Expression and characterization of UDPGlc dehydrogenase (KfiD), which is encoded in the type-specific region 2 of the Escherichia coli K5 capsule genes

Sieberth, Veit; Rigg, Gordon P.; Roberts, Ian S.; Jann, Klaus

doi:10.1128/jb.177.15.4562-4565.1995

Cited by 35 publications

(26 citation statements)

References 32 publications

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“…The purified fusion proteins were used t o successfully generate rabbit polyclonal antisera. The KfiD protein had previously been purified and antisera to the KfiD protein generated (Sieberth et al, 1995).…”

Section: Localization Of the Kfia Kfic Kfid And The Kpsc Kpss And mentioning

confidence: 99%

“…Mutations in any of these genes abolish the production of any detectable K5 polysaccharide, indicating essential roles for these proteins in the biosynthesis of the K5 polysaccharide (Petit et al, 1995). The KfiC protein has been demonstrated to be a glucosyltransferase which adds alternating glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) residues to the non-reducing end of the K5 polysaccharide chain (Petit et al, 1995;Sieberth et al, 1995). The KfiD protein has been purified and shown to be a UDP-glucose dehydrogenase and is responsible for generating UDP-GlcA, which is a substrate for K5 polysaccharide biosynthesis (Petit et al, 1995 ;Sieberth et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

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The localization of KpsC, S and T, and KfiA, C and D proteins involved in the biosynthesis of the Escherichia coli K5 capsular polysaccharide: evidence for a membrane-bound complex

Rigg¹,

Barrett²,

Roberts³

1998

Microbiology

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SUMMARY: Biosynthesis of the Escherichis coli K5 polysaccharide requires the Kf iA, Kf iB, KfiC and KfiD proteins. The subsequent transport of the polysaccharide onto the cell surface requires the KpsC, KpsD, KpsE, KpsM, KpsS and KpsT proteins, which are conserved between different group II capsular polysaccharides. The KfiA and KfiC, together with the KpsC, KpsS and KpsT proteins, were purified and polyclonal antisera to each protein generated. These antisera, together with one previously generated (by others) against the purified KfiD protein, were used in Western blot analysis to locate the corresponding proteins within the cell. Analysis of membrane fractions revealed that KfiA (involved in initiation of polysaccharide synthesis), Kf iC (K5 glycosyl transferase) and the Kf iD protein (UDP-glucose dehydrogenase) were associated with the inner membrane. The KpsC, KpsS and KpsT proteins involved in polysaccharide transport were associated with the inner membrane and this membrane association occurred in the absence of any other capsule-related proteins. The effect of mutations in individual kps genes on the localization of each protein was determined. Mutations in the kpC# kpsM, kpsS and kpsT genes resulted in a loss of membrane targeting for KfiA and KfiC, suggesting some form of hetero-oligomeric membrane-bound biosynthetic complex. Osmotic shock caused the release of KfiA, KfiC, KpsC and KpsS from the inner membrane into the periplasm, suggesting that the polysaccharide biosynthetic complex may be associated with sites of adhesion between the inner and outer membrane.

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Section: Localization Of the Kfia Kfic Kfid And The Kpsc Kpss And mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The localization of KpsC, S and T, and KfiA, C and D proteins involved in the biosynthesis of the Escherichia coli K5 capsular polysaccharide: evidence for a membrane-bound complex

Rigg¹,

Barrett²,

Roberts³

1998

Microbiology

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“…Cps2K contains the same strictly conserved active site signature sequence of GGXCXXXD, as well as extensive homology to the signature NAD ϩ -and UDPsugar binding domains, found in other UDP-GlcDHs (14). UDPGlcDHs play critical roles in the formation of many microbial capsules, including those of Streptococcus pyogenes (25,66), Escherichia coli K5 (52), Cryptococcus neoformans (29), and many S. pneumoniae serotypes (57), as well as mammalian polymers such as hyaluronan, chondroitin sulfate, and heparan sulfate. In many of these polymers, GlcUA is part of the backbone structure, and for capsules such as type 3 in S. pneumoniae, mutations affecting the synthesis of UDP-GlcUA have severe effects on polysaccharide production and virulence (22,31,61).…”

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confidence: 99%

Mutations Blocking Side Chain Assembly, Polymerization, or Transport of a Wzy-DependentStreptococcus pneumoniaeCapsule Are Lethal in the Absence of Suppressor Mutations and Can Affect Polymer Transfer to the Cell Wall

