The expression of lipopolysaccharide by strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii and their cross-reacting strains of Escherichia coli

Strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii express lipopolysaccharides, that enable the serotyping of strains based on their antigenic structures. Certain strains of S. dysenteriae, S. flexneri and S. boydii are known to share epitopes with strains of Escherichia coli ; however, the lipopolysaccharide profiles of the cross-reacting organisms have not been compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) lipopolysaccharides profiling. In the present study, type strains of these bacteria were examined using SDS-PAGE/silver staining to compare their respective lipopolysaccharide profiles. Strains of S. dysenteriae, S. boydii and S. flexneri all expressed long-chain lipopolysaccharide, with distinct profile patterns. The majority of strains of Shigella spp., known to cross-react with strains of E. coli , had lipopolysaccharide profiles quite distinct from the respective strain of E. coli . It was concluded that while cross-reacting strains of Shigella spp. and E. coli may express shared lipopolysaccharide epitopes, their lipopolysaccharide structures are not identical.     

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 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-->     

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.     

Nucleotide sequences of the trpG regions of Escherichia coli,Shigella dysenteriae,Salmonella typhimurium and Serratia marcescens

The nucleotide sequences of Serratia marcescens trpG and the corresponding regions of Escherichia coli, Shigella dysenteriae and Salmonella typhimurium trpD have been determined. Analysis of the nucleotide sequence divergence suggests the following evolutionary relationships: Serratia-[Salmonella, (Escherichia, Shigella)]. Partial reconstruction of ancestral nucleotide sequences and subsequent analysis of nucleotide substitutions show that the majority of nucleotide substitutions in the evolution of trp(G)D are transitions that result in a reduction of G + C content. Since most of the nucleotide substitutions are in the third position of codons, bias in synonymous codon usage also reflects G + C content. The trpE-trp(G)D junction in the four organisms is characterized by overlapping translation termination and initiation codons. The relative positions of trpE and trp(G)D thus became fixed in evolution before the fusion of trpG and trpD. Nucleotide sequences representing the fusion of trpG and trpD in Escherichia, Shigella and Salmonella are not more nor less divergent than other portions of the trp(G)D coding sequences.     

Use of coagglutination for serotyping Shigella dysenteriae and Shigella boydii

The coagglutination test was used to identify Shigella boydii and Shigella dysenteriae. A trial was carried out with 13 native rabbit antisera to S. boydii and 10 antisera to S. dysenteriae, as well as with coagglutinating reagents prepared from these antisera. The use of coagglutinating reagents was shown to ensure the complete specificity of the results, to prevent the adsorption of diagnostic antisera and to decrease their consumption 50 times. The importance of the coagglutination test for the identification of shigellae is discussed.     

Phosphate regulon in members of the family Enterobacteriaceae: comparison of the phoB-phoR operons of Escherichia coli, Shigella dysenteriae, and Klebsiella pneumoniae.

The structure and function of the phoB and phoR genes of Shigella dysenteriae strains and Klebsiella pneumoniae, which are involved in regulation of the phosphate regulon, were analyzed. Complementation tests among the genes of Escherichia coli, S. dysenteriae strains, and K. pneumoniae for production of alkaline phosphatase indicate that S. dysenteriae serotype 2 and serotype 3 strains and K. pneumoniae are phoA+ phoB+ phoR+ but S. dysenteriae Sh and serotype 1 strains are phoA phoB+ phoR. Nucleotide sequences of phoB and phoR of S. dysenteriae Sh and K. pneumoniae are highly homologous to those of E. coli, except for a single base insertion found in phoR of S. dysenteriae Sh.     

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.     

The detection of Vero cytotoxin-producing Escherichia coli and Shigella dysenteriae type 1 in faecal specimens using polymerase chain reaction gene amplification

Fifty consecutive faecal specimens received by the LEP were examined for the presence of Vero cytotoxin (VT) genes by polymerase chain reaction (PCR) gene amplification. Nineteen were positive by PCR and from 16 of these, VT positive Escherichia coli O157 were isolated. The remaining three samples were positive for VT genes by PCR but VTEC were not isolated. In a preliminary experiment, Shigella dysenteriae type 1 was isolated from a case of bloody diarrhoea following a positive amplification result.     

