Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Staphylococcal SCCmec elements encode an active MCM-like helicase and thus may be replicative

Abstract

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Cartoon of the conserved cores of SCC and related mobile elements.
Figure 2: Cch forms a hexameric ring.
Figure 3: Potential DNA-binding surfaces of Cch.
Figure 4: Comparison of DNA helicase and translocase AAA+ domains.
Figure 5: Helicase and DNA-binding activities of Cch.
Figure 6: LP1413 binds ssDNA.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Moellering, R.C. Jr. MRSA: the first half century. J. Antimicrob. Chemother. 67, 4–11 (2012).

    Article  CAS  Google Scholar 

  2. 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).

    Article  CAS  Google Scholar 

  3. Misiura, A. et al. Roles of two large serine recombinases in mobilizing the methicillin-resistance cassette SCCmec. Mol. Microbiol. 88, 1218–1229 (2013).

    Article  CAS  Google Scholar 

  4. 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).

    Article  CAS  Google Scholar 

  5. 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).

    Article  CAS  Google Scholar 

  6. 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).

  7. 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).

    Article  CAS  Google Scholar 

  8. Grindley, N.D.F., Whiteson, K.L. & Rice, P.A. Mechanisms of site-specific recombination. Annu. Rev. Biochem. 75, 567–605 (2006).

    Article  CAS  Google Scholar 

  9. 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).

    Article  Google Scholar 

  10. Mir-Sanchis, I. et al. Control of Staphylococcus aureus pathogenicity island excision. Mol. Microbiol. 85, 833–845 (2012).

    Article  CAS  Google Scholar 

  11. 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).

    Article  Google Scholar 

  12. 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).

    Article  CAS  Google Scholar 

  13. 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).

    Article  CAS  Google Scholar 

  14. Hickman, A.B. & Dyda, F. Binding and unwinding: SF3 viral helicases. Curr. Opin. Struct. Biol. 15, 77–85 (2005).

    Article  CAS  Google Scholar 

  15. Marchler-Bauer, A. et al. CDD: NCBI's conserved domain database. Nucleic Acids Res. 43, D222–D226 (2015).

    Article  CAS  Google Scholar 

  16. Wang, H.-C. et al. Staphylococcus aureus protein SAUGI acts as a uracil-DNA glycosylase inhibitor. Nucleic Acids Res. 42, 1354–1364 (2014).

    Article  CAS  Google Scholar 

  17. 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).

    Article  CAS  Google Scholar 

  18. 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).

    Article  CAS  Google Scholar 

  19. 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).

    Article  Google Scholar 

  20. 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).

    CAS  PubMed  Google Scholar 

  21. 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).

    Article  Google Scholar 

  22. 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).

    Article  CAS  Google Scholar 

  23. 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).

    Article  CAS  Google Scholar 

  24. Holm, L. & Sander, C. Dali: a network tool for protein structure comparison. Trends Biochem. Sci. 20, 478–480 (1995).

    Article  CAS  Google Scholar 

  25. Trakselis, M.A. Structural mechanisms of hexameric helicase loading, assembly, and unwinding. F1000Res. 5, 111 (2016).

    Article  Google Scholar 

  26. Bleichert, F., Botchan, M.R. & Berger, J.M. Crystal structure of the eukaryotic origin recognition complex. Nature 519, 321–326 (2015).

    Article  CAS  Google Scholar 

  27. Erzberger, J.P. & Berger, J.M. Evolutionary relationships and structural mechanisms of AAA+ proteins. Annu. Rev. Biophys. Biomol. Struct. 35, 93–114 (2006).

    Article  CAS  Google Scholar 

  28. 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).

    Article  CAS  Google Scholar 

  29. Enemark, E.J. & Joshua-Tor, L. Mechanism of DNA translocation in a replicative hexameric helicase. Nature 442, 270–275 (2006).

    Article  CAS  Google Scholar 

  30. 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).

    Article  CAS  Google Scholar 

  31. 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).

    Article  CAS  Google Scholar 

  32. LeBowitz, J.H. & McMacken, R. The Escherichia coli dnaB replication protein is a DNA helicase. J. Biol. Chem. 261, 4738–4748 (1986).

    CAS  PubMed  Google Scholar 

  33. 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).

    Article  CAS  Google Scholar 

  34. Ziegelin, G. & Lanka, E. Bacteriophage P4 DNA replication. FEMS Microbiol. Rev. 17, 99–107 (1995).

    Article  CAS  Google Scholar 

  35. Duderstadt, K.E., Chuang, K. & Berger, J.M. DNA stretching by bacterial initiators promotes replication origin opening. Nature 478, 209–213 (2011).

    Article  CAS  Google Scholar 

  36. 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).

    Article  CAS  Google Scholar 

  37. 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).

    Article  CAS  Google Scholar 

  38. Mkrtchyan, H.V., Xu, Z. & Cutler, R.R. Diversity of SCCmec elements in Staphylococci isolated from public washrooms. BMC Microbiol. 15, 120 (2015).

    Article  Google Scholar 

  39. Smyth, D.S., Wong, A. & Robinson, D.A. Cross-species spread of SCCmec IV subtypes in staphylococci. Infect. Genet. Evol. 11, 446–453 (2011).

    Article  Google Scholar 

  40. Crossley, K.B., Jefferson, K.K., Archer, G.L. & Fowler, V.G. Staphylococci in Human Disease (Wiley, 2009).

  41. Samuels, M. et al. A biochemically active MCM-like helicase in Bacillus cereus. Nucleic Acids Res. 37, 4441–4452 (2009).

    Article  CAS  Google Scholar 

  42. 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).

    Article  Google Scholar 

  43. Li, N. et al. Structure of the eukaryotic MCM complex at 3.8 Å. Nature 524, 186–191 (2015).

    Article  CAS  Google Scholar 

  44. 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).

    Article  CAS  Google Scholar 

  45. Putnam, C.D. et al. Structure and mechanism of the RuvB Holliday junction branch migration motor. J. Mol. Biol. 311, 297–310 (2001).

    Article  CAS  Google Scholar 

  46. 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).

    Article  CAS  Google Scholar 

  47. 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).

    Article  CAS  Google Scholar 

  48. 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).

    Article  Google Scholar 

  49. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  50. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  51. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  52. Wittig, I., Braun, H.-P. & Schägger, H. Blue native PAGE. Nat. Protoc. 1, 418–428 (2006).

    Article  CAS  Google Scholar 

  53. 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).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

P.A.R. conceived and directed the project and carried out most of the bioinformatics. I.M.-S. carried out all of the crystallography and biochemistry shown in the figures as well as some of the bioinformatics. S.B.-V. advised on staphylococcal molecular biology, suggested examining the cch gene and carried out the initial cloning of cch. A.M. contributed to the cloning and initial stages of the Cch biochemistry; C.A.R. worked out the purification and initial characterization of LP1413; and Y.Z.P. assisted with protein purification and crystallization. I.M.-S. and P.A.R. wrote the paper.

Corresponding author

Correspondence to Phoebe A Rice.

Ethics declarations

Competing interests

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 information

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

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.3286

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing