FIGURE 4.

Fragmentation of BlaR1 in CBAP-induced NRS128 and in E. coli and the shedding of its sensor domain in antibiotic-induced S. aureus NRS128. A, full-length BlaR1 in CBAP-induced NRS128 (right lane) and in E. coli (left lane) have comparable sizes, with that in E. coli larger by the size of the His₆ tag moiety. BlaR1 immunoprecipitated from CBAP-induced NRS128 and from E. coli was separated by SDS-PAGE (10% gel), transferred to PVDF membrane, and detected by Western blot using the anti-BlaR^S antibodies and the ONE-HOUR IP-Western^TM kit (GenScript). B, recombinant BlaR1 (∼60-kDa) obtained from E. coli whole-cell extracts by means of the preparative purification for Edman degradation, visualized in the Coomassie-stained PVDF membrane. Numbers on the left of A and B indicate the position of migration of the molecular mass markers (kDa). C, detection of the C-terminal domain of BlaR1 (BlaR^S or BlaR1_332–585) in media of the S. aureus NRS128 culture after induction with different β-lactam antibiotics (CBAP, PEN, AMP, and OXA) for different times (15 min, 30 min, 1 h, and 3 h). The results for the three other strains are given in the supplemental material. The proteins were separated by SDS-PAGE (12% gels) and transferred to nitrocellulose membrane, and BlaR1 was detected by Western blot using antibodies specific to recombinant BlaR^S and the ONE-HOUR IP-Western^TM kit. Numbers on the right indicate the position of migration of the molecular mass markers (kDa).