Non-canonical Staphylococcus aureus pathogenicity island repression.

Mobile genetic elements control their life cycles by the expression of a master repressor, whose function must be disabled to allow the spread of these elements in nature. Here, we describe an unprecedented repression-derepression mechanism involved in the transfer of Staphylococcus aureus pathogenicity islands (SaPIs). Contrary to the classical phage and SaPI repressors, which are dimers, the SaPI1 repressor StlSaPI1 presents a unique tetrameric conformation never seen before. Importantly, not just one but two tetramers are required for SaPI1 repression, which increases the novelty of the system. To derepress SaPI1, the phage-encoded protein Sri binds to and induces a conformational change in the DNA binding domains of StlSaPI1, preventing the binding of the repressor to its cognate StlSaPI1 sites. Finally, our findings demonstrate that this system is not exclusive to SaPI1 but widespread in nature. Overall, our results characterize a novel repression-induction system involved in the transfer of MGE-encoded virulence factors in nature.

Cleavage and Structural Transitions during Maturation of Staphylococcus aureus Bacteriophage 80α and SaPI1 Capsids.

In the tailed bacteriophages, DNA is packaged into spherical procapsids, leading to expansion into angular, thin-walled mature capsids. In many cases, this maturation is accompanied by cleavage of the major capsid protein (CP) and other capsid-associated proteins, including the scaffolding protein (SP) that serves as a chaperone for the assembly process. Staphylococcus aureus bacteriophage 80α is capable of high frequency mobilization of mobile genetic elements called S. aureus pathogenicity islands (SaPIs), such as SaPI1. SaPI1 redirects the assembly pathway of 80α to form capsids that are smaller than those normally made by the phage alone. Both CP and SP of 80α are N-terminally processed by a host-encoded protease, Prp. We have analyzed phage mutants that express pre-cleaved or uncleavable versions of CP or SP, and show that the N-terminal sequence in SP is absolutely required for assembly, but does not need to be cleaved in order to produce viable capsids. Mutants with pre-cleaved or uncleavable CP display normal viability. We have used cryo-EM to solve the structures of mature capsids from an 80α mutant expressing uncleavable CP, and from wildtype SaPI1. Comparisons with structures of 80α and SaPI1 procapsids show that capsid maturation involves major conformational changes in CP, consistent with a release of the CP N-arm by SP. The hexamers reorganize during maturation to accommodate the different environments in the 80α and SaPI1 capsids.

Competing scaffolding proteins determine capsid size during mobilization of Staphylococcus aureus pathogenicity islands.

Staphylococcus aureus pathogenicity islands (SaPIs), such as SaPI1, exploit specific helper bacteriophages, like 80α, for their high frequency mobilization, a process termed 'molecular piracy'. SaPI1 redirects the helper's assembly pathway to form small capsids that can only accommodate the smaller SaPI1 genome, but not a complete phage genome. SaPI1 encodes two proteins, CpmA and CpmB, that are responsible for this size redirection. We have determined the structures of the 80α and SaPI1 procapsids to near-atomic resolution by cryo-electron microscopy, and show that CpmB competes with the 80α scaffolding protein (SP) for a binding site on the capsid protein (CP), and works by altering the angle between capsomers. We probed these interactions genetically and identified second-site suppressors of lethal mutations in SP. Our structures show, for the first time, the detailed interactions between SP and CP in a bacteriophage, providing unique insights into macromolecular assembly processes.

Derepression of SaPIbov1 Is Independent of φNM1 Type 2 dUTPase Activity and Is Inhibited by dUTP and dUMP.

Staphylococcus aureus is an opportunistic human pathogen able to transfer virulence genes to other cells through the mobilization of S. aureus pathogenicity islands (SaPIs). SaPIs are derepressed and packaged into phage-like transducing particles by helper phages like 80α or φNM1. Phages 80α and φNM1 encode structurally distinct dUTPases, Dut80α (type 1) and DutNM1 (type 2). Both dUTPases can interact with the SaPIbov1 Stl master repressor, leading to derepression and mobilization. That two structurally distinct dUTPases bind the same repressor led us to speculate that dUTPase activity may be important to the derepression process. In type 1 dUTPases, Stl binding is inhibited by dUTP. The purpose of this study was to assess the involvement of dUTP binding and dUTPase activity in derepression by DutNM1. DutNM1 activity mutants were created and tested for dUTPase activity using a novel NMR-based assay. We found that all DutNM1 null activity mutants interacted with the SaPIbov1 Stl C-terminal domain, formed DutNM1-Stl heterodimers, and caused the release of the Pstr promoter. However, promoter release was inhibited in the presence of dUTP or dUMP. We tested two φNM1 mutant phages that had null enzyme activity and found that they could still mobilize SaPIbov1. These results show that only the apo form of DutNM1 is active in Stl derepression and that dUTPase activity is not necessary for the mobilization of SaPIbov1 by DutNM1.

