Methicillin-resistant Staphylococcus aureus (MRSA) is a public-health threat worldwide. Although the mobile genomic island responsible for this phenotype, staphylococcal cassette chromosome (SCC), has been thought to be nonreplicative, we predicted DNA-replication-related functions for some of the conserved proteins encoded by SCC. We show that one of these, Cch, is homologous to the self-loading initiator helicases of an unrelated family of genomic islands, that it is an active 3′-to-5′ helicase and that the adjacent ORF encodes a single-stranded DNA–binding protein. Our 2.9-Å crystal structure of intact Cch shows that it forms a hexameric ring. Cch, like the archaeal and eukaryotic MCM-family replicative helicases, belongs to the pre–sensor II insert clade of AAA+ ATPases. Additionally, we found that SCC elements are part of a broader family of mobile elements, all of which encode a replication initiator upstream of their recombinases. Replication after excision would enhance the efficiency of horizontal gene transfer.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Staphylococcus sciuri bacteriophages double-convert for staphylokinase and phospholipase, mediate interspecies plasmid transduction, and package mecA gene
Scientific Reports Open Access 13 April 2017
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Get just this article for as long as you need it
Prices may be subject to local taxes which are calculated during checkout
Moellering, R.C. Jr. MRSA: the first half century. J. Antimicrob. Chemother. 67, 4–11 (2012).
Ito, T., Katayama, Y. & Hiramatsu, K. Cloning and nucleotide sequence determination of the entire mec DNA of pre-methicillin-resistant Staphylococcus aureus N315. Antimicrob. Agents Chemother. 43, 1449–1458 (1999).
Misiura, A. et al. Roles of two large serine recombinases in mobilizing the methicillin-resistance cassette SCCmec. Mol. Microbiol. 88, 1218–1229 (2013).
Katayama, Y., Ito, T. & Hiramatsu, K. A new class of genetic element, staphylococcus cassette chromosome mec, encodes methicillin resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 44, 1549–1555 (2000).
Ito, T. et al. Novel type V staphylococcal cassette chromosome mec driven by a novel cassette chromosome recombinase, ccrC. Antimicrob. Agents Chemother. 48, 2637–2651 (2004).
Ito, T., Tsubakishita, S., Kuwahara-Arai, K., Han, X. & Hiramatsu, K. Staphylococcal cassette chromosome (scc): a unique gene transfer system in staphylococci. in Bacterial Integrative Mobile Genetic Elements (eds. Roberts, A.P. & Mullany, P.) Ch. 18 (CRC Press, 2013).
Novick, R.P., Christie, G.E. & Penadés, J.R. The phage-related chromosomal islands of Gram-positive bacteria. Nat. Rev. Microbiol. 8, 541–551 (2010).
Grindley, N.D.F., Whiteson, K.L. & Rice, P.A. Mechanisms of site-specific recombination. Annu. Rev. Biochem. 75, 567–605 (2006).
Úbeda, C. et al. SaPI mutations affecting replication and transfer and enabling autonomous replication in the absence of helper phage. Mol. Microbiol. 67, 493–503 (2008).
Mir-Sanchis, I. et al. Control of Staphylococcus aureus pathogenicity island excision. Mol. Microbiol. 85, 833–845 (2012).
Úbeda, C., Barry, P., Penadés, J.R. & Novick, R.P. A pathogenicity island replicon in Staphylococcus aureus replicates as an unstable plasmid. Proc. Natl. Acad. Sci. USA 104, 14182–14188 (2007).
Ubeda, C., Tormo-Más, M.Á., Penadés, J.R. & Novick, R.P. Structure-function analysis of the SaPIbov1 replication origin in Staphylococcus aureus. Plasmid 67, 183–190 (2012).
Ziegelin, G., Linderoth, N.A., Calendar, R. & Lanka, E. Domain structure of phage P4 alpha protein deduced by mutational analysis. J. Bacteriol. 177, 4333–4341 (1995).
Hickman, A.B. & Dyda, F. Binding and unwinding: SF3 viral helicases. Curr. Opin. Struct. Biol. 15, 77–85 (2005).
Marchler-Bauer, A. et al. CDD: NCBI's conserved domain database. Nucleic Acids Res. 43, D222–D226 (2015).
Wang, H.-C. et al. Staphylococcus aureus protein SAUGI acts as a uracil-DNA glycosylase inhibitor. Nucleic Acids Res. 42, 1354–1364 (2014).
