FIG 5.

Characterization of GlnH as a lipoprotein. (A to C) Western blot analysis of crude extracts of C. glutamicum ΔglnH/pAN6-glnH (A and B, lanes 1 to 4; C, lane 2) and C. glutamicum ΔglnH/pAN6-glnH-C27A (C, lane 1). The plasmid-encoded GlnH contains a C-terminal StrepTag-II, which was detected by Streptactin-alkaline phosphatase conjugate. Panel A shows the control samples without globomycin, and panel B shows the samples where globomycin was added during exponential growth. Samples were taken immediately before (A and B, lane 1) and 1 h, 2 h, and 3 h after globomycin addition (A and B lanes 2, 3, and 4). In panel C, a comparison of the GlnH-C27A variant (lane 1) and wild-type GlnH (lane 2) is shown. Mutated GlnH-C27A cannot be modified by prolipoprotein diacylglycerol transferase or cleaved by the lipoprotein signal peptidase. In panels A and B, 10 μg protein was applied per lane. In panel C, 50 μg protein of crude extract was applied in lane 1 and 5 μg membrane protein was applied in lane 2. Lane S shows the molecular mass standards. The lanes in panels A, B, and C were derived from a single blot in each case and arranged for better visibility of the relevant features.