An eightresidue deletion in Escherichia coli FabG causes temperature-sensitive growth and lipid synthesis plus resistance to the calmodulin inhibitor trifluoperazine

Swaminath Srinivas, John E. Cronan

Research output: Contribution to journalArticle

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 (fabGΔ8) 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 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 Ca2+ 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.

Original languageEnglish (US)
Article numbere00074-17
JournalJournal of bacteriology
Volume199
Issue number10
DOIs
StatePublished - May 1 2017

Keywords

  • Antibiotic resistance
  • Calcium
  • Deletion mutant
  • Dimer interface
  • Fatty acid synthesis
  • Lipid A
  • Temperature sensitive

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

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