FIGURE 2.

(A) ArgR binds in vitro to the operating sites of argC, argG, arcA and sigB, and activates the argG operon promoter. Gel mobility shift assay (EMSA) experiments were employed to examine binding of the recombinant ArgR from L. monocytogenes (ArgR) and its amino acid mutant protein (ArgR_S42AR43A) to the argC, argG, arcA, and sigB promoter region DNA. The promoter fragments were obtained by PCR with primers specified in Supplementary Table S1, and incubated with recombinant proteins for 30 min at room temperature. Gel retardation by DNA–protein complexes was monitored after ethidium bromide staining. The housekeeping gene gyrB was used as a negative control (NC) for the EMSAs. Arrows indicate DNA-protein complexes. (B,C) ArgR activates the argG operon promoter. The fluorescence intensity was observed by confocal laser scanning microscopy of overnight grown L. monocytogenes wild-type and ArgR mutant strains carrying a gfp3 reporter fused with the promoter of argG, and then stress-treated for an additional 1 h under pH 7.0 and 5.5 conditions (B). Bars represent the relative fluorescence units (RFU) after subtracting the absolute values for the PBS control (C). Data shown represents the Mean ± SD of three independent experiments, each performed in duplicate. ^∗∗P < 0.01 for comparisons between the wild-type and mutant strains.