Assembly of lipopolysaccharide in Escherichia coli requires the essential LapB heat shock protein.
Here, we describe two new heat shock proteins involved in the assembly of LPS in Escherichia coli, LapA and LapB (lipopolysaccharide assembly protein A and B). lapB mutants were identified based on an increased envelope stress response. Envelope stress-responsive pathways control key steps in LPS biogenesis and respond to defects in the LPS assembly. Accordingly, the LPS content in ΔlapB or Δ(lapA lapB) mutants was elevated, with an enrichment of LPS derivatives with truncations in the core region, some of which were pentaacylated and exhibited carbon chain polymorphism. Further, the levels of LpxC, the enzyme that catalyzes the first committed step of lipid A synthesis, were highly elevated in the Δ(lapA lapB) mutant. Δ(lapA lapB) mutant accumulated extragenic suppressors that mapped either to lpxC, waaC, and gmhA, or to the waaQ operon (LPS biosynthesis) and lpp (Braun's lipoprotein). Increased synthesis of either FabZ (3-R-hydroxymyristoyl acyl carrier protein dehydratase), slrA (novel RpoE-regulated non-coding sRNA), lipoprotein YceK, toxin HicA, or MurA (UDP-N-acetylglucosamine 1-carboxyvinyltransferase) suppressed some of the Δ(lapA lapB) defects. LapB contains six tetratricopeptide repeats and, at the C-terminal end, a rubredoxin-like domain that was found to be essential for its activity. In pull-down experiments, LapA and LapB co-purified with LPS, Lpt proteins, FtsH (protease), DnaK, and DnaJ (chaperones). A specific interaction was also observed between WaaC and LapB. Our data suggest that LapB coordinates assembly of proteins involved in LPS synthesis at the plasma membrane and regulates turnover of LpxC, thereby ensuring balanced biosynthesis of LPS and phospholipids consistent with its essentiality.
From the Faculty of Chemistry, Gdansk University of Technology, Narutowicza 11/12, 80-233 Gdansk, Poland and the Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 22, 23845 Borstel, Germany.
Gracjana
G
Klein
From the Faculty of Chemistry, Gdansk University of Technology, Narutowicza 11/12, 80-233 Gdansk, Poland and.
Natalia
N
Kobylak
the Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 22, 23845 Borstel, Germany.
Buko
B
Lindner
From the Faculty of Chemistry, Gdansk University of Technology, Narutowicza 11/12, 80-233 Gdansk, Poland and.
Anna
A
Stupak
From the Faculty of Chemistry, Gdansk University of Technology, Narutowicza 11/12, 80-233 Gdansk, Poland and the Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 22, 23845 Borstel, Germany satish.raina@pg.gda.pl.
Satish
S
Raina
D049069:ATP-Dependent Proteases / Q000235:genetics / Q000378:metabolism; D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D020043:Genes, Essential / Q000235:genetics; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D008070:Lipopolysaccharides / Q000096:biosynthesis* / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020133:Reverse Transcriptase Polymerase Chain Reaction
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008070:Lipopolysaccharides; D008565:Membrane Proteins; C586514:YciM protein, E coli; D016695:Glycosyltransferases; C080934:lipooligosaccharide 1,5-heptosyltransferase; D049069:ATP-Dependent Proteases; C513383:FtsH protein, E coli; D000581:Amidohydrolases; C097307:UDP-3-O-acyl-N-acetylglucosamine deacetylase; C063842:dnaK protein, E coli
Endotoxin; Glycobiology; Glycolipid Structure; Glycosyltransferases; Heptosyltransferase; Lipid A; Lipopolysaccharide (LPS); Myristoyltransferase; RpoH; Tetratricopeptide Repeat (TPR)