Bradyrhizobium ottawaense efficiently reduces nitrous oxide through high nosZ gene expression.

N2O is an important greenhouse gas influencing global warming, and agricultural land is the predominant (anthropogenic) source of N2O emissions. Here, we report the high N2O-reducing activity of Bradyrhizobium ottawaense, suggesting the potential for efficiently mitigating N2O emission from agricultural lands. Among the 15 B. ottawaense isolates examined, the N2O-reducing activities of most (13) strains were approximately five-fold higher than that of Bradyrhizobium diazoefficiens USDA110T under anaerobic conditions. This robust N2O-reducing activity of B. ottawaense was confirmed by N2O reductase (NosZ) protein levels and by mitigation of N2O emitted by nodule decomposition in laboratory system. While the NosZ of B. ottawaense and B. diazoefficiens showed high homology, nosZ gene expression in B. ottawaense was over 150-fold higher than that in B. diazoefficiens USDA110T, suggesting the high N2O-reducing activity of B. ottawaense is achieved by high nos expression. Furthermore, we examined the nos operon transcription start sites and found that, unlike B. diazoefficiens, B. ottawaense has two transcription start sites under N2O-respiring conditions, which may contribute to the high nosZ expression. Our study indicates the potential of B. ottawaense for effective N2O reduction and unique regulation of nos gene expression towards the high performance of N2O mitigation in the soil.

Evolution of rhizobial symbiosis islands through insertion sequence-mediated deletion and duplication.

Symbiosis between organisms influences their evolution via adaptive changes in genome architectures. Immunity of soybean carrying the Rj2 allele is triggered by NopP (type III secretion system [T3SS]-dependent effector), encoded by symbiosis island A (SymA) in B. diazoefficiens USDA122. This immunity was overcome by many mutants with large SymA deletions that encompassed T3SS (rhc) and N2 fixation (nif) genes and were bounded by insertion sequence (IS) copies in direct orientation, indicating homologous recombination between ISs. Similar deletion events were observed in B. diazoefficiens USDA110 and B. japonicum J5. When we cultured a USDA122 strain with a marker gene sacB inserted into the rhc gene cluster, most sucrose-resistant mutants had deletions in nif/rhc gene clusters, similar to the mutants above. Some deletion mutants were unique to the sacB system and showed lower competitive nodulation capability, indicating that IS-mediated deletions occurred during free-living growth and the host plants selected the mutants. Among 63 natural bradyrhizobial isolates, 2 possessed long duplications (261-357 kb) harboring nif/rhc gene clusters between IS copies in direct orientation via homologous recombination. Therefore, the structures of symbiosis islands are in a state of flux via IS-mediated duplications and deletions during rhizobial saprophytic growth, and host plants select mutualistic variants from the resultant pools of rhizobial populations. Our results demonstrate that homologous recombination between direct IS copies provides a natural mechanism generating deletions and duplications on symbiosis islands.

Symbiotic incompatibility between soybean and Bradyrhizobium arises from one amino acid determinant in soybean Rj2 protein.

Cultivated soybean (Glycine max) carrying the Rj2 allele restricts nodulation with specific Bradyrhizobium strains via host immunity, mediated by rhizobial type III secretory protein NopP and the host resistance protein Rj2. Here we found that the single isoleucine residue I490 in Rj2 is required for induction of symbiotic incompatibility. Furthermore, we investigated the geographical distribution of the Rj2-genotype soybean in a large set of germplasm by single nucleotide polymorphism (SNP) genotyping using a SNP marker for I490. By allelic comparison of 79 accessions in the Japanese soybean mini-core collection, we suggest substitution of a single amino acid residue (R490 to I490) in Rj2 induces symbiotic incompatibility with Bradyrhizobium diazoefficiens USDA 122. The importance of I490 was verified by complementation of rj2-soybean by the dominant allele encoding the Rj2 protein containing I490 residue. The Rj2 allele was also found in Glycine soja, the wild progenitor of G. max, and their single amino acid polymorphisms were associated with the Rj2-nodulation phenotype. By SNP genotyping against 1583 soybean accessions, we detected the Rj2-genotype in 5.4% of G. max and 7.7% of G. soja accessions. Distribution of the Rj2-genotype soybean plants was relatively concentrated in the temperate Asian region. These results provide important information about the mechanism of host genotype-specific symbiotic incompatibility mediated by host immunity and suggest that the Rj2 gene has been maintained by environmental conditions during the process of soybean domestication.