Specificity of the Escherichia coli proline transport system.

The presence of both the carbonyl portion of the carboxyl group at position 2 of the pyrrolidine ring and a secondary amine was essential for uptake of a compound by the proline permease of Escherichia coli. The permease possessed a high affinity for azetidine-2-carboxylic acid and for compounds with ring structures smaller than the pyrrolidine ring. Pipecolic acid, the higher homologue of proline, and its derivatives were not transported. Cis- and trans-3,4-methano-prolines, also six-membered ring structures, behaved anomolously in that they possessed a high affinity for the permease. The difference between the methano-prolines and other six-membered ring structures probably resides in the fact that the former exist in the "boat" configuration whereas the latter possess the "chair" configuration. In general, substituted prolines in the cis configuration displayed a higher affinity for the permease than did corresponding trans isomers, though the affinity for substituted prolines was influenced by the position, size, and polar or nonpolar nature of the substituent group. At O C many analogues with affinity for proline permease exchanged with intracellular proline, but some analogues, notably trans-3-methyl- and trans-4-methyl-L-prolines, though possessing high affinity for the permease, showed an almost complete inability to exchange with intracellular proline.     

Characterization of cryptic flagellin genes in Shigella boydii and Shigella dysenteriae.

Flagellin (fliC) genes of 12 Shigella boydii and five Shigella dysenteriae strains were characterized. Though these strains are nonmotile, the cryptic fliCSB gene, cloned from S. boydii strain C3, is functional for expression of flagellin. It consists of 1,704 bp, and encodes 568 amino acid residues (57,918 Da). The fliCSD gene from S. dysenteriae strain 16 consists of 1,650 bp encoding 549 amino acid residues (57,591 Da) and contains an IS1 element inserted in its 3' end. The two genes are composed of the 5'-constant, central variable and 3'-constant sequences, like other known fliC genes. The two genes share high homology in nucleotide and amino acid sequences with each other and also with the Escherichia coli fliCE gene, indicating that both genes are closely related to the fliCE gene. Comparison of the central variable sequences of six different fliC genes showed that the fliCSB and fliCSD genes share low homology in amino acid sequence with the other fliC genes, suggesting that they encode antigenic determinants intrinsic to respective subgroups. However, Southern blotting using as probes the central variable sequences of several fliC genes showed that four of 12 S. boydii strains have a fliC gene similar to that of Shigella flexneri, and that among five fliC genes from S. dysenteriae strains, one is similar to that of S. flexneri, two are similar to that of S. boydii, and only one is unique to S. dysenteriae. Some of these variant alleles were verified by immunoblotting with flagellins produced from cloned fliC genes. The presence of variant fliC alleles in S. boydii and S. dysenteriae indicates that subdivision into subgroups does not reflect the ancestral flagella H antigenic relationships. These data will be useful in considering the evolutionary divergence of the Shigella spp..     

Specificity of the Transport of Lipid II by FtsW in Escherichia coli

Synthesis of biogenic membranes requires transbilayer movement of lipid-linked sugar molecules. This biological process, which is fundamental in prokaryotic cells, remains as yet not clearly understood. In order to obtain insights into the molecular basis of its mode of action, we analyzed the structure-function relationship between Lipid II, the important building block of the bacterial cell wall, and its inner membrane-localized transporter FtsW. Here, we show that the predicted transmembrane helix 4 of Escherichia coli FtsW (this protein consists of 10 predicted transmembrane segments) is required for the transport activity of the protein. We have identified two charged residues (Arg¹⁴⁵ and Lys¹⁵³) within this segment that are specifically involved in the flipping of Lipid II. Mutating these two amino acids to uncharged ones affected the transport activity of FtsW. This was consistent with loss of in vivo activity of the mutants, as manifested by their inability to complement a temperature-sensitive strain of FtsW. The transport activity of FtsW could be inhibited with a Lipid II variant having an additional size of 420 Da. Reducing the size of this analog by about 274 Da resulted in the resumption of the transport activity of FtsW. This suggests that the integral membrane protein FtsW forms a size-restricted porelike structure, which accommodates Lipid II during transport across the bacterial cytoplasmic membrane.