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1.  Cryo-Electron Microscopy Structure of Lactococcal Siphophage 1358 Virion 
Journal of Virology  2014;88(16):8900-8910.
ABSTRACT
Lactococcus lactis, a Gram+ lactic acid-producing bacterium used for the manufacture of several fermented dairy products, is subject to infection by diverse virulent tailed phages, leading to industrial fermentation failures. This constant viral risk has led to a sustained interest in the study of their biology, diversity, and evolution. Lactococcal phages now constitute a wide ensemble of at least 10 distinct genotypes within the Caudovirales order, many of them belonging to the Siphoviridae family. Lactococcal siphophage 1358, currently the only member of its group, displays a noticeably high genomic similarity to some Listeria phages as well as a host range limited to a few L. lactis strains. These genomic and functional characteristics stimulated our interest in this phage. Here, we report the cryo-electron microscopy structure of the complete 1358 virion. Phage 1358 exhibits noteworthy features, such as a capsid with dextro handedness and protruding decorations on its capsid and tail. Observations of the baseplate of virion particles revealed at least two conformations, a closed and an open, activated form. Functional assays uncovered that the adsorption of phage 1358 to its host is Ca2+ independent, but this cation is necessary to complete its lytic cycle. Taken together, our results provide the complete structural picture of a unique lactococcal phage and expand our knowledge on the complex baseplate of phages of the Siphoviridae family.
IMPORTANCE Phages of Lactococcus lactis are investigated mainly because they are sources of milk fermentation failures in the dairy industry. Despite the availability of several antiphage measures, new phages keep emerging in this ecosystem. In this study, we provide the cryo-electron microscopy reconstruction of a unique lactococcal phage that possesses genomic similarity to particular Listeria phages and has a host range restricted to only a minority of L. lactis strains. The capsid of phage 1358 displays the almost unique characteristic of being dextro handed. Its capsid and tail exhibit decorations that we assigned to nonspecific sugar binding modules. We observed the baseplate of 1358 in two conformations, a closed and an open form. We also found that the adsorption to its host, but not infection, is Ca2+ independent. Overall, this study advances our understanding of the adhesion mechanisms of siphophages.
doi:10.1128/JVI.01040-14
PMCID: PMC4136242  PMID: 24872584
2.  Comparison of Five Bacteriophages as Models for Viral Aerosol Studies 
Applied and Environmental Microbiology  2014;80(14):4242-4250.
Bacteriophages are perceived to be good models for the study of airborne viruses because they are safe to use, some of them display structural features similar to those of human and animal viruses, and they are relatively easy to produce in large quantities. Yet, only a few studies have investigated them as models. It has previously been demonstrated that aerosolization, environmental conditions, and sampling conditions affect viral infectivity, but viral infectivity is virus dependent. Thus, several virus models are likely needed to study their general behavior in aerosols. The aim of this study was to compare the effects of aerosolization and sampling on the infectivity of five tail-less bacteriophages and two pathogenic viruses: MS2 (a single-stranded RNA [ssRNA] phage of the Leviviridae family), Φ6 (a segmented double-stranded RNA [dsRNA] phage of the Cystoviridae family), ΦX174 (a single-stranded DNA [ssDNA] phage of the Microviridae family), PM2 (a double-stranded DNA [dsDNA] phage of the Corticoviridae family), PR772 (a dsDNA phage of the Tectiviridae family), human influenza A virus H1N1 (an ssRNA virus of the Orthomyxoviridae family), and the poultry virus Newcastle disease virus (NDV; an ssRNA virus of the Paramyxoviridae family). Three nebulizers and two nebulization salt buffers (with or without organic fluid) were tested, as were two aerosol sampling devices, a liquid cyclone (SKC BioSampler) and a dry cyclone (National Institute for Occupational Safety and Health two-stage cyclone bioaerosol sampler). The presence of viruses in collected air samples was detected by culture and quantitative PCR (qPCR). Our results showed that these selected five phages behave differently when aerosolized and sampled. RNA phage MS2 and ssDNA phage ΦX174 were the most resistant to aerosolization and sampling. The presence of organic fluid in the nebulization buffer protected phages PR772 and Φ6 throughout the aerosolization and sampling with dry cyclones. In this experimental setup, the behavior of the influenza virus resembled that of phages PR772 and Φ6, while the behavior of NDV was closer to that of phages MS2 and ΦX174. These results provide critical information for the selection of appropriate phage models to mimic the behavior of specific human and animal viruses in aerosols.
doi:10.1128/AEM.00767-14
PMCID: PMC4068686  PMID: 24795379
3.  Molecular Insights on the Recognition of a Lactococcus lactis Cell Wall Pellicle by the Phage 1358 Receptor Binding Protein 
Journal of Virology  2014;88(12):7005-7015.
