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

Zhonghao Shi,Kin-Fu Chak,Hanna S Yuan

doi:10.1074/jbc.m501216200

Abstract

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.

Highlights

Protein transport mechanisms across cell or organelle membranes have been studied extensively because these processes are essential for cell survival and defense
It has been shown that nuclease colicin Colicin E7 (ColE7) is likely processed in the periplasm during translocation with only the C-terminal cytotoxic nuclease domain transported into the cytoplasm [18]
A result of cleavage of the Periplasmic Proteins Cleave ColE7 at Arg447 in the Presence or Absence of Im7—To ascertain the location of ColE7 processing sites, a full-length ColE7-Im7 complex was incubated with E. coli periplasmic extracts prepared by osmotic shock [21]

Summary

Introduction

Protein transport mechanisms across cell or organelle membranes have been studied extensively because these processes are essential for cell survival and defense. Examples include the vitamin B12 receptor BtuB for all the E-group colicins and iron siderophore receptor FepA for colicin B (ColB) and colicin D (ColD) [7] They are imported into cells by two different routes, one depending on Ton proteins (ExbB, ExbD, and TonB) and the other depending on Tol proteins (TolA, -B, -Q, and -R) [8, 9]. Specific Cleavage at ColE7 during Translocation tRNase activity and protect ColD against LepB-mediated cleavage during export Based on these results, it is generally accepted that nuclease colicins are processed during translocation but the cleavage sites in colicins and the component proteins involved in the processing have not yet been clearly elucidated. A conserved basic residue was identified in a number of nuclease colicins, implying that a similar cleavage process may be involved for all these colicins during translocation

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Conclusion