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• W2896023929 abstract "•Protein-targeting ADP-ribosyltransferases (ARTs) act as interbacterial toxins•Immunity proteins protect from ARTs by toxin binding and ADP-ribose (ADPr) cleavage•A type VI secretion effector from Serratia acts via ADPr addition to FtsZ•FtsZ-ADPr blocks polymerization and cell division, leading to cell death ADP-ribosylation of proteins can profoundly impact their function and serves as an effective mechanism by which bacterial toxins impair eukaryotic cell processes. Here, we report the discovery that bacteria also employ ADP-ribosylating toxins against each other during interspecies competition. We demonstrate that one such toxin from Serratia proteamaculans interrupts the division of competing cells by modifying the essential bacterial tubulin-like protein, FtsZ, adjacent to its protomer interface, blocking its capacity to polymerize. The structure of the toxin in complex with its immunity determinant revealed two distinct modes of inhibition: active site occlusion and enzymatic removal of ADP-ribose modifications. We show that each is sufficient to support toxin immunity; however, the latter additionally provides unprecedented broad protection against non-cognate ADP-ribosylating effectors. Our findings reveal how an interbacterial arms race has produced a unique solution for safeguarding the integrity of bacterial cell division machinery against inactivating post-translational modifications. ADP-ribosylation of proteins can profoundly impact their function and serves as an effective mechanism by which bacterial toxins impair eukaryotic cell processes. Here, we report the discovery that bacteria also employ ADP-ribosylating toxins against each other during interspecies competition. We demonstrate that one such toxin from Serratia proteamaculans interrupts the division of competing cells by modifying the essential bacterial tubulin-like protein, FtsZ, adjacent to its protomer interface, blocking its capacity to polymerize. The structure of the toxin in complex with its immunity determinant revealed two distinct modes of inhibition: active site occlusion and enzymatic removal of ADP-ribose modifications. We show that each is sufficient to support toxin immunity; however, the latter additionally provides unprecedented broad protection against non-cognate ADP-ribosylating effectors. Our findings reveal how an interbacterial arms race has produced a unique solution for safeguarding the integrity of bacterial cell division machinery against inactivating post-translational modifications. Microbial communities are of fundamental importance to virtually all natural ecosystems. Although the term “communities” implies cooperation, it is now appreciated that antagonistic behavior directed against closely interacting microbes serves as a key driver of composition and resilience within these consortia (Foster and Bell, 2012Foster K.R. Bell T. Competition, not cooperation, dominates interactions among culturable microbial species.Curr. Biol. 2012; 22: 1845-1850Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). Reflecting this central role of antagonism, pathways dedicated to interspecies antibacterial toxin delivery occur broadly throughout both Gram-positive and -negative bacteria (Jones et al., 2017Jones A.M. Low D.A. Hayes C.S. Can’t you hear me knocking: contact-dependent competition and cooperation in bacteria.Emerg. Top. Life Sci. 2017; 1: 75-83Crossref PubMed Scopus (10) Google Scholar, Russell et al., 2014Russell A.B. Wexler A.G. Harding B.N. Whitney J.C. Bohn A.J. Goo Y.A. Tran B.Q. Barry N.A. Zheng H. Peterson S.B. et al.A type VI secretion-related pathway in Bacteroidetes mediates interbacterial antagonism.Cell Host Microbe. 2014; 16: 227-236Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, Whitney et al., 2017Whitney J.C. Peterson S.B. Kim J. Pazos M. Verster A.J. Radey M.C. Kulasekara H.D. Ching M.Q. Bullen N.P. Bryant D. et al.A broadly distributed toxin family mediates contact-dependent antagonism between gram-positive bacteria.eLife. 