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

Publication type:
Journal article
Metadata:
Autoren
  • SL Tran
  • M Rao
  • C Simmers
  • S Gebhard
  • K Olsson
  • GM Cook
Autoren-URL
https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=fis-test-1&SrcAuth=WosAPI&KeyUT=WOS:000227707100006&DestLinkType=FullRecord&DestApp=WOS_CPL
DOI
10.1099/mic.0.27624-0
eISSN
1465-2080
Externe Identifier
  • Clarivate Analytics Document Solution ID: 907HM
  • PubMed Identifier: 15758213
ISSN
1350-0872
Zeitschrift
MICROBIOLOGY-SGM
Paginierung
665 - 672
Datum der Veröffentlichung
2005
Status
Published
Titel
Mutants of <i>Mycobacterium smegmatis</i> unable to grow at acidic pH in the presence of the protonophore carbonyl cyanide <i>m</i>-chlorophenylhydrazone
Sub types
  • Article
Ausgabe der Zeitschrift
151

Data source: Web of Science (Lite)

Other metadata sources:
Abstract
<jats:p> <jats:italic>Mycobacterium smegmatis</jats:italic> 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 <jats:italic>M. smegmatis</jats:italic>. To identify genes encoding proteins involved in protecting cells from acid stress, a screening method was developed using the electrogenic protonophore carbonyl cyanide <jats:italic>m</jats:italic>-chlorophenylhydrazone (CCCP). CCCP was used to suppress intrinsic acid resistance of <jats:italic>M. smegmatis</jats:italic>. 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 (Tn<jats:italic>611</jats:italic>) insertion mutants screened, eight acid-sensitive <jats:italic>M. smegmatis</jats:italic> 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 mc<jats:sup>2</jats:sup>155. The DNA flanking the site of Tn<jats:italic>611</jats:italic> was identified using marker rescue in <jats:italic>Escherichia coli</jats:italic>, and DNA sequencing to identify the disrupted locus. Acid-sensitive mutants of <jats:italic>M. smegmatis</jats:italic> 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 <jats:italic>M. smegmatis</jats:italic>.</jats:p>
Autoren
  • Sieu L Tran
  • Min Rao
  • Cameron Simmers
  • Susanne Gebhard
  • Karen Olsson
  • Gregory M Cook
DOI
10.1099/mic.0.27624-0
eISSN
1465-2080
ISSN
1350-0872
Ausgabe der Veröffentlichung
3
Zeitschrift
Microbiology
Sprache
en
Paginierung
665 - 672
Datum der Veröffentlichung
2005
Status
Published
Herausgeber
Microbiology Society
Herausgeber URL
http://dx.doi.org/10.1099/mic.0.27624-0
Datum der Datenerfassung
2020
Titel
Mutants of Mycobacterium smegmatis unable to grow at acidic pH in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone
Ausgabe der Zeitschrift
151

Data source: Crossref

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 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.
Addresses
  • Department of Microbiology, Otago School of Medical Sciences, University of Otago, PO Box 56, Dunedin, New Zealand.
Autoren
  • Sieu L Tran
  • Min Rao
  • Cameron Simmers
  • Susanne Gebhard
  • Karen Olsson
  • Gregory M Cook
DOI
10.1099/mic.0.27624-0
eISSN
1465-2080
Externe Identifier
  • PubMed Identifier: 15758213
Open access
false
ISSN
1350-0872
Ausgabe der Veröffentlichung
Pt 3
Zeitschrift
Microbiology (Reading, England)
Schlüsselwörter
  • Mycobacterium smegmatis
  • Carbonyl Cyanide m-Chlorophenyl Hydrazone
  • Bacterial Proteins
  • DNA Transposable Elements
  • Ionophores
  • Culture Media
  • Mutagenesis, Insertional
  • Cell Membrane Permeability
  • Gene Expression Regulation, Bacterial
  • Mutation
  • Hydrogen-Ion Concentration
Sprache
eng
Medium
Print
Paginierung
665 - 672
Datum der Veröffentlichung
2005
Status
Published
Datum der Datenerfassung
2005
Titel
Mutants of Mycobacterium smegmatis unable to grow at acidic pH in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone.
Sub types
  • Research Support, Non-U.S. Gov't
  • Journal Article
Ausgabe der Zeitschrift
151

Data source: Europe PubMed Central

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 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.
Autoren
  • Sieu L Tran
  • Min Rao
  • Cameron Simmers
  • Susanne Gebhard
  • Karen Olsson
  • Gregory M Cook
Autoren-URL
https://www.ncbi.nlm.nih.gov/pubmed/15758213
DOI
10.1099/mic.0.27624-0
ISSN
1350-0872
Ausgabe der Veröffentlichung
Pt 3
Zeitschrift
Microbiology (Reading)
Schlüsselwörter
  • Bacterial Proteins
  • Carbonyl Cyanide m-Chlorophenyl Hydrazone
  • Cell Membrane Permeability
  • Culture Media
  • DNA Transposable Elements
  • Gene Expression Regulation, Bacterial
  • Hydrogen-Ion Concentration
  • Ionophores
  • Mutagenesis, Insertional
  • Mutation
  • Mycobacterium smegmatis
Sprache
eng
Country
England
Paginierung
665 - 672
Datum der Veröffentlichung
2005
Status
Published
Datum, an dem der Datensatz öffentlich gemacht wurde
2005
Titel
Mutants of Mycobacterium smegmatis unable to grow at acidic pH in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone.
Sub types
  • Journal Article
  • Research Support, Non-U.S. Gov't
Ausgabe der Zeitschrift
151

Data source: PubMed

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