Plasmids of enterotoxigenic Escherichia coli H10407: evidence for two heat-stable enterotoxin genes and a conjugal transfer system

Three species of plasmids, associated with virulence and conjugal transfer, were identified in a clinically isolated enterotoxigenic Escherichia coli strain, H10407 (serotype O78:H11). pCS1, a non-self-transmissible plasmid species with a molecular weight of 62 X 10(6) and a 47 mol% guanine-plus-cytosine content, specified colonization factor antigen I and heat-stable enterotoxin (ST) production, as reported by others previously. A second non-self-transmissible plasmid species, designated pJY11, with a molecular weight of 42 X 10(6) and a 51 mol% guanine-plus-cytosine content, specified ST and heat-labile enterotoxin production and manifested T5/T6 phage restriction. The third plasmid species, pTRA1, also had a molecular weight of 42 X 10(6) and had a guanine-plus-cytosine content of 51 mol%; this species was self-transmissible and promoted transfer of both pCS1 and pJY11 to other bacterial cells. pCS1 may have originated from species of bacteria with a lower guanine-plus-cytosine content than E. coli. Finally, although demonstrating some heterogeneity with each other, both STs encoded by pCS1 and pJY11 belonged to the STa group.     

Amino-acid sequence of a heat-stable enterotoxin produced by human enterotoxigenic Escherichia coli

A heat-stable enterotoxin produced by a human strain of enterotoxigenic Escherichia coli was extensively purified by reverse-phase high-performance liquid chromatography. The minimum effective dose of the purified toxin to cause fluid accumulation in suckling mice was 2.5 ng. The amino acid sequence of the purified toxin was determined by Edman degradation and a combination of fast atom bombardment mass spectrometry and carboxypeptidase digestion to be Asn-Ser-Ser-Asn-Tyr-Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr. This sequence was identical to that deduced from the nucleotide sequence encoding a human heat-stable enterotoxin, reported by Moseley et al., except for the C-terminal Tyr residue.     

Catabolite repression of Escherichia coli heat-stable enterotoxin activity.

The Escherichia coli heat-stable enterotoxin (ST) coded for by plasmid pYK007 (Apr ST+) showed a dependence for cyclic adenosine 3',5'-monophosphate (cAMP) to express ST activity in an adenyl cyclase (cya) deletion mutant; no ST activity was detected in the presence of cAMP in a cAMP receptor protein (crp) deletion mutant or in a double deletion mutant (delta cya delta crp). The cya-crp effect on ST activity could not be accounted for by a modification of the copy number of plasmid deoxyribonucleic acid per chromosome equivalent or by an alteration in the secretion of an active intracellular enterotoxin.     

Preparative purification of Escherichia coli heat-stable enterotoxin

Heat-stable enterotoxin (STa) isolated from bovine Escherichia coli strains was purified to homogeneity by growing the bacterial strains in a chemically defined medium, desalting, and concentrating the culture filtrate by batch adsorption chromatography on Amberlite XAD-2 resin, batch adsorption chromatography on reversed-phase silica, and preparative reversed-phase high-performance liquid chromatography. This rapid preparative purification scheme gave high recovery yields of pure STa which exhibited biochemical homology to STa purified by more complicated procedures.     

Purification and characterization of Escherichia coli heat-stable enterotoxin II.

Escherichia coli heat-stable enterotoxin II (STII) was purified to homogeneity by successive column chromatographies from the culture supernatant of a strain harboring the plasmid encoding the STII gene. The purified STII evoked a secretory response in the suckling mouse assay and ligated rat intestinal loop assay in the presence of protease inhibitor, but the response was not observed in the absence of the inhibitor. Analyses of the peptide by the Edman degradation method and fast atom bombardment mass spectrometry revealed that purified STII is composed of 48 amino acid residues and that its amino acid sequence was identical to the 48 carboxy-terminal amino acids of STII predicted from the DNA sequence (C. H. Lee, S. L. Mosely, H. W. Moon, S. C. Whipp, C. L. Gyles, and M. So, Infect. Immun. 42:264-268, 1983). STII has four cysteine residues which form two intramolecular disulfide bonds. Two disulfide bonds were determined to be formed between Cys-10-Cys-48 and Cys-21-Cys-36 by analyzing tryptic hydrolysates of STII.     

Vibrio cholerae enterotoxin genes: nucleotide sequence analysis of DNA encoding ADP-ribosyltransferase.

The nucleotide sequence of the DNA encoding the ADP-ribosyltransferase (A1) fragment of cholera enterotoxin was determined. A putative precursor of the A1 peptide contains an 18-amino acid leader peptide, and the mature A1 peptide contains 194 amino acids. The primary structure of the A1 fragment from cholera enterotoxin is more related to that from a human enterotoxigenic Escherichia coli than to that from a porcine enterotoxigenic E. coli.     

Thermoactivation of a periplasmic heat-stable enterotoxin of Escherichia coli.

