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

Abstract

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...

Substrate specificity of bacterial oligosaccharyltransferase suggests a common transfer mechanism for the bacterial and eukaryotic systems

“…Immunoblotting was performed as described (41). Antisera specific for AcrA, R12 (17), K30 capsular polysaccharide (42), and O9a polysaccharide (20) have been described. Con A-horseradish peroxidase was from Sigma.…”

“…The O9a polymannan is linked to the lipid A core, whereas the K30 antigen forms the capsular polysaccharide. The O9a antigen is synthesized by the ABC transporter-dependent mechanism (18)(19)(20). The K30 capsular polysaccharide is the established prototype for group 1 capsules (21).…”

A Membrane-located Glycosyltransferase Complex Required for Biosynthesis of the d-Galactan I Lipopolysaccharide O Antigen in Klebsiella pneumoniae

“…When a ⌬wbbN version of the pWQ289 construct was examined, the cells lacked D-galactan I but had a normal cell size and shape (data not shown). Transport defects in the E. coli O9a O-PS assembly system also give rise to pleomorphic cells, which lose viability (15,17). Although the underlying mechanisms may differ, both instances could reflect either some toxicity resulting from accumulation of D-galactan I intermediates, or depletion of the cellular pool of the essential und-P carrier, which participates in peptidoglycan biosynthesis (36).…”

“…The completed O-PS structure is then exported across the membrane by the ABC transporter, which is composed of two Wzm (transmembrane domain) polypeptides, and two Wzt (nucleotide-binding domain) polypeptides. In E. coli O8/O9a, Wzt contains an additional carbohydrate-binding module that recognizes the precise export signal on the nascent polymer (16,17). In contrast, in the biosynthesis of D-galactan I in K. pneumoniae O2a, chain termination is determined by an interaction between the transporter and the glycosyltransferases (or their product) in a manner that is not yet determined (18).…”

“…Chain extension in the ABC transporter-dependent pathways is then terminated by one of two remarkably different strategies. In a model exemplified by the polymannose O-PSs from Escherichia coli O8 and O9a, a residue not found in the repeat unit structure is added to the non-reducing terminus and this serves as an export signal (15)(16)(17). The completed O-PS structure is then exported across the membrane by the ABC transporter, which is composed of two Wzm (transmembrane domain) polypeptides, and two Wzt (nucleotide-binding domain) polypeptides.…”