We recently identified the genes responsible for the serotype c-specific glucose side chain formation of rhamnose-glucose polysaccharide (RGP) in Streptococcus mutans. These genes were located downstream from the rgpA through rgpF locus that is involved in the synthesis of RGP. In the present study, the corresponding chromosomal regions were isolated from serotype e and f strains and characterized. The rgpA through rgpF homologs were well conserved among the three serotypes. By contrast, the regions downstream from the rgpF homolog differed considerably among the three serotypes. Replacement of these regions in the different serotype strains converted their serotypic phenotypes, suggesting that these regions participated in serotypespecific glucose side chain formation in each serotype strain. Based on the differences among the DNA sequences of these regions, a PCR method was developed to determine serotypes. S. mutans was isolated from 198 of 432 preschool children (3 to 4 years old). The serotypes of all but one S. mutans isolate were identified by serotyping PCR. Serotype c predominated (84.8%), serotype e was the next most common (13.3%), and serotype f occured rarely (1.9%) in Japanese preschool children. Caries experience in the group with a mixed infection by multiple serotypes of S. mutans was significantly higher than that in the group with a monoinfection by a single serotype.Streptococcus mutans strains are classified into three serotypes (c, e, and f), and the serologic specificity is defined by rhamnose-glucose polysaccharide (RGP) on the cell wall (6). We have characterized the genes involved in RGP synthesis in S. mutans Xc (serotype c) in the course of our previous studies. Four rml genes (rmlA through rmlD) are directly related to the synthesis of dTDP-L-rhamnose (12, 13), and the gluA gene encodes the enzyme producing UDP-D-glucose (18). The rgpG gene is implicated in the initiation of RGP synthesis by transfer of N-acetylglucosamine-1-phosphate to a lipid carrier (16). Furthermore, six other genes (rgpA through rgpF) required for RGP synthesis were identified in the region downstream from rmlD, and these genes are likely to be involved in the transport and assembly of RGP (11,19).The RGPs are composed of ␣1,2-and ␣1,3-linked rhamnan backbones with glucose side chains linked to alternate rhamnoses. Each serotype-specific polysaccharide has unique linkages of its glucose side chains (serotype c, ␣1,2-linkage; serotype e, ␤1,2-linkage; and serotype f, ␣1,3-linkage) (5, 10). Recently, we identified and characterized the genes required for glucose side chain formation of the serotype c-specific RGP (9). However, the loci responsible for the determination of the other serotypes have not yet been elucidated.In this study, we identified the loci involved in the glucose side chain formation of RGP in serotypes e and f of S. mutans and confirmed that these regions determine serotype specificities. Furthermore, we designed three pairs of primers from specific DNA sequences within each serotype determinan...

Six genes (rgpA through rgpF) that were involved in assembling the rhamnose-glucose polysaccharide (RGP) in Streptococcus mutans were previously identified (Y. Yamashita, Y. Tsukioka, K. Tomihisa, Y. Nakano, and T. Koga, J. Bacteriol. 180:5803-5807, 1998). The group-specific antigens of Lancefield group A, C, and E streptococci and the polysaccharide antigen of Streptococcus sobrinus have the same rhamnan backbone as the RGP of S. mutans. Escherichia coli harboring plasmid pRGP1 containing all six rgp genes did not synthesize complete RGP. However, E. coli carrying a plasmid with all of the rgp genes except for rgpE synthesized the rhamnan backbone of RGP without glucose side chains, suggesting that in addition to rgpE, another gene is required for glucose side-chain formation. Synthesis of the rhamnan backbone in E. coli required the initiation of transfer of N-acetylglucosamine to a lipid carrier and the expression of the rgpC and rgpD genes encoding the putative ABC transporter specific for RGP. The similarities in RGP synthesis between E. coli and S. mutans suggest common pathways for rhamnan synthesis. Therefore, we evaluated the rhamnosyl polymerization process in E. coli by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the lipooligosaccharide (LOS). An E. coli transformant harboring rgpA produced the LOS modified by the addition of a single rhamnose residue. Furthermore, the rgpA, rgpB, and rgpF genes of pRGP1 were independently mutated by an internal deletion, and the LOS chemotypes of their transformants were examined. The transformant with an rgpA deletion showed the same LOS profile as E. coli without a plasmid. The transformant with an rgpB deletion showed the same LOS profile as E. coli harboring rgpA alone. The transformant with an rgpF deletion showed the LOS band with the most retarded migration. On the basis of these results, we speculated that RgpA, RgpB, and RgpF, in that order, function in rhamnan polymerization.Polysaccharides are the major constituents of streptococcal cell walls and are useful for the serological classification and identification of streptococci. The group-specific polysaccharide antigens of Lancefield group A, C, and E streptococci (3, 26), the serotype-specific antigen of Streptococcus mutans (18,27), and the rhamnose-glucose polysaccharide (RGP) antigen of Streptococcus sobrinus (19) share a common structural relationship. The backbones of these polysaccharides are polymers of ␣1,2-and ␣1,3-linked rhamnose units. Although the rhamnan backbone has been identified in many streptococci, little is known about the mechanism of its synthesis. Rhamnan is also present in O polysaccharides of phytopathogenic bacteria (Xanthomonas, Pseudomonas, and Stenotrophomonas), Yersinia enterocolitica, and Pseudomonas aeruginosa, and these O polysaccharides are regarded as pathogenic factors (1,6,25,28,32,40). However, the only report dealing with the assembly of rhamnan is that describing the synthesis of the A band, Drhamnan polysaccharide, of P. aeruginosa ...

We have cloned two genes (rgpH and rgpI) that encode proteins for the formation of the glucose side-chains of the Streptococcus mutans rhamnose-glucose polysaccharide (RGP), which consists of a rhamnan backbone with glucose side-chains. The roles of rgpH and rgpI were evaluated in a rhamnan-synthesizing Escherichia coli. An E. coli strain that harbored rgpH reacted with antiserum directed against complete RGP, whereas the E. coli strain that carried rgpI did not react with this antiserum. Although E. coli:rgpH reacted strongly with rhamnanspeci¢c antiserum, co-transformation of this strain with rgpI increased the number of glucose side-chains and decreased immunoreactivity with the rhamnan-speci¢c antiserum signi¢-cantly. These results suggest that two genes are involved in side-chain formation during S. mutans RGP synthesis in E. coli: one gene encodes a glucosyltransferase, and the other gene probably controls the frequency of branching. This is the ¢rst report to identify a gene that is involved in regulation of branching frequency in polysaccharide synthesis.

We have proposed a novel structure of holographic data storage media, employing a phase-change (PC) reflector for optical disc applications. The PC layer, which is initially amorphous and has a low reflectivity in a write process, works as an absorber and inhibits reflected stray beam exposure. After the write process, the PC layer is crystallized and its reflectivity is switched to a high state, realizing a sufficient signal beam intensity in a read process. Experimental results show a low noise and a high signal-to-noise ratio, and a potential to have a large multiplexing number in the PC reflector.