DNA Sequence of Genes for Detoxification of Fusaric Acid, a Wilt-inducing Agent Produced by Fusarium Species

Isolation and nucleotide sequence determination of fusaric acid-detoxification genes are described in this paper. For screening the genes, bacteria collected from soil were positively selected in a selective medium containing fusaric acid. The capability of fusaric acid-resistant isolates to detoxify the toxin was assayed by examining the survival of tomato callus cells in culture filtrates prepared from the bacterial culture, in the presence of fusaric acid. The isolate (HY-1) showing the highest detoxification was selected and identified as Klebsiella oxytoca. Chromosomal DNA of this isolate was digested with Bam HI and shotgun-cloned to fusaric acid-sensitive E. coli. The DNA fragment carrying fusaric acid-detoxification genes was further shortened by enzyme digestion and the open reading frames in the fragment were analyzed by determining total nucleotide sequences of the fragment. Finally, three open reading frames were shown to be essential for expressing the detoxification of fusaric acid. These frames possessed a single promoter sequence at the upstream region of the first open reading frame. Northern blot analysis showed that these genes were polycistronically transcribed to express the fusaric acid detoxification, strongly supporting th results of DNA sequence analysis.