Molecular studies of the Escherichia coli K5 capsule gene cluster.

Group II K antigens such as the K5 are associated with Escherichia coli causing serious extraintestinal infections. Genes for the production of group II capsules (kps) are organised into three functional regions 1, 2 and 3. The central region 2 encodes proteins involved in the biosynthesis of type specific polysaccharide. Proteins encoded by the flanking regions 1 and 3 are involved in the export of polysaccharide across the bacterial membranes unto the cell surface. Translocation of polysaccharide across the inner membrane is mediated by two region 3 encoded proteins, KpsM and KpsT which belong to the ATP-binding cassette (ABC) superfamily of transporters. Region 1 encodes six proteins, KpsF-E-D-U-C-S. The KpsE protein was shown to localise in the inner membrane and mutants lacking the encoded protein were unable to export polysaccharide to the cell surface. To understand the export role of the KpsE, its membrane topology was determined using TnphoA mutagenesis and beta-lactamase fusions. The topology was confirmed by accessibility of fusion proteins to proteinase K. These results indicated that the KpsE protein adopts a type II bitopic membrane topology consisting of an N-terminus in the cytoplasm followed by a membrane spanning domain, a large periplasmic segment and a C-terminus that may be associated with the outer membrane. On the basis of structural similarity, the KpsE protein is proposed as the membrane fusion protein (MFP) component in the KpsM and T-mediated export of group II capsular polysaccharide and it is postulated to function in the late stages of export across the periplasmic space unto the cell surface. The determined membrane topology contradicts a second C-terminal transmembrane domain in the proposed model based on secondary structure predictions. This points to the importance of experimental data in the establishment of membrane topological models. In addition, the cloning and analysis of a bacteriophage-borne lyase enzyme specific for the E. coli K5 capsular polysaccharide is described. The activity of the K5 lyase enzyme was demonstrated in vitro and its applications are discussed.