Takuji Ohwada

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.

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.

Enhancement of the nitrogen fixation efficiency of genetically-engineered Rhizobium with high catalase activity.

The vktA catalase gene, which had been cloned from Vibrio rumoiensis S-1T having extraordinarily high catalase activity, was introduced into the root nodule bacterium, Rhizobium leguminosarum bv. phaseoli USDA 2676. The catalase activity of the vktA-transformed R. leguminosarum cells (free-living) was three orders in magnitude higher than that of the parent cells and this transformant could grow in a higher concentration of exogenous hydrogen peroxide (H2O2). The vktA-transformant was inoculated to the host plant (Phaseolus vulgaris L.) and the nodulation efficiency was evaluated. The results showed that the nitrogen-fixing activity of nodules was increased 1.7 to 2.3 times as compared to the parent. The levels of H2O2 in nodules formed by the vktA-transformant were decreased by around 73%, while those of leghemoglobins (Lba and Lbb) were increased by 1.2 (Lba) and 2.1 (Lbb) times compared with the parent. These results indicated that the increase of catalase activity in rhizobia could be useful to improve the nitrogen-fixing efficiency of nodules by the reduction of H2O2 content concomitantly with the enhancement of leghemoglobins contents.

Enhanced heterologous production of eicosapentaenoic acid in Escherichia coli cells that co-express eicosapentaenoic acid biosynthesis pfa genes and foreign DNA fragments including a high-performance catalase gene, vktA.

Cellular eicosapentaenoic acid (EPA) makes up approximately 3% of total fatty acids in Escherichiacoli DH5α, a strain that carries EPA biosynthesis genes (pEPAΔ1). EPA was increased to 12% of total fatty acids when the host cell co-expressed the vector pGBM3::sa1(vktA), which carried the high-performance catalase gene, vktA. Where this vector was co-expressed, the transformant accumulated a large amount of VktA protein. However, the EPA production of cells carrying the vector, that included the insert lacking almost the entire vktA gene, was approximately 6%. This suggests that the retention of a large DNA insert in the vector and the accumulation of the resulting protein, but not the catalytic activity of VktA catalase, would potentially be able to increase the content of EPA.

Direct formation of heart-shaped embryos from differentiated single carrot cells in culture

Abstract Single cells of a variety of shapes and sizes, which had been released from regenerated carrot plantlets, divided with irregular patterns to form cell clusters of various shapes and sizes with uneven surfaces. Once these cell clusters had become isodiametric globular embryos with a smooth surface, they developed to heart-shaped embryos that were bilaterally symmetrical along the apical–basal axis. The transition of single cells to globular embryos was developmentally different from the transition of zygotes to globular embryos, which is caused by regular longitudinal and transverse divisions. Serial observations revealed a divergent sequence of morphological stages, from cell clusters to heart-shaped embryos during somatic embryogenesis. In some parts of cell clusters, globular embryos with cotyledonary primordia were formed, but cell clusters also developed directly to heart-shaped embryos without going through the typical globular stage.

Correlation between NaCl Sensitivity of Rhizobium Bacteria and Ineffective Nodulation of Leguminous Plants

A sodium chloride (NaCl)-sensitive mutant of Rhizobium fredii USDA191, which contained a single copy of Tn5-Mob transposed into chromosomal DNA, was obtained by Tn5-Mob random insertion. The growth rate of this mutant was lower than that of the wild type in the presence of 0.2 M NaCl and it seemed to lack the inductive ATP production in response to the addition of NaCl. This mutant induced the formation of small and whitish nodules on lateral roots of soybeans, which were negative for acetylene reduction activity, indicating that the nodules were ineffective for nitrogen fixation. The mutant also reduced the weight of above-ground portions and roots to 64 and 55%, respectively, compared with the weight of the plants inoculated with the wild-type cells. These results suggest that NaCl sensitivity of Rhizobium bacteria is one of the important factors for nodule formation and nitrogen fixation.

An assessment of the diversity of culturable bacteria from main root of sugar beet.

The partial sequences of the 16S rRNA genes of 531 bacteria isolated from the main root of the sugar beet (Beta vulgaris L.) were determined and subsequently grouped into 155 operational taxonomic units by clustering analysis (≥99% identity). The most abundant phylum was Proteobacteria (72.5–77.2%), followed by Actinobacteria (9.8–16.6%) and Bacteroidetes (4.3– 15.4%). Alphaproteobacteria (46.7–64.8%) was the most dominant class within Proteobacteria. Four strains belonging to Verrucomicrobia were also isolated. Phylogenetic analysis revealed that the Verrucomicrobia bacterial strains were closely related to Haloferula or Verrucomicrobium.

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.

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

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 (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.