Polysialic acid export in Escherichia coli K1: the role of KpsT, the ATP-binding component of an ABC transporter, in chain translocation.
The polysialic acid (polySia) capsule of Escherichia coli K1 is a key virulence determinant of the organism, allowing it to evade host defenses. The proteins necessary for expression of the capsule are encoded by the 17 kb kps gene cluster. This cluster contains two genes, kpsM and kpsT, that are required for polySia transport across the cytoplasmic membrane. KpsM is a hydrophobic integral inner membrane protein, while KpsT is a peripheral inner membrane protein that binds ATP. They belong to the ATP-binding cassette (ABC) superfamily of transporters. To study the role of KpsT in polySia translocation, we used PCR mutagenesis to isolate dominant negative mutations of plasmid-encoded kpsT. All mutations mapped to the same glutamic acid residue at position 150, adjacent to Walker motif B of KpsT. Wild-type (kps+) cells harboring one such allele, E150G, did not transport polySia to the cell surface but accumulated intracellular polysaccharide and produced small colonies containing cells that grew as long filaments. The E150G protein still bound ATP as shown by 8-azidoATP photolabeling assays. We combined the E150G allele with each of five mutations isolated previously in kpsT. Mutations that disrupt ATP-binding (K44E) or alter regions of the protein thought to interact with KpsM (G84D, S126F) suppressed the dominant negative phenotype while mutations in the C-terminal portion of the protein (C163Y, H181Y) did not suppress. These studies have allowed the development of a working model for the role of KpsT in polySia chain translocation.
Bliss
J M
JM
Department of Microbiology and Immunology, University of Rochester Medical Center, NY 14642, USA.
Garon
C F
CF
Silver
R P
RP
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000483:Alleles; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008855:Microscopy, Electron, Scanning; D016296:Mutagenesis; D010641:Phenotype; D012794:Sialic Acids / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012794:Sialic Acids; C021319:polysialic acid; C068103:kpsT protein, E coli; D000255:Adenosine Triphosphate