Fig 4.

Induction of van gene expression by vancomycin is significantly reduced in cells where d-Ala-d-Lac-containing PG precursors are more abundant than d-Ala-d-Ala-containing PG precursors. (A) Relative abundance of the d-Ala-d-Ala-containing PG precursor (UDP-MurNAc-pentapeptide, peak 1, as shown in Fig. 3B) and the d-Ala-d-Lac-containing PG precursor (UDP-MurNAc-pentadepsipeptide, peak 2, as shown in Fig. 3B) in the wild type (H2077), ΔddlA mutant (H2027), and the ΔddlA mutant complemented with ermEp-ddlA (H2009) or ermEp-vanA (H2012) when grown in the presence (gray bars) or absence (white bars) of a 10 mM d-Lac supplement. Cells grown to the mid-late exponential phase in NMMP liquid cultures were extracted and analyzed by HPLC-MS/MS. Relative abundances were calculated by normalizing each sample so that the sum of the two UDP-MurNAC derivatives equaled 100. Strain H2027 (ΔddlA) is viable only in the presence of vancomycin and was therefore grown in NMMP containing 20 μg/ml vancomycin, as indicated by “+Van.” (B) Bioassay for kanamycin resistance induced from the vancomycin-inducible vanJ promoter in the reporter strains. Approximately 10⁵ spores were spotted onto agar plates containing 20 μg/ml vancomycin, kanamycin at a range of concentrations between 0 and 80 μg/ml in 20 μg/ml increments, and in the presence (+) or absence (−) of 10 mM d-Lac. The result was scored after 4 days of incubation at 30°C. (C) Induction of vanH and vanK transcription in ΔddlA mutant complemented with ermEp-ddlA (H2009) and ermEp-vanA (H2012) in response to vancomycin, as determined by qRT-PCR. Each strain was grown to the mid-late exponential phase in two different NMMP liquid media, one with 10 mM d-Lac (+) and one without (−). Samples were then taken immediately before the addition of 10 μg/ml vancomycin (−) and 30 min after treatment (+). Total RNAs were extracted from each sample, and qRT-PCR was carried out as described in Materials and Methods.