Figure 1. Regulation of hmsCDE by Rcs.

(a) β-galactosidase activities of hmsC::lacZ reporter in Y. pestis. Y. pestis KIM6+ (WT) transformed with empty vector (vector) and functional RcsA (p-rcsA), RcsB deletion mutant transformed with empty vector (vector), functional RcsA (p-rcsA), wild-type RcsB (p-rcsB), inactive RcsB (p-rcsB (D56Q)) and active RcsB (p-rcsB (D56E)). (b) mRNA levels of hmsD regulated by Rcs in Y. pestis. hmsD mRNA levels were determined by qRT- PCR (Methods), and normalized to wild type. The mean and standard deviation of three independent experiments with three replicates are indicated. *P < 0.05. (c) Expression of HmsD regulated by Rcs in Y. pestis. Western blots of total protein-matched lysates prepared from stationary phase LB cultures and probed with anti-Myc antibody. Levels of HmsD were quantitated by densitometry using ImageJ from at least two independent experiments: numbers below blots indicate the ratio of HmsD from the indicated strain compared to that from wild-type hmsD-Myc₂ strain (WT). ns, none specific band. Strain designations (Supplementary Table S1) are: Control, KIM6+ without Myc tag; WT, hmsD-Myc₂; rcsA+, functional rcsA hmsD-Myc₂; ΔrcsB, ΔrcsB hmsD-Myc₂; ΔrcsB Vector, ΔrcsB hmsD-Myc₂/pUC19; ΔrcsB p-rcsB, ΔrcsB hmsD-Myc₂/pYC332; ΔrcsD Vector, ΔrcsD-N-terminal hmsD- Myc₂/pET-32a; ΔrcsD p-rcsD, ΔrcsD-N-terminal hmsD- Myc₂/pYC225. Full-length blots are presented in Supplementary Fig. S5 online.