Mechanism of Staphylococcal Multiresistance Plasmid Replication Origin Assembly by the Repa Protein

Mechanism of staphylococcal multiresistance plasmid replication origin assembly by the RepA protein

Maria A. Schumachera,1, Nam K. Tonthata, Stephen M. Kwongb, Naga babu Chinnama, Michael A. Liub, Ronald A. Skurrayb, and Neville Firthb

aDepartment of Biochemistry, Duke University Medical Center, Durham, NC 27710; and bSchool of Biological Sciences, University of Sydney, Sydney, NSW 2006, Australia

Edited by James M. Berger, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved May 15, 2014 (received for review April 1, 2014)

The staphylococcal multiresistance plasmids are key contributors (16). In Gram-negative bacteria the primosome includes DnaG to the alarming rise in bacterial multidrug resistance. A conserved primase, the replicative helicase (DnaB), and DnaC (17). Rep- replication initiator, RepA, encoded on these plasmids is essential lication initiation in Gram-positive bacteria involves DnaG pri- for their propagation. RepA proteins consist of flexibly linked mase and helicase (DnaC) and the proteins DnaD, DnaI, and N-terminal (NTD) and C-terminal (CTD) domains. Despite their essen- DnaB (18–22). DnaD binds first to DnaA at the origin. This is tial role in replication, the molecular basis for RepA function is followed by binding of DnaI/DnaB and DnaG, which together unknown. Here we describe a complete structural and functional recruit the replicative helicase (23, 24). Instead of DnaA, plas- dissection of RepA proteins. Unexpectedly, both the RepA NTD and mids encode and use their own specific replication initiator CTD show similarity to the corresponding domains of the bacterial binding protein. Structures are only available for RepA proteins primosome protein, DnaD. Although the RepA and DnaD NTD both (F, R6K, and pPS10 Rep) harbored in Gram-negative bacteria. These proteins contain winged helix-turn-helix (winged HTH) contain winged helix-turn-helices, the DnaD NTD self-assembles domains and bind iteron DNA as monomers to, in some still into large scaffolds whereas the tetrameric RepA NTD binds DNA unclear manner, drive replicon assembly (25–27). iterons using a newly described DNA binding mode. Strikingly, Replication mechanisms used by plasmids harbored in Gram- structural and atomic force microscopy data reveal that the NTD positive bacteria are less well understood and are distinct from tetramer mediates DNA bridging, suggesting a molecular mecha- their Gram-negative counterparts. Indeed, most plasmid RepA BIOCHEMISTRY nism for origin handcuffing. Finally, data show that the RepA CTD proteins in Gram-negative and Gram-positive bacteria show interacts with the host DnaG primase, which binds the replicative no sequence homology and seem to be unrelated. The multi- helicase. Thus, these combined data reveal the molecular mechanism resistance RepA proteins are arguably among the most abundant by which RepA mediates the specific replicon assembly of staphy- of plasmid Rep proteins, yet how they function is not known. lococcal multiresistant plasmids. Data suggest that these proteins are composed of three main regions: an N-terminal domain (NTD) consisting of ∼120 aa, a long RepA_N | replication initiation | S. aureus | two hybrid and variable linker region (∼30–50 residues), and a C-terminal