ABSTRACT
The Gram-positive bacterium Lactococcus lactis is used for the production of cheeses and other fermented dairy products. Accidental infection of L. lactis cells by virulent lactococcal tailed phages is one of the major risks of fermentation failures in industrial dairy factories. Lactococcal phage 1358 possesses a host range limited to a few L. lactis strains and strong genomic similarities to Listeria phages. We report here the X-ray structures of phage 1358 receptor binding protein (RBP) in complex with monosaccharides. Each monomer of its trimeric RBP is formed of two domains: a “shoulder” domain linking the RBP to the rest of the phage and a jelly roll fold “head/host recognition” domain. This domain harbors a saccharide binding crevice located in the middle of a monomer. Crystal structures identified two sites at the RBP surface, ∼8 Å from each other, one accommodating a GlcNAc monosaccharide and the other accommodating a GlcNAc or a glucose 1-phosphate (Glc1P) monosaccharide. GlcNAc and GlcNAc1P are components of the polysaccharide pellicle that we identified at the cell surface of L. lactis SMQ-388, the host of phage 1358. We therefore modeled a galactofuranose (Galf) sugar bridging the two GlcNAc saccharides, suggesting that the trisaccharidic motif GlcNAc-Galf-GlcNAc (or Glc1P) might be common to receptors of genetically distinct lactococcal phages p2, TP091-1, and 1358. Strain specificity might therefore be elicited by steric clashes induced by the remaining components of the pellicle hexasaccharide. Taken together, these results provide a first insight into the molecular mechanism of host receptor recognition by lactococcal phages.
IMPORTANCE Siphophages infecting the Gram-positive bacterium Lactococcus lactis are sources of milk fermentation failures in the dairy industry. We report here the structure of the pellicle polysaccharide from L. lactis SMQ-388, the specific host strain of phage 1358. We determined the X-ray structures of the lytic lactococcal phage 1358 receptor binding protein (RBP) in complex with monosaccharides. The positions and nature of monosaccharides bound to the RBP are in agreement with the pellicle structure and suggest a general binding mode of lactococcal phages to their pellicle saccharidic receptor.
doi:10.1128/JVI.00739-14
PMCID: PMC4054337  PMID: 24719416
4.  Functional and Structural Basis for a Bacteriophage Homolog of Human RAD52 
Current biology : CB  2008;18(15):1142-1146.
Summary
In eukaryotes, homologous recombination proteins such as RAD51 and RAD52 play crucial roles in DNA repair and genome stability. Human RAD52 is a member of a large single-strand annealing protein (SSAP) family [1] and stimulates Rad51-dependent recombination [2, 3]. In prokaryotes and phages, it has been difficult to establish the presence of RAD52 homologs with conserved sequences. Putative SSAPs were recently found in several phages that infect strains of Lactococcus lactis [4]. One of these SSAPs was identified as Sak and was found in the virulent L. lactis phage ul36, which belongs to the Siphoviridae family [4, 5]. In this study, we show that Sak is homologous to the N terminus of human RAD52. Purified Sak binds single-stranded DNA (ssDNA) preferentially over double-stranded DNA (dsDNA) and promotes the renaturation of long complementary ssDNAs. Sak also binds RecA and stimulates homologous recombination reactions. Mutations shown to modulate RAD52 DNA binding [6] affect Sak similarly. Remarkably, electron-microscopic reconstruction of Sak reveals an undecameric (11) subunit ring, similar to the crystal structure of the N-terminal fragment of human RAD52 [7, 8]. For the first time, we propose a viral homolog of RAD52 at the amino acid, phylogenic, functional, and structural levels.
doi:10.1016/j.cub.2008.06.071
PMCID: PMC4149281  PMID: 18656357
5.  Complete Genome Sequence of a Staphylococcus epidermidis Bacteriophage Isolated from the Anterior Nares of Humans 
Genome Announcements  2014;2(4):e00549-14.
