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1.  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.
PMCID: PMC3754610  PMID: 23813728
2.  The double-edged sword of CRISPR-Cas systems 
Cell Research  2012;23(1):15-17.
PMCID: PMC3541657  PMID: 22945354
3.  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.
PMCID: PMC3697491  PMID: 23666331
4.  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.
PMCID: PMC3623202  PMID: 23396328
5.  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.
PMCID: PMC3502894  PMID: 23042172
6.  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.
PMCID: PMC3457483  PMID: 22820334
7.  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.
PMCID: PMC3597502  PMID: 23516369
8.  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
PMCID: PMC3370485  PMID: 22522686
9.  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.
PMCID: PMC3302630  PMID: 22287009
10.  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.
PMCID: PMC3401199  PMID: 22911717
11.  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.
PMCID: PMC3391216  PMID: 22792219
12.  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.
PMCID: PMC3530524  PMID: 23275866
bacteriophage; dairy industry; milk fermentation; lactic acid bacteria; phage control strategy
13.  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.
PMCID: PMC3380444  PMID: 21552286
14.  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.
PMCID: PMC3209164  PMID: 21926216
15.  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.
PMCID: PMC3133188  PMID: 21515781
16.  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.
PMCID: PMC3231927  PMID: 21995802
17.  Genome Annotation and Intraviral Interactome for the Streptococcus pneumoniae Virulent Phage Dp-1▿ ¶  
Journal of Bacteriology  2010;193(2):551-562.
Streptococcus pneumoniae causes several diseases, including pneumonia, septicemia, and meningitis. Phage Dp-1 is one of the very few isolated virulent S. pneumoniae bacteriophages, but only a partial characterization is currently available. Here, we confirmed that Dp-1 belongs to the family Siphoviridae. Then, we determined its complete genomic sequence of 56,506 bp. It encodes 72 open reading frames, of which 44 have been assigned a function. We have identified putative promoters, Rho-independent terminators, and several genomic clusters. We provide evidence that Dp-1 may be using a novel DNA replication system as well as redirecting host protein synthesis through queuosine-containing tRNAs. Liquid chromatography-mass spectrometry analysis of purified phage Dp-1 particles identified at least eight structural proteins. Finally, using comprehensive yeast two-hybrid screens, we identified 156 phage protein interactions, and this intraviral interactome was used to propose a structural model of Dp-1.
PMCID: PMC3019816  PMID: 21097633
18.  Detection of Airborne Lactococcal Bacteriophages in Cheese Manufacturing Plants▿  
The dairy industry adds starter bacterial cultures to heat-treated milk to control the fermentation process during the manufacture of many cheeses. These highly concentrated bacterial populations are susceptible to virulent phages that are ubiquitous in cheese factories. In this study, the dissemination of these phages by the airborne route and their presence on working surfaces were investigated in a cheese factory. Several surfaces were swabbed, and five air samplers (polytetrafluoroethylene filter, polycarbonate filter, BioSampler, Coriolis cyclone sampler, and NIOSH two-stage cyclone bioaerosol personal sampler) were tested. Samples were then analyzed for the presence of two Lactococcus lactis phage groups (936 and c2), and quantification was done by quantitative PCR (qPCR). Both lactococcal phage groups were found on most swabbed surfaces, while airborne phages were detected at concentrations of at least 103 genomes/m3 of air. The NIOSH sampler had the highest rate of air samples with detectable levels of lactococcal phages. This study demonstrates that virulent phages can circulate through the air and that they are ubiquitous in cheese manufacturing facilities.
PMCID: PMC3020544  PMID: 21115712
19.  A reverse transcriptase-related protein mediates phage resistance and polymerizes untemplated DNA in vitro 
Nucleic Acids Research  2011;39(17):7620-7629.
