Elsevier

Plasmid

Volume 67, Issue 2, March 2012, Pages 183-190
Plasmid

Structure–function analysis of the SaPIbov1 replication origin in Staphylococcus aureus

https://doi.org/10.1016/j.plasmid.2012.01.006Get rights and content

Abstract

The SaPIs and their relatives are phage satellites and are unique among the known bacterial pathogenicity islands in their ability to replicate autonomously. They possess a phage-like replicon, which is organized as two sets of iterons arrayed symmetrically to flank an AT-rich region that is driven to melt by the binding of a SaPI-specific initiator (Rep) to the flanking iterons. Extensive deletion analysis has revealed that Rep can bind to a single iteron, generating a simple shift in a gel mobility assay; when bound on both sides, a second retarded band is seen, suggesting independent binding. Binding to both sites of the ori is necessary but not sufficient to melt the AT-rich region and initiate replication. For these processes, virtually the entire origin must be present. Since SaPI replication can be initiated on linear DNA, it is suggested that bilateral binding may be necessary to constrain the intervening DNA to enable Rep-driven melting.

Highlights

► Demonstration of iterons in a replication origin from a Gram-positive organism. ► Symmetrical arrangement of iterons flanking an AT-rich region. ► Requirement of flanking iterons for melting of the AT-rich region. ► High frequency SaPI transfer in the absence of replication.

Introduction

The staphylococcal pathogenicity islands are prototypes of a large family of phage-related chromosomal islands that are widely distributed among Gram-positive bacteria (Novick and Subedi, 2007). Following infection of their host organism by any of several helper phages, the SaPI genome excises, replicates autonomously, and is encapsidated in small infectious phage-like particles composed of phage virion proteins (Lindsay et al., 1998, Tormo-Más et al., 2010, Tormo et al., 2008, Ubeda et al., 2005, Ubeda et al., 2008). In previous studies, we have analyzed several components of the SaPI lifecycle, including genome organization, phage induction, excision and integration, and packaging (review: Novick et al., 2010). In this report, we describe the sequence requirements for the initiation of replication.

Classically, the initiation of replication in prokaryotes involves specific binding of a replication initiator protein to a unique replication origin followed by melting at an AT-rich region within or adjacent to the origin, which enables helicase-driven unwinding preparatory to the start of polymerization. This paradigm applies fully to the SaPIs, as the specific components of the SaPI replicon include a specific replication origin (ori) and an initiator protein (Rep) that recognizes and binds to it. All of the 16 known SaPI replication origins have a common, though rather unusual structure, consisting of two sets of short repeated sequences (iterons) flanking an AT-rich region of about 80 bp (Ubeda et al., 2007). The Rep protein, like analogous proteins of various phages and viruses (Briani et al., 2001), has helicase activity, which is required for initiation (Ubeda et al., 2007), and is predicted to be hexameric. It binds specifically to the isolated ori region, showing multiple bands in a gel mobility shift assay (Ubeda et al., 2007). The Rep–ori interaction is SaPI-specific and is determined by a matching interaction between the iterons and a specificity determinant in the C-terminal region of the Rep protein (Ubeda et al., 2007). Following initiation, replication is continued by host polymerization functions, probably aided by a SaPI-coded primase. The product of SaPI replication is a linear concatemer (Ubeda et al., 2007) which is packaged by the headful mechanism (Ruzin et al., 2001), initiated by a complex between the phage terminase large subunit and a SaPI-encoded version of the terminase small subunit.

In this study, we have sought to ascertain the roles of the several sequence elements in the unusual SaPI replication origin and to see how they interact with the Rep protein. We show that although Rep can bind to a single iteron segment, it can induce melting, which occurs within the AT-rich region as one might have expected, and can initiate replication only when essentially the entire ori is present.

Section snippets

Bacterial strains and growth conditions

Bacterial strains used in this study are listed in Table S1 (supplementary data). Bacteria were grown at 32 or 37 °C overnight on glycerol–lactate agar medium (Novick, 1991), supplemented with antibiotics as appropriate. Broth cultures were grown at 32 or 43 °C in casamino acids–yeast extract broth (Novick, 1991) or TSB with shaking (240 rpm). Procedures for transduction and transformation in Staphylococcus aureus were performed essentially as described (Novick, 1991).

DNA methods

General DNA manipulations

Deletion analysis of Rep–ori binding

The SaPIbov1-ori contains 11 hexanucleotide iterons, 10 with the sequence gtaccc and one with a single mismatch, (gtatcc), flanking the AT-rich region (Fig. 1A). To determine the role of these iterons in the initiation of replication, we constructed a series of deletions in the cloned replication origin and tested these for Rep binding, for melting and for their ability to support replication. In the first series of tests (Fig. 1B), we performed an electrophoretic mobility shift assay (EMSA)

Discussion

The overall SaPI replication process is similar to that of a bacteriophage: there is a SaPI-specific replication initiation protein with helicase activity that binds to the SaPI origin in a sequence-specific manner and initiates melting within the origin, followed by helicase-driven unwinding (Ubeda et al., 2007). As the SaPIs do not encode homologs of any other replication proteins, it is assumed that all of the polymerization functions are provided by the host cell. The focus of the present

References (22)

  • F. Briani

    The plasmid status of satellite bacteriophage P4

    Plasmid

    (2001)
  • R.P. Novick

    Genetic systems in staphylococci

    Methods Enzymol.

    (1991)
  • S. Zzaman et al.

    Oligomeric initiator protein-mediated DNA looping negatively regulates plasmid replication in vitro by preventing origin melting

    Mol. Cell.

    (2005)
  • Ausubel, F. et al., 1987. Current Protocols in Molecular Biology. New York:...
  • D. Bates

    The DnaA box R4 in the minimal oriC is dispensable for initiation of Escherichia coli chromosome replication

    Nucleic Acids Res.

    (1995)
  • K. Denniston-Thompson

    Physical structure of the replication origin of bacteriophage lambda

    Science

    (1977)
  • K. Horiuchi

    Initiation mechanisms in replication of filamentous phage DNA

    Genes Cells

    (1997)
  • J.A. Lindsay

    The gene for toxic shock toxin is carried by a family of mobile pathogenicity islands in Staphylococcus aureus

    Mol. Microbiol.

    (1998)
  • Y.B. Lu

    Mechanistic studies of initiator–initiator interaction and replication initiation

    EMBO J.

    (1998)
  • R.P. Novick

    The phage-related chromosomal islands of Gram-positive bacteria

    Nat. Rev. Microbiol.

    (2010)
  • R.P. Novick et al.

    The SaPIs: mobile pathogenicity islands of Staphylococcus

    Chem. Immunol. Allergy

    (2007)
  • Cited by (12)

    View all citing articles on Scopus
    1

    Present address: Department of Genomics and Health, Center for Advanced Research in Public Health, Valencia 46020, Spain.

    View full text