Single amino acid substitutions in either YhjD or MsbA confer viability to 3-deoxy-d-manno-oct-2-ulosonic acid-depleted Escherichia coli.
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 IV(A), 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 IV(A) from the inner membrane (IM), thus relieving toxic side-effects of lipid IV(A) 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 DeltaKdo phenotype. Further, the yhjD400 suppressor allele renders the LPS transporter MsbA dispensable for lipid IV(A) 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.
Mamat
Uwe
U
Division of Structural Biochemistry, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, D-23845 Borstel, Germany.
Meredith
Timothy C
TC
Aggarwal
Parag
P
Kühl
Annika
A
Kirchhoff
Paul
P
Lindner
Buko
B
Hanuszkiewicz
Anna
A
Sun
Jennifer
J
Holst
Otto
O
Woodard
Ronald W
RW
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D002462:Cell Membrane / Q000737:chemistry; D048168:Escherichia coli K12 / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000502:physiology*; D020043:Genes, Essential; D006017:Glycolipids / Q000032:analysis / Q000737:chemistry; D008050:Lipid A / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D050296:Microbial Viability; D008958:Models, Molecular; D013400:Sugar Acids / Q000378:metabolism*; D013489:Suppression, Genetic
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006017:Glycolipids; D008050:Lipid A; D008565:Membrane Proteins; C079726:MsbA protein, Bacteria; D013400:Sugar Acids; C528319:YhjD protein, E coli; C040142:lipid A precursors, bacterial; C120356:3-deoxy-manno-oct-2-ulopyranosonic acid