FIG. 5.

Complementation of the ΔccaR::tsrA1 mutant with pSET152 constructs. Western blot analysis and bioassay for production of cephamycin C were used to assess complementation. The designations A and B represent two independent clones generated during the same transformation. Five micrograms of cell extract protein from each strain, harvested after 48 h of growth, was separated by SDS-PAGE (10% gel) and transferred to PVDF membranes. The resulting Western blots were developed with polyclonal antibodies specific for the CcaR, LAT, IPNS, and DAOCS proteins. Each strain’s ability to produce cephamycin (Ceph) C was determined by bioassay, and the results were scored as + (production) and − (lack of production).