tolC is Required for the Activity of a Tetracycline Efflux Pump (tetA)

DE CRISTÓBAL, R.E; VINCENT P.A; SALOMÓN R.A

Iguazú-Misiones del 5 al 8 de Diciembre de 2004

Congreso; XL Reunión Nacional SAIB; 2004

Sociedad Argentina de Investigaciones Bioquímicas y Biología Molecular

Tetracycline (Tc) is an antibiotic which inhibits bacterial growth through the inhibition of protein synthesis. One mechanism of resistance among pathogenic strains are Tc efflux pumps which maintain the intracellular concentration at a low level. One of these is encoded by tetA gene of transposon Tn10. We observed that the E coli strain MC4100 tolC::Tn10 grew poorly, forming small colonies in LB medium with 15 mg/ml of Tc. The tolC mutation in this strain decreased the MIC for Tc by 7 fold, compared with MC4100 thr::Tn10. This reduction in the MIC was also observed both in other tolC mutants with a Tn10 insertion elsewhere and MC4100 tolC::Tn5 transformed with pBR322 (TcR). The reduction in the MIC was, in all cases, reverted by  plasmid pAX629 carrying wild type tolC gene. We next investigated whether inhibition of the TetA efflux pump was responsible for the enhanced Tc activity on these bacterial strains. Spectrofluorometric analyses suggested an efflux inhibition. Moreover, total accumulation of Tc was increased. On the other hand, this Tc sensitivity phenotype was also seen with another Tc derivative, chlortetracycline (CTc). All tolC mutants failed to form colonies in LB medium with a low concentration (10 mg/ml) of CTc. Even autoclaved CTc (50 mg/ml) inhibited grow. Again, this phenotype was reverted by pAX629. Based on these results,we conclude that TolC is required for the activity of the tetracycline efflux pump TetA.