Capsid size determination by Staphylococcus aureus pathogenicity island SaPI1 involves specific incorporation of SaPI1 proteins into procapsids

The Staphylococcus aureus pathogenicity island SaPI1 carries the gene for the toxic shock syndrome toxin (TSST-1) and can be mobilized by infection with S. aureus helper phage 80α. SaPI1 depends on the helper phage for excision, replication and genome packaging. The SaPI1-transducing particles comprise proteins encoded by the helper phage, but have a smaller capsid commensurate with the smaller size of the SaPI1 genome. Previous studies identified only 80α-encoded proteins in mature SaPI1 virions, implying that the presumptive SaPI1 capsid size determination function(s) must act transiently during capsid assembly or maturation. In this study, 80α and SaPI1 procapsids were produced by induction of phage mutants lacking functional 80α or SaPI1 small terminase subunits. By cryo-electron microscopy, these procapsids were found to have a round shape and an internal scaffolding core. Mass spectrometry was used to identify all 80α-encoded structural proteins in 80α and SaPI1 procapsids, including several that had not previously been found in the mature capsids. In addition, SaPI1 procapsids contained at least one SaPI1-encoded protein that has been implicated genetically in capsid size determination. Mass spectrometry on full-length phage proteins showed that the major capsid protein and the scaffolding protein are N-terminally processed in both 80α and SaPI1 procapsids.     

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.     

Molecular genetics of SaPI1--a mobile pathogenicity island in Staphylococcus aureus

The Staphylococcus aureus gene for toxic shock toxin (tst) is carried by a 15 kb mobile pathogenicity island, SaPI1, that has an intimate relationship with temperate staphylococcal phage 80alpha. During phage growth, SaPI1 is excised from its unique chromosomal site, attC, replicates autonomously, interferes with phage growth, and is efficiently encapsidated into special small phage heads commensurate with its size. Upon transfer to a recipient organism, SaPI1 integrates at attC by means of a self-coded integrase. One or more phage functions are required for excision, autonomous replication and encapsidation of the element and, thus, the overall relationship between SaPI1 and 80alpha is similar to that between coliphages P4 and P2. Among other staphylococcal phages tested, only phi13 interacts with SaPI1, inducing excision but not replication or transfer of the element.     

Specificity of staphylococcal phage and SaPI DNA packaging as revealed by integrase and terminase mutations

SaPI1 and SaPIbov1 are chromosomal pathogenicity islands in Staphylococcus aureus that carry tst and other superantigen genes. They are induced to excise and replicate by certain phages, are efficiently encapsidated in SaPI-specific small particles composed of phage virion proteins and are transferred at very high frequencies. In this study, we have analysed three SaPI genes that are important for the phage–SaPI interaction, int (integrase) terS (phage terminase small subunit homologue) and pif (phage interference function). SaPI1 int is required for SaPI excision, replication and packaging in a donor strain, and is required for integration in a recipient. A SaPI1 int mutant, following phage induction, produces small SaPI-specific capsids which are filled with partial phage genomes. SaPIbov1 DNA is efficiently packaged into full-sized phage heads as well as into SaPI-specific small ones, whereas SaPI1 DNA is found almost exclusively in the small capsids. TerS, however, determines DNA packaging specificity but not the choice of large versus small capsids. This choice is influenced by SaPIbov1 gene 12, which prevents phage DNA packaging into small capsids, and which is also primarily responsible for interference by SaPIbov1 with phage reproduction.     

