Virus satellites drive viral evolution and ecology

Virus satellites are widespread subcellular entities, present both in eukaryotic and in prokaryotic cells. Their modus vivendi involves parasitism of the life cycle of their inducing helper viruses, which assures their transmission to a new host. However, the evolutionary and ecological implications of satellites on helper viruses remain unclear. Here, using staphylococcal pathogenicity islands (SaPIs) as a model of virus satellites, we experimentally show that helper viruses rapidly evolve resistance to their virus satellites, preventing SaPI proliferation, and SaPIs in turn can readily evolve to overcome phage resistance. Genomic analyses of both these experimentally evolved strains as well as naturally occurring bacteriophages suggest that the SaPIs drive the coexistence of multiple alleles of the phage-coded SaPI inducing genes, as well as sometimes selecting for the absence of the SaPI depressing genes. We report similar (accidental) evolution of resistance to SaPIs in laboratory phages used for Staphylococcus aureus typing and also obtain the same qualitative results in both experimental evolution and phylogenetic studies of Enterococcus faecalis phages and their satellites viruses. In summary, our results suggest that helper and satellite viruses undergo rapid coevolution, which is likely to play a key role in the evolution and ecology of the viruses as well as their prokaryotic hosts

Staphylococcal self-loading helicases couple the staircase mechanism with inter domain high flexibility

Replication is a crucial cellular process. Replicative helicases unwind DNA providing the template strand to the polymerase and promoting replication fork progression. Helicases are multi-domain proteins which use an ATPase domain to couple ATP hydrolysis with translocation, however the role that the other domains might have during translocation remains elusive. Here, we studied the unexplored self-loading helicases called Reps, present in Staphylococcus aureus pathogenicity islands (SaPIs). Our cryoEM structures of the PriRep5 from SaPI5 (3.3 Å), the Rep1 from SaPI1 (3.9 Å) and Rep1-DNA complex (3.1Å) showed that in both Reps, the C-terminal domain (CTD) undergoes two distinct movements respect the ATPase domain. We experimentally demonstrate both in vitro and in vivo that SaPI-encoded Reps need key amino acids involved in the staircase mechanism of translocation. Additionally, we demonstrate that the CTD's presence is necessary for the maintenance of full ATPase and helicase activities. We speculate that this high interdomain flexibility couples Rep's activities as initiators and as helicases

A novel dna primase-helicase pair encoded by SCC<i>mec</i> elements

Mobile genetic elements (MGEs) are a rich source of new enzymes, and conversely, understanding the activities of MGE-encoded proteins can elucidate MGE function. Here we biochemically characterize 3 proteins encoded by a conserved operon carried by the Staphylococcal Cassette Chromosome (SCCmec), an MGE that confers methicillin resistance to Staphylococcus aureus, creating MRSA strains. The first of these proteins, CCPol, is an active A-family DNA polymerase. The middle protein, MP, binds tightly to CCPol and confers upon it the ability to synthesize DNA primers de novo. The CCPol-MP complex is therefore a unique primase-polymerase enzyme unrelated to either known primase family. The third protein, Cch2, is a 3’-to-5’ helicase. Cch2 additionally binds specifically to a dsDNA sequence downstream of its gene that is also a preferred initiation site for priming by CCPol MP. Taken together, our results suggest that this is a functional replication module for SCCmec

A novel DNA primase-helicase pair encoded by SCCmec elements

Mobile genetic elements (MGEs) are a rich source of new enzymes, and conversely, understanding the activities of MGE-encoded proteins can elucidate MGE function. Here, we biochemically characterize three proteins encoded by a conserved operon carried by the Staphylococcal Cassette Chromosome (SCCmec), an MGE that confers methicillin resistance to Staphylococcus aureus, creating MRSA strains. The first of these proteins, CCPol, is an active A-family DNA polymerase. The middle protein, MP, binds tightly to CCPol and confers upon it the ability to synthesize DNA primers de novo. The CCPol-MP complex is therefore a unique primase-polymerase enzyme unrelated to either known primase family. The third protein, Cch2, is a 3'-to-5' helicase. Cch2 additionally binds specifically to a dsDNA sequence downstream of its gene that is also a preferred initiation site for priming by CCPol-MP. Taken together, our results suggest that this is a functional replication module for SCCmec