2017; 6: e26938Crossref PubMed Scopus (84) Google Scholar). These include the Esx pathway, prevalent in the phyla Firmicutes and Actinobacteria, and the type VI secretion system (T6SS), which is widespread in the phyla Proteobacteria and Bacteroidetes. Pathogenic bacteria may use these pathways to facilitate invasion of or dominance within polymicrobial infections, whereas for commensal organisms they can dictate compatibility within consortia (Sana et al., 2016Sana T.G. Flaugnatti N. Lugo K.A. Lam L.H. Jacobson A. Baylot V. Durand E. Journet L. Cascales E. Monack D.M. Salmonella Typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut.Proc. Natl. Acad. Sci. USA. 2016; 113: E5044-E5051Crossref PubMed Scopus (185) Google Scholar, Speare et al., 2018Speare L. Cecere A.G. Guckes K.R. Smith S. Wollenberg M.S. Mandel M.J. Miyashiro T. Septer A.N. Bacterial symbionts use a type VI secretion system to eliminate competitors in their natural host.Proc. Natl. Acad. Sci. USA. 2018; 115: E8528-E8537Crossref PubMed Scopus (92) Google Scholar, Verster et al., 2017Verster A.J. Ross B.D. Radey M.C. Bao Y. Goodman A.L. Mougous J.D. Borenstein E. The landscape of type vi secretion across human gut microbiomes reveals its role in community composition.Cell Host Microbe. 2017; 22: 411-419Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Contact-dependent interbacterial competition is generally mediated by secreted toxic effector proteins. Complex export machinery enables these effectors to access target molecules that reside within the cell envelope of competitor cells (Russell et al., 2011Russell A.B. Hood R.D. Bui N.K. LeRoux M. Vollmer W. Mougous J.D. Type VI secretion delivers bacteriolytic effectors to target cells.Nature. 2011; 475: 343-347Crossref PubMed Scopus (469) Google Scholar, Whitney et al., 2017Whitney J.C. Peterson S.B. Kim J. Pazos M. Verster A.J. Radey M.C. Kulasekara H.D. Ching M.Q. Bullen N.P. Bryant D. et al.A broadly distributed toxin family mediates contact-dependent antagonism between gram-positive bacteria.eLife. 2017; 6: e26938Crossref PubMed Scopus (84) Google Scholar). The T6SS and Esx pathways deliver effectors indiscriminately; therefore, bacteria harboring effectors transported by these systems require a means of inhibiting self-intoxication. To date, protection against all characterized effectors of these systems has been shown to derive from the production of specific immunity determinants that bind cognate toxins and inhibit their enzymatic function (Alcoforado Diniz et al., 2015Alcoforado Diniz J. Liu Y.C. Coulthurst S.J. Molecular weaponry: diverse effectors delivered by the Type VI secretion system.Cell. Microbiol. 2015; 17: 1742-1751Crossref PubMed Scopus (97) Google Scholar). Cognate effector and immunity proteins, herein referred to as E-I pairs, are without known exception encoded adjacently. Identification and characterization of toxic effectors from a growing number of species has revealed that these proteins exhibit tremendous sequence and biochemical diversity. For instance, large superfamilies of toxins that share little homology beyond conserved catalytic residues target amide or glycosidic bonds in the bacterial cell wall, DNA, membrane phospholipids, and the cellular metabolite NAD+ (Alcoforado Diniz et al., 2015Alcoforado Diniz J. Liu Y.C. Coulthurst S.J. Molecular weaponry: diverse effectors delivered by the Type VI secretion system.Cell. Microbiol. 2015; 17: 1742-1751Crossref PubMed Scopus (97) Google Scholar, Whitney et al., 2015Whitney J.C. Quentin D. Sawai S. LeRoux M. Harding B.N. Ledvina H.E. Tran B.Q. Robinson H. Goo Y.A. Goodlett D.R. et al.An interbacterial NAD(P)(+) glycohydrolase toxin requires elongation factor Tu for delivery to target cells.Cell. 2015; 163: 607-619Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). By targeting highly conserved, essential primary metabolites, interbacterial effectors allow bacteria to compete effectively against a wide phylogenetic cross-section of organisms. Multiple toxins targeting a broad array of cellular structures are often employed simultaneously by a single delivery pathway, which promotes bacterial competitiveness via synergistic toxigenic activities and widens the environmental conditions under which at least one effector is efficacious (LaCourse et al., 2018LaCourse K.D. Peterson S.B. Kulasekara H.D. Radey M.C. Kim J. Mougous J.D. Conditional toxicity and synergy drive diversity among antibacterial effectors.Nat. Microbiol. 