Genetic analysis of chromosomal mutations in the polysialic acid gene cluster of Escherichia coli K1

Abstract

The kps gene cluster of Escherichia coli K1 encodes functions for sialic acid synthesis, activation, polymerization, and possibly translocation of polymer to the cell surface. The size and complexity of this membrane polysaccharide biosynthetic cluster have hindered genetic mapping and functional descriptions of the kps genes. To begin a detailed investigation of the polysialic acid synthetic mechanism, acapsular mutants were characterized to determine their probable defects in polymer synthesis. The mutants were tested for complementation with kps fragments subcloned from two separately isolated, functionally intact kps gene clusters. Complementation was assayed by immunological and biochemical methods and by sensitivity to the K1-specific bacteriophage K1F. The kps cluster consisted of a central 5.8-kilobase region that contained at least two genes coding for sialic acid synthetic enzymes, a gene encoding the sialic acid-activating enzyme, and a gene encoding the sialic acid polymerase. This biosynthetic region is flanked on one side by an approximately 2.8-kilobase region that contains a potential regulatory locus and at least one structural gene for a polypeptide that appears to function in polysialic acid assembly. Flanking the biosynthetic region on the opposite side is a 6- to 8.4-kilobase region that codes for at least three proteins which may also function in polymer assembly and possibly in translocating polymer to the outer cell surface. Results of transduction crosses supported these conclusions and indicated that some of the kps genes flanking the central biosynthetic region may not function directly in transporting polymer to the cell surface. The results also demonstrate that the map position and probable function of most of the kps cluster genes have been identified.