Molecular cloning of a gene for indole-3-acetamide hydrolase from Bradyrhizobium japonicum.
A pLAFR1 cosmid genomic library of wild-type Bradyrhizobium japonicum J1063 was constructed. A cosmid clone designated pBjJ4, containing a 26-kilobase (kb) DNA insert, was identified as being able to confer the ability to convert alpha-naphthaleneacetamide acid on B. japonicum J1B7 Rifr, which cannot perform this conversion. The gene coding for the enzyme that converts alpha-naphthaleneacetamide to alpha-naphthaleneacetic acid was localized in the 3.5-kb region of pBjJ4 by recloning in plasmid pSUP202. The gene coding for the enzyme was also mapped by Tn5 insertion mutagenesis to a region of ca. 2.3 kb. When the gene was placed behind the lacZ promoter and used to transform Escherichia coli, a high level of expression of indole-3-acetamide hydrolase activity was found. Since there have been no reports of this activity in E. coli, we have thus confirmed that the gene cloned here is a structural gene for indole-3-acetamide hydrolase and have designated it as the bam (Bradyrhizobium amidehydrolase) gene. Southern hybridization with the central region of the bam gene indicated that a high degree of similarity exists among the bam gene, the iaaH gene from Pseudomonas savastonoi, and the tms-2 gene from Agrobacterium tumefaciens. The result suggests that there is a common origin for the gene that encodes the enzyme that catalyzes the biosynthesis of indoleacetic acid.
Sekine
M
M
Department of Pure and Applied Sciences, College of Arts and Sciences, University of Tokyo, Japan.
Watanabe
K
K
Syono
K
K
D000581:Amidohydrolases / Q000235:genetics*; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D009154:Mutation; D010957:Plasmids; D015183:Restriction Mapping; D012231:Rhizobium / Q000201:enzymology / Q000235:genetics*
D000581:Amidohydrolases; C058772:indole-3-acetamide hydrolase