A Pseudomonas stutzeri gene cluster encoding the biosynthesis of the CCl4-dechlorination agent pyridine-2,6-bis(thiocarboxylic acid)

A spontaneous mutant of Pseudomonas stutzeri strain KC lacked the carbon tetrachloride (CCl4) transformation ability of wild-type KC. Analysis of restriction digests separated by pulsed-field gel electrophoresis (PFGE) indicated that the mutant strain CTN1 differed from strain KC by deletion of approximately 170 kb of chromosomal DNA. CTN1 did not produce pyridine-2,6-bis(thiocarboxylic acid) (PDTC), the agent determined to be responsible for CCl4 dechlorination in cultures of strain KC. Cosmids from a genomic library of strain KC containing DNA from within the deleted region were identified by hybridization with a 148 kb genomic Spel fragment absent in strain CTN1. Several cosmids identified in this manner were further screened for complementation of the PDTC biosynthesis-negative (Pdt(-)) phenotype. One cosmid (pT31) complemented the Pdt- phenotype of CTN1 and conferred CCl4 transformation activity and PDTC production upon other pseudomonads. Southern analysis showed that none of three other P. stutzeri strains representing three genomovars contained DNA that would hybridize with the 25 746 bp insert of pT31. Transposon mutagenesis of pT31 identified open reading frames (ORFs) whose disruption affected the ability to make PDTC in the strain CTN1 background. These data describe the pdt locus of strain KC as residing in a non-essential region of the chromosome subject to spontaneous deletion. The pdt locus is necessary for PDTC biosynthesis in strain KC and is sufficient for PDTC biosynthesis by other pseudomonads but is not a common feature of P. stutzeri strains.