Crystal Structure of ORF12 from Lactococcus lactis Phage p2 Identifies a Tape Measure Protein Chaperone

We report here the characterization of the nonstructural protein ORF12 of the virulent lactococcal phage p2, which belongs to the Siphoviridae family. ORF12 was produced as a soluble protein, which forms large oligomers (6- to 15-mers) in solution. Using anti-ORF12 antibodies, we have confirmed that ORF12 is not found in the virion structure but is detected in the second half of the lytic cycle, indicating that it is a late-expressed protein. The structure of ORF12, solved by single anomalous diffraction and refined at 2.9-Å resolution, revealed a previously unknown fold as well as the presence of a hydrophobic patch at its surface. Furthermore, crystal packing of ORF12 formed long spirals in which a hydrophobic, continuous crevice was identified. This crevice exhibited a repeated motif of aromatic residues, which coincided with the same repeated motif usually found in tape measure protein (TMP), predicted to form helices. A model of a complex between ORF12 and a repeated motif of the TMP of phage p2 (ORF14) was generated, in which the TMP helix fitted exquisitely in the crevice and the aromatic patches of ORF12. We suggest, therefore, that ORF12 might act as a chaperone for TMP hydrophobic repeats, maintaining TMP in solution during the tail assembly of the lactococcal siphophage p2.During industrial milk fermentation, Lactococcus lactis cells are added to transform milk into an array of fermented products such as cheese. However, this manufacturing process may be impaired by lytic phages present in the factory environment as well as in the milk itself (30). Due to the destructive effects of phage infections on bacterial fermentation, much effort has been undertaken to isolate and study the biodiversity of these bacteriophages. Lactococcal bacteriophages belong to at least 10 different genetically distinct species of double-stranded DNA viruses (9). Of them, three lactococcal phage species, all belonging to the Siphoviridae family, are the major source of problems in milk fermentation, namely, the 936, P335, and c2 species (7, 28, 29). Furthermore, members of the 936 species are by far responsible for the majority of infections (50 to 80%) (1, 24, 41). Numerous phages of the 936 species have been isolated, and several have been characterized at the genome level (25). However, little is known concerning their molecular mechanisms of infection, although we recently solved the structure of the receptor-binding protein (RBP) of our model 936-like phage, namely, the virulent phage p2 (38, 43), and of phages belonging to the P335 species (27, 34, 37, 38).As with all viruses, bacteriophage genomes are quite compact, leaving little room for noncoding sequences (4). In fact, phage genes are disposed in an operon-type organization (4), and the order of genes corresponds to the different phases of the infection cycle. Moreover, genes are often in clusters (referred to as modules), with gene products from adjacent genes generally found to interact with each other. Interestingly, phage genome organization, including individual gene order, is often conserved within a given species, particularly within the Siphoviridae family. In the case of L. lactis virulent phages belonging to the 936 or P335 species, this principle applies particularly to the morphogenesis gene module, which includes all the genes coding for the phage structural protein genes. For the tail assembly, a module comprises a set of genes between the portal protein, which is connecting the tail to the capsid, and the RBP, which is located at the tip of the tail and is involved in host recognition (39, 43).The characterization of tail assembly genes of lactococcal phages has been more extensive for temperate siphophages belonging to the P335 species (27, 34, 37, 38). Because of the similarities in genome organization, the findings in this phage species can, in some cases, be used as clues toward understanding the morphology of 936-like phages. For the temperate phage Tuc2009 (P335 species), all structural proteins required for tail and baseplate assembly have been identified (27, 34, 37, 38). Genes located between those coding for the tape measure protein (TMP) and BppL (RBP) were identified as corresponding to components of the baseplate structure, located at the tail distal end. Furthermore, a gene coding for the major tail protein (MTP) was also identified at a position upstream from tmp. Between the genes coding for the MTP and those coding for the TMP in Tuc2009 are two gene products identified as gpG and gpGT, which are not present in the phage particle. These two proteins were named based on their likely role analogous to the tail assembly proteins present in coliphage lambda, a model virus belonging to the Siphoviridae family (21, 27, 47). gpGT has an essential role in lambda tail assembly, acting prior to tail shaft assembly, while the role of gpG in tail assembly is not known (21). Both gpG and gpGT are also absent from mature lambda virions (21). It has been argued that they may act as assembly chaperones (47).A close examination of 936 genomes indicates the presence of two genes coding for gpG and gpGT-like proteins. Analysis of the phage p2 genome, closely related to that of lactococcal phage sk1 (6), revealed that the putative tail assembly proteins could correspond to gene products ORF12 and ORF13. These two genes are followed by the TMP gene corresponding to orf14, other genes coding for other structural proteins, and the RBP gene orf18. During our ongoing investigation of the structure of phage p2, we report here the cloning, expression, and crystal structure of ORF12 in order to decipher its role in the tail assembly process.     