domain (CTD) of ∼120 residues (28–31). The NTD and CTD CHEMISTRY he emergence of multidrug-resistant bacteria is a mounting are both essential for replication. The NTD exhibits the highest global health crisis. In particular, multidrug-resistant Staph- level of sequence conservation, which has resulted in the desig- T nation of plasmids that encode these proteins as the RepA_N ylococcus aureus is a major cause of nosocomial and community- replicon family (10). Although not as well conserved as the NTD, acquired infections and is resistant to most antibiotics commonly RepA CTD regions show homology between plasmids found in used for patient treatment (1). Hospital intensive care units in many countries, including the United States, now report methi- Significance cillin-resistant S. aureus infection rates exceeding 50% (2, 3). Antibiotic resistance in contemporary infectious S. aureus strains, such as in hospitals, is often encoded by plasmids that can be The large staphylococcal multiresistance plasmids harbored in transmitted between strains via horizontal DNA transfer mech- Gram-positive pathogens contribute significantly to the spread anisms. These plasmids are typically classified as small (<5 kb) of multidrug-resistant bacteria and are typified by the presence multicopy plasmids, which usually encode only a single resistance of a highly conserved replication initiator protein, RepA, which gene; medium-sized (8–40 kb) multirestance plasmids that con- is required for plasmid retention. RepA proteins contain N-terminal (NTD) and C-terminal (CTD) domains, which are both fer resistance to multiple antibiotics, disinfectants, and/or heavy required for replication. We show that the RepA NTD and CTD metals; and large (>40 kb) conjugative multiresistance plasmids show striking homology to the host primosome protein DnaD that additionally encode a conjugative DNA transfer mechanism yet perform distinct functions; the NTD binds origin DNA in (4–6). Importantly, sequence analyses have shown that most a novel manner and the CTD recruits the replicative helicase. staphylococcal conjugative and nonconjugative multiresistance Moreover, NTD–DNA structures reveal the first mechanism of plasmids encode a highly conserved replication initiation protein, – origin handcuffing. Combined, the data unveil the minimal denoted RepA_N (5 15). RepA_N proteins are also encoded by mechanism by which multiresistance plasmids mediate origin plasmids from other Gram-positive bacteria as well as by some assembly via the highly conserved RepA protein. phage, underscoring their ubiquitous nature (10). These RepA proteins are essential for replication of multiresistance plasmids, Author contributions: M.A.S., S.M.K., R.A.S., and N.F. designed research; M.A.S., N.K.T., and hence plasmid carriage and dissemination, yet the mech- S.M.K., N.b.C., and M.A.L. performed research; M.A.S., N.K.T., and N.F. analyzed data; and anisms by which these proteins function in replication are cur- M.A.S. wrote the paper. rently unknown. The authors declare no conflict of interest. The DNA replication cycle can be divided into three stages: This article is a PNAS Direct Submission. initiation, elongation, and termination. Replication initiation Data deposition: Coordinates and structure factor amplitudes have been deposited in the proteins (RepA) mediate the crucial first step of initiation. Bac- Protein Data Bank (PDB), www.pdb.org (PDB ID codes 4PQK, 4PQL, 4PT7, and 4PTA). terial chromosome replication is initiated by the chromosomal 1To whom correspondence should be addressed. E-mail: [email protected]. replication initiator protein, DnaA, which binds the origin and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. recruits the replication components known as the primosome 1073/pnas.1406065111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1406065111 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 genus-specific clusters, suggesting that this domain may perform a host-specific role (28–32). Although the function of the RepA CTD remains enigmatic, recent studies have indicated that the NTD mediates DNA binding and interacts with iterons that re- side within the plasmid origin (30). The essential roles played by RepA proteins in multiresistance plasmid retention marks them as attractive targets for the development of specific chemotherapeutics. However, the successful design of such com- pounds necessitates structural and mechanistic insight. Here, we describe a detailed dissection of the RepA proteins from the multiresistance plasmids pSK41 and pTZ2126. The combined data reveal the molecular underpinnings of a minimalist replication assembly mediated by multiresistance RepA proteins. Results and Discussion Structure of the RepA NTD Contains a Winged HTH with a Unique Dimer-of-Dimers Arrangement. To gain insight into the function of the RepA NTD, which is conserved in more than 100 proteins, structures were determined of the pSK41 and pTZ2162 RepA NTDs. The pSK41 NTD structure was determined first to 2.60 Å (Table S1). The structure revealed that the RepA NTD has an overall fold composed of a central winged HTH (residues 33– 112) flanked by an N-terminal helix-strand-helix (residues 1–27) and C-terminal helix-loop motif (residues 116–132), with overall topology [α1(residues 8–14)-β1(16–20)-α2(21–27)-α3(33–53)- α4(67–76)-α5(79–94)-β2(94–100)-β3(106–112)-α6(116–129)] (Fig. 1A). The helix-strand-helix and C-terminal helix function as oligomerization elements to create an extensive dimer-of-dimers or tetramer (Fig. 1 B and C). To form dimers, the β1 strands from two NTDs interdigitate via antiparallel interactions. The dimer is further stabilized by multiple contacts between residues in helices α1andα6(Fig.1B). The resultant dimer interface is extensive and buries 2,005 Å2 of protein surface from solvent. The RepA tetramer is created by the orthogonal packing of two Fig. 1. Structure of the multiresistance RepA NTD. (A) Ribbon diagram of NTD dimers. Key to the formation of this compact tetramer is the pTZ2162 RepA NTD. The winged HTH is colored gray and secondary the deep insertion of the N-terminal tails of each subunit into structural elements and the N and C termini are labeled. Ribbon diagrams complementary hydrophobic cavities located at the junction of were made using PyMOL (39). (B) RepA NTD dimer. The pink subunit is β2–β3 and α3 (Fig. 1C). The residues mediating oligomerization shown in the same orientation as in A.(C) RepA NTD tetramer. The pink and are highly conserved (Fig. S1). Moreover, tetramer formation yellow subunits are in the same position as in B.