2018; 3: 440-446Crossref PubMed Scopus (62) Google Scholar). Despite a high degree of toxin diversity, genes encoding effector proteins of interbacterial antagonistic systems are often readily recognizable in bacterial genomes. These genes are typically characterized by 5′ regions encoding conserved effector targeting domains and by polymorphic toxin modules encoded at their 3′ ends. Targeting domains linked to T6SS effectors include PAAR, RHS, and VgrG, whereas LXG is the sole domain so far implicated in the recognition of antibacterial substrates by the Esx pathway (Cianfanelli et al., 2016Cianfanelli F.R. Monlezun L. Coulthurst S.J. Aim, Load, Fire: The Type VI Secretion System, a Bacterial Nanoweapon.Trends Microbiol. 2016; 24: 51-62Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, Whitney et al., 2017Whitney J.C. Peterson S.B. Kim J. Pazos M. Verster A.J. Radey M.C. Kulasekara H.D. Ching M.Q. Bullen N.P. Bryant D. et al.A broadly distributed toxin family mediates contact-dependent antagonism between gram-positive bacteria.eLife. 2017; 6: e26938Crossref PubMed Scopus (84) Google Scholar). Interestingly, there is substantial overlap in the modes of action of bacterial toxins that target eukaryotic host cells and those that target other bacteria. For instance, structurally related NADases of Pseudomonas aeruginosa and Mycobacterium tuberculosis are employed to neutralize competing Gram-negative bacterial cells and to induce necrotic cell death in human macrophages, respectively (Sun et al., 2015Sun J. Siroy A. Lokareddy R.K. Speer A. Doornbos K.S. Cingolani G. Niederweis M. The tuberculosis necrotizing toxin kills macrophages by hydrolyzing NAD.Nat. Struct. Mol. Biol. 2015; 22: 672-678Crossref PubMed Scopus (88) Google Scholar, Whitney et al., 2015Whitney J.C. Quentin D. Sawai S. LeRoux M. Harding B.N. Ledvina H.E. Tran B.Q. Robinson H. Goo Y.A. Goodlett D.R. et al.An interbacterial NAD(P)(+) glycohydrolase toxin requires elongation factor Tu for delivery to target cells.Cell. 2015; 163: 607-619Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). In some cases, the same toxin may be capable of targeting cells of both domains, as described for T6SS-delivered phospholipases (Bleves, 2016Bleves S. Game of trans-kingdom effectors.Trends Microbiol. 2016; 24: 773-774Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). These results point toward the important role that interbacterial antagonism can play in shaping the toxin repertoire and emergence of bacterial pathogens. Here, we describe an interbacterial toxin that inactivates the critical cell division protein, FtsZ, via ADP-ribosyltransferase (ART) activity. This toxin is the first characterized member of a large family of related ART toxins, with phylogenetically broadly distributed representatives bearing hallmarks of interbacterial effectors exported by the T6SS and the Esx pathway. Proteins with this activity, such as cholera and diphtheria toxins, are known to be widely utilized by pathogens (Simon et al., 2014Simon N.C. Aktories K. Barbieri J.T. Novel bacterial ADP-ribosylating toxins: structure and function.Nat. Rev. Microbiol. 2014; 12: 599-611Crossref PubMed Scopus (148) Google Scholar); however, proteins with this activity have not previously been demonstrated to participate in interbacterial antagonism. Finally, structural analyses reveal that protection against ART toxins is conferred by bifunctional immunity determinants, which concomitantly utilize active site occlusion to inactivate cognate effector proteins and a promiscuous ADP-ribosylhydrolase (ARH) activity to reverse ART-catalyzed modifications more broadly. During the course of our efforts to define mechanisms of interbacterial competition, we found an uncharacterized protein domain common to predicted effectors of diverse contact-dependent antagonism pathways. In Gram-negative bacteria, this domain is found in proteins also containing PAAR, RHS, or both of these domains, indicative of intercellular delivery via the T6SS pathway (Figure 1A; Table S1) (Cianfanelli et al., 2016Cianfanelli F.R. Monlezun L. Coulthurst S.J. Aim, Load, Fire: The Type VI Secretion System, a Bacterial Nanoweapon.Trends Microbiol. 