Agrobacterium tumefaciens Deploys a Superfamily of Type VI Secretion DNase Effectors as Weapons for Interbacterial Competition In Planta

摘要

class="head no_bottom_margin" id="sec1title">IntroductionBacteria produce diverse toxic compounds, including diffusible small molecules such as antibiotics, that allow them to thrive in a competitive environment. They can also produce and secrete enzymatic toxins targeting nucleic acids, membrane lipids, or the peptidoglycan of competing bacterial cells (). The type VI secretion system (T6SS) is a molecular machine found in most Proteobacteria () and can deliver effectors to both eukaryotic () and prokaryotic cells, which appear to be the major targets ().Functional and structural studies have shown that the T6SS nanomachine shares remarkable similarities with the bacteriophage tail structure (). The system contains a TssB-TssC contractile sheath, which is proposed to accommodate the Hcp-VgrG tail tube/puncturing device. The contraction of the sheath leads to the propelling of Hcp, VgrG, and T6SS effectors across bacterial membranes (). Time-lapse fluorescent experiments highlighted the dynamics of this mechanism by revealing “T6SS dueling” between interacting cells ().To date, only a few toxins have been biochemically characterized and shown to contribute to the bactericidal activity mediated by the T6SS (). The most remarkable examples are the cell-wall-degrading effectors that include the type VI secretion amidase effector (Tae) and type VI secretion glycoside hydrolase effector (Tge) superfamilies (). The Tae family includes Tse1 from Pseudomonas aeruginosa () and Ssp1 or Ssp2 from Serratia marcescens (). The Tge family includes the Tse3 muramidase from P. aeruginosa () and Tge2 and Tge3 from Pseudomonas protegens (). VgrG3 from Vibrio cholerae () represents another effector family with a distinct muramidase fold unrelated to the Tge family (href="#bib45" rid="bib45" class=" bibr popnode">Russell et al., 2014). These enzymes are injected into the periplasm of target cells, where they hydrolyze the peptidoglycan, thereby inducing cell lysis (href="#bib11" rid="bib11 bib15 bib42 bib51" class=" bibr popnode">Brooks et al., 2013; English et al., 2012; Russell et al., 2011; Whitney et al., 2013). The phospholipase Tle superfamilies represent an additional set of T6SS toxins. By degrading phosphotidylethanolamine, a major constituent of bacterial membranes, these effectors challenge the membrane integrity of target cells (href="#bib44" rid="bib44" class=" bibr popnode">Russell et al., 2013).A recent study reported the nuclease activity of two proteins, RhsA and RhsB from Dickeya dadantii, containing NS_2 and HNH endonuclease domains, respectively, which cause the degradation of cellular DNA and confer an intraspecies competitive advantage (href="#bib30" rid="bib30" class=" bibr popnode">Koskiniemi et al., 2013). However, whether the D. dadantii antibacterial activity mostly relies on the DNase activity, and whether Rhs proteins are delivered by a dedicated T6SS machine remains to be determined (href="#bib45" rid="bib45" class=" bibr popnode">Russell et al., 2014).Agrobacterium tumefaciens is a soil bacterium that triggers tumorigenesis in plants by delivering T-DNA from bacterial cells into host plant cells through a type IV secretion system (T4SS) (href="#bib2" rid="bib2 bib19" class=" bibr popnode">Alvarez-Martinez and Christie, 2009; Gelvin, 2010). Although not essential for tumorigenesis (href="#bib53" rid="bib53" class=" bibr popnode">Wu et al., 2008), the A. tumefaciens T6SS is activated at both transcriptional (href="#bib54" rid="bib54" class=" bibr popnode">Wu et al., 2012) and posttranslational levels (href="#bib35" rid="bib35" class=" bibr popnode">Lin et al., 2014) when sensing acidity, a signal enriched in the plant wound site and apoplast. Here, using A. tumefaciens as a model organism, we report the discovery of a type VI DNase effector (Tde) family that exhibits potent antibacterial activity. The toxic activity of the Tde DNase is counteracted by a cognate immunity protein, here called Tdi. The T6SS increases the fitness of A. tumefaciens during in planta colonization, and the bacterium uses Tde to attack both intraspecies and interspecies bacterial competitors. The widespread conservation of the Tde toxin and Tdi immunity across bacterial genomes suggests that an appropriate combination of a functional T6SS and a broad toxin repertoire is key to niche colonization within a polymicrobial environment.