An enterotoxin-producing strain of Escherichia coli isolated from a case of cholera-like diarrhea (E. coli strain H-10407) was found to possess a surfaceassociated colonization factor. Colonization was manifested as the ability of small inocula (105 bacteria) to attain large (10w) populations in the infant rabbit intestine with a concomitant diarrheal response. A laboratory-passed derivative of E. coli H-10407, designated H-10407-P, failed to exhibit an increase in population in the infant rabbit and also failed to induce diarrhea. Cell-free culture supernatant fluids of E. coli H-10407 and H-10407-P produced equivalent enterotoxic responses in infant and in adult rabbits. Specific anti-colonization factor antiserum was produced by adsorbing hyperimmune anti-H-10407 serum with both heat-killed and living cells of E. coli H-10407-P. This specific adsorbed serum protected infant rabbits from challenge with living E. coli H-10407 although the serum did not possess bactericidal activity. The anti-colonization factor serum did not agglutinate a strain of E. coli K-12 possessing the K88 colonization factor peculiar to E. coli enterotoxigenic for swine. By electron microscopy it was demonstrated that E. coli H-10407, but not H-10407-P, possessed pilus-like surface structures which agglutinated with the specific adsorbed (anti-colonization factor) antiserum. E. coli H-10407 possessed three species of plasmid deoxyribonucleic acid, measuring 60 x 10X, 42 x 106, and 3.7 x 10' daltons, respectively. E. coli H-10407-P possessed only the 42 x 106and the 3.7 x 106-dalton plasmid species. Spontaneous loss of the specific H-10407 surface-associated antigen was accompanied by loss of the 60 x 106-dalton species of

SummaryCampylobacter jejuni strain 81-176 (HS36, 23) synthesizes two distinct glycan structures, as visualized by immunoblotting of proteinase K-digested whole-cell preparations. A site-specific insertional mutant in the kpsM gene results in loss of expression of a highmolecular-weight (HMW) glycan (apparent M r 26 kDa to . 85 kDa) and increased resolution of a second ladder-like glycan (apparent M r 26±50 kDa). The kpsM mutant of 81-176 is no longer typeable in either HS23 or HS36 antisera, indicating that the HMW glycan structure is the serodeterminant of HS23 and HS36. Both the kpsM-dependent HMW glycan and the kpsMindependent ladder-like structure appear to be capsular in nature, as both are attached to phospholipid rather than lipid A. Additionally, the 81-176 kpsM gene can complement a deletion in Escherichia coli kpsM, allowing the expression of an a2,8 polysialic acid capsule in E. coli. Loss of the HMW glycan in 81-176 kpsM also increases the surface hydrophobicity and serum sensitivity of the bacterium. The kpsM mutant is also significantly reduced in invasion of INT407 cells and reduced in virulence in a ferret diarrhoeal disease model. The expression of the kpsM-dependent capsule undergoes phase variation at a high frequency.

Variable properties among Escherichia coli isolates include serotype, electrophoretic migration of major outer membrane proteins, metabolic properties, production of hemolysin or colicin or both, and plasmid content. These characteristics were compared in E. coli strains of capsular types Kl, K5, K92, and K100 and in non-encapsulated isolates. The 234 bacterial strains from the United States and Europe which we studied had been isolated from healthy or diseased individuals recently or as long ago as 1941. Regardless of source, most 07:K1, 016:K1, and 075:K100 isolates could be assigned to three unique, serotypespecific groups, which were interpreted as representing three bacterial clones. Two bacterial (sub)clones each were discerned among the 018:K1 and 018:K5 isolates, and two further, distinct clones were discerned among the 01:K1 isolates. The implications of these results for epidemiological analyses and for MATERIALS AND METHODS Bacterial strains. Bacterial strains isolated recently in several countries were obtained from the following individuals:

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...

