Hirohito Tsurumaru

NaCl-activated nucleoside diphosphate kinase from extremely halophilic archaeon, Halobacterium salinarum, maintains native conformation without salt

Enzymes from extremely halophilic archaea are readily denatured in the absence of a high salt concentration. However, we have observed here that a nucleoside diphosphate kinase prepared from Halobacterium salinarum was active and stable in the absence of salt, though it has the amino acid composition characteristic of halophilic enzymes. Recombinant nucleoside diphosphate kinase expressed in Escherichia coli requires salt for activation in vitro, but once it acquires the proper folding, it no longer requires the presence of salts for its activity and stability.

Metaproteomic Identification of Diazotrophic Methanotrophs and Their Localization in Root Tissues of Field-Grown Rice Plants

ABSTRACT In a previous study by our group, CH4 oxidation and N2 fixation were simultaneously activated in the roots of wild-type rice plants in a paddy field with no N input; both processes are likely controlled by a rice gene for microbial symbiosis. The present study examined which microorganisms in rice roots were responsible for CH4 oxidation and N2 fixation under the field conditions. Metaproteomic analysis of root-associated bacteria from field-grown rice (Oryza sativa Nipponbare) revealed that nitrogenase complex-containing nitrogenase reductase (NifH) and the alpha subunit (NifD) and beta subunit (NifK) of dinitrogenase were mainly derived from type II methanotrophic bacteria of the family Methylocystaceae, including Methylosinus spp. Minor nitrogenase proteins such as Methylocella, Bradyrhizobium, Rhodopseudomonas, and Anaeromyxobacter were also detected. Methane monooxygenase proteins (PmoCBA and MmoXYZCBG) were detected in the same bacterial group of the Methylocystaceae. Because these results indicated that Methylocystaceae members mediate both CH4 oxidation and N2 fixation, we examined their localization in rice tissues by using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). The methanotrophs were localized around the epidermal cells and vascular cylinder in the root tissues of the field-grown rice plants. Our metaproteomics and CARD-FISH results suggest that CH4 oxidation and N2 fixation are performed mainly by type II methanotrophs of the Methylocystaceae, including Methylosinus spp., inhabiting the vascular bundles and epidermal cells of rice roots.

Tn5 mutants of Bradyrhizobium japonicum Is-1 with altered compatibility with Rj 2-soybean cultivars

Abstract Bradyrhizobium japonicum Is-1 is incompatible with soybean (Glycine max L. Merr.) cv. CNS (Rj 2 Rj 3) and compatible with cv. Hill (Rj 4) and is, therefore, classified into nodulation-type B. Tn5 mutants (1C1, 1C2, 5C1, 6C1, 7C1, 7C2, 10C1 and 10C2) were isolated from eight nodules produced on CNS that had been inoculated with the kanamycin-resistant (Kmr) transconjugants of strain Is-1. To investigate the shift of host range, Tn5 mutants were inoculated to soybean cvs. CNS, D-51 (Rj 3), Hardee (Rj 2 Rj 3), Hill and IAC-2 (Rj 2 Rj 3). They acquired the ability to nodulate Rj 2 Rj 3 soybeans without losing the ability to nodulate both Rj 3 and Rj 4. These results suggest that the Tn5 mutation in B. japonicum strain Is-1 is only related to the ability to nodulate Rj 2 soybean. Although the host ranges were common to Tn5 mutants and all sections of nodules produced by Tn5 mutants indicate a red color, the nodule numbers and nitrogen fixation activities were different for each mutant. These results suggest that Tn5 insertion sites are different from each other. Tn5-flanking sequences in these mutants were specifically amplified. The electrophoresis analyses of the polymerase chain reaction (PCR) products showed that Tn5 mutants contained a single copy of Tn5, except for 10C1. These PCR products were cloned and sequenced. The sequence analysis of Tn5-flanking sequences revealed that these Tn5 insertion sites were different from each other and all homologous sequences to them were found in the complete genome sequence of B. japonicum USDA 110. These results are in good accordance with our expectation that Tn5 insertion sites are different for each mutant. Most Tn5-inserted gene products relate to the cell membrane structure. This suggests that the change in the cell surface structure in Tn5 mutants may overcome nodulation restriction conditioned by Rj 2 soybean.