Highly potent dUTPase inhibition by a bacterial repressor protein reveals a novel mechanism for gene expression control

Transfer of phage-related pathogenicity islands of Staphylococcus aureus (SaPI-s) was recently reported to be activated by helper phage dUTPases. This is a novel function for dUTPases otherwise involved in preservation of genomic integrity by sanitizing the dNTP pool. Here we investigated the molecular mechanism of the dUTPase-induced gene expression control using direct techniques. The expression of SaPI transfer initiating proteins is repressed by proteins called Stl. We found that Φ11 helper phage dUTPase eliminates SaPIbov1 Stl binding to its cognate DNA by binding tightly to Stl protein. We also show that dUTPase enzymatic activity is strongly inhibited in the dUTPase:Stl complex and that the dUTPase:dUTP complex is inaccessible to the Stl repressor. Our results disprove the previously proposed G-protein-like mechanism of SaPI transfer activation. We propose that the transfer only occurs if dUTP is cleared from the nucleotide pool, a condition promoting genomic stability of the virulence elements.     

Genetic transformation in Staphylococcus aureus: isolation and characterization of a competence-conferring factor from bacteriophage 80 alpha lysates.

The competence-conferring activity in crude lysates of the staphylococcal bacteriophage 80 alpha was concentrated and purified by (NH4)2SO4 precipitation, differential ultracentrifugation and rate-zonal centrifugation through Ficoll. This concentrated preparation exhibited lytic activity toward assay cells of Staphylococcus aureus 8325-4 that could not be attributed to the residual 80 alpha infectious particles present. Electron microscopic examination of the concentrated competence-conferring activity revealed an occasional intact but empty virion and large numbers of free phage tails. Sodium dodecyl sulfate-polyacrylamide gel analysis of this material confirmed that the competence-conferring activity contained only some, but not all, of the major virion proteins. The competence-conferring activity exhibited single-hit kinetics when assay cells and 80 alpha transfecting deoxyribonucleic acid were present in excess. The competence-conferring activity thus seems to be a unique morphogenic precursor of the 80 alpha virion that mediates transfection and transformation in the presence of 0.1 M CaCl2.     

Transformation in Staphylococcus aureus: role of bacteriophage and incidence of competence among strains.

When used in a helper phage capacity, phages 29, 52, 52A, 79, 80, 55, 71, 53, 83A, 85, 95, 96, phi11, and 80 alpha, all serological group B Staphylococcus phages, conferred competence for transformation to S. aureus 8325-4, a strain that does not normally become competent. Of the serological group A phages tested, only phage 3A showed significant competence-conferring activity. Phages 29, 55, 53, 83A, .85, 95, phi11, and 80 alpha showed an enhancement of competence-conferring activity if exposure to the cells occurred in the presence of nromal rabbit serum. All of the propagating strains for the Staphylococcus reference typing phages were rendered competent for transformation by exposure to at least one of these helper phages. The use of a helper phage to confer competence to S. aureus did not result in distortion of the genetic linkages observed in an inherently competent strain. Lysogenization by phages phi11 or 83A is shown not to be required for the expression of competence, and evidence is presented which indicates that competence in the inherently competent 8325 strain is due to a helper phage effect initiated by the adsorption to cells of phi11 virion parts [or phi11 particles in the case of the single lysogen 8325-4(phi11)] that have been liberated by prophage induction.     

Staphylococcus aureus pathogenicity island DNA is packaged in particles composed of phage proteins

Staphylococcus aureus pathogenicity islands (SaPIs) have an intimate relationship with temperate staphylococcal phages. During phage growth, SaPIs are induced to replicate and are efficiently encapsidated into special small phage heads commensurate with their size. We have analyzed by amino acid sequencing and mass spectrometry the protein composition of the specific SaPI particles. This has enabled identification of major capsid and tail proteins and a putative portal protein. As expected, all these proteins were phage encoded. Additionally, these analyses suggested the existence of a protein required for the formation of functional phage but not SaPI particles. Mutational analysis demonstrated that the phage proteins identified were involved only in the formation and possibly the function of SaPI or phage particles, having no role in other SaPI or phage functions.     

