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Mutants of Mycobacterium smegmatis unable to grow at acidic pH in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone

Tran, Sieu L.; Rao, Min; Simmers, Cameron; Gebhard, Susanne; Olsson, Karen and Cook, Gregory M. (2005). Mutants of Mycobacterium smegmatis unable to grow at acidic pH in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone. Microbiology, 151(3) pp. 665–672.

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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 microM 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.

Item Type: Journal Article
Copyright Holders: 2005 SGM
ISSN: 1465-2080
Academic Unit/Department: Science > Environment, Earth and Ecosystems
Interdisciplinary Research Centre: Centre for Earth, Planetary, Space and Astronomical Research (CEPSAR)
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Item ID: 33187
Depositing User: Karen Olsson-Francis
Date Deposited: 15 Mar 2012 09:51
Last Modified: 18 Jan 2016 14:01
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