Complete WO Phage Sequences Reveal Their Dynamic Evolutionary Trajectories and Putative Functional Elements Required for Integration into the Wolbachia Genome

Wolbachia endosymbionts are ubiquitously found in diverse insects including many medical and hygienic pests, causing a variety of reproductive phenotypes, such as cytoplasmic incompatibility, and thereby efficiently spreading in host insect populations. Recently, Wolbachia-mediated approaches to pest control and management have been proposed, but the application of these approaches has been hindered by the lack of genetic transformation techniques for symbiotic bacteria. Here, we report the genome and structure of active bacteriophages from a Wolbachia endosymbiont. From the Wolbachia strain wCauB infecting the moth Ephestia kuehniella two closely related WO prophages, WOcauB2 of 43,016 bp with 47 open reading frames (ORFs) and WOcauB3 of 45,078 bp with 46 ORFs, were characterized. In each of the prophage genomes, an integrase gene and an attachment site core sequence were identified, which are putatively involved in integration and excision of the mobile genetic elements. The 3′ region of the prophages encoded genes with sequence motifs related to bacterial virulence and protein-protein interactions, which might represent effector molecules that affect cellular processes and functions of their host bacterium and/or insect. Database searches and phylogenetic analyses revealed that the prophage genes have experienced dynamic evolutionary trajectories. Genes similar to the prophage genes were found across divergent bacterial phyla, highlighting the active and mobile nature of the genetic elements. We suggest that the active WO prophage genomes and their constituent sequence elements would provide a clue to development of a genetic transformation vector for Wolbachia endosymbionts.Members of the genus Wolbachia are endosymbiotic bacteria belonging to the Alphaproteobacteria and infecting a wide range of arthropods, including over 60% of insect species, and some filarial nematodes. They are vertically transmitted through the maternal germ line of their host and are known to distort host reproduction by causing cytoplasmic incompatibility (CI), parthenogenesis, male killing, or feminization. The ability of Wolbachia to cause these reproductive phenotypes is thought to be responsible for their efficient and rapid spread into host populations (5, 21, 35, 51).Recently, Wolbachia-mediated pest control approaches have been proposed. A number of insect pests that have important medical and hygienic consequences, such as tsetse flies and mosquitoes that vector devastating human pathogens including African sleeping disease trypanosomes, malaria plasmodia, dengue viruses, Japanese encephalitis viruses, and others, often also carry Wolbachia infections (8, 24, 25, 34). In theory, if maternally transmitted genetic elements coinherited with a CI-inducing Wolbachia, such as mitochondria, the Wolbachia itself, or other coinfecting endosymbionts, are transformed with a gene of interest (like a gene that confers resistance of the vector insect against the pathogen infection), the genetic trait is expected to be spread and fixed in the host insect population, driven by the symbiont-induced reproductive phenotype (1, 2, 10, 11, 13, 32, 43, 44). The paratransgenesis and Wolbachia-driven population replacement approaches are, although potentially promising in controlling such insect-borne diseases, still at a conceptual stage mainly because no technique has been available for Wolbachia transformation.For genetic transformation of bacteria, mobile genetic elements such as plasmids, bacteriophages, and transposons have been used successfully. For example, pUC plasmids, λ phages, and transposons