2016; 24: 51-62Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). In Gram-positive species, proteins with this domain additionally contain LXG domains, indicative of transit through the Esx pathway (Whitney et al., 2017Whitney J.C. Peterson S.B. Kim J. Pazos M. Verster A.J. Radey M.C. Kulasekara H.D. Ching M.Q. Bullen N.P. Bryant D. et al.A broadly distributed toxin family mediates contact-dependent antagonism between gram-positive bacteria.eLife. 2017; 6: e26938Crossref PubMed Scopus (84) Google Scholar). In all cases, the uncharacterized domain resides at the C terminus of the protein—the stereotyped position in each of these systems for the toxin domain of their substrates. Subsequent sequence analyses showed that the domain possesses a constellation of conserved amino acids characteristic of RSE family ART enzymes (Figure 1B) (Cohen and Chang, 2018Cohen M.S. Chang P. Insights into the biogenesis, function, and regulation of ADP-ribosylation.Nat. Chem. Biol. 2018; 14: 236-243Crossref PubMed Scopus (154) Google Scholar). Bacterial members of this family include eukaryotic cell-targeting toxins; however, members of this family are not known to act between bacterial cells. ADP-ribosylation can be a reversible modification, and enzymes belonging to the ARH family are known to catalyze ADP-ribose removal (Cohen and Chang, 2018Cohen M.S. Chang P. Insights into the biogenesis, function, and regulation of ADP-ribosylation.Nat. Chem. Biol. 2018; 14: 236-243Crossref PubMed Scopus (154) Google Scholar). Interestingly, we noted that the genes immediately downstream of predicted antibacterial RSE family genes encode ARH family proteins (Figure 1C; Table S1). Immunity determinants of interbacterial toxins are typically encoded in this position, suggesting that the role of these proteins may be to provide protection from ART-based intoxication via hydrolysis of ADP-ribose adducts. The active site of ARH proteins contains metal ions coordinated by a network of conserved, predominately acidic, residues (Berthold et al., 2009Berthold C.L. Wang H. Nordlund S. Högbom M. Mechanism of ADP-ribosylation removal revealed by the structure and ligand complexes of the dimanganese mono-ADP-ribosylhydrolase DraG.Proc. Natl. Acad. Sci. USA. 2009; 106: 14247-14252Crossref PubMed Scopus (31) Google Scholar). These amino acids are conserved in the ARH family proteins we identified, further suggesting an enzymatic mechanism of immunity (Figure 1D). Taken together with prior bioinformatic analyses (Aravind et al., 2015Aravind L. Zhang D. de Souza R.F. Anand S. Iyer L.M. The natural history of ADP-ribosyltransferases and the ADP-ribosylation system.Curr. Top. Microbiol. Immunol. 2015; 384: 3-32PubMed Google Scholar), these observations led us to hypothesize that toxins with ART activity are widespread mediators of bacterial competition. To investigate the potential for ART enzymes to mediate interbacterial antagonism, we initially focused on a predicted ART toxin of Serratia proteamaculans. This protein, which we termed Tre1 (type VI secretion ADP-ribosyltransferase effector 1), contains an N-terminal PAAR domain, a configuration consistent with known T6SS effector proteins (Figure 1A). Relative to control E. coli cultures, those expressing Tre1 or its c-terminal toxin domain (Tre1tox) displayed a significant loss in colony forming units (Figure 2A). Time-lapse microscopy revealed that this loss in viability was associated with both cellular elongation and lysis (Figures 2B and S1A; Videos S1 and S2). To determine whether the toxicity of Tre1 derives from its predicted ART activity, we expressed a Tre1tox protein in which the catalytic glutamic acid within the RSE motif is substituted with glutamine (Tre1toxE415Q) (Tsuge et al., 2003Tsuge H. Nagahama M. Nishimura H. Hisatsune J. Sakaguchi Y. Itogawa Y. Katunuma N. Sakurai J. Crystal structure and site-directed mutagenesis of enzymatic components from Clostridium perfringens iota-toxin.J. Mol. Biol. 2003; 325: 471-483Crossref PubMed Scopus (88) Google Scholar). Despite expression levels matching that of Tre1tox, this predicted catalytically inactive protein did not reduce