Mobile genetic elements and bacterial toxinoses: the superantigen-encoding pathogenicity islands of Staphylococcus aureus

It is a remarkable observation that virtually all bacterial toxins associated with specific clinical conditions (toxinoses) are encoded by mobile (and therefore variable) genetic elements. Remarkably, these rarely, if ever, carry determinants of antibiotic resistance. Examples are the toxins responsible for diphtheria, anthrax, tetanus, botulism, cholera, toxic shock, scarlet fever, exfoliative dermatitis, food poisoning, travelers' diarrhea, shigella dysentery, necrotizing pneumonia, and others. A recently discovered example of this phenomenon is the family of related staphylococcal pathogenicity islands encoding superantigens (SAgs). These are 15-20kb elements that occupy constant positions in the chromosomes of toxigenic strains, and are characterized by certain phage-related features, namely genes encoding integrases, helicases, and terminases, and the presence of flanking direct repeats. The prototype, SaPI1 of Staphylococcus aureus, encodes TSST-1 plus two newly described SAgs, SEK and SEL. Other members of the family encode enterotoxins B (SaPI3) and C (SaPI4), plus at least two other SAgs each. SaPI1 and SaPI2, also encoding TSST-1, are excised and induced to replicate by certain staphylococcal phages, and are then encapsidated at high efficiency into phage-like infectious particles with heads about 1/3 the size of the helper phage heads, commensurate with the sizes of the respective genomes. This results in transfer frequencies of the order of 10(8)/ml, and is presumably responsible for the spread of these elements as well as for their acquisition in the first place. In the absence of a helper phage, these two islands are highly stable; neither excision, loss, or transfer occurs at detectable frequency. Several general implications of this phenomenon will be discussed. One is that the determinants of these toxins have been imported from other species and therefore are not components of the basic genome of the extant producing organisms. This raises the question of the biological (adaptive?) roles of these toxins. Another is that the toxin-carrying units can spread among different (though probably related) species. An interesting question is that of the biological basis for the separation of toxin and resistance determinants.     

Molecular relationships among serogroup B bacteriophages of Staphylococcus aureus.

The typing bacteriophages 55, 80, 83A, and 85 of Staphylococcus aureus, representative of the three major lytic groups of serological group B aureophages, have been examined for relatedness of their genomes and virion proteins. Phages 11 and 80 alpha were also examined to determine the relationship of phage 80 alpha to phages 11 and 80. Total genome hybridization measurements divided the phages into two groups. Phages 55 and 80, in the first group, had DNA homology of 50%. Phages 11, 80 alpha, 83A, and 85 formed a second group with 27 to 65% homology. Homology between the two groups was in the range of 14 to 22%. Phage 80 alpha is more closely related to phage 11 than to phage 80, though it is probably not a simple recombinant of phages 11 and 80. Restriction enzyme digestion and phage [32P]DNA hybridization analysis of the endonuclease-generated fragments from each phage DNA confirmed the findings of the DNA homology measurements. The endonuclease fragment patterns generated by EcoRI and HindIII were distinctive for each phage, confirming that none of the phages are closely related. Common sequences were present in most fragments from the phage DNAs when the labeled probe DNA was from a different phage in the same group. Cross-group probing of endonuclease fragments revealed both a diminished level of homology when similar sequences were present and the probable absence of some sequences. Virion proteins, examined by polyacrylamide gel electrophoresis, were similar in number and molecular weight for phages 11, 80 alpha, 83A, and 85, reflecting the DNA homology analyses. The virion proteins from phages 55 and 80, however, were more distinctive, and both differed from the phages in the other group.     

