Synthesis of the K5 (group II) capsular polysaccharide in transport-deficient recombinant<i>Escherichia coli</i>

Bronner, D; Sieberth, Veit; Pazzani, Carlo; Smith, A. N.; Boulnois, Graham J.; Roberts, Ian S.; Jann, Barbara; Jann, Klaus

doi:10.1111/j.1574-6968.1993.tb06527.x

Cited by 30 publications

(3 citation statements)

References 21 publications

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“…Mutants in kpsT in both E. coli K1 and K5 are unable to incorporate sialic acid onto endogenous polysialosyl acceptor in vitro (50,60), providing further evidence for a biosynthesis complex. It is interesting to note that whereas mutants in kpsM or kpsT are viable and maintain normal cellular morphology in the K1 and K5 systems (20), introduction of a dominant negative allele of kpsT K1 causes cells to take on an elongated morphology (61) similar to that observed in the E. coli O8 and O9a wzm and wzt mutants.…”

Section: Discussionmentioning

confidence: 98%

The C-terminal Domain of the Nucleotide-binding Domain Protein Wzt Determines Substrate Specificity in the ATP-binding Cassette Transporter for the Lipopolysaccharide O-antigens in Escherichia coli Serotypes O8 and O9a

Cuthbertson¹,

Powers²,

Whitfield

2005

Journal of Biological Chemistry

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The polymannan O-antigenic polysaccharides (O-PSs) of Escherichia coli O8 and O9a are synthesized via an ATP-binding cassette (ABC) transporter-dependent pathway. The group 2 capsular polysaccharides of E. coli serve as prototypes for polysaccharide synthesis and export via this pathway. Here, we show that there are some fundamental differences between the ABC transporter-dependent pathway for O-PS biosynthesis and the capsular polysaccharide paradigm. In the capsule system, mutants lacking the ABC transporter are viable, and membranes isolated from these strains are no longer able to synthesize polymer using an endogenous acceptor. In contrast, E. coli strains carrying mutations in the membrane component (Wzm) and/or the nucleotide-binding component (Wzt) of the O8 and O9a polymannan transporters are nonviable under conditions permissive to O-PS biosynthesis and take on an aberrant elongated cell morphology. Whereas the ABC transporters for capsular polysaccharides with different structures are functionally interchangeable, the O8 and O9a exporters are specific for their cognate polymannan substrates. The E. coli O8 and O9a Wzt proteins contain a C-terminal domain not present in the corresponding nucleotide-binding protein (KpsT) from the capsule exporter. Whereas the Wzm components are functionally interchangeable, albeit with reduced efficiency, the Wzt components are not, indicating a specific role for Wzt in substrate specificity. Chimeric Wzt proteins were constructed in order to localize the region involved in substrate specificity to the C-terminal domain. ATP-binding cassette (ABC)1 transporters, or traffic ATPases, are responsible for the import and export of a variety of molecules across membranes. In the Escherichia coli K-12 genome, 79 known or putative ABC transporters have been identified indicating the importance of this protein superfamily in cellular physiology (1). The prototypical transporter consists of four domains (two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs)), which may be organized in a variety of ways (2). The TMD components between different systems share low sequence similarity and contain a variable number of transmembrane segments, whereas the NBD proteins of different systems share a higher overall sequence similarity as a result of the conserved sequence motifs required for ATP hydrolysis (3). X-ray structures are available for a number of NBD proteins as well as four complete transporters (reviewed in Refs. 4 -7). The structures reveal a common organization of the NBD monomers into two subdomains: a RecAlike domain and a helical domain not found in other ATPhydrolyzing proteins (4). The ATP-binding site is found along the NBD dimer interface and is made up of conserved residues from each monomer, namely the Walker A motif from one monomer and the ABC signature motif from the other monomer (8). Because the ABC transporter TMD components differ greatly between systems, whereas the NBD components remain highly similar, ABC transporters have been said to...

