FIGURE 4.

Tat-independent export of overproduced YkuE to the cell wall. To overproduce YkuE, a subtilin-inducible copy of the ykuE gene was expressed from the plasmid pNZ8910ykuE-strep in B. subtilis cells expressing the SpaRK two-component regulatory system. YkuE-overproducing cells grown in LB medium were subjected to subcellular fractionation as described for Fig. 1. The presence of YkuE was monitored by Western blotting with specific polyclonal antibodies. The lanes with samples from YkuE (over)producing cells are labeled as follows: 168, B. subtilis 168; AyCy, B. subtilis tatAyCy; AdCd, B. subtilis tatAdCd; total-tat, B. subtilis lacking all tat genes. YkuE-specific protein bands are marked with an arrow.