(D) Superimposition of RepA buries 6,865 Å2 of surface area, supporting the notion that RepA NTD structures determined in this study. The pSK41 tetramers are colored is tetrameric. shades of blue and the pTZ2162 tetramers, orange. (Right) Overlay of the A second pSK41 RepA NTD structure was solved to 3.20 Å RepA NTD tetramer and dimer (cyan) onto the DnaD NTD tetramer and di- and two structures of the pTZ2162 RepA NTD (70% identical mer (red). The dimers overlay well with the exception of the C-terminal helix. The differences in these C-terminal helices leads to alternate tetramer for- to pSK41 NTD) were determined to 2.35 Å and 2.45 Å. Notably, mation of the proteins. (E) SEC analyses. The blue cross represents the all of the structures revealed the same tetramer, which can be – D standard curve and pTZ2162 and pSK41 RepA NTD samples are represented superimposed with rmsds of 0.56 0.78 Å (Fig. 1 ). The expan- as a green circle and yellow square, respectively. (F) SAXS curves of pTZ2162 sive surface area buried by tetramer formation as well as the fact and pSK41 RepA NTD samples plotted against calculated curves based on the that four different crystal structures revealed the same tetramer crystal structure tetramers. provided compelling support that the RepA NTD is tetrameric. However, to assess the NTD oligomeric state in solution, size- exclusion chromatography (SEC) and small-angle X-ray scat- two Rep families; in contrast to pSK41 and pTZ2162 RepA, the tering (SAXS) studies were carried out. SEC experiments F, R6K, and pPS10 RepA proteins consist of two tandem winged revealed that both the pTZ2162 and pSK41 RepA NTDs eluted HTH domains and function as monomers (26, 27). Thus, these as tetramers (Fig. 1E). SAXS data analysis produced radius-of- RepA proteins belong to distinct structural families. To identify gyration (Rg) values for the pTZ2162 RepA NTD and pSK41 RepA NTD of ∼29 Å and 30 Å, which compares well with the possible structural homologs of the RepA NTD, database searches were performed. The only protein that showed ho- calculated tetrameric Rgs of 27 and 26 Å, but not with the Rg values for monomers and dimers of 18 Å and 20 Å, respectively mology with RepA was the NTD of the Geobacillus kaustophilus (Fig. 1F). Thus, the collective data all support that the RepA DnaD protein (PDB ID code 2VN2), which although only NTD is tetrameric. The large number of conserved RepA pro- sharing 13% sequence identity with RepA forms similar oligo- teins, and specifically the RepA NTD, has led the National Center meric arrangements (33). The DnaD and RepA subunits and for Biotechnology Information (NCBI) Conserved Domain dimers can be superimposed with rmsds of 3.0 Å and 3.5 Å, Database to classify RepA_N as its own NCBI category, pfam-6970 respectively (Fig. 1D). The C-terminal regions of the protein, (10). The unique tetrameric fold revealed by the RepA NTD however, differ, leading to the formation of distinct tetramers structures can now be assigned to this ubiquitous and important (rmsd of 5.0 Å) (Fig. 1D, Center). Although the DnaD and RepA protein superfamily. It is notable that the multiresistance RepA proteins contain NTDs display structural similarities they perform diverse func- a winged HTH, because structures of the F, R6K, and pPS10 tions. Specifically, the DnaD NTD does not bind DNA but self- RepA proteins encoded on plasmids harbored in Gram-negative assembles into large scaffold structures, aiding primosome as- bacteria also contain this motif (26, 27). The winged HTH ele- sembly, and the RepA NTD carries out the key role of DNA ments, however, are the only shared structural feature of these iteron binding at the plasmid origin (30).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1406065111 Schumacher et al. Downloaded by guest on September 29, 2021 RepA NTD–DNA Structure Reveals Basis for Iteron Recognition. Iter- The distorted DNA adopts an optimal shape for interacting ons boxes bound by the RepA_N family of proteins consist of with the electropositive RepA NTD, as well as for permitting repeats that are ∼20–25 bp (10, 29, 30). Plasmid origins typically direct protein–DNA contacts by the wings and recognition he- contain three and sometimes four such boxes. Although boxes lices of the HTHs (Fig. 2 B and D). The deep insertion of the bound by different RepA proteins are divergent, they all contain wing is made possible by the presence of Gly101 at the tip of the extended AT tracts, usually located in the center of each repeat wing. As expected given its key role in DNA binding, this glycine (10). DNase I protection studies demonstrated that pSK41 RepA is one of the most conserved residues among these RepA pro- binding led to a periodic and nearly complete protection of the teins (Fig. S1). The long side chain of wing residue Arg99 rec- iteron region, and electrophoretic mobility shift experiments, ognizes O2 moieties on adjacent pyrimidine nucleotides (Fig. which revealed a single supershift upon RepA addition, in- 2D). Wing residues Leu102 and Asn103 make additional base dicated that RepA binding is cooperative (30). To gain insight interactions. Resides from the recognition helices provide fur- into how multiresistance RepA proteins recognize iteron DNA, ther specificity in RepA DNA recognition by reading nucleo- the structure of the pTZ2162 RepA NTD bound to a 32mer bases in the major grooves. Lys79 hydrogen bonds to the O6 based on pTZ2162 iteron 2 was determined to 3.09 Å (Fig. 2A) atom of conserved guanines and Glu80 reads the N6 atoms of (SI Methods and Table S2). To obtain crystals, excess protein adenosine bases and also the O2 atom of a thymine. Finally, over DNA was necessary and in the structure only one dimer of Thr83 is positioned to make van der Waals contacts to thymines. the tetramer is bound to the 32mer DNA. The recognition he- In sum, the pTZ2162 RepA NTD-iteron structure indicates that lices, α5, of each HTH (α4–α5) of the RepA dimer dock onto the base-specific contacts and DNA deformability, in particular that DNA major grooves, and the wings insert into the adjacent mi- mediated by the central AT-rich region, are key for RepA DNA nor grooves (Fig. 2 A and B). Except for folding of the wings, binding specificity. The pTZ2162 RepA NTD-iteron structure which are largely disordered in the apo structures, there are no was obtained using a 32-bp element that contained the 23-bp structural changes in the RepA NTD tetramer upon DNA predicted iteron box as well as regions 5′ and 3′ of the repeat binding (rmsd of 0.8 Å for Cα superimposition of DNA-bound because the minimal motif required for specific binding by a and apo forms). However, to accommodate binding of consec- single RepA unit had been unclear (29–31). Unexpectedly, the utive RepA winged HTH elements, the DNA is significantly bent structure revealed that the minimal site required to permit op- at the central AT-rich region by 30°. Because AT tracts are timal contacts to the DNA extended beyond the 23mer motif known to be intrinsically bent, the finding that RepA-bound such that the wings of adjacently bound dimers would insert into iterons must be distorted to bind RepA_N proteins suggests an the same minor groove (Fig. 2E, Top). Modeling indicates that explanation for the conservation of AT tracts in their origins these consecutively bound wings could occupy the same minor BIOCHEMISTRY (10, 34). Consistent with this supposition, fluorescence po- groove without overlap or clash. Interestingly, this modeling larization (FP) DNA binding assays revealed that substitution of exercise also suggests the possibility that, in addition to possible the AT-rich region to a GC tract reduced the binding affinity of wing–wing contacts, adjacently bound RepA proteins could in- RepA by ∼10-fold (Fig. 2C). In addition to the compressed teract (Fig. 2E). Thus, DNA binding cooperativity could arise central minor groove, the major grooves where the recogni- from minor groove widening by binding the first wing (allowing tion helices dock are widened (13 Å compared with 11 Å for the second to bind more readily), wing–wing contacts, and/or

