Figure 6. Iron and zinc inhibit B. mallei T6SS-1 expression.

Whole cell lysates were prepared from overnight cultures of B. mallei SR1A grown in: (A) M9CG, M9TG or M9TG-C; (B) M9CG alone (none), M9CG supplemented with copper, iron, magnesium, manganese, nickel and zinc (pooled; 10 µM each), M9CG individually supplemented with copper, iron, magnesium, manganese, nickel or zinc (Cu, Fe, Mg, Mn, Ni or Zn; 10 µM each) or; (C) M9CG alone (none), M9CG supplemented with iron and zinc (Fe/Zn, 10 µM each), 2x iron (2x Fe, 20 µM) or 2x zinc (2x Zn, 20 µM); 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. (D) 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. mallei strains grown for 8 h in M9CG (none) or M9CG plus iron and zinc (Fe/Zn; 10 µM each). 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.