have been widely utilized for transforming Escherichia coli and other model bacterial species (38). While few plasmids and transposons have been reported from Wolbachia, a family of bacteriophages, called WO phages, has been detected from a diverse array of Wolbachia strains (3, 6, 7, 12, 17, 18, 31, 39, 49). For example, in the genomes of the Wolbachia strains wMel from the fruit fly Drosophila melanogaster and wPip from the mosquito Culex quinquefasciatus, three and five WO prophages are present, respectively (26, 52). Many of the prophages are pseudogenized and inactive while some are active and capable of producing phage particles (4, 7, 15, 17, 30, 40). Such active WO phage elements may provide tools for genetic transformation of Wolbachia endosymbionts.λ phage and many other temperate bacteriophages alternate between lytic phase and lysogenic phase in their life cycles. In the lytic phase, phage particles are produced and released via host cell lysis for infection to new host cells. In the lysogenic phase, the phage genome is integrated into the host genome via a site-specific recombination process, and the integrated phage genome, called prophage, is maintained in the host genome and multiplies together with the host DNA replication (38). Upon infection and lysogenic integration of λ phage, both ends of the linear phage genomic DNA are connected by DNA ligase, and the resultant circular phage genome is inserted into the E. coli genome by site-specific recombination at a region containing a core sequence of an attachment (att) site (28). att sites on the phage genome and the bacterial genome are called attP (phage att site) and attB (bacterial att site), respectively. After integration, attP and attB are located on both ends of the prophage, called attL (left prophage att site) and attR (right prophage att site), respectively. The integration and excision processes are mediated by a site-specific recombinase, called λ integrase, encoded in the phage genome (see Fig. S1 in the supplemental material) (27, 50). Hence, the att site and the integrase are the pivotal functional elements that mediate site-specific integration and excision of λ phage. Considering the structural similarity between λ phage and WO phage (31), identification of the att site and integrase from WO phage is of interest in that these elements could be utilized for delivering foreign genes into the Wolbachia genome.In order to identify a functional att site and integrase of WO phage, the complete genome sequences of active prophage elements producing phage particles should be determined. Here, the Wolbachia strain wCauB derived from the almond moth Cadra cautella was investigated because wCauB was reported to actively produce phage particles, and a partial genome sequence of its WO phage has been determined (15). In the original host insect, C. cautella, wCauB coexists with another Wolbachia strain wCauA, and both cause CI phenotypes and produce phage particles (15, 41). Not to be confounded by the coinfecting Wolbachia strains, we used a transfected line of the Mediterranean flour moth Ephestia kuehniella infected with wCauB only, which was generated by interspecific ooplasm transfer (42). It should be noted that a mass preparation procedure for WO phage particles by centrifugation has been established for the wCauB-infected E. kuehniella (15).In this study, we determined the complete genome sequences of two active WO prophages, named WOcauB2 and WOcauB3, that are capable of producing phage particles and that are located on the genome of the Wolbachia strain wCauB. Furthermore, we identified core sequences of att sites and integrase genes of these WO phages that are putatively involved in integration of the genetic elements into the Wolbachia genome.     

