Salmonella typhimurium mutants defective in UDP-D-galactose:lipopolysaccharide alpha 1,6-D-galactosyltransferase. Structural, immunochemical, and enzymologic studies of rfaB mutants.

Wollin, Ralfh; Creeger, E S; Rothfield, Lawrence; Stocker, B. A. D.; Lindberg, Alf A.

doi:10.1016/s0021-9258(18)32731-5

Cited by 37 publications

(2 citation statements)

References 17 publications

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“…This is also consistent with previous observations with an S. typhimurium waaB mutant. 26 Despite lacking the branched galactose, additional core sugars were detected in a fraction of the isolated LPS molecules. Our work clearly shows that the branched galactose added by WaaB is not absolutely necessary for the action of other enzymes involved in outer core biosynthesis, consistent with the LPS structures observed in waaB mutants.…”

Section: ■ Discussionmentioning

confidence: 99%

“…This suggests that the more glycosylated LPS species detected in the waaB and Δ gal mutants are due to WaaI adding glucose to Glc-Hep 2 -1-deP-KLA. This is also consistent with previous observations with an S. typhimurium waaB mutant . Despite lacking the branched galactose, additional core sugars were detected in a fraction of the isolated LPS molecules.…”

Section: Discussionmentioning

confidence: 99%

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In Vitro Assembly of the Outer Core of the Lipopolysaccharide from Escherichia coli K-12 and Salmonella typhimurium

2014

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There are five distinct core structures in the lipopolysaccharides of Escherichia coli and at least two in Salmonella isolates, which vary principally in the outer core oligosaccharide. Six outer core glycosyltransferases, E. coli K-12 WaaG, WaaB, and WaaO and Salmonella typhimurium WaaI, WaaJ, and WaaK, were cloned, overexpressed, and purified. A novel substrate for WaaG was isolated from ΔwaaG E. coli overexpressing the lipid A phosphatase lpxE and the lipid A late acyltransferase lpxM. The action of lpxE and lpxM in the ΔwaaG background yielded heptose2-1-dephospho Kdo2-lipid A, a 1-dephosphorylated hexa-acylated lipid A with the inner core sugars that is easily isolated by organic extraction. Using this structurally defined acceptor and commercially available sugar nucleotides, each outer core glycosyltransferases was assayed in vitro. We show that WaaG and WaaB add a glucose and galactose sequentially to heptose2-1-dephospho Kdo2-lipid A. E. coli K-12 WaaO and S. typhimurium WaaI add a galactose to the WaaG/WaaB product but can also add a galactose to the WaaG product directly without the branched core sugar added by WaaB. Both WaaI and WaaO require divalent metal ions for optimal activity; however, WaaO, unlike WaaI, can add several glucose residues to its lipid acceptor. Using the product of WaaG, WaaB, and WaaI, we show that S. typhimurium WaaJ and WaaK transfer a glucose and N-acetylglucosamine, respectively, to yield the full outer core. This is the first demonstration of the in vitro assembly of the outer core of the lipopolysaccharide using defined lipid A-oligosaccharide acceptors and sugar donors.

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Section: ■ Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

In Vitro Assembly of the Outer Core of the Lipopolysaccharide from Escherichia coli K-12 and Salmonella typhimurium

2014

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Interaction between phage G13 and its oligosaccharide receptor studied by equilibrium dialysis

Bruse¹,

Wollin²,

Lindberg

1989

J of Molecular Recognition

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The reversible binding of phage G13, a phi X174-like single-strand DNA phage, to a 3H-labelled nonasaccharide from the lipopolysaccharide of its natural host Escherichia coli C was studied with equilibrium dialysis. The binding constant (Ka) was determined to 1.3 x 10(7) M-1 in Scatchard and Lineweaver-Burk plots. Approximately one saccharide bound per G13 phage particle which suggests that only one of the 12 spikes in each G13 virion was engaged in the phage/receptor saccharide interaction. Equilibrium dialysis inhibition experiments with saccharides from lipopolysaccharides of an isogenic series of Salmonella typhimurium mutants showed that hepta- and pentasaccharides from two G13-sensitive bacteria, i.e., with efficiencies of plating of 0.1-1.0 compared to E. coli C, were efficient inhibitors with Ka-values greater than or equal to 1.2 x 10(7) M-1. The octa- and hexasaccharides from two G13 resistant strains, with efficiency of plating less than or equal to x 10(-4), were either greater than 1000-fold or greater than 15-fold less efficient as inhibitors with Ka-values less than or equal to 8.8 x 10(5) M-1. The results show that phage G13 binds in a specific and reversible way to penta-, hepta-, and nonasaccharides from G13 sensitive bacteria with the specificity residing in the hexose and heptose region of the core lipopolysaccharide.

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Definition of the phage G13 receptor as structural domains of trisaccharides in Salmonella and Escherichia coli core oligosaccharides

Wollin¹,

Bruse²,

Jansson

et al. 1989

J of Molecular Recognition

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The interaction between phage G13 and different bacterial and synthetic oligosaccharides has been studied using equilibrium dialysis inhibition. The results, and conformational analysis of the oligosaccharides, make us conclude that the phage G13 carbohydrate receptor is a conformational domain involving three sugar residues. The following trisaccharide elements contain the domain: alpha-D-Galp-(1----3)-[alpha-D-Galp-(1----6)]-alpha-D-Glcp, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)-alpha-D-Manp , and alpha-D-Glcp-(1----3)-[L-gly-alpha-D-man-Hepp-(1----7)]-L-gly-alph a-D- man-Hepp. Thus two structures, either a hexose substituted with alpha-D-glycopyranosyl groups in the 3- and 6-positions, or a heptose substituted with such groups in the 3- and 7-positions are functional G13 binding sites. Such domains are present in several cores of lipopolysaccharides from Salmonella and Escherichia coli species. Some cores, e.g. those from S. typhimurium chemotypes Ra, Rb1 and Rb2, contain two such domains. The identification of two G13 receptor domains within different core saccharides could explain the broad host range of this phage.

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Salmonella typhimurium mutants defective in UDP-D-galactose:lipopolysaccharide alpha 1,6-D-galactosyltransferase. Structural, immunochemical, and enzymologic studies of rfaB mutants.

Cited by 37 publications

References 17 publications

In Vitro Assembly of the Outer Core of the Lipopolysaccharide from Escherichia coli K-12 and Salmonella typhimurium

In Vitro Assembly of the Outer Core of the Lipopolysaccharide from Escherichia coli K-12 and Salmonella typhimurium

Interaction between phage G13 and its oligosaccharide receptor studied by equilibrium dialysis

Definition of the phage G13 receptor as structural domains of trisaccharides in Salmonella and Escherichia coli core oligosaccharides

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