Type IV secretion systems (T4SSs) are multiprotein complexes that transport effector proteins and protein-DNA complexes through bacterial membranes to the extracellular milieu or directly into the cytoplasm of other cells. Many bacteria of the family Xanthomonadaceae, which occupy diverse environmental niches, carry a T4SS with unknown function but with several characteristics that distinguishes it from other T4SSs. Here we show that the Xanthomonas citri T4SS provides these cells the capacity to kill other Gram-negative bacterial species in a contact-dependent manner. The secretion of one type IV bacterial effector protein is shown to require a conserved C-terminal domain and its bacteriolytic activity is neutralized by a cognate immunity protein whose 3D structure is similar to peptidoglycan hydrolase inhibitors. This is the first demonstration of the involvement of a T4SS in bacterial killing and points to this special class of T4SS as a mediator of both antagonistic and cooperative interbacterial interactions.6 Burkinshaw, B.J., Strynadka, N.C., Assembly and structure of the T3SS (2014) Biochim. Biophys. Acta, 1843, pp. 1649-1663Alvarez-Martinez, C.E., Christie, P.J., Biological diversity of prokaryotic type IV secretion systems (2009) Microbiol. Mol. Biol. Rev, 73, pp. 775-808Silverman, J.M., Brunet, Y.R., Cascales, E., Mougous, J.D., Structure and regulation of the type VI secretion system (2012) Annu. Rev. Microbiol, 66, pp. 453-472Nivaskumar, M., Francetic, O., Type II secretion system: A magic beanstalk or a protein escalator (2014) Biochim. Biophys. Acta, 1843, pp. 1568-1577Van Ulsen, P., Rahman, S., Jong, W.S., Daleke-Schermerhorn, M.H., Luirink, J., Type v secretion: From biogenesis to biotechnology (2014) Biochim. Biophys. Acta, 1843, pp. 1592-1611Hayes, C.S., Koskiniemi, S., Ruhe, Z.C., Poole, S.J., Low, D.A., Mechanisms and biological roles of contact-dependent growth inhibition systems (2014) Cold Spring Harb. Perspect Med, 4, p. a010025Hood, R.D., A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria (2010) Cell. Host. Microbe, 7, pp. 25-37Backert, S., Meyer, T.F., Type IV secretion systems and their effectors in bacterial pathogenesis (2006) Curr. Opin. Microbiol, 9, pp. 207-217Dehio, C., Infection-associated type IV secretion systems of Bartonella and their diverse roles in host cell interaction (2008) Cell. Microbiol, 10, pp. 1591-1598Locht, C., Coutte, L., Mielcarek, N., The ins and outs of pertussis toxin (2011) FEBS J, 278, pp. 4668-4682De Jong, M.F., Tsolis, R.M., Brucellosis and type IV secretion (2012) Future Microbiol, 7, pp. 47-58Tegtmeyer, N., Wessler, S., Backert, S., Role of the cag-pathogenicity island encoded type IV secretion system in Helicobacter pylori pathogenesis (2011) FEBS J, 278, pp. 1190-1202Ge, J., Shao, F., Manipulation of host vesicular trafficking and innate immune defence by Legionella Dot/Icm effectors (2011) Cell. Microbiol, 13, pp. 1870-1880Hofreuter, D., Odenbreit, S., Haas, R., Natural transformation competence in Helicobacter pylori is mediated by the basic components of a type IV secretion system (2001) Mol. Microbiol, 41, pp. 379-391Hamilton, H.L., Dominguez, N.M., Schwartz, K.J., Hackett, K.T., Dillard, J.P., Neisseria gonorrhoeae secretes chromosomal DNA via a novel type IV secretion system (2005) Mol. Microbiol, 55, pp. 1704-1721Pitzschke, A., Hirt, H., New insights into an old story: Agrobacterium-induced tumour formation in plants by plant transformation (2010) EMBO J, 29, pp. 1021-1032Low, H.H., Structure of a type IV secretion system (2014) Nature, 508, pp. 550-553Waksman, G., Orlova, E.V., Structural organisation of the type IV secretion systems (2014) Curr. Opin. Microbiol, 17, pp. 24-31Trokter, M., Felisberto-Rodrigues, C., Christie, P.J., Waksman, G., Recent advances in the structural and molecular biology of type IV secretion systems (2014) Curr. Opin. Struct. Biol, 27, pp. 16-23Alegria, M.C., Identification of