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Export and processing analysis of a fusion between the extracellular heat‐stable enterotoxin and the periplasmic B subunit of the heat‐labile enterotoxin in Escherichia coli
Author(s) -
GuzmánVerduzco L.M.,
Kupersztoch Y. M.
Publication year - 1990
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1990.tb00592.x
Subject(s) - periplasmic space , enterotoxin , heat stable enterotoxin , escherichia coli , extracellular , biology , protein subunit , secretion , biochemistry , fusion protein , signal peptide , heat labile enterotoxin , bacterial outer membrane , lysis , microbiology and biotechnology , peptide sequence , recombinant dna , gene
Summary As an initial approach in the study of the mechanism of secretion of the extracellular heat‐stable enterotoxin of Escherichia coli (ST A ), and in order to use this polypeptide as an extracellular carrier we previously constructed a fusion between the complete ST A toxin (pre‐pro‐ST A ) and the mature B subunit of the periplasmic heat‐labile enterotoxin (LT B ); the resulting ST A ‐LT B hybrid was not secreted to the extracellular environment, and cells expressing the hybrid lysed at temperatures above 35°C. In this work we have established that the hybrid is initially detected as pre‐pro‐ST A ‐LT B and converted to pro‐ST A ‐LT B , which lacks the 19amino acids that share the properties of a signal peptide; the sequenced 17 amino‐terminal residues of pro‐ST A ‐LT B defined the processing site of pre‐pro‐ST A ‐LT B at pro_3phe_2ala_1 ↓ gln + 1. This process was sensitive to an energy uncoupler (CCCP) and was correlated with translocation of pro‐ST A ‐LT B across the inner membrane. Additionally, we are able to show that although pre‐pro‐ST A ‐LT B is processed at 37°C and 29°C, it is more efficiently processed at the latter temperature. At 37°C, pro‐ST A ‐LT B was poorly released into the periplasm, resulting in accumulation of this protein, pre‐pro‐ST A ‐LT B , and pre‐β‐lactamase in the inner membrane, and in cell lysis. In contrast, at 29°C pro‐ST A ‐LT B was localized in the periplasm and in the inner membrane, and pre‐pro‐ST A ‐LT B and pre‐β‐lactamase did not accumulate; however, translocation of periplasmic pro‐ST A ‐LT B across the outer membrane still did not occur, and a second processing step that would eliminate the pro segment from pro‐ST A ‐LT B was never observed. Thus, the fusion of pre‐pro‐ST A and LT B resulted in a polypeptide that, while incompatible with secretion to the extracellular medium, is exported to the periplasm in a temperature‐conditional fashion. This latter observation is consistent with an ST A secretion pathway whereby pre‐pro‐ST A is first processed to periplasmic pro‐ST A by the removal of 19‐amino‐acid signal peptide.