Molecular characterization of a negative regulator of Streptococcus sobrinus surface protein antigen gene

Mutans streptococci have been shown to give rise to variants in terms of expression of surface protein antigens by repeated subculturing of the organisms, which in turn induces changes in colonial morphologies. A 2,850-bp upstream region of the gene (pag) for a surface protein antigen, PAg, of Streptococcus sobrinus MT3791 was determined. Analysis of the nucleotide sequence revealed the existence of three open reading frames (ORFs) located upstream of the pag gene. ORF1 extended from an undetermined further upstream sequence to the termination codon TAG lying 1,943 bp upstream of the pag gene. ORF2, consisting of 609 bp lying 1,689 bp upstream of the pag gene, encoded a protein of 23,347 Da and a protein of 22,792 Da. The synthesis of these proteins (protein antigen regulators) was demonstrated by using the in vitro T7 RNA polymerase/promoter system. ORF3, extending from 314 bp upstream of the pag gene to 712 bp upstream of the pag gene, encoded a protein of 14,802 Da. Disruption of chromosomal ORF2 of parent strain MT3791 by allelic exchange resulted in isogenic mutants, termed PAREm-1 and PAREm-2, that synthesized larger amounts of cell-free and cell-associated PAg than did the parent strain. RNA dot blot analysis demonstrated that expression of PAg-specific mRNA transcripts by mutants PAREm-1 and PAREm-2 was about 32-fold higher than that by strain 3791. Mutants PAREm-1 and PAREm-2 were found to be more hydrophobic than strain MT3791. Resting cells of these mutants attached in larger numbers to saliva-coated hydroxyapatite than did those of the parent strain. These results suggest that protein antigen regulator regulates the expression of PAg gene in a negative fashion, affecting the colonization of tooth surfaces by the organism. Thus, ORF2 is concluded to be a negative regulator gene of PAg synthesis and was designated par.