SpxA1 involved in hydrogen peroxide production, stress tolerance and endocarditis virulence in Streptococcus sanguinis.

Streptococcus sanguinis is one of the most common agents of infective endocarditis. Spx proteins are a group of global regulators that negatively or positively control global transcription initiation. In this study, we characterized the spxA1 gene in S. sanguinis SK36. The spxA1 null mutant displayed opaque colony morphology, reduced hydrogen peroxide (H(2)O(2)) production, and reduced antagonistic activity against Streptococcus mutans UA159 relative to the wild type strain. The ΔspxA1 mutant also demonstrated decreased tolerance to high temperature, acidic and oxidative stresses. Further analysis revealed that ΔspxA1 also exhibited a ∼5-fold reduction in competitiveness in an animal model of endocarditis. Microarray studies indicated that expression of several oxidative stress genes was downregulated in the ΔspxA1 mutant. The expression of spxB and nox was significantly decreased in the ΔspxA1 mutant compared with the wild type. These results indicate that spxA1 plays a major role in H(2)O(2) production, stress tolerance and endocarditis virulence in S. sanguinis SK36. The second spx gene, spxA2, was also found in S. sanguinis SK36. The spxA2 null mutant was found to be defective for growth under normal conditions and showed sensitivity to high temperature, acidic and oxidative stresses.

Identification of hydrogen peroxide production-related genes in Streptococcus sanguinis and their functional relationship with pyruvate oxidase.

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.

Genome-wide essential gene identification in Streptococcus sanguinis.

A clear perception of gene essentiality in bacterial pathogens is pivotal for identifying drug targets to combat emergence of new pathogens and antibiotic-resistant bacteria, for synthetic biology, and for understanding the origins of life. We have constructed a comprehensive set of deletion mutants and systematically identified a clearly defined set of essential genes for Streptococcus sanguinis. Our results were confirmed by growing S. sanguinis in minimal medium and by double-knockout of paralogous or isozyme genes. Careful examination revealed that these essential genes were associated with only three basic categories of biological functions: maintenance of the cell envelope, energy production, and processing of genetic information. Our finding was subsequently validated in two other pathogenic streptococcal species, Streptococcus pneumoniae and Streptococcus mutans and in two other gram-positive pathogens, Bacillus subtilis and Staphylococcus aureus. Our analysis has thus led to a simplified model that permits reliable prediction of gene essentiality.