FIG 5.

metJ deletion and MTA treatment of S. Typhimurium reduce SPI-1 secretion. (A) SPI-1 genes are suppressed in ΔmetJ mutants. RNA was extracted from wild-type S. Typhimurium or ΔmetJ mutant bacterial cultures grown to late log phase in LB broth (n = 5) and analyzed by qPCR, with the ribosomal rrs gene serving as the endogenous control. Each dot represents an independent biological replicate. P values, indicated at the top, were calculated by a one-way analysis of variance with Dunnett's multiple-comparison test. (B) SipA secretion is suppressed in ΔmetJ mutants. The SipA protein was measured by Western blotting of cell lysates or cell-free supernatants collected at late log phase of growth. (C) SipA secretion is suppressed in S. Typhimurium treated with exogenous MTA. SipA protein was measured by Western blotting of cell lysates or cell-free supernatants collected at late log phase of growth from bacteria grown in either 0.5% DMSO or 300 μM MTA. For panels E and F, cell lysates were normalized by the total protein content. Cell-free supernatants were spiked with 100 ng/μl of BSA as a loading control, concentrated by TCA precipitation, and normalized by volume and total protein. Data represent those from three independent experiments and are normalized to wild-type expression. P values were calculated by a one-way analysis of variance with Tukey's multiple-comparison test or a Student's t test. All error bars represent the standard error of the mean.