FIG 5.

GFP secretion from B. subtilis Tat deletion strains. (A) Secretion of total GFP in single or double Tat mutants. The indicated strains (in a Δ7 3610 background) were grown overnight in HPDM supplemented with 1 mM IPTG. The next day, the cells were washed and resuspended in LPDM supplemented with 1 mM IPTG. At the indicated time points, the cells were lysed and the medium was precipitated with TCA. The cell lysates and precipitated medium were examined by Western blot probing with α-GFP and α-SigA antibodies. (B) Secretion of folded GFP. The indicated strains were grown overnight as described for panel A. At the indicated time points, the medium was harvested and examined for GFP fluorescence. Relative fluorescent units were normalized to account for differences in cell density. Results from one representative experiment are shown. (C) Expression and secretion of endogenous PhoD in the single and double Tat mutants. Samples were prepared as described for panel A. The migration of molecular mass standards is indicated to the right of each blot. (D) Secretion of total GFP in a quintuple Tat mutant in a Δ7 3610 background. Samples were prepared as described for panel A. The cell lysates and precipitated medium were examined by Western blot probing with α-GFP, α-PhoD, and α-SigA antibodies.