The proposed research aims to further the understanding on a genetic level of the synthesis of lipoteichoic acid (LTA), an important wall polymer found within the cell wall envelope of many Gram-positive bacteria, including the human pathogen Staphylococcus aureus. Functions of LTA are scavenging of Mg+2 ions required for the proper function of cell wall-associated enzymes and regulation of autolysins required for cell growth and septation. More recently, LTA has been recognized as immunostimulatory molecule of Gram-positive bacterial pathogens. Despite a detailed knowledge of the biochemical structure of LTA, only a few gene products have been shown to be required for LTA synthesis, which together cannot account fully for the synthesis of the polyglycerolphosphate polymer. No mutations that completely abrogate LTA biosynthesis have been described for Gram-positive bacteria and it has been speculated that LTA is an essential component of the cell wall. The experiments proposed aim to identify and characterize additional S. aureus genes required for LTA biosynthesis. We have identified two S. aureus mutants with transposon insertions in previously uncharacterized genes that produced LTA of altered structure as judged by western-blot analysis. An experimental design is described herein to characterize these mutants using established biochemical assays. In addition, we propose to construct a plasmid library for expression of S. aureus genes in E. coli and to screen for plasmid clones, which confer to the heterologous host the ability to produce polyglycerolphosphate polymers. LTA is produced by many Gram-positive pathogens, including Group A and B streptococci, Enterococcus faecalis, and B. anthracis. By studying the biosynthesis pathway of this conserved, possibly essential surface molecule in S. aureus, we will further our understanding of the synthesis of an important element of the bacterial envelope. [unreadable] [unreadable] In summary, the bacterium Staphylococcus aureus causes a wide range of human diseases. The bacterial surface harbors many important molecules that allow the bacterium to adhere and invade host cells and cause disease. The goal of this research is to understanding how a particular surface molecule is produced with the goal to inhibit its synthesis, prevent colonization and disease. [unreadable] [unreadable] [unreadable] [unreadable]