B-DNA) and the wings that bind near the ends of the DNA interactions between RepA molecules outside the wing region. CHEMISTRY cause distortion, including DNA untwisting. These DNA defor- The cooperative interaction of multiple RepA proteins to suc- mations suggest that RepA may participate in or facilitate DNA cessive Rep boxes could amplify local untwisting of the DNA. melting. Indeed, FP studies show that the NTD can bind ssDNA, Because excess protein was required to obtain the pTZ2162 which could aid and stabilize origin strand opening (Fig. S2). RepA NTD-iteron crystals, only one NTD dimer of the tetramer

Fig. 2. Structure of the pTZ2162 RepA–iteron DNA complex. (A) pTZ2162 RepA NTD–iteron DNA complex. (B) Electrostatic surface representation of the pTZ2162 RepA NTD–iteron complex. (C) Semi-log plot of FP RepA NTD binding to the WT iteron (blue) and one in which the AT tract was replaced by a GC tract (red). (D, Upper) RepA–DNA contacts. (D, Lower) Schematic showing the RepA NTD residues that contact bases and the phosphate backbone. (E) Sequence of the pTZ2162 origin site showing four Rep boxes (Top). Repeats originally identified are colored and the regions bound by each RepA complex are under- and overlined. pTZ2162 RepA NTD iteron binding model constructed by docking suc- cessive RepA NTDs onto consecutive Rep boxes as would be located within the pTZ2162 origin. Shown as close up view is the modeled wing–wing interaction. (Middle) RepA NTD binding to two distinct Rep boxes that would be located on different DNA substrates. (Bottom) A model of RepA tetramer binding between Rep boxes on one duplex origin site. This is only possible if the tetramer bridges between boxes 1 and 3 or 1 and 4 (or 2 and 4). However, this model is not supported by DNase I protection studies (indicated by the cross through the model) (30).

Schumacher et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 was bound to DNA. However, modeling shows that each dimer of the tetramer could bind Rep boxes located on distinct DNA duplexes simultaneously to mediate bridging (Fig. 2E, Middle). By contrast, one RepA tetramer could not wrap two consecutive Rep boxes on the same strand about itself because, as noted, RepA molecules bind close in space to directly abutted iterons. Hence, looping within a single origin would require that RepA proteins bind to nonconsecutive iterons (e.g., iterons 1–3, 1–4, or 2–4). However, DNase I protection studies on RepA–origin interactions clearly revealed that when RepA binds, every box is protected, which would not be consistent with nonconsecutive looping (28–30). Moreover, the pattern of protection in the footprints is strikingly periodic, which indicates essentially identical modes of RepA binding to each iteron, in particular for boxes 2–4 in the pSK41 origin (Fig. S3) (30). Box 1 is also protected but compared with boxes 2–4 shows a slightly altered pattern, likely owing to its departure from the iteron consensus. Nonetheless, the combined data indicate that the origin iterons are bound by adjacent RepA proteins. Interestingly, however, the RepA proteins bound to all of the iterons within an origin would each have one of its dimer faces free for potential interaction with DNA at a distance or on a separate duplex. Hence, to probe the possible higher-order structures that may be formed by RepA on DNA, atomic force microscopy (AFM) was used.