new protein-protein interactions involving the products of the chromosome-and plasmid-encoded type IV secretion loci of the phytopathogen Xanthomonas axonopodis pv. Citri (2005) J. Bacteriol, 187, pp. 2315-2325Souza, D.P., A component of the Xanthomonadaceae type IV secretion system combines a VirB7 motif with a N0 domain found in outer membrane transport proteins (2011) PLoS Pathog, 7, p. e1002031Frederiksen, R.F., Bacterial chitinases and chitin-binding proteins as virulence factors (2013) Microbiology, 159, pp. 833-847Russell, A.B., Type VI secretion delivers bacteriolytic effectors to target cells (2011) Nature, 475, pp. 343-347Russell, A.B., Diverse type VI secretion phospholipases are functionally plastic antibacterial effectors (2013) Nature, 496, pp. 508-512Van Herreweghe, J.M., Lysozyme inhibitor conferring bacterial tolerance to invertebrate type lysozyme (2010) Cell. Mol. Life Sci, 67, pp. 1177-1188Leysen, S., Molecular basis of bacterial defense against host lysozymes: X-ray structures of periplasmic lysozyme inhibitors PliI and PliC (2011) J. Mol. Biol, 405, pp. 1233-1245Ding, J., Wang, W., Feng, H., Zhang, Y., Wang, D.C., Structural insights into the Pseudomonas aeruginosa type VI virulence effector Tse1 bacteriolysis and self-protection mechanisms (2012) J. Biol. Chem, 287, pp. 26911-26920Fronzes, R., Structure of a type IV secretion system core complex (2009) Science, 323, pp. 266-268Basler, M., Ho, B.T., Mekalanos, J.J., Tit-for-tat: Type VI secretion system counterattack during bacterial cell-cell interactions (2013) Cell, 152, pp. 884-894Leroux, M., Quantitative single-cell characterization of bacterial interactions reveals type VI secretion is a double-edged sword (2012) Proc. Natl Acad. Sci. USA, 109, pp. 19804-19809Macintyre, D.L., Miyata, S.T., Kitaoka, M., Pukatzki, S., The Vibrio cholerae type VI secretion system displays antimicrobial properties (2010) Proc. Natl Acad. Sci. USA, 107, pp. 19520-19524Murdoch, S.L., The opportunistic pathogen Serratia marcescens utilizes type VI secretion to target bacterial competitors (2011) J. Bacteriol, 193, pp. 6057-6069Schwarz, S., Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions (2010) PLoS Pathog, 6, p. e1001068Casas-Godoy, L., Duquesne, S., Bordes, F., Sandoval, G., Marty, A., Lipases: An overview (2012) Methods Mol. Biol, 861, pp. 3-30Lovering, A.L., Safadi, S.S., Strynadka, N.C., Structural perspective of peptidoglycan biosynthesis and assembly (2012) Annu. Rev. Biochem, 81, pp. 451-478Li, G., Miller, A., Bull, H., Howard, S.P., Assembly of the type II secretion system: Identification of ExeA residues critical for peptidoglycan binding and secretin multimerization (2011) J. Bacteriol, 193, pp. 197-204Scheurwater, E., Reid, C.W., Clarke, A.J., Lytic transglycosylases: Bacterial space-making autolysins (2008) Int. J. Biochem. Cell Biol, 40, pp. 586-591Firczuk, M., Bochtler, M., Folds and activities of peptidoglycan amidases (2007) FEMS Microbiol. Rev, 31, pp. 676-691Wohlkonig, A., Huet, J., Looze, Y., Wintjens, R., Structural relationships in the lysozyme superfamily: Significant evidence for glycoside hydrolase signature motifs (2010) PLoS One, 5, p. e15388Anantharaman, V., Aravind, L., Evolutionary history, structural features and biochemical diversity of the NlpC/P60 superfamily of enzymes (2003) Genome. Biol, 4, p. R11Mutschler, H., Gebhardt, M., Shoeman, R.L., Meinhart, A., A novel mechanism of programmed cell death in bacteria by toxin-antitoxin systems corrupts peptidoglycan synthesis (2011) PLoS Biol, 9, p. e1001033Zhang, D., Iyer, L.M., Aravind, L., A novel immunity system for bacterial nucleic acid degrading toxins and its recruitment in various eukaryotic and DNA viral systems (2011) Nucleic Acids Res, 39, pp. 4532-4552Zhang, D., De Souza, R.F., Anantharaman, V., Iyer, L.M., Aravind, L., Polymorphic toxin systems: Comprehensive characterization of trafficking modes, processing, mechanisms of action, immunity