REGULATION OF STREPTOCOCCUS GORDONII GLUCOSYLTRANSFERASE

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Streptococcus gordonii have a single glucosyltransferase (GTF) enzyme that produces glucans from sucrose. Differences in GTF activity, which are affected by environmental and growth conditions, affect the ability of S. gordonii to colonize surfaces in vitro and therefore, may affect their ability to establish in ecological niches in vivo. Since S. gordonii cells and their products are present in early dental plaque, they act as a substratum for the attachment of other oral organisms, including potential pathogens, and therefore may play important roles in determining mature plaque composition. Unlike mutans streptococci, which have multiple gtf genes with as yet undescribed genetic regulatory mechanisms, S. gordonii has a single gtf gene. The expression of the GTF structural gene, gtfG, is positively regulated by the upstream gene rgg, the only described regulatory determinant for a gtf gene. S. gordonii undergo a spontaneous, reversible phase variation between high (Spp+) and low (Spp-) levels of GTF activity. rgg can act in trans to increase GTF activity in both Spp+ and Spp- strains. The molecular basis for changes in gtfG expression is unknown but there is preliminary evidence that more than one mechanism is involved. These could include frameshifts or rearrangements in rgg or gtfG; alternatively, distally located regulatory gene(s) may affect rgg and/or gtfG expression. The proposed studies will attempt to determine factors associated with regulation of gtfG and to help elucidate mechanisms underlying phase variation at the DNA level. Nucleotide sequences of Spp+ and Spp- phase variants will be compared for structural differences in the rgg and gtfG genes and their flanking regions, which may affect their expression. Distal regions of S. gordonii DNA that affect expression of rgg and/or gtfG will be identified in an E. faecalis system that allows efficient screening of many plasmid clones. Plasmid DNA containing rgg and/or gtfG lacZ transcriptional fusions will be introduced into E. faecalis and the resulting strains will be transformed with compatible plasmids carrying randomly cloned & gordonii DNA. Any clones affecting expression of rgg and/or gtfG, as monitored by changes in colony color reflecting changes in beta- galactosidase activity, will be characterized further. Because lacZ transcriptional fusions are only weakly expressed in S. gordonii, another reporter system is necessary to study the identified regulatory elements in the S. gordonii genetic background. Therefore, chloramphenicol acetyltransferase (CAT) transcriptional fusions will be integrated into the chromosome of S. gordonii to monitor expression of rgg and/or gtfG by CAT activity. Plasmids carrying the characterized distal regulatory segments of DNA will be transformed into these S. gordonii fusion strains for additional genetic studies. Finally, S. gordonii strains with chromosomal CAT fusions will be used to study environmental influences on expression of gtfG and its regulatory gene(s) at the DNA level. It hoped that these studies, aimed at better understanding the regulation of gtfG expression, will provide insights into the oral microbial ecology and dental health.