Serrano-Heras, G., Bravo, A. & Salas, M. Phage phi29 protein p56 prevents viral DNA replication impairment caused by uracil excision activity of uracil-DNA glycosylase. Proc. Natl. Acad. Sci. USA 105, 19044–19049 (2008).
Lina, G. et al. Staphylococcal chromosome cassette evolution in Staphylococcus aureus inferred from ccr gene complex sequence typing analysis. Clin. Microbiol. Infect. 12, 1175–1184 (2006).
Iyer, L.M., Abhiman, S. & Aravind, L. A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases. Biol. Direct 3, 39 (2008).
Chen, Y., Narendra, U., Iype, L.E., Cox, M.M. & Rice, P.A. Crystal structure of a Flp recombinase-Holliday junction complex: assembly of an active oligomer by helix swapping. Mol. Cell 6, 885–897 (2000).
Bjørkeng, E.K. et al. ccrABEnt serine recombinase genes are widely distributed in the Enterococcus faecium and Enterococcus casseliflavus species groups and are expressed in E. faecium. Microbiology 156, 3624–3634 (2010).
Oliveira, D.C., Tomasz, A. & de Lencastre, H. The evolution of pandemic clones of methicillin-resistant Staphylococcus aureus: identification of two ancestral genetic backgrounds and the associated mec elements. Microb. Drug Resist. 7, 349–361 (2001).
David, M.Z. & Daum, R.S. Community-associated methicillin-resistant Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic. Clin. Microbiol. Rev. 23, 616–687 (2010).
Holm, L. & Sander, C. Dali: a network tool for protein structure comparison. Trends Biochem. Sci. 20, 478–480 (1995).
Trakselis, M.A. Structural mechanisms of hexameric helicase loading, assembly, and unwinding. F1000Res. 5, 111 (2016).
Bleichert, F., Botchan, M.R. & Berger, J.M. Crystal structure of the eukaryotic origin recognition complex. Nature 519, 321–326 (2015).
Erzberger, J.P. & Berger, J.M. Evolutionary relationships and structural mechanisms of AAA+ proteins. Annu. Rev. Biophys. Biomol. Struct. 35, 93–114 (2006).
Iyer, L.M., Leipe, D.D., Koonin, E.V. & Aravind, L. Evolutionary history and higher order classification of AAA+ ATPases. J. Struct. Biol. 146, 11–31 (2004).
Enemark, E.J. & Joshua-Tor, L. Mechanism of DNA translocation in a replicative hexameric helicase. Nature 442, 270–275 (2006).
Soni, R.K., Mehra, P., Choudhury, N.R., Mukhopadhyay, G. & Dhar, S.K. Functional characterization of Helicobacter pylori DnaB helicase. Nucleic Acids Res. 31, 6828–6840 (2003).
Li, Y. & Araki, H. Loading and activation of DNA replicative helicases: the key step of initiation of DNA replication. Genes Cells 18, 266–277 (2013).
LeBowitz, J.H. & McMacken, R. The Escherichia coli dnaB replication protein is a DNA helicase. J. Biol. Chem. 261, 4738–4748 (1986).
Singleton, M.R., Sawaya, M.R., Ellenberger, T. & Wigley, D.B. Crystal structure of T7 gene 4 ring helicase indicates a mechanism for sequential hydrolysis of nucleotides. Cell 101, 589–600 (2000).
Ziegelin, G. & Lanka, E. Bacteriophage P4 DNA replication. FEMS Microbiol. Rev. 17, 99–107 (1995).
Duderstadt, K.E., Chuang, K. & Berger, J.M. DNA stretching by bacterial initiators promotes replication origin opening. Nature 478, 209–213 (2011).
Dueber, E.L.C., Corn, J.E., Bell, S.D. & Berger, J.M. Replication origin recognition and deformation by a heterodimeric archaeal Orc1 complex. Science 317, 1210–1213 (2007).
Gaudier, M., Schuwirth, B.S., Westcott, S.L. & Wigley, D.B. Structural basis of DNA replication origin recognition by an ORC protein. Science 317, 1213–1216 (2007).
Mkrtchyan, H.V., Xu, Z. & Cutler, R.R. Diversity of SCCmec elements in Staphylococci isolated from public washrooms. BMC Microbiol. 15, 120 (2015).
Smyth, D.S., Wong, A. & Robinson, D.A. Cross-species spread of SCCmec IV subtypes in staphylococci. Infect. Genet. Evol. 11, 446–453 (2011).