E. coli viability, nor did it promote cell elongation and lysis to levels approaching the wild-type (Figures 2A, 2C, S1A, and S1B; Videos S1 and S2). Co-expression of Tre1tox with the predicted ARH family protein encoded downstream, which we subsequently refer to as Tri1, restored the viability of E. coli to that of the control (Figures 2A and S1A). Together, these data demonstrate that Tre1 and Tri1 are an E-I pair, and they are consistent with the toxicity of Tre1 deriving from its ART-like domain.Figure S1Induction of Tre1tox Expression Leads to Death of E. coli Cells, Related to Figure 2Show full caption(A) Viable E. coli cells recovered following induction of expression of the indicated proteins. Expression was induced at T = 3 hr. Data represent means ± SD, n = 3.(B) Anti-Tre1 western blot indicating levels of the indicated proteins produced by E. coli cells after 3-5 h of induction (until OD600 = 1) from inducible expression plasmids. Wild-type Tri1-Sp was coexpressed to prevent cytotoxicity caused by expression of Tre1 and variants. Control band is a non-specific target of the Tre1 antibody, used to indicate equal loading.(C) Schematic depicting the tre1 (yellow) tri1 (blue) locus of wild-type S. proteamaculans and the strain bearing an in-frame deletion of the two genes. Numbers indicate last nucleotide remaining on either side of the deletion.(D) Anti-Tre1 western blot indicating levels of the protein variants shown produced from the native locus in S. proteamaculans grown in liquid LB for 18 h.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Viable E. coli cells recovered following induction of expression of the indicated proteins. Expression was induced at T = 3 hr. Data represent means ± SD, n = 3. (B) Anti-Tre1 western blot indicating levels of the indicated proteins produced by E. coli cells after 3-5 h of induction (until OD600 = 1) from inducible expression plasmids. Wild-type Tri1-Sp was coexpressed to prevent cytotoxicity caused by expression of Tre1 and variants. Control band is a non-specific target of the Tre1 antibody, used to indicate equal loading. (C) Schematic depicting the tre1 (yellow) tri1 (blue) locus of wild-type S. proteamaculans and the strain bearing an in-frame deletion of the two genes. Numbers indicate last nucleotide remaining on either side of the deletion. (D) Anti-Tre1 western blot indicating levels of the protein variants shown produced from the native locus in S. proteamaculans grown in liquid LB for 18 h. https://www.cell.com/cms/asset/7dc75035-a864-4cbe-9ea4-2f1cfcf6fce9/mmc2.mp4Loading ... Download .mp4 (10.18 MB) Help with .mp4 files Video S1. Time-Lapse Phase Contrast Microscopy Series of E. coli Expressing Tre1toxE415Q (Left) or Tre1tox (Right), Related to Figure 2 eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIwMDAyMDM0NTcwNWNhNmFhNjk4YTAxZGExYTAxNzhhZSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc5MDM1MzY3fQ.VHROEtA1bJyt7CUnp_23sfodV5T1Pi5Smn6ImijWtKWufPaIDtWOFusZLSFCExWfnQmts6UnYSHhvJlS1FwgsFFQXBHwmac62B70uARgtLQm_vVvGEhghf4dzL23D-epBf-0OO9qUpHoTaK_69Y-Zr9USry1IKy2sc8oT58e87GY-EiK0UMz9GThuOfXUnyGQ8HzwHsZccDAx0d0sXq76FALN5ZOFxYl2Q161Dc92OxlBD-vkaTEI8JzlI5rpnFUAESqQfg5HAXFEJexj-wqIpVL1e8gSGj7sHHNZGo8S3HiY4TCwJKdC_HKoMmlAvodzZqhSaFyqWfKV6j-Mg4O5A Download .mp4 (6.41 MB) Help with .mp4 files Video S2. Time-Lapse Phase Contrast Microscopy Series of E. coli Containing an Empty Vector (Left) or Expressing Tre1tox (Right), Related to Figure 2 We next sought to determine whether Tre1 functions as an interbacterial T6SS toxin. To this end, we used allelic exchange to generate a strain of S. proteamaculans bearing a deletion of the tre1 tri1 locus (Figure S1C). Growth competition assays performed under contact-promoting conditions revealed that this strain exhibits a fitness defect when incubated with wild-type, but not with S. proteamaculans lacking a functional T6SS (ΔicmF) (Figure 2D). To determine the contribution of the putative catalytic activity of Tre1 to this phenotype, we generated a S. proteamaculans strain expressing the E415Q allele from the native tre1 chromosomal locus (tre1E415Q). Tre1E415Q was produced at the same level as the native protein, yet unlike wild-type S. proteamaculans, the strain