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. 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.
Yasuta
T
T
Institute of Genetic Ecology, Tohoku University, Katahira, Aoba-ku, Sendai 980-8577, Japan.
Okazaki
S
S
Mitsui
H
H
Yuhashi
K
K
Ezura
H
H
Minamisawa
K
K
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D020369:Bradyrhizobium / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D002853:Chromatography, Liquid; D003470:Culture Media; D044943:Fatty Acid Desaturases / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D009586:Nitrogen Fixation / Q000187:drug effects; D016366:Open Reading Frames; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011412:Propanolamines / Q000378:metabolism* / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D000637:Transaminases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D003470:Culture Media; D009097:Multienzyme Complexes; D011412:Propanolamines; D010088:Oxidoreductases; D044943:Fatty Acid Desaturases; C443998:dihydrorhizobitoxine desaturase; D000637:Transaminases; C443997:rtxA protein, Bradyrhizobium elkanii; C041618:rhizobitoxine