Staphylococcus aureus is an important human pathogen, capable of causing acute and chronic infections such as septicemia and carditis. Half of all hospital-associated infections are now caused by S. aureus strains, which often carry determinants encoding resistance to many antimicrobial agents. One of these resistance determinants is the mercury resistance determinant (mer operon) contained on the S. aureus plasmid pI258. The goal of this proposal is to understand the regulatory and mercury detoxification mechanisms encoded by the pI258 mer operon. The DNA sequence of the pI258 mer operon has been determined and significant progress has been made in determining the function(s) of four of the six mer operon genes. The proposed experimental plan consists of the following parts: i) Transcriptional analysis of the mer operon will be done. Specifically, a detailed analysis of the efficiency of the pI258 mer operon termination sequence will be done using northern blot analysis, S1 mapping of the 3'-end of the two pI258 mer transcripts, and transcriptional fusions (using the beta-lactamase reporter system). S1 mapping of the 3'-end of the two pI258 mer transcripts will be done. The relationship between gene dosage and the total number of transcripts produced will also be examined. ii) Each protein encoded by the pI258 mer operon will be over-expressed using a T7 expression system, and then the over-expressed proteins will be isolated and purified. iii) Gene/translational fusion experiments using the beta-lactamase reporter gene will be done to study the promoter/operator site and which substances are capable of inducing the mer operon. iv) A low copy number vector will be constructed and subsequently utilized in physiological studies on the function(s) of individual genes in the pI258 mer operon. The specific goal of this group of experiments will be to investigate the significance of the pI258 mer operon mercuric ion transport system and to determine if the system can also transport organomercurials. The long-term objectives of this research program are to understand the genetics and mechanism of resistance to mercuric compounds, the mechanisms responsible for the distribution of mercury resistance determinants among different strains, and ultimately, the evolution/development of mercury resistance determinants in S. aureus.
