An Eight-Residue Deletion in Escherichia coli FabG Causes Temperature-Sensitive Growth and Lipid Synthesis Plus Resistance to the Calmodulin Inhibitor Trifluoperazine

Abstract

ABSTRACT FabG performs the NADPH-dependent reduction of β-keto acyl-acyl carrier protein substrates in the elongation cycle of fatty acid synthesis. We report the characterization of a temperature-sensitive mutation ( fabG Δ 8 ) in Escherichia coli fabG that results from an in-frame 8-amino-acid residue deletion in the α6/α7 subdomain. This region forms part of one of the two dimerization interfaces of this tetrameric enzyme and is reported to undergo significant conformational changes upon cofactor binding, which define the entrance to the active-site cleft. The activity of the mutant enzyme is extremely thermolabile and is deficient in forming homodimers at nonpermissive temperatures with a corresponding decrease in fatty acid synthesis both in vivo and in vitro . Surprisingly, the fabG Δ 8 strain reverts to temperature resistance at a rate reminiscent of that of a point mutant with intragenic pseudorevertants located either on the 2-fold axes of symmetry or at the mouth of the active-site cleft. The fabG Δ 8 mutation also confers resistance to the calmodulin inhibitor trifluoperazine and renders the enzyme extremely sensitive to Ca 2+ in vitro . We also observed a significant alteration in the lipid A fatty acid composition of fabG Δ 8 strains but only in an lpxC background, probably due to alterations in the permeability of the outer membrane. These observations provide insights into the structural dynamics of FabG and hint at yet another point of regulation between fatty acid and lipid A biosynthesis. IMPORTANCE Membrane lipid homeostasis and its plasticity in a variety of environments are essential for bacterial survival. Since lipid biosynthesis in bacteria and plants is fundamentally distinct from that in animals, it is an ideal target for the development of antibacterial therapeutics. FabG, the subject of this study, catalyzes the first cofactor-dependent reduction in this pathway and is active only as a tetramer. This study examines the interactions responsible for tetramerization through the biochemical characterization of a novel temperature-sensitive mutation caused by a short deletion in an important helix-turn-helix motif. The mutant strain has altered phospholipid and lipid A compositions and is resistant to trifluoperazine, an inhibitor of mammalian calmodulin. Understanding its structural dynamics and its influence on lipid A synthesis also allows us to explore lipid homeostasis as a mechanism for antibiotic resistance.