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 the information necessary for the expression of capsular polysaccharide. Region 3 of the cluster encodes two genes, kpsM and kpsT, whose products belong to the ATP-Binding Cassette (ABC)-transporter protein family. The KpsMT system is involved with the export of capsular polysaccharide in E. coli. Earlier work indicated that interaction between KpsT and ATP is important for transport. In this study, we report that KpsT, a peripheral inner membrane protein, can be photolabeled by the ATP analog, 8-N3[gamma-32P]ATP. The derivatization of KpsT by this reagent is inhibited by cold ATP or ATP gamma S. Furthermore, the protein seems to require a membrane environment for efficient photolabeling, but does not require any other kps gene products. Results obtained from saturation mutagenesis of the ATP-binding consensus sequence of KpsT and the phenotypes of strains with defined mutations in the chromosomal gene, are consistent with the view that ATP-binding by KpsT is required for transport of polymer across the inner membrane. The structure of KpsT was compared to a model developed for other ABC-transport proteins, and important functional regions were determined. The results obtained from chemical mutagenesis of kpsT are consistent with the model and revealed characteristics particular to capsule transporters.
Pavelka
M S
MS
Department of Microbiology and Immunology, University of Rochester Medical Center, New York 14642.
Hayes
S F
SF
Silver
R P
RP
D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001386:Azides; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D005810:Multigene Family; D011487:Protein Conformation; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012794:Sialic Acids / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D000345:Affinity Labels; D001386:Azides; D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D012794:Sialic Acids; C021319:polysialic acid; C068103:kpsT protein, E coli; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate