| Actinomyces are high G+C content, gram-positive, facultatively anaerobic rods, which, through the action of fructosyltransferase (FTF), produce cell-associated polymers of D-fructose (levans) from sucrose. Levans are believed to contribute to the persistence and pathogenic potential of Actinomyces and other bacteria in the oral cavity. A gene encoding FTF was isolated from a bacteriophage lambdaGEM12 library of A. naeslundii WVU45 and the deduced amino acid sequence of the A. naeslundii FTF showed high homologies to levansucrases of gram-negative plant pathogens. A transcriptional start site was mapped by primer extension and an atypical promoter was found to drive transcription of the ftf gene. The ftf gene was constituitively transcribed and its expression was unaffected by carbohydrate source, similar to many of the plant-colonizing bacteria. The ftf promoter was fused to a cat reporter gene and results indicated that there is a functional promoter driving ftf expression, and that regions upstream of the transcription initiation site may be important for optimal expression of ftf. Biochemically, the enzyme shared characteristics with all FTFs, with an optimum pH near neutrality, temperature optimum at 45°C and enzyme activity inhibited by Cu 2+ and Zn2+. In addition to studying the levan-synthesizing abilities of A. naeslundii, the potential to hydrolyze fructans via levanases was explored. A levanase-defective strain was created and was found to be almost entirely devoid of levanase and inulinase activity, however, there was some detectable levanase and inulinase activity when bacteria were grown with fructose as the sole carbohydrate source. An FTF-defective A. naeslundii was constructed by allelic exchange and was shown to be capable of implanting into a rat caries model. This strain may be used in combination with other mutants with defects in fructan metabolism in future animal studies to assess the role of fructans in oral diseases. |
