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

نویسندگان

  • R M Green
  • A Seth
  • N D Connell
چکیده

Oligopeptides play important roles in bacterial nutrition and signaling. Using sequences from the available genome database for Mycobacterium tuberculosis H37Rv, the oligopeptide permease operon (oppBCDA) of Mycobacterium bovis BCG was cloned from a cosmid library. An opp mutant strain was constructed by homologous recombination with an allele of oppD interrupted by kanamycin and streptomycin resistance markers. The deletion was complemented with a wild-type copy of the opp operon. Two approaches were taken to characterize the peptide transporter defect in this mutant strain. First, growth of wild-type and mutant strains was monitored in media containing a wide variety 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 supporting measurable growth as the sole carbon source in either wild-type or mutant strains. The second approach exploited the resistance of permease mutants to toxic substrates. The tripeptide glutathione (gamma-glutamyl-L-cyteinylglycine [GSH]) is toxic to wild-type BCG and was used successfully to characterize peptide uptake in the opp mutant. In 2 mM GSH, growth of the wild-type strain is inhibited, whereas the opp mutant is resistant to concentrations as high as 10 mM. Similar results were found with the tripeptide S-nitrosoglutathione (GSNO), thought to be a donor of NO in mammalian cells. Using incorporation of [(3)H]uracil to monitor the effects of GSH and GSNO on macromolecular 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 uptake measurements, incorporation of [(3)H]GSH is reduced in the mutant compared with wild type and the complemented mutant. Finally, growth of the three strains in the tripeptides suggests that GSH is bacteriostatic, whereas GSNO is bacteriocidal.

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عنوان ژورنال:
  • Infection and immunity

دوره 68 2  شماره 

صفحات  -

تاریخ انتشار 2000