Nucleotide sequence and promoter mapping of the Escherichia coli Shiga-like toxin operon of bacteriophage H-19B

Abstract

We determined the nucleotide sequence of the Shiga-like toxin-1 (SLT-1) genes carried by the toxinconverting bacteriophage H-19B. Two open reading frames were identified; these were separated by 12 base pairs and encoded proteins of 315 (A subunit) and 89 (B subunit) amino acids. The predicted protein subunits had N-terminal hydrophobic signal sequences of 22 and 20 amino acids, respectively. The predicted amino acid sequence of the B subunit was identical to that of the B subunit of Shiga toxin. The A chain of ricin was found to be significantly related to the predicted Al fragment of the SLT-1 A subunit. SI nuclease protection experiments showed that the two cistrons formed a single transcriptional unit, with the A subunit being proximal to the promoter. A probable promoter was identified by primer extension, and transcription was found to increase dramatically under conditions of iron starvation. A 21-base-pair sequence with dyad symmetry was found in the region of the SLT-1 -10 sequence, which was found to be 68% homologous to a region of dyad symmetry found in the -35 region of the promoter of the iucA gene op plasmid ColV-K30, which specifies the 74,000-dalton ferric-aerobactin receptor protein. Shigella dysenteriae 1 produces a toxin which is cytotoxic to eucaryotic cell lines (8). Binding to the glycolipid globotriaosylceramide (Gb3) membrane receptor is mediated by a pentamer of 7-kilodalton (kDa) B subunits, while the 31-kDa A subunit, after proteolytic nicking and reduction, inhibits protein synthesis by catalytic inactivation of the 60S ribosomal subunit (8,20,34,35). O'Brien and LaVeck (30,31) have shown that some Escherichia coli strains produce large amounts of a cytotoxin which appeared very similar to Shiga toxin with respect to its subunit structure and mechanism of action. It was completely neutralized by antiserum raised against Shiga toxin and was named Shiga-like toxin 1 (SLT-1) (28, 42). Recently, Strockbine et al. (42) have characterized a second cytotoxin, also produced by E. coli, which is related to SLT-1 at the DNA sequence level but is not neutralized by antiserum raised against Shiga toxin or SLT-

Characterization of verocytotoxin-producingEscherichia coliO157 isolates from patients with haemolytic uraemic syndrome in Western Europe

“…The higher prevalence of infection among HUS-patients with VTEC producing only VT2 than infection with VTEC producing either only VT1 or a combination of VT1 and VT2, is consistent with other studies carried out in Western Europe and North-America [2,31]. However, Rowe and co-workers [32] recently found that 22 of the 26 0157. H7 VTEC isolates from Canadian HUSpatients produced both VT1 and VT2 rather than VT2 only.…”

“…Fifty-two human faecal VTEC isolates were studied, including 30 isolates from 19 children with D + HUS, nine isolates from family contacts (n = 9) of five of these HUS-patients, 11 2687-2706 * The position numbers are those in the published nucleotide sequences of VT1 [22], VT2 [23] and eae [24].…”

Molecular cloning and nucleotide sequence of another variant of the Escherichia coli Shiga-like toxin II family

“…This mechanism of action is shared by plant toxins such as ricin (Endo et al, 1987). Homology of the amino acid sequence of the processed A subunit of SLTs with ricin A chain has been noted by several workers (De Grandis et al, 1987;Calderwood et al, 1987;Weinstein et al, 1988) and recently, Hovde et al (1988) have demonstrated, with oligonucleotide-directed mutagenesis, that the glutamic acid residue at position 167 of SLTI is necessary for full enzymic activity of the toxin subunit. A number of common amino acids were noted from positions 165 to 206 of the processed SLTIIva A subunit and 175 to 215 of the ricin A chain (see Fig.…”

“…Two open reading frames corresponding to the genes encoding the A and B subunits were located within this DNA fragment. Homology to the published DNA sequences and the predicted amino acid sequences of SLTs (Calderwood et al, 1987;De Grandis et al, 1987;Jackson et al, 1987a;Weinstein et al, 1988;Gyles et al, 1988) is summarized in Table 4. The DNA sequence of the B subunit gene had the closest relationship (98 %) to the B subunit region of sZtIIv while the A subunit gene had a similar level of homology to the A subunit genes of sZtIIv (70.6%) and sltII (6905%).…”

Alteration of the carbohydrate binding specificity of verotoxins from Gal alpha 1-4Gal to GalNAc beta 1-3Gal alpha 1-4Gal and vice versa by site-directed mutagenesis of the binding subunit.

“…The toxin family can be divided into two main groups, the first of which consists of Shiga toxin and VT-1. These are fully crossneutralizable and differ only in a single conservative amino acid substitution in the A subunit (6,7). The second group includes SLT-II, VT-2, and VTE which are partially crossneutralizable but are not neutralized at all by antiserum raised against Shiga toxin (3).…”