Visualization of RepA-Mediated DNA Bridging Between DNA Duplexes and Atomic Model for Plasmid Copy Number Control by Handcuffing. For AFM studies, DNA sites containing the pSK41 and pTZ2162 iteron boxes embedded at the center of 700-bp DNA fragments were used (SI Methods). Samples containing pTZ2162 or pSK41 RepA NTD formed similar complexes. Consistent with modeling, the images reveal no looping within one DNA duplex and the entire central, iteron-containing regions are occupied by RepA proteins (Fig. S4). Strikingly, however, when complexes were imaged with high DNA concentrations (SI Methods) all images revealed that two separate DNA strands are linked together between RepA molecules, indicating that the RepA- bound tetramers can mediate DNA bridging (Fig. 3 A–C, i–iii). The DNA bridging visualized by AFM suggests a molecular mechanism for an important means of plasmid copy number regulation termed “handcuffing.” Regulation of replication is essential for stable plasmid retention in host cells. Plasmids must be kept to a minimum to avert metabolic overburdening of the host cell and out-competition by cells with fewer plasmids. RepA_N replicons are known to be regulated via antisense RNA molecules, which prevent excess RepA expression and as a result the overproduction of plasmids (10). However, studies indicate that multiple mechanisms of replicon regulation provide optimal control of replication and copy number production (35). Hand- cuffing sterically hinders the initiation process and prevents overreplication. Although Chattoraj and coworkers (35) pro- posed this mechanism decades ago, how the monomeric F, R6K, Fig. 3. RepA NTD bridges two replication origins; insight into replicon as- and psS10 RepA proteins could mediate this process has been sembly and direct visualization of handcuffing. (A) Top shows the design of unclear. RepA tetramer-mediated bridging suggests an ideal the DNA used in AFM imaging for pTZ2162 and pSK41 studies. AFM images of molecular mechanism to explain handcuffing. Further evidence pTZ2162 RepA NTD in complex with DNA700. (i) A RepA NTD–DNA complex for RepA DNA bridging was provided by a pTZ2162 RepA composed of two DNA strands and RepA NTD tetramers. Also shown is NTD-15mer crystal structure (3.6 Å resolution), which showed a strand of DNA with no protein bound. (B) AFM images of pSK41 RepA two DNA duplexes sandwiched between a RepA tetramer NTD in complex with DNA700. (C) AFM images of pTZ2162 RepA NTD and (Fig. S5). RepA CTD incubated with DNA700. (D) Representative ITC binding isotherm Isothermal titration calorimetry (ITC), which can provide of pTZ2162 and pSK41 RepA NTD titrated into 23-bp DNA encoding a single binding stoichiometries, was next used to probe DNA binding Rep box. (E) Molecular model for RepA-mediated plasmid handcuffing. and possible DNA bridging by the pTZ2162 and pSK41 RepA NTDs. The resulting isotherms of the NTDs binding to single the best fit to the SAXS data (Rg of the model was 87 Å and the iteron DNA sites revealed stoichiometries of one RepA NTD ± SI Methods dimer:one DNA duplex, or one RepA NTD tetramer binding to data were 87 5Å)( and Fig. S6). Thus, AFM, X-ray two DNA molecules, for both pTZ2162 and pSK41 RepA, con- crystallography, ITC, and SAXS analyses all provide support for sistent with DNA bridging (Fig. 3D). To provide a final assessment a RepA_N-mediated DNA handcuffing model (Fig. 3E). of RepA NTD-mediated DNA bridging, SAXS experiments were carried out on the pTZ2162 RepA NTD in complex with a 100-bp Structure and Function of the RepA CTD: Homology to the DnaD CTD iteron-containing DNA site. A model constructed of a RepA and DnaG Recruitment. The combined data reveal the basis for spe- NTD tetramer bridged between two 100mer DNA sites provided cific and cooperative origin binding by the RepA NTD. However,

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1406065111 Schumacher et al. Downloaded by guest on September 29, 2021 the RepA CTD is also required for replication, yet its role(s) S. aureus Y2H prey library to identify candidate CTD interaction in this process are unknown. To assess whether the CTD inter- partners. Screening of ∼320,000 transformants (approximately acts with the NTD and whether it may play a role in DNA fivefold library coverage) yielded only two strongly positive binding or organization of the Rep boxes, AFM was performed interacting prey constructs that did not self-activate. Strikingly, on samples containing separated domains of pTZ2162 RepA (the sequencing revealed that both contained a DNA fragment en- NTD and CTD) with DNA700. The resulting images were in- coding residues 81–313 of DnaG primase (Fig. 4E). However, distinguishable from those obtained from the RepA NTD–DNA700 to directly assay for a RepA CTD–DnaG interaction, FP ex- samples (Fig. 3C, i–iii). These data are consistent with previous periments were carried out. These studies examining the ability studies demonstrating that the CTD binds neither DNA nor the of DnaG to bind to a preformed RepA–DNA complex revealed NTD (30). Our FP studies also indicated that the CTD does not a clear second binding event, indicating that DnaG binds to bind either DNA or the RepA NTD–DNA complex (Fig. S7). The RepA–DNA (Fig. 4F). CTD sequence revealed no insight into its function. However, DnaG primase is a key component of the chromosomal DNA although not as generally conserved as the NTD, the RepA CTD replication machinery, synthesizing RNA primers for leading- shows strong sequence homology between RepA_N plasmids strand DNA synthesis, and is an essential component of the in genus-specific clusters, suggesting that it may perform host- primosome in lagging-strand DNA synthesis. Moreover, DnaG is specific functions necessary for replication. To gain insight into known to interact with the replicative helicase DnaC; this in- possible role(s) of the RepA CTD, the structure of the pTZ2162 teraction regulates the activities of both proteins and is critical to CTD was solved to 3.4 Å resolution. To obtain well-diffracting primosome formation (23, 36). Y2H baits corresponding to crystals, a fusion protein was produced in which the pTZ2162 pSK41 RepA NTD (residues 1–120) and CTD (201–319) were CTD was attached to the C terminus of the maltose binding constructed and tested against the DnaG prey. An interaction protein (MBP) (Table S2). The structure shows that the RepA was only detected with CTD, thereby corroborating the previous CTD folds into a compact structure composed of five helices. YH studies. Thus, our data indicate that staphylococcal RepA_N Helices α1 and α2 pack against a three-helix bundle formed by proteins mediate replication initiation by anchoring RepA to the α3, α4, and α5 (Fig. 4A and Fig. S8). No CTD oligomers were initiation site via NTD–iteron interactions and recruiting the observed in the structure, suggesting that this domain may be host primase and hence helicase proteins via its CTD. Whereas monomeric. Indeed, SAXS studies, revealing an experimentally this is the first example to our knowledge of a plasmid RepA derived Rg value of 16 Å, were consistent with the CTD monomer protein loading the primase, previous work by the Bastia labo- Rg (17 Å) (Fig. 4B). Finally, the SAXS data were of sufficient ratory has shown that the monomeric plasmid RepA proteins can