Metagenomic Analysis of the Bacterial Community Associated with the Taproot of Sugar Beet

We analyzed a metagenome of the bacterial community associated with the taproot of sugar beet (Beta vulgaris L.) in order to investigate the genes involved in plant growth-promoting traits (PGPTs), namely 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, indole acetic acid (IAA), N2 fixation, phosphate solubilization, pyrroloquinoline quinone, siderophores, and plant disease suppression as well as methanol, sucrose, and betaine utilization. The most frequently detected gene among the PGPT categories encoded β-1,3-glucanase (18 per 105 reads), which plays a role in the suppression of plant diseases. Genes involved in phosphate solubilization (e.g., for quinoprotein glucose dehydrogenase), methanol utilization (e.g., for methanol dehydrogenase), siderophore production (e.g. isochorismate pyruvate lyase), and ACC deaminase were also abundant. These results suggested that such PGPTs are crucially involved in supporting the growth of sugar beet. In contrast, genes for IAA production (iaaM and ipdC) were less abundant (~1 per 105 reads). N2 fixation genes (nifHDK) were not detected; bacterial N2 -fixing activity was not observed in the 15N2 -feeding experiment. An analysis of nitrogen metabolism suggested that the sugar beet microbiome mainly utilized ammonium and nitroalkane as nitrogen sources. Thus, N2 fixation and IAA production did not appear to contribute to sugar beet growth. Taxonomic assignment of this metagenome revealed the high abundance of Mesorhizobium, Bradyrhizobium, and Streptomyces, suggesting that these genera have ecologically important roles in the taproot of sugar beet. Bradyrhizobium-assigned reads in particular were found in almost all categories of dominant PGPTs with high abundance. The present study revealed the characteristic functional genes in the taproot-associated microbiome of sugar beet, and suggest the opportunity to select sugar beet growth-promoting bacteria.

Elevated atmospheric CO2 levels affect community structure of rice root-associated bacteria

A number of studies have shown that elevated atmospheric CO2 ([CO2]) affects rice yields and grain quality. However, the responses of root-associated bacteria to [CO2] elevation have not been characterized in a large-scale field study. We conducted a free-air CO2 enrichment (FACE) experiment (ambient + 200 μmol.mol−1) using three rice cultivars (Akita 63, Takanari, and Koshihikari) and two experimental lines of Koshihikari [chromosome segment substitution and near-isogenic lines (NILs)] to determine the effects of [CO2] elevation on the community structure of rice root-associated bacteria. Microbial DNA was extracted from rice roots at the panicle formation stage and analyzed by pyrosequencing the bacterial 16S rRNA gene to characterize the members of the bacterial community. Principal coordinate analysis of a weighted UniFrac distance matrix revealed that the community structure was clearly affected by elevated [CO2]. The predominant community members at class level were Alpha-, Beta-, and Gamma-proteobacteria in the control (ambient) and FACE plots. The relative abundance of Methylocystaceae, the major methane-oxidizing bacteria in rice roots, tended to decrease with increasing [CO2] levels. Quantitative PCR revealed a decreased copy number of the methane monooxygenase (pmoA) gene and increased methyl coenzyme M reductase (mcrA) in elevated [CO2]. These results suggest elevated [CO2] suppresses methane oxidation and promotes methanogenesis in rice roots; this process affects the carbon cycle in rice paddy fields.

Metagenomic Analysis Revealed Methylamine and Ureide Utilization of Soybean-Associated Methylobacterium.

