Two Novel Point Mutations in Clinical Staphylococcus aureus Reduce Linezolid Susceptibility and Switch on the Stringent Response to Promote Persistent Infection
A) Alignment of the N-terminal Rsh domains of RelA/SpoT from S. mutans (Rsh_SM) and RelA from S. aureus JKD6210 (Rsh_SA). The triangles are regions shown by Hogg et al.  that-when mutated-affect hydrolase function. Indicated by star and grey shading is the F128Y amino acid substitution that occurs in SCV JKD6229 (Rsh_SA_SCV). B and C) Analysis of ppGpp levels in JKD6210 (MRSA), JKD6229 (SCV) and JKD6301 (JKD6210 with relA F128Y mutation) using the fluorescent chemosensor PyDPA . B) Five, two-fold serial dilutions (1/2–1/32) of test strains demonstrate increased ppGpp levels by increased fluorescence in JKD6229 and JKD6301 compared with the parental strain JKD6210. Control 1 is JKD6210 exposed to serine hydroxymate; control 2 is JKD6210 without the addition of PyDPA; control 3 is buffer alone with PyDPA added. (C) Results confirmed in a 96-well plate format, analysed with a fluorescent plate reader. Results are presented as the mean±SD of biological replicates with a significant increase in fluorescence found for JKD6229 and JKD6301 compared to the parental strain JKD6210 (P<0.0001).