Amino Acid Metabolism Controls Fatty Acid Structure in Staphylococcus aureus

Abstract

Staphylococcus aureus produces a combination of branched and straight chain fatty acids to maintain membrane homeostasis. Recently, we showed that the branched:straight chain ratio in fatty acids is controlled by amino acid metabolism that supplies the branched‐chain acyl‐CoA primers for fatty acid biosynthesis. The fatty acid composition is determined when FabH catalyzes the initial condensation using the available short‐chain acyl‐CoA primers with malonyl‐acyl carrier protein. Branched‐chain ketoacid dehydrogenase (Bkd) is recognized as the primary enzyme responsible for the formation of branched‐chain acyl‐CoA primers. Most experiments with S. aureus are carried out in media with an abundant supply of branched‐chain amino acids where the Bkd pathway for the utilization of Ile dominates. However, branched‐chain fatty acids are still produced in bkd knockout strains and the enzyme(s) responsible for this alternate biosynthetic pathway is unknown. It is important to understand how S. aureus copes with limited availability of Ile because multiple laboratories conclude that isoleucine is a limiting nutrient at S. aureus infection sites that regulates metabolism and virulence.Mass tracing experiments with heavy amino acids show that Bkd is the key step in a pathway that selectively converts extracellular Ile into 2‐methyl‐butyryl‐CoA (anteiso‐C5‐CoA) that FabH uses to initiate anteiso fatty acid synthesis. The phosphatidylglycerol (PG) molecular species in wild‐type strains consists of a mixture of anteiso‐17:0 and ‐19:0 and 18:0 and 20:0 straight chain fatty acids in the 1‐position paired with anteiso‐15:0 in the 2‐position. If Ile is removed from the medium, Leu metabolism results in the introduction of iso‐15:0 into the 2‐position. Val is not significantly metabolized by the Bkd pathway. The analysis of the PG molecular species in strains lacking an active Bkd complex have a straight chain fatty acid in the 1‐position paired with either iso‐14:0 or anteiso‐15:0 in the 2‐position. This same switch in PG molecular species composition is observed when wild‐type strains are grown in defined medium lacking both Ile and Leu. Our metabolomics analyses show that C5‐CoA derived from the Bkd pathway is the most abundant acyl‐CoA primer available for FabH in wild‐type strains; however, when Ile and Leu are not present in the medium, iso‐C4‐CoA increases and anateiso‐C5‐CoA decreases. In the bkd knockout strains, the C4‐CoA pool is even more elevated, and the C5‐CoA pool is substantially lower. This means that in the absence of Bkd, a second pathway creates a supply of iso‐C4‐CoA for FabH to prime fatty acid synthesis and produce iso‐14:0 for the 2‐position. Thus, there are two pathways for the formation of branched‐chain acyl‐CoA substrates for FabH. The Bkd pathway is responsible for the utilization of extracellular Ile (and to a lesser extent, Leu) to initiate fatty acid synthesis, whereas the alternate, de novo biosynthetic pathway makes a significant contribution in wild‐type strain growth without exogenous Ile and is the only process operating in the bkd knockout strains. We are testing the hypothesis that the key enzyme in the de novo pathway to iso‐C4‐CoA is CidC, an enzyme related to pyruvate oxidase.