Identification of an Essential Cleavage Site in ColE7 Required for Import and Killing of Cells

Zhonghao Shi‘Þ¡±, Kin-Fu Chak‘Þ, and Hanna S. Yuan‘Þ¡±‘ø

From the ‘ÞInstitute of Biochemistry, National Yang-Ming University, Taipei 11221, Taiwan and ¡±Institute of

Molecular Biology, Academia Sinica, Taipei 11529, Taiwan

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 Lys446 and Arg447 in the presence or absence of its inhibitor Im7 protein. Several residues near cleavage sites were mutated, but only mutants of Arg447 completely lost in vivo cell-killing activity. Both the full-length and the nuclease domain of Arg447 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 Arg447 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 Arg447 have also been found in other nuclease colicins, implying that the processing at this position may be common to other

colicins during translocation.

J. Biol. Chem., 280, 24663-24668 (2005)

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