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..     

Survival of Shigella in Sewage: II. Effect of Glycerol on Shigella flexneri and Shigella Bacteriophage1

Glycerol (30%) inhibited or delayed the adsorption of Shigella bacteriophage on its host organism, S. flexneri II; glycerol also inhibited or delayed the burst of phage, whether or not adsorption was carried out in the presence of glycerol. Studies of the mechanisms of these effects showed that viscosity and osmotic shock probably were not responsible for either phenomenon. The inhibition of adsorption, however, was proportional to the concentration of glycerol, and appeared to be a function of the hydroxyl groups on the glycerol molecule. The inhibition of burst seemed to be related to the osmotic pressure outside the bacterial cells.     

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.     

Aerobactin genes in Shigella spp.

Aerobactin, a hydroxamate iron transport compound, is synthesized by some, but not all, Shigella species. Conjugation and hybridization studies indicated that the genes for the synthesis and transport of aerobactin are linked and are found on the chromosome of Shigella flexneri, S. boydii, and S. sonnei. The genes were not found in S. dysenteriae. A number of aerobactin synthesis mutants and transport mutants have been isolated. The most common mutations are deletions of the biosynthesis or biosynthesis and transport genes. The Shigella aerobactin genes share considerable homology with the E. coli ColV aerobactin genes. On the ColV plasmid and in the Shigella chromosome, the aerobactin genes are associated with a repetitive sequence which has been identified as IS1.     

Genetic basis of virulence in Shigella species.

Shigella species and enteroinvasive strains of Escherichia coli cause disease by invasion of the colonic epithelium, and this invasive phenotype is mediated by genes carried on 180- to 240-kb plasmids. In addition, at least eight loci on the Shigella chromosome are necessary for full expression of virulence. The products of these genes can be classified as (i) virulence determinants that directly affect the ability of shigellae to survive in the intestinal tissues, e.g., the aerobactin siderophore (iucABCD and iutA), superoxide dismutase (sodB), and somatic antigen expression (rfa and rfb); (ii) cytotoxins that contribute to the severity of disease, e.g., the Shiga toxin (stx) and a putative analog of this toxin (flu); and (iii) regulatory loci that affect the expression of plasmid genes, e.g., ompR-envZ, which mediates response to changes in osmolarity, virR (osmZ), which mediates response to changes in temperature, and kcpA, which affects the translation of the plasmid virG (icsA) gene which is associated with intracellular bacterial mobility and intracellular bacterial spread. A single plasmid regulatory gene (virF) controls a virulence-associated plasmid regulon including virG (icsA) and two invasion-related loci, i.e., (i) ipaABCD, encoding invasion plasmid antigens that may be structural components of the Shigella invasion determinant; and (ii) invAKJH (mxi), which is necessary for insertion of invasion plasmid antigens into the outer membrane.     

PCR for Detection of Shigella spp. in Mayonnaise

The use of PCR to amplify a specific virA gene fragment serves as a highly specific and sensitive method to detect virulent bacteria of the genus Shigella and enteroinvasive Escherichia coli. Amplification of a 215-bp DNA band was obtained by using isolated genomic DNA of Shigella, individual cells of Shigella dysenteriae, and mayonnaise contaminated with S. dysenteriae. Moreover, a multiplex PCR with specific (virA) and bacterium-restricted (16S ribosomal DNA) primers generated an amplification product of approximately 755 bp for all bacteria tested and an additional 215-bp product for Shigella and enteroinvasive E. coli.     

To Shigella sonnei phage typing.

The epidemiological significance of phage types changes in the course of years. In Czechoslovakia, in the years 1967-1971, the most frequent phage types were 2,6, 3, 30 and 65, and, in the Middle-Bohemian Region, 2,30, 6, 3 and 65. In the epidemiological years 1972-1973 the sequence of the most frequent S. sonnet phage types changed in the Middle-Bohemian region to: 65, 23, 6, 2 and 12. Some problems concerning the instability of phage types and the part of further auxiliary tests (biochemical differentiation according to Bojlen and drug sensitivity pattern) are discussed.     

Somatic antigens of Shigella. The strucuture of the specific polysaccharide chain of Shigella dysenteriae type 5 lipopolysaccharide.

The specific polysaccharide was released from Shigella dysenteriae type 5 lipopolysaccharide by mild acidic hydrolysis and then purified by gel chromatography on Sephadex G-50. The polysaccharide was built up of residues of D-mannose, 2-acetamido-2-deoxy-D-glucose, 3-0-(D-1-carboxyethyl)-L-rhamnose (rhamnolactylic acid) and 0-acetyl groups in a ratio 2:1:1:1. On the basis of radiospectroscopy, methylation analysis, Smith degradation, and chromium trioxide oxidation, the repeating oligosaccharide unit of the polysaccharide can be assigned the following structure: (formula: see text) where GlcNAc is 2-acetamido-2-deoxy-D-glucopyranose, Manp is mannopyranose, RhaLcA is rhammolacytic acid and Ac is an acetyl group. The serological properties of Sh. dysenteriae somatic antigens are discussed in relation to the chemical structures of their specific polysaccharides.     

Somatic antigens of Shigella. Structural investigation on the O-specific polysaccharide chain of Shigella dysenteriae type-2 lipopolysaccharide.

The O-specific polysaccharide obtained from Shigella dysenteriae type-2 lipopolysaccharide by mild acid hydrolysis consisted of N-acetylgalactosamine, N-acetylglucosamine, D-galactose, D-glucose, and O-acetyl group in the ratio of 2:1:1:1:1. A number of oligosaccharides were obtained by deamination of the N-deacetylated polysaccharide and by Smith degradation of the both native and O-deacetylated polysaccharides. The identification of oligosaccharides along with methylation analysis and chromic anhydride oxidation showed that the polysaccharide was built up of the repeating pentasaccharide units whose proposed structure is given below: (see article) Serological properties of Sh. dysenteriae O-specific polysaccharides are discussed.     

Analysis of mobilization elements in plasmids from Shigella flexneri.

The mobilization properties of three plasmids were examined after cotransfer from Shigella flexneri to Escherichia coli. The largest plasmid, pCN1, was shown to be a conjugative R factor that could promote its own transfer and allow cotransfer of a 4.1-kilobase plasmid, pCN3; mobilization of the third plasmid, pCN2 (6.3 kilobases), required the presence of both pCN1 and pCN3. Sequences from pCN2 and pCN3 homologous to the bom (basis of mobilization) sites of ColE1 and pBR322 were localized by analysis of site-specific deletion derivatives generated in vivo during the transfer of composite plasmids and were characterized by DNA sequencing.