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 mc2155

Abstract

Transposon mutagenesis of Mycobacterium smegmatis mc2155 enabled the isolation of a mutant strain (called LGM1) altered in the regulation of piperidine and pyrrolidine utilization. The complete nucleotide sequence of the gene inactivated in mutant LGM1 was determined from the wild-type strain. This gene (pipR) encoded a member of the GntR family of bacterial regulatory proteins. An insertion element (IS1096), previously described for M. smegmatis, 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 experiment, it was demonstrated that the cytochrome P450 pipA gene is required for piperidine and pyrrolidine utilization in M. smegmatis mc2155. Genes homologous to pipA were detected by hybridization in several, previously isolated, morpholine-degrading mycobacterial strains. A gene encoding a putative [3Fe-4S] ferredoxin (orf1) and a truncated gene encoding a putative glutamine synthetase (orf2') were found downstream of pipA.

FIG. 1

Reduced CO difference spectra of crude extracts of glucose-grown M. smegmatis cells. Spectrum a, Tn611 insertion mutant LGM1; spectrum b, wild-type, M. smegmatis mc²155 strain. The protein concentrations were 10 mg ml⁻¹.

FIG. 2

Map of the 5.8-kb EcoRI DNA region of M. smegmatis mc²155 containing the piperidine-inducible cytochrome P450 (pipA) and its regulatory gene (pipR). The DNA regions sequenced on both strands are indicated in shaded boxes. The other ORFs shown correspond to the putative resolvase (TnpR) and transposase (TnpA) of IS1096 insertion element and to the putative ferredoxin (ORF1) and glutamine synthetase (truncated ORF2′) of M. smegmatis mc²155. Only the restriction sites mentioned in the text are indicated. The position of Tn611 insertion (triangle) into the pipR gene, determined from sequence analysis, is shown.

FIG. 3

Nucleotide sequence of the 1,215-bp fragment containing the regulatory protein (PipR) gene. The presumptive ribosome binding site aaggaaga (italic), the stop codon (asterisk), and the nucleotides potentially involved in the formation of a stem-loop structure (underlined) are indicated. The GenBank accession number is AF102509. Bases shown in lowercase letters are noncoding sequences.

FIG. 4

Nucleotide sequence of the 2,177-bp SacI-EcoRI region encoding a cytochrome P450 (PipA), a putative ferredoxin (ORF1), and a putative glutamine synthetase (truncated ORF2′). The two sets of possible inverted repeats (underlined) in the putative promoter region of pipA, the putative ribosome binding sites ggagg (italic), and stop codons (asterisks) are indicated. The first 182 nucleotides of this sequence are identical to the end of the IS1096 insertion element (positions 2086 to 2268) except for one nucleotide: the base A, in position 2147 in IS1096, is missing. The GenBank accession number is AF102510. Bases shown in lowercase letters are noncoding sequences.

FIG. 5

Amino acid sequence comparisons of S. griseolus (Fd-1 and Fd-2) and M. smegmatis (Fdpip) ferredoxins. The three cysteine residues, which are supposed to be involved in the attachment of a [3Fe-4S] cluster and are conserved in all three proteins, are indicated in bold. Hyphens indicate gaps introduced to optimize alignments. Residues that are identical in at least two of the three sequences are boxed.