Characterization of Pseudomonas aeruginosa DesT (PA4890), a transcriptional factor sensing long‐chain acyl‐CoAs

Abstract

Cells regulate their membrane phospholipid biophysical properties by coordinated synthesis of saturated and unsaturated fatty acids. In bacteria, unsaturated fatty acids are produced by the de novo fatty acid biosynthetic pathway anaerobically, or by oxidative desaturation of the existing fatty acids catalyzed by desaturases. In this study, we have identified and characterized a unique transcriptional repressor in Pseudomonas aeruginosa, DesT (PA4890), which regulates the expression of an acyl-CoA desaturase operon (desCB, PA4889 and PA4888). The desCB operon is located immediately upstream of desT and is transcribed to the opposite direction to desT. Both Affymetrix array and quantitative real-time PCR analyses demonstrated that desC and desB expression were strongly and selectively up-regulated in a ΔdesT deletion strain. The purified recombinant DesT bound to the desCB promoter region at two distinct sites with different affinities. The interaction of DesT with the high-affinity binding site (Kd = 0.35 ± 0.03 nM) was enhanced by unsaturated acyl-CoAs such as palmitoleoyl-CoA and oleoyl-CoA (Kd = 0.10 ± 0.01 nM), whereas saturated acyl-CoAs such as myristoyl-CoA and palmitoyl-CoA prevented DesT interaction with the high-affinity site. CoA, short-chain acyl-CoAs, free fatty acids and acyl-ACPs did not influence DesT-DNA interactions in vitro. Binding to the low-affinity site was not regulated. Accordingly, the expression of desB was up-regulated in cells grown in the presence of stearate and repressed when oleate was present. Thus, DesT senses the composition of the cellular acyl-CoA pool to either increase or decrease the expression of the acyl-CoA Δ9 desaturase system to adjust fatty acid metabolism to the composition of the exogenous fatty acid pool.