Catalytic domain of plasmid pAD1 relaxase TraX defines a group of relaxases related to restriction endonucleases María Victoria Franciaa,1, Don B. Clewellb,c, Fernando de la Cruzd, and Gabriel Moncaliánd aServicio de Microbiología, Hospital Universitario Marqués de Valdecilla e Instituto de Formación e Investigación Marqués de Valdecilla, Santander 39008, Spain; bDepartment of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, MI 48109; cDepartment of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109; and dDepartamento de Biología Molecular e Instituto de Biomedicina y Biotecnología de Cantabria, Universidad de Cantabria–Consejo Superior de Investigaciones Científicas–Sociedad para el Desarrollo Regional de Cantabria, Santander 39011, Spain Edited by Roy Curtiss III, Arizona State University, Tempe, AZ, and approved July 9, 2013 (received for review May 30, 2013) Plasmid pAD1 is a 60-kb conjugative element commonly found in known relaxases show two characteristic sequence motifs, motif I clinical isolates of Enterococcus faecalis. The relaxase TraX and the containing the catalytic Tyr residue (which covalently attaches to primary origin of transfer oriT2 are located close to each other and the 5′ end of the cleaved DNA) and motif III with the His-triad have been shown to be essential for conjugation. The oriT2 site essential for relaxase activity (facilitating the cleavage reaction contains a large inverted repeat (where the nic site is located) by activation of the catalytic Tyr). This His-triad has been used as adjacent to a series of short direct repeats. TraX does not show a relaxase diagnostic signature (12). fi any of the typical relaxase sequence motifs but is the prototype of Plasmid pAD1 relaxase, TraX, was identi ed (9) and shown to oriT2 a unique family of relaxases (MOB ). The present study focuses on cleave within . It does not share overall homology with pre- C viously known relaxases. Together with MobC, from Enterobacter the genetic, biochemical, and structural analysis of TraX, whose 3D cloacae structure could be predicted by protein threading. The structure mobilizable plasmid CloDF13 (14), they form a unique consists of two domains: (i) an N-terminal domain sharing the group of relaxase family, known as MOBC (3), which contains invariant motifs unrelated to those of His-hydrophobe-His (HUH) topology of the DNA binding domain of the MarR family of tran- ii relaxases. An in-frame deletion of such motifs (of pAD1) resulted scriptional regulators and ( ) a C-terminal catalytic domain related in complete loss of relaxase activity, suggesting an important to the PD-(D/E)XK family of restriction endonucleases. Alignment function, at or near an active site of the protein (9). MOBC of MOBC relaxase amino acid sequences pointed to several con- contains relaxases belonging to two well differentiated clades, served polar amino acid residues (E28, D152, E170, E172, K176, clade MOBC1 comprised of Gammaproteobacteria mobile genetic R180, Y181, and Y203) that were mutated to alanine. Functional elements and clade MOBC2 composed of relaxases from Gram- analysis of these mutants (in vivo DNA transfer and cleavage positive bacteria. MobC_CloDF13 and TraX_pAD1 are the assays) revealed the importance of these residues for relaxase respective prototypes. activity and suggests Y181 as a potential catalytic residue similarly In this paper, we present data concerning the characterization to His-hydrophobe-His relaxases. We also show that TraX binds of TraX, which include the identification of a relaxase domain specifically to dsDNA containing the oriT2 direct repeat sequences, essential for the catalytic activity of TraX. This characterization fi fi con rming their role in transfer speci city. The results provide provides a catalytic model for the MOBC family of relaxases. insights into the catalytic mechanism of MOBC relaxases, which Moreover, our study widens the general model of the DNA differs radically from that of His-hydrophobe-His relaxases. processing for bacterial conjugation. – Results lasmid pAD1 is a conjugative sex-pheromone responding fi Pplasmid originally identified in Enterococcus faecalis (1, 2). It Identi cation of Two Structural Domains in TraX. The TraX relaxase of plasmid pAD1 belongs to the MOBC relaxase family (3). Fig. 1 is the prototype conjugative plasmid of the MOBC relaxase family (3). In the pheromone-mediated conjugative process, shows an alignment of some MOBC relaxase sequences. The plasmid-free bacteria secrete multiple pheromones (small, hy- conserved signature D-x6–17-E-x-E-(RL)-x2-K-x3-R-Y is apparent drophobic, linear peptides) that induce a process in which spe- in the alignment, as shown in Fig. 1. This signature has no re- cific plasmid-containing cells become activated by a particular lationship with the invariant motifs in HUH relaxases (3). To pheromone for adherence to potential recipients and plasmid further characterize this MOBC family of proteins, we carried transfer functions (4–8). The related conjugative DNA pro- out a structural