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 qf 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 Kl-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.The polysialic acid capsule of Escherichia coli K1 is an unbranched homopolymer of 200 sialic acid residues in a-2,8-ketosidic linkage (19). Molecules with more than three ao-2,8-linked internal sialic acid residues are rare in nature and, until recently, only were known in E. coli K1 and Neisseria meningitidis group B (26). Poly-x-2,8-sialic acid chains similar to the K1 antigen are now known to be widely distributed on the vertebrate cell adhesion molecule NCAM (20, 31). The rarity of sialic acids in bacteria and the association of polysialic acid capsules with virulence suggested that the bacterial polysaccharide may mimic polysialic acid moieties on mammalian host glycoconjugates (5). Repeated failures to develop effective vaccines for the a-2,8-linked capsule may thus be due to immune tolerance in the host (5). Through a better understanding of the genetics and biochemistry of capsule biosynthesis, it may be possible to develop alternative therapeutic approaches for treating infections caused by polysialic acid-encapsulated bacteria.Polysialic acid synthesis in E. coli K1 has been one of the best-characterized capsular polysaccharide biosynthetic systems. The broad outlines of polysialic acid synthesis are understood at a biochemi...

Reduced polysialic acid capsule expression in Escherichia coli K1 mutants with chromosomal defects in kpsF

“…Several previous attempts to overproduce and purify KpsF using commercially available expression systems failed owing to the apparent marked propensity of the KpsF fusion polypeptides to aggregate, thus preventing the use of affinity columns that depend on tertiary conformation of the fusion tag for matrix a. The n superscript refers to the null genotype of E. coli K-12 strains, which lack the kps locus (Vimr et al, 1989). b.…”

“…K1 capsule mutants harbouring chromosomal kps defects are usually selected as survivors of infection by the lytic polysialic acid-specific bacteriophage K1F (Vimr and Troy, 1985a;Vimr et al, 1989). K1F binding to and hydrolysis of the polysialic acid receptor are obligatory processes for productive phage infection.…”

KpsF Is the Arabinose-5-phosphate Isomerase Required for 3-Deoxy-d-manno-octulosonic Acid Biosynthesis and for Both Lipooligosaccharide Assembly and Capsular Polysaccharide Expression in Neisseria meningitidis

“…To determine whether the meningococcal kpsF::aphA-3 mutant accumulated intracellular capsular polysaccharide, mutant NMB206 was lysed by freezethaw treatment or by an EDTA-HEPES method described by Moe et al (44). An E. coli strain, EV94 (kpsS::Tn10), which has been shown to accumulate intracellular capsule (45), was included as a positive control for lysis. Capsular polysaccharide released into the supernatant was quantified by ELISA.…”

Functional Relationships of the Sialyltransferases Involved in Expression of the Polysialic Acid Capsules ofEscherichia coli K1 and K92 and Neisseria meningitidis Groups B or C

“…1). Epistasis analysis and complementation experiments showed that polymerase activity is dependent on the structural integrity of the polysaccharide translocation and capsule assembly apparatus (9,10), suggesting the multiprotein complex diagramed below in Fig. 1.…”