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Staphylococcal pathogenicity islands (SaPIs) are approximately 15 kb chromosomally located mobile elements that parasitize "helper" phages which provide a de-repressor protein plus virion and lysis proteins which enable the release of infectious SaPI particles in very high titers. All SaPIs interfere with the reproduction of their helper phages, using 3 different mechanisms. The logic of SaPI reproduction requires that these interference mechanisms do not totally block phage production, as this would be lethal for them as well as for the phage. The discovery of 2 SaPI2 proteins that totally block phage 80 by interfering with late phage transcription was inconsistent with this principle and led to the discovery of a third protein that binds to one of the interference proteins and modulates its activity, thus preventing complete inhibition of the phage. These systems permit the SaPIs to engage in horizontal transfer of unlinked chromosomal genes as well as their own.

Bacteriophages are types of viruses that infect bacteria. They are the most abundant and diverse entities in the biosphere, and influence the evolution of most bacterial species by promoting gene transfer, sometimes in unexpected ways. Although pac-type phages can randomly package and transfer bacterial DNA by a process called generalized transduction, some mobile genetic elements have developed elegant and sophisticated strategies to hijack the phage DNA-packaging machinery for their own transfer. Moreover, phage-like particles (gene transfer agents) have also evolved, that can package random pieces of the producing cell's genome. The purpose of this review is to give an overview of some of the various ways by which phages and phage-like particles can transfer bacterial genes, driving bacterial evolution and promoting the emergence of novel pathogens.

In recent decades, the notorious pathogen Staphylococcus aureus has become progressively more contagious, more virulent, and more resistant to antibiotics. This implies a rather dynamic evolutionary capability, representing a remarkable level of genomic plasticity, most probably maintained by horizontal gene transfer. Here we report that the staphylococcal pathogenicity islands have a dual role in gene transfer: they not only mediate their own transfer, but they can independently direct the transfer of unlinked chromosomal segments containing virulence genes. While transfer of the island itself requires specific helper phages, transfer of unlinked chromosomal segments does not, so potentially any pac-type phage will serve. These results reveal that SaPIs can increase the horizontal exchange of accessory genes associated with disease and may shape pathogen genomes beyond the confines of their attachment sites.

We have previously reported the construction of Staphylococcus aureus integration vectors based on the staphylococcal pathogenicity island 1 (SaPI1) site-specific recombination system. These are shuttle vectors that can be propagated in Escherichia coli, which allows for standard DNA manipulations. In S. aureus, these vectors are temperature-sensitive and can only be maintained at non-permissive (42 degrees C) temperatures by integrating into the chromosome. However, most S. aureus strains are sensitive to prolonged incubations at higher temperatures and will rapidly accumulate mutations, making the use of temperature-sensitive integration vectors impractical for single-copy applications. Here we describe improved versions of these vectors, which are maintained only in single-copy at the SaPI1 attachment site. In addition, we introduce several additional cassettes containing resistance markers, expanding the versatility of integrant selection, especially in strains that are resistant to multiple antibiotics.

Having gone to great evolutionary lengths to develop resistance to bacteriophages, bacteria have come up with resistance mechanisms directed at every aspect of the bacteriophage life cycle. Most genes involved in phage resistance are carried by plasmids and other mobile genetic elements, including bacteriophages and their relatives. A very special case of phage resistance is exhibited by the highly mobile phage satellites, staphylococcal pathogenicity islands (SaPIs), which carry and disseminate superantigen and other virulence genes. Unlike the usual phage-resistance mechanisms, the SaPI-encoded interference mechanisms are carefully crafted to ensure that a phage-infected, SaPI-containing cell will lyse, releasing the requisite crop of SaPI particles as well as a greatly diminished crop of phage particles. Previously described SaPI interference genes target phage functions that are not required for SaPI particle production and release. Here we describe a SaPI-mediated interference system that affects expression of late phage gene transcription and consequently is required for SaPI and phage. Although when cloned separately, a single SaPI gene totally blocks phage production, its activity in situ is modulated accurately by a second gene, achieving the required level of interference. The advantage for the host bacteria is that the SaPIs curb excessive phage growth while enhancing their gene transfer activity. This activity is in contrast to that of the clustered regularly interspaced short palindromic repeats (CRISPRs), which totally block phage growth at the cost of phage-mediated gene transfer. In staphylococci the SaPI strategy seems to have prevailed during evolution: The great majority of Staphylococcus aureus strains carry one or more SaPIs, whereas CRISPRs are extremely rare.

