Published online 10 April 2017 Nucleic Acids Research, 2017, Vol. 45, No. 9 5013–5025 doi: 10.1093/nar/gkx230 NAR Breakthrough Article The CDI toxin of Yersinia kristensenii is a novel bacterial member of the RNase A superfamily Ga¨ elle Batot 1,† , Karolina Michalska 2,3,† , Greg Ekberg 4,† , Ervin M. Irimpan 1 , Grazyna Joachimiak 2 , Robert Jedrzejczak 2 , Gyorgy Babnigg 2 , Christopher S. Hayes 4,5 , Andrzej Joachimiak 2,3,6 and Celia W. Goulding 1,7,* 1 Department of Molecular Biology & Biochemistry, University of California Irvine, Irvine, CA 92697, USA, 2 Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne, IL 60439, USA, 3 Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, USA, 4 Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA 93106-9625, USA, 5 Biomolecular Science and Engineering Program, University of California, Santa Barbara, Santa Barbara, CA 93106-9625, USA, 6 Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA and 7 Department of Pharmaceutical Sciences, University of California Irvine, Irvine, CA 92697, USA Received January 25, 2017; Revised March 21, 2017; Editorial Decision March 22, 2017; Accepted March 31, 2017 ABSTRACT Contact-dependent growth inhibition (CDI) is an im- portant mechanism of inter-bacterial competition found in many Gram-negative pathogens. CDI + cells express cell-surface CdiA proteins that bind neigh- boring bacteria and deliver C-terminal toxin domains (CdiA-CT) to inhibit target-cell growth. CDI + bacteria also produce CdiI immunity proteins, which specif- ically neutralize cognate CdiA-CT toxins to prevent self-inhibition. Here, we present the crystal structure of the CdiA-CT/CdiI Ykris complex from Yersinia kris- tensenii ATCC 33638. CdiA-CT Ykris adopts the same fold as angiogenin and other RNase A paralogs, but the toxin does not share sequence similarity with these nucleases and lacks the characteristic disul- fide bonds of the superfamily. Consistent with the structural homology, CdiA-CT Ykris has potent RNase activity in vitro and in vivo. Structure-guided mu- tagenesis reveals that His175, Arg186, Thr276 and Tyr278 contribute to CdiA-CT Ykris activity, suggest- ing that these residues participate in substrate bind- ing and/or catalysis. CdiI Ykris binds directly over the putative active site and likely neutralizes toxicity by blocking access to RNA substrates. Significantly, CdiA-CT Ykris is the first non-vertebrate protein found to possess the RNase A superfamily fold, and ho- mologs of this toxin are associated with secretion systems in many Gram-negative and Gram-positive bacteria. These observations suggest that RNase A- like toxins are commonly deployed in inter-bacterial competition. INTRODUCTION Bacteria have evolved several mechanisms to sense, com- municate and compete with other microbes in the envi- ronment. Contact-dependent growth inhibition (CDI) is a common mode of inter-cellular interaction in which Gram- negative bacteria exchange protein toxins upon direct cell- to-cell contact (1–3). CDI toxin delivery is mediated by the CdiB/CdiA family of two-partner secretion proteins. CdiB is an Omp85 -barrel protein that exports and dis- plays CdiA effectors on the cell surface. CdiA proteins are very large (180–630 kDa), and each effector molecule is pre- dicted to form an elongated flament that extends several hundred angstroms from the cell (3–5). CdiA recognizes specifc receptors on neighboring bacteria, then transfers its C-terminal toxin region (CdiA-CT) into the target cell through an incompletely understood translocation path- way (6,7). The CdiA-CT region varies widely in sequence between effector proteins, but is typically demarcated by highly conserved peptide motifs such as VENN in enter- obacteria and ELYN in the Burkholderiales (1,8,9). In ac- cordance with CdiA-CT sequence diversity, CdiA effectors carry toxins with distinct activities, many of which are nu- * To whom correspondence should be addressed. Tel: +1 949 824 0337; Fax: +1 949 824 8551 Email: celia.goulding@uci.edu † These authors contributed equally to this work as frst authors. C  The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com