Previous work suggested that the underlying mechanisms by which the Streptococcus mutans ClpXP protease affects virulence traits are associated with accumulation of two orthologues of the Spx regulator, named SpxA and SpxB. Here, a thorough characterization of strains lacking the spx genes (Delta spxA, Delta spxB, and Delta spxA Delta spxB) revealed that Spx, indeed, participates in the regulation of processes associated with S. mutans pathogenesis. The Delta spxA strain displayed impaired ability to grow under acidic and oxidative stress conditions and had diminished long-term viability at low pH. Although the Delta spxB strain did not show any inherent stress-sensitive phenotype, the phenotypes observed in Delta spxA were more pronounced in the Delta spxA Delta spxB double mutant. By using two in vivo models, we demonstrate for the first time that Spx is required for virulence in a Gram-positive pathogen. Microarrays confirmed the global regulatory role of SpxA and SpxB. In particular, SpxA was shown to positively regulate genes associated with oxidative stress, a finding supported by enzymatic assays. SpxB had a secondary role in regulation of oxidative stress genes but appeared to play a larger role in controlling processes associated with cell wall homeostasis. Given the high degree of conservation between Spx proteins of low-GC Gram-positive bacteria, these results are likely to have broad implications Ver menos