Manabu Itakura

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.

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

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 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.

Symbiotic Bradyrhizobium japonicum reduces N2O surrounding the soybean root system via nitrous oxide reductase

ABSTRACT N2O reductase activity in soybean nodules formed with Bradyrhizobium japonicum was evaluated from N2O uptake and conversion of 15N-N2O into 15N-N2. Free-living cells of USDA110 showed N2O reductase activity, whereas a nosZ mutant did not. Complementation of the nosZ mutant with two cosmids containing the nosRZDFYLX genes of B. japonicum USDA110 restored the N2O reductase activity. When detached soybean nodules formed with USDA110 were fed with 15N-N2O, they rapidly emitted 15N-N2 outside the nodules at a ratio of 98.5% of 15N-N2O uptake, but nodules inoculated with the nosZ mutant did not. Surprisingly, N2O uptake by soybean roots nodulated with USDA110 was observed even in ambient air containing a low concentration of N2O (0.34 ppm). These results indicate that the conversion of N2O to N2 depends exclusively on the respiratory N2O reductase and that soybean roots nodulated with B. japonicum carrying the nos genes are able to remove very low concentrations of N2O.

Complete genome sequence of Bradyrhizobium sp. S23321: insights into symbiosis evolution in soil oligotrophs.

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 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.

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

ABSTRACT 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.

N2O Emission from Degraded Soybean Nodules Depends on Denitrification by Bradyrhizobium japonicum and Other Microbes in the Rhizosphere

A model system developed to produce N2O emissions from degrading soybean nodules in the laboratory was used to clarify the mechanism of N2O emission from soybean fields. Soybean plants inoculated with nosZ-defective strains of Bradyrhizobium japonicum USDA110 (ΔnosZ, lacking N2O reductase) were grown in aseptic jars. After 30 days, shoot decapitation (D, to promote nodule degradation), soil addition (S, to supply soil microbes), or both (DS) were applied. N2O was emitted only with DS treatment. Thus, both soil microbes and nodule degradation are required for the emission of N2O from the soybean rhizosphere. The N2O flux peaked 15 days after DS treatment. Nitrate addition markedly enhanced N2O emission. A 15N tracer experiment indicated that N2O was derived from N fixed in the nodules. To evaluate the contribution of bradyrhizobia, N2O emission was compared between a nirK mutant (ΔnirKΔnosZ, lacking nitrite reductase) and ΔnosZ. The N2O flux from the ΔnirKΔnosZ rhizosphere was significantly lower than that from ΔnosZ, but was still 40% to 60% of that of ΔnosZ, suggesting that N2O emission is due to both B. japonicum and other soil microorganisms. Only nosZ-competent B. japonicum (nosZ+ strain) could take up N2O. Therefore, during nodule degradation, both B. japonicum and other soil microorganisms release N2O from nodule N via their denitrification processes (N2O source), whereas nosZ-competent B. japonicum exclusively takes up N2O (N2O sink). Net N2O flux from soybean rhizosphere is likely determined by the balance of N2O source and sink.

Aerobic Vanillate Degradation and C1 Compound Metabolism in Bradyrhizobium japonicum

ABSTRACT 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, a methoxylated lignin monomer, as an energy source. A transcriptome analysis indicated that one set of vanA1B, pcaG1H1, and genes for C1 compound catabolism was upregulated in B. japonicum USDA110 cells grown in vanillate (N. Ito, M. Itakura, S. Eda, K. Saeki, H. Oomori, T. Yokoyama, T. Kaneko, S. Tabata, T. Ohwada, S. Tajima, T. Uchiumi, E. Masai, M. Tsuda, H. Mitsui, and K. Minamisawa, Microbes Environ. 21:240-250, 2006). To examine the functions of these genes in vanillate degradation, we tested cell growth and substrate consumption in 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.

Soybean Seed Extracts Preferentially Express Genomic Loci of Bradyrhizobium japonicum in the Initial Interaction with Soybean, Glycine max (L.) Merr

Initial interaction between rhizobia and legumes actually starts via encounters of both partners in the rhizosphere. In this study, the global expression profiles of Bradyrhizobium japonicum USDA 110 in response to soybean (Glycine max) seed extracts (SSE) and genistein, a major soybean-released isoflavone for nod genes induction of B. japonicum, were compared. SSE induced many genomic loci as compared with genistein (5.0 µM), nevertheless SSE-supplemented medium contained 4.7 µM genistein. SSE markedly induced four predominant genomic regions within a large symbiosis island (681 kb), which include tts genes (type III secretion system) and various nod genes. In addition, SSE-treated cells expressed many genomic loci containing genes for polygalacturonase (cell-wall degradation), exopolysaccharide synthesis, 1-aminocyclopropane-1-carboxylate deaminase, ribosome proteins family and energy metabolism even outside symbiosis island. On the other hand, genistein-treated cells exclusively showed one expression cluster including common nod gene operon within symbiosis island and six expression loci including multidrug resistance, which were shared with SSE-treated cells. Twelve putatively regulated genes were indeed validated by quantitative RT-PCR. Several SSE-induced genomic loci likely participate in the initial interaction with legumes. Thus, these results can provide a basic knowledge for screening novel genes relevant to the B. japonicum- soybean symbiosis.

Genome Analysis Suggests that the Soil Oligotrophic Bacterium Agromonas oligotrophica (Bradyrhizobium oligotrophicum) Is a Nitrogen-Fixing Symbiont of Aeschynomene indica

ABSTRACT Agromonas oligotrophica (Bradyrhizobium oligotrophicum) S58T 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.

Generation of Bradyrhizobium japonicum Mutants with Increased N2O Reductase Activity by Selection after Introduction of a Mutated dnaQ Gene

ABSTRACT 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 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 into 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% (vol/vol) or 20% (vol/vol) N2O atmosphere. At the 10th cultivation cycle, cell populations of USDA110(pKQ2) showed higher N2OR activity than the wild-type strains. Four bacterial mutants lacking pKQ2 obtained by plant passage showed 7 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.