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

Abstract

A genetic locus from Staphylococcus xylosus involved in maltose-maltotriose utilization has been characterized. The chromosomal region was identified by screening a genomic library of S. xylosus in Escherichia coli for sucrose hydrolase activity. Nucleotide sequence analysis yielded two open reading frames (malR and malA) encoding proteins of 37.7 and 62.5 kDa, respectively. MalR was found to be homologous to the LacI-GalR family of transcriptional regulators, and MalA showed high similarity to yeast ␣-1,4-glucosidases and bacterial ␣-1,6-glucosidases. Inactivation of malA in the genome of S. xylosus led to a maltose-maltotriose-negative phenotype. In cell extracts of the mutant, virtually no glucose release from maltose and short maltodextrins was detectable. Inactivation of malA in a sucrose-6-phosphate hydrolase-deficient S. xylosus strain resulted in the complete loss of the residual sucrose hydrolase activity. The MalA enzyme has a clear preference for maltose but is also able to release glucose from short maltosaccharides. It cannot cleave isomaltose. Therefore, malA encodes an ␣-1,4-glucosidase or maltase, which also liberates glucose from sucrose. Subcloning experiments indicated that malA does not possess its own promoter and is cotranscribed with malR. Its expression could not be stimulated when maltose was added to the growth medium. Chromosomal inactivation of malR led to reduced maltose utilization, although ␣-glucosidase activity in the malR mutant was slightly higher than in the wild type. In the mutant strain, maltose uptake was reduced and inducibility of the transport activity was partially lost. It seems that MalR participates in the regulation of the gene(s) for maltose transport and is needed for their full expression. Thus, the malRA genes constitute an essential genetic locus for maltosaccharide utilization in S. xylosus.Maltose-maltodextrin utilization has been studied mainly in gram-negative enteric bacteria (38). The maltose regulon of Escherichia coli, which is controlled by a positive regulator MalT (30), consists of a number of genes encoding proteins responsible for uptake and metabolism of maltosaccharides. These enter the periplasmic space through a diffusion pore, the product of lamB (38). Uptake of maltose and maltodextrins up to maltoheptaose into the cytoplasm is mediated by a multicomponent, binding protein-dependent transport system encoded by malE, malF, malG, and malK (38). Degradation of maltodextrins is achieved by the action of three cytoplasmic enzymes, amylomaltase (21), maltodextrin phosphorylase (34), and maltodextrin glucosidase (29, 43), the gene products of malQ, malP, and malZ, respectively, and a periplasmic ␣-amylase (24, 36) specified by malS, which is required only for growth on long maltodextrins. None of these enzymes is able to cleave maltose itself, but they recognize maltotriose or longer maltodextrins as substrates. Thus, maltose utilization in E. coli depends on the formation of maltotriose as the shortest substrate for the degradative enzymes and, eq...

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…CcpA-mediated CCA of the B. subtilis als operon occurs probably via an indirect mechanism, as no obvious cre is located in front of the als promoter (882). The cre of the CCR-sensitive S. xylosus malRA operon is also located in front of the promoter (205). However, the mal cre and the mal promoter are separated by only 8 bp.…”

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

“…S. xylosus possesses two enzymes that hydrolyze sucrose. One is the sucrose-6-phosphate hydrolase ScrB, which is essential for sucrose catabolism (6); the other constitutes the ␣-glucosidase or maltase MalA, which is needed for maltose utilization (12). To study the regulation of the sucrose utilization system of S. xylosus, we intended to take sucrase activity as a measure of gene expression.…”

“…Fragments of the scrB region from the S. xylosus chromosome were isolated from plasmid pRA7 (6). Plasmid pSH5 (12) served as the source of malRA fragments used to construct S. xylosus malA deletion strains. Inactivation of scrR and disruption of the operator sequence were performed by using the E. coliStaphylococcus shuttle vector pBT1.…”

“…The second gene, scrB, codes for the sucrase responsible for hydrolysis of sucrose-6-phosphate. A second enzyme showing sucrose-hydrolyzing activity was found to be an ␣-glucosidase that is needed for maltose-maltotriose catabolism, but it is dispensable for sucrose utilization (12). The two sucrose genes are not clustered in the genome of S. xylosus (42).…”

Maltose and Maltodextrin Transport in the Thermoacidophilic Gram-Positive Bacterium Alicyclobacillus acidocaldarius Is Mediated by a High-Affinity Transport System That Includes a Maltose Binding Protein Tolerant to Low pH

“…Genes encoding homologous proteins from other gram-positive bacteria are usually transcribed from their own promoter (10,43,45,56). One exception appears to be the malR gene of S. pneumoniae, which probably belongs to the same transcriptional unit as malA, necessary for growth on maltotetraose (40).…”