ISOLATION OF A GENE (PBSC) REQUIRED FOR SIDEROPHORE BIOSYNTHESIS IN FLUORESCENT PSEUDOMONAS SP STRAIN M114

An iron-regulated gene, pbsC, required for siderophore production in f luorescent Pseudomonas sp. strain M114 has been identified. A kanamyci n-resistance cassette was inserted at specific restriction sites withi n a 7 kb genomic fragment of M114 DNA and by marker exchange two sider ophore-negative mutants, designated M1 and M2, were isolated. The nucl eotide sequence of approximately 4 kb of the region flanking the inser tion sites was determined and a large open reading frame (ORF) extendi ng for 2409 bp was identified. This gene was designated pbsC (pseudoba ctin synthesis C) and its putative protein product termed PbsC. PbsC w as found to be homologous to a family of enzymes involved in the biosy nthesis of secondary metabolites, including EntF of Escherichia coli. These enzymes are believed to act via ATP-dependent binding of AMP to their substrate. Several areas of high sequence homology between these proteins and PbsC were observed, including a conserved AMP-binding do main. The expression of pbsC is iron-regulated as revealed when a DNA fragment containing the upstream region was cloned in a promoter probe vector and conjugated into the wild-type strain, M114. The nucleotide sequence upstream of the putative translational start site contains a region homologous to previously defined -16 to -25 sequences of iron- regulated genes but did not contain an iron-box consensus sequence. It was noted that inactivation of the pbsC gene also affected other iron -regulated phenotypes of Pseudomonas M114.