Capsules are well-studied components of the bacterial surface that modulate interactions between the cell and its environment. Generally composed of polysaccharide, they are key virulence determinants in invasive infections in humans and other animals. Genetic determinants involved in capsule expression have been isolated from a number of organisms, but perhaps the best characterized is the kps cluster of Escherichia coli K1. In this review, the current understanding of the functions of the kps gene products is summarized. Further, a proposed mechanistic model for capsule expression is presented and discussed. The model is based on the premise that the numerous components of the kps cluster form a hetero-oligomeric complex responsible for synthesis and concurrent translocation of the capsular polysialic acid through sites of inner and outer membrane fusion. We view the ATP-binding cassette (ABC) transporter, KpsMT, to be central to the functioning of the complex, interacting with the biosynthetic apparatus as well as the extracytoplasmic components of the cluster to co-ordinate synthesis and translocation. The model provides the basis for additional experimentation and reflects emerging similarities among systems responsible for macromolecular export in Gram-negative bacteria.

The polysialic acid capsule of Escherichia coli K1, a causative agent of neonatal septicemia and meningitis, is an essential virulence determinant. The 17-kb kps gene cluster, which is divided into three functionally distinct regions, encodes proteins necessary for polymer synthesis and expression at the cell surface. The central region, 2, encodes products required for synthesis, activation, and polymerization of sialic acid, while flanking regions, 1 and 3, are thought to be involved in polymer assembly and transport. In this study, we identified two genes in region 3, kpsM and kpsT, which encode proteins with predicted sizes of 29.6 and 24.9 kDa, respectively. The hydrophobicity profile of KpsM suggests that it is an integral membrane protein, while KpsT contains a consensus ATP-binding domain. KpsM and KpsT belong to a family of prokaryotic and eukaryotic proteins involved with a variety of biological processes, including membrane transport. A previously described kpsT chromosomal mutant that accumulates intracellular polysialic acid was characterized and could be complemented in trans. Results of site-directed mutagenesis of the putative ATP-binding domain of KpsT are consistent with the view that KpsT is a nucleotide-binding protein. KpsM and KpsT have significant similarity to BexB and BexA, two proteins that are essential for polysaccharide capsule expression in Haemophilus influenzae type b. We propose that KpsM and KpsT constitute a system for transport of polysialic acid across the cytoplasmic membrane.

Recently, M. Dmitrova et al. (Mol. Gen. Genet. 257:205-212, 1998) described a LexA-based genetic system to monitor protein-protein interactions in an Escherichia coli background. However, the plasmids used in this system, pMS604 and pDP804, were not readily amenable for general use. In this report, we describe modifications of both plasmids that allow fragments of DNA to be fused to either vector in any reading frame. Homodimerization and heterodimerization of full-length proteins involved in polysialic acid synthesis in E. coli K1, as well as heterodimerization between a full-length protein and a protein fragment, demonstrate the usefulness of the modified plasmids for investigating bacterial protein-protein interactions in vivo.

Escherichia coli K1 produces a capsular polysaccharide of alpha(2-8) poly-N-acetylneuraminic acid. This polysaccharide is an essential virulence factor of these neuropathogenic bacteria. The genes necessary for the synthesis of neuNAc were localized to a plasmid containing the neuBAC genes of the K1 gene cluster. Cells harboring the neuB+ allele in an aldolase (nanA-) negative background produce neuNAc in vivo. Enzymatic synthesis of neuNAc could be demonstrated in extracts of cells harboring an expression plasmid (pNEUB) containing the neuB gene alone. NeuNAc synthetase was purified to homogeneity from extracts of cells harboring pNEUB. The molecular weight of the purified enzyme is 40 kDa, similar to that predicted by the nucleotide sequence of the neuB gene. The amino terminal sequence of the purified protein matches that predicted by the nucleotide sequence of the neuB gene. NeuNAc synthetase catalyzes the formation of neuNAc as indicated by its coupling to the CMP-neuNAc synthetase reaction. The enzyme condenses manNAc and PEP with the release of phosphate. The E. coli neuNAc synthetase is specific for manNAc and PEP, unlike rat liver enzyme that utilizes N-acetylmannosamine-6-phosphate to form neuNAc-9-PO4. This represents the first report of a purification of a sialic acid synthetase from either a eukaryotic or prokaryotic source to homogeneity. These experiments clearly demonstrate an aldolase-independent sialic acid synthetase activity in E. coli K1.