quality to use in de novo envelope calculations and docking of recruit host replication proteins, including DnaA and DnaB, BIOCHEMISTRY the CTD into the envelope produced an excellent fit (Fig. 4B). suggesting that plasmid RepA proteins have evolved to co-opt Thus, the combined data support that the RepA CTD exists as the host replication machinery (37, 38). a monomeric entity, flexibly tethered to the DNA-bound NTD. Database searches revealed that the RepA CTD shared the Conclusions: A “Minimalist” Replication Initiation Model for RepA_N strongest structural homology to the Enterococcus faecalis DnaD Replicons. RepA_N initiator proteins are employed by nearly all CTD (rmsd of 2.9 Å for overlay of 67 Cα atoms) (PDB ID code characterized large staphylococcal multiresistance plasmids as 2I5U) (Fig. 4 C and D). This was a striking finding given that the well as plasmids from Gram-positive bacteria including the en- RepA NTD was found to harbor structural similarity to the terococcal pheromone-responsive plasmids and plasmids from CHEMISTRY DnaD NTD. The CTD RepA and DnaD regions show stronger bacilli, lactococci, and lactobacilli. However, how these ubiquitous homology than the corresponding NTDs, which, as noted, have RepA proteins function in replication has been unknown. Here we different functions. Whether the roles of the RepA and DnaD reveal the structures of the RepA protein domains as well as the CTDs have also diverged is unclear. In Bacillus subtilis, DnaD structure of a complex of RepA bound to an iteron DNA site. plays a central role in replication origin assembly by mediating Remarkably, both RepA domains show structural similarity to recruitment of essential replication proteins. To determine the corresponding domains of the S. aureus host primosome whether the RepA CTD interacts with replication factors or any protein DnaD. This unexpected finding suggests the intriguing proteins in its S. aureus host, yeast two-hybrid (Y2H) analyses possibility that multiresistance plasmids may have, through hori- were carried out. First, Y2H showed that both full-length (FL) zontal gene transfer, acquired the host DnaD gene and mod- RepA and NTD self-interact, whereas the CTD does not, con- ified it for plasmid-specific functions. Indeed, the RepA NTD sistent with our structural and biochemical data. An FL pSK41 function diverged significantly from DnaD to bind DNA rather RepA Y2H bait construct was then used to screen a genomic than mediate self-assembly, as does the corresponding domain in

Fig. 4. Structure of pTZ2162 RepA CTD. (A) The MBP–RepA CTD structure. The MBP is green, the maltose molecules are shown as sticks, and the RepA CTD is blue. (B) SAXS scattering plot of pTZ2162 CTD compared with the calculated curve based on the crystal structure. Also shown is the CTD crystal structure docked into the de novo SAXS envelope. (C) RepA CTD structure with secondary structure elements labeled. (D) Superimposition of the RepA CTD (cyan) onto the DnaD CTD structure (red) revealing their structural homology. (E) Identification of the pSK41 RepA–DnaG interaction by Y2H analysis. Two prey clones (5A and 6A) strongly activated expression of the selectable marker and reporter genes only in the presence of the RepA bait construct. DNA sequencing revealed that they were independent clones expressing DnaG. All other prey clones (3A and 7A) self- activated in the presence of empty bait vector, hence repre- senting false positives. (F) FP binding of pSK41 RepA to an iteron site until saturation, followed by addition of increasing concentrations of DnaG (indicated by arrow). A saturable second binding event was evident.

