Analysis of regulatory elements and genes required for carbon tetrachloride degradation in Pseudomonas stutzeri strain KC

Previously, we described the generation and initial characterization of four Tn5 mutants of Pseudomonas stutzeri strain KC with impaired ability to degrade carbon tetrachloride (Sepúlveda-Torres et al., 1999). In this study, we show cloning and sequencing of an 8.3 kbp region in which all four transposons were located. This fragment encodes eight potential genes and is located in the central part of the 25 kbp fragment recently identified by Lewis et al. (2000) and shown by them to be sufficient to confer carbon tetrachloride transformation capability upon other pseudomonads. The four transposon insertion mutants mapped in ORF's F and I designated by Lewis et al. (2000). This is consistent with the results by Lewis et al. (2000) that orfF is required for carbon tetrachloride degradation. We further established that orfI is required for CCl₄ degradation since the three mutants in this ORF were unable to degrade carbon tetrachloride. We present our analysis of the gene and protein sequences from the 8.3 kbp region and propose a tentative model for the role of different genes in the synthesis and activity of pyridine-2,6-bis(thiocarboxylate) (PDTC), the secreted factor responsible for carbon tetrachloride dechlorination. We also found a putative promoter that overlaps with a Fur-box-like sequence in the region upstream of mutated genes. To test this putative promoter region and Fur-box, we generated and ligated DNA fragments containing wild-type and mutant Fur-boxes to a lacZ reporter. The wild-type fragment showed promoter activity that is regulated by the concentration of iron in the medium. Finally, we screened a selection of Pseudomonas strains, including P. putida DSMZ 3601 - a strain known to produce PDTC - for the presence of the genes characterized in this study. None of the strains tested positive, suggesting that Pseudomonas stutzeri strain KC may possess a distinct biosynthetic pathway for PDTC production.