Structural modeling and functional analysis of the essential ribosomal processing protease Prp from Staphylococcus aureus.

In Firmicutes and related bacteria, ribosomal large subunit protein L27 is encoded with a conserved N-terminal extension that is removed to expose residues critical for ribosome function. Bacteria encoding L27 with this N-terminal extension also encode a sequence-specific cysteine protease, Prp, which carries out this cleavage. In this work, we demonstrate that L27 variants with an un-cleavable N-terminal extension, or lacking the extension (pre-cleaved), are unable to complement an L27 deletion in Staphylococcus aureus. This indicates that N-terminal processing of L27 is not only essential but possibly has a regulatory role. Prp represents a new clade of previously uncharacterized cysteine proteases, and the dependence of S. aureus on L27 cleavage by Prp validates the enzyme as a target for potential antibiotic development. To better understand the mechanism of Prp activity, we analyzed Prp enzyme kinetics and substrate preference using a fluorogenic peptide cleavage assay. Molecular modeling and site-directed mutagenesis implicate several residues around the active site in catalysis and substrate binding, and support a structural model in which rearrangement of a flexible loop upon binding of the correct peptide substrate is required for the active site to assume the proper conformation. These findings lay the foundation for the development of antimicrobials that target this novel, essential pathway.

Bacteriophage P2.

P2 is the original member of a highly successful family of temperate phages that are frequently found in the genomes of gram-negative bacteria. This article focuses on the organization of the P2 genome and reviews current knowledge about the function of each open reading frame.

The Phage-Inducible Chromosomal Islands: A Family of Highly Evolved Molecular Parasites.

The phage-inducible chromosomal islands (PICIs) are a family of highly mobile genetic elements that contribute substantively to horizontal gene transfer, host adaptation, and virulence. Initially identified in Staphylococcus aureus, these elements are now thought to occur widely in gram-positive bacteria. They are molecular parasites that exploit certain temperate phages as helpers, using a variety of elegant strategies to manipulate the phage life cycle and promote their own spread, both intra- and intergenerically. At the same time, these PICI-encoded mechanisms severely interfere with helper phage reproduction, thereby enhancing survival of the bacterial population. In this review we discuss the genetics and the life cycle of these elements, with special emphasis on how they interact and interfere with the helper phage machinery for their own benefit. We also analyze the role that these elements play in driving bacterial and viral evolution.

Specific N-terminal cleavage of ribosomal protein L27 in Staphylococcus aureus and related bacteria.

Ribosomal protein L27 is a component of the eubacterial large ribosomal subunit that has been shown to play a critical role in substrate stabilization during protein synthesis. This function is mediated by the L27 N-terminus, which protrudes into the peptidyl transferase center. In this report, we demonstrate that L27 in Staphylococcus aureus and other Firmicutes is encoded with an N-terminal extension that is not present in most Gram-negative organisms and is absent from mature ribosomes. We have identified a cysteine protease, conserved among bacteria containing the L27 N-terminal extension, which performs post-translational cleavage of L27. Ribosomal biology in eubacteria has largely been studied in the Gram-negative bacterium Escherichia coli; our findings indicate that there are aspects of the basic biology of the ribosome in S. aureus and other related bacteria that differ substantially from that of the E. coli ribosome. This research lays the foundation for the development of new therapeutic approaches that target this novel pathway.

Sequence determinants for DNA packaging specificity in the S. aureus pathogenicity island SaPI1.

The SaPIs and their relatives are a family of genomic islands that exploit helper phages for high frequency horizontal transfer. One of the mechanisms used by SaPIs to accomplish this molecular piracy is the redirection of the helper phage DNA packaging machinery. SaPIs encode a small terminase subunit that can be substituted for that of the phage. In this study we have determined the initial packaging cleavage sites for helper phage 80α, which uses the phage-encoded small terminase subunit, and for SaPI1, which uses the SaPI-encoded small terminase subunit. We have identified a 19nt SaPI1 sequence that is necessary and sufficient to allow high frequency 80α transduction of a plasmid by a terminase carrying the SaPI1-encoded small subunit. We also show that the hybrid enzyme with the SaPI1 small terminase subunit is capable of generalized transduction.

Genomic characterization of six novel Bacillus pumilus bacteriophages.

Twenty-eight bacteriophages infecting the local host Bacillus pumilus BL-8 were isolated, purified, and characterized. Nine genomes were sequenced, of which six were annotated and are the first of this host submitted to the public record. The 28 phages were divided into two groups by sequence and morphological similarity, yielding 27 cluster BpA phages and 1 cluster BpB phage, which is a BL-8 prophage. Most of the BpA phages have a host range restricted to distantly related strains, B. pumilus and B. simplex, reflecting the complexities of Bacillus taxonomy. Despite isolation over wide geographic and temporal space, the six cluster BpA phages share most of their 23 functionally annotated protein features and show a high degree of sequence similarity, which is unique among phages of the Bacillus genera. This is the first report of B. pumilus phages since 1981.