Schumacher et al. PNAS Early Edition | 5of6 Downloaded by guest on September 29, 2021 DnaD. Our data show that the RepA CTD is similar to the Methods DnaD CTD in that it harbors a primosome assembly role. The genes encoding the pSK41 and pTZ2162 RepA proteins were purchased However, distinct from DnaD, the RepA CTD binds DnaG, from Genscript Corporation and subcloned into pET15b. Proteins were purified forming a plasmid-specific scaffold for helicase recruitment and via multiple column chromatography steps. Biochemical, structural, and Y2H primosome assembly. studies were performed as described in SI Methods. In summary, our combined data indicate that RepA_N repli- ACKNOWLEDGMENTS. We thank Dr. Charles W. Pemble IV for assisting N.K.T. cons are minimalist replicons that require only the plasmid with data processing and the Nanoimaging Core Facility at the University RepA protein to bind to the origin and facilitate replicon as- of Nebraska Medical Center (UNMC). The facility is supported by National sembly. Binding of the RepA NTD to the origin recruits its at- Institutes of Health (NIH) Grant 1S10 RR023400-01 (Shared Instrument Grant tached CTD, which binds the host primosome DnaG, and hence program), the UNMC Program of Excellence, and the Nebraska Research Initiative. Small-angle X-ray scattering and X-ray crystallographic data were the replicative helicase (23). Thus, RepA_N proteins alone can collected at the Advance Light Source (ALS) and Advanced Photon Source assemble a functional replicon. A further striking finding from (APS). ALS is a national user facility operated by Lawrence Berkeley National the RepA NTD–DNA structure is its bridging capability, which Laboratory on behalf of the Department of Energy (DOE), Office of Basic Energy Sciences, through the Integrated Diffraction Analysis Technologies presents an optimal conformation for plasmid origin handcuffing. program, supported by DOE Office of Biological and Environmental Re- Given that RepA_N proteins are essential and conserved in search. Additional support comes from project MINOS (Macromolecular staphylococcal multiresistance plasmids, this molecular dissection Insights on Nucleic Acids Optimized by Scattering) Grant R01GM105404. of RepA_N proteins may enable the development of urgently APS is supported by the US DOE, Office of Science, and the Office of Basic Energy Sciences under Contract W-31-109-Eng-38. This work was also sup- needed arsenals of new and highly specific chemotherapeutics ported by NIH Grant GM074815 (to M.A.S.) and National Health and Medical to combat the growing threat of multidrug-resistant bacteria. Research Council (Australia) Grant 571029 (to N.F. and S.M.K.).