Reverse transcriptases (RTs) are RNA-dependent DNA polymerases that usually function in the replication of selfish DNAs such as retrotransposons and retroviruses. Here, we have biochemically characterized a RT-related protein, AbiK, which is required for abortive phage infection in the Gram-positive bacterium Lactococcus lactis. In vitro, AbiK does not exhibit the properties expected for an RT, but polymerizes long DNAs of ‘random’ sequence, analogous to a terminal transferase. Moreover, the polymerized DNAs appear to be covalently attached to the AbiK protein, presumably because an amino acid serves as a primer. Mutagenesis experiments indicate that the polymerase activity resides in the RT motifs and is essential for phage resistance in vivo. These results establish a novel biochemical property and a non-replicative biological role for a polymerase.
PMCID: PMC3177184  PMID: 21676997
20.  Lactococcal Abortive Infection Protein AbiV Interacts Directly with the Phage Protein SaV and Prevents Translation of Phage Proteins▿  
Applied and Environmental Microbiology  2010;76(21):7085-7092.
AbiV is an abortive infection protein that inhibits the lytic cycle of several virulent phages infecting Lactococcus lactis, while a mutation in the phage gene sav confers insensitivity to AbiV. In this study, we have further characterized the effects of the bacterial AbiV and its interaction with the phage p2 protein SaV. First, we showed that during phage infection of lactococcal AbiV+ cells, AbiV rapidly inhibited protein synthesis. Among early phage transcripts, sav gene transcription was slightly inhibited while the SaV protein could not be detected. Analyses of other phage p2 mRNAs and proteins suggested that AbiV blocks the activation of late gene transcription, probably by a general inhibition of translation. Using size exclusion chromatography coupled with on-line static light scattering and refractometry, as well as fluorescence quenching experiments, we also demonstrated that both AbiV and SaV formed homodimers and that they strongly and specifically interact with each other to form a stable protein complex.
PMCID: PMC2976256  PMID: 20851990
21.  Characterization of Lactococcus lactis Phage 949 and Comparison with Other Lactococcal Phages▿  
Applied and Environmental Microbiology  2010;76(20):6843-6852.
The virulent Lactococcus lactis phage 949 was isolated in 1975 from cheese whey in New Zealand. This phage is a member of the Siphoviridae family and of a rare lactococcal phage group that bears its name (949 group). It has an icosahedral capsid (79-nm diameter) and a very long noncontractile tail (length, 500 nm; width, 12 nm). It infected 7 of 59 tested L. lactis strains, a somewhat expanded host range for a rare lactococcal phage. The abortive phage infection defense mechanisms AbiQ and AbiT strongly inhibited the multiplication of phage 949, but AbiK and AbiV did not. Its double-stranded DNA (dsDNA) genome of 114,768 bp is, to date, the largest among lactococcal phages. Its GC content was calculated at 32.7%, which is the lowest reported for a lactococcal phage. Its 154 open reading frames (ORFs) share limited identity with database sequences. In addition, terminal redundancy was observed as well as the presence of six tRNAs, one group I intron, and putative recombinases. SDS-PAGE coupled with mass spectrometry identified 13 structural proteins. The genomes of the members of the 10 currently known L. lactis phage groups were used to construct a proteomic tree. Each L. lactis phage group separated into distinct genetic clusters, validating the current classification scheme. Of note, members of the polythetic P335 groups were clearly separated into subgroups.
PMCID: PMC2953028  PMID: 20802084
22.  Genome Organization and Characterization of the Virulent Lactococcal Phage 1358 and Its Similarities to Listeria Phages▿  
Virulent phage 1358 is the reference member of a rare group of phages infecting Lactococcus lactis. Electron microscopy revealed a typical icosahedral capsid connected to one of the smallest noncontractile tails found in a lactococcal phage of the Siphoviridae family. Microbiological characterization identified a burst size of 72 virions released per infected host cell and a latent period of 90 min. The host range of phage 1358 was limited to 3 out of the 60 lactococcal strains tested. Moreover, this phage was insensitive to four Abi systems (AbiK, AbiQ, AbiT, and AbiV). The genome of phage 1358 consisted of a linear, double-stranded, 36,892-bp DNA molecule containing 43 open reading frames (ORFs). At least 14 ORFs coded for structural proteins, as identified by SDS-PAGE coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The genomic organization was similar to those of other siphophages. All genes were on the same coding strand and in the same orientation. This lactococcal phage was unique, however, in its 51.4% GC content, much higher than those of other phages infecting this low-GC Gram-positive host. A bias for GC-rich codons was also observed. Comparative analyses showed that several phage 1358 structural proteins shared similarity with two Listeria monocytogenes phages, P35 and P40. The possible origin and evolution of lactococcal phage 1358 is discussed.