We report here the complete genome sequence of a virulent Staphylococcus epidermidis siphophage, phage 6ec, isolated from the anterior nares of a human. This viral genome is 93,794 bp in length, with a 3′ overhang cos site of 10 nucleotides, and it codes for 142 putative open reading frames.
doi:10.1128/genomeA.00549-14
PMCID: PMC4125762  PMID: 25103751
6.  Improving the Safety of Staphylococcus aureus Polyvalent Phages by Their Production on a Staphylococcus xylosus Strain 
PLoS ONE  2014;9(7):e102600.
Team1 (vB_SauM_Team1) is a polyvalent staphylococcal phage belonging to the Myoviridae family. Phage Team1 was propagated on a Staphylococcus aureus strain and a non-pathogenic Staphylococcus xylosus strain used in industrial meat fermentation. The two Team1 preparations were compared with respect to their microbiological and genomic properties. The burst sizes, latent periods, and host ranges of the two derivatives were identical as were their genome sequences. Phage Team1 has 140,903 bp of double stranded DNA encoding for 217 open reading frames and 4 tRNAs. Comparative genomic analysis revealed similarities to staphylococcal phages ISP (97%) and G1 (97%). The host range of Team1 was compared to the well-known polyvalent staphylococcal phages phi812 and K using a panel of 57 S. aureus strains collected from various sources. These bacterial strains were found to represent 18 sequence types (MLST) and 14 clonal complexes (eBURST). Altogether, the three phages propagated on S. xylosus lysed 52 out of 57 distinct strains of S. aureus. The identification of phage-insensitive strains underlines the importance of designing phage cocktails with broadly varying and overlapping host ranges. Taken altogether, our study suggests that some staphylococcal phages can be propagated on food-grade bacteria for biocontrol and safety purposes.
doi:10.1371/journal.pone.0102600
PMCID: PMC4111496  PMID: 25061757
7.  CRISPR-Cas: an efficient tool for genome engineering of virulent bacteriophages 
Nucleic Acids Research  2014;42(14):9504-9513.
Bacteriophages are now widely recognized as major players in a wide variety of ecosystems. Novel genes are often identified in newly isolated phages as well as in environmental metavirome studies. Most of these novel viral genes have unknown functions but appear to be coding for small, non-structural proteins. To understand their biological role, very efficient genetic tools are required to modify them, especially in the genome of virulent phages. We first show that specific point mutations and large deletions can be engineered in the genome of the virulent phage 2972 using the Streptococcus thermophilus CRISPR-Cas Type II-A system as a selective pressure to increase recombination efficiencies. Of significance, all the plaques tested contained recombinant phages with the desired mutation. Furthermore, we show that the CRISPR-Cas engineering system can be used to efficiently introduce a functional methyltransferase gene into a virulent phage genome. Finally, synthetic CRISPR bacteriophage insensitive mutants were constructed by cloning a spacer-repeat unit in a low-copy vector illustrating the possibility to target multiple regions of the phage genome. Taken together, this data shows that the CRISPR-Cas system is an efficient and adaptable tool for editing the otherwise intractable genomes of virulent phages and to better understand phage-host interactions.
doi:10.1093/nar/gku628
PMCID: PMC4132740  PMID: 25063295
8.  First Complete Genome Sequence of Staphylococcus xylosus, a Meat Starter Culture and a Host to Propagate Staphylococcus aureus Phages 
Genome Announcements  2014;2(4):e00671-14.
Staphylococcus xylosus is a bacterial species used in meat fermentation and a commensal microorganism found on animals. We present the first complete circular genome from this species. The genome is composed of 2,757,557 bp, with a G+C content of 32.9%, and contains 2,514 genes and 79 structural RNAs.
doi:10.1128/genomeA.00671-14
PMCID: PMC4110768  PMID: 25013142
9.  Structure, Adsorption to Host, and Infection Mechanism of Virulent Lactococcal Phage p2 
Journal of Virology  2013;87(22):12302-12312.
