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Journal of Bacteriology, October 2009, p. 6029-6039, Vol. 191, No. 19
0021-9193/09/$08.00+0     doi:10.1128/JB.00720-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Mutagenesis and Functional Characterization of the RNA and Protein Components of the toxIN Abortive Infection and Toxin-Antitoxin Locus of Erwinia{triangledown}

T. R. Blower,1 P. C. Fineran,1,{dagger} M. J. Johnson,1 I. K. Toth,2 D. P. Humphreys,3 and G. P. C. Salmond1*

Department of Biochemistry, Tennis Court Road, University of Cambridge, Cambridge CB2 1QW, United Kingdom,1 Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, United Kingdom,2 UCB-Celltech, 216 Bath Road, Slough, Berkshire SL1 4EN, United Kingdom3

Received 3 June 2009/ Accepted 20 July 2009

Bacteria are constantly challenged by bacteriophage (phage) infection and have developed multiple adaptive resistance mechanisms. These mechanisms include the abortive infection systems, which promote "altruistic suicide" of an infected cell, protecting the clonal population. A cryptic plasmid of Erwinia carotovora subsp. atroseptica, pECA1039, has been shown to encode an abortive infection system. This highly effective system is active across multiple genera of gram-negative bacteria and against a spectrum of phages. Designated ToxIN, this two-component abortive infection system acts as a toxin-antitoxin module. ToxIN is the first member of a new type III class of protein-RNA toxin-antitoxin modules, of which there are multiple homologues cross-genera. We characterized in more detail the abortive infection phenotype of ToxIN using a suite of Erwinia phages and performed mutagenesis of the ToxI and ToxN components. We determined the minimal ToxI RNA sequence in the native operon that is both necessary and sufficient for abortive infection and to counteract the toxicity of ToxN. Furthermore, site-directed mutagenesis of ToxN revealed key conserved amino acids in this defining member of the new group of toxic proteins. The mechanism of phage activation of the ToxIN system was investigated and was shown to have no effect on the levels of the ToxN protein. Finally, evidence of negative autoregulation of the toxIN operon, a common feature of toxin-antitoxin systems, is presented. This work on the components of the ToxIN system suggests that there is very tight toxin regulation prior to suicide activation by incoming phage.

* Corresponding author. Mailing address: Department of Biochemistry, Tennis Court Road, University of Cambridge, Cambridge CB2 1QW, United Kingdom. Phone: (44) 1223 333650. Fax: (44) 1223 766108. E-mail: gpcs{at}mole.bio.cam.ac.uk

{triangledown} Published ahead of print on 24 July 2009.

{dagger} Present address: Department of Microbiology and Immunology, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand.

Journal of Bacteriology, October 2009, p. 6029-6039, Vol. 191, No. 19
0021-9193/09/$08.00+0     doi:10.1128/JB.00720-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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