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

We cloned and sequenced a cluster of genes involved in the biosynthesis of rhizobitoxine, a nodulation enhancer produced by Bradyrhizobium elkanii. Th e nucleotide sequence of the cloned 28.4-kb DNA region encompassing rtxA sh owed that several open reading frames (ORFs) were located downstream of rtx A. A large-deletion mutant of B. elkanii, USDA94 Delta rtx::Ohm1, which lac ks rtxA, ORF1 (rtxC), ORF2, and ORF3, did not produce rhizobitoxine, dihydr orhizobitoxine, or serinol. The broad-host-range cosmid pLAFR1, which conta ins rtxA and these ORFs, complemented rhizobitoxine production in USDA94 De lta rtx::Ohm1. Further complementation experiments involving cosmid derivat ives obtained by random mutagenesis with a kanamycin cassette revealed that at least rtxA and rtxC are necessary for rhizobitoxine production. Inserti onal mutagenesis of the N-terminal and C-terminal regions of rtxA indicated that rtxA is responsible for two crucial steps, serinol formation and dihy drorhizobitoxine biosynthesis. An insertional mutant of rtxC produced serin ol and dihydrorhizobitoxine but no rhizobitoxine. Moreover, the rtxC produc t was highly homologous to the fatty acid desaturase of Pseudomonas syringa e and included the copper-binding signature and eight histidine residues co nserved in membrane-bound desaturase. This result suggested that rtxC encod es dihydrorhizobitoxine desaturase for the final step of rhizobitoxine prod uction. In light of results from DNA sequence comparison, gene disruption e xperiments, and dihydrorhizobitoxine production from various substrates, we discuss the biosynthetic pathway of rhizobitoxine and its evolutionary sig nificance in bradyrhizobia.