Development of Genetic Tools for Analysis of Streptococcus sanguinis

Objectives: Streptococcus sanguinis is a member of the normal oral flora and a common cause of infective endocarditis (IE). Completion of the genome sequence of S. sanguinis SK36 necessitates tools for further characterization of this species. It is often desirable to insert antibiotic resistance markers and other exogenous genes into the chromosome; therefore, we sought to identify a chromosomal site for ectopic expression of foreign genes, and verify that insertion into this site did not affect important cellular phenotypes. Methods: An erm resistance gene controlled by a synthetic promoter was cloned into a genomic region encoding only a small (65aa) hypothetical protein to create strain JFP36. To determine whether this insertion affected important cellular properties, SK36 and JFP36 were compared for: (i) growth, by measuring growth curves in a microplate reader; (ii) competence, by measuring transformation frequency and efficiency; (iii) biofilm formation using a standard crystal violet staining assay; and (iv) virulence for endocarditis in the rabbit model of IE. Results: Insertion of erm did not affect growth or competence of JFP36. Average transformation frequencies for JFP36 and SK36 were 27.13% and 27.38%, respectively, while transformation efficiencies were 8.25x10⁷ and 8.6 x10⁷ CFU/µg DNA. Biofilm formation of both strains was similar whether glucose (P=0.2464) or sucrose (P=0.1145) was provided as a carbon source. Virulence in the rabbit model of IE, as assessed by recovery of bacteria from heart valves of co-inoculated rabbits, was similar (P=0.5237), indicating that virulence was not significantly affected. Conclusions: Our results suggest that insertion of the erm gene does not alter in vitro or in vivo phenotypes that typify S. sanguinis SK36; therefore, this chromosomal site is a good candidate for further manipulations of S. sanguinis. We are currently developing strains with other alterations in this region. Supported by NIH grants R01AI47841 and K02AI054908 (TK).