In vitro synthesis of a lipid-linked trisaccharide involved in synthesis of enterobacterial common antigen

Barr, Kathleen; Nunes-Edwards, P; Rick, Paul D.

doi:10.1128/jb.171.3.1326-1332.1989

Cited by 25 publications

(20 citation statements)

References 31 publications

(16 reference statements)

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Section: Methodsmentioning

confidence: 99%

“…The total concentration of ECA CYC in samples was adjusted to a final concentration of 5 ϫ 10 2 pmol/l by the addition of 5 mM MgSO 4 . Serial twofold dilutions of the sample in 5 mM MgSO 4 were then prepared to give final concentrations ranging from 5 ϫ 10 2 pmol/l to 0.049 pmol/l. The appropriate amounts of each of these samples were then added to empty wells in a multititer plate to give a row of wells containing serial twofold decreasing amounts of ECA CYC that ranged from 100 pmol to approximately 0.1 pmol.…”

Section: Methodsmentioning

confidence: 99%

“…Many of the genes and enzymes involved in the synthesis and assembly of both ECA PG and ECA CYC have been identified, and this information has been described in detail in previous reports (4,5,15,19,43,44). Briefly, most of the genes known to be involved in the assembly of ECA polysaccharide chains are located in the wec gene cluster, which includes 12 genes located at 85.4 min on the E. coli chromosome (3,10,30,39).…”

mentioning

confidence: 99%

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O Acetylation of the Enterobacterial Common Antigen Polysaccharide Is Catalyzed by the Product of the yiaH Gene of Escherichia coli K-12

et al. 2006

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The carbohydrate component of the enterobacterial common antigen (ECA) of Escherichia coli K-12 occurs primarily as a water-soluble cyclic polysaccharide located in the periplasm (ECACYC) and as a phosphoglyceride-linked linear polysaccharide located on the cell surface (ECAPG). The polysaccharides of both forms are comprised of the amino sugars N-acetyl-d-glucosamine (GlcNAc), N-acetyl-d-mannosaminuronic acid (ManNAcA), and 4-acetamido-4,6-dideoxy-d-galactose (Fuc4NAc). These amino sugars are linked to one another to form trisaccharide repeat units with the structure →3-α-d-Fuc4NAc-(1→4)-β-d-ManNAcA-(1→4)-α-d-GlcNAc-(1→. The hydroxyl group in the 6 position of the GlcNAc residues of both ECACYC and ECAPG are nonstoichiometrically esterified with acetyl groups. Random transposon insertion mutagenesis of E. coli K-12 resulted in the generation of a mutant defective in the incorporation of O-acetyl groups into both ECACYC and ECAPG. This defect was found to be due to an insertion of the transposon into the yiaH locus, a putative gene of unknown function located at 80.26 min on the E. coli chromosomal map. Bioinformatic analyses of the predicted yiaH gene product indicate that it is an integral inner membrane protein that is a member of an acyltransferase family of enzymes found in a wide variety of organisms. The results of biochemical and genetic experiments presented here strongly support the conclusion that yiaH encodes the O-acetyltransferase responsible for the incorporation of O-acetyl groups into both ECACYC and ECAPG. Accordingly, we propose that this gene be designated wecH.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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O Acetylation of the Enterobacterial Common Antigen Polysaccharide Is Catalyzed by the Product of the yiaH Gene of Escherichia coli K-12

et al. 2006

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“…The number of bactoprenyl moieties per cell is small relative to the number of glycerophospholipid molecules (6), but this issue needs to be reinvestigated. The partitioning of bactoprenyl pyrophosphate during the biosynthesis of peptidoglycan (110,111), O-antigen (18,98), enterobacterial common antigen (169), and membrane-derived oligosaccharides (MDO) (170) may be regulated . The biochemistry of isoprene polymerization in bacteria is poorly characterized, and is assumed to be similar to that of [ManRhaGalln-PP-Bactoprenol…”

Section: Biosynthesis Of O-antigenmentioning

confidence: 99%

Biochemistry Of Endotoxins

Raetz¹

1990

Annual Review of Biochemistry

166

192

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“…The trisaccharide repeat unit of ECA PG is assembled as the lipid carrier-linked intermediate Fuc4NAc-ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid III; Fuc4NAc-ManNAcA-GlcNAc-PP-Und) (3,4,37,38). The genes involved in the assembly of lipid III, as well many of the other genes involved in the utilization of lipid III for the assembly of ECA PG , are located in the wec gene cluster at approximately 85.4 min on the E. coli chromosome (2,7,24,32).…”

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confidence: 99%

Assembly of Cyclic Enterobacterial Common Antigen in Escherichia coli K-12

2005

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We describe here the purification and quantification of a water-soluble cyclic form of enterobacterial common antigen (ECA CYC ) from Escherichia coli K-12 as well as information regarding its subcellular location and the genetic loci involved in its assembly. Structural characterization of purified ECA CYC molecules obtained from E. coli K-12 revealed that they uniformly contained four trisaccharide repeat units, and they were substituted with from zero to four O-acetyl groups. Cells from overnight cultures contained approximately 2 g ECA CYC per milligram (dry weight), and cell fractionation studies revealed that these molecules were localized exclusively in the periplasm. The synthesis and assembly of ECA CYC were found to require the wzxE and wzyE genes of the wec gene cluster. These genes encode proteins involved in the transmembrane translocation of undecaprenylpyrophosphate-linked ECA trisaccharide repeat units and the polymerization of trisaccharide repeat units, respectively. Surprisingly, synthesis of ECA CYC was dependent on the wzzE gene, which is required for the modulation of the polysaccharide chain lengths of phosphoglyceride-linked ECA (ECA PG ). The presence of ECA CYC in extracts of several other gram-negative enteric organisms was also demonstrated; however, it was not detected in cell extracts of Pseudomonas aeruginosa. These data suggest that in addition to ECA PG , ECA CYC may be synthesized in many, if not all, members of the Enterobacteriaceae.

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In vitro synthesis of a lipid-linked trisaccharide involved in synthesis of enterobacterial common antigen

Cited by 25 publications

References 31 publications

O Acetylation of the Enterobacterial Common Antigen Polysaccharide Is Catalyzed by the Product of the yiaH Gene of Escherichia coli K-12

O Acetylation of the Enterobacterial Common Antigen Polysaccharide Is Catalyzed by the Product of the yiaH Gene of Escherichia coli K-12

Biochemistry Of Endotoxins

Assembly of Cyclic Enterobacterial Common Antigen in Escherichia coli K-12

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