PMCID: PMC2832367  PMID: 20061452
23.  Evolution of Lactococcus lactis Phages within a Cheese Factory▿  
Applied and Environmental Microbiology  2009;75(16):5336-5344.
We have sequenced the double-stranded DNA genomes of six lactococcal phages (SL4, CB13, CB14, CB19, CB20, and GR7) from the 936 group that were isolated over a 9-year period from whey samples obtained from a Canadian cheese factory. These six phages infected the same two industrial Lactococcus lactis strains out of 30 tested. The CB14 and GR7 genomes were found to be 100% identical even though they were isolated 14 months apart, indicating that a phage can survive in a cheese plant for more than a year. The other four genomes were related but notably different. The length of the genomes varied from 28,144 to 32,182 bp, and they coded for 51 to 55 open reading frames. All five genomes possessed a 3′ overhang cos site that was 11 nucleotides long. Several structural proteins were also identified by nano-high-performance liquid chromatography-tandem mass spectrometry, confirming bioinformatic analyses. Comparative analyses suggested that the most recently isolated phages (CB19 and CB20) were derived, in part, from older phage isolates (CB13 and CB14/GR7). The organization of the five distinct genomes was similar to the previously sequenced lactococcal phage genomes of the 936 group, and from these sequences, a core genome was determined for lactococcal phages of the 936 group.
PMCID: PMC2725462  PMID: 19542338
24.  Crystal Structure of a Chimeric Receptor Binding Protein Constructed from Two Lactococcal Phages▿ † 
Journal of Bacteriology  2009;191(10):3220-3225.
Lactococcus lactis, a gram-positive bacterium widely used by the dairy industry to manufacture cheeses, is subject to infection by a diverse population of virulent phages. We have previously determined the structures of three receptor binding proteins (RBPs) from lactococcal phages TP901-1, p2, and bIL170, each of them having a distinct host range. Virulent phages p2 and bIL170 are classified within the 936 group, while the temperate phage TP901-1 is a member of the genetically distinct P335 polythetic group. These RBPs comprise three domains: the N-terminal domain, binding to the virion particle; a β-helical linker domain; and the C-terminal domain, bearing the receptor binding site used for host recognition. Here, we have designed, expressed, and determined the structure of an RBP chimera in which the N-terminal and linker RBP domains of phage TP901-1 (P335) are fused to the C-terminal RBP domain of phage p2 (936). This chimera exhibits a stable structure that closely resembles the parental structures, while a slight displacement of the linker made RBP domain adaptation efficient. The receptor binding site is structurally indistinguishable from that of native p2 RBP and binds glycerol with excellent affinity.
PMCID: PMC2687176  PMID: 19286807
25.  Activation and Transfer of the Chromosomal Phage Resistance Mechanism AbiV in Lactococcus lactis▿  
Applied and Environmental Microbiology  2009;75(10):3358-3361.
AbiV is a chromosomally encoded phage resistance mechanism that is silent in the wild-type phage-sensitive strain Lactococcus lactis subsp. cremoris MG1363. Spontaneous phage-resistant mutants of L. lactis MG1363 were analyzed by reverse transcriptase PCR and shown to express AbiV. This expression was related to a reorganization in the upstream region of abiV. Transfer of abiV between two lactococcal strains, most likely by conjugation, was also demonstrated. To our knowledge, this is the first report of natural transfer of a chromosomally encoded phage resistance mechanism.
PMCID: PMC2681626  PMID: 19286782

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