Expression of Streptococcus mutans gtf genes in Streptococcus milleri.
The Streptococcus mutans glucosyltransferase (GTF) genes gtfB and gtfC were ligated into Escherichia coli-streptococcus shuttle plasmids and introduced into Streptococcus milleri. gtfB transformant KSB8 formed an S. mutans-like rough colony on mitis salivarius agar and expressed an extracellular GTF-I, of 158 kDa, and two cell-bound GTF-Is, of 158 and 135 kDa. gtfC transformant KSC43 formed a semirough colony on mitis salivarius agar and expressed primarily an extracellular GTF-SI, of 146 kDa, and two cell-bound GTF-SIs, of 146 and 152 kDa. The extracellular GTFs from KSB8 and KSC43 were purified and characterized. The two types of GTF also reacted specifically with monoclonal antibodies directed against each enzyme. Both enzymes synthesized significant amounts of oligosaccharides, consisting primarily of alpha-1,6-glucosidic linkages, as well as water-insoluble glucans, containing alpha-1,3-glucosidic linkages. Insoluble-glucan-synthesizing activities of both enzymes were stimulated (three- to sixfold) by the addition of dextran T10 and were inhibited in the presence of 1.5 M ammonium sulfate. The Km(s) for sucrose and the optimal pHs were also similar for both enzymes. However, when the transformants were grown in Todd-Hewitt broth supplemented with sucrose, KSC43 cells, expressing GTF-SI activity, adhered to glass surfaces in vitro, while KSB8 cells, expressing GTF-I activity, did not. These results are discussed relative to the potential role of the gtfB and gftC genes in S. mutans cariogenicity.
Fukushima
K
K
Department of Microbiology, School of Dentistry at Matsudo, Nihon University, Chiba, Japan.
Ikeda
T
T
Kuramitsu
H K
HK
D000645:Ammonium Sulfate / Q000494:pharmacology; D001422:Bacterial Adhesion / Q000187:drug effects / Q000235:genetics; D015139:Blotting, Southern; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D002874:Chromosome Mapping; D003911:Dextrans / Q000494:pharmacology; D004587:Electrophoresis, Agar Gel; D005936:Glucans / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D010957:Plasmids; D013295:Streptococcus mutans / Q000235:genetics* / Q000472:pathogenicity; D013395:Sucrose / Q000494:pharmacology; D014169:Transformation, Bacterial
D003911:Dextrans; D005936:Glucans; D013395:Sucrose; D000645:Ammonium Sulfate