Crossley, K.B., Jefferson, K.K., Archer, G.L. & Fowler, V.G. Staphylococci in Human Disease (Wiley, 2009).
Samuels, M. et al. A biochemically active MCM-like helicase in Bacillus cereus. Nucleic Acids Res. 37, 4441–4452 (2009).
Miller, J.M., Arachea, B.T., Epling, L.B. & Enemark, E.J. Analysis of the crystal structure of an active MCM hexamer. eLife 3, e03433 (2014).
Li, N. et al. Structure of the eukaryotic MCM complex at 3.8 Å. Nature 524, 186–191 (2015).
Yuan, Z. et al. Structure of the eukaryotic replicative CMG helicase suggests a pumpjack motion for translocation. Nat. Struct. Mol. Biol. 23, 217–224 (2016).
Putnam, C.D. et al. Structure and mechanism of the RuvB Holliday junction branch migration motor. J. Mol. Biol. 311, 297–310 (2001).
Altschul, S.F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).
Kelley, L.A. & Sternberg, M.J.E. Protein structure prediction on the Web: a case study using the Phyre server. Nat. Protoc. 4, 363–371 (2009).
Kearse, M. et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28, 1647–1649 (2012).
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).
Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).
Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).
Wittig, I., Braun, H.-P. & Schägger, H. Blue native PAGE. Nat. Protoc. 1, 418–428 (2006).
Folta-Stogniew, E. Oligomeric states of proteins determined by size-exclusion chromatography coupled with light scattering, absorbance, and refractive index detectors. Methods Mol. Biol. 328, 97–112 (2006).
We thank R. Daum (Department of Pediatrics and MRSA Center, University of Chicago) for providing USA300 MRSA strain 923, from which we cloned Cch and LP1413, J.R. Penadés and M.R. Boocock for insightful discussions, J. Herrou for assistance with crystallization, Y.-L. Chan for biochemical advice, the CCP4 summer school for crystallographic advice and the staff of the Structural Biology Center at Argonne National Laboratory for assistance with data collection. This work was funded by grant R21AI117593 (NIAID, NIH ) to P.A.R.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Alignment of Cch and SaPIbovI Rep primary sequences and secondary structures.
Cch sequences from S. aureus USA300 strain (SAUSA300_0039), S. cohnii (ADM43452), S. hominis (BAB83484), M. caseolyticus (BAI83364) and Rep from SaPIBov1 (CAI80039) were aligned using ClustalW 59. Asterisks indicate positions that have a fully conserved residue, colon indicates strong conservation between groups of strongly similar properties and period indicates partial conservation. Secondary structure predictions (using the jpred server 60) of Cch_USA300 and Rep_SaPIbov1 are displayed above and below their sequences respectively. Predicted alpha helices and beta strands are shown as orange cylinders and green arrows respectively. Real secondary structure, based on our crystal structure, is displayed above the Cch_USA300 sequence as open cylinders and open arrows. Plus signs indicate residues that could not be modeled due to disorder. Helices and arrows have been numbered accordingly to their domain. N-terminal domain: N1, N2, N3…ATPase domain: A1, A2, A3… C-terminal domain: C1, C2, C3…Note that secondary structure elements in the ATPase domain have been labeled according to a standard system (article reference 27). Catalytic residues are highlighted in red rectangles: WA, Walker A; WB, Walker B; S-I, sensor-I; RF, arginine finger; S-II, sensor-II. Black rectangles within the top line mark the Pre-sensor I (PS-I) and Pre-sensor II insertions. Color code of the residues is according to ClustalX default values.
59. Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).
60. Drozdetskiy, A., Cole, C., Procter, J. & Barton, G. J. JPred4: a protein secondary structure prediction server. Nucleic Acids Res. gkv332 (2015). doi:10.1093/nar/gkv332
Supplementary Figure 2 Alignment of Cch2 and SaPI5-type Rep protein sequences and secondary structure.
CchC sequences (named Cch2 in Figure 1) from S. aureus strain WIS (AB121219), SCCmec type VII (AB373032), strain TSGH17 (AB512767) and Pri-Rep sequences from SaPImw2 (BA000033), SaPI5 (NC_007793), strain WP_001670264, SaPI from S. pseudointermedius (WP_015728773) were used for alignment comparison using ClustalW 59. Note that the primase domain of the Pri-Rep fused proteins and the extreme N-terminus of the Cch proteins were excluded for the alignment (aligned portions began with Gly73 for CchC type V and Gly342 for SaPI5 Rep). The secondary structure prediction (from the jpred server 60) of CchC type VII and SaPI5 Pri-Rep proteins is displayed above and below the sequences respectively. Alpha helices, orange cylinders; beta-sheets, green arrows. Predicted catalytic residues have been highlighted as in Supplementary Fig. 1. Color code of the residues is according to ClustalX default values. See references 59 and 60 in Supplementary legend Fig. 1.
