The K1 capsule is an essential virulence determinant of Escherichia coli strains that cause meningitis in neonates. Biosynthesis and transport of the capsule, an α-2,8-linked polymer of sialic acid, are encoded by the 17-kb kps gene cluster. We deleted neuC, a K1 gene implicated in sialic acid synthesis, from the chromosome of EV36, a K-12-K1 hybrid, by allelic exchange. Exogenously added sialic acid restored capsule expression to the deletion strain (∆neuC), confirming that NeuC is necessary for sialic acid synthesis. The deduced amino acid sequence of NeuC showed similarities to those of UDP-N-acetylglucosamine (GlcNAc) 2-epimerases from both prokaryotes and eukaryotes. The NeuC homologue from serotype III Streptococcus agalactiae complements ∆neuC. We cloned the neuC gene into an intein expression vector to facilitate purification. We demonstrated by paper chromatography that the purified neuC gene product catalyzed the formation of [2-(14)C]acetamidoglucal and [N-(14)C]acetylmannosamine (ManNAc) from UDP-[(14)C]GlcNAc. The formation of reaction intermediate 2-acetamidoglucal with the concomitant release of UDP was confirmed by proton and phosphorus nuclear magnetic resonance spectroscopy. NeuC could not use GlcNAc as a substrate. These data suggest that neuC encodes an epimerase that catalyzes the formation of ManNAc from UDP-GlcNAc via a 2-acetamidoglucal intermediate. The unexpected release of the glucal intermediate and the extremely low rate of ManNAc formation likely were a result of the in vitro assay conditions, in which a key regulatory molecule or protein was absent.

biosynthesis, virulence, gene cluster, capsule, Escherichia coli K1

NCBI PubMed ID: 14729696
Journal NLM ID: 2985120R
Publisher: American Society for Microbiology
Correspondence: wvannhelix.nih.gov
Institutions: Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York 14642, USA
 

• Compound ID: 844
  

  -8)-a-Neup5Ac-(2-

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  Structure type: homopolymer
  Trivial name: colominic acid sodium salt, colominic acid, type B polysaccharide, polysialic acid, PSA, oligosaccharide repeating unit, α-2,8-linked polysialic acid
  Compound class: CPS, O-polysaccharide, K-antigen, O-antigen
  Reference(s) to other database(s): GTC:G30588ZL, GlycomeDB:677
  
   
  
  Escherichia coli K1
  CSDB ID 8985 (all data & tools)