Repression and catabolite repression of the lactose operon of Staphylococcus aureus

Abstract

The lacR gene encodes the repressor of the lactose operon of S. aureus. The nucleotide sequence of this gene and the promoter-operator region of the operon are reported. The lacR gene encodes a protein with a molecular weight of 28,534. This protein was found to share sequence homology with the DeoR protein, the repressor of the E. coli deoxyribonucleotide operon. Directly and invertedly repeated sequences were found associated with the promoter for the structural genes of the operon. These sequences were examined by site-directed mutagenesis and found to be important in repressor binding and in the binding of a catabolite repressor. Evidence is presented in support of a model for catabolite repression of the operon which involves a negative-acting transcriptional regulator which binds to the promoter region of the operon and prevents transcription.The uptake of lactose by Staphylococcus aureus takes place via a phosphoenol pyruvate-dependent phosphotransferase system (reviewed in references 25 and 28). Lactose is phosphorylated upon entry into the cell by the joint action of two sugar-specific components, enzyme 11Lac (EIILaC) and enzyme I11Lac (ElIlLac). The intracellular phosphorylated lactose is then cleaved into glucose and galactose-6-phosphate by phospho-fi-galactosidase (14). The galactose-6-phosphate generated is further metabolized via a tagatosephosphate pathway (2).The lactose (lac) genes of S. aureus have been shown to be inducible with the addition of either lactose or galactose to the culture medium. Galactose-6-phosphate was found to be the actual intracellular inducer (22). Additionally, the lac genes are subject to catabolite repression (20). The significance of the glucose effect lies in the fact that cyclic AMP, which plays a critical role in catabolite repression in Escherichia coli, is not found, at least in physiologically significant concentrations, in S. aureus (3; unpublished data). The mechanism of catabolite repression in gram-positive bacteria is unknown.Our laboratory has reported the cloning of the structural genes of the staphylococcal lac operon (5-7, 29). These genes are arranged as a heptacistronic operon, according to data obtained by nucleotide sequence analysis. The organization of the operon is presented in Fig. 1. The terminal three genes in the operon, lacFEG, encode EIITLaC, EIILaC, and phospho-p-galactosidase, respectively (5, 7). The functions of the lacA-D determinants are currently under investigation (29). We previously reported the cloning of the gene encoding the repressor of the lac operon (lacR), which was found to lie immediately upstream of the promoter-proximal end of the lacA-G genes (24).In this report, the nucleotide and deduced amino acid sequences of lacR are presented, as are experiments to define the repressor target site, the lac operator. In addition, the promoter of the operon has been identified through deletion and gene fusion analysis. We will also present data pertaining to the mechanism of catabolite repression of this * Corresponding author. o...

Identification of Residues in the Staphylococcus aureus Fibrinogen-binding MSCRAMM Clumping Factor A (ClfA) That Are Important for Ligand Binding

“…Electrocompetent S. aureus cells were prepared by the method of Oskouian and Stewart (28). The pCU1-derived plasmids were initially introduced into S. aureus strain RN4220 by electroporation (29) with a Gene Pulser II set at 2.3 kV, 25 microfarads, and 100 ohms in a 0.2-cm cuvette and were subsequently transduced to strain DU5941 using phage 85 (30).…”

The dipeptide repeat region of the fibrinogen‐binding protein (clumping factor) is required for functional expression of the fibrinogen‐binding domain on the Staphylococcus aureus cell surface

“…Recombinant plasmids were introduced into S. aureus by electroporation. Electrocompetent S. aureus cells were prepared by the method of Oskouian and Stewart (1990). E. coli XL-1 Blue cells were made competent by CaCl 2 treatment (Sambrook et al, 1989).…”

Lactose metabolism by Staphylococcus aureus: characterization of lacABCD, the structural genes of the tagatose 6-phosphate pathway

“…Our laboratory has reported the cloning and nucleotide sequences of the structural genes of the heptacistronic staphylococcal lac operon (8)(9)(10)24; this report) as well as that of a closely linked gene, lacR, which encodes the repressor of the lac operon (22,23). The organization of the operon is shown in Fig.…”