Strains of Escherichia coli that host a plasmid that codes for the heat-stable (ST) enterotoxin showed 160 times more extracellular enterotoxin than intracellular activity. However, when washed bacteria were sonicated and incubated at between 50 and 85 degrees C, an activity similar to that of the ST enterotoxin was detected. No such effect was present in strains lacking the plasmid, in a plasmid ST- mutant, or in chromosomal mutants that lack a cyclic AMP-linked positive regulatory system which previously were shown to yield an ST- phenotype. The thermoactivation was inhibited by iodoacetamide and N-ethylmaleimide; chloramphenicol did not affect the phenomenon. The heat-activated ST-like enterotoxin was localized in the periplasmic space. The results are discussed in relation to the export of the toxin from the periplasm to the outside of the cell.     

Amino acid sequence of heat-stable enterotoxin produced by Escherichia coli pathogenic for man

The primary structure of a heat-stable toxin produced by a strain of Escherichia coli pathogenic for man has been established and is given below: Asn-Thr-Phe-Tyr-Cys-Cys-Glu-Leu-Cys-Cys-Tyr-Pro-Ala-Cys-Ala-Gly-Cys-Asn.     

A modified bioassay for heat-stable Escherichia coli enterotoxin1.

Infant mice were injected orally with preparations containing Escherichia coli heat-stable enterotoxin (ST) and Evans blue dye, and incubated at 22 degrees C. With enterotoxin-positive samples, the stomach was distended and contained essentially all of the dye. With enterotoxin-negative samples, the stomach remained normal in size and the dye passed freely into the intestines. The time required to obtain the maximum ratio of gut weight to body weight varied from 30 to 90 min and was dependent upon the concentration of enterotoxin. Heat-labile enterotoxin (LT) had no effect during this period. Based on these findings, the mouse incubation time was reduced from 4 h to 90 min, and the heating of test samples was retained only for confirmation of ST. The location of the dye and stomach distention served as an indicator of positive responses to ST. Incubation of the mice at room temperature (22 degrees C) was found satisfactory.     

Recovery, growth, and production of heat-stable enterotoxin by Escherichia coli after copper-induced injury.

Exposure of enterotoxigenic Escherichia coli strains to a sublethal concentration (0.75 mg/liter) of copper for 3 days at 4 degrees C induced sensitivity to deoxycholate (0.1%). When placed in a complex (brain heart infusion) or a defined amino acid salt medium, the copper-injured cells recovered their tolerance to deoxycholate in 3 and 6 h, respectively, and commenced active growth. Growth and heat-stable enterotoxin production of uninjured and copper-injured cells were studied in brain heart infusion medium. A slightly altered growth curve and an initial slow rate of toxin production were observed in injured cells when compared with those corresponding uninjured controls. However, maximum heat-stable enterotoxin levels in injured cultures were comparable to those produced by uninjured cells, suggesting that the enterotoxigenic potential of copper-injured cells was fully retained.     

Disulfide bond formation and secretion of Escherichia coli heat-stable enterotoxin II.

The Escherichia coli heat-stable enterotoxin II (STII) is a typical extracellular toxin consisting of 48 amino acid residues, of which 4 are cysteine. There are two disulfide bonds, one between Cys-10 and Cys-48 and one between Cys-21 and Cys-36. We examined the involvement of DsbA in the formation of the disulfide bonds of STII and the role of each in the secretion of STII. A dsbA mutant was transformed with a plasmid harboring the STII gene, and STII was not detected either in the cells or in the culture supernatant. Reducing the level of STII brought about the dsbA mutation restored by introducing the wild-type dsbA gene into the mutant strain. These results showed that DsbA is involved in forming the disulfide bonds of STII and that STII without these disulfide bonds is degraded during secretion. We substituted these four cysteine residues in vivo by oligonucleotide-directed site-specific mutagenesis. The amino acid sequence of the purified STII (C48S) and pulse-chase studies revealed that two intermolecular disulfide bonds must be formed to be efficiently secreted and that cleavage between amino acid residues 14 and 15 is probably the first step in the proteolytic degradation of STII.     

Nucleotide sequence of the pntA and pntB genes encoding the pyridine nucleotide transhydrogenase of Escherichia coli

A 3240-base-pair DNA fragment spanning the pyridine nucleotide transhydrogenase (pnt) genes of Escherichia coli has been sequenced. The sequence contains two open-reading frames, pntA and pntB of 1506 and 1386 base pairs, coding for the transhydrogenase alpha and beta subunits, respectively. The coding sequences are preceded by a promoter-like structure and are most likely co-transcribed. Each coding sequence is preceded by a Shine-Dalgarno sequence. The amino-terminal amino acid sequences were determined from the purified alpha and beta subunits of the transhydrogenase. These sequences agree with those predicted from the nucleotide sequences of the pntA and pntB genes. The predicted relative molecular masses of 53906 (alpha) and 48667 (beta) are close to the values obtained by analysis of the subunits by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Several hydrophobic regions large enough to span the cytoplasmic membrane were observed in each subunit. These results indicate that transhydrogenase is an intrinsic membrane protein.