prediction and modeling of TraX by the Protein cessing machinery includes a specific relaxase that generates Homology/analogY Recognition Engine (PHYRE) Web Server a nick in the strand of the plasmid to be transferred (9), which (15). Two structural domains within TraX were predicted, an ultimately results in the acquisition of the plasmid by the re- N-terminal domain, between residues 1 and 90, and a C-terminal domain encompassing the 170 C-terminal residues (residues cipient cell. When the recipient cells have acquired the plasmid, – shutdown or masking of the corresponding endogenous phero- 132 261). The homology model of TraX N-terminal domain was mone takes place (10, 11). This serves to prevent self-induction directly obtained by submission of the full-length sequence of caused by residual amounts of endogenous pheromone, thus TraX. The structural prediction of TraX C-terminal domain was ensuring that induction occurs only in the presence of recipient generated by Phyre2 One-to-one threading using the model of cells. So, although pheromone responding plasmids are highly transmissible among E. faecalis populations, plasmid transfer is highly regulated and induced only in the presence of sufficient Author contributions: M.V.F., D.B.C., F.d.l.C., and G.M. designed research; M.V.F. and G.M. pheromone concentrations that reflect the presence of nearby performed research; M.V.F., D.B.C., F.d.l.C., and G.M. analyzed data; and M.V.F., D.B.C., potential recipients. F.d.l.C., and G.M. wrote the paper. The term relaxase is used to define proteins involved in initi- The authors declare no conflict of interest. ation and termination of DNA conjugative transfer (12, 13). This article is a PNAS Direct Submission. They recognize a specific sequence called nic located within the 1To whom correspondence should be addressed. E-mail: [email protected]. origin of transfer (oriT2 in the case of pAD1) and cleave it in a This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. strand- and site-specific manner to initiate DNA transfer. Most 1073/pnas.1310037110/-/DCSupplemental. 13606–13611 | PNAS | August 13, 2013 | vol. 110 | no. 33 www.pnas.org/cgi/doi/10.1073/pnas.1310037110 Downloaded by guest on September 29, 2021 MICROBIOLOGY Fig. 1. Alignment of representative MOBC relaxases. (Upper) Sequence alignment of representative MOBC relaxases obtained with ClustalW. Accession numbers: MobC_CloDF13, CAB62410; MobC_PI, AAP70292; MobC_Y1, CAD58576; MobC_Y2, NP_995437; Orf8_pAM373, NP_072012; and TraX_pAD1, AAL59457. Color code: red on yellow, invariant amino acids; blue on blue, strongly conserved; black on green, similar; green on white, weakly similar; black on white, not conserved. The violet bar below the alignment indicates the region deleted in the in-frame traX deletion mutant resulting in a defective relaxase (9). The arrows refer to the conserved polar amino acid residues that were selected for site-directed mutagenesis. (Lower) Conserved sequence motif in MOBC relaxases. The height of each letter is proportional to the frequency of the amino acid residue. The logo was obtained by using WebLogo (36). the C-terminal domain of CloDF13 MobC. MobC_CloDF13 is active site residues of BamHI, suggesting a putative function for the only member of the MOBC family of relaxases biochemically this domain as the TraX catalytic domain. Moreover, the con- characterized to date (3). Its C-terminal domain model was served basic residues K176 and R180, both in α2, are located in obtained by submission of MobC C-terminal sequence to Phyre2, a similar position as the RE residues that interact with the DNA as template. Both TraX domains were modeled at >90% confi- molecule to be cleaved. To evaluate the functional importance of dence. The N-terminal domain shares the topology of the MarR such residues along with other conserved amino acid residues in – family of transcriptional regulators, including a winged helix MOBC relaxases (alignment shown in Fig. 1), site-directed mu- turn–helix DNA binding motif (16, 17). TraX N-terminal domain tagenesis of the traX structural gene was performed. The amino is predicted to be formed by four helices (α2, α3, α4, and α5) and acid residues selected for replacement are shown in Fig. 1, and a two-strand β-sheet (β1 and β2) (Fig. 2). As in other winged- all contain polar side chains. The selected residues were all helix DNA-binding proteins, the loop adjacent to the HTH motif changed to Ala, and four different approaches were applied to connects two antiparallel strands and forms like a wing that binds characterize the properties of the mutant proteins: (i) in vivo the DNA minor groove. Accordingly, the α4 helix would be re- DNA transfer assays (overnight filter matings), (ii) in vivo sponsible for the interaction with the DNA major groove. TraX nicking assays on supercoiled plasmid DNA containing pAD1 C-terminal domain shares structural homology with the PD- oriT or run-off assays (9), (iii) site-specific cleavage assays of (D/E)XK family of restriction endonucleases (REs; Fig.