Nonfimbrial blood group N-specific adhesin (NFA-3) from Escherichia coli O20: KX104: H−, causing systemic infection

Abstract From unfimbriated Escherichia coli O20:KX104: H⁻, which caused systemic infection in man, and adhesin (NFA-3) was isolated by extraction from the bacteria in phosphate buffered saline at 70°C. It was purified by ammonium sulfate precipitation and anion exchange chromatography in the presence of urea. The adhesin had an apparent molecular weight in excess to 10⁶ daltons and consisted of 17.500-d peptide subunits. It had an isoelectric point of 3.9 and contained one cysteine and no methionine. In haemagglutination assays and adhesion immunoassays, NFA-3 showed a preferred specificity for blood group N.     

Analysis of the K1 capsule biosynthesis genes of Escherichia coli: definition of three functional regions for capsule production

Summary Transposon and deletion analysis of the cloned K1 capsule biosynthesis genes of Escherichia coli revealed that approximately 17 kb of DNA, split into three functional regions, is required for capsule production. One block (region 1) is required for translocation of polysaccharide to the cell surface and mutations in this region result in the intracellular appearance of polymer indistinguishable on immunoelectrophoresis to that found on the surface of K1 encapsulated bacteria. This material was released from the cell by osmotic shock indicating that the polysaccharide was probably present in the periplasmic space. Insertions in a second block (region 2) completely abolished polymer production and this second region is believed to encode the enzymes for the biosynthesis and polymerisation of the K1 antigen. Addition of exogenous N-acetylneuraminic acid to one insertion mutant in this region restored its ability to express surface polymer as judged by K1 phage sensitivity. This insertion probably defines genes involved in biosynthesis of N-acetylneuraminic acid. Insertions in a third block (region 3) result in the intracellular appearance of polysaccharide with a very low electrophoretic mobility. The presence of the cloned K1 capsule biosynthesis genes on a multicopy plasmid in an E. coli K-12 strain did not increase the yields of capsular polysaccharide produced compared to the K1⁺ isolate from which the genes were cloned.     

Activity of CMP-2-keto-3-deoxyoctulosonic acid synthetase in Escherichia coli strains expressing the capsular K5 polysaccharide implication for K5 polysaccharide biosynthesis.

The activity of the cytoplasmic CMP-2-keto-3-deoxyoctulosonic acid synthetase (CMP-KDO synthetase), which is low in Escherichia coli rough strains such as E. coli K-12 and in uncapsulated strains such as E. coli O111, was significantly elevated in encapsulated E. coli O10:K5 and O18:K5. This enzyme activity was even higher in an E. coli clone expressing the K5 capsule. This and the following findings suggest a correlation between elevated CMP-KDO synthetase activity and the biosynthesis of the capsular K5 polysaccharide. (i) Expression of the K5 polysaccharide and elevated CMP-KDO synthetase activity were observed with bacteria grown at 37 degrees C but not with cells grown at 20 degrees C or below. (ii) The recovery kinetics of capsule expression of intact bacteria, in vitro K5 polysaccharide-synthesizing activity of bacteria, and CMP-KDO synthetase activity of bacteria after temperature upshift from 18 to 37 degrees C were the same. (iii) Chemicals which inhibit capsule (polysaccharide) expression also inhibited the elevation of CMP-KDO synthetase activity. The chromosomal location of the gene responsible for the elevation of this enzyme activity was narrowed down to the distal segment of the transport region of the K5 expression genes.     

Structure and serological characteristics of the capsular K4 antigen of Escherichia coli O5:K4:H4, a fructose-containing polysaccharide with a chondroitin backbone

The chemical structure of the K4-specific capsular polysaccharide (K4 antigen) of Escherichia coli O5:K4:H4 was elucidated by composition, carboxyl reduction periodate oxidation methylation nuclear-magnetic-resonance spectroscopy and enzymatic cleavage. The polysaccharide consists of a backbone with the structure----3)-beta-D-glucuronyl-(1,4)-beta-D-N-acetylgalactosaminyl(1- to which beta-fructofuranose is linked at C-3 of glucuronic acid. Mild acid hydrolysis liberated fructose and converted the K4 antigen into a polysaccharide which has the same structure as chondroitin. The defructosylated polysaccharide was a substrate for hyaluronidase and chondroitinase. The serological reactivity of the K4 polysaccharide was markedly reduced after defructosylation.     

Characterization and localization of the KpsE protein of Escherichia coli K5, which is involved in polysaccharide export.

In Escherichia coli with group II capsules, the synthesis and cellular expression of capsular polysaccharide are encoded by the kps gene cluster. This gene cluster is composed of three regions. The central region 2 encodes proteins involved in polysaccharide synthesis, and the flanking regions 1 and 3 direct the translocation of the finished polysaccharide across the cytoplasmic membrane and its surface expression. The kps genes of the K5 polysaccharide, which is a group II capsular polysaccharide, have been cloned and sequenced. Region 1 contains the kpsE, -D, -U, -C, and -S genes. In this communication we describe the KpsE protein, the product of the kpsE gene. A truncated kpsE gene was fused with a truncated beta-galactosidase gene to generate a fusion protein containing the first 375 amino acids of beta-galactosidase and amino acids 67 to 382 of KpsE (KpsE'). This fusion protein was isolated and cleaved with factor Xa, and the purified KpsE' was used to immunize rabbits. Intact KpsE was extracted from the membranes of a KpsE-overexpressing recombinant strain with octyl-beta-glucoside. It was purified by affinity chromatography with immobilized anti-KpsE antibodies. Cytofluorometric analysis using the anti-KpsE antibodies with whole cells and spheroplasts, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) of proteins from spheroplasts and membranes before and after treatment with proteinase K, indicated that the KpsE protein is associated with the cytoplasmic membrane and has an exposed periplasmic domain. By TnphoA mutagenesis and by constructing beta-lactamase fusions to the KpseE protein, it was possible to determine the topology of the KpsE protein within the cytoplasmic membrane.     

