Localization of stx, a determinant essential for high-level production of shiga toxin by Shigella dysenteriae serotype 1, near pyrF and generation of stx transposon mutants

Abstract

Hfr strains of Shigella dysenteriae serotype 1 were constructed by transient integration of an RP4 plasmid derivative carrying transposon TnSOI into the Shigella chromosome through TnSOl-mediated cointegration. The Hfr strains were mated with Escherichia coli K-12 recipients carrying various auxotrophic markers, and E. coli recombinants which had received prototrophic Shigella genes were selected. Some of the E. coli transconjugants produced high levels of a cytotoxin which was neutralized by both polyclonal and monoclonal anti-Shiga toxin sera. The determinant for Shiga toxin production, designated stx, was first transferred to E. coli K-12 and then mapped by Hfr crosses to the trp-pyrF region located at 30 min on the E. coli chromosome. Bacteriophage Pl-mediated transduction analysis of stx gave the following gene order: trp-pyrF-stx. The level of Shiga toxin production in E. coli Stx+ transconjugants and transductants was as high as that of the parental S. dysenteriae 1 strain. Stxmutants of an Stx+ E. coli transductant were generated by random in vivo insertion mutagenesis with a TnlO derivative transposon, Tn-mini-kan, followed by P1 cotransduction of the kanamycin resistance and PyrF+ markers into a pyrF Stx+ E. coli K-12 recipient. One stx::Tn-mini-kan transposon mutation was transferred by P1 transduction from this E. coli Stxmutant to an E. coli K-12 Hfr strain and in turn transferred by conjugation to the original S. dysenteriae 1 strain plus two others. All kanamycinresistant recombinants of S. dysenteriae 1 had lost their ability to produce high levels of Shiga toxin. A gene that specifies high-level Shiga toxin production is thus located near pyrF on the chromosome of S. dysenteriae 1. Stx mutants of S. dysenteriae 1 exhibited full virulence in the Sereny test.

Spontaneous tandem amplification and deletion of the Shiga toxin operon in Shigella dysenteriae 1

“…Genetic studies have mapped stx near pyrF, a chromosomal locus at 28 min that is not linked to other known virulence determinants of Shigella spp. (Sekizaki et al, 1987). This approximate location was corroborated by studies with chlorate-resistant mutants of S. dysenteriae 1.…”

“…In E. coli , this region encompasses ≈ 56 kbp. This location correlates well with prior genetic mapping, which placed the stx operon near pyrF at min 28 and near the trp and putative chl loci at min 27 (Gemski et al ., 1972; Sekizaki et al ., 1987; Neill et al ., 1988).…”

Construction of stable LamB-Shiga toxin B subunit hybrids: analysis of expression in Salmonella typhimurium aroA strains and stimulation of B subunit-specific mucosal and serum antibody responses

“…The B subunit binds to its cell surface receptor, a glycolipid, globotriosyl ceramide (Gb3), carrying a terminal Galoa(1-4)Gal moiety (20,25). Chromosomal sixA and stxB genes which encode the Shiga toxin A and B subunits, respectively, have been mapped, cloned (45,48), and sequenced (48). The role of Shiga toxin in pathogenesis has recently been explored by Fontaine and colleagues (12), who demonstrated that it influences the severity of bacillary dysentery by inducing colonic ischemia and inflation of the polymorphonuclear intestinal compartment during the infection process.…”