and ecology using comparative genomics (2012) Biol. Direct, 7, p. 18Salomon, D., Marker for type VI secretion system effectors (2014) Proc. Natl Acad. Sci. USA, 111, pp. 9271-9276Ho, B.T., Basler, M., Mekalanos, J.J., Type 6 secretion system-mediated immunity to type 4 secretion system-mediated gene transfer (2013) Science, 342, pp. 250-253Guimaraes, B.G., The MX2 macromolecular crystallography beamline: A wiggler X-ray source at the LNLS (2009) J. Synchrotron Radiat, 16, pp. 69-75Pflugrath, J.W., The finer things in X-ray diffraction data collection (1999) Acta. Crystallogr. D Biol. Crystallogr, 55, pp. 1718-1725Otwinowski, Z., Minor, W., Processing of X-ray diffraction data collected in oscillation mode (1997) Methods Enzymol, 276, pp. 307-326Sheldrick, G.M., A short history of SHELX (2008) Acta. Crystallogr, 64, pp. 112-122Vonrhein, C., Blanc, E., Roversi, P., Bricogne, G., Automated structure solution with autoSHARP (2007) Methods Mol. Biol, 364, pp. 215-230Langer, G., Cohen, S.X., Lamzin, V.S., Perrakis, A., Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7 (2008) Nat. Protoc, 3, pp. 1171-1179Emsley, P., Lohkamp, B., Scott, W.G., Cowtan, K., Features and development of Coot (2010) Acta. Crystallogr. D Biol. Crystallogr, 66, pp. 486-501Painter, J., Merritt, E.A., Optimal description of a protein structure in terms of multiple groups undergoing TLS motion (2006) Acta. Crystallogr. D Biol. Crystallogr, 62, pp. 439-450Murshudov, G.N., REFMAC5 for the refinement of macromolecular crystal structures (2011) Acta. Crystallogr. D Biol. Crystallogr, 67, pp. 355-367The Ccp4 suite-Programs for protein crystallography (1994) Acta. Crystallogr. D Biol. Crystallogr, 50, pp. 760-763. , Collaborative Computational Project Number 4Davis, I.W., MolProbity: All-atom contacts and structure validation for proteins and nucleic acids (2007) Nucleic Acids Res, 35, pp. W375-W383Lovell, S.C., Structure validation by Calpha geometry: Phi,psi and Cbeta deviation (2003) Proteins, 50, pp. 437-450Laskowski, R.A., Macarthur, M.W., Moss, D.S., Thornton, J.M., Procheck-A program to check the stereochemical quality of protein structures (1993) J. Appl. Crystallogr, 26, pp. 283-291Bond C. ., S., TopDraw: A sketchpad for protein structure topology cartoons (2003) Bioinformatics, 19, pp. 311-312Holm, L., Rosenstrom, P., Dali server: Conservation mapping in 3D (2010) Nucleic Acids Res, 38, pp. W545-W549Barbosa, L.R., The importance of protein-protein interactions on the pHinduced conformational changes of bovine serum albumin: A small-angle X-ray scattering study (2010) Biophys. J, 98, pp. 147-157Jacques, D.A., Trewhella, J., Small-angle scattering for structural biology-expanding the frontier while avoiding the pitfalls (2010) Protein Sci, 19, pp. 642-657Petoukhov, M.V., New developments in the ATSAS program package for small-angle scattering data analysis (2012) J. Appl. Crystallogr, 45, pp. 342-350Svergun, D.I., Determination of the regularization parameter in indirecttransform methods using perceptual criteria (1992) J. Appl. Crystallogr, 25, pp. 495-503Svergun, D.I., Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing (1999) Biophys. J, 76, pp. 2879-2886Volkov, V.V., Svergun, D.I., Uniqueness of ab initio shape determination in small-angle scattering (2003) J. Appl. Crystallogr, 36, pp. 860-864Svergun, D., Barberato, C., Koch, M.H.J., CRYSOL-A program to evaluate x-ray solution scattering of biological macromolecules from atomic coordinates (1995) J. Appl. Crystallogr, 28, pp. 768-773Babu, M.M., A database of bacterial lipoproteins (DOLOP) with functional assignments to predicted lipoproteins (2006) J. Bacteriol, 188, pp. 2761-2773Da Silva, A.C., Comparison of the genomes of two Xanthomonas pathogens with differing host specificities (2002) Nature, 417, pp. 459-463Marchler-Bauer, A., Cdd: A conserved domain database for the functional annotation of proteins (2011) Nucleic Acids Res, 39, pp. D225-D229