Coexpression of colanic acid and serotype-specific capsular polysaccharides in Escherichia coli strains with group II K antigens.

In Escherichia coli K-12, the rcsA and rcsB gene products are positive regulators in expression of the slime polysaccharide colanic acid. We have previously demonstrated the presence of rcsA sequences in E. coli K1 and K5, strains with group II capsular K antigens, and shown that introduction of multicopy rcsA into these strains results in the expression of colanic acid. We report here the presence of rcsB sequences in E. coli K1 and K5 and demonstrate that RcsB also plays a role in the biosynthesis of colanic acid in strains with group II K antigens. In E. coli K1 and K5 grown at 37 degrees C, multicopy rcsB and the resulting induction of colanic acid synthesis had no significant effect on synthesis of the group II K antigens. K-antigen-specific sugar transferase activities were not significantly different in the presence or absence of multicopy rcsB, and introduction of a cps mutation to eliminate colanic acid biosynthesis in a K1-derivative strain did not influence the activity of the polysialyltransferase enzyme responsible for synthesis of the K1 polymer. Furthermore, immunoelectron microscopy showed no detectable difference in the size or distribution of the group II K-antigen capsular layer in cells which produced colanic acid. Colanic acid expression therefore does not appear to significantly affect synthesis of the group II K-antigen capsule and, unlike for group I K antigens, expression of group II K antigens is not positively regulated by the rcs system.     

Identification and analysis of a lipopolysaccharide in cell walls of the blue-green alga Anacystis nidulans

Summary A lipopolysaccharide was isolated from cell walls of Anacystis nidulans by extraction with 45% aqueous phenol at 65°, and further purified by repeated high speed centrifugation. It contains 30–40% of lipid and about 60% of carbohydrate components. The carbohydrate moiety contains predominantly mannose and smaller amounts of galactose, glucose, fucose, rhamnose, 2-keto-3-deoxyoctonate, glucosamine and a second aminosugar. The latter was identified as a 2-amino-2-deoxyheptose with the gluco-configuration from C3 to C7. Thelipid moiety contains glucosamine and fatty acids (C22:0, C18:2, C16:0, C12:0 and C14:OH). The lipopolysaccharide has a very low phosphate content and does not contain heptose. It shows low pyrogenicity in rabbits and it is not toxic in mice.Abbreviations KDO 2-Keto-3-deoxy-octonate - LPS Lipopolysaccharide     

Effect of cations on structure-linked sedimentability of lysosomal hydrolases

1. A partially purified lysosomal preparation was obtained from mouse liver sucrose homogenates by differential and discontinuous gradient centrifugation. 2. Triton X-100 or repeated freezing and thawing of the lysosomal suspension (subfraction B) allowed comparison of free and activated values for acid phosphohydrolase, beta-glucuronidase and N-acetylglucosaminidase in the presence and absence of ascorbate. 3. The distribution of hydrolase activities between supernatant and pellet after high-speed centrifugation was measured and the percentages of total enzyme found in the supernatant were: acid phosphohydrolase, 40.7; beta-glucuronidase, 51; N-acetylglucosaminidase, 39.4. 4. Differential rates of elution of the three hydrolases from the membrane fraction occurred with increasing Na(+) and K(+) concentrations, whereas complex biphasic elution curves were obtained as a function of bivalent cation concentration with Ca(2+) and Mg(2+). 5. Sucrose-density-gradient centrifugation of frozen-and-thawed subfraction B demonstrated highly significant changes in the protein gradient profile in the presence of a low concentration of bivalent cation, indicating membrane aggregation and enzyme-membrane association. 6. The data provide further evidence for the nature of lysosomal enzyme binding and indicate the presence of different enzyme-membrane bonds conferring structure-linked latency upon individual lysosomal enzymes.     

Further electron microscopic studies on the expression of Escherichia coli group II capsules.

The de novo expression of Escherichia coli K1, K5, and K12 capsules was analyzed with immunoelectron microscopy in temperature upshift experiments, with upshift from 18 degrees C (capsule restrictive) to 37 degrees C (capsule permissive). Newly produced capsular polysaccharides appeared at the cell surface atop membrane adhesion sites (Bayer's junctions). After plasmolysis of the bacteria at an early expression stage, the capsular polysaccharides were labeled at discrete sites in the periplasm by the immunogold technique. After temperature upshift in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or chloramphenicol, the polysaccharides were labeled in the cytoplasm.     

CMP-KDO-synthetase activity in Escherichia coli expressing capsular polysaccharides

The temperature-regulated expression of capsular group II polysaccharides of Escherichia coli (B. Jann and K. Jann, (1990) Curr. Top. Microbiol. Immunol. 150: 19-42) depends on an elevated concentration of CMP-KDO, as evidenced by an increased activity of CMP-KDO synthetase. The increase in activity of CMP-KDO synthetase is observed only in cytoplasmic fractions of bacteria which had been grown at 37 degrees C but not after growth at 18 degrees C. The activity of CMP-KDO synthetase thus parallels the activity of the (membrane-associated) system synthesizing capsules of group II in E. coli. No such dependence of capsule expression on CMP-KDO was observed with E. coli with capsules of group I. A number of E. coli strains with capsular polysaccharides, which on the basis of genetic determination and chemical characteristics are considered as group II capsules, show no temperature regulation of their capsules and do not depend on an elevated CMP-KDO concentration for capsule expression. The capsular polysaccharides of these E. coli strains, which possibly represent a new group of E. coli capsules are tentatively classified as group I/II.