2007

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Extracellular polysaccharides of many bacteria are synthesized by the Wzy polymerase-dependent mechanism, where long-chain polymers are assembled from undecaprenyl-phosphate-linked repeat units on the outer face of the cytoplasmic membrane. In gram-positive bacteria, Wzy-dependent capsules remain largely cell associated via membrane and peptidoglycan linkages. Like many Wzy-dependent capsules, the Streptococcus pneumoniae serotype 2 capsule is branched. In this study, we found that deletions of cps2K, cps2J, or cps2H, which encode a UDP-glucose dehydrogenase necessary for side chain synthesis, the putative Wzx transporter (flippase), and the putative Wzy polymerase, respectively, were obtained only in the presence of suppressor mutations. Most of the suppressor mutations were in cps2E, which encodes the initiating glycosyltransferase for capsule synthesis. The cps2K mutants containing the suppressor mutations produced low levels of highmolecular-weight polymer that was detected only in membrane fractions. cps2K-repaired mutants exhibited only modest increases in capsule production due to the effect of the secondary mutation, but capsule was detectable in both membrane and cell wall fractions. Lethality of the cps2K, cps2J, and cps2H mutations was likely due to sequestration of undecaprenyl-phosphate in the capsule pathway and either preclusion of its turnover for utilization in essential pathways or destabilization of the membrane due to an accumulation of lipid-linked intermediates. The results demonstrate that proper polymer assembly requires not only a functional transporter and polymerase but also complete repeat units. A central role for the initiating glycosyltransferase in controlling capsule synthesis is also suggested.

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“…In addition, in vitro capsule synthesis assays (14) using genetically well-characterized UDP-GlcDH mutants do not allow the direct analysis of the biochemical product encoded by cap3A. Sonicated cell lysates prepared from E. coli JM109(DE3)(pTVU1) were assayed for UDP-GlcDH activity by following a modified protocol based on a method previously described (18). Unless stated otherwise, portions (5 to 40 l, containing 20 to 160 g of protein) of cytosol fractions were incubated in a buffer containing 100 mM Tris (pH 9.0), 10 mM sodium thioglycolate, 1 mM UDP-Glc, and 2 mM NAD, in a (14) Mg 2ϩ was apparently not required for full pneumococcal UDP-GlcDH activity, since the addition of 10 mM EDTA did not inhibit the reaction (not shown).…”

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confidence: 99%

Demonstration of UDP-glucose dehydrogenase activity in cell extracts of Escherichia coli expressing the pneumococcal cap3A gene required for the synthesis of type 3 capsular polysaccharide

1996

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The gene cluster of Streptococcus pneumoniae coding for the type 3 capsular polysaccharide contains four genes (cap3ABCD). A DNA fragment containing the cap3A gene was amplified by PCR and cloned under the control of a T7 RNA polymerase-dependent promoter. Overexpression of this gene in Escherichia coli resulted both in a 47-kDa protein in the cytoplasm of isopropyl-␤-D-thiogalactopyranoside-induced bacteria and in high levels of UDP-glucose dehydrogenase activity. These data demonstrate, in a direct experimental way, that cap3A encodes the UDP-glucose dehydrogenase of pneumococcus type 3.

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Expression and characterization of UDPGlc dehydrogenase (KfiD), which is encoded in the type-specific region 2 of the Escherichia coli K5 capsule genes

Cited by 35 publications

References 32 publications

The localization of KpsC, S and T, and KfiA, C and D proteins involved in the biosynthesis of the Escherichia coli K5 capsular polysaccharide: evidence for a membrane-bound complex

The localization of KpsC, S and T, and KfiA, C and D proteins involved in the biosynthesis of the Escherichia coli K5 capsular polysaccharide: evidence for a membrane-bound complex

Mutations Blocking Side Chain Assembly, Polymerization, or Transport of a Wzy-DependentStreptococcus pneumoniaeCapsule Are Lethal in the Absence of Suppressor Mutations and Can Affect Polymer Transfer to the Cell Wall

Demonstration of UDP-glucose dehydrogenase activity in cell extracts of Escherichia coli expressing the pneumococcal cap3A gene required for the synthesis of type 3 capsular polysaccharide

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