A peptide permease mutant of Mycobacterium bovis BCG resistant to the toxic peptides glutathione and S-nitrosoglutathione

Oligopeptides play important roles in bacterial nutrition and signaling. Us ing sequences from the available genome database for Mycobacterium tubercul osis H37Rv, the oligopeptide permease operon (oppBCDA) of Mycobacterium bov is BCG was cloned from a cosmid library. An opp mutant strain was construct ed by homologous recombination with an allele of oppD interrupted by kanamy cin and streptomycin resistance markers, The deletion was complemented with a wild-type copy of the opp operon, Two approaches were taken to character ize the peptide transporter defect in this mutant strain. First, growth of wild-type and mutant strains was monitored in media containing a wide varie ty of peptides as sole source of carbon and/or nitrogen. Among 25 peptides ranging from two to six amino acids in length, none was capable of supporti ng measurable growth as the sole carbon source in either wild-type or mutan t strains. The second approach exploited the resistance of permease mutants to toxic substrates. The tripeptide glutathione (gamma-glutamyl-L-cyteinyl glycine [GSH]) is toxic to wild-type BCG and was used successfully to chara cterize peptide uptake in the opp mutant. In 2 mM GSH, growth of the wild-t ype strain is inhibited, whereas the opp mutant is resistant to concentrati ons as high as 10 mM, Similar results were found with the tripeptide S-nitr osoglutathione (GSNO), thought to be a donor of NO in mammalian cells, Usin g incorporation of [H-3] uracil to monitor the effects of GSH and GSNO on m acromolecular synthesis in growing cells, it was demonstrated that the opp mutant is resistant, whereas the wild type and the mutant complemented with a wild-type copy of the operon are sensitive to both tripeptides, In uptak e measurements, incorporation of [H-3]GSH is reduced in the mutant compared with wild type and the complemented mutant. Finally, growth of the three s trains in the tripeptides suggests that GSH is bacteriostatic, whereas GSNO is bacteriocidal.