Functional and genetic characterization of the tap efflux pump in Mycobacterium bovis BCG.
Efflux pumps extrude a wide variety of chemically unrelated compounds conferring multidrug resistance and participating in numerous physiological processes. Mycobacterium tuberculosis possesses many efflux pumps, and their roles in drug resistance and physiology are actively investigated. In this work we found that tap mutant cells showed changes in morphology and a progressive loss of viability upon subcultivation in liquid medium. Transcriptome analysis in Mycobacterium bovis BCG revealed that disruption of the Rv1258c gene, encoding the Tap efflux pump, led to an extensive change in gene expression patterns during stationary phase, with no changes during exponential growth. In stationary phase, Tap inactivation triggered a general stress response and led to a general repression of genes involved in cell wall biosynthesis, in particular the formation of the peptidoglycan; this suggested the accumulation of an unknown Tap substrate that reaches toxic concentrations during stationary phase. We also found that both disruption and overexpression of tap altered susceptibility to many clinically approved antibiotics in M. bovis BCG. Acriflavine and tetracycline accumulation assays and carbonyl cyanide m-chlorophenylhydrazone (CCCP) potentiation experiments demonstrated that this phenotype was due to an active efflux mechanism. These findings emphasize the important role of the Tap efflux pump in bacterial physiology and intrinsic drug resistance.
Departamento de Microbiología, Medicina Preventiva y Salud Pública, Universidad de Zaragoza, Zaragoza, Spain. ramon@mail.ubc.ca
Santiago
S
Ramón-García
Virginie
V
Mick
Elisa
E
Dainese
Carlos
C
Martín
Charles J
CJ
Thompson
Edda
E
De Rossi
Riccardo
R
Manganelli
José A
JA
Aínsa
D000167:Acriflavine / Q000378:metabolism / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001576:Benzophenoneidum; D015139:Blotting, Southern; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005456:Fluorescent Dyes; D041722:Genes, Transgenic, Suicide; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D046228:Microarray Analysis; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009163:Mycobacterium bovis / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D013752:Tetracycline / Q000378:metabolism / Q000494:pharmacology; D014475:Uncoupling Agents / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D026901:Membrane Transport Proteins; D012329:RNA, Bacterial; C115861:Tap protein, Mycobacterium; D014475:Uncoupling Agents; D000167:Acriflavine; D001576:Benzophenoneidum; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D013752:Tetracycline