Lactococcal siphophages from the 936 and P335 groups infect the Gram-positive bacterium Lactococcus lactis using receptor binding proteins (RBPs) attached to their baseplate, a large multiprotein complex at the distal part of the tail. We have previously reported the crystal and electron microscopy (EM) structures of the baseplates of phages p2 (936 group) and TP901-1 (P335 group) as well as the full EM structure of the TP901-1 virion. Here, we report the complete EM structure of siphophage p2, including its capsid, connector complex, tail, and baseplate. Furthermore, we show that the p2 tail is characterized by the presence of protruding decorations, which are related to adhesins and are likely contributed by the major tail protein C-terminal domains. This feature is reminiscent of the tail of Escherichia coli phage λ and Bacillus subtilis phage SPP1 and might point to a common mechanism for establishing initial interactions with their bacterial hosts. Comparative analyses showed that the architecture of the phage p2 baseplate differs largely from that of lactococcal phage TP901-1. We quantified the interaction of its RBP with the saccharidic receptor and determined that specificity is due to lower koff values of the RBP/saccharidic dissociation. Taken together, these results suggest that the infection of L. lactis strains by phage p2 is a multistep process that involves reversible attachment, followed by baseplate activation, specific attachment of the RBPs to the saccharidic receptor, and DNA ejection.
doi:10.1128/JVI.02033-13
PMCID: PMC3807928  PMID: 24027307
10.  Effect of the Abortive Infection Mechanism and Type III Toxin/Antitoxin System AbiQ on the Lytic Cycle of Lactococcus lactis Phages 
Journal of Bacteriology  2013;195(17):3947-3956.
To survive in phage-containing environments, bacteria have evolved an array of antiphage systems. Similarly, phages have overcome these hurdles through various means. Here, we investigated how phages are able to circumvent the Lactococcus lactis AbiQ system, a type III toxin-antitoxin with antiviral activities. Lactococcal phage escape mutants were obtained in the laboratory, and their genomes were sequenced. Three unrelated genes of unknown function were mutated in derivatives of three distinct lactococcal siphophages: orf38 of phage P008, m1 of phage bIL170, and e19 of phage c2. One-step growth curve experiments revealed that the phage mutations had a fitness cost while transcriptional analyses showed that AbiQ modified the early-expressed phage mRNA profiles. The L. lactis AbiQ system was also transferred into Escherichia coli MG1655 and tested against several coliphages. While AbiQ was efficient against phages T4 (Myoviridae) and T5 (Siphoviridae), escape mutants of only phage 2 (Myoviridae) could be isolated. Genome sequencing revealed a mutation in gene orf210, a putative DNA polymerase. Taking these observations together, different phage genes or gene products are targeted or involved in the AbiQ phenotype. Moreover, this antiviral system is active against various phage families infecting Gram-positive and Gram-negative bacteria. A model for the mode of action of AbiQ is proposed.
doi:10.1128/JB.00296-13
PMCID: PMC3754610  PMID: 23813728
11.  The double-edged sword of CRISPR-Cas systems 
Cell Research  2012;23(1):15-17.
doi:10.1038/cr.2012.124
PMCID: PMC3541657  PMID: 22945354
12.  Identification of a New P335 Subgroup through Molecular Analysis of Lactococcal Phages Q33 and BM13 
Applied and Environmental Microbiology  2013;79(14):4401-4409.
Lactococcal dairy starter strains are under constant threat from phages in dairy fermentation facilities, especially by members of the so-called 936, P335, and c2 species. Among these three phage groups, members of the P335 species are the most genetically diverse. Here, we present the complete genome sequences of two P335-type phages, Q33 and BM13, isolated in North America and representing a novel lineage within this phage group. The Q33 and BM13 genomes exhibit homology, not only to P335-type, but also to elements of the 936-type phage sequences. The two phage genomes also have close relatedness to phages infecting Enterococcus and Clostridium, a heretofore unknown feature among lactococcal P335 phages. The Q33 and BM13 genomes are organized in functionally related clusters with genes encoding functions such as DNA replication and packaging, morphogenesis, and host cell lysis. Electron micrographic analysis of the two phages highlights the presence of a baseplate more reminiscent of the baseplate of 936 phages than that of the majority of members of the P335 group, with the exception of r1t and LC3.