Identification of ORF636 in Phage φSLT Carrying Panton-Valentine Leukocidin Genes,Acting as an Adhesion Protein for a Poly(Glycerophosphate) Chain of Lipoteichoic Acid on the Cell Surface of Staphylococcus aureus

The temperate phage φSLT of Staphylococcus aureus carries genes for Panton-Valentine leukocidin. Here, we identify ORF636, a constituent of the phage tail tip structure, as a recognition/adhesion protein for a poly(glycerophosphate) chain of lipoteichoic acid on the cell surface of S. aureus. ORF636 bound specifically to S. aureus; it did not bind to any other staphylococcal species or to several gram-positive bacteria.Staphylococcus aureus, a ubiquitous and harmful human pathogen, produces three types of bicomponent pore-forming cytotoxins, namely, γ-hemolysin (LukF and Hlg2), leukocidin (LukF and LukS), and Panton-Valentine leukocidin (PVL) (LukF-Pv and LukS-Pv) (16). Of these, PVL has been investigated as a virulence-related factor of some S. aureus infectious diseases (7, 11, 23, 24, 31, 37). PVL shows high cytolytic specificity against human polymorphonuclear leukocytes and macrophages, and it is closely associated with most cutaneous necrotic lesions, such as furuncles or primary abscesses, and severe necrotic skin infection (24, 31), as well as with severe necrotic hemorrhagic pneumonia (11, 23). LukF-Pv and LukS-Pv are expressed by the PVL locus (pvl), which is distinct from the γ-hemolysin locus (hlg) (16, 32). In previous research, we found that pvl genes are located in the genome of the lysogenic bacteriophage φPVL (17, 18). We also found another PVL-carrying temperate elongated-head Siphoviridae phage, φSLT, which has the ability to convert S. aureus to the PVL-producing strain from a clinical isolate (29). These findings indicated that at least two types of staphylococcal temperate phages are involved in the horizontal transfer of pvl genes among S. aureus strains (16, 29). Recently, the emergence of a single clonal community-acquired methicillin-resistant S. aureus (CA-MRSA), which produces PVL, was reported (7). Most CA-MRSA strains isolated in the United States and Australia carry the staphylococcal cassette chromosome mec (SCCmec) IV, and they were divided into five clonal complexes by multilocus sequence typing (30). The analysis of the CA-MRSA clones confirmed the presence of PVL genes and SCCmec IV in CA-MRSA and suggested that various CA-MRSA strains have arisen from the diverse genetic backgrounds associated with each geographic origin, rather than from the worldwide spread of a single clone (30, 37). Although there is great debate as to whether PVL is an important virulence factor, numerous studies support the hypothesis that PVL plays an important role in the pathogenesis of CA-MRSA necrotizing pneumonia (3, 6). In regard to the acquisition of PVL gene clusters and the proliferation of PVL-carrying CA-MRSA, the horizontal transfer of PVL via PVL-carrying phages, as well as that of SCCmec, has become the focus of intense research interest. To understand the horizontal transfer of PVL, the analysis of the infection ability of a PVL-carrying phage is important. If the phage has a wide host range, the PVL-carrying phage might threaten to become a source of emerging PVL-positive bacteria. Phage infection starts from an interaction between a phage virion and its host cell surface receptor. Nevertheless, little is known about phage receptors on the surface of S. aureus, and the mechanism of host cell-specific binding of staphylococcal phages has been poorly characterized. In addition, there is no information about staphylococcal phage proteins involved in host cell recognition and/or binding. Here, we identify ORF636, with a mass of 66 kDa, as a structural protein of the φSLT tail and determine that it acts as a protein for recognition/adhesion of a poly(glycerophosphate) moiety of lipoteichoic acid (LTA) on the cell surface of the host S. aureus in the first stage of infection by φSLT.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

A Conserved Acetyl Esterase Domain Targets Diverse Bacteriophages to the Vi Capsular Receptor of Salmonella enterica Serovar Typhi

