Characterization of the Staphylococcus aureus fatty acid degradation pathway - PROJECT SUMMARY/ABSTRACT Bacteria require fatty acids for a variety of biological functions, including the construction of phospholipids. Staphylococcus aureus, like other bacteria, synthesize fatty acids using a fatty acid biosynthesis pathway referred to as FASII, but can also scavenge fatty acids from the environment using the recently described fatty acid kinase (Fak) pathway. In addition to fatty acid biosynthesis and acquisition, it is common for bacteria to degrade fatty acids using fatty acid degradation (Fad) enzymes. Bacteria can degrade fatty acids for energy production (known as b-oxidation) but can also degrade fatty acids to alter the length of the acyl chain. S. aureus has been thought to not possess the capacity to degrade or perform b-oxidation of fatty acids. This is due, in part, to the absence of a key crotonase enzyme. This is surprising considering that S. aureus is annotated to encode all the other necessary Fad enzymes, though their function has not been confirmed. During RNAseq studies of the Fak pathway, we observed an ~17-fold increased expression of the group of genes annotated to encode Fad proteins when the Fak pathway was inactivated. Indeed, no crotonase enzyme was apparent, though all the other Fad functionalities were annotated. Using bioinformatics, we identified a putative crotonase enzyme in S. aureus that we now call FadB based on nomenclature in other systems. When co-expressed with S. aureus FadA, FadB can functionally complement an E. coli fadA and fadB mutants on minimal media with fatty acid as a sole carbon source. This demonstrates that 1) S. aureus FadB can substitute for the E. coli crotonase activity-containing enzyme, and 2) S. aureus does possess a complete Fad pathway and likely can degrade fatty acids. We seek to characterize the regulation of this pathway, demonstrate the saFadA and saFadB proteins form a complex like the E. coli proteins, and demonstrate the functionality of the S. aureus Fad pathway in three Specific Aims. Aim 1 will identify regulatory proteins of the Fad operon. Aim 2 determines if the S. aureus FadA and FadB proteins form a complex. In addition, Aim 3 examines the ability for S. aureus to degrade fatty acids and use them as alternative carbon sources using controlled expression of the Fad genes using a combination of 13C- labeled fatty acids with mass spectrometry and growth assays. The Fad-encoding genes have been identified in a variety of transcriptomic studies but have remained unstudied. This is likely due to the known dogma in the field that S. aureus does not possess a complete Fad pathway and cannot degrade fatty acids. We anticipate that the completion of this application will redefine fatty acid metabolism in S. aureus and determine for the first time that S. aureus can degrade fatty acids. This will change how the field understands S. aureus metabolism and will set the stage for future applications examining the role of this pathway in cell physiology and pathogenesis.
