Derepression of Phosphomannose Isomerase by Regulator Gene Mutations Involved in Capsular Polysaccharide Synthesis in Escherichia coli K-12

A regulator gene mutation (capR) that causes increased synthesis of capsular polysaccharide and derepressed synthesis of several enzymes involved in polysaccharide synthesis also derepresses phosphomannose isomerase (PMI) synthesis. In contrast, a second mutation (capS, which maps separately from capR) that causes increased production of the same polysaccharide does not lead to increased synthesis of PMI (nor of several of the other enzymes involved in polysaccharide synthesis). Introduction of the capR9 allele by transduction or mutation of capR(+) to capR can change the phenotype of a mannose-negative nonmucoid strain to a mannose-positive mucoid phenotype. Thus, genotype capR(+)man-2 is mannose-negative and nonmucoid, but genotype capR9 man-2 is mannose positive and mucoid. Other interactions between these alleles in the synthesis of capsular polysaccharide are recorded.     

Mutation Preventing Capsular Polysaccharide Synthesis in Escherichia coli K-12 and Its Effect on Bacteriophage Resistance

A mutant strain of Escherichia coli K-12 was found in which spontaneous mutation to phage T7 resistance occurred at a very low frequency. T7 resistance in the parental strain from which this mutant was derived resulted from a mutation to excess capsular polysaccharide synthesis. The mutation preventing T7 resistance, non-9, inhibited capsule formation when transduced into capsulated strains. The non-9 mutation was cotransducible with his, the gene order in this region being non-9 his Su-1.     

Structural studies of the O-antigenic polysaccharides from Shigella dysenteriae type 3 and Escherichia coli O124, a reinvestigation

The structures of the O-antigenic part of the lipopolysaccharides from Shigella dysenteriae type 3 and Escherichia coli O124 have been reinvestigated. (1)H and (13)C NMR spectroscopy in combination with selected 2D NMR techniques were used to determine the O-antigen pentasaccharide repeating units with the following structure: [see text]. From biosynthetic considerations this should also be the biological repeating unit. The structures of the repeating units also explain the previously observed cross-reactivity between the strains and to E. coli O164, which only differs in the terminal sugar residue that is lacking the (R)-1-carboxyethyl group.     

Ultraviolet Sensitivity Gene of Escherichia coli B

The ultraviolet sensitivity gene of Escherichia coli B was introduced into a K-12 recipient by transduction with phage P1. The uvs gene of E. coli B is cotransducible with the proC locus of K-12, is closely linked to tsx, is not linked to lacZ, and only rarely to purE. The transductants are mucoid, filamentous on irradiation, and show plating-medium response. The order of markers is lacZ proC tsx uvs purE.     

Gene cloning and characterization of alanine racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei.

Alanine racemase genes (alr) from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei were cloned and expressed in Escherichia coli JM109. All genes encoded a polypeptide of 359 amino acids, and showed more than 99% sequence identities with each other. In particular, the S. dysenteriae alr was identical with the S. flexneri alr. Differences in the amino acid sequences between the four Shigella enzymes were only two residues: Gly138 in S. dysenteriae and S. flexneri (Glu138 in the other) and Ile225 in S. sonnei (Thr225 in the other). The S. boydii enzyme was identical with the E. coli K12 alr enzyme. Each Shigella alr enzyme purified to homogeneity has an apparent molecular mass about 43,000 by SDS-gel electrophoresis, and about 46,000 by gel filtration. However, all enzymes showed an apparent molecular mass about 60,000 by gel filtration in the presence of a substrate, 0.1 M l-alanine. These results suggest that the Shigella alr enzymes having an ordinary monomeric structure interact with other monomer in the presence of the substrate. The enzymes were almost identical in the enzymological properties, and showed lower catalytic activities (about 210 units/mg) than those of homodimeric alanine racemases reported.     

Properties and regulation of the beta-D-galactosidase in Shigella dysenteriae and in Escherichia coli-Shigella dysenteriae hybrids

Sarkar, S. (Massachusetts Institute of Technology, Cambridge). Properties and regulation of the beta-d-galactosidase in Shigella dysenteriae and in Escherichia coli-Shigella dysenteriae hybrids. J. Bacteriol. 91:1477-1488. 1966.-Shigella dysenteriae strain 60 has a beta-d-galactosidase related to that of Escherichia coli but more heat-sensitive and with a turnover number about 10 times lower. Hybridization by transduction produces strains with enzymes of intermediate properties by recombination within the z gene. Both E. coli and S. dysenteriae have a regulatory i(+) gene. Recombination between i(-) mutants of the two organisms leads to restoration of the i(+) genotype. In S. dysenteriae 60, most of the i(-) mutants are subject to genetic suppression by suppressor mutations at unlinked loci. The effect of these suppressors on the products of the suppressed i(-) genes is discussed.     