Identification of Genes Regulated by the Antitermination Factor NasT during Denitrification in Bradyrhizobium diazoefficiens.

The soybean symbiont Bradyrhizobium diazoefficiens grows anaerobically in the presence of nitrate using the denitrification pathway, which involves the nap, nir, nor, and nos genes. We previously showed that NasT acts as a transcription antitermination regulator for nap and nos gene expression. In the present study, we investigated the targets of NasT in B. diazoefficiens during denitrifying growth by performing transcription profiling with RNA-seq and quantitative reverse-transcription PCR. Most of the genes with altered expression in the absence of NasT were related to nitrogen metabolism, specifically several systems for branched-chain amino acid transport. The present results suggest that the reduced expression of genes involved in nitrogen acquisition leads to the induction of alternative sets of genes with similar functions. The ΔnasT mutant of B. diazoefficiens grew better than the wild type under denitrifying conditions. However, this enhanced growth was completely abolished by an additional loss of the narK or bjgb genes, which encode cytoplasmic systems for nitrite and nitric oxide detoxification, respectively. Since the expression of narK and bjgb was increased in the ΔnasT mutant, the growth of the ΔnasT mutant may be promoted by increased detoxification activity.

Variation in bradyrhizobial NopP effector determines symbiotic incompatibility with Rj2-soybeans via effector-triggered immunity.

Genotype-specific incompatibility in legume-rhizobium symbiosis has been suggested to be controlled by effector-triggered immunity underlying pathogenic host-bacteria interactions. However, the rhizobial determinant interacting with the host resistance protein (e.g., Rj2) and the molecular mechanism of symbiotic incompatibility remain unclear. Using natural mutants of Bradyrhizobium diazoefficiens USDA 122, we identified a type III-secretory protein NopP as the determinant of symbiotic incompatibility with Rj2-soybean. The analysis of nopP mutations and variants in a culture collection reveal that three amino acid residues (R60, R67, and H173) in NopP are required for Rj2-mediated incompatibility. Complementation of rj2-soybean by the Rj2 allele confers the incompatibility induced by USDA 122-type NopP. In response to incompatible strains, Rj2-soybean plants activate defense marker gene PR-2 and suppress infection thread number at 2 days after inoculation. These results suggest that Rj2-soybeans monitor the specific variants of NopP and reject bradyrhizobial infection via effector-triggered immunity mediated by Rj2 protein.

Expression of Two RpoH Sigma Factors in Sinorhizobium meliloti upon Heat Shock.

The plant symbiotic α-proteobacterium Sinorhizobium meliloti has two RpoH-type sigma factors, RpoH1 and RpoH2. The former induces the synthesis of heat shock proteins and optimizes interactions with the host. Using a Western blot analysis, we examined time course changes in the intracellular contents of these factors upon a temperature upshift. The RpoH1 level was relatively high and constant, suggesting that its regulatory role in the heat shock response is attained through the activation of the pre-existing RpoH1 protein. In contrast, the RpoH2 level was initially undetectable, and gradually increased. These differential patterns reflect the functional diversification of these factors.

Regulation of nitrous oxide reductase genes by NasT-mediated transcription antitermination in Bradyrhizobium diazoefficiens.

In Bradyrhizobium diazoefficiens, maximal expression of the nitrous oxide reductase gene (nosZ) requires oxygen limitation and the presence of a nitrogen oxide. The putative transcription antiterminator NasT is a positive regulator of nosZ; but in the absence of nitrate, NasT is counteracted by the nitrate sensor NasS. Here, we examined the NasT-mediated mechanism of nosRZDFYLX gene cluster expression. We mapped two transcription start sites of nosR and identified two potential hairpins, H1 and H2, within the 5'-leader of nosR transcripts. Electrophoretic mobility shift assay showed that NasT specifically bound the nosR-leader RNA and deletion of H1 abolished such binding. Under aerobic nitrate-deficient conditions, deletion of H1 or H2 increased the level of nosRZD transcripts. Under denitrifying conditions (anaerobiosis with nitrate supply), the level of nosRZD transcripts was severely impaired in the nasT mutant; in the nasT background, deletions of either hairpin led to increased level of nosRZD transcripts. In contrast to nosRZD coding region, nosR-leader transcript level was not affected by nasS or nasT mutations under aerobic or denitrifying conditions respectively. These results suggest that the two-hairpin RNA structure acts for transcription termination upstream of nosR and the binding of NasT to H1 facilitates read-through transcription to induce nos expression.