Methylobacterium inhabits the phyllosphere of a large number of plants. We herein report the results of comparative metagenome analyses on methylobacterial communities of soybean plants grown in an experimental field in Tohoku University (Kashimadai, Miyagi, Japan). Methylobacterium was identified as the most dominant genus (33%) among bacteria inhabiting soybean stems. We classified plant-derived Methylobacterium species into Groups I, II, and III based on 16S rRNA gene sequences, and found that Group I members (phylogenetically close to M. extorquens) were dominant in soybean-associated Methylobacterium. By comparing 29 genomes, we found that all Group I members possessed a complete set of genes for the N-methylglutamate pathway for methylamine utilization, and genes for urea degradation (urea carboxylase, urea amidolyase, and conventional urease). Only Group I members and soybean methylobacterial isolates grew in a culture supplemented with methylamine as the sole carbon source. They utilized urea or allantoin (a urea-related compound in legumes) as the sole nitrogen source; however, group III also utilized these compounds. The utilization of allantoin may be crucial in soybean-bacterial interactions because allantoin is a transported form of fixed nitrogen in legume plants. Soybean-derived Group I strain AMS5 colonized the model legume Lotus japonicus well. A comparison among the 29 genomes of plant-derived and other strains suggested that several candidate genes are involved in plant colonization such as csgG (curli fimbriae). Genes for the N-methylglutamate pathway and curli fimbriae were more abundant in soybean microbiomes than in rice microbiomes in the field. Based on these results, we discuss the lifestyle of Methylobacterium in the legume phyllosphere.

A Putative Type III Secretion System Effector Encoded by the MA20_12780 Gene in Bradyrhizobium japonicum Is-34 Causes Incompatibility with Rj4 Genotype Soybeans

ABSTRACT The nodulation of Bradyrhizobium japonicum Is-34 is restricted by Rj 4 genotype soybeans (Glycine max). To identify the genes responsible for this incompatibility, Tn5 mutants of B. japonicum Is-34 that were able to overcome this nodulation restriction were obtained. Analysis of the Tn5 mutants revealed that Tn5 was inserted into a region containing the MA20_12780 gene. In addition, direct disruption of this gene using marker exchange overcame the nodulation restriction by Rj 4 genotype soybeans. The MA20_12780 gene has a tts box motif in its upstream region, indicating a possibility that this gene encodes a type III secretion system (T3SS) effector protein. Bioinformatic characterization revealed that the MA20_12780 protein contains the small ubiquitin-like modifier (SUMO) protease domain of the C48 peptidase (ubiquitin-like protease 1 [Ulp1]) family. The results of the present study indicate that a putative T3SS effector encoded by the MA20_12780 gene causes the incompatibility with Rj 4 genotype soybeans, and they suggest the possibility that the nodulation restriction of B. japonicum Is-34 may be due to Rj 4 genotype soybeans recognizing the putative T3SS effector (MA20_12780 protein) as a virulence factor.

Nitrogen fixation and nifH diversity in human gut microbiota.

It has been hypothesized that nitrogen fixation occurs in the human gut. However, whether the gut microbiota truly has this potential remains unclear. We investigated the nitrogen-fixing activity and diversity of the nitrogenase reductase (NifH) genes in the faecal microbiota of humans, focusing on Papua New Guinean and Japanese individuals with low to high habitual nitrogen intake. A 15N2 incorporation assay showed significant enrichment of 15N in all faecal samples, irrespective of the host nitrogen intake, which was also supported by an acetylene reduction assay. The fixed nitrogen corresponded to 0.01% of the standard nitrogen requirement for humans, although our data implied that the contribution in the gut in vivo might be higher than this value. The nifH genes recovered in cloning and metagenomic analyses were classified in two clusters: one comprising sequences almost identical to Klebsiella sequences and the other related to sequences of Clostridiales members. These results are consistent with an analysis of databases of faecal metagenomes from other human populations. Collectively, the human gut microbiota has a potential for nitrogen fixation, which may be attributable to Klebsiella and Clostridiales strains, although no evidence was found that the nitrogen-fixing activity substantially contributes to the host nitrogen balance.

Draft genome sequence of Bradyrhizobium japonicum Is-34, which is incompatible with Rj4 genotype soybeans

ABSTRACT We report the draft genome sequence of Bradyrhizobium japonicum Is-1, which is incompatible with Rj2 genotype soybeans. The estimated genome size of this strain is 8.9 Mb. Genome sequence information of this strain will help to identify a causal gene for this incompatibility.

Draft Genome Sequences of Bradyrhizobium elkanii Strains BLY3-8 and BLY6-1, Which Are Incompatible with Rj3 Genotype Soybean Cultivars

ABSTRACT We report here the draft genome sequences of Bradyrhizobium elkanii strains BLY3-8 and BLY6-1, which are incompatible with Rj3 genotype soybean cultivars. The genome sequences of these strains will be useful to identify a causal gene for this incompatibility.