doi:10.1128/AEM.00832-13
PMCID: PMC3697491  PMID: 23666331
13.  Characterization of a Novel Panton-Valentine Leukocidin (PVL)-Encoding Staphylococcal Phage and Its Naturally PVL-Lacking Variant 
A new siphophage (LH1) was isolated from raw milk using a Staphylococcus aureus ST352 host. Its genome (46,048 bp, 57 open reading frames) includes the two genes encoding Panton-Valentine leukocidin (PVL), a virulence factor usually harbored by S. aureus prophages. Nine structural proteins were identified, including a tail protein generated through a +1 frameshift. A phage lytic mutant was isolated, and its analysis revealed the deletion of genes coding for the PVL and an integrase. The deletion likely occurred through recombination between direct repeats.
doi:10.1128/AEM.03852-12
PMCID: PMC3623202  PMID: 23396328
14.  Characterization of Two Virulent Phages of Lactobacillus plantarum 
Applied and Environmental Microbiology  2012;78(24):8719-8734.
We characterized two Lactobacillus plantarum virulent siphophages, ATCC 8014-B1 (B1) and ATCC 8014-B2 (B2), previously isolated from corn silage and anaerobic sewage sludge, respectively. Phage B2 infected two of the eight L. plantarum strains tested, while phage B1 infected three. Phage adsorption was highly variable depending on the strain used. Phage defense systems were found in at least two L. plantarum strains, LMG9211 and WCSF1. The linear double-stranded DNA genome of the pac-type phage B1 had 38,002 bp, a G+C content of 47.6%, and 60 open reading frames (ORFs). Surprisingly, the phage B1 genome has 97% identity with that of Pediococcus damnosus phage clP1 and 77% identity with that of L. plantarum phage JL-1; these phages were isolated from sewage and cucumber fermentation, respectively. The double-stranded DNA (dsDNA) genome of the cos-type phage B2 had 80,618 bp, a G+C content of 36.9%, and 127 ORFs with similarities to those of Bacillus and Lactobacillus strains as well as phages. Some phage B2 genes were similar to ORFs from L. plantarum phage LP65 of the Myoviridae family. Additionally, 6 tRNAs were found in the phage B2 genome. Protein analysis revealed 13 (phage B1) and 9 (phage B2) structural proteins. To our knowledge, this is the first report describing such high identity between phage genomes infecting different genera of lactic acid bacteria.
doi:10.1128/AEM.02565-12
PMCID: PMC3502894  PMID: 23042172
15.  Involvement of the Major Capsid Protein and Two Early-Expressed Phage Genes in the Activity of the Lactococcal Abortive Infection Mechanism AbiT 
Applied and Environmental Microbiology  2012;78(19):6890-6899.
The dairy industry uses the mesophilic, Gram-positive, lactic acid bacterium (LAB) Lactococcus lactis to produce an array of fermented milk products. Milk fermentation processes are susceptible to contamination by virulent phages, but a plethora of phage control strategies are available. One of the most efficient is to use LAB strains carrying phage resistance systems such as abortive infection (Abi) mechanisms. Yet, the mode of action of most Abi systems remains poorly documented. Here, we shed further light on the antiviral activity of the lactococcal AbiT system. Twenty-eight AbiT-resistant phage mutants derived from the wild-type AbiT-sensitive lactococcal phages p2, bIL170, and P008 were isolated and characterized. Comparative genomic analyses identified three different genes that were mutated in these virulent AbiT-insensitive phage derivatives: e14 (bIL170 [e14bIL170]), orf41 (P008 [orf41P008]), and orf6 (p2 [orf6p2] and P008 [orf6P008]). The genes e14bIL170 and orf41P008 are part of the early-expressed genomic region, but bioinformatic analyses did not identify their putative function. orf6 is found in the phage morphogenesis module. Antibodies were raised against purified recombinant ORF6, and immunoelectron microscopy revealed that it is the major capsid protein (MCP). Coexpression in L. lactis of ORF6p2 and ORF5p2, a protease, led to the formation of procapsids. To our knowledge, AbiT is the first Abi system involving distinct phage genes.
