Figure 1: Phenotypic screening of walK^EC-PAS mutants.

(a) No impact on haemolysis was observed for the WalK^D119A mutation in either Newman or NRS384 on sheep blood agar. (b) Growth kinetics were identical for the newly created WalK^D119A mutants in TSB at 37 °C with aeration (200 r.p.m.) when compared with the parental strain. Optical density (○) and colony-forming units (□) were enumerated. (c) In a lysostaphin growth sensitivity assay, the UoC WalK^D119A exhibited a loss of viability, while the parent or UoM WalK^D119A did not. The WalK^D119A mutation in the NRS384 background enhanced sensitivity to lysostaphin when compared with the parental strain. Error bars depict the s.d. of the mean from three independent experiments. (d) P1 sae promoter activity with a DsRED reporter. Left: no promoter. Right: P1 sae driving RFP expression; (1) Newman (2) UoM WalK^D119A (3) UoC WalK^D119A. No expression of sae was observed in UoC WalK^D119A. (e) By allelic exchange, the UoC saeS mutation was introduced into Newman or UoM WalK^D119A (saeS^STOP) with haemolysis abolished, while introduction of the wild-type saeS gene into UoC WalK^D119A (saeS^FIX) restored haemolysis.