Single amino acid substitutions in either YhjD or MsbA confer viability to 3-deoxy-D-manno-oct-2-ulosonic acid-depleted Escherichia coli

Uwe Mamat, Timothy C. Meredith, Parag Aggarwal, Annika Kühl, Paul Kirchhoff, Buko Lindner, Anna Hanuszkiewicz, Jennifer Sun, Otto Holst, Ronald W. Woodard

Research output: Contribution to journalArticlepeer-review

35 Scopus citations

Abstract

The Escherichia coli K-12 strain KPM22, defective in synthesis of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo), is viable with an outer membrane (OM) composed predominantly of lipid IVA, a precursor of lipopolysaccharide (LPS) biosynthesis that lacks any glycosylation. To sustain viability, the presence of a second-site suppressor was proposed for transport of lipid IVA from the inner membrane (IM), thus relieving toxic side-effects of lipid IVA accumulation and providing sufficient amounts of LPS precursors to support OM biogenesis. We now report the identification of an arginine to cysteine substitution at position 134 of the conserved IM protein YhjD in KPM22 that acts as a compensatory suppressor mutation of the lethal ΔKdo phenotype. Further, the yhjD400 suppressor allele renders the LPS transporter MsbA dispensable for lipid IVA transmembrane trafficking. The independent derivation of a series of non-conditional KPM22-like mutants from the Kdo-dependent parent strain TCM15 revealed a second class of suppressor mutations localized to MsbA. Proline to serine substitutions at either residue 18 or 50 of MsbA relieved the Kdo growth dependence observed in the isogenic wild-type strain. The possible impact of these suppressor mutations on structure and function are discussed by means of a computationally derived threading model of MsbA.

Original languageEnglish (US)
Pages (from-to)633-648
Number of pages16
JournalMolecular Microbiology
Volume67
Issue number3
DOIs
StatePublished - Feb 2008

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology

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