doi:10.1128/AEM.01755-12
PMCID: PMC3457483  PMID: 22820334
16.  The Population and Evolutionary Dynamics of Phage and Bacteria with CRISPR–Mediated Immunity 
PLoS Genetics  2013;9(3):e1003312.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), together with associated genes (cas), form the CRISPR–cas adaptive immune system, which can provide resistance to viruses and plasmids in bacteria and archaea. Here, we use mathematical models, population dynamic experiments, and DNA sequence analyses to investigate the host–phage interactions in a model CRISPR–cas system, Streptococcus thermophilus DGCC7710 and its virulent phage 2972. At the molecular level, the bacteriophage-immune mutant bacteria (BIMs) and CRISPR–escape mutant phage (CEMs) obtained in this study are consistent with those anticipated from an iterative model of this adaptive immune system: resistance by the addition of novel spacers and phage evasion of resistance by mutation in matching sequences or flanking motifs. While CRISPR BIMs were readily isolated and CEMs generated at high rates (frequencies in excess of 10−6), our population studies indicate that there is more to the dynamics of phage–host interactions and the establishment of a BIM–CEM arms race than predicted from existing assumptions about phage infection and CRISPR–cas immunity. Among the unanticipated observations are: (i) the invasion of phage into populations of BIMs resistant by the acquisition of one (but not two) spacers, (ii) the survival of sensitive bacteria despite the presence of high densities of phage, and (iii) the maintenance of phage-limited communities due to the failure of even two-spacer BIMs to become established in populations with wild-type bacteria and phage. We attribute (i) to incomplete resistance of single-spacer BIMs. Based on the results of additional modeling and experiments, we postulate that (ii) and (iii) can be attributed to the phage infection-associated production of enzymes or other compounds that induce phenotypic phage resistance in sensitive bacteria and kill resistant BIMs. We present evidence in support of these hypotheses and discuss the implications of these results for the ecology and (co)evolution of bacteria and phage.
Author Summary
The evidence that the CRISPR regions of the genomes of archaea and bacteria play a role in the ecology and (co)evolution of these microbes and their viruses is overwhelming: (i) the spacers (variable sequences of 26–72 bp of DNA between the repeats of this region) of these prokaryotes are homologous to the DNA of viruses in their communities; (ii) experimentally, the acquisition and incorporation of spacers of viral DNA can protect these organisms from subsequent infection by these viruses; (iii) experimentally, viruses evade this immunity by mutation in homologous protospacers or protospacer-adjacent motifs (PAMs). Not so clear are the nature and magnitude of the role CRISPR plays in this ecology and evolution. Here, we use mathematical models, experiments with Streptococcus thermophilus and the phage 2972, and DNA sequence analyses to explore the contribution of CRISPR–cas immunity to the ecology and (co)evolution of bacteria and their viruses. The results of this study suggest that the contribution of CRISPR to the ecology of bacteria and phage is more modest and limited, and the conditions for a CRISPR–mediated coevolutionary arms race between these organisms more restrictive, than anticipated from models based on the canonical view of phage infection and CRISPR–cas immunity.
doi:10.1371/journal.pgen.1003312
PMCID: PMC3597502  PMID: 23516369
17.  Multilocus Sequence Typing Scheme for the Characterization of 936-Like Phages Infecting Lactococcus lactis 
Applied and Environmental Microbiology  2012;78(13):4646-4653.
Lactococcus lactis phage infections are costly for the dairy industry because they can slow down the fermentation process and adversely impact product safety and quality. Although many strategies have been developed to better control phage populations, new virulent phages continue to emerge. Thus, it is beneficial to develop an efficient method for the routine identification of new phages within a dairy plant to rapidly adapt antiphage tactics. Here, we present a multilocus sequence typing (MLST) scheme for the characterization of the 936-like phages, the most prevalent phage group infecting L. lactis strains worldwide. The proposed MLST system targets the internal portion of five highly conserved genomic sequences belonging to the packaging, morphogenesis, and lysis modules. Our MLST scheme was used to analyze 100 phages with different restriction fragment length polymorphism (RFLP) patterns isolated from 11 different countries between 1971 and 2010. PCR products were obtained for all the phages analyzed, and sequence analysis highlighted the high discriminatory power of the MLST system, detecting 93 different sequence types. A conserved locus within the lys gene (coding for endolysin) was the most discriminative, with 65 distinct alleles. The locus within the mcp gene (major capsid protein) was the most conserved (54 distinct alleles). Phylogenetic analyses of the concatenated sequences exhibited a strong concordance of the clusters with the phage host range, indicating the clonal evolution of these phages. A public database has been set up for the proposed MLST system, and it can be accessed at http://pubmlst.org/bacteriophages/.
