Cloning and expression of the genes specifying Shiga-like toxin production in Escherichia coli H19

Abstract

Some strains of Escherichia coli produce a protein which is cytotoxic for Vero cell and HeLa cell monolayers. This toxin is very similar to the toxin of Shigella dysenteriae 1 and has been named verotoxin or E. coli Shiga-like toxin. It has been shown that toxin conversion is due to a group of bacteriophages, one of which has been designated H-19B. In this study we report hybridization experiments showing that part of the H-19B genome is homologous to phage lambda. We have cloned a 1.7-kilobase BalI-BglII fragment from the genome of H-19B into pUC18. The recombinant plasmid confers the ability to produce high levels of Shiga-like toxin on transformed E. coli cells. We demonstrate using an in vitro transcription/translation system that the cloned fragment specifies the two verotoxin subunit peptides which have masses of 31 and 5.5 kilodaltons. The identity of peptides was confirmed by immunoprecipitation with verotoxin antiserum and protein A-Sepharose beads.

Vero cytotoxin-producing strains of Escherichia coli

“…VT genes have been cloned in E. coli strain K12 from phages originating in strains of serogroups 026 and 0157 (Newland et al, 1985;Willshaw et al, 1985Huang et al, 1986). The VT1 region cloned from a strain of serogroup 0157 was very similar to the VT1 sequences cloned from strain H19 in the distribution of sites for several restriction enzymes.…”

Lipid modulation of glycolipid receptor function

“…Goat antimouse and antirabbit IgG horseradish peroxidase conjugates were from Sigma. Verotoxin-1 was purified from the recombinant strain pJLB28 [24] as described [25]. Toxin was radioiodinated using iodobeads (Pierce) [13].…”

Diphtheria Disease and Genes Involved in Formation of Diphthamide, Key Effector of the Diphtheria Toxin

“…The latter scenario is predominantly found in bacterial species and accounts for lysogenic conversions, phenomena in which phenotypes of the microbial host including pathogenicity, growth performance and production of virulence factors and toxins can be significantly affected by expression of prophage encoded genes. Prominent examples for phage-dependent toxin expression and disease formation include exotoxin-associated scarlet fever by Streptocoocus pyogenes (Johnson et al, 1986;Broudy et al, 2001), dysentery causing Shiga toxins Stx1 and Stx2 from Shigella dysenteriae (Newland et al, 1985;Willshaw et al, 1985;Huang, et al, 1986), phage CTXφ encoded cholera toxin from Vibrio cholera (Waldor & Mekalanos, 1996;Faruque & Nair, 2002) and diphtheria toxin encoded on a beta prophage from lysogens of Corynebacterium diphtheriae (Holmes & Barksdale, 1969;Bishai & Murphy 1988). Together 334 with tRNase ribotoxins and anticodon nucleases from prokaryal and eukaryal microbes that have been shown to be encoded by transposable elements as well as circular and nonconventional linear DNA plasmids (Tokunaga et al, 1990;Kaufmann, 2000;Schaffrath & Meinhardt, 2005;Schaffrath et al, 1999), all of these genetic constellations implicate scenarios in which killer phenotypes have been evolved and spread by way of viral DNA transduction pathways or other forms of horizontal gene transfer.…”