Complete Genome Sequence of Bradyrhizobium diazoefficiens USDA 122, a Nitrogen-Fixing Soybean Symbiont.

We report the complete genome sequence of Bradyrhizobium diazoefficiens USDA 122, a nitrogen-fixing soybean symbiont. The genome consists of a 9.1 Mb circular chromosome, and 8,551 coding sequences (CDSs) were predicted on the genome. The sequence will provide insight into the evolution of rhizobial genome, and the symbiotic compatibility with host plants.

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 Sinorhizobium meliloti RpoH-Regulated Gene Is Involved in Iron-Sulfur Protein Metabolism and Effective Plant Symbiosis under Intrinsic Iron Limitation.

UNLABELLED: In Sinorhizobium meliloti, RpoH-type sigma factors have a global impact on gene expression during heat shock and play an essential role in symbiosis with leguminous plants. Using mutational analysis of a set of genes showing highly RpoH-dependent expression during heat shock, we identified a gene indispensable for effective symbiosis. This gene, designated sufT, was located downstream of the sufBCDS homologs that specify the iron-sulfur (Fe/S) cluster assembly pathway. The identified transcription start site was preceded by an RpoH-dependent promoter consensus sequence. SufT was related to a conserved protein family of unknown molecular function, of which some members are involved in Fe/S cluster metabolism in diverse organisms. A sufT mutation decreased bacterial growth in both rich and minimal media, tolerance to stresses such as iron starvation, and activities of some Fe/S cluster-dependent enzymes. These results support the involvement of SufT in SUF (sulfur mobilization) system-mediated Fe/S protein metabolism. Furthermore, we isolated spontaneous pseudorevertants of the sufT mutant with partially recovered growth; each of them had a mutation in rirA This gene encodes a global iron regulator whose loss increases the intracellular iron content. Deletion of rirA in the original sufT mutant improved growth and restored Fe/S enzyme activities and effective symbiosis. These results suggest that enhanced iron availability compensates for the lack of SufT in the maintenance of Fe/S proteins. IMPORTANCE: Although RpoH-type sigma factors of the RNA polymerase are present in diverse proteobacteria, their role as global regulators of protein homeostasis has been studied mainly in the enteric gammaproteobacterium Escherichia coli In the soil alphaproteobacterium Sinorhizobium meliloti, the rpoH mutations have a strong impact on symbiosis with leguminous plants. We found that sufT is a unique member of the S. meliloti RpoH regulon; sufT contributes to Fe/S protein metabolism and effective symbiosis under intrinsic iron limitation exerted by RirA, a global iron regulator. Our study provides insights into the RpoH regulon function in diverse proteobacteria adapted to particular ecological niches and into the mechanism of conserved Fe/S protein biogenesis.

Complete Genome Sequence of Methylobacterium sp. Strain AMS5, an Isolate from a Soybean Stem.

Nonrhizobial Methylobacterium spp. inhabit the phyllosphere of a wide variety of plants. We report here the complete genome sequence of Methylobacterium sp. AMS5, which was isolated from a soybean stem. The information is useful for understanding the molecular mechanisms of the interaction between nonrhizobial Methylobacterium spp. and plants.

Bacterial clade with the ribosomal RNA operon on a small plasmid rather than the chromosome.

rRNA is essential for life because of its functional importance in protein synthesis. The rRNA (rrn) operon encoding 16S, 23S, and 5S rRNAs is located on the "main" chromosome in all bacteria documented to date and is frequently used as a marker of chromosomes. Here, our genome analysis of a plant-associated alphaproteobacterium, Aureimonas sp. AU20, indicates that this strain has its sole rrn operon on a small (9.4 kb), high-copy-number replicon. We designated this unusual replicon carrying the rrn operon on the background of an rrn-lacking chromosome (RLC) as the rrn-plasmid. Four of 12 strains close to AU20 also had this RLC/rrn-plasmid organization. Phylogenetic analysis showed that those strains having the RLC/rrn-plasmid organization represented one clade within the genus Aureimonas. Our finding introduces a previously unaddressed viewpoint into studies of genetics, genomics, and evolution in microbiology and biology in general.