A number of bacteriophages have been identified that target the Vi capsular antigen of Salmonella enterica serovar Typhi. Here we show that these Vi phages represent a remarkably diverse set of phages belonging to three phage families, including Podoviridae and Myoviridae. Genome analysis facilitated the further classification of these phages and highlighted aspects of their independent evolution. Significantly, a conserved protein domain carrying an acetyl esterase was found to be associated with at least one tail fiber gene for all Vi phages, and the presence of this domain was confirmed in representative phage particles by mass spectrometric analysis. Thus, we provide a simple explanation and paradigm of how a diverse group of phages target a single key virulence antigen associated with this important human-restricted pathogen.Bacteriophages are dependent for their survival on the presence of susceptible host bacteria in their environment. The first stage of recognition of the bacterial host normally involves binding of a specific phage attachment protein to a receptor molecule on the bacterial surface. Bacteria can evade phage infection by various mechanisms, including accumulating escape mutations in the receptor, acquiring phage inhibitory proteins, or directly modifying the receptor, for example, lipopolysaccharide (LPS) (43). In addition, phage can adapt to recognize different receptors through a number of genetic mechanisms involving evolution of their attachment proteins (20) or by tropism switching (21, 22).Phage can exploit capsular exopolysaccharides as receptors, some of which are associated with virulence in pathogens (5, 23, 35). A notable example is the Vi capsule found in Salmonella enterica serovar Typhi (S. Typhi) and some isolates of S. Dublin and Citrobacter freundii (29). The Vi capsule of S. Typhi is an important virulence factor, facilitating the bacteria to escape opsonization and other forms of immune surveillance (14, 30) as well as potentially helping the bacteria to evade phage that would otherwise target the O:9 LPS, which the Vi capsule can, at least in part, mask (27). In the middle of the last century, a set of lytic phages were isolated that utilized the Vi capsule as a receptor (6). These Vi phages were exploited in diagnostic laboratories as a “typing set” to distinguish between different strains of S. Typhi isolated from typhoid patients (8). A secondary typing set was generated from Vi typing phage II by adapting this phage to grow on different S. Typhi hosts (6). At this time, typhoid was still common in many parts of Europe and North America, and clinicians tested some of these Vi phages for their potential in phage therapy experiments with human typhoid patients (11). Although this work showed significant promise, phage therapy gradually disappeared from clinical practice in many countries as antibiotics became readily available.S. Typhi is a monophyletic serovar of the broad enteric species S. enterica (16, 31). Interestingly, S. Typhi is host restricted to humans and has no known zoonotic source. Unlike many other S. enterica serovars, S. Typhi normally causes a systemic infection and does not persist in the intestine efficiently, where high levels of bacteriophage are present. Although it is rare in developed countries, S. Typhi is still a significant cause of mortality in many developing countries (26). Most current clinical isolates are Vi positive when first isolated (2), but it is noteworthy that the Vi capsule biosynthesis and export genes are carried by an operon within a potentially unstable island called Salmonella pathogenicity island 7 (SPI-7) (29).Although some phenotypic characterization of the Vi phage has been undertaken (1), very little has been performed at the molecular level. We previously showed that Vi typing phage II-E1 is related to the S. Typhimurium phage ES18 (4, 28), with synteny in many capsid and tail proteins. We have now further characterized the other members of this S. Typhi Vi phage collection, designated types I, III, IV, V, VI, and VII (abbreviated from here on as Vi phages I, III, IV, etc.) (6, 11), by utilizing electron microscopy and genomic analysis. This analysis shows that this collection of Vi phages represents a diverse group of bacteriophages that have adapted to growth on S. Typhi through convergent evolution within their tail spike protein genes and the acquisition of conserved acetyl esterase domains.     

The Autolysin LytA Contributes to Efficient Bacteriophage Progeny Release in Streptococcus pneumoniae

Most bacteriophages (phages) release their progeny through the action of holins that form lesions in the cytoplasmic membrane and lysins that degrade the bacterial peptidoglycan. Although the function of each protein is well established in phages infecting Streptococcus pneumoniae, the role—if any—of the powerful bacterial autolysin LytA in virion release is currently unknown. In this study, deletions of the bacterial and phage lysins were done in lysogenic S. pneumoniae strains, allowing the evaluation of the contribution of each lytic enzyme to phage release through the monitoring of bacterial-culture lysis and phage plaque assays. In addition, we assessed membrane integrity during phage-mediated lysis using flow cytometry to evaluate the regulatory role of holins over the lytic activities. Our data show that LytA is activated at the end of the lytic cycle and that its triggering results from holin-induced membrane permeabilization. In the absence of phage lysin, LytA is able to mediate bacterial lysis and phage