INDUCIBLE ANTIBIOTIC RESISTANCE

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The long-term general objective of this research program is to study regulation of genes whose expression is induced or modified by antibiotics. The emphasis is on a group of genes that confer antibiotic resistance. The specific aims of this proposal are to study regulation of erythromycin-inducible resistance to the macrolide, lincosamide, and streptogramin B (MLS) antibiotics conferred by Staphylococcus aureus plasmid pEl94. An inducible 23S rRNA methylase confers resistance by methylating a specific adenine residue in 23S rRNA, and its synthesis is induced by erythromycin. Factors that enter into the control of MLS resistance can be classified according to whether they act on: (1) the rate of synthesis of the message (transcriptional control), (2) the rate of degradation of the message (mRNA stability), (3) the efficiency of utilization of the message (translational control), and (4) the number of plasmid DNA copies that are available from which to transcribe the message (plasmid copy number control). Two new aspects of the problem that will serve as the focus of the present work include: (1) a study of the interaction between the ErmC methylase, a member of the family of enzymes that confer MLS resistance, with its 23S rRNA substrate; and (2) a study of the interaction between the pEl94 Cop protein and its putative binding site in plasmid DNA, a reaction that is proposed to regulate the expression of erythromycin resistance by controlling plasmid copy number. In the first case, we shall attempt to distinguish between structural aspects of the methylase amino acid sequence that are responsible for the ability to recognize the 23S rRNA substrate and distinguish it from other potential substrates such as 16S rRNA as well as to identify the parts of the sequence that are responsible for catalytic (i.e., methylase) activity. In the second case, we shall study the interaction between a recently identified Cop protein specified by plasmid pEl94 and its presumed target plasmid pEl94 DNA - asking specifically, where does Cop protein bind and how does this binding ultimately contribute to control the concentration of plasmid DNA?