CHARACTERIZATION OF KPST, THE ATP-BINDING COMPONENT OF THE ABC-TRANSPORTER INVOLVED WITH THE EXPORT OF CAPSULAR POLYSIALIC ACID IN ESCHERICHIA-COLI K1

The 17-kilobase kps gene cluster of Escherichia coli K1 contains all t he information necessary for the expression of capsular polysaccharide . Region 3 of the cluster encodes two genes, kpsM and kpsT, whose prod ucts belong to the (A) under bar TP-(B) under bar inding (C) under bar assette (ABC)-transporter protein family. The KpsMT system is involve d with the export of capsular polysaccharide in E. coli. Earlier work indicated that interaction between KpsT and ATP is important for trans port. In this study, we report that KpsT, a peripheral inner membrane protein, can be photolabeled by the ATP analog, 8-N-3[gamma-P-32]ATP. The derivatiza- tion of KpsT by this reagent is inhibited by cold ATP or ATP gamma S. Furthermore, the protein seems to require a membrane e nvironment for efficient photolabeling, but does not require any other hps gene products. Results obtained from saturation mutagenesis of th e ATP-binding consensus sequence of KpsT and the phenotypes of strains with defined mutations in the chromosomal gene, are consistent with t he view that ATP-binding by KpsT is required for transport of polymer across the inner membrane. The structure of KpsT was compared to a mod el developed for other ABC-transport proteins, and important functiona l regions were determined. The results obtained from chemical mutagene sis of kpsT are consistent with the model and revealed characteristics particular to capsule transporters.