Klaus Jann

Region 2 of the Escherichia coli K5 capsule gene cluster encoding proteins for the biosynthesis of the K5 polysaccharide

The nucleotide sequence of region 2 of the Escherichia coli K5 capsule gene cluster has been determined. This region, essential for the biosynthesis of the K5 poly‐saccharide, contained four genes, termed kfiA‐D. The G+C ratio was 33.4%, which was lower than the typical G+C ratio for E. coli and that of the flanking regions 1 and 3 in the K5 capsule gene cluster. Three major RNA transcripts were detected within region 2 by Northern blotting and three promoters located by transcript mapping. Promoter activity was confirmed by promoter‐probe analysis. The predicted amino acid sequence of KfiC had homology to a number of glycosyl transferase enzymes and overexpression of the kfiC gene resulted in increased K5 transferase activity. The predicted amino acid sequence of KfiD had homology to a number of NAD‐dependent dehydrogenase enzymes and was demonstrated to be a UDP‐glucose dehydrogenase that catalyses the formation of UDP‐glucuronic acid from UDP‐glucose.

Analysis of genes coding for the sialic acid‐binding adhesin and two other minor fimbrial subunits of the S‐fimbrial adhesin determinant of Escherichia coli

The S fimbrial adhesin (Sfa) enables Escherichia coli to attach to sialic acid‐containing receptor molecules of eukaryotic cells. As previously reported, the genetic determinant coding for the Sfa of an E. coli O6 strain was cloned, the gene coding for the major fimbrial subunit was identified and sequenced and the S specific adhesin was detected. Here we present evidence that in addition to the major subunit protein SfaA three other minor subunit proteins, SfaG (17kD), SfaS (14 kD) and SfaH (31 kD) can be isolated from the S‐specific fimbrial adhesin complex. The genes coding for these minor subunits were identified, mutagenized separately and sequenced. Using haemagglutination tests, electron‐microscopy and quantitative ELISA assays with monoclonal anti‐SfaA and anti‐SfaS antibodies the functions of the minor subunits were determined. It was determined that SfaS is identical to the S‐specific adhesin, which also plays a role in determination of the degree of fimbriation of the cell. The minor subunit SfaH also had some influence on the level of fimbriation of the cell, while SfaG is necessary for full expression of S‐specific binding. It was further shown that the amino‐terminal protein sequence of the isolated SfaS protein was identical to the protein sequence calculated from the DNA sequence of the sfaS gene locus.

Structural studies of the O-specific side chain of the lipopolysaccharide from Escherichia coli O:7.

The structure of the O-specific side-chain of the lipopolysaccharide from Escherichia coli O:7 has been investigated, using n.m.r. spectroscopy, methylation analysis, partial hydrolysis, and Smith degradation as the principal methods. It is concluded that the polysaccharide is constructed of repeating pentasaccharide units having the structure (formula; see text) where D-QuipNAc stands for 4-acetamido-4,6-dideoxy-D-glucopyranose. The 13C-n.m.r. spectrum of the polysaccharide has been interpreted completely.

On a bacteriophage T3 and T4 receptor region within the cell wall lipopolysaccharide of escherichia coli B

Abstract † Mutants of Escherichia coli B with increasing structural defects in the cell wall lipopolysaccharide (above) were tested for adsorption resistance to bacteriophages T3 and T4, and the lipopolysaccharide extracted from these mutants was assayed for loss of ability to inactivate the viruses. Absence of terminal glucose, or of any of the substituents (branch heptose, phosphate and pyrophosphorylethanolamine) on the two chain heptoses was found to have little effect on T3 and T4 susceptibility. However, all mutants lacking both glucoses were resistant to both phages.

Bacterial polysaccharide capsule synthesis, export and evolution of structural diversity

Elaboration of a capsule composed of one of a range of acidic polysaccharides is a common feature of many bacteria, particularly those capable of causing serious infections in humans. Biochemical and genet‐cal analyses of capsule biogenesis in Escherichia coli are beginning to reveal new aspects of polysaccharide biosynthesis. Genes have been identified which are thought to encode products responsible for the trans‐location of these high molecular‐weight polysaccharides across the cytoplasmic and outer membranes, and the organization of exported polysaccharide into a capsule. Their further analysis should provide new insights into membrane biology, particularly since the genes in question are absent from the often used laboratory strains of E. coli. Genetic analysis of capsule diversity is beginning to suggest possible mechanisms for the generation of the structural diversity of polysaccharides.

Induction of inflammation by Escherichia coli on the mucosal level: requirement for adherence and endotoxin.