1. Goetghebeur M, Landry PA, Han D, Vicente C (2007) Methicillin-resistant Staphylo- 21. Smits WK, Goranov AI, Grossman AD (2010) Ordered association of helicase loader coccus aureus: A public health issue with economic consequences. Can J Infect Dis proteins with the Bacillus subtilis origin of replication in vivo. Mol Microbiol 75(2): Med Microbiol 18(1):27–34. 452–461. 2. Cardo D, et al.; National Nosocomial Infections Surveillance System (2004) National 22. Ishigo-Oka D, Ogasawara N, Moriya S (2001) DnaD protein of Bacillus subtilis interacts Nosocomial Infections Surveillance (NNIS) system report, data summary from January with DnaA, the initiator protein of replication. J Bacteriol 183(6):2148–2150. 1992 through June 2004, issued October 2004. Am J Infect Control 32(8):470–485. 23. Corn JE, Berger JM (2006) Regulation of bacterial priming and daughter strand syn- 3. Rosenthal VD, et al.; INICC members (2012) International Nosocomial Infection Con- thesis through helicase-primase interactions. Nucleic Acids Res 34(15):4082–4088. trol Consortium (INICC) report, data summary of 36 countries, for 2004-2009. Am J 24. Rymer RU, et al. (2012) Binding mechanism of metal·NTP substrates and stringent- – Infect Control 40(5):396 407. response alarmones to bacterial DnaG-type primases. Structure 20(9):1478–1489. 4. Lacey RW (1975) Antibiotic resistance plasmids of Staphylococcus aureus and their 25. Abhyankar MM, Reddy JM, Sharma R, Büllesbach E, Bastia D (2004) Biochemical in- clinical importance. Bacteriol Rev 39(1):1–32. vestigations of control of replication initiation of plasmid R6K. J Biol Chem 279(8): 5. Firth N, Skurray R (2006) Genetics: Accessory elements and genetic exchange. Gram- 6711–6719. Positive Pathogens, eds Fischetti VA, Novick RP, Ferretti JJ, Portnoy DA, Rood JI (ASM, 26. Swan MK, Bastia D, Davies C (2006) Crystal structure of pi initiator protein-iteron Washington), pp 413–426. complex of plasmid R6K: Implications for initiation of plasmid DNA replication. Proc 6. Novick RP (1989) Staphylococcal plasmids and their replication. Annu Rev Microbiol Natl Acad Sci USA 103(49):18481–18486. 43:537–565. 27. Giraldo R, Fernández-Tresguerres ME (2004) Twenty years of the pPS10 replicon: In- 7. del Solar G, Espinosa M (2000) Plasmid copy number control: An ever-growing story. Mol Microbiol 37(3):492–500. sights on the molecular mechanism for the activation of DNA replication in iteron- – 8. Diep BA, et al. (2006) Complete genome sequence of USA300, an epidemic clone of containing bacterial plasmids. Plasmid 52(2):69 83. community-acquired methicillin–resistant Staphylococcus aureus. Lancet 367(9512): 28. Kwong SM, Lim R, Lebard RJ, Skurray RA, Firth N (2008) Analysis of the pSK1 replicon, 731–739. a prototype from the staphylococcal multiresistance plasmid family. Microbiology 9. Weigel LM, et al. (2003) Genetic analysis of a high-level vancomycin-resistant isolate 154(Pt 10):3084–3094. of Staphylococcus aureus. Science 302(5650):1569–1571. 29. Kwong SM, Skurray RA, Firth N (2004) Staphylococcus aureus multiresistance plasmid 10. Weaver KE, Kwong SM, Firth N, Francia MV (2009) The RepA_N replicons of Gram- pSK41: Analysis of the replication region, initiator protein binding and antisense RNA positive bacteria: A family of broadly distributed but narrow host range plasmids. regulation. Mol Microbiol 51(2):497–509. Plasmid 61(2):94–109. 30. Liu MA, Kwong SM, Pon CK, Skurray RA, Firth N (2012) Genetic requirements for 11. Kuroda M, et al. (2001) Whole genome sequencing of methicillin-resistant Staphy- replication initiation of the staphylococcal multiresistance plasmid pSK41. Microbi- lococcus aureus. Lancet 357(9264):1225–1240. ology 158(Pt 6):1456–1467. 12. Baba T, et al. (2002) Genome and virulence determinants of high virulence commu- 31. Nakaminami H, Noguchi N, Nishijima S, Kurokawa I, Sasatsu M (2008) Characteriza- nity-acquired MRSA. Lancet 359(9320):1819–1827. tion of the pTZ2162 encoding multidrug efflux gene qacB from Staphylococcus au- 13. Gill SR, et al. (2005) Insights on evolution of virulence and resistance from the com- reus. Plasmid 60(2):108–117. plete genome analysis of an early methicillin-resistant Staphylococcus aureus strain 32. Liu MA, Kwong SM, Jensen SO, Brzoska AJ, Firth N (2013) Biology of the staphylo- and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain. coccal conjugative multiresistance plasmid pSK41. Plasmid 70(1):42–51. – J Bacteriol 187(7):2426 2438. 33. Schneider S, Zhang W, Soultanas P, Paoli M (2008) Structure of the N-terminal olig- 14. Holden MT, et al. (2004) Complete genomes of two clinical Staphylococcus aureus omerization domain of DnaD reveals a unique tetramerization motif and provides strains: Evidence for the rapid evolution of virulence and drug resistance. Proc Natl insights into scaffold formation. J Mol Biol 376(5):1237–1250. Acad Sci USA 101(26):9786–9791. 34. Carrera P, Azorín F (1994) Structural characterization of intrinsically curved AT-rich 15. Mwangi MM, et al. (2007) Tracking the in vivo evolution of multidrug resistance in DNA sequences. Nucleic Acids Res 22(18):3671–3680. Staphylococcus aureus by whole-genome sequencing. Proc Natl Acad Sci USA 104(22): 35. Paulsson J, Chattoraj DK (2006) Origin inactivation in bacterial DNA replication con- 9451–9456. trol. Mol Microbiol 61(1):9–15. 16. del Solar G, Giraldo R, Ruiz-Echevarría MJ, Espinosa M, Díaz-Orejas R (1998) Replica- 36. Koepsell SA, Larson MA, Griep MA, Hinrichs SH (2006) Staphylococcus aureus helicase tion and control of circular bacterial plasmids. Microbiol Mol Biol Rev 62(2):434–464. 17. Soultanas P (2012) Loading mechanisms of ring helicases at replication origins. Mol but not Escherichia coli helicase stimulates S. aureus primase activity and maintains – Microbiol 84(1):6–16. initiation specificity. J Bacteriol 188(13):4673 4680. 18. Konieczny I (2003) Strategies for helicase recruitment and loading in bacteria. EMBO 37. Sharma R, Kachroo A, Bastia D (2001) Mechanistic aspects of DnaA-RepA interaction Rep 4(1):37–41. as revealed by yeast forward and reverse two-hybrid analysis. EMBO J 20(16): 19. Bruand C, Ehrlich SD, Jannière L (1995) Primosome assembly site in Bacillus subtilis. 4577–4587. EMBO J 14(11):2642–2650. 38. Datta HJ, Khatri GS, Bastia D (1999) Mechanism of recruitment of DnaB helicase to the 20. Bruand C, Farache M, McGovern S, Ehrlich SD, Polard P (2001) DnaB, DnaD and DnaI replication origin of the plasmid pSC101. Proc Natl Acad Sci USA 96(1):73–78. proteins are components of the Bacillus subtilis replication restart primosome. Mol 39. DeLano WL (2002) The PyMOL Molecular Graphics System (DeLano Scientific, Microbiol 42(1):245–255. San Carlos, CA).

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1406065111 Schumacher et al. Downloaded by guest on September 29, 2021