Control of Staphylococcus aureus pathogenicity island excision

Staphylococcus aureus pathogenicity islands (SaPIs) are a group of related 15–17 kb mobile genetic elements that commonly carry genes for superantigen toxins and other virulence factors. The key feature of their mobility is the induction of SaPI excision and replication by certain phages and their efficient encapsidation into specific small‐headed phage‐like infectious particles. Previous work demonstrated that chromosomal integration depends on the SaPI‐encoded recombinase, Int. However, although involved in the process, Int alone was not sufficient to mediate efficient SaPI excision from chromosomal sites, and we expected that SaPI excision would involve an Xis function, which could be encoded by a helper phage or by the SaPI, itself. Here we report that the latter is the case. In vivo recombination assays with plasmids in Escherichia coli demonstrate that SaPI‐coded Xis is absolutely required for recombination between the SaPI att_L and att_R sites, and that both sites, as well as their flanking SaPI sequences, are required for SaPI excision. Mutational analysis reveals that Xis is essential for efficient horizontal SaPI transfer to a recipient strain. Finally, we show that the master regulator of the SaPI life cycle, Stl, blocks expression of int and xis by binding to inverted repeats present in the promoter region, thus controlling SaPI excision.     

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.     

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

Tst , the gene for toxic shock syndrome toxin-1 (TSST-1), is part of a 15.2 kb genetic element in Staphylococcus aureus that is absent in TSST-1-negative strains. The prototype, in RN4282, is flanked by a 17 nucleotide direct repeat and contains genes for a second possible superantigen toxin, a Dichelobacter nodosus VapE homologue and a putative integrase. It is readily transferred to a recA ⁻ recipient, and it always inserts into a unique chromosomal copy of the 17 nucleotide sequence in the same orientation. It is excised and circularized by staphylococcal phages φ13 and 80α and replicates during the growth of the latter, which transduces it at very high frequency. Because of its site and orientation specificity and because it lacks other identifiable phage-like genes, we consider it to be a pathogenicity island (PI) rather than a transposon or a defective phage. The tst element in RN4282, near tyrB , is designated SaPI1. That in RN3984 in the trp region is only partially homologous to SaPI1 and is excised by phage 80 but not by 80α. It is designated SaPI2. These PIs are the first in any Gram-positive species and the first for which mobility has been demonstrated. Their mobility may be responsible for the spread of TSST-1 production among S. aureus strains.     

DNA Genome Size Affects the Stability of the Adenovirus Virion

Replication-defective adenovirus (Ad) vectors can vary considerably in genome length, but whether this affects virion stability has not been investigated. Helper-dependent Ad vectors with a genome size of ~30 kb were 100-fold more sensitive to heat inactivation than their parental helper virus (>36 kb), and increasing the genome size of the vector significantly improved heat stability. A similar relationship between genome size and stability existed for Ad with early region 1 deleted. Loss of infectivity was due to release of vertex proteins, followed by disintegration of the capsid. Thus, not only does the viral DNA encode all of the heritable information essential for virus replication, it also plays a critical role in maintaining capsid strength and integrity.     

Induction and biological properties of defective interfering particles of rabies virus.

A method for obtaining large quantities of defective interfering (DI) rabies virus particles that fulfill all the criteria delineated by Huang and Baltimore (1970) is described. The purified rabies DI virion was found to be much shorter (60 to 80 nm) than the complete virion (180 nm) and to have a viral genome of about half the size of normal rabies RNA but with all of the structural proteins of standard virions. Rabies DI virions were noninfectious for both cells in culture and for animals. As determined by in vitro and in vivo techniques, interference with the replication of standard virus was specific to rabies virus. The possible role of rabies DI virion in the pathogenicity of rabies virus infection and in the establishment of attenuated strains for use as live rabies vaccines is discussed.     

Structural Proteins of Adenovirus-Associated Virus Type 3

Three major structural proteins were found in adenovirus-associated virus (AAV) type 3H virions which were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of the polypeptides were determined to be approximately 66,000 (VP1), 80,000 (VP2), and 92,000 (VP3). The component having a molecular weight of 66,000 comprised about 80% of the total virion protein in the major AAV-3H particle, and the other two components comprised about 10% each. Proteins of the same molecular weight were found in the minor dense AAV-3H virion, but the 80,000- and 92,000-molecular-weight components were present at about one-half the concentration. The AAV-3H virion contains about 72 molecules of VP1 and 8 and 7 molecules of VP2 and VP3, respectively.