To examine whether the system also enables the controlled expression of a secreted heterologous protein, we placed the gene encoding the C. perfringens β-toxin (cpb) behind the abrB promoter and combined this construct with the strain carrying the spo0A mutation (Δspo0A) and the inducible Spo0A*.
The full coding sequence for β-toxin (cpb
) was amplified by PCR with plasmid pXB10 as a template. C. perfringens
β-toxin is a secreted protein with a Sec-type signal sequence and is an important component in animal vaccines against C. perfringens
types B and C (7
To visualize β-toxin secretion, total medium proteins were 10× concentrated by trichloroacetic acid precipitation and separated by SDS-PAGE as described previously (14
). β-Toxin was detected using Western blotting as described previously (7
). As shown in Fig. , at high IPTG induction levels, no β-toxin could be detected. Upon derepression from Spo0A*, β-toxin accumulated in the growth medium. These results show the versatility of the derepression system and demonstrate that (heterologous) gene expression can be accurately controlled.
FIG. 3. Controlled secretion of β-toxin via derepression. abrB derepression results in reduced extracellular proteolytic activity. (A) Detection of 35-kDa β-toxin secreted by strain 0A*/Δ0A/A-β (PabrB-cpb Pspac-spo0A* (more ...)
To examine whether the described derepression system can be activated in cultures with a high cellular density, which is required when producing toxic products, we grew strains A-β (PabrB-cpb) and A-β/0A*/Δ0A (PabrB-cpb Pspac-spo0A* Δspo0A) to dense cultures in TY medium containing 250 μM IPTG (full repression). Next, cells were spun down, washed once, and resuspended in fresh TY medium without IPTG. Medium fractions were collected at timely intervals and assayed for β-toxin secretion. As shown in Fig. , within 20 min after resuspension, PabrB was derepressed in A-β/0A*/Δ0A and β-toxin could be detected in the growth medium. β-Toxin continued to accumulate in the medium up to 2 h after derepression in this dense culture, and we were able to recover β-toxin until 5.5 h after suspension. In the presence of a functional spo0A gene, however, secreted protein could not be observed after 3.5 h.