Bacterial cell surfaces interact with the host to form a pathogenic or symbiotic association. These interactions are directly influenced by the outer membrane of Gram negative bacteria (such as Escherichia coli) which serves as the interface between the environment and the interior of the cell. It is becoming increasingly clear that the outer membrane is extremely important in making bacteria resistant to host defence factors by providing an effective barrier against the detergent action of bile salts, degradation by digestive enzymes, and killing by hydrophobic antibiotics such as novobiocin and erythromycin. Small water-soluble nutrients and antibiotics such as penicillin and cephalothin penetrate the cell by diffusing through pores or channels formed by a unique set of proteins, called porins. OmpF is an example of a porin that forms nonspecific pores, whereas LamB forms pores that specifically facilitate diffusion of maltodextrins (maltooligomers up to 7 glucose units). These porin proteins also provide receptor sites for bacterial viruses. Porin proteins exist in tight association with other membrane components such as lipopolysaccharide (LPS) and peptidoglycan. The long term goal of this research is to understand the biogenesis of the outer membrane and molecular interactions between the various membrane components. The specific aim of this proposal is to examine how proteins (OmpF and LamB) are targeted to the outer membrane and what role, if any, LPS plays in this process. These objectives will be achieved by utilizing a combination of genetic and biochemical approaches. We have devised a genetic selection scheme to isolate temperature sensitive folding mutations that conditionally affect assembly of OmpF and LamB. The conditional accumulation of assembly intermediates will allow us to determine their structure and cellular location. The experimental approach described is novel for studying membrane proteins and will reveal the key residues involved in the targeting and assembly of outer membrane proteins. These studies will be extended to examine the role of other components through suppressor analysis.