Mutants of Mycobacterium smegmatis unable to grow at acidic pH in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone
Open Access
- 1 March 2005
- journal article
- Published by Microbiology Society in Microbiology
- Vol. 151 (3), 665-672
- https://doi.org/10.1099/mic.0.27624-0
Abstract
Mycobacterium smegmatis is able to grow and survive at acidic pH, and exhibits intracellular pH homeostasis under these conditions. In this study, the authors have identified low proton permeability of the cytoplasmic membrane, and high cytoplasmic buffering capacity, as determinants of intrinsic acid resistance of M. smegmatis. To identify genes encoding proteins involved in protecting cells from acid stress, a screening method was developed using the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). CCCP was used to suppress intrinsic acid resistance of M. smegmatis. The screen involved exposing cells to pH 5·0 in the presence of CCCP, and survivors were rescued at various time intervals on solid medium at pH 7·5. Cells capable of responding to intracellular acidification (due to CCCP-induced proton equilibration) will survive longer under these conditions than acid-sensitive cells. From a total pool of 5000 transposon (Tn611) insertion mutants screened, eight acid-sensitive M. smegmatis mutants were isolated. These acid-sensitive mutants were unable to grow at pH 5·0 in the presence of 1–5 μM CCCP, a concentration not lethal to the wild-type strain mc2155. The DNA flanking the site of Tn611 was identified using marker rescue in Escherichia coli, and DNA sequencing to identify the disrupted locus. Acid-sensitive mutants of M. smegmatis were disrupted in genes involved in phosphonate/phosphite assimilation, methionine biosynthesis, the PPE multigene family, xenobiotic-response regulation and lipid biosynthesis. Several of the acid-sensitive mutants were also defective in stationary-phase survival, suggesting that overlapping stress protection systems exist in M. smegmatis.Keywords
This publication has 30 references indexed in Scilit:
- Purification and Biochemical Characterization of the F 1 F o -ATP Synthase from Thermoalkaliphilic Bacillus sp. Strain TA2.A1Journal of Bacteriology, 2003
- Microarray Analysis of the Mycobacterium tuberculosis Transcriptional Response to the Acidic Conditions Found in PhagosomesJournal of Bacteriology, 2002
- Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequenceNature, 1998
- Stability and proton-permeability of liposomes composed of archaeal tetraether lipidsBiochimica et Biophysica Acta (BBA) - Biomembranes, 1994
- Acid-sensitive mutants of Salmonella typhimurium identified through a dinitrophenol lethal screening strategyJournal of Bacteriology, 1994
- Adaptation of Escherichia coli to the uncoupler of oxidative phosphorylation 2,4-dinitrophenolJournal of Bacteriology, 1993
- Modulation of the heat shock response by one-carbon metabolism in Escherichia coliJournal of Bacteriology, 1993
- Immunopathogenesis of Pulmonary TuberculosisHospital Practice, 1993
- Inducible pH homeostasis and the acid tolerance response of Salmonella typhimuriumJournal of Bacteriology, 1991
- Adaptive acidification tolerance response of Salmonella typhimuriumJournal of Bacteriology, 1990