Low nitrogen fertilization adapts rice root microbiome to low nutrient environment by changing biogeochemical functions.

Reduced fertilizer usage is one of the objectives of field management in the pursuit of sustainable agriculture. Here, we report on shifts of bacterial communities in paddy rice ecosystems with low (LN), standard (SN), and high (HN) levels of N fertilizer application (0, 30, and 300 kg N ha(-1), respectively). The LN field had received no N fertilizer for 5 years prior to the experiment. The LN and HN plants showed a 50% decrease and a 60% increase in biomass compared with the SN plant biomass, respectively. Analyses of 16S rRNA genes suggested shifts of bacterial communities between the LN and SN root microbiomes, which were statistically confirmed by metagenome analyses. The relative abundances of Burkholderia, Bradyrhizobium and Methylosinus were significantly increased in root microbiome of the LN field relative to the SN field. Conversely, the abundance of methanogenic archaea was reduced in the LN field relative to the SN field. The functional genes for methane oxidation (pmo and mmo) and plant association (acdS and iaaMH) were significantly abundant in the LN root microbiome. Quantitative PCR of pmoA/mcrA genes and a (13)C methane experiment provided evidence of more active methane oxidation in the rice roots of the LN field. In addition, functional genes for the metabolism of N, S, Fe, and aromatic compounds were more abundant in the LN root microbiome. These results suggest that low-N-fertilizer management is an important factor in shaping the microbial community structure containing key microbes for plant associations and biogeochemical processes in paddy rice ecosystems.

A rice gene for microbial symbiosis, Oryza sativa CCaMK, reduces CH4 flux in a paddy field with low nitrogen input.

Plants have mutualistic symbiotic relationships with rhizobia and fungi by the common symbiosis pathway, of which Ca(2+)/calmodulin-dependent protein kinase (encoded by CCaMK) is a central component. Although Oryza sativa CCaMK (OsCCaMK) is required for fungal accommodation in rice roots, little is known about the role of OsCCaMK in rice symbiosis with bacteria. Here, we report the effect of a Tos17-induced OsCCaMK mutant (NE1115) on CH4 flux in low-nitrogen (LN) and standard-nitrogen (SN) paddy fields compared with wild-type (WT) Nipponbare. The growth of NE1115 was significantly decreased compared with that of the WT, especially in the LN field. The CH4 flux of NE1115 in the LN field was significantly greater (156 to 407% in 2011 and 170 to 816% in 2012) than that of the WT, although no difference was observed in the SN field. The copy number of pmoA (encodes methane monooxygenase in methanotrophs) was significantly higher in the roots and rhizosphere soil of the WT than in those of NE1115. However, the mcrA (encodes methyl coenzyme M reductase in methanogens) copy number did not differ between the WT and NE1115. These results were supported by a (13)C-labeled CH4-feeding experiment. In addition, the natural abundance of (15)N in WT shoots (3.05‰) was significantly lower than in NE1115 shoots (3.45‰), suggesting greater N2 fixation in the WT because of dilution with atmospheric N2 (0.00‰). Thus, CH4 oxidation and N2 fixation were simultaneously activated in the root zone of WT rice in the LN field and both processes are likely controlled by OsCCaMK.

Impact of Azospirillum sp. B510 inoculation on rice-associated bacterial communities in a paddy field.

Rice seedlings were inoculated with Azospirillum sp. B510 and transplanted into a paddy field. Growth in terms of tiller numbers and shoot length was significantly increased by inoculation. Principal-coordinates analysis of rice bacterial communities using the 16S rRNA gene showed no overall change from B510 inoculation. However, the abundance of Veillonellaceae and Aurantimonas significantly increased in the base and shoots, respectively, of B510-inoculated plants. The abundance of Azospirillum did not differ between B510-inoculated and uninoculated plants (0.02-0.50%). These results indicate that the application of Azospirillum sp. B510 not only enhanced rice growth, but also affected minor rice-associated bacteria.

