We propose to develop a system to study the mechanisms of extracellular secretion in enteric bacteria. The genetic approaches used in Escherichia coli to study protein targeting and localization have proven extremely powerful, but E. coli does not normally secrete protein out of the cell. Serratia marcescens does secrete a number of proteins abundantly into the growth media. Being an enterobacteria it is closely related to E. coli and many of the plasmid and phage vectors developed for E. coli will function equally well in S. marcescens, as the exoproteins of S. marcescens will function and be secreted in E. coli. We propose to use secreted exonuclease from S. marcescens as a model to develop secretion systems for genetic study in both E. coli and S. marcescens. We have already identified the exonuclease gene and it is presently being sequenced. We have shown that it is also secreted in E. coli as well as in S. marcescens. By constructing protein fusion between the exoprotein and a marker gene such as alkaline phosphatase or beta-galactosidase, we hope to identify regions involved in protein export. Selection schemes based on the fusion proteins, or based on properties intrinsic to the nuclease, will be used to isolate mutations which alter or abolish extracellular secretion. These mutations could either be within the nuclease gene itself, thus identifying specific signals for secretion, or could be within host genes required for secretion. Mutations affecting protein export in E. coli will be tested to determine how they effect extracellular secretion; this should help characterize the relationship between these processes. Using LacZ fusions which inhibit normal protein export in E. coli when overexpressed, will be introduced into S. marcescens to test their effect on secretion of the exoproteins of S. marcescens. Experiments designed to probe the path of exonuclease secretion are planned. We hope to use variety of approaches to elucidate the interactions between the exonuclease and the host export mechanism and to determine the path of secretion. Competition experiments can determine the common steps in the secretion process. Components which are limiting might be identified by these competition experiments. The long term goals of this research are to understand the mechanism of this one type of extracellular secretion, to compare and contrast extracellular secretion and export of envelope proteins, and to determine if the exoproteins of S. marcescens utilize a unique path for extracellular secretion distinct from the secretion of exoproteins by other gram negative bacteria.
Showing the most recent 10 out of 16 publications