release, although exclusive dependence on the autolysin results in reduced virion egress and altered kinetics that may impair phage fitness. Under normal conditions, activation of bacterial LytA, together with the phage lysin, leads to greater phage progeny release. Our findings demonstrate that S. pneumoniae phages use the ubiquitous host autolysin to accomplish an optimal phage exiting strategy.Streptococcus pneumoniae (pneumococcus), a common and important human pathogen, is characterized by the high incidence of lysogeny in isolates associated with infection (34, 44). Pneumococcal bacteriophages (phages) share with the majority of bacteriophages infecting other bacterial species the “holin-lysin” system to lyse the host cell and release their progeny at the end of the lytic cycle. Genes encoding both holins and lysins (historically termed “endolysins”) are indeed found in the genomes of all known pneumococcal phages (8, 28, 31, 37). Supporting this mechanism, a lytic phenotype in the heterologous Escherichia coli system was achieved only by the simultaneous expression of the Ejh holin and the Ejl endolysin of pneumococcal phage EJ-1 (8). When these proteins were independently expressed, cellular lysis was not perceived. Similar results were shown for pneumococcal phage Cp-1, not only in E. coli, but also in the pneumococcus itself (28).Phage lysins destroy the pneumococcal peptidoglycan network due to their muralytic activity, whereas holins have been shown in S. pneumoniae to form nonspecific lesions (8), most likely upon a process of oligomerization in the cytoplasmic membrane, as observed for the E. coli phage λ (13, 14, 43). It was generally proposed that holin lesions allow access of phage lysins to the cell wall (52, 54), as the majority of phage lysins, including the pneumococcal endolysins, lack a typical N-terminal secretory signal sequence and transmembrane domains (8). However, recent evidence also highlights the possibility for a holin-independent targeting of phage lysins to the cell wall, where holin lesions seem to be crucial for the activation of the already externalized phage lysins (42, 50, 51). Regardless of the mechanism operating in S. pneumoniae to activate phage lysins, holin activity compromises membrane integrity.Pneumococcal cells present their own autolytic activity, mainly due to the presence of a powerful bacterial cell wall hydrolase, LytA (an N-acetylmuramoyl-l-alanine-amidase), responsible for bacterial lysis under certain physiological conditions (47). Although other bacterial species also encode peptidoglycan hydrolases, the extensive lysis shortly after entering stationary phase caused by LytA is a unique feature of S. pneumoniae. Interestingly, LytA is translocated across the cytoplasmic membrane to the cell wall—where it remains inactive—in spite of the absence of a canonical N-terminal sequence signal (7). In the cell wall, autolysin activities are tightly regulated by mechanisms that seem to be related to the energized state of the cell membrane. In fact, depolarizing agents are able to trigger autolysis in Bacillus subtilis (16, 17), and bacteriocin-induced depletion of membrane potential triggers autolysis of some species of the genera Lactococcus and Lactobacillus, closely related to streptococci (29). It is therefore possible that the holin-inflicted perturbations of the S. pneumoniae cytoplasmic membrane upon the induction of the lytic cycle may trigger not only the lytic activity of the phage lysin, but also that of inactive LytA located in the cell wall. Accordingly, LytA could also participate in the release of phage particles at the end of the infectious cycle, especially considering its powerful autolytic activity. Previous studies have suggested a role for the host autolytic enzyme in the release of phage progeny (11, 38), but in fact, the evidence is unclear and dubious, considering that the existence of phage-encoded lysins was unknown or very poorly understood and some of the experimental conditions used to show a role of LytA could have also affected the activity of the phage lysin (38).To clarify the possible role of the bacterial autolysin in host lysis, we used the S. pneumoniae strain SVMC28, lysogenic for the SV1 prophage (34), which contains a typical “holin-lysin” cassette, and a different host strain lysogenized with the same SV1 phage. Our results show that LytA is activated by the holin-induced membrane disruption, just like the phage endolysin. In the absence of the endolysin, LytA is capable of mediating host lysis, releasing functional phage particles able to complete their life cycle. Still, sole dependence on LytA results in an altered pattern of phage release that may reduce phage fitness. Importantly, we also show that, together with the endolysin, the concurrent LytA activation is critical for optimal phage progeny release.     

