DNA Sequence and Mutational Analysis of Rhizobitoxine Biosynthesis Genes in Bradyrhizobium elkanii

Tsuyoshi Yaslta, Shin Okazaki, Hisayuki Mitsui, Ken Ichi Yuhashi, Hiroshi Ezura, Kiwamu Minamisawa

Research output: Contribution to journalArticlepeer-review

51 Citations (Scopus)

Abstract

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 ft elkanii, USDA94Δrtx::Ω1, which lacks rtxA, ORF1 (rtrC), 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 USDA94Δrtx::Ω1, 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.

Original languageEnglish
Pages (from-to)4999-5009
Number of pages11
JournalApplied and environmental microbiology
Volume67
Issue number3-12
DOIs
Publication statusPublished - 2001

ASJC Scopus subject areas

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology

Fingerprint Dive into the research topics of 'DNA Sequence and Mutational Analysis of Rhizobitoxine Biosynthesis Genes in Bradyrhizobium elkanii'. Together they form a unique fingerprint.

Cite this