Natural genetic transformation of Streptococcus mutans growing in biofilms

Streptococcus mutans is a bacterium that has evolved to be dependent upon a biofilm "lifestyle" for survival and persistence in its natural ecosystem, dental plaque. We initiated this study to identify the genes involved in t he development of genetic competence in S. mutans and to assay the natural genetic transformability of biofilm-grown cells, Using genomic analyses, we identified a quorum-sensing peptide pheromone signaling system similar to those previously found in other streptococci, The genetic locus of this sys tem comprises three genes, comC, comD, and comE, that encode a precursor to the peptide competence factor, a histidine kinase, and a response regulato r, respectively. We deduced the sequence of comC and its active pheromone p roduct and chemically synthesized the corresponding 21-amino-acid competenc e-stimulating peptide (CSP). Addition of CSP to noncompetent cells facilita ted increased transformation frequencies, with typically Ire of the total c ell population transformed. To further confirm the roles of these genes in genetic competence, we inactivated them by insertion-duplication mutagenesi s or allelic replacement followed by assays of transformation efficiency, W e also demonstrated that biofilm-grown S. mutans cells were transformed at a rate 10- to 600-fold higher than planktonic S, mutans cells. Donor DNA in cluded a suicide plasmid, S. mutans chromosomal DNA harboring a heterologou s erythromycin resistance gene, and a replicative plasmid, The cells were o ptimally transformed during the formation of 8- to 16-h-old biofilms primar ily consisting of microcolonies on solid surfaces. We also found that dead cells in the biofilms could act as donors of a chromosomally encoded antibi otic resistance determinant. This work demonstrated that a peptide pheromon e system controls genetic competence in S. mutans and that the system funct ions optimally when the cells are living in actively growing biofilms.