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.
As an initial approach in the study of the mechanism of secretion of the extracellular heat-stable enterotoxin of Escherichia coli (STA), and in order to use this polypeptide as an extracellular carrier we previously constructed a fusion between the complete STA toxin (pre-pro-STA) and the mature B subunit of the periplasmic heat-labile enterotoxin (LTB); the resulting STA-LTB hybrid was not secreted to the extracellular environment, and cells expressing the hybrid lysed at temperatures above 35 degrees C. In this work we have established that the hybrid is initially detected as pre-pro-STA-LTB and converted to pro-STA-LTB, which lacks the 19 amino acids that share the properties of a signal peptide; the sequenced 17 amino-terminal residues of pro-STA-LTB defined the processing site of pre-pro-STA-LTB at pro-3phe-2ala-1 decreases gln+1. This process was sensitive to an energy uncoupler (CCCP) and was correlated with translocation of pro-STA-LTB across the inner membrane. Additionally, we are able to show that although pre-pro-STA-LTB is processed at 37 degrees C and 29 degrees C, it is more efficiently processed at the latter temperature. At 37 degrees C, pro-STA-LTB was poorly released into the periplasm, resulting in accumulation of this protein, pre-pro-STA-LTB, and pre-beta-lactamase in the inner membrane, and in cell lysis. In contrast, at 29 degrees C pro-STA-LTB was localized in the periplasm and in the inner membrane, and pre-pro-STA-LTB and pre-beta-lactamase did not accumulate; however, translocation of periplasmic pro-STA-LTB across the outer membrane still did not occur, and a second processing step that would eliminate the pro segment from pro-STA-LTB was never observed. Thus, the fusion of pre-pro-STA and LTB 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 STA secretion pathway whereby pre-pro-STA is first processed to periplasmic pro-STA by the removal of a 19-amino-acid signal peptide.
Department of Microbiology, University of Texas, Southwestern Medical Center, Dallas 75235.
L M
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Guzmán-Verduzco
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Kupersztoch
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D001433:Bacteriolysis / Q000502:physiology; D001692:Biological Transport / Q000235:genetics; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D004768:Enterotoxins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013696:Temperature; D014357:Trypsin
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C034151:heat stable toxin (E coli); D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; C027538:heat-labile enterotoxin, E coli; D014357:Trypsin