Structural studies of the O-antigen polysaccharide from the enteroinvasive Escherichia coli O164 cross-reacting with Shigella dysenteriae type 3.

The structure of the O-antigen polysaccharide from Escherichia coli O164 has been determined. Nuclear magnetic resonance spectroscopy together with component and methylation analyses of lipid free polysaccharide were the principal methods used. The sequence of the sugar residues could be determined by NOESY and heteronuclear multiple bond connectivity NMR experiments. It is concluded that the polysaccharide is composed of a pentasaccharide repeating unit with the following structure: [structure: see text]. Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) was performed on intact lipopolysaccharide and from the resulting molecular mass, the O-antigen part was estimated to contain approximately 24 repeating units. The nature of the previously reported cross-reactivity of this O-antigen to those of Escherichia coli O124 and Shigella dysenteriae type 3 is discussed.     

Purification of a cell-associated hemagglutinin from Shigella dysenteriae type 1

Abstract A cell-associated hemagglutinin (HA) was isolated and purified from a clinical isolate of Shigella dysenteriae type 1 by affinity chromatography on a fetuin-agarose column. The purified hemagglutinin produced a single-stained protein band of around 66 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). In an immunodiffusion test, HA-antisera produced a single precipitin band against the purified HA without exhibiting any reactivity towards lipopolysaccharide (LPS) of S. dysenteriae type 1 strain. Inhibition of the hemagglutination by the glycoproteins fetuin, asialofetuin and a sugar derivative N -acetyl-neuraminic acid but not by simple sugars, suggested the specific requirement of complex carbohydrate for binding. Electron micrographs of the purified HA revealed a morphology typical of globular protein.     

Synthesis of the tetrasaccharide related to the repeating unit of the antigen from Shigella dysenteriae type 5

Starting from L-rhamnose, D-mannose and 2-amino-2-deoxy-D-glucose hydrochloride, two disaccharide blocks, namely, ethyl 2,4-di-O-benzyl-3-O-[(R)-1-(methoxycarbonyl)ethyl]-alpha-L-rhamnopyranos yl-(1-->3)-2-O-acetyl-4,6-di-O-benzyl-1-thio-alpha-D-mannopyranoside and 2-(trimethylsilyl)ethyl 2-O-acetyl-3,6-di-O-benzyl-alpha-D-mannopyranosyl-(1-->3)-4,6-di-O-benzy l-2-deoxy-2-phthalimido-beta-D-glucopyranoside, were synthesised and then allowed to react in the presence of N-iodosuccinimide and trifluoromethane sulfonic acid to give a tetrasaccharide derivative. This compound was converted into 2-(trimethylsilyl)ethyl 2,4-di-O-benzyl-3-O-[(R)-1-(methoxycarbonyl)ethyl]-alpha-L-rhamno- pyranosyl-(1-->3)-2-O-acetyl-4,6-di-O-benzyl-alpha-D-mannopyranosyl-(1-- >4)-2-O-acetyl-3,6-di-O-benzyl-alpha-D-mannopyranosyl-(1-->3)-2-acetamid o-4,6-di-O-benzyl-2-deoxy-beta-D-glucopyranoside, which on hydrogenolysis, afforded the methyl ester 2-(trimethylsilyl)ethyl glycoside of the tetrasaccharide related to the repeating unit of the O-antigen from Shigella dysenteriae type 5.     

Mapping of a Structural Gene for Valyl-Transfer Ribonucleic Acid Synthetase in Escherichia coli by Transduction

A structural gene, valS, for the valyl-transfer ribonucleic acid synthetase of Escherichia coli has been mapped on the clockwise side of pyrB and is closely linked to it.     

Structural relation of the antigenic polysaccharides of Escherichia coli O40, Shigella dysenteriae type 9, and E. coli K47

O-polysaccharides were isolated from the lipopolysaccharides of Escherichia coli O40 and Shigella dysenteriae type 9 and studied by chemical analyses along with (1)H and (13)C NMR spectroscopy. The following new structure of the O-polysaccharide of E. coli O40 was established: -->2)-beta-D-Galp-(1-->4)-beta-D-Manp-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1--> TheO-polysaccharide structure of S. dysenteriae type 9 established earlier was revised and found to be identical to the reported structure of the capsular polysaccharide of E. coli K47 and to differ from that of the E. coli O40 polysaccharide in the presence of a 3,4-linked pyruvic acid acetal having the (R)-configuration (RPyr): -->2)-beta-D-Galp3,4(RPyr)-(1-->4)-beta-D-Manp-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->