Regulatory circuits controlling Spx levels inStreptococcus mutans

Spx is a major regulator of stress responses in Firmicutes. InStreptococcus mutans, two Spx homologues, SpxA1 and SpxA2, were identified as mediators of oxidative stress responses but the regulatory circuits controlling their levels and activity are presently unknown. Comparison of SpxA1 and SpxA2 protein sequences revealed differences at the C-terminal end, with SpxA1 containing an unusual number of acidic residues. Here, we showed that a green fluorescence protein (GFP) reporter becomes unstable when fused to the last 10 amino acids of SpxA2 but remained stable when fused to the C-terminal acidic tail of SpxA1. Inactivation ofclpPor simultaneous inactivation ofclpCandclpEstabilized the GFP::SpxA2(tail)fusion protein. Addition of acidic amino acids to the GFP::SpxA2(tail)chimera stabilized GFP, while deletion of the acidic residues destabilized GFP::SpxA1(tail). Promoter reporter fusions revealed thatspxA1transcription is co-repressed by the metalloregulators PerR and SloR whilespxA2transcription is largely dependent on the envelope stress regulator LiaFSR. In agreement withspxA2being part of the LiaR regulon, SpxA2 was found to be critical for the growth ofS. mutansunder envelope stress conditions. Finally, we showed that redox sensing is essential for SpxA1-dependent activation of oxidative stress responses but dispensable for SpxA2-mediated envelope stress responses.