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J. Biol. Chem., Vol. 280, Issue 26, 24663-24668, July 1, 2005

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

Zhonghao Shi, Kin-Fu Chak, and Hanna S. Yuan¶

From the Institute 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.

Received for publication, February 2, 2005 , and in revised form, April 27, 2005.

^* This work was supported by an Academia Sinica research grant and National Science Council of the Republic of China Grant NSC92-2321-B001-012 (to H. S. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Inst. of Molecular Biology, Academia Sinica, Taipei, Taiwan 11529. Tel.: 886-2-27884151; Fax: 886-2-27826085; E-mail: hanna{at}sinica.edu.tw.

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