Bacterial infection of the mouse urinary tract is followed by the recruitment of leukocytes to the mucosal surface. This study examined the bacterial components involved in the induction of this response. Escherichia coli of serotype O75:K5:H- expressing adhesins specific for the Gal alpha 1-4Gal beta- (Gal, galactose) and mannose-containing receptors were instilled into the urinary bladder of lipopolysaccharide responder (C3H/HcN) and lipopolysaccharide nonresponder (C3H/HeJ) mice. The inflammation was quantitated as the number of leukocytes excreted into the urine at various times after infection. The response was first shown to depend on the Lps genotype of the mouse. The leukocyte excretion that occurred within 24 h after infection of C3H/HeN mice was absent in C3H/HeJ mice. The components triggering the response were present on both live and Formalin-killed bacterial cells, and the response was mimicked by intravesical inoculation of isolated lipid A. Pretreatment of bacteria with soluble receptor oligosaccharides resulted in inhibition of attachment in vitro and of the inflammation in vivo. A direct synergy between adhesins specific for Gal alpha 1-4Gal beta receptors and lipid A was demonstrated. Mixtures of these components induced a leukocyte response higher than the sum of the responses to each component alone. These results suggest that the inflammation induced by gram-negative bacteria in the urinary tract can be triggered at the level of the epithelial cells by endotoxin presented by an attaching bacterial cell and that intact function at the Lps locus of the host is required for this to occur.

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

SummaryTransposon 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.

Genetic and biochemical analysis of Shigella dysenteriae 1 O antigen polysaccharide biosynthesis in Escherichia coli K-12: structure and functions of the rfb gene cluster

The genetic organization and functions of the Shigella dysenteriae 1 rfb gene cluster, which specifies the somatic O antigen in this organism, have been studied in Escherichia coli K-12 by insertion and deletion mutagenesis of pSS9, a pBR322 hybrid containing the Shigella rfb genes. On the basis of the sensitivity/resistance to rough-specific bacteriophage T3 of E. coli K-12 derivatives containing mutant pSS9 plasmids, of the banding patterns and immunoreactivity of LPS isolated from such derivatives and electrophoresed on SDS-polyacrylamide gels, and of the sugar composition of the polysaccharide portion of the LPS determined by chemical analysis, six determinants for O antigen production were identified and localized. At least two determinants are involved in synthesis of TDP-rhamnose and the transfer of a rhamnose residue to the galactose-substituted core. One of these functions is probably TDP-rhamnose synthetase. A third function effects the transfer of a second rhamnose residue to the rha----gal-substituted core. A fourth function, for which evidence was obtained for two determinants (cistrons), is N-acetylglucosamine transferase, whereas a sixth determinant is necessary for extension of the first completed side chain repeat unit to the full O antigen polymer. These results confirmed the previously-determined chemical composition of the S. dysenteriae 1 O antigen and demonstrated that the order of the sugars is glcNAc----rha----rha----gal with gal as the first sugar linked to the core. Evidence was obtained for at least two transcriptional units in the rfb gene cluster and the approximate locations of two promoters are suggested. The detection of new electrophoretic species of LPS that may correspond to LPS biosynthetic intermediates, and the finding on the cell surfaces of structures corresponding to LPS core substituted with one or more O-specific sugars, appear to be novel findings.

Structure of the amino acid-containing capsular polysaccharide (K54 antigen) from escherichia coli O6:K54:H10

The structure of the K54-antigenic polysaccharide (K54 antigen) of Escherichia coli O6:K54:H10 was elucidated by determination of the composition, 1H- and 13C-n.m.r. spectroscopy, periodate oxidation, and a study of the oligosaccharides obtained by partial hydrolysis with acid. The K54 polysaccharide consists of----3)-beta-D-glucosyluronic acid-(1----3)-alpha-L-rhamnosyl-(1----repeating-units. Of the glucuronic acid residues, approximately 85% are substituted in the ratio 9:1 with L-threonine and L-serine amidically linked to the carboxyl group. The K54 polysaccharide has a molecular weight of approximately 160,000, corresponding to approximately 380 repeating-units.

The three‐dimensional structure of capsule‐specific CMP: 2‐keto‐3‐deoxy‐manno‐octonic acid synthetase from Escherichia coli

CMP‐Kdo synthetases from Gram‐negative bacteria activate Kdo for incorporation into lipo‐ and capsule‐polysaccharides. Here we report the crystal structure of the capsule‐specific synthetase from E. coli at 2.3 Å resolution. The enzyme is a dimer of 2 × 245 amino acid residues assuming C2 symmetry. It contains a central predominantly parallel β‐sheet with surrounding helices. The chain fold is novel; it is remotely related to a double Rossmann fold. A large pocket at the carboxyl terminal ends of the central β‐strands most likely accommodates the catalytic center. A putative phosphate binding site at the loop between the first β‐strand and the following helix is indicated by a bound iridium hexachloride anion.