Identification of an essential cleavage site in ColE7 required for import and killing of cells.
Colicin E7 (ColE7), a nuclease toxin released from Escherichia coli, kills susceptible bacteria under environmental stress. Nuclease colicins are processed during translocation with only the cytotoxic nuclease domains traversing the inner membrane to cleave tRNA, rRNA, or DNA in the cytoplasm of target cells. In this study, we show that the E. coli periplasmic extract cleaves ColE7 between Lys(446) and Arg(447) in the presence or absence of its inhibitor Im7 protein. Several residues near cleavage sites were mutated, but only mutants of Arg(447) completely lost in vivo cell-killing activity. Both the full-length and the nuclease domain of Arg(447) mutants retained their nuclease activities, indicating that failure to kill cells was not a consequence of damage to the endonuclease activity of the enzyme. Moreover, the R447E ColE7 mutant was not cleaved at its 447 site by periplasmic extracts or transported into the cytoplasm of target cells. Collectively, these results suggest that ColE7 is cleaved at Arg(447) during translocation and that cleavage is an essential step for ColE7 import into the cytoplasm of target cells and its cell-killing activity. Conserved basic residues aligned with Arg(447) have also been found in other nuclease colicins, implying that the processing at this position may be common to other colicins during translocation.
Shi
Zhonghao
Z
Institute of Biochemistry, National Yang-Ming University, Taipei 11221, Taiwan and Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan.
Chak
Kin-Fu
KF
Yuan
Hanna S
HS
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D002942:Circular Dichroism; D003087:Colicins / Q000737:chemistry*; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D003851:Deoxyribonucleases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry; D009154:Mutation; D019897:Periplasm / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
D003087:Colicins; D029968:Escherichia coli Proteins; D046912:Multiprotein Complexes; D014364:Tryptophan; D004247:DNA; D001120:Arginine; D003851:Deoxyribonucleases; D004720:Endonucleases; D008239:Lysine