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

Abstract

FabG performs the NADPH-dependent reduction of ␤-keto acyl-acyl carrier protein substrates in the elongation cycle of fatty acid synthesis. We report the characterization of a temperature-sensitive mutation (fabGΔ8) in Escherichia coli fabG that results from an in-frame 8-amino-acid residue deletion in the ␣6/␣7 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 fabGΔ8 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 fabGΔ8 mutation also confers resistance to the calmodulin inhibitor trifluoperazine and renders the enzyme extremely sensitive to Ca 2ϩ in vitro. We also observed a significant alteration in the lipid A fatty acid composition of fabGΔ8 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.KEYWORDS fatty acid synthesis, lipid A, temperature sensitive, antibiotic resistance, calcium, deletion mutant, dimer interface B acteria and plants utilize type II fatty acid synthesis (FAS), a dissociated system of individual enzymes, to generate the entire fatty acid repertoire from acetyl coenzyme A (acetyl-CoA). The intermediates are elongated by a series of four enzymes while

Tachyplesin Causes Membrane Instability That Kills Multidrug-Resistant Bacteria by Inhibiting the 3-Ketoacyl Carrier Protein Reductase FabG

“…N-terminal His-tagged FabG from E. coli and C-terminal His-tagged FabG from Staphylococcus aureus were cloned into the NcoI and XhoI cloning sites of the vector pET28a, expressed in BL21 DE3 E. coli and further purified with Ni resin ( Kristan et al, 2009 ; Srinivas and Cronan, 2017 ). The binding interaction between tachyplesin III and FabG was analyzed by surface plasmon resonance (SPR) with a Biocore 3000 (Biocore, Piscataway, NJ, United States).…”

Genomic and cytogenetic analysis of the Ceratitis capitata temperature-sensitive lethal region

“…1992 ; Varadarajan and Richards 1992 ; Matthews 1995 ; Mela et al . 2009 ; Srinivas and Cronan 2017 ) located in intergenic and promoter regions can be responsible for temperature-sensitive lethal phenotypes. In addition, it was recently shown in the yeast Saccharomyces cerevisiae ( Shen et al .…”

Escherichia coli vectors having stringently repressible replication origins allow a streamlining of Crispr/Cas9 gene editing

“…Most genes in the E. coli fatty acid biosynthetic pathway are essential and have generally been studied using temperature-sensitive (Ts) mutants that grow at lower temperatures but lose function at higher temperatures. (Srinivas and Cronan, 2017). Using plasmid pSgRNA-fabG encoding a single guide RNA that targeted base 714 of fabG (Table 2) and an oligonucleotide targeted at the sequence of the lagging strand, we successfully introduced the E233K mutation into fabG with an efficiency of 99% to give the expected Ts strain (Table 3).…”

“…We used a sgRNA and an oligonucleotide to target and delete a short 32 bp sequence from the fabG gene that corresponds to an 8 residue in-frame deletion in the gene. This deletion results in severe temperature sensitivity with growth seen only at 23 °C (Srinivas and Cronan, 2017). Of the 72 colonies tested 94% contained the deletion (Table 3).…”