Virus Satellites Drive Viral Evolution and Ecology

19 páginas, 4 figuras, 4 tablasVirus satellites are widespread subcellular entities, present both in eukaryotic and in prokaryotic cells. Their modus vivendi involves parasitism of the life cycle of their inducing helper viruses, which assures their transmission to a new host. However, the evolutionary and ecological implications of satellites on helper viruses remain unclear. Here, using staphylococcal pathogenicity islands (SaPIs) as a model of virus satellites, we experimentally show that helper viruses rapidly evolve resistance to their virus satellites, preventing SaPI proliferation, and SaPIs in turn can readily evolve to overcome phage resistance. Genomic analyses of both these experimentally evolved strains as well as naturally occurring bacteriophages suggest that the SaPIs drive the coexistence of multiple alleles of the phage-coded SaPI inducing genes, as well as sometimes selecting for the absence of the SaPI depressing genes. We report similar (accidental) evolution of resistance to SaPIs in laboratory phages used for Staphylococcus aureus typing and also obtain the same qualitative results in both experimental evolution and phylogenetic studies of Enterococcus faecalis phages and their satellites viruses. In summary, our results suggest that helper and satellite viruses undergo rapid coevolution, which is likely to play a key role in the evolution and ecology of the viruses as well as their prokaryotic hostsThis work was supported by grants Consolider-Ingenio CSD2009-00006, BIO2011- 30503-C02-01, ERAnet-Pathogenomics PIM2010EPA-00606 and MR/M003876/1 from the Medical Research Council (UK) to JRP, BIO2010-15424 to AM, and BFU2012-30805 to SFE, all from the Spanish Ministerio de Economía y Competitividad (MINECO), and grant R01AI022159 to RPN and JRP, from the USA National Institutes of Health. AB acknowledges support from the Royal Society, NERC, BBSRC and AXA research fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscriptPeer reviewe

Sak and Sak4 recombinases are required for bacteriophage replication in Staphylococcus aureus

Medical Research Council (UK) [MR/M003876/1]; Biotechnology and Biological Sciences Research Council (BBSRC, UK) [BB/N002873/1]; from European Union [ERC-ADG-2014 Proposal n° 670932 Dut-signal to J.R.P.]; MINECO (Spain) [BFU2012-39879-C02-02, BFU2015-67065-P to S.A.]; MINECO (Spain) [BIO2013-42619-P]; Valencian Government [Prometeo II/2014/029 to A.M.]. Funding for open access charge: Medical Research Council (UK).DNA-single strand annealing proteins (SSAPs) are recombinases frequently encoded in the genome of many bacteriophages. As SSAPs can promote homologous recombination among DNA substrates with an important degree of divergence, these enzymes are involved both in DNA repair and in the generation of phage mosaicisms. Here, analysing Sak and Sak4 as representatives of two different families of SSAPs present in phages infecting the clinically relevant bacterium Staphylococcus aureus, we demonstrate for the first time that these enzymes are absolutely required for phage reproduction. Deletion of the genes encoding these enzymes significantly reduced phage replication and the generation of infectious particles. Complementation studies revealed that these enzymes are required both in the donor (after prophage induction) and in the recipient strain (for infection). Moreover, our results indicated that to perform their function SSAPs require the activity of their cognate single strand binding (Ssb) proteins. Mutational studies demonstrated that the Ssb proteins are also required for phage replication, both in the donor and recipient strain. In summary, our results expand the functions attributed to the Sak and Sak4 proteins, and demonstrate that both SSAPs and Ssb proteins are essential for the life cycle of temperate staphylococcal phages.Publisher PDFPeer reviewe

Pirating conserved phage mechanisms promotes promiscuous staphylococcal pathogenicity island transfer

Targeting conserved and essential processes is a successful strategy to combat enemies. Remarkably, the clinically important Staphylococcus aureus pathogenicity islands (SaPIs) use this tactic to spread in nature. SaPIs reside passively in the host chromosome, under the control of the SaPI-encoded master repressor, Stl. It has been assumed that SaPI de-repression is effected by specific phage proteins that bind to Stl, initiating the SaPI cycle. Different SaPIs encode different Stl repressors, so each targets a specific phage protein for its de-repression. Broadening this narrow vision, we report here that SaPIs ensure their promiscuous transfer by targeting conserved phage mechanisms. This is accomplished because the SaPI Stl repressors have acquired different domains to interact with unrelated proteins, encoded by different phages, but in all cases performing the same conserved function. This elegant strategy allows intra-and inter-generic SaPI transfer, highlighting these elements as one of nature’s most fascinating subcellular parasites

The phage-coded SaPI inducer proteins are under purifying selection^a.

<p>^aTable shows the statistical analysis of the <i>d</i>_<i>N</i><i>−d</i>_<i>S</i> values obtained comparing the SaPI inducing proteins analysed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005609#pgen.1005609.s006" target="_blank">S1 Table</a>.</p><p>^bRepresents the overall mean distance of the values shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005609#pgen.1005609.s006" target="_blank">S1 Table</a> (± standard error).</p><p>^c<i>z</i> = mean distance / standard error.</p><p>^dStatistical significance of the null hypothesis H₀: <i>d</i>_<i>N</i><i>−d</i>_<i>S</i> = 0.</p><p>The phage-coded SaPI inducer proteins are under purifying selection<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005609#t001fn001" target="_blank">^a</a>.</p