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ABSTRACT Chemotaxis and motility are important traits that support bacterial survival in various ecological niches and in pathogenic and symbiotic host interaction. Chemotactic stimuli are sensed by chemoreceptors or m ethyl-accepting c hemotaxis p roteins (MCPs), which direct the swimming behavior of the bacterial cell. In this study, we present evidence that the cellular abundance of chemoreceptors in the plant symbiont Sinorhizobium meliloti can be altered by the addition of several to as few as one amino acid residues and by including common epitope tags such as 3×FLAG and 6×His at their C termini. To further dissect this phenomenon and its underlying molecular mechanism, we focused on a detailed analysis of the amino acid sensor McpU. Controlled proteolysis is important for the maintenance of an appropriate stoichiometry of chemoreceptors and between chemoreceptors and chemotactic signaling proteins, which is essential for an optimal chemotactic response. We hypothesized that enhanced stability is due to interference with protease binding, thus affecting proteolytic efficacy. Location of the protease recognition site was defined through McpU stability measurements in a series of deletion and amino acid substitution mutants. Deletions in the putative protease recognition site had similar effects on McpU abundance, as did extensions at the C terminus. Our results provide evidence that the programmed proteolysis of chemotaxis proteins in S. meliloti is cell cycle regulated. This posttranslational control, together with regulatory pathways on the transcriptional level, limits the chemotaxis machinery to the early exponential growth phase. Our study identified parallels to cell cycle-dependent processes during asymmetric cell division in Caulobacter crescentus . IMPORTANCE The symbiotic bacterium Sinorhizobium meliloti contributes greatly to growth of the agriculturally valuable host plant alfalfa by fixing atmospheric nitrogen. Chemotaxis of S. meliloti cells toward alfalfa roots mediates this symbiosis. The present study establishes programmed proteolysis as a factor in the maintenance of the S. meliloti chemotaxis system. Knowledge about cell cycle-dependent, targeted, and selective proteolysis in S. meliloti is important to understand the molecular mechanisms of maintaining a suitable chemotaxis response. While the role of regulated protein turnover in the cell cycle progression of Caulobacter crescentus is well understood, these pathways are just beginning to be characterized in S. meliloti . In addition, our study should alert about the cautionary use of epitope tags for protein quantification. 

ABSTRACT Staphylococcus aureus can utilize exogenous fatty acids for phospholipid synthesis. The fatty acid kinase FakA is essential for this utilization by phosphorylating exogenous fatty acids for incorporation into lipids. How FakA impacts the lipid membrane composition is unknown. In this study, we used mass spectrometry to determine the membrane lipid composition and properties of S. aureus in the absence of fakA . We found the fakA mutant to have increased abundance of lipids containing longer acyl chains. Since S. aureus does not synthesize unsaturated fatty acids, we utilized oleic acid (18:1) to track exogenous fatty acid incorporation into lipids. We observed a concentration-dependent incorporation of exogenous fatty acids into the membrane that required FakA. We also tested how FakA and exogenous fatty acids impact membrane-related physiology and identified changes in membrane potential, cellular respiration, and membrane fluidity. To mimic the host environment, we characterized the lipid composition of wild-type and fakA mutant bacteria grown in mouse skin homogenate. We show that wild-type S. aureus 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 unclear. Our results reveal lipid composition changes in response to fatty acid addition and when S. aureus is unable to activate fatty acids through FakA. We identify concentration-dependent utilization of oleic acid that, when combined with previous work, provides evidence that fatty acids can serve as a signal to S. aureus . Furthermore, using mouse skin homogenates as a surrogate for in vivo conditions, we showed that S. aureus can incorporate host fatty acids. This study highlights how exogenous fatty acids impact bacterial membrane composition and function.