doi:10.1128/AEM.00931-12
PMCID: PMC3370485  PMID: 22522686
18.  Biology and Genome Sequence of Streptococcus mutans Phage M102AD 
M102AD is the new designation for a Streptococcus mutans phage described in 1993 as phage M102. This change was necessitated by the genome analysis of another S. mutans phage named M102, which revealed differences from the genome sequence reported here. Additional host range analyses confirmed that S. mutans phage M102AD infects only a few serotype c strains. Phage M102AD adsorbed very slowly to its host, and it cannot adsorb to serotype e and f strains of S. mutans. M102AD adsorption was blocked by c-specific antiserum. Phage M102AD also adsorbed equally well to heat-treated and trypsin-treated cells, suggesting carbohydrate receptors. Saliva and polysaccharide production did not inhibit plaque formation. The genome of this siphophage consisted of a linear, double-stranded, 30,664-bp DNA molecule, with a GC content of 39.6%. Analysis of the genome extremities indicated the presence of a 3′-overhang cos site that was 11 nucleotides long. Bioinformatic analyses identified 40 open reading frames, all in the same orientation. No lysogeny-related genes were found, indicating that phage M102AD is strictly virulent. No obvious virulence factor gene candidates were found. Twelve proteins were identified in the virion structure by mass spectrometry. Comparative genomic analysis revealed a close relationship between S. mutans phages M102AD and M102 as well as with Streptococcus thermophilus phages. This study also highlights the importance of conducting research with biological materials obtained from recognized microbial collections.
doi:10.1128/AEM.07726-11
PMCID: PMC3302630  PMID: 22287009
19.  Cleavage of Phage DNA by the Streptococcus thermophilus CRISPR3-Cas System 
PLoS ONE  2012;7(7):e40913.
Streptococcus thermophilus, similar to other Bacteria and Archaea, has developed defense mechanisms to protect cells against invasion by foreign nucleic acids, such as virus infections and plasmid transformations. One defense system recently described in these organisms is the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats loci coupled to CRISPR-associated genes). Two S. thermophilus CRISPR-Cas systems, CRISPR1-Cas and CRISPR3-Cas, have been shown to actively block phage infection. The CRISPR1-Cas system interferes by cleaving foreign dsDNA entering the cell in a length-specific and orientation-dependant manner. Here, we show that the S. thermophilus CRISPR3-Cas system acts by cleaving phage dsDNA genomes at the same specific position inside the targeted protospacer as observed with the CRISPR1-Cas system. Only one cleavage site was observed in all tested strains. Moreover, we observed that the CRISPR1-Cas and CRISPR3-Cas systems are compatible and, when both systems are present within the same cell, provide increased resistance against phage infection by both cleaving the invading dsDNA. We also determined that overall phage resistance efficiency is correlated to the total number of newly acquired spacers in both CRISPR loci.
doi:10.1371/journal.pone.0040913
PMCID: PMC3401199  PMID: 22911717
20.  Phage Morphology Recapitulates Phylogeny: The Comparative Genomics of a New Group of Myoviruses 
PLoS ONE  2012;7(7):e40102.