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Section: Discussionmentioning

confidence: 98%

The C-terminal Domain of the Nucleotide-binding Domain Protein Wzt Determines Substrate Specificity in the ATP-binding Cassette Transporter for the Lipopolysaccharide O-antigens in Escherichia coli Serotypes O8 and O9a

Cuthbertson¹,

Powers²,

Whitfield

2005

Journal of Biological Chemistry

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“…The attachment site of the reducing termini of the bacterial polysaccharides during polymerization has not been conclusively identified. K5 polysaccharides isolated from the bacterial capsule have been shown to contain 2-keto-3-deoxyoctonate (KDO) and phosphatidic acid at the reducing end; however, polysaccharide isolated from K5 bacteria deficient in translocation mechanisms and thus with the polysaccharide appearing inside the cell lacked KDO at the reducing end [17]. It therefore seems likely that KDO is added to the polymer during the translocation of the polysaccharide out of the cell.…”

Section: Biosynthesis Of Bacterial Gagsmentioning

confidence: 99%

Biosynthesis of glycosaminoglycans in mammalian cells and in bacteria

Lidholt

1997

Biochemical Society Transactions

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Studies on the biosynthesis of glycosaminoglycans (GAGs) in mammalian and bacterial systems have revealed similarities with regard to polymerization mechanisms and the enzymes involved. Several of the basic features of the biosynthetic machinery in diverse organisms seem to be conserved, such that bacteria may provide potential model systems for the investigation of polysaccharide biosynthesis in eukaryotic systems. Structure of GAGsHeparin1 heparan sulphate (HS), chondroitin sulphate (CS)/dermatan sulphate (DS) and hyaluronan (HA) are all mammalian GAGs [l] that have analogues in bacterial capsular polysaccharides. The GAGs consist of long unbranched carbohydrates with alternating Abbreviations used: CS, chondroitin sulphate; DS, dermatan sulphate; HA, hyaluronan; HS, heparan sulphate; KDO, 2-keto-3-deoxyoctonate; PG, proteoglycan; UDPGDH, UDP-glucose dehydrogenase. hexosamine and uronic acid units. They are classified according to their hexosamine units, where heparinlHS and HA are glucosaminoglycans and CS/DS are galactosaminoglycans. Heparin/HS and CS/DS are sulphated polymers, which together with the uronic acids, give rise to highly negatively charged chains ( Figure 1A).Heparin has the highest negative-charge density of any known biological macromolecule. The different sulphation patterns contribute to the enormous heterogeneity found within and between the polymers. Heparin/HS can be N-sulphated on glucosamine and 0-sulphated at various positions on the monosaccharide units, and the hexuronic acid can be either glucuronic or iduronic acid. All 'sequences' that can be found in heparin also occur in HS, which differs from heparin in the presence of non-sulphated regions, which leads to a lower total sulphate content [2]. CS and DS are 0-sulphated at various positions and the polymer is defined CS if glucuronic acid is the only hexuronic acid in the polymer and DS if there are both glucuronic and iduronic acid units. Both the heparinlHS and the CS/DS Structures of mammalian and bacterial capsular polysaccharides (A) The anti-thrombin-binding pentasaccharide in heparin/HS, a CS polysaccharide and a mammalian HA. (6) Escherichia coli K5 polysaccharide, E. coli K4 polysaccharide and HA from Streptococci.

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“…The group 2 K antigen gene cluster consists of 3 regions, 1-3 ( Fig.1) (5). Regions 1 and 3 are conserved between different group 2 antigens and encode 8 proteins responsible for the transport of the polysaccharide from its site of synthesis in the cytoplasm to the external cell surface (6)(7)(8). Region 2 is serotype-specific and encodes proteins essential in the synthesis of the particular capsular polysaccharide (1).…”

Section: Introductionmentioning

confidence: 99%

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

Griffiths

Cook

et al. 2000

Journal of Biological Chemistry

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Synthesis of the K5 (group II) capsular polysaccharide in transport-deficient recombinantEscherichia coli

Cited by 30 publications

References 21 publications

The C-terminal Domain of the Nucleotide-binding Domain Protein Wzt Determines Substrate Specificity in the ATP-binding Cassette Transporter for the Lipopolysaccharide O-antigens in Escherichia coli Serotypes O8 and O9a

The C-terminal Domain of the Nucleotide-binding Domain Protein Wzt Determines Substrate Specificity in the ATP-binding Cassette Transporter for the Lipopolysaccharide O-antigens in Escherichia coli Serotypes O8 and O9a

Biosynthesis of glycosaminoglycans in mammalian cells and in bacteria

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

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