The flow rate was 500 l min?1, using a column heat of 30C. homogenate. We show that wild-type can incorporate exogenous unsaturated fatty acids from host tissue, highlighting the importance of FakA in the presence of host skin tissue. In conclusion, FakA is important for maintaining the composition and properties of the phospholipid membrane in the presence of exogenous fatty acids, impacting overall cell physiology. IMPORTANCE Environmental fatty acids can be harvested to supplement endogenous fatty acid synthesis to produce membranes and circumvent fatty acid biosynthesis inhibitors. However, how the inability to use these fatty acids impacts lipids is usually unclear. Our results reveal lipid composition changes in response to fatty acid addition and when is unable to activate fatty acids through FakA. We identify concentration-dependent utilization of Droxinostat oleic acid that, when combined with previous work, provides evidence that fatty acids can serve as a signal to conditions, we showed that can incorporate host fatty acids. This study highlights how exogenous fatty acids impact bacterial membrane composition and function. remains a tremendous cause of contamination and morbidity in the human population (1). Approximately 30% of the population are asymptomatic carriers of (2); however, this bacterium can cause contamination in numerous anatomical sites, including skin and soft tissues, bones, lungs, and the heart, as well as foreign implants, such as catheters and prosthetic joints (3). While contamination was originally characterized as a typically hospital-acquired contamination, the incidence of infections Droxinostat in the community has increased concern and awareness of this pathogen, as community-associated strains have become dominant in the United States (4, 5). Thus, a thorough understanding of how can establish contamination, fend off the immune system, and maintain contamination is needed to combat this pathogen. Phospholipids lie at the interface of the host-pathogen conversation. Membrane-associated products, such as lipopolysaccharides, lipoteichoic acids, and lipoproteins, are sensed by the germ line-encoded pattern recognition receptors that induce the activity of numerous host immune cells (6, 7). In addition to these membrane products, phospholipids themselves can play a role in evading the immune system. For example, lysyl-phosphatidylglycerol (LPG) has been shown to be important for evading neutrophils and antimicrobial peptides (8, 9). The composition of the phospholipid membranes of bacteria can also dictate if antimicrobial treatment during contamination is successful. Resistance to daptomycin, a lipopeptide antimicrobial, can result from the mutation of cardiolipin (CL) synthase (endogenously synthesizes fatty acids via the fatty acid synthesis type II system (FASII) (13). Due to the differences between fatty acid synthesis enzymes of bacteria and humans, FASII has been the subject of antimicrobial targeting (14,C17). Bacteria, including synthesizes predominantly three classes of phospholipids: phosphatidylglycerol (PG), LPG, and CL (8, 19). One interesting caveat to fatty acid and lipid synthesis in is the inability of this bacterium to synthesize unsaturated fatty acids (20). Instead, utilizes branched-chain fatty acids (BCFAs), derived from the branched-chain amino acids isoleucine, leucine, and valine, to help modulate the membrane in response to environmental stimuli (21, 22). A large portion of the BCFAs produced by include odd-numbered iso and anteiso BCFAs, with an acyl chain length of 15 being the most abundant (22, 23). FakA was first identified as a regulator of virulence due to the decrease in -hemolysin activity, increased protease activity, and increased dermonecrosis in a murine model of contamination (24). Originally named virulence factor regulator B Droxinostat (VfrB) due to this altered virulence, FakA was eventually identified to be a fatty acid kinase (18). Subsequently, the altered virulence factor profile of a mutant was OPD2 identified to be due, in part, to altered activity of the SaeRS two-component system (25, 26). The current model for the FakA-dependent alteration of SaeRS signaling is due to the accumulation of fatty acids within the cell (26). A mechanism for how these accumulated fatty acids within the cell decrease SaeRS signaling is still Droxinostat undetermined. The absence of FakA affects global metabolism (27) and increases the resistance of to toxic fatty acids (28, 29). How the inability to use exoFAs affects the overall membrane lipid composition has not been evaluated. In the current study, we aimed to determine the changes in membrane lipid composition in the absence of FakA. We tested how these overall membrane lipid changes affect the properties of the membrane.