Fig 2.

Saturation of the d-Ala-d-Ala termini of immature PG by pretreatment of cells with (A) vancomycin or (B) teicoplanin prevents the sensing of vancomycin by VanS. S. coelicolor M600 cells were grown to the mid-late exponential phase in NMMP liquid medium and pretreated at time zero by addition of 10 μg/ml vancomycin (A) or 50 μg/ml teicoplanin (B), as indicated by the vertical arrows. Samples were taken at the intervals shown before addition of a further 10 μg/ml vancomycin (arrowed) after 90 min (A) or 30 min (B). RNA was extracted from each sample, and vanH transcript abundance was determined by S1 nuclease protection. Transcription of sigE is shown as a positive control for cell viability and RNA integrity and as an indicator of cell wall stress. Where shown, “V” represents vancomycin and “T” indicates teicoplanin.