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J. Biol. Chem., Vol. 282, Issue 43, 31389-31397, October 26, 2007

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The Role of Electrostatics in Colicin Nuclease Domain Translocation into Bacterial Cells^*

Daniel Walker¹, Khédidja Mosbahi¹, Mireille Vankemmelbeke, Richard James, and Colin Kleanthous²

From the Department of Biology, University of York, York YO10 5YW, United Kingdom and the Institute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom

The mechanism(s) by which nuclease colicins translocate distinct cytotoxic enzymes (DNases, rRNases, and tRNases) to the cytoplasm of Escherichia coli is unknown. Previous in vitro investigations on isolated colicin nuclease domains have shown that they have a strong propensity to associate with anionic phospholipid vesicles, implying that electrostatic interactions with biological membranes play a role in their import. In the present work we set out to test this hypothesis in vivo. We show that cell killing by the DNase toxin colicin E9 of E. coli HDL11, a strain in which the level of anionic phospholipid and hence inner membrane charge is regulated by isopropyl -D-thiogalactopyranoside induction, is critically dependent on the level of inducer, whereas this is not the case for pore-forming colicins that take the same basic route into the periplasm. Moreover, there is a strong correlation between the level and rate of HDL11 cell killing and the net positive charge on a colicin DNase, with similar effects seen for wild type E. coli cells, data that are consistent with a direct, electrostatically mediated interaction between colicin nucleases and the bacterial inner membrane. We next sought to identify how membrane-associated colicin nucleases might be translocated into the cell. We show that neither the Sec or Tat systems are involved in nuclease colicin uptake but that nuclease colicin toxicity is instead dependent on functional FtsH, an inner membrane AAA⁺ ATPase and protease that dislocates misfolded membrane proteins to the cytoplasm for destruction.

Received for publication, July 18, 2007 , and in revised form, August 24, 2007.

^* This work was supported by grants from the Wellcome Trust and the Biotechnology and Biological Sciences Research Council. 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.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 44-1904-328820; Fax: 44-1904-328825; E-mail: ck11{at}york.ac.uk.

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