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 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.
Cuthbertson
Leslie
L
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Powers
Jacqueline
J
Whitfield
Chris
C
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D005838:Genotype; D008070:Lipopolysaccharides / Q000737:chemistry; D008358:Mannose / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D019081:O Antigens / Q000737:chemistry*; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013997:Time Factors
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D008070:Lipopolysaccharides; D026901:Membrane Transport Proteins; D019081:O Antigens; D011108:Polymers; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; C108433:Wzt protein, bacteria; D000255:Adenosine Triphosphate; D004247:DNA; D008358:Mannose