Identification that KfiA, a Protein Essential for the Biosynthesis of the Escherichia coli K5 Capsular Polysaccharide, is an alpha -UDP-GlcNAc Glycosyltransferase: The Formation of a Membrane Associated K5 Biosynthetic Complex Requires KfiA, KfiB and KfiC

Hodson, Nigel; Griffiths, Gary L.; Cook, Nicola; Pourhossein, Meraj; Gottfridson, Eva; Lind, Thomas; Lidholt, Kerstin; Roberts, Ian S.

doi:10.1074/jbc.m004426200

Cited by 31 publications

(42 citation statements)

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“…Region 2, flanked by regions 1 and 3, contains the highly variable serotype-specific genes involved in the biosynthesis of the particular polysaccharide (12, 13). In the case of the K5 capsule gene cluster, this involves the kfiABCD genes (9).The biosynthesis of the K5 polysaccharide occurs through the sequential addition of GlcA and GlcNAc residues to the nonreducing end of the polysaccharide chain catalyzed by two glycosyltransferases, KfiA and KfiC (4,6). Polysaccharide biosynthesis occurs at the cytoplasmic face of the inner membrane and involves a hetero-oligomeric complex, consisting of proteins involved in both biosynthesis and export, that is localized at the pole of the cell (8).…”

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“…The biosynthesis of the K5 polysaccharide occurs through the sequential addition of GlcA and GlcNAc residues to the nonreducing end of the polysaccharide chain catalyzed by two glycosyltransferases, KfiA and KfiC (4,6). Polysaccharide biosynthesis occurs at the cytoplasmic face of the inner membrane and involves a hetero-oligomeric complex, consisting of proteins involved in both biosynthesis and export, that is localized at the pole of the cell (8).…”

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The Escherichia coli K5 Capsule Is Not Synthesized in a Protected Compartment within the Cytoplasm

2009

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The intracellular expression of the K5 lyase enzyme, which degrades the K5 polysaccharide, decreased cell surface expression of the Escherichia coli K5 capsule. This indicates that biosynthesis of K5 polysaccharide in the cytoplasm is accessible to the action of K5 lyase and is not synthesized within a protected cytoplasmic compartment.The polysaccharide capsules of bacteria have been studied in most detail in Escherichia coli (13). E. coli has over 80 chemically and serologically distinct polysaccharide capsules, which are designated K antigens and classified into four groups (13). Group 2 polysaccharide capsules, of which K1 and K5 have been most studied (12, 13), are commonly expressed in pathogenic extraintestinal E. coli (1, 3, 7) and closely resemble the capsules of Neisseria meningitidis and Haemophilus influenzae (13). Group 2 capsule gene clusters have a conserved genetic organization comprising three regions. Regions 1 and 3 are common to all group 2 capsule gene clusters and encode the Kps proteins involved in polysaccharide export across the inner membrane, periplasm, and outer membrane. Region 2, flanked by regions 1 and 3, contains the highly variable serotype-specific genes involved in the biosynthesis of the particular polysaccharide (12, 13). In the case of the K5 capsule gene cluster, this involves the kfiABCD genes (9).The biosynthesis of the K5 polysaccharide occurs through the sequential addition of GlcA and GlcNAc residues to the nonreducing end of the polysaccharide chain catalyzed by two glycosyltransferases, KfiA and KfiC (4,6). Polysaccharide biosynthesis occurs at the cytoplasmic face of the inner membrane and involves a hetero-oligomeric complex, consisting of proteins involved in both biosynthesis and export, that is localized at the pole of the cell (8). Such a complex would facilitate a linkage between polysaccharide synthesis and export, although at this stage the mechanism by which synthesis and export are linked is unclear. A recent paper in which the K1-specific endosialidase was expressed in the cytoplasm of a K1-expressing strain indicated that K1 polysaccharide synthesis may occur within a protected cytoplasmic compartment that is inaccessible to endosialidase cleavage (10). To test whether this was also true for the synthesis of the K5 polysaccharide, we expressed the K5-specific lyase, an enzyme that specifically degrades K5 and is associated with the tail spike of K5-specific bacteriophage (2, 5), in the cytoplasm of a K5-encoding strain. In contrast to the situation with K1, we found that expression of the K5 lyase in the cytoplasm reduced the cell surface expression of K5 polysaccharide, suggesting that unlike K1 polysaccharide synthesis, K5 polysaccharide is not synthesized within a protected cytoplasmic compartment.Expression and purification of K5 lyase within MS101. To investigate whether K5 lyase could be expressed and purified from E. coli expressing K5 polysaccharide, the K5-positive strain MS101 (11) was transformed with plasmid pLYA100, an arabinose-inducible p...