Involvement of a novel genistein-inducible multidrug efflux pump of Bradyrhizobium japonicum early in the interaction with Glycine max (L.) Merr.

The early molecular dialogue between soybean and the bacterium Bradyrhizobium japonicum is crucial for triggering their symbiotic interaction. Here we found a single large genomic locus that is widely separated from the symbiosis island and was conspicuously induced within minutes after the addition of genistein. This locus (named BjG30) contains genes for the multidrug efflux pump, TetR family transcriptional regulator, and polyhydroxybutyrate (PHB) metabolism. The induction of BjG30 by genistein was competitively inhibited by daidzein, although both genistein and daidzein are soybean-derived inducers of nodulation (nod) genes. Such a differential expression pattern is also observed in some legume-derived flavonoids, which structurally differ in the hydroxy/deoxy group at the 5-position. In addition, not only did the induction start far in advance of nodW and nodD1 after the addition of genistein, but the levels showed distinct concentration dependence, indicating that the induction pattern of BjG30 is completely different from that of nod genes. The deletion of genes encoding either the multidrug efflux pump or PHB metabolism, especially the former, resulted in defective nodulation performance and nitrogen-fixing capability. Taken together, these results indicate that BjG30, and especially its multidrug efflux pump, may play a key role in the early stage of symbiosis by balancing the dual functions of genistein as both a nod gene inducer and toxicant.

Linked expressions of nap and nos genes in a Bradyrhizobium japonicum mutant with increased N(2)O reductase activity.

To understand the mechanisms underlying the increased N2O reductase activity in the Bradyrhizobium japonicum 5M09 mutant from enrichment culture under N2O respiration, we analyzed the expression of genes encoding denitrification reductases and regulators. Our results suggest a common regulation of nap (encoding periplasmic nitrate reductase) and nos (encoding N2O reductase).

Genome analysis suggests that the soil oligotrophic bacterium Agromonas oligotrophica (Bradyrhizobium oligotrophicum) is a nitrogen-fixing symbiont of Aeschynomene indica.

Agromonas oligotrophica (Bradyrhizobium oligotrophicum) S58(T) is a nitrogen-fixing oligotrophic bacterium isolated from paddy field soil that is able to grow in extra-low-nutrient environments. Here, the complete genome sequence of S58 was determined. The S58 genome was found to comprise a circular chromosome of 8,264,165 bp with an average GC content of 65.1% lacking nodABC genes and the typical symbiosis island. The genome showed a high level of similarity to the genomes of Bradyrhizobium sp. ORS278 and Bradyrhizobium sp. BTAi1, including nitrogen fixation and photosynthesis gene clusters, which nodulate an aquatic legume plant, Aeschynomene indica, in a Nod factor-independent manner. Although nonsymbiotic (brady)rhizobia are significant components of rhizobial populations in soil, we found that most genes important for nodule development (ndv) and symbiotic nitrogen fixation (nif and fix) with A. indica were well conserved between the ORS278 and S58 genomes. Therefore, we performed inoculation experiments with five A. oligotrophica strains (S58, S42, S55, S72, and S80). Surprisingly, all five strains of A. oligotrophica formed effective nitrogen-fixing nodules on the roots and/or stems of A. indica, with differentiated bacteroids. Nonsymbiotic (brady)rhizobia are known to be significant components of rhizobial populations without a symbiosis island or symbiotic plasmids in soil, but the present results indicate that soil-dwelling A. oligotrophica generally possesses the ability to establish symbiosis with A. indica. Phylogenetic analyses suggest that Nod factor-independent symbiosis with A. indica is a common trait of nodABC- and symbiosis island-lacking strains within the members of the photosynthetic Bradyrhizobium clade, including A. oligotrophica.

The type III Secretion System of Bradyrhizobium japonicum USDA122 mediates symbiotic incompatibility with Rj2 soybean plants.

The rhcJ and ttsI mutants of Bradyrhizobium japonicum USDA122 for the type III protein secretion system (T3SS) failed to secrete typical effector proteins and gained the ability to nodulate Rj2 soybean plants (Hardee), which are symbiotically incompatible with wild-type USDA122. This suggests that effectors secreted via the T3SS trigger incompatibility between these two partners.

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.