Exogenous fatty acids are directly incorporated into bacterial membranes, heavily influencing cell envelope properties, antibiotic susceptibility, and bacterial ecology. Here, we quantify fatty acid biosynthesis metabolites and enzymes of the fatty acid synthesis pathway to determine how exogenous fatty acids inhibit fatty acid synthesis in Escherichia coli. We find that acyl-CoA synthesized from exogenous fatty acids rapidly increases concentrations of long-chain acyl-acyl carrier protein (acyl-ACP), which inhibits fatty acid synthesis initiation. Accumulation of long-chain acyl-ACP is caused by competition with acyl-CoA for phospholipid synthesis enzymes. Furthermore, we find that transcriptional regulation rebalances saturated and unsaturated acyl-ACP while maintaining overall expression levels of fatty acid synthesis enzymes. Rapid feedback inhibition of fatty acid synthesis by exogenous fatty acids thus allows E. coli to benefit from exogenous fatty acids while maintaining fatty acid synthesis capacity. We hypothesize that this indirect feedback mechanism is ubiquitous across bacterial species.

All free-living microorganisms homeostatically maintain the fluidity of their membranes by adapting lipid composition to environmental temperatures. Here, we quantify enzymes and metabolic intermediates of the Escherichia coli fatty acid and phospholipid synthesis pathways, to describe how this organism measures temperature and restores optimal membrane fluidity within a single generation after a temperature shock. A first element of this regulatory system is a temperature-sensitive metabolic valve that allocates flux between the saturated and unsaturated fatty acid synthesis pathways via the branchpoint enzymes FabI and FabB. A second element is a transcription-based negative feedback loop that counteracts the temperature-sensitive valve. The combination of these elements accelerates membrane adaptation by causing a transient overshoot in the synthesis of saturated or unsaturated fatty acids following temperature shocks. This strategy is comparable to increasing the temperature of a water bath by adding water that is excessively hot rather than adding water at the desired temperature. These properties are captured in a mathematical model, which we use to show how hard-wired parameters calibrate the system to generate membrane compositions that maintain constant fluidity across temperatures. We hypothesize that core features of the E. coli system will prove to be ubiquitous features of homeoviscous adaptation systems.

Every cell must produce enough membrane to contain itself. However, the mechanisms by which the rate of membrane synthesis is coupled with the rate of cell growth remain unresolved. By comparing substrate and enzyme concentrations of the fatty acid and phospholipid synthesis pathways of Escherichia coli across a 3-fold range of carbon-limited growth rates, we show that the rate of membrane phospholipid synthesis during steady-state growth is determined principally through allosteric control of a single enzyme, PlsB. Due to feedback regulation of the fatty acid pathway, PlsB activity also indirectly controls synthesis of lipopolysaccharide, a major component of the outer membrane synthesized from a fatty acid synthesis intermediate. Surprisingly, concentrations of the enzyme that catalyzes the committed step of lipopolysaccharide synthesis (LpxC) do not differ across steady-state growth conditions, suggesting that steady-state lipopolysaccharide synthesis is modulated primarily via indirect control by PlsB. In contrast to steady-state regulation, we found that responses to environmental perturbations are triggered directly via changes in acetyl coenzyme A (acetyl-CoA) concentrations, which enable rapid adaptation. Adaptations are further modulated by ppGpp, which regulates PlsB activity during slow growth and growth arrest. The strong reliance of the membrane synthesis pathway upon posttranslational regulation ensures both the reliability and the responsiveness of membrane synthesis. IMPORTANCE How do bacterial cells grow without breaking their membranes? Although the biochemistry of fatty acid and membrane synthesis is well known, how membrane synthesis is balanced with growth and metabolism has remained unclear. This is partly due to the many control points that have been discovered within the membrane synthesis pathways. By precisely establishing the contributions of individual pathway enzymes, our results simplify the model of membrane biogenesis in the model bacterial species Escherichia coli. Specifically, we found that allosteric control of a single enzyme, PlsB, is sufficient to balance growth with membrane synthesis and to ensure that growing E. coli cells produce sufficient membrane. Identifying the signals that activate and deactivate PlsB will resolve the issue of how membrane synthesis is synchronized with growth.