Staphylococcal pathogenicity islands (SaPIs) are the prototypical members of a widespread family of chromosomally located mobile genetic elements that contribute substantially to intra- and interspecies gene transfer, host adaptation, and virulence. The key feature of their mobility is the induction of SaPI excision and replication by certain helper phages and their efficient encapsidation into phage-like infectious particles. Most SaPIs use the headful packaging mechanism and encode small terminase subunit (TerS) homologs that recognize the SaPI-specific pac site and determine SaPI packaging specificity. Several of the known SaPIs do not encode a recognizable TerS homolog but are nevertheless packaged efficiently by helper phages and transferred at high frequencies. In this report, we have characterized one of the non-terS-coding SaPIs, SaPIbov5, and found that it uses two different, undescribed packaging strategies. SaPIbov5 is packaged in full-sized phage-like particles either by typical pac-type helper phages, or by cos-type phages-i.e., it has both pac and cos sites-a configuration that has not hitherto been described for any mobile element, phages included-and uses the two different phage-coded TerSs. To our knowledge, this is the first example of SaPI packaging by a cos phage, and in this, it resembles the P4 plasmid of Escherichia coli. Cos-site packaging in Staphylococcus aureus is additionally unique in that it requires the HNH nuclease, carried only by cos phages, in addition to the large terminase subunit, for cos-site cleavage and melting.

Staphylococcus aureus virulence is regulated when secreted autoinducing peptides (AIPs) are recognized by a membrane-bound receptor histidine kinase (RHK), AgrC. Some AIPs are agonists of virulence gene expression, while others are antagonists. It is unclear how AIP binding regulates AgrC activity. Here, we reconstitute an AgrC family member, AgrC-I, using nanometer-scale lipid bilayer discs. We show that AgrC-I requires membranes rich in anionic lipids to function. The agonist, AIP-I, binds AgrC-I noncooperatively in a 2:2 stoichiometry, while an antagonist ligand, AIP-II, functions as an inverse agonist of the kinase activity. We also demonstrate the kinase and sensor domains in AgrC are connected by a helical linker whose conformational state exercises rheostat-like control over the kinase activity. Binding of agonist or inverse-agonist peptides results in twisting of the linker in different directions. These two observations provide a view of the molecular motions triggered by ligand binding in an intact membrane-bound RHK.

Methicillin-resistant Staphylococcus aureus (MRSA) continues to be a serious human pathogen, and particularly the spread of community associated (CA)-MRSA strains such as USA300 is a concern, as these strains can cause severe infections in otherwise healthy adults. Recently, we reported that a cyclodepsipeptide termed Solonamide B isolated from the marine bacterium, Photobacterium halotolerans strongly reduces expression of RNAIII, the effector molecule of the agr quorum sensing system. Here we show that Solonamide B interferes with the binding of S. aureus autoinducing peptides (AIPs) to sensor histidine kinase, AgrC, of the agr two-component system. The hypervirulence of USA300 has been linked to increased expression of central virulence factors like alpha-hemolysin and the phenol soluble modulins (PSMs). Importantly, in strain USA300 Solonamide B dramatically reduced the activity of alpha-hemolysin and the transcription of psma encoding PSMs with an 80% reduction in toxicity of supernatants towards human neutrophils and rabbit erythrocytes. To our knowledge this is the first report of a compound produced naturally by a Gram-negative marine bacterium that interferes with agr and affects both RNAIII and AgrA controlled virulence gene expression in S. aureus.

The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria.