Our aim is to determine how the binding of a beta-lactam antibiotic to the membrane of a sensitive microorganism leads to the production of the enzyme beta-lactamase which destroys the antibiotic. These enzymes are a major cause of clinical resistance to the penicillin/cephalosporin antibiotics, and information concerning the regulation of their production would be of considerable value. In addition, the beta-lactamases are closely related to some of the enzymes important in cell wall formation and are useful vectors for the secretion of foreign proteins by bacteria. Despite much attention to the genetics of these enzymes, little is known about their regulation at the molecular level. We will concentrate on the Bacillus licheniformis enzyme, both as regulated in the bacillus and when cloned in E. coli. Results should have general applicability to the beta-lactamases of other microorganisms. Analysis by Southern hybridizaton will be used to detect and characterize the mRNAs produced in the region of the beta-lactamase gene and to correlate their presence with induction of the enzyme. Secondly, a more limited system in E. coli will be used to characterize the repressor and the target sites in the regulatory regions. Two plasmids are present, one contains only the repressor and other potential regulatory regions, the other a complete gene region, but one from a mutant that does not produce an active repressor. With the two systems available, the requirements for both repressor and promotor (target) functions can be studied, and modified forms of one or the other can be introduced into the system. The important regulatory areas will be sequenced, and the repressor will be isolated, characterized and its function examined. No repressor for a chromosomal gene from a gram-positive bacterium has yet been sequenced and studied. The final stage will be to identify the additional components needed to transfer the signal for induction from the cell membrane, where finding of the beta-lactam occurs, to the regulatory region of the DNA. A variety of mutants showing aberrant regulation are available and should be of value in establishing the role of these additional components.
