Membrane homeostasis: Fabulous feedback

Lipidomics Gateway (30 December 2009) [doi:10.1038/lipidmaps.2009.36]

A bacterial transcription factor responds to the ratio of unsaturated versus saturated fatty acids to balance de novo phospholipid synthesis.

Conserved pathway for the formation of phosphatidic acid in bacteria. From: Zhang, Y.-M. & Rock, C. R. Nat. Rev. Micro. 6, 222-233 (2008) doi:10.1038/nrmicro1839

Regulation of de novo fatty acid synthesis is essential to control the biophysical properties of bacterial membranes. Membrane phospholipids that have an unsaturated fatty acyl chain pack together more loosely than saturated molecules, increasing membrane fluidity. In Eschericia coli, fatty acid and phospholipid biosynthetic pathways are well characterized, but the mechanism for 'sensing' fatty acid composition of the membrane was unknown. Now, Charles Rock and colleagues report in the Journal of Biological Chemistry that monounsaturated fatty acids (UFAs) increase binding of the transcription factor FabR to promoters that control expression of UFA biosynthetic enzymes. Saturated fatty acids (SFAs), by contrast, prevent FabR-mediated repression of UFA synthesis. This feedback mechanism balances the availability of UFAs and SFAs for incorporation into membrane phospholipids.

Bacterial membrane phospholipid synthesis involves an iterative process of elongation of fatty acid chains tethered to acyl carrier protein (ACP). Mature, long-chain acyl-ACPs are substrates for acyltransferases, which attach the fatty acids to glycerol-3-phosphate to form phospholipids. The enzymes that initiate synthesis, and the dehydratase and condensing enzymes that produce saturated fatty acids, are universally expressed in bacteria. Some bacteria, including E. coli, also express alternative isoforms that can introduce a double bond at the 10-carbon stage to form UFAs. The bifunctional FabA dehydratase/ isomerase generates a cis isomer that can only be elongated by the FabB condensing enzyme.

The overall synthesis of fatty acids is regulated by product inhibition at several stages, whereas the production of UFAs depends on the expression of FabA and FabB. Expression of the fabA and fabB genes is repressed by FabR binding to the promoter sequence. To find out how this binding is controlled, Rock and colleagues developed a procedure to recover soluble FabR from E. coli inclusion bodies, and then carried out ligand binding screens. They expected to find a limiting intracellular ligand based on data from expression of a FabR plasmid. In fabR knockout strains UFA content was raised and could be restored to normal by introduction of the plasmid. However, overexpression of FabR in a wildtype strain did not decrease UFA content, indicating that it does not bind DNA in the absence of ligand.

Gel shift experiments revealed that long chain UFA-ACP, but not short-chain or saturated acyl-ACPs, increased FabR binding to the fabA and fabB promoters, and this was antagonized by the presence of SFA-ACP. E. coli can save energy by using exogenous fatty acids for phospholipid synthesis, converting them to acyl-CoA thioesters. UFA-CoA and SFA-CoA behaved in the same way as their ACP counterparts. These data are consistent with induction of a conformational change in FabR by UFA ligands, which does not occur with binding of SFA ligands. The data also suggested that FabR responds to the UFA:SFA ratio rather than the concentration of UFA. To test this, the authors perturbed the ratio through inhibition of an enzyme that would normally use about a third of new SFAs for lipid A biosynthesis. The inhibition abruptly decreased the UFA:SFA ratio, which gradually recovered only in the presence of FabR.

This study adds FabR to a growing list of bacterial transcriptional regulators that respond to fatty acid structure, and reveals how E. coli can adjust the proportion of UFA formed by de novo synthesis to balance the composition of fatty acids from the environment and preserve membrane integrity.

Emma Leah