Hydrogen peroxide (H(2)O(2)), an important substance produced by many members of the genus Streptococcus, plays important roles in virulence and antagonism within a microbial community such as oral biofilms. The spxB gene, which encodes pyruvate oxidase, is involved in H(2)O(2) production in many streptococcal species. However, knowledge about its regulation and relation with other genes putatively involved in the same pathway is limited. In this study, three genes--ackA, spxR and tpk--were identified as contributing to H(2)O(2) production in Streptococcus sanguinis by screening mutants for opaque colony appearance. Mutations in all three genes resulted in significant decreases in H(2)O(2) production, with 16-31% of that of the wild-type. H(2)O(2) production was restored in the complemented strains. Antagonism against Streptococcus mutans by these three S. sanguinis mutants was reduced, both on plates and in liquid cultures, indicating the critical roles of these three genes for conferring the competitive advantage of S. sanguinis. Analysis by qPCR indicated that the expression of spxB was decreased in the ackA and spxR mutants and significantly increased in the tpk mutant.