A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria.

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.

Pirates of the Caudovirales.

Molecular piracy is a biological phenomenon in which one replicon (the pirate) uses the structural proteins encoded by another replicon (the helper) to package its own genome and thus allow its propagation and spread. Such piracy is dependent on a complex web of interactions between the helper and the pirate that occur at several levels, from transcriptional control to macromolecular assembly. The best characterized examples of molecular piracy are from the E. coli P2/P4 system and the S. aureus SaPI pathogenicity island/helper system. In both of these cases, the pirate element is mobilized and packaged into phage-like transducing particles assembled from proteins supplied by a helper phage that belongs to the Caudovirales order of viruses (tailed, dsDNA bacteriophages). In this review we will summarize and compare the processes that are involved in molecular piracy in these two systems.

Staphylococcal pathogenicity island interference with helper phage reproduction is a paradigm of molecular parasitism.

Staphylococcal pathogenicity islands (SaPIs) carry superantigen and resistance genes and are extremely widespread in Staphylococcus aureus and in other Gram-positive bacteria. SaPIs represent a major source of intrageneric horizontal gene transfer and a stealth conduit for intergeneric gene transfer; they are phage satellites that exploit the life cycle of their temperate helper phages with elegant precision to enable their rapid replication and promiscuous spread. SaPIs also interfere with helper phage reproduction, blocking plaque formation, sharply reducing burst size and enhancing the survival of host cells following phage infection. Here, we show that SaPIs use several different strategies for phage interference, presumably the result of convergent evolution. One strategy, not described previously in the bacteriophage microcosm, involves a SaPI-encoded protein that directly and specifically interferes with phage DNA packaging by blocking the phage terminase small subunit. Another strategy involves interference with phage reproduction by diversion of the vast majority of virion proteins to the formation of SaPI-specific small infectious particles. Several SaPIs use both of these strategies, and at least one uses neither but possesses a third. Our studies illuminate a key feature of the evolutionary strategy of these mobile genetic elements, in addition to their carriage of important genes-interference with helper phage reproduction, which could ensure their transferability and long-term persistence.

Assembly of bacteriophage 80α capsids in a Staphylococcus aureus expression system.

80α is a temperate, double-stranded DNA bacteriophage of Staphylococcus aureus that can act as a "helper" for the mobilization of S. aureus pathogenicity islands (SaPIs), including SaPI1. When SaPI1 is mobilized by 80α, the SaPI genomes are packaged into capsids that are composed of phage proteins, but that are smaller than those normally formed by the phage. This size determination is dependent on SaPI1 proteins CpmA and CpmB. Here, we show that co-expression of the 80α capsid and scaffolding proteins in S. aureus, but not in E. coli, leads to the formation of procapsid-related structures, suggesting that a host co-factor is required for assembly. The capsid and scaffolding proteins also undergo normal N-terminal processing upon expression in S. aureus, implicating a host protease. We also find that SaPI1 proteins CpmA and CpmB promote the formation of small capsids upon co-expression with 80α capsid and scaffolding proteins in S. aureus.

The roles of SaPI1 proteins gp7 (CpmA) and gp6 (CpmB) in capsid size determination and helper phage interference.

SaPIs are molecular pirates that exploit helper bacteriophages for their own high frequency mobilization. One striking feature of helper exploitation by SaPIs is redirection of the phage capsid assembly pathway to produce smaller phage-like particles with T=4 icosahedral symmetry rather than T=7 bacteriophage capsids. Small capsids can accommodate the SaPI genome but not that of the helper phage, leading to interference with helper propagation. Previous studies identified two proteins encoded by the prototype element SaPI1, gp6 and gp7, in SaPI1 procapsids but not in mature SaPI1 particles. Dimers of gp6 form an internal scaffold, aiding fidelity of small capsid assembly. Here we show that both SaPI1 gp6 (CpmB) and gp7 (CpmA) are necessary and sufficient to direct small capsid formation. Surprisingly, failure to form small capsids did not restore wild-type levels of helper phage growth, suggesting an additional role for these SaPI1 proteins in phage interference.

The Staphylococcus aureus pathogenicity island 1 protein gp6 functions as an internal scaffold during capsid size determination.

