FIG 5.
tssA1 was repressed by NrtR directly. (A, C) Real-time qPCR assay. (A) The relative mRNA levels of tssA1 in the PAK, ΔnrtR, and ΔnrtR complement strains. (C) The relative mRNA levels of egfp in BL21 containing both PtssA1-EGFP (egfp gene driven by tssA1 promoter) and pET28a or pET28a-nrtR plasmids. Total RNA was isolated from bacteria at an OD600 of 1.0, and the indicated mRNA levels were examined by real-time qPCR using rpsL as an internal control. **, P < 0.01; ***, P < 0.001, by Student's t test. (B, D) Western blot assay. PAK and ΔnrtR containing a PtssA1-EGFP plasmid (B) or BL21 containing both PtssA1-EGFP and pET28a or pET28a-nrtR plasmids (D) were grown to an OD600 of 1.0 in LB medium. Proteins from an equivalent number of bacterial cells of the indicated strains were separated on a 12% SDS-PAGE and probed with an antibody against EGFP or RpoA. Relative densities represent the density of EGFP/density of RpoA with the first lane as 1.