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The Escherichia coli K5 Capsule Is Not Synthesized in a Protected Compartment within the Cytoplasm

2009

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“…The newly discovered E. coli chondroitin polymerase, KfoC, is homologous to the pmHAS and pmCS enzymes (ϳ60% identical) and appears to operate in a similar fashion (Ninomiya et al, 2002) (note that this enzyme was termed a polymerase rather than a synthase, because of its apparent requirement for an acceptor oligosaccharide). On the other hand, heparosan made in E. coli K5 is polymerized by a complex containing at least two polypeptides, KfiA and KfiC, each of which is responsible for a single sugar linkage (Hodson et al, 2000).…”

Section: Enzymes That Produce Glycosaminoglycansmentioning

confidence: 99%

“…These bacteria make an unsulfated heparan (N-acetylheparosan or heparosan) using a distinct enzyme, pmHS, that is not similar to the other Pasteurella synthases or to the animal heparan enzymes (DeAngelis and White, 2001). It is extremely likely that the Gram-negative E. coli K5 dual glycosyltransferase complex of KfiA and KfiC is related to pmHS (Hodson et al, 2000). It is not clear yet whether a fusion or a scission event created a dual-action synthase or two single-action transferases.…”

Section: Evolution Of Gag Synthases Of Bacterial Pathogensmentioning

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Evolution of glycosaminoglycans and their glycosyltransferases: Implications for the extracellular matrices of animals and the capsules of pathogenic bacteria

DeAngelis

2002

Anat. Rec.

101

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Glycosaminoglycans (linear polysaccharides with a repeating disaccharide backbone containing an amino sugar) are essential components of extracellular matrices of animals. These complex molecules play important structural, adhesion, and signaling roles in mammals. Direct detection of glycosaminoglycans has been reported in a variety of organisms, but perhaps more definitive tests for the glycosyltransferase genes should be utilized to clarify the distribution of glycosaminoglycans in metazoans. Recently, glycosyltransferases that form the hyaluronan, heparin/heparan, or chondroitin backbone were identified at the molecular level. The three types of glycosyltransferases appear to have evolved independently based on sequence comparisons and other characteristics. All metazoans appear to possess heparin/heparan. Chondroitin is found in some worms, arthropods, and higher animals. Hyaluronan is found only in two of the three main branches of chordates. The presence of several types of glycosaminoglycans in the body allows multiple communication channels and adhesion systems to operate simultaneously. Certain pathogenic bacteria produce extracellular coatings, called capsules, which are composed of glycosaminoglycans that increase their virulence during infection. The capsule helps shield the microbe from the host defenses and/or modulates host physiology. The bacterial and animal polysaccharides are chemically identical or at least very similar. Therefore, no immune response is generated, in contrast to the vast majority of capsular polymers from other bacteria. In microbial systems, it appears that in most cases functional convergent evolution of glycosaminoglycan glycosyltransferases occurred, rather than direct horizontal gene transfer from their vertebrate hosts. Anat Rec 268: 317-326, 2002.

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“…17,18 The synthesis of heparosan chain may be coupled or at least correlated to the transportation of heparosan to the cell surface. [19][20][21][22] The heparosan export pathway involves six proteins: KpsC, KpsD, KpsE, KpsM, KpsS and KpsT.…”

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Escherichia coliK5 heparosan fermentation and improvement by genetic engineering

Wang¹,

Dordick²,

Linhardt³

2011

Bioengineered Bugs

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Identification that KfiA, a Protein Essential for the Biosynthesis of the Escherichia coli K5 Capsular Polysaccharide, is an alpha -UDP-GlcNAc Glycosyltransferase: The Formation of a Membrane Associated K5 Biosynthetic Complex Requires KfiA, KfiB and KfiC

Cited by 31 publications

References 20 publications

The Escherichia coli K5 Capsule Is Not Synthesized in a Protected Compartment within the Cytoplasm

The Escherichia coli K5 Capsule Is Not Synthesized in a Protected Compartment within the Cytoplasm

Evolution of glycosaminoglycans and their glycosyltransferases: Implications for the extracellular matrices of animals and the capsules of pathogenic bacteria

Escherichia coliK5 heparosan fermentation and improvement by genetic engineering

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