Regulation of expression of the fructan hydrolase gene of Streptococcus mutans GS-5 by induction and carbon catabolite repression

The polymers of fructose, levan and inulin, as well as sucrose and raffinos e, are substrates for the product of the fruA gene of Streptococcus mutans GS-5. The purpose of this study was to characterize the DNA immediately fla nking fruA, to explore the regulation of expression of fruA by the carbohyd rate source, and to begin to elucidate the molecular basis for differential expression of the gene. Located 3' to fruA was an open reading frame (ORF) with similarity to beta-fructosidases which was cotranscribed with fruA. A transcriptional initiation site, located an appropriate distance from an e xtended -10-like promoter, was mapped at 165 bp 5' to the fruA structural g ene. By the use of computer algorithms, two overlapping, stable stem-loop s equences with the potential to function as rho-independent terminators,were found in the 5' untranslated region. Catabolite response elements (CREs), which have been shown to govern carbon catabolite repression (CCR) by funct ioning as negative cis elements in gram-positive bacteria, were located clo se to the promoter. The levels of production of fruA mRNA and FruA were ele vated in cells growing on levan, inulin, or sucrose as the sole carbohydrat e source, and repression was observed when cells were grown on readily meta bolizable hexoses. Deletion derivatives containing fusions of fruA promoter regions, lacking sequences 5' or 3' to the promoter, and a promoterless ch loramphenicol acetyltransferase gene were used (i) to demonstrate the funct ionality of the promoter mapped by primer extension, (ii) to demonstrate th at CCR of the fru operon requires the CRE that is located 3' to the promote r region, and (iii) to provide preliminary evidence that supports the invol vement of an antitermination mechanism in fruA induction.