A rapidly increasing number of membrane proteins in bacteria have been found to contain an N- terminal lipoamino acid, N-acyl-diacylglycerylcysteine, the hallmark of all bacterial lipoproteins. As of 1993, more than 130 lipoproteins have been identified in both gram positive and gram negative bacteria covering a wide spectrum of genera and species. The biosynthesis of lipoproteins in bacteria is both temporally and spatially related to the export of these membrane proteins across the cytoplasmic membranes. These proteins are made as precursor proteins with N-terminal signal sequences which contain a "lipobox" sequence at the junction of the signal sequences and the mature proteins. Following the interaction of the precursor proteins with the export machinery defined by the SecA, SecY SecD and SecF proteins, the prolipoproteins are modified and processed by three enzymes located in the cytoplasmic membrane. These three enzymes are prolipoprotein diacylglyceral transferase, prolipoprotein signal peptidase and apolipoprotein N- acyltransferase encoded by the lgt, lsp, and lnt genes, respectively, in Escherichia coli and Salmonella typhimurium. Mutants defective in any of these three genes are conditionally lethal. In addition, lnt mutants are also copper sensitive, and the lnt gene is allelic with the cutE gene, one of six genes postulated to be involved in copper transport and homeostasis in E. coli. Using the E. coli major outer membrane lipoprotein as the model system, the investigator proposes; (i) to elucidate the biochemical basis for the essential nature of the lipoprotein biosynthetic pathway in E. coli or S. typhimurium; (ii) to identify the gene encoding lipoprotein:peptidoglycan ligase in E. coli; (iii) to study the structure, function and assembly of the prolipoprotein modification and processing enzymes; and (iv) to study the interrelationship between apolipoprotein N-acylation and the transport and homeostasis of divalent cations such as Cu++ in E. coli. The results of the proposed studies will provide new information regarding the structures, functions and biogenesis of lipoproteins in bacteria. These studies will also provide a rational basis for the search or design of antibacterial agents which utilize the enzymes of this pathway as targets.