producing this variant displayed equal fitness with Δtre1 Δtri1 in co-culture (Figures 2D and S1D). These data show that Tre1 and Tri1 can function as a T6S-dependent E-I pair between cells of S. proteamaculans. In other bacteria, T6S-exported toxins can be delivered to competing Gram-negative organisms and thus enhance the fitness of the producing organism during co-culture with other species. Indeed, we found that inactivation of the S. proteamaculans T6SS significantly diminished fitness in co-culture with E. coli (Figure 2E). We next measured the contribution of the toxic activity of Tre1 to this phenotype. The S. proteamaculans tre1E415Q strain was employed in this experiment, because removing a PAAR domain-containing effector can lead to a generalized structural perturbation of the secretory apparatus. Our results showed that Tre1 inactivation yields a strain with fitness intermediate to that of wild-type and ΔicmF strains, consistent with Tre1 constituting one component of a multi-part effector payload delivered by this S. proteamaculans T6S pathway (Figure 2E). Ectopic expression of Tri1 in E. coli abrogated the tre1-dependent fitness advantage of wild-type, further confirming the contribution of this toxin to antagonism of E. coli by S. proteamaculans (Figure 2E). Bioinformatic analysis of Tri1 suggested that, unlike previously characterized immunity determinants of toxins delivered between bacteria, it could provide protection from intoxication via an enzymatic mechanism. However, we found that Tri1 stably interacts with Tre1, a feature shared with immunity proteins that function by inhibiting the active site of their cognate toxin (Figures S2A and S2B). To gain insight into both the mechanism of Tre1-induced toxicity and of Tri1-mediated immunity, we determined a 2.3 Å X-ray crystal structure of Tre1tox in complex with Tri1 (Figure 3A). Phases were obtained experimentally using selenomethionine-substituted protein, and molecular replacement with the resulting model was used to solve the structure of the native crystal in a second space group (Table S3). In our structure, the exclusively α-helical Tri1 and mixed α,β-Tre1 share an extensive interaction surface (2,604 Å2) consisting of two distinct regions: the plane formed between the two globular domains of the proteins (1,178 Å2) and an interface formed by a 65-amino acid N-terminal extension (NTE) of Tri1 (1,550 Å2). This striking structural feature of Tri1 extends away from the core of the immunity protein, reaches into the active site of the toxin, and at its distal end returns to pack into the Tre1tox -Tri1 globular domain interface (Figure 3A and 3B).Figure 3Tri1 Provides Immunity to Intoxication by Tre1 through Two Structurally Distinct MechanismsShow full caption(A) Ribbon diagram representation of the X-ray crystal structure of Tre1tox (yellow) in complex with Tri1 (blue). Mg2+ ion and coordinating residues in the Tri1 active site shown in red.(B) Space filling depiction of Tre1 showing occlusion of active site residues R356, S381, and E415 (red) by the N-terminal extension (NTE) of Tri1 (blue).(C and D) Structural alignments of Tre1 (C) and Tri1 (D) with previously characterized ART and ARH proteins, respectively (gray). Region of Tri1 comprising the NTE is indicated.(E) Diagram of regions shared between Tri1 proteins and previously characterized ARH proteins (blue; NTE found only in predicted ARH domain-containing immunity proteins). Gaps present in sequence alignments not shown. Conservation at each position (15-amino acid window) is shown above for all NTE-containing ARH domain proteins.(F) Proportion of the NTE segment consisting of the indicated secondary structure elements. For Tri1-Sp (dark gray), percentages determined from current structural analysis (PDB:6DRH, 6DRE); for other Tri1 homologs (light gray), percentages represent average values from prediction analyses performed using GeneSilico Metaserver (n = 22 representative sequences).(G) Magnification of the active site of Tre1tox in complex with Tri1, showing electrostatic and hydrogen bond interactions between Arg32 of Tri1 and the catalytic glutamic acid (Glu415) of Tre1tox.