Characterization of a sucrase gene from Staphylococcus xylosus

Abstract

The Staphylococcus xylosus gene scrB, encoding a sucrase, has been isolated from a genomic library of S. xylosus constructed in Escherichia coli. The gene was detected by its ability to confer utilization of the glucose and fructose residues of raffinose in an E. coli strain that is not able to metabolize galactose. It was found to reside within a 1.8-kb DNA fragment, the nucleotide sequence of which was determined. One large open reading frame, which is preceded by a ribosome binding site, is encoded on the fragment. Its deduced amino acid sequence yields a protein with a molecular mass of 57.377 kDa which shows significant homology with bacterial sucrose-6-phosphate hydrolases and sucrases. Overexpression of scrB in E. coli by the bacteriophage T7 polymerase promoter system resulted in the production of a protein with an apparent molecular mass of 58 kDa. Disruption of the scrB gene in the S. xylosus genome rendered S. xylosus unable to utilize sucrose. Thus, the ScrB sucrase is essential for sucrose metabolism in S. xylosus.The disaccharide sucrose can serve as a carbon source for a wide variety of bacteria. Sucrose can be cleaved by sucrases (invertases) or sucrose-6-phosphate hydrolases after uptake into the bacterial cytoplasm (22,52). In many bacteria, sucrose is transported across cytoplasmic membranes by means of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) (31). By this mechanism, sucrose enters the cell as sucrose-6-phosphate, which is then hydrolyzed to glucose-6-phosphate and fructose. Sucrose can also be split in the culture medium by extracellular enzymes such as levansucrase, levanase, and glucosyl-and fructosyltransferases (26,29,49). In some bacteria, more than one sucrose-hydrolyzing activity is present (24,29).At the molecular level, genes encoding secreted sucrosecleaving enzymes of Bacillus subtilis and Streptococcus mutans (20,26,44,45,49,53) and systems mediating sucrose uptake and metabolism of B. subtilis, S. mutans, Streptococcus sobrinus, Lactococcus lactis, Salmonella typhimurium, Klebsiella pneumoniae, and Vibrio alginolyticus have been characterized (4,8,10,12,13,17,32,36,37,39,42,47,55). These sucrose utilization systems belong to the PTS, and their genes are clustered on the chromosome, on conjugative transposons, or on plasmids. Genes for sucrose-specific transport enzymes (enzyme IIScr) and sucrose hydrolases are essential components of all these gene clusters. On the S. typhimurium plasmid pUR400 and in the genome of K pneumoniae, genes for a sucrose-specific outer membrane porin and an ATP-dependent fructokinase are found (3,18,40). The latter is also encoded in the V. alginolyticus system (4). The gram-negative sucrose PTS genes constitute operons that are controlled by repressors (5, 39). In B. subtilis, the sucrose PTS operon is regulated by an antitermination mechanism which is part of a complex regulatory network controlling sucrose metabolism (1, 48).To get an insight into the molecular organization and regulation of the sucrose utiliza...

Transcriptional regulation of the sucrase gene of Staphylococcus xylosus by the repressor ScrR

“…In Staphylococcus xylosus, a gram-positive, nonpathogenic bacterium originally isolated from human skin (36), two genes essential for sucrose utilization were identified (6,42). One of the two genes, scrA, encodes a PTS permease required for transport and phosphorylation of the sugar.…”

Characterization of a genetic locus essential for maltose-maltotriose utilization in Staphylococcus xylosus

“…During the characterization of scrB, the gene encoding the sucrose-6-phosphate hydrolase of S. xylosus, we detected a residual sucrose hydrolase activity in a scrB null mutant (4). To clone the gene encoding this minor sucrase of S. xylosus, aliquots of a genomic library ligation, which had been prepared to clone the scrB gene (4), were plated to yield about 5,000 E. coli transformants.…”

“…In an attempt to characterize the components of the sucrose utilization system of S. xylosus, a gene mediating sucrase activity was isolated from a genomic library of S. xylosus DNA in E. coli. It was distinct from the previously characterized gene for the sucrose-6-phosphate hydrolase of that organism (4). The genetic locus isolated in this way turned out to be essential for maltose utilization in S. xylosus.…”

“…Sucrase activity in bacterial extracts prepared by disrupting the cells with glass beads was quantified by measuring the release of glucose from sucrose as described previously (4). The amount of glucose was determined by means of a commercially available test kit (Boehringer Mannheim).…”

“…Chromosomal DNA from S. xylosus was prepared by the method of Marmur (15). Construction of the genomic library of S. xylosus in E. coli and screening for sucrase activity have been described previously (4). Plasmids were introduced into S. xylosus and S. carnosus by protoplast transformation (7).…”