Inhibition ofStreptococcus mutansbytheAntibiotic Streptozotocin: Mechanisms ofUptakeandtheSelection of Carbohydrate-Negative

an analogofNacetylglucosamine (NAG),hasbeenshowntobeuseful fortheselection ofcarbohydrate-negative and auxotrophic bacterial mutants. We haveadapted this methodforusewiththeoralpathogen Streptococcus mutans, a gram-positive, aerotolerant anaerobe that usespredominantly carbohydrates ascarbon sourcesfor growth. Streptozotocin selectively kills growing cells ofS.mutansGS-5,andunderappropriate conditions it canreduce thenumberofviable cells inactively growing cultures bya factor of103to104. However, unlike inenteric bacteria, whichtake up this antibiotic bya single NAG-specific transport system, streptozotocin appearstobetaken up inS.mutansbyboth aNAG-specific systemanda relatively nonspecific systemthatis alsoinvolved inglucose, fructose, andmannose uptake. Combining streptozotocin selection anda screening procedure involving indicator plates containing triphenyl-tetrazolium chloride, we developed ageneral method fortheisolation ofcarbohydrate-negative andauxotrophic mutantsofS.mutans.A preliminary characterization ofbothpleiotropic andspecific carbohydrate-negative mutantsisolated byusingthis procedure is presented. Theoral pathogen Streptococcus mutansisbelieved tobe theprimary etiologic agentofdental caries inhumansand other animals, andthemetabolism ofsucroseappearstoplay acentral role inthecariogenicity ofthis organism. Sucrose is botha substrate forextracellular andmembrane-bound glucosyltransferases, whichsynthesize extracellular glucans important foradhesion ofthis organism totooth surfaces, andan efficient growth substrate forS.mutans,leading to demineralization oftooth enamel byvirtue ofitsfermentativeproduction oflactic acid(for reviews, seereferences 9 and17). Sucrose, glucose, fructose, mannose,lactose, maltose,mannitol, andglucitol (sorbitol) haveall beenshownto betaken upinS.mutansbyahigh-affinity phosphoenolpyruvate(PEP)-dependent carbohydrate phosphotransferase sys