(H) E. coli cells recovered following induction of heterologously expressed Tre1 and the indicated Tri1 alleles or empty vector control. Left (dark gray): expression of both proteins controlled by the pBAD promoter and induced by 0.2% arabinose. Right (light gray): Tre1 expression controlled by the T7 promoter and induced by IPTG (0.01 mM), and Tri1 controlled by pBAD and induced by arabinose (indicated concentrations).Data are presented as means ± SD. Asterisks indicate significant differences in viability between populations expressing the indicated Tri1 variant proteins and the wild-type Tri1 control, when induced with 0.1% arabinose (p < 0.05, n ≥ 3).See also Figure S2 and Tables S2 and S3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Ribbon diagram representation of the X-ray crystal structure of Tre1tox (yellow) in complex with Tri1 (blue). Mg2+ ion and coordinating residues in the Tri1 active site shown in red. (B) Space filling depiction of Tre1 showing occlusion of active site residues R356, S381, and E415 (red) by the N-terminal extension (NTE) of Tri1 (blue). (C and D) Structural alignments of Tre1 (C) and Tri1 (D) with previously characterized ART and ARH proteins, respectively (gray). Region of Tri1 comprising the NTE is indicated. (E) Diagram of regions shared between Tri1 proteins and previously characterized ARH proteins (blue; NTE found only in predicted ARH domain-containing immunity proteins). Gaps present in sequence alignments not shown. Conservation at each position (15-amino acid window) is shown above for all NTE-containing ARH domain proteins. (F) Proportion of the NTE segment consisting of the indicated secondary structure elements. For Tri1-Sp (dark gray), percentages determined from current structural analysis (PDB:6DRH, 6DRE); for other Tri1 homologs (light gray), percentages represent average values from prediction analyses performed using GeneSilico Metaserver (n = 22 representative sequences). (G) Magnification of the active site of Tre1tox in complex with Tri1, showing electrostatic and hydrogen bond interactions between Arg32 of Tri1 and the catalytic glutamic acid (Glu415) of Tre1tox. (H) E. coli cells recovered following induction of heterologously expressed Tre1 and the indicated Tri1 alleles or empty vector control. Left (dark gray): expression of both proteins controlled by the pBAD promoter and induced by 0.2% arabinose. Right (light gray): Tre1 expression controlled by the T7 promoter and induced by IPTG (0.01 mM), and Tri1 controlled by pBAD and induced by arabinose (indicated concentrations). Data are presented as means ± SD. Asterisks indicate significant differences in viability between populations expressing the indicated Tri1 variant proteins and the wild-type Tri1 control, when induced with 0.1% arabinose (p < 0.05, n ≥ 3). See also Figure S2 and Tables S2 and S3. The structure revealed that Tre1 shares a high degree of similarly to the catalytic domains of RSE-type ART enzymes (Figure 3C) (Cohen and Chang, 2018Cohen M.S. Chang P. Insights into the biogenesis, function, and regulation of ADP-ribosylation.Nat. Chem. Biol. 2018; 14: 236-243Crossref PubMed Scopus (154) Google Scholar). Among this large and diverse group of proteins found in both bacteria and eukaryotes, Tre1 is most similar to a subset of those which ADP-ribosylate arginine residues, including iota toxin from C. perfringens (2.1 Å root-mean-square deviation [RMSD], 148 residues) (Tsuge et al., 2003Tsuge H. Nagahama M. Nishimura H. Hisatsune J. Sakaguchi Y. Itogawa Y. Katunuma N. Sakurai J. Crystal structure and site-directed mutagenesis of enzymatic components from Clostridium perfringens iota-toxin.J. Mol. Biol. 2003; 325: 471-483Crossref PubMed Scopus (88) Google Scholar). Like these proteins and ARTs more broadly, the active site of Tre1 is formed at the interface" @default.
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• W2896023929 date "2018-11-01" @default.
• W2896023929 modified "2023-10-11" @default.
• W2896023929 title "Bifunctional Immunity Proteins Protect Bacteria against FtsZ-Targeting ADP-Ribosylating Toxins" @default.
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• W2896023929 doi "https://doi.org/10.1016/j.cell.2018.09.037" @default.
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