Supplementary Figure 3 Structure determination.
a. Experimental electron density map in stereo view. ATPase domain residues S217 to A227, AMP-PNP and magnesium (cyan sphere) are shown with the experimental density map at contour levels 1.5 σ (grey) and 4 σ (purple), carve=1.6 Å. The map was subjected to automated solvent modification and twofold averaging in Phenix Autosol, but contains no information from the model.
b. An anomalous difference Fourier map is shown contoured at 4 σ and carved at 4 Å. The two monomers in the asymmetric unit are displayed as green and cyan ribbons and SeMet residues are displayed as sticks.
c. Close-up of the N-terminal N-domains (displayed as lines) showing the agreement of the SeMet residues (shown as sticks) with the anomalous difference Fourier map contoured at 10 σ with carve set at 10 Å.
Supplementary Figure 4 The N-terminal DUF927-containing domain of Cch.
The N-terminal DUF927-containing domain of Cch shaded from blue (L5) to red (L154). Three Helices (N1, N4 and N5) and two strands (N3 and N4) have been labeled according to Supplementary Fig. 1.
Supplementary Figure 5 Oligomerization state of Cch.
a. Size exclusion chromatography (SEC). Orange line: 400 microliters of 32 micromolar Cch was loaded onto a Superdex 200 increase 10/300 column that was both pre-equilibrated and eluted with buffer containing 20 mM tris pH 7.8, 0.5 mM EDTA, 5% glycerol and 0.5 M NaCl. Blue line: Same, except that a 1.5 molar excess of LP1413 was added. Green line: LP1413 alone.
b. Elution volumes for standards and samples plotted as a function of molecular weight. Standards (red symbols) were (name, MW in Da, volume in ml): Thyroglobulin 670,000 10.043; γ-globulin 158,000 13.311; ovalbumin 44,000 15.921; Myoglobin 17,000 17.788; VitaminB12 1350 20.265. Elution volumes for the 3 Cch + LP peaks (blue symbols) were 10.64, 14.50 and 18.52 ml; for Cch alone (orange symbols, occluded by blue) were 10.62 and 14.48; for LP1413 alone the major peak eluted at 18.51 ml (green symbol, occluded by blue). The molecular weights plotted for the experimental samples were those of hexamers (407.4 kDa) and monomers (67.9 kDa) for Cch and of monomers for LP1413 (10.9 kDa). The shoulder on the LP1413 elution peak may represent a higher-order species, but under these conditions, it did not appear to interact with Cch.
c. SEC-MALS (SEC followed by multi angle light scattering) was carried out with150 microliters of 30 micromolar Cch (in the same buffer than a) on a Wyatt SEC-MALS GE AKTAPurifier UPC-10 with a DAWN HELEOS II detector. The molecular weights derived from the two peaks in this experiment were 380KDa for Cch hexamer and 64KDa for Cch monomer.
d. Blue Native Polyacrylamide Gel Electrophoresis shows that Cch (MW 67.9kDa) forms an oligomer consistent with a hexamer in solution. M, marker; 1, Cch (~3μM); 2, Cch and single stranded DNA. See ONLINE METHODS for details.
Supplementary Figure 6 Similarities and differences between ORC and Cch architectures.
In both cases the C-terminal WH domains make protein-protein contacts to one another and to a neighboring subunit’s ATPase domain (article reference 26).
Supplementary Text and Figures
Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Note (PDF 2085 kb)
Supplementary Data Set 1
Uncropped gel images (PDF 1135 kb)
Rights and permissions
About this article
Cite this article
Mir-Sanchis, I., Roman, C., Misiura, A. et al. Staphylococcal SCCmec elements encode an active MCM-like helicase and thus may be replicative. Nat Struct Mol Biol 23, 891–898 (2016). https://doi.org/10.1038/nsmb.3286
This article is cited by
Staphylococcus sciuri bacteriophages double-convert for staphylokinase and phospholipase, mediate interspecies plasmid transduction, and package mecA gene
Scientific Reports (2017)
Replicating methicillin resistance?
Nature Structural & Molecular Biology (2016)