Structure–function analysis of the SaPIbov1 replication origin in Staphylococcus aureus
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)
The plasmid status of satellite bacteriophage P4
Plasmid
(2001)Genetic systems in staphylococci
Methods Enzymol.
(1991)- 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:...
The DnaA box R4 in the minimal oriC is dispensable for initiation of Escherichia coli chromosome replication
Nucleic Acids Res.
(1995)Physical structure of the replication origin of bacteriophage lambda
Science
(1977)Initiation mechanisms in replication of filamentous phage DNA
Genes Cells
(1997)The gene for toxic shock toxin is carried by a family of mobile pathogenicity islands in Staphylococcus aureus
Mol. Microbiol.
(1998)Mechanistic studies of initiator–initiator interaction and replication initiation
EMBO J.
(1998)The phage-related chromosomal islands of Gram-positive bacteria
Nat. Rev. Microbiol.
(2010)
The SaPIs: mobile pathogenicity islands of Staphylococcus
Chem. Immunol. Allergy
Cited by (12)
Sequence determinants for DNA packaging specificity in the S. aureus pathogenicity island SaPI1
2014, PlasmidCitation Excerpt :The SaPI excision–replication–packaging cycle is induced either by phage infection or by induction of a helper prophage in a SaPI-containing strain. SaPI induction involves derepression by specific phage-encoded antirepressors (Tormo-Más et al., 2010), which leads to expression of SaPI excision and replication functions (Mir-Sanchis et al., 2012; Ubeda et al., 2007, 2008, 2012). SaPI DNA is then encapsidated in virions comprised of phage-encoded structural proteins (Tallent et al., 2007; Tormo et al., 2008).
SV40 T-antigen uses a DNA shearing mechanism to initiate origin unwinding
2022, Proceedings of the National Academy of Sciences of the United States of AmericaStaphylococcal self-loading helicases couple the staircase mechanism with inter domain high flexibility
2022, Nucleic Acids ResearchPathogenicity islands and their role in staphylococcal biology
2019, Microbiology SpectrumPathogenicity islands and their role in staphylococcal biology
2019, Gram-Positive Pathogens
- 1
Present address: Department of Genomics and Health, Center for Advanced Research in Public Health, Valencia 46020, Spain.