Cloning and characterization of the genes encoding a cytochrome P450 (PipA) involved in piperidine and pyrrolidine utilization and its regulatory protein (PipR) in Mycobacterium smegmatis mc(2)155

Transposon mutagenesis of Mycobacterium smegmatis mc(2)155 enabled the isol ation of a mutant strain (called LGM1) altered in the regulation of piperid ine and pyrrolidine utilization. The complete nucleotide sequence of the ge ne inactivated in mutant LGM1 was determined from the wild-type strain. Thi s gene (pipR) encoded a member of the GntR family of bacterial regulatory p roteins. An insertion element (IS1096), previously described for M. smegmat is, was detected downstream of the gene pipR. Three additional open reading frames were found downstream of IS1096. The first open reading frame (pipA ) appeared to encode a protein identified as a cytochrome P450 enzyme. This gene is the first member of a new family, CYP151. By a gene replacement ex periment, it was demonstrated that the cytochrome P450 pipA gene is require d for piperidine and pyrrolidine utilization in M. smegmatis mc2155. Genes homologous to pipA were detected by hybridization in several, previously is olated, morpholine-degrading mycobacterial strains. A gene encoding a putat ive [3Fe-4S] ferredoxin (orf1) and a truncated gene encoding a putative glu tamine synthetase (orf2') were found downstream of pipA.