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1.
J Bacteriol ; 186(3): 706-12, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14729696

ABSTRACT

The K1 capsule is an essential virulence determinant of Escherichia coli strains that cause meningitis in neonates. Biosynthesis and transport of the capsule, an alpha-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 (DeltaneuC), 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 DeltaneuC. 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.


Subject(s)
Carbohydrate Epimerases/physiology , Escherichia coli Proteins/physiology , Carbohydrate Epimerases/chemistry , Carbohydrate Epimerases/genetics , Magnetic Resonance Spectroscopy
2.
FEMS Microbiol Lett ; 224(1): 113-8, 2003 Jul 15.
Article in English | MEDLINE | ID: mdl-12855177

ABSTRACT

The K1 capsule, an alpha(2,8)-linked polymer of sialic acid, is an important virulence determinant of invasive Escherichia coli. The 17-kb kps gene cluster of E. coli K1 encodes the information necessary for capsule expression at the cell surface. Two proteins, KpsM and KpsT, play a role in the transport of capsular polysaccharide across the cytoplasmic membrane, utilizing the energy from ATP hydrolysis. They belong to the ATP-binding cassette superfamily of transport proteins. In this study, we purified KpsT in its native form and show that the purified protein is able to bind ATP, undergo an ATP-dependent conformational change and hydrolyze ATP. Protease accessibility studies demonstrate the in vivo interaction between KpsM and KpsT.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Adenosine Triphosphate/analogs & derivatives , Bacterial Capsules/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/isolation & purification , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacokinetics , Azides/pharmacokinetics , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/isolation & purification , Membrane Transport Proteins/metabolism , Phosphorus Radioisotopes , Photochemistry , Plasmids , Protein Structure, Tertiary
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