Cloning and characterization of the repressor gene of the Staphylococcus aureus lactose operon

Abstract

The genes responsible for utilization of lactose in Staphylococcus aureus are organized as an inducible operon, with galactose 6-phosphate being the intracellular inducer. To clone the repressor gene of this operon, we constructed an integration vehicle carrying 1.9 kilobases (kb) of DNA sequences from a region upstream of the structural genes of the operon. Through integration and subsequent rescue of this plasmid, we were able to clone approximately 7 kb of staphylococcal chromosomal DNA. We have shown that the plasmid insert complemented lac constitutive mutants. This repressor activity was localized to a 1.8-kb DNA fragment and, through maxicell analysis, was shown to correlate with the presence of a polypeptide with an apparent molecular weight of 32,000. Furthermore, a region between the repressor gene and the other genes of the operon was identified which, when carried on multicopy plasmids, resulted in expression of the operon in the absence of any exogenous induction. This region may represent an operator-type element capable of titrating repressor molecules away from chromosomal operator, allowing transcription of the operon in the absence of induction.