INSERTIONAL INACTIVATION OF BINDING DETERMINANTS OF STREPTOCOCCUS-CRISTA CC5A USING TN916

Intermicrobial binding plays an important role in the ecology of the oral cavity because it represents one mechanism by which specific bacteria colonize dental plaque. The formation of ''corncobs'', a morphologically distinct microbial unit composed of Streprococcus crista and Fusobacterium nucleatum, is a highly specific binding interaction that depends on the presence of polar tufts of fimbriae on the streptococci. We have used a genetic approach to examine the role of streptococcal cell surface components involved in the binding of S. crista to F. nucleatum. Such binding may be an important component of corncob formation. A method for the genetic transformation of S. crista was used to transfer the broad host range transposon, Tn916, into the bacteria. Cells were grown to early log phase in brain heart infusion broth containing 10% fetal calf serum. The competent cells were mixed with purified DNA from pDL916, a plasmid construct consisting of Tn916 and the streptococcal/Escherichia coli shuttle vector pDL278. Over 300 transformants were screened for a reduction in binding to F. nucleatum. Five of the transformants showed a change in binding ranging from 59% to 29% of the positive control values, Southern blots revealed that the binding-deficient transformants contained the Tn916 element integrated into one of 4 different sites in the chromosome, The transposon, integrated into 4 different sites, appeared to be stable in the absence of selective pressure. Based on these findings, it appears that some strains of S. crista are naturally competent and that insertional inactivation methods can be used to facilitate the study of binding receptors in this group of oral streptococci.