities of culturable bacterial endophytes from stems of field-grown soybeans.

  Authors: Takashi Okubo, Seishi Ikeda, Takakazu Kaneko, Shima Eda, Hisayuki Mitsui, Shusei Sato, Satoshi Tabata, Kiwamu Minamisawa

  Microbes and environments / JSME. 01/2009; 24(3):253-8.

  Endophytic bacteria (247 isolates) were randomly isolated from surface-sterilized stems of non-nodulated (Nod(-)), wild-type nodulated (Nod(+)), and hypernodulated (Nod(++)) soybeans (Glycine maxEndophytic bacteria (247 isolates) were randomly isolated from surface-sterilized stems of non-nodulated (Nod(-)), wild-type nodulated (Nod(+)), and hypernodulated (Nod(++)) soybeans (Glycine max [L.] Merr) on three agar media (R2A, nutrient agar, and potato dextrose agar). Their diversity was compared on the basis of 16S rRNA gene sequences. The phylogenetic composition depended on the soybean nodulation phenotype, although diversity indexes were not correlated with nodulation phenotype. The most abundant phylum throughout soybean lines tested was Proteobacteria (58-79%). Gammaproteobacteria was the dominant class (21-72%) with a group of Pseudomonas sp. significantly abundant in Nod(+) soybeans. A high abundance of Alphaproteobacteria was observed in Nod(-) soybeans, which was explained by the increase in bacterial isolates of the families Rhizobiaceae and Sphingomonadaceae. A far greater abundance of Firmicutes was observed in Nod(-) and Nod(++) mutant soybeans than in Nod(+) soybeans. An impact of culture media on the diversity of isolated endophytic bacteria was also observed: The highest diversity indexes were obtained on the R2A medium, which enabled us to access Alphaproteobacteria and other phyla more frequently. The above results indicated that the extent of nodulation changes the phylogenetic composition of culturable bacterial endophytes in soybean stems.

• Bradyrhizobium japonicum mutants with increased N2O reductase activity by selection after the introduction of mutated dnaQ gene.

  Authors: Manabu Itakura, Kazufumi Tabata, Shima Eda, Hisayuki Mitsui, Kiriko Murakami, Junichi Yasuda, Kiwamu Minamisawa

  Applied and environmental microbiology. 11/2008;

  We obtained two beneficial mutants of Bradyrhizobium japonicum USDA110 with increased nitrous oxide (N2O) reductase (N2OR) activity by introducing a plasmid containing a mutated B. japonicum dnaQWe obtained two beneficial mutants of Bradyrhizobium japonicum USDA110 with increased nitrous oxide (N2O) reductase (N2OR) activity by introducing a plasmid containing a mutated B. japonicum dnaQ gene (pKQ2) and performing enrichment culture under selection pressure for N2O respiration. Mutation of dnaQ, which encodes the epsilon subunit of DNA polymerase III, gives a strong mutator phenotype in Escherichia coli. pKQ2 introduction in B. japonicum USDA110 increased the frequency of occurrence of colonies spontaneously resistant to kanamycin. A series of repeated cultivations of USDA110 with and without pKQ2 was conducted in anaerobic conditions under 5% (v/v) or 20% (v/v) N2O atomosphere. At the 10th cultivation cycle, cell populations of USDA110 (pKQ2) showed higher N2OR activity than that of the wild-type strains. Four bacterial mutants lacking pKQ2 obtained by plant passage showed seven to 12 times the N2OR activity of the wild-type USDA110. Although two mutants had a weak or null fix phenotype for symbiotic nitrogen fixation, the remaining two (5M09 and 5M14) had the same symbiotic nitrogen fixation ability and heterotrophic growth in culture as wild-type USDA110.

• Genomic comparison of Bradyrhizobium japonicum strains with different symbiotic nitrogen-fixing capabilities and other Bradyrhizobiaceae members.

  Authors: Manabu Itakura, Kazuhiko Saeki, Hirofumi Omori, Tadashi Yokoyama, Takakazu Kaneko, Satoshi Tabata, Takuji Ohwada, Shigeyuki Tajima, Toshiki Uchiumi, Keina Honnma, Konosuke Fujita, Hiroyoshi Iwata, Yuichi Saeki, Yoshino Hara, Seishi Ikeda, Shima Eda, Hisayuki Mitsui, Kiwamu Minamisawa

  The ISME journal. 11/2008;

  Comparative genomic hybridization (CGH) was performed with nine strains of Bradyrhizobium japonicum (a symbiotic nitrogen-fixing bacterium associated with soybean) and eight other members of theComparative genomic hybridization (CGH) was performed with nine strains of Bradyrhizobium japonicum (a symbiotic nitrogen-fixing bacterium associated with soybean) and eight other members of the Bradyrhizobiaceae by DNA macroarray of B. japonicum USDA110. CGH clearly discriminated genomic variations in B. japonicum strains, but similar CGH patterns were observed in other members of the Bradyrhizobiaceae. The most variable regions were 14 genomic islands (4-97 kb) and low G+C regions on the USDA110 genome, some of which were missing in several strains of B. japonicum and other members of the Bradyrhizobiaceae. The CGH profiles of B. japonicum were classified into three genome types: 110, 122 and 6. Analysis of DNA sequences around the boundary regions showed that at least seven genomic islands were missing in genome type 122 as compared with type 110. Phylogenetic analysis for internal transcribed sequences revealed that strains belonging to genome types 110 and 122 formed separate clades. Thus genomic islands were horizontally inserted into the ancestor genome of type 110 after divergence of the type 110 and 122 strains. To search for functional relationships of variable genomic islands, we conducted linear models of the correlation between the existence of genomic regions and the parameters associated with symbiotic nitrogen fixation in soybean. Variable genomic regions including genomic islands were associated with the enhancement of symbiotic nitrogen fixation in B. japonicum USDA110.The ISME Journal advance online publication, 30 October 2008; doi:10.1038/ismej.2008.88.