Cell death caused by hyper-expression of a secretory exoglucanase in Escherichia coli.
Induced expression of a gene fusion between the ompA leader sequence and the Cellulomonas fimi cex gene encoding a secretory exoglucanase, Exg, engineered in the Tac-cassette excretion vector was lethal to Escherichia coli. An exponentially growing culture harboring the recombinant construct suffered slow growth and 99.9% of its cells died within 60-100 min after induction. This abnormality was found to have a close correlation with the rapid increase in the relative amount of the OmpA/Exg fusion precursor (Pre-Exg) compared to its processed product (Mat-Exg). Analysis of subcellular fractions revealed the presence of Pre-Exg in the inner membrane of cultures expressing high levels but not low levels of Pre-Exg. As only Pre-Exg but not Mat-Exg was detectable in the cytoplasm, and Exg was shown by cross-linking experiments to be physically associated with the Sec proteins, it was concluded that secretion and processing of Pre-Exg took place in the SecYEG translocation machinery. The results were in line with the previous speculation that accumulation of unprocessed precursor proteins in the cytoplasmic membrane was detrimental, and supported the idea that cell death was caused by some unusual tie-up of Pre-Exg with the SecYEG translocation machinery, thus imposing an inhibitory effect on the secretion of endogenous secretory proteins. A new model, designated "Saturated Translocation," was proposed to explain the interchangeable lethal and non-lethal properties of Pre-Exg, and to address the possible scenarios that might occur in the course of cell death triggered by secretion of Pre-Exg.
Fu
Z B
ZB
Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, China.
Ng
K L
KL
Lam
T L
TL
Wong
W K R
WK
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D002455:Cell Division / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D040143:Cellulomonas / Q000201:enzymology* / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics*; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism; D008954:Models, Biological; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; C078167:OMPA outer membrane proteins; D006026:Glycoside Hydrolases