Publications

• DNA sequence and mutational analysis of rhizobitoxine biosynthesis genes in Bradyrhizobium elkanii.

  Yasuta T., Okazaki S., Mitsui H., Yuhashi K., Ezura H., Minamisawa K.

  We cloned and sequenced a cluster of genes involved in the biosynthesis of rhizobitoxine, a nodulation enhancer produced by Bradyrhizobium elkanii. The nucleotide sequence of the cloned 28.4-kb DNA region encompassing rtxA showed that several open reading frames (ORFs) were located downstream of r ... >> More

  We cloned and sequenced a cluster of genes involved in the biosynthesis of rhizobitoxine, a nodulation enhancer produced by Bradyrhizobium elkanii. The nucleotide sequence of the cloned 28.4-kb DNA region encompassing rtxA showed that several open reading frames (ORFs) were located downstream of rtxA. A large-deletion mutant of B. elkanii, USDA94 Delta rtx::Omega 1, which lacks rtxA, ORF1 (rtxC), ORF2, and ORF3, did not produce rhizobitoxine, dihydrorhizobitoxine, or serinol. The broad-host-range cosmid pLAFR1, which contains rtxA and these ORFs, complemented rhizobitoxine production in USDA94 Delta rtx::Omega 1. Further complementation experiments involving cosmid derivatives obtained by random mutagenesis with a kanamycin cassette revealed that at least rtxA and rtxC are necessary for rhizobitoxine production. Insertional mutagenesis of the N-terminal and C-terminal regions of rtxA indicated that rtxA is responsible for two crucial steps, serinol formation and dihydrorhizobitoxine biosynthesis. An insertional mutant of rtxC produced serinol and dihydrorhizobitoxine but no rhizobitoxine. Moreover, the rtxC product was highly homologous to the fatty acid desaturase of Pseudomonas syringae and included the copper-binding signature and eight histidine residues conserved in membrane-bound desaturase. This result suggested that rtxC encodes dihydrorhizobitoxine desaturase for the final step of rhizobitoxine production. In light of results from DNA sequence comparison, gene disruption experiments, and dihydrorhizobitoxine production from various substrates, we discuss the biosynthetic pathway of rhizobitoxine and its evolutionary significance in bradyrhizobia. << Less

  Appl. Environ. Microbiol. 67:4999-5009(2001) [PubMed] [EuropePMC]

• Bradyrhizobium elkanii rtxC gene is required for expression of symbiotic phenotypes in the final step of rhizobitoxine biosynthesis.

  Okazaki S., Sugawara M., Minamisawa K.

  We disrupted the rtxC gene on the chromosome of Bradyrhizobium elkanii USDA94 by insertion of a nonpolar aph cartridge. The rtxC mutant, designated DeltartxC, produced serinol and dihydrorhizobitoxine but no rhizobitoxine, both in culture and in planta. The introduction of cosmids harboring the rt ... >> More

  We disrupted the rtxC gene on the chromosome of Bradyrhizobium elkanii USDA94 by insertion of a nonpolar aph cartridge. The rtxC mutant, designated DeltartxC, produced serinol and dihydrorhizobitoxine but no rhizobitoxine, both in culture and in planta. The introduction of cosmids harboring the rtxC gene into the DeltartxC mutant complemented rhizobitoxine production, suggesting that rtxC is involved in the final step of rhizobitoxine biosynthesis in B. elkanii USDA94. Glycine max cv. Lee inoculated with DeltartxC or with a null mutant, Deltartx::Omega1, showed no foliar chlorosis, whereas the wild-type strain USDA94 caused severe foliar chlorosis. The two mutants showed significantly less nodulation competitiveness than the wild-type strain on Macroptilium atropurpureum. These results indicate that dihydrorhizobitoxine, the immediate precursor of the oxidative form of rhizobitoxine, has no distinct effect on nodulation phenotype in these legumes. Thus, desaturation of dihydrorhizobitoxine by rtxC-encoded protein is essential for the bacterium to show rhizobitoxine phenotypes in planta. In addition, complementation analysis of rtxC by cosmids differing in rtxC transcription levels suggested that rhizobitoxine production correlates with the amount of rtxC transcript. << Less

  Appl. Environ. Microbiol. 70:535-541(2004) [PubMed] [EuropePMC]