Reinhold Brückner

Catabolite repression mediated by the catabolite control protein CcpA in Staphylococcus xylosus

The gene ccpA encoding the catabolite control protein CcpA of Staphylococcus xylosus has been cloned and characterized. The CcpA protein belongs to the LacI/GalR family of bacterial regulators and is comprised of 329 amino acids, with a molecular mass of 36.3 kDa. It shows 56% identity with the CcpA proteins of Bacillus subtilis and Bacillus megaterium. Inactivation of the ccpA gene in the genome of S. xylosus relieved the activities of three enzymes, α‐glucosidase, β‐glucuronidase, and β‐galactosidase, from catabolite repression by several carbohydrates. Concomitantly, transcription initiation of the maltose‐utilization operon malRA, including the α‐glucosidase gene malA, was no longer subject to glucose‐specific control. Carbon source‐dependent malRA regulation was also lost upon deletion of a palindromic sequence in the malRA promoter region resembling the catabolite‐responsive elements essential for CcpA‐dependent catabolite repression in Bacillus. These results strongly suggest that S. xylosus CcpA controls transcription of catabolite‐repressible genes and operons by binding to catabolite‐responsive operators when rapidly metabolizable carbohydrates are available. Accordingly, the cloned S. xylosusccpA gene could complement the ccpA mutation in B. subtilis. The ccpA gene of S. xylosus is transcribed from two promoters, one of which is subject to autogenous repression by CcpA. Autoregulation results in a slight reduction of CcpA protein in glucose‐grown cells. The characterization of the role of CcpA in carbon catabolite repression in S. xylosus demonstrates that a regulatory mechanism originally detected in Bacillus applies to another Gram‐positive bacterium with low GC content.

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

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 11 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.

Identification of a New Repetitive Element in Staphylococcus aureus

ABSTRACT The Staphylococcus aureus repeat (STAR) element is a sequence identified in two intergenic regions in S. aureus. The element is found in 13 to 21 copies in individual S. aureus strains, and elements in the homologous intergenic location are variable in length. The element sequence consists of several small and unusually GC-rich direct repeats with recurring intervening sequences. In addition, STAR-like elements may be present in related staphylococcal species.

Characterization of the single superoxide dismutase of Staphylococcus xylosus.

ABSTRACT Staphylococcus xylosus is a facultative anaerobic bacterium used as a starter culture for fermented meat products. In an attempt to analyze the antioxidant capacities of this organism, the superoxide dismutase (SOD) was characterized.S. xylosus contains a single cytoplasmic SOD, which was not inhibited by H2O2. The SOD activity in crude extracts was completely lost upon metal depletion, but it could be recovered by manganese and very weakly by iron. It is therefore suggested that the S. xylosus SOD is a manganese-preferring enzyme. The corresponding gene, sod, was isolated from a genomic library of S. xylosus DNA and complemented the growth defect of an Escherichia coli SOD-deficient mutant. As deduced from the nucleotide sequence, sod encodes a protein of 199 amino acids with a molecular mass of 22.5 kDa. Two transcriptional start sites 25 and 120 bp upstream of thesod start codon were identified. A terminator-like structure downstream of the gene suggested a monocistronicsod mRNA. Regulation of sod expression was studied using fusions of the sod promoters to a genomic promoterless β-galactosidase gene. The sod expression was not affected by manganese and increased slightly with paraquat. It was induced during stationary phase in a complex medium but not in a chemically defined medium. To investigate the physiological role of SOD, a mutant devoid of SOD activity was constructed. Growth experiments showed that sod is not essential for aerobic growth in complex medium. However, in chemically defined medium without leucine, isoleucine, and valine, the sod mutant hardly grew, in contrast to the wild-type strain. In addition, the mutant was sensitive to hyperbaric oxygen and to paraquat. Therefore,sod plays an important role in the protection of S. xylosus from oxidative stress.

Carbon Catabolite Repression of Sucrose Utilization in Staphylococcus xylosus: Catabolite Control Protein CcpA Ensures Glucose Preference and Autoregulatory Limitation of Sucrose Utilization

Sucrose utilization in Staphylococcus xylosus is dependent on two genes, scrA and scrB; encoding a PTS permease and a sucrose phosphate hydrolase, respectively. The genes are encoded on separate loci and are transcribed from two promoters, PscrA and PscrB, both of which are controlled by the repressor ScrR by binding to the operator sequences OA and OB. In the scrA promoter region, a catabolite-responsive element (cre), operator for the global catabolite control protein CcpA, is also present, but its contribution to scrA regulation has not been determined. Using an integrative promoter probe plasmid, the activities of the promoters PscrA and PscrB were determined under different growth conditions. Both promoters are induced by sucrose and induction is prevented when glucose is also present. Without a functional CcpA, glucose-mediated prevention of induction is lost, clearly demonstrating that CcpA ensures hierarchical sugar utilization with glucose as preferred substrate. Measurements of promoter activities in the absence of a functional ScrR repressor indicated that CcpA also acts upon the operators OA and OB, albeit not as efficiently as on the genuine cre in PsrcA. Besides determining the choice of the carbon source, CcpA has a second effect on sucrose gene expression. When sucrose is the sole carbon source, sucrose catabolism activates carbon catabolite repression and CcpA prevents full induction of the sucrose utilization genes by partially repressing the scrA promoter. Thus, CcpA-dependent regulation serves as a built-in autoregulatory device to restrict sucrose uptake.

Development of a Food-Grade Vector System for Staphylococcus carnosus

Summary The characterization of components for a food-grade vector for Staphylococcus carnosus employing utilization of sucrose as a selection marker is described. The genes mediating sucrose uptake and hydrolysis were isolated from Staphylococcus xylosus . The replicon for the vector was derived from the S. xylosus plasmid pSX267. The applicability of the replicon and the selection principle in S. carnosus is discussed.