Staphylococcus aureus pathogenicity island 1 (SaPI1) is a mobile genetic element that carries genes for several superantigen toxins. SaPI1 is normally stably integrated into the host genome but can become mobilized by "helper" bacteriophage 80α, leading to the packaging of SaPI1 genomes into phage-like transducing particles that are composed of structural proteins supplied by the helper phage but having smaller capsids. We show that the SaPI1-encoded protein gp6 is necessary for efficient formation of small capsids. The NMR structure of gp6 reveals a dimeric protein with a helix-loop-helix motif similar to that of bacteriophage scaffolding proteins. The gp6 dimer matches internal densities that bridge capsid subunits in cryo-electron microscopy reconstructions of SaPI1 procapsids, suggesting that gp6 acts as an internal scaffolding protein in capsid size determination.

RinA controls phage-mediated packaging and transfer of virulence genes in Gram-positive bacteria.

Phage-mediated transfer of microbial genetic elements plays a crucial role in bacterial life style and evolution. In this study, we identify the RinA family of phage-encoded proteins as activators required for transcription of the late operon in a large group of temperate staphylococcal phages. RinA binds to a tightly regulated promoter region, situated upstream of the terS gene, that controls expression of the morphogenetic and lysis modules of the phage, activating their transcription. As expected, rinA deletion eliminated formation of functional phage particles and significantly decreased the transfer of phage and pathogenicity island encoded virulence factors. A genetic analysis of the late promoter region showed that a fragment of 272 bp contains both the promoter and the region necessary for activation by RinA. In addition, we demonstrated that RinA is the only phage-encoded protein required for the activation of this promoter region. This region was shown to be divergent among different phages. Consequently, phages with divergent promoter regions carried allelic variants of the RinA protein, which specifically recognize its own promoter sequence. Finally, most Gram-postive bacteria carry bacteriophages encoding RinA homologue proteins. Characterization of several of these proteins demonstrated that control by RinA of the phage-mediated packaging and transfer of virulence factor is a conserved mechanism regulating horizontal gene transfer.

A conformational switch involved in maturation of Staphylococcus aureus bacteriophage 80α capsids.

Bacteriophages are involved in many aspects of the spread and establishment of virulence factors in Staphylococcus aureus, including the mobilization of genetic elements known as S. aureus pathogenicity islands (SaPIs), which carry genes for superantigen toxins and other virulence factors. SaPIs are packaged into phage-like transducing particles using proteins supplied by the helper phage. We have used cryo-electron microscopy and icosahedral reconstruction to determine the structures of the procapsid and the mature capsid of 80α, a bacteriophage that can mobilize several different SaPIs. The 80α capsid has T=7 icosahedral symmetry with the capsid protein organized into pentameric and hexameric clusters that interact via prominent trimeric densities. The 80α capsid protein was modeled based on the capsid protein fold of bacteriophage HK97 and fitted into the 80α reconstructions. The models show that the trivalent interactions are mediated primarily by a 22-residue ß hairpin structure called the P loop that is not found in HK97. Capsid expansion is associated with a conformational switch in the spine helix that is propagated throughout the subunit, unlike the domain rotation mechanism in phage HK97 or P22.

The phage-related chromosomal islands of Gram-positive bacteria.

The phage-related chromosomal islands (PRCIs) were first identified in Staphylococcus aureus as highly mobile, superantigen-encoding genetic elements known as the S. aureus pathogenicity islands (SaPIs). These elements are characterized by a specific set of phage-related functions that enable them to use the phage reproduction cycle for their own transduction and inhibit phage reproduction in the process. SaPIs produce many phage-like infectious particles; their streptococcal counterparts have a role in gene regulation but may not be infectious. These elements therefore represent phage satellites or parasites, not defective phages. In this Review, we discuss the shared genetic content of PRCIs, their life cycle and their ability to be transferred across large phylogenetic distances.

Moonlighting bacteriophage proteins derepress staphylococcal pathogenicity islands.

Staphylococcal superantigen-carrying pathogenicity islands (SaPIs) are discrete, chromosomally integrated units of approximately 15 kilobases that are induced by helper phages to excise and replicate. SaPI DNA is then efficiently encapsidated in phage-like infectious particles, leading to extremely high frequencies of intra- as well as intergeneric transfer. In the absence of helper phage lytic growth, the island is maintained in a quiescent prophage-like state by a global repressor, Stl, which controls expression of most of the SaPI genes. Here we show that SaPI derepression is effected by a specific, non-essential phage protein that binds to Stl, disrupting the Stl-DNA complex and thereby initiating the excision-replication-packaging cycle of the island. Because SaPIs require phage proteins to be packaged, this strategy assures that SaPIs will be transferred once induced. Several different SaPIs are induced by helper phage 80alpha and, in each case, the SaPI commandeers a different non-essential phage protein for its derepression. The highly specific interactions between different SaPI repressors and helper-phage-encoded antirepressors represent a remarkable evolutionary adaptation involved in pathogenicity island mobilization.