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...
Bordetella pertussis, the aetiological agent of whooping cough, coordinately regulates the expression of many virulence-associated determinants, including filamentous haemagglutinin, pertussis toxin, adenylyl cyclase toxin, dermonecrotic toxin and haemolysin. The coordinate regulation is apparent in the repression of synthesis of these determinants in response to environmental stimuli; a phenomenon known as antigenic or phenotypic modulation. B. pertussis also varies between metastable genetic states, or phases. There is a virulent phase in which virulence-associated determinants are synthesized, and an avirulent phase in which they are not. Previous studies have shown that a genetic locus, vir, is required for expression from many virulence-associated loci, and that replacing the cloned vir locus in trans can restore the virulent phase phenotype to spontaneously occurring avirulent phase strains. Here, we show that phase variation in one series of strains is due to a frameshift mutation within an open reading frame that is predicted to code for a Vir protein product. The deduced protein sequence is similar to both components of the 'two-component' regulatory system which control gene expression in response to environmental stimuli in a range of bacterial species.
Epidemiological and immunological evidence indicates that the K1 capsular polysaccharide confers the property of virulence on Escherichia coli. E coli K1 is associated with invasive diseases in humans and in laboratory and domesticated animals. K1 isolates account for 80% of E. coli neonatal meningitis and comprise the majority of capsular types in neonatal septicaemia without meningitis and in childhood pyelonephritis. Passive administration of K1 antibodies prevented bacteraemia and meningitis in infant rats fed E. coli K1. Nonencapsulated derivatives of these invasive K1 strains did not cause bacteraemia in infant rats, although intestinal colonization was similar to that of the parent strains (M. Achtman and R.P.S., unpublished results). Several reports propose that the E. coli K1 capsular polysaccharide exerts an anti-phagocytic effect similar to that observed with other pathogenic encapsulated bacteria. One approach to studying whether the K1 antigen is sufficient to confer virulence of if other E. coli structures are necessary is to isolate the K1 genes for genetic and biochemical analysis. Recombinant DNA methodology provides a powerful tool for such an approach. Here, we report the molecular cloning of the E. coli K1 antigen genes. The cloned K1 genes synthesize a capsule in E. coli K12 indistinguishable chemically and immunologically from that of wild-type K1 strains.
An 11.6-kilobase (kb) region of a 34-kb fragment of Escherichia coli DNA that encodes-the Kl capsular polysaccharide genes is necessary for translocation of the Kl polysaccharide to the bacterial cell surface. This 11.6-kb region contains a gene, kpsD, encoding a 60-kilodalton protein. The kpsD gene was localized to a 2.4-kb PstI-BamHI fragment. Cells harboring a TnlOOO insertion in kpsD did not synthesize the 60-kilodalton protein and did not express polysaccharide on the cell surface. Immunodiffusion and rocket immunoelectrophoresis of cell extracts, however, demonstrated that Kl polysaccharide was synthesized by these cells. We present evidence that the kpsD gene product is synthesized as a precursor and that the processed form is located in the periplasmic space. Analysis of alkaline phosphatase activity of a kpsD-phoA fusion demonstrated that kpsD expression was under positive regulation. A 260-base-pair AluI fragment located within the kpsD coding sequence was used as a probe and was found to hybridize to chromosomal DNA from E. coli that synthesizes the K2, K5, K7, K12, and K13 capsular polysaccharides but not K3 and K100. These results suggest that the kpsD gene product may be required for export not only of Kl but for other K antigens as well.The Kl capsular polysaccharide is an important virulence determinant of invasive Escherichia coli (35). Kl-producing isolates account for 80% of E. coli neonatal meningitis cases and are common in cases of neonatal septicemia and childhood pyelonephritis (21, 33). The Kl polysaccharide is an a-2,8-linked linear homopolymer of sialic acid (N-acetylneuraminic acid [NeuAc]; 2, 9). The initial events in the synthesis of the Kl polysaccharide have been extensively investigated (40, 42). A membrane-associated sialyltransferase complex catalyzes the polymerization of NeuAc from CMP-NeuAc. These reactions involve a NeuAc-Pundecaprenol intermediate, and synthesis requires endogenous sialyl acceptors within the membrane (41, 43). In contrast, the mechanism of translocation of the polysaccharide from its site of synthesis on the cytoplasmic membrane to the bacterial cell surface is poorly understood. It has been postulated that transfer occurs at sites of adhesion between the inner and outer membranes (Bayer junctions; 3). There is, however, a paucity of information about these events at the genetic and molecular levels.The genes encoding proteins required for the synthesis and expression of the E. coli Kl capsular polysaccharide have been cloned (11,38) and analyzed by genetic techniques (34, 39). These genes are organized in three coordinately regulated clusters. Deletion of one of these clusters, an 11.6-kilobase (kb) region, inactivated the expression but not the synthesis of the Kl polysaccharide (39). We therefore proposed that this one cluster was responsible for transport of the polysaccharide to the cell surface. Five protein species with molecular masses of 80, 77, 60, 40, and 37 kilodaltons (kDa), encoded within the 11.6-kb region, were postulated to be associated ...
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