The pac gene of the serotype c strain Streptococcus mutans MT8148 encodes a cell surface protein antigen (PAc) of approximate 190 kilodaltons. The serotype c strain S. mutans GS-5 does not produce the 190-kilodalton PAc but produces a lower-molecular-weight protein that reacts with anti-PAc serum. The SphI-BamHI fragment of the pac gene was ligated with the S. mutans-Escherichia coli shuttle vector pSA3. The chimeric shuttle vector was transformed into strain GS-5, and two transformants (TK15 and TK18) were isolated. These transformants produced a large amount of cell-free and cell-bound PAc of 190 kilodaltons. No plasmid was isolated from these transformants, and the EcoRI fragments of their chromosomal DNA hybridized with the erythromycin resistance gene in the shuttle vector DNA, indicating insertion of the chimeric shuttle vector DNA into the chromosomal DNA. The cell hydrophobicity of strains TK15 and TK18 as well as PAc-defective mutants constructed by inserting an erythromycin resistance gene into the pac gene of strain MT8148 was analyzed. Strains MT8148, TK15, and TK18 were hydrophobic. On the other hand, strain GS-5 and PAc-defective MT8148 transformants were hydrophilic. Resting cells of the hydrophobic strains attached in larger numbers to saliva-coated hydroxyapatite than did the hydrophilic strains. Human whole saliva induced the aggregation of cells of the hydrophobic strains but not that of cells of the hydrophilic strains. These results suggest that cell surface PAc of S. mutans serotype c participates in attachment of the streptococcal cell to experimental pellicles.

Four strains of serotype c Streptococcus mutans differing in glucosyltransferase (GTase) and fructosyltransferase (FTase) activities were examined. These strains had been made resistant to streptomycin. FTase activity of an S . mutans clinical variant, MT6801 R, which forms large mucoid colonies on sucrose-containing agar, was considerably higher than that of a typical serotype c strain, MT8148R, which forms small, rough colonies on the same agar. Two mutants, NG 14 and NG7183, were induced from strain MT6801 R by N-methyl-N'-nitro-N-nitrosoguanidine, and were found to be streptomycin-resistant. GTase and FTase activities of mutant NG 14 were similar to those of the typical serotype c strain, while in mutant NG7183 the two enzyme activities were very low. Growing cells of these strains (except NG7183) adhered firmly to a glass surface in sucrose broth. Resting cells of all strains attached in small numbers to salivacoated hydroxyapatite in the absence of sucrose. On the other hand, the presence of sucrose markedly enhanced the attachment of cells of strains MT8148R, MT6801 R and NG 14, but not NG7 183. Cell-surface hydrophobicity and acid production of all strains were similar. Both strain MT8148R and NG14 colonized tooth surfaces and produced significant dental caries in specific-pathogen-free rats. Strain MT6801 R had lower colonization ability and cariogenicity when compared with strains MT8148R and NG14. Furthermore, mutant NG7183 was able to colonize the tooth surfaces in small numbers, but failed to cause dental caries. These results indicate that sucrose-dependent cell adherence mediated by de no00 glucan synthesis is necessary for the accumulation of serotype c S . mutans cells on the tooth surface and the induction of dental caries.

Two freshly isolated strains, Xc and Yc, of Streptococcus mutans serotype c from human dental plaque were subcultured 100 times in Brain Heart Infusion broth. The cell-surface hydrophobicity of strain Xc markedly decreased after subculturing 60 times, but that of strain Yc remained unaltered. Radioimmunoassay showed a close correlation between surface hydrophobicity and the amount of a cell-surface protein antigen (PAc) of Mr 190,000. One hydrophilic variant (strain Xc100L), one relatively hydrophobic variant (strain Xc100H), and two hydrophobic variants (strains Yc100H1 and Yc100H2) were isolated from the 100-fold subcultures of hydrophobic strains Xc and Yc, respectively. SDS-PAGE showed that the amount of cell-associated and cell-free PAc of strain Xc100L was smaller than that of strains Xc and Xc100H. Strain Yc100H2 produced larger amounts of cell-associated PAc than strains Yc and Yc100H1. Resting cells of hydrophilic strain Xc100L attached in smaller numbers to saliva-coated hydroxyapatite than did other hydrophobic strains. RNA dot-blot analysis demonstrated a significant decrease in PAc-specific mRNA in strain Xc100L, as compared with strains Xc and Xc100H. Neither rearrangement nor deletion in the structural gene (pac) for PAc of these strains was observed by Southern blot analysis. These findings suggest that a mechanism which regulates the transcription of the pac gene participates in the quantitative variation of PAc after repeated subculturing.

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The ultrastructure of extracellular polysaccharides produced in colonies by two clinical isolates and that of a nitrosoguanidine-induced mutant of serotype c Streptococcus mutans with different polysaccharide-synthesizing abilities were compared electron-microscopically. A large amount of polysaccharide was produced from sucrose by colonies of typical serotype c strain MT8148R and a clinical variant MT6801R with an enhanced fructan-synthesizing ability. Transmission electron-microscopy (TEM) revealed that the polysaccharides consisted of three structural components, i.e., globular, single-stranded filamentous, and double-stranded fibrillar structures. These structures were ascribed to production of fructan, water-soluble glucan, and water-insoluble glucan, respectively. On the other hand, two kinds of structures, a globular body and an amorphous substance, were observed by scanning electron-microscopy (SEM). The former was composed of fructan, while the latter contained a mixture of water-soluble and water-insoluble glucans which formed filamentous and double-stranded fibrillar structures under TEM. Very small quantities of polysaccharides were formed in colonies of mutant NG7183, which was derived from S. mutans MT6801R. This strain was found to possess low glucan- and no fructan-synthesizing abilities. The polysaccharides produced in colonies of mutant NG7183 were composed only of filamentous and double-stranded fibrils under TEM. A small amount of amorphous substance was observed by SEM in colonies of NG7183.