Staphylococcus aureus is a significant human pathogen, which is the leading cause of surgical-wound infections and the second most frequent cause of nosocomial bloodstream infections in the United States. A significant number of cases of food poisoning are also linked to contamination of foodstuffs with S. aureus. Almost every tissue and organ within the human body is susceptible to infections by this species. Many of the current infections are caused by staphylococcal strains that are resistant to one or more antibiotics. Eighty to ninety percent of all S. aureus strains are resistant to the antibiotic penicillin and up to fifty percent of all strains isolated from patients in hospitals are resistant to methicillin. Recent outbreaks of community-acquired S. aureus possessing methicillin resistance and the emergence of vancomycin-resistant S. aureus strains mean that some strains may be untreatable by any antibiotic. Because S. aureus is able to infect so many different tissues within the human body, this grant proposes to study proline transport in S. aureus as a means to study the role proline transporters play in the pathogenesis of the bacteria. At least two proline transport systems are known for S. aureus. This grant proposes to identify the homolog of the ProP low affinity proline transporter and mutate the prop gene by allelic exchange or transposon mutagenesis with Tn917. With this proP mutant, a proPputP double mutant will then be created. Both the single and double mutants will be tested for proline transport differences in vitro and for their attenuation in animal models of infection. The regulation of the high affinity proline transport gene, putP, also will be tested in vitro in proline uptake assays and in vivo in several animal models of infection using a putP transcriptional fusion. The results of this study will help us understand the role of proline transport in S. aureus infections.