Search

Random transposon mutagenesis led to the isolation of a novel Mesorhizobium loft mutant that is defective in nitrogen fixation during symbiosis with Lotus japonicus. The mutated locus, designated cep, encodes a putative cell-envelope protein displaying no significant sequence similarity to proteins with known functions. This mutant elicits the formation of nodule-like bumps and root-hair curling, but not the elongation of infection threads, on L. japonicus roots. This is reminiscent of the phenotypes of rhizobial mutants impaired in cyclic [beta]-glucan biosynthesis. The cep mutant exhibits partially reduced content of cell-associated glucans and intermediate deficiency of motility under hypo-osmotic conditions as compared to a glucan-deficient mutant. Second-site pseudorevertants of the cep mutant were isolated by selecting for restoration of symbiotic nitrogen fixation. A subset of pseudorevertants restored both symbiotic capability and glucan content to levels comparable to that of the wild-type. These results suggest that the Cep product acts on a successful symbiosis by affecting cell-associated glucan content.

The expression of nitrogenase genes of Herbaspirillum sp. B501 associated in shoot of wild rice, Oryza officinalis, is explored by means of reverse transcription-PCR (RT-PCR) targeted at the nifH gene. The results indicate that in planta nitrogen fixation by the endophyte shows a daily rhythm determined by the plant's light environment.

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.

Researchers describe an endophytic diazotroph that colonizes wild rice and fixes nitrogen within plant tissues. Unfortunately, it did not substantially colonized cultivated rice. This is critical because sustainable agriculture requires the use of nitrogen-fixing bacteria in crops rather than nitrogen fertilizer.

The genes involved in the biosynthesis of rhizobitoxine in Bradyrhizobium elkanii have been cloned and sequenced. Rhizobitoxine is considered a plant toxin but it also enhances nodulation and competitiveness in some legumes.

Highly reiterated sequence-possessing (HRS) strains of Bradyrhizobium japonicum have the IS1631 insertion sequence-like element whereas non-HRS strains do not. IS elements are mobile DNA elements that occur in the genomes, plasmids, and bacteriophages of many bacteria.

The POP2 (Caf1) protein in Saccharomyces cerevisiae affects a variety of transcriptional processes and is a component of the Ccr4p complex. We have isolated five multicopy suppressor genes of a pop2 deletion mutation: CCR4, DHH1 (a putative RNA helicase), PKC1, STM1, and MPT5 (multicopy suppressor of pop two). Overexpression of either the CCR4 or DHH1 genes effectively suppressed phenotypes associated with pop2 mutant cells; overexpression of PKC1, STM1, or MPT5 genes produced only partial suppression. Disruption of the CCR4 or DHH1 genes resulted in phenotypes similar to those observed for pop2 cells. In addition, overexpression of the DHH1 gene also suppressed the ccr4 mutation, suggesting a close relationship between the POP2, CCR4, and DHH1 genes. Two-hybrid analysis and coimmunoprecipitation experiments revealed that Pop2p and Dhh1p interact physically, and these and other data suggest that Dhh1p is also a component of the Ccr4p complex. Finally, we investigated the genetic interaction between factors associated with POP2 and the PKC1 pathway. The temperature-sensitive growth defect of dhh1 or mpt5 cells was suppressed by overexpression of PKC1, and the defect of mpk1 cells was suppressed by overexpression of MPT5. These results and phenotypic analysis of double mutants from the POP2 and PKC1 pathways suggested that the POP2 and the PKC1 pathways are independent but have some overlapping functions.

Phylogenetic analysis was performed to understand the association of thiosulfate oxidation capability in members of Brudyrhizobiaceae with the presence of homologous for sox locus I genes of B. japonicum USDA110. The results suggested that USDA110 was able to grow chemolithoautotrophically using thiosulfate as an electron donor, oxygen as an electron acceptor, and carbon dioxide as a carbon source which possibly depends on sox locus I including the sox[Y.sub.1] gene on USDA110 genome.

The functions of the vanA1B, pcaG1H1 and mxaF genes are examined during vanillate degradation by disrupting the Burkholderia japonicum USDA110 genes that are upregulated in the transcriptome analysis. The mxaF mutant has showed normal nodulation and [N.sub.2] fixation phenotypes with soybeans, which is not similar to symbiotic phenotypes of methylotrophic rhizobia.

A plasmid containing a mutated Bradyrhizobium japonicum dnaQ gene (pKQ2) is used to obtain two beneficial mutants of B. japonicum USDA110 with increased nitrous oxide ([N.sub.2]O) reductase ([N.sub.2]OR) activity. Four bacterial mutants lacking pKQ2 obtained by plant passage has shown 7 to 12 times the [N.sub.2]OR activity of the wild-type USDA110.

Ribosomal intergenic transcribed spacer analysis (RISA) and automated RISA (ARISA) are used for analyzing microorganisms associated with stems and roots of nonnodulated ([Nod.sup.-]), wild-type nodulated ([Nod.sup.+]) and hypernodulated ([Nod.sup.++]) soybeans. The results have shown that the microbial community in [Nod.sup.-] soybeams is similar to that in [Nod.sup.++] soybeans than to that in [Nod.sup.+] soybeans.

[N.sub.2]O reductase activity in soybean nodules formed with Bradyrhizobium japonicum was evaluated from [N.sub.2]O uptake and conversion of [sup.15.N]-[N.sub.2]O into [sup.15.N]-[N.sub.2]. Results indicate that the conversion of [N.sub.2]O to [N.sub.2] depends exclusively on the respiratory [N.sub.2]O reductase and that soybean roots nodulated with B. japanicum carrying the 'nos' genes are able to remove very low concentration of [N.sub.2]O.