Figure 7. B. pseudomallei T6SS-1 expression is suppressed by iron and zinc.

(A) Whole cell lysates were prepared from overnight cultures of B. pseudomallei K96243 and 1026b grown in M9CG alone (none), M9CG supplemented with copper, iron, magnesium, manganese, nickel and zinc (pooled; 10 µM each), or M9CG individually supplemented with copper, iron, magnesium, manganese, nickel or zinc (Cu, Fe, Mg, Mn, Ni or Zn; 10 µM each), and then assayed for Hcp1 production by Western immunoblotting using anti-BmHcp1 polyclonal rat serum. The protein band corresponding to Hcp1 is indicated by the black arrowheads. (B) Transcript levels of bimA, tssA, hcp1, virG, tssM and dnaK were determined by qRT-PCR using gene specific primers and dual-labeled Taqman probes. RNA was harvested from B. pseudomallei K96243 grown for 8 h in M9CG (none), M9CG plus iron and zinc (Fe/Zn; 10 µM each), M9CG plus iron (Fe; 10 µM), or M9CG plus zinc (Zn; 10 µM). Relative mRNA levels were calculated using the ΔΔC_T method and represent fold changes in comparison to M9CG. All values have been normalized to the internal control, rpoA. Results represent the means and standard deviations of three independent experiments performed in triplicate.