Expression of the gene of the methanol-soluble, heat-stable enterotoxin of Escherichia coli (STA) allowed the identification by SDS-PAGE of a cell-associated 7500 Dalton STA-related peptide; when similar experiments were performed with a phosphate buffer SDS-PAGE system, an additional Mr 9800 band became apparent. The 9800 Dalton form, pre-pro-STA, accumulated as an intracellular species when the experiments were performed in the presence of the proton ionophore CCCP (carbonylcyanide m-chlorophenylhydrazone); by pulse-chase experiments, it was shown that pre-pro-STA became a periplasmic Mr 7500 pro-STA and this form was chased to the culture supernatant; periplasmic and extracellular pro-STA showed the same electrophoretic mobility. A short time after the pulse, pro-STA was converted extracellularly to mature STA (Mr 4500). It is proposed that STA is synthesized as pre-pro-STA, a 72-amino-acid peptide that is subsequently cleaved between amino acids 19 and 20 as it is translocated across the inner membrane. The resulting 53-amino-acid pro-STA is first detected in the periplasm and is then secreted to the culture supernatant. Pro-STA is cleaved extracellularly to yield mature STA (Mr 4500).

The 3' terminus of the DNA coding for the extracellular Escherichia coli heat-stable enterotoxin (ST) devoid of transcription and translation stop signals was fused to the 5' terminus of the DNA coding for the periplasmic B subunit of the heat-labile enterotoxin (LTB) deleted of ribosomal binding sites and leader peptide. By RNA-DNA hybridization analysis, it was shown that the fused DNA was transcribed in vivo into an RNA species in close agreement with the expected molecular weight inferred from the nucleotide sequence. The translation products of the fused DNA resulted in a hybrid molecule recognized in Western blots (immunoblots) with antibodies directed against the heat-labile moiety. Anti-LTB antibodies coupled to a solid support bound ST and LTB simultaneously when incubated with ST-LTB cellular extracts. By [35S]cysteine pulse-chase experiments, it was shown that the fused ST-LTB polypeptide was converted from a precursor with an equivalent electrophoretic mobility of 20,800 daltons to an approximately 18,500-dalton species, which accumulated within the cell. The data suggest that wild-type ST undergoes at least two processing steps during its export to the culture supernatant. Blocking the natural carboxy terminus of ST inhibited the second proteolytic step and extracellular delivery of the hybrid molecule.Enterotoxigenic Escherichia coli strains have been isolated from 9 to 30% of cases of diarrheal disease in infants (12). Two kinds of plasmid-encoded enterotoxins have been shown to induce secretory diarrhea in humans and animals: the heat-stable (ST) and the heat-labile (LT) families of enterotoxins (32, 37). ST subtype A (STA) is an extracellular (13), methanol-soluble, 18-to 19-amino-acid peptide; due to its low molecular weight, it is not naturally immunogenic. The genetic information encoded by ST DNA results in the extracellular delivery of the ST enterotoxin; the amino acid sequence inferred from the DNA sequence (11, 27, 38; H. Stieglitz, R. Robledo, R. Fonseca, L. Cervantes, L. Covarrubias, F. Bolivar, and Y. M. Kupersztoch, manuscript in preparation) shows an initial 19-amino-acid region that has the properties of a leader peptide (31), followed by a 53-amino-acid-long peptide that includes the active 19-aminoacid ST enterotoxin (2) thought to be decisive for the final localization of the toxin. The gene is followed by proposed translation and transcription termination signals (27,38 harboring the fused DNA produced both ST and LTB. It is also shown that the fused ST-LTB is first detected as a precursor which after chasing is accumulated as a cellassociated form apparently 2,300 daltons smaller. The data support the hypothesis that the ST enterotoxin undergoes two proteolytic steps during its export process. The results are discussed in relation to the export of ST and the use of the fusion product for immunoprotection against both toxins and for their detection.MATERIALS AND METHODS Bacterial strains, plasmids, and media. The E. coli strains used were JM83 [ara A(lac-pro) rpsL thi 4)80d ...

We have used a combination of nucleotide and N-terminal-amino-acid-sequence analyses to determine the primary structure of the 37-kilodalton (kDa) endoflageliar outer layer, or sheath, protein. Initially, a Agtll clone (designated AA34) expressing a portion of the 37-kDa protein was selected from a Treponema pallidum genomic library with a murine monoclonal antibody (H9-2) directed against an epitope of the 37-kDa protein.The insert from AA34 provided a probe with which a chimeric plasmid (pRI4) encoding all but the nine N-terminal amino acids of the entire protein was selected from a T. palldium(pBR322) genomic library. The nine N-terminal amino acids determined by amino acid sequencing were combined with the DNA sequence encoded by pRI4 to determine the primary structure of the entire 37-kDa protein; the combined sequence made up a polypeptide with a calculated molecular mass of 36,948 Da. Approximately one-third of the deduced sequence was confirmed by N-terminal amino acid analysis of tryptic peptides from the purified 37-kDa protein. Repeated attempts to clone upstream portions of the gene (flaA) by using a variety of strategies were unsuccessful, suggesting that unregulated expression of the intact sheath protein or of its most amino-terminal portions is toxic in Escherichia coli. These studies should provide the basis for further molecular investigations of the endoflagellar apparatus and of treponemal motility.

Summary The methanol‐soluble, heat‐stable enterotoxin of Escherichia coli is a protease‐resistant extracellular peptide which is synthesized as a 72‐amino‐acid precursor Pre‐Pro‐STA3. The specific roles of Fre (19 amino acids), Pro (34 amino acids) and STA3 (19 amino acids) in the secretion process were studied by functionally deleting each of the three domains. Deletion of the Pre signal sequence resulted in a short‐lived cell‐associated molecule with an Mr equivalent to that of Pro‐STA3. Deletion of Pro (i.e., Pre‐STA3) resulted in the rapid extracellular accumulation of STA3 the periplasmic intermediate found in the secretion of the wild‐type toxin was undetected. Deletion of the STA3 domain resulted in a cell‐associated Pre‐Pro peptide; with time this form converted to periplasmic Pro which later became extracellular. When DNA encoding either STA3, by itself, or Pro‐STA3 (lacking the signal peptide) was expressed, these peptides were degraded intracellularly, with no periplasmic or extracellular forms detected. The results presented demonstrate that the signal peptide (Pre) is essential even for the export of small peptides to the periplasm, and that its absence causes the STAS domain to become susceptible to intracellular proteases. The rapid degradation of intracetlular STA3 indicates that its proteolytic resistance is acquired in a compartment other than the cytoplasm. The results also show that after the Pre domain is proteolytically cleaved from Pre‐STA3 and Pre‐Pro, the STA3 and Pro peptides can exit to the culture supernatant. Since STA3 and Pro have neither sequence nor conformational homology, it appears

Resequencing estA3, an allele of the methanol-soluble heat-stable enterotoxin of Escherichia coli showed that the proline triplet 19 is in fact an alanine codon; thus, estA alleles 3 and 4 were shown to be identical. Resequencing has also shown that the carboxy terminus of another allele, estA2, is not the previously inferred histidine triplet but the same tyrosine codon reported for all other estA alleles. The improperly inferred histidine codon was used in constructions to fuse estA2 to the B subunit of the heat-labile enterotoxin gene, and the fused gene products as well as three amino acid insertional mutants containing histidine-72 were not efficiently secreted. We show that the defective secretion is not due to histidine as a carboxy-terminal residue, since site-directed mutagenesis of wild-type tyrosine-72 to histidine did not influence the localization of the activity of the methanol-soluble heat-stable enterotoxin.