The objective of the present study was to investigate whether Streptococcus sanguinis SpxA2 plays a role in competence development and endogenous H2O2 generation, and whether the SpxA2 Cys10-XX-Cys13 (CXXC) motif is involved in competence development.Methodology.
The competence development of wild-type S. sanguinis (SK36) and its derivatives was compared by transformation efficiency assay and real-time RT-PCR. The spx allele mutants, spxA2 (C10A) and spxA2 (C13A), were constructed by site-directed mutagenesis. The Δpox mutant was treated with 1 mM H2O2 to exclude the effect of other Pox products on competence development.Results.
Compared with the wild-type (4.42±0.58×10−4), the ΔspxA2 mutant showed decreased transformation efficiency (0.07±0.03×10−4). Furthermore, there was a 2- to 15-fold reduction in ΔspxA2 mutant com gene expression. SpxA2 was able to down-regulate endogenous H2O2 generation by repressing pox expression. Additionally, endogenous H2O2 negatively regulated competence without affecting spxA2 expression. The Δpox mutant increased com gene expression (2- to 8-fold), but the 1 mM H2O2-treated Δpox mutant showed decreased com gene expression. Interestingly, the ΔspxA2Δpox mutant showed enhanced competence-associated parameters. The fact that spxA2 (C10A) and spxA2 (C13A) behaved like the ΔspxA2 mutant revealed the role of the CXXC motif in competence development.Conclusion.
Although the intricate relationship between SpxA2, pox-mediated H2O2 production and competence development was clarified in S. sanguinis, it would be worthwhile to explore further whether H2O2 is involved in competence development through oxidizing the SpxA2 CXXC motif.