Cloning and characterization of the scrA gene encoding the sucrose-specific Enzyme II of the phosphotransferase system from Staphylococcus xylosus

Abstract

By insertional mutagenesis with the staphylococcal transposon Tn551, mutants of Staphylococcus xylosus were isolated that were unable to utilize sucrose. One of these was found to be deficient in sucrose uptake. The genomic region containing this sucrose uptake gene of Staphylococcus xylosus (scrA) was cloned in Staphylococcus carnosus. The scrA gene was further localized to a 4.4 kb DNA fragment by complementation of the sucrose transport-deficient S. xylosus mutant. The DNA sequence analysis of the scrA region revealed three open reading frames, one of which encodes a protein of 480 amino acids (51.335 kDa) with significant similarity to sucrose-specific Enzymes II of phosphoenolpyruvate-dependent carbohydrate phosphotransferase systems (PTS). A protein with an apparent molecular weight of 50 kDa was obtained in Escherichia coli by expression of scrA with the bacteriophage T7 RNA polymerase promoter system. Transcriptional start sites of the scrA gene were localized by primer extension analysis to positions 46 and 49 nucleotides upstream of the scrA start codon. No additional sucrose utilization genes are encoded close to scrA on the S. xylosus chromosome.