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 K1is a key virulence determinant of the organism, allowing itto evade host defenses. The proteins necessary for expressionof the capsule are encoded by the 17 kb kps gene cluster. Thiscluster contains two genes, kpsM and kpsT, that are requiredfor polySia transport across the cytoplasmic membrane. KpsMis a hydrophobic integral inner membrane protein, while KpsTis a peripheral inner membrane protein that binds ATP. Theybelong to the ATP-binding cassette (ABC) superfamily of transporters.To study the role of KpsT in polySia translocation, we usedPCR mutagenesis to isolate dominant negative mutations of plasniid-encodedkpsT. All mutations mapped to the same glutamic acid residueat position 150, adjacent to Walker motif B of KpsT. Wild-type(kps⁺) cells harboring one such allele, E150G, did not transportpolySia to the cell surface but accumulated intracellular polysaccharideand produced small colonies containing cells that grew as longfilaments. The E150G protein still bound ATP as shown by 8-azidoATPphotolabeling assays. We combined the E150G allele with eachof five mutations isolated previously in kpsT. Mutations thatdisrupt ATP-binding (K44E) or alter regions of the protein thoughtto interact with KpsM (G84D, S126F) suppressed the dominantnegative phenotype while mutations in the C-terminal portionof the protein (C163Y, H181Y) did not suppress. These studieshave allowed the development of a working model for the roleof KpsT in polySia chain translocation. ABC-transporter dominant negative mutation Escherichia coli Kl KpsT polysialic acid