Among dsDNA tailed bacteriophages (Caudovirales), members of the Myoviridae family have the most sophisticated virion design that includes a complex contractile tail structure. The Myoviridae generally have larger genomes than the other phage families. Relatively few “dwarf” myoviruses, those with a genome size of less than 50 kb such as those of the Mu group, have been analyzed in extenso. Here we report on the genome sequencing and morphological characterization of a new group of such phages that infect a diverse range of Proteobacteria, namely Aeromonas salmonicida phage 56, Vibrio cholerae phages 138 and CP-T1, Bdellovibrio phage φ1422, and Pectobacterium carotovorum phage ZF40. This group of dwarf myoviruses shares an identical virion morphology, characterized by usually short contractile tails, and have genome sizes of approximately 45 kb. Although their genome sequences are variable in their lysogeny, replication, and host adaption modules, presumably reflecting differing lifestyles and hosts, their structural and morphogenesis modules have been evolutionarily constrained by their virion morphology. Comparative genomic analysis reveals that these phages, along with related prophage genomes, form a new coherent group within the Myoviridae. The results presented in this communication support the hypothesis that the diversity of phages may be more structured than generally believed and that the innumerable phages in the biosphere all belong to discrete lineages or families.
doi:10.1371/journal.pone.0040102
PMCID: PMC3391216  PMID: 22792219
21.  Bacteriophages and dairy fermentations 
Bacteriophage  2012;2(3):149-158.
This review highlights the main strategies available to control phage infection during large-scale milk fermentation by lactic acid bacteria. The topics that are emphasized include the factors influencing bacterial activities, the sources of phage contamination, the methods available to detect and quantify phages, as well as practical solutions to limit phage dispersion through an adapted factory design, the control of air flow, the use of adequate sanitizers, the restricted used of recycled products, and the selection and growth of bacterial cultures.
doi:10.4161/bact.21868
PMCID: PMC3530524  PMID: 23275866
bacteriophage; dairy industry; milk fermentation; lactic acid bacteria; phage control strategy
22.  Evolution and classification of the CRISPR-Cas systems 
Nature Reviews. Microbiology  2011;9(6):467-477.
The CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) modules are adaptive immunity systems that are present in many archaea and bacteria. These defence systems are encoded by operons that have an extraordinarily diverse architecture and a high rate of evolution for both the cas genes and the unique spacer content. Here, we provide an updated analysis of the evolutionary relationships between CRISPR–Cas systems and Cas proteins. Three major types of CRISPR–Cas system are delineated, with a further division into several subtypes and a few chimeric variants. Given the complexity of the genomic architectures and the extremely dynamic evolution of the CRISPR–Cas systems, a unified classification of these systems should be based on multiple criteria. Accordingly, we propose a `polythetic' classification that integrates the phylogenies of the most common cas genes, the sequence and organization of the CRISPR repeats and the architecture of the CRISPR–cas loci.
doi:10.1038/nrmicro2577
PMCID: PMC3380444  PMID: 21552286
23.  Staphylococcus epidermidis Bacteriophages from the Anterior Nares of Humans▿ 
Applied and Environmental Microbiology  2011;77(21):7853-7855.
The role of virulent bacteriophages in staphylococcal colonization of the human anterior nares is not known. This report of lytic bacteriophages against Staphylococcus epidermidis in the anterior nares of 5.5% of human subjects (n = 202) suggests their potential role in modulating staphylococcal colonization in this ecological niche.
doi:10.1128/AEM.05367-11
PMCID: PMC3209164  PMID: 21926216
24.  The Proteome and Interactome of Streptococcus pneumoniae Phage Cp-1 ▿ † 
Journal of Bacteriology  2011;193(12):3135-3138.
Mass spectrometry analysis of Streptococcus pneumoniae bacteriophage Cp-1 identified a total of 12 proteins, and proteome-wide yeast two-hybrid screens revealed 17 binary interactions mainly among these structural proteins. On the basis of the resulting linkage map, we suggest an improved structural model of the Cp-1 virion.
doi:10.1128/JB.01481-10
PMCID: PMC3133188  PMID: 21515781
25.  Bacteriophages of lactic acid bacteria and their impact on milk fermentations 
Microbial Cell Factories  2011;10(Suppl 1):S20.
Every biotechnology process that relies on the use of bacteria to make a product or to overproduce a molecule may, at some time, struggle with the presence of virulent phages. For example, phages are the primary cause of fermentation failure in the milk transformation industry. This review focuses on the recent scientific advances in the field of lactic acid bacteria phage research. Three specific topics, namely, the sources of contamination, the detection methods and the control procedures will be discussed.
doi:10.1186/1475-2859-10-S1-S20
PMCID: PMC3231927  PMID: 21995802

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