Mutagenesis and Functional Characterization of the RNA and Protein Components of the toxIN Abortive Infection and Toxin-Antitoxin Locus of Erwinia

Bacteria are constantly challenged by bacteriophage (phage) infection and have developed multiple adaptive resistance mechanisms. These mechanisms include the abortive infection systems, which promote “altruistic suicide” of an infected cell, protecting the clonal population. A cryptic plasmid of Erwinia carotovora subsp. atroseptica, pECA1039, has been shown to encode an abortive infection system. This highly effective system is active across multiple genera of gram-negative bacteria and against a spectrum of phages. Designated ToxIN, this two-component abortive infection system acts as a toxin-antitoxin module. ToxIN is the first member of a new type III class of protein-RNA toxin-antitoxin modules, of which there are multiple homologues cross-genera. We characterized in more detail the abortive infection phenotype of ToxIN using a suite of Erwinia phages and performed mutagenesis of the ToxI and ToxN components. We determined the minimal ToxI RNA sequence in the native operon that is both necessary and sufficient for abortive infection and to counteract the toxicity of ToxN. Furthermore, site-directed mutagenesis of ToxN revealed key conserved amino acids in this defining member of the new group of toxic proteins. The mechanism of phage activation of the ToxIN system was investigated and was shown to have no effect on the levels of the ToxN protein. Finally, evidence of negative autoregulation of the toxIN operon, a common feature of toxin-antitoxin systems, is presented. This work on the components of the ToxIN system suggests that there is very tight toxin regulation prior to suicide activation by incoming phage.Interactions between bacteria and their natural parasites, bacteriophages (phage), have global-scale effects (42). Although the vast majority of the phage infections, which occur at a rate of 10²⁵ infections per s (26), are overlooked by humans, en masse they affect environmental nutrient cycling (18) and have long been known to be vital to the spread and continued diversity of microbial genes (11). A tiny proportion of this activity can directly affect our everyday activities; the lysis of bacteria following phage infection has potential medical benefits, such as use in phage therapy (30), or can be economically damaging, as it is in cases of bacterial fermentation failure (for instance, in the dairy industry [31]).Gram-positive lactococcal strains used in dairy fermentation have been shown to naturally harbor multiple phage resistance mechanisms (16). These mechanisms can be broadly classed as systems which (i) prevent phage adsorption, (ii) interfere with phage DNA injection, (iii) restrict unmodified DNA, and (iv) induce abortive infection. There is also an increasing amount of research that focuses on new systems that use clustered regularly interspaced short palindromic repeats to mediate phage resistance (3). Clustered regularly interspaced short palindromic repeats and associated proteins, although widespread in archaea and bacteria (39), have not been identified yet in lactococcal strains (23).The abortive infection (Abi) systems induce cell death upon phage infection and often rely on a toxic protein to cause “altruistic cell suicide” in the infected host (16). Although Abi systems have been studied predominantly using lactococcal systems, because of their potential economic importance (8) they have been identified in some gram-negative species, such as Escherichia coli, Vibrio cholerae, Shigella dysenteriae, and Erwinia carotovora (9, 14, 36, 38). The prr and lit systems of E. coli have been studied at the molecular level, and their mode of action and mode of activation by incoming phage have been identified (2, 37, 38). In contrast, lactococcal Abi systems have been characterized mainly by the range of phages actively aborted and the scale of these effects, and the Abi systems have been grouped based on general modes of action (8, 12). More recently, research has begun to identify more specific lactococcal Abi activities at the molecular level (12, 17) and has revealed phage activation of two such Abi systems (6, 21).An Abi system was identified on plasmid pECA1039, which was isolated from a strain of the phytopathogen E. carotovora subsp. atroseptica (14). Designated ToxIN, this two-component Abi system operates as a novel protein-RNA toxin-antitoxin (TA) system to abort phage infection in multiple gram-negative bacteria. The toxic activity of the ToxN protein was inhibited by ToxI RNA, which consists of 5.5 direct repeats of 36 nucleotides. It is now recognized that TA loci, which were originally characterized as “plasmid addiction” modules (43), are widely distributed in the chromosomes of archaea and bacteria (19) and in phage genomes, such as that of the extrachromosomal prophage P1 (27). As a result, the precise biological role of TA systems is under debate (29). It is clear, however, that they can be effective phage resistance systems, as is the case for toxIN in E. carotovora subsp. atroseptica (14) and hok/sok and mazEF in E. coli (22, 33). Previously characterized TA systems operate with both components interacting as either RNAs (e.g., hok/sok) (type I) or proteins (e.g., MazE and MazF) (type II). In this study, a mutagenesis approach was used to further characterize the ToxI and ToxN components of the new (type III) protein-RNA TA Abi system. The regulation of the operon and the mode of phage activation were also examined.