Simon J. Labrie

Bacteriophage resistance mechanisms

Phages are now acknowledged as the most abundant microorganisms on the planet and are also possibly the most diversified. This diversity is mostly driven by their dynamic adaptation when facing selective pressure such as phage resistance mechanisms, which are widespread in bacterial hosts. When infecting bacterial cells, phages face a range of antiviral mechanisms, and they have evolved multiple tactics to avoid, circumvent or subvert these mechanisms in order to thrive in most environments. In this Review, we highlight the most important antiviral mechanisms of bacteria as well as the counter-attacks used by phages to evade these systems.

Abortive Infection Mechanisms and Prophage Sequences Significantly Influence the Genetic Makeup of Emerging Lytic Lactococcal Phages

In this study, we demonstrated the remarkable genome plasticity of lytic lactococcal phages that allows them to rapidly adapt to the dynamic dairy environment. The lytic double-stranded DNA phage ul36 was used to sequentially infect a wild-type strain of Lactococcus lactis and two isogenic derivatives with genes encoding two phage resistance mechanisms, AbiK and AbiT. Four phage mutants resistant to one or both Abi mechanisms were isolated. Comparative analysis of their complete genomes, as well as morphological observations, revealed that phage ul36 extensively evolved by large-scale homologous and nonhomologous recombination events with the inducible prophage present in the host strain. One phage mutant exchanged as much as 79% of its genome compared to the core genome of ul36. Thus, natural phage defense mechanisms and prophage elements found in bacterial chromosomes contribute significantly to the evolution of the lytic phage population.

Genomes of marine cyanopodoviruses reveal multiple origins of diversity

The marine cyanobacteria Prochlorococcus and Synechococcus are highly abundant in the global oceans, as are the cyanophage with which they co-evolve. While genomic analyses have been relatively extensive for cyanomyoviruses, only three cyanopodoviruses isolated on marine cyanobacteria have been sequenced. Here we present nine new cyanopodovirus genomes, and analyse them in the context of the broader group. The genomes range from 42.2 to 47.7 kb, with G+C contents consistent with those of their hosts. They share 12 core genes, and the pan-genome is not close to being fully sampled. The genomes contain three variable island regions, with the most hypervariable genes concentrated at one end of the genome. Concatenated core-gene phylogeny clusters all but one of the phage into three distinct groups (MPP-A and two discrete clades within MPP-B). The outlier, P-RSP2, has the smallest genome and lacks RNA polymerase, a hallmark of the Autographivirinae subfamily. The phage in group MPP-B contain photosynthesis and carbon metabolism associated genes, while group MPP-A and the outlier P-RSP2 do not, suggesting different constraints on their lytic cycles. Four of the phage encode integrases and three have a host integration signature. Metagenomic analyses reveal that cyanopodoviruses may be more abundant in the oceans than previously thought.

Morphology, Genome Sequence, and Structural Proteome of Type Phage P335 from Lactococcus lactis

ABSTRACT Lactococcus lactis phage P335 is a virulent type phage for the species that bears its name and belongs to the Siphoviridae family. Morphologically, P335 resembled the L. lactis phages TP901-1 and Tuc2009, except for a shorter tail and a different collar/whisker structure. Its 33,613-bp double-stranded DNA genome had 50 open reading frames. Putative functions were assigned to 29 of them. Unlike other sequenced genomes from lactococcal phages belonging to this species, P335 did not have a lysogeny module. However, it did carry a dUTPase gene, the most conserved gene among this phage species. Comparative genomic analyses revealed a high level of identity between the morphogenesis modules of the phages P335, ul36, TP901-1, and Tuc2009 and two putative prophages of L. lactis SK11. Differences were noted in genes coding for receptor-binding proteins, in agreement with their distinct host ranges. Sixteen structural proteins of phage P335 were identified by liquid chromatography-tandem mass spectrometry. A 2.8-kb insertion was recognized between the putative genes coding for the activator of late transcription (Alt) and the small terminase subunit (TerS). Four genes within this region were autonomously late transcribed and possibly under the control of Alt. Three of the four deduced proteins had similarities with proteins from Streptococcus pyogenes prophages, suggesting that P335 acquired this module from another phage genome. The genetic diversity of the P335 species indicates that they are exceptional models for studying the modular theory of phage evolution.

Characterization of two polyvalent phages infecting Enterobacteriaceae.

Bacteriophages display remarkable genetic diversity and host specificity. In this study, we explore phages infecting bacterial strains of the Enterobacteriaceae family because of their ability to infect related but distinct hosts. We isolated and characterized two novel virulent phages, SH6 and SH7, using a strain of Shigella flexneri as host bacterium. Morphological and genomic analyses revealed that phage SH6 belongs to the T1virus genus of the Siphoviridae family. Conversely, phage SH7 was classified in the T4virus genus of the Myoviridae family. Phage SH6 had a short latent period of 16 min and a burst size of 103 ± 16 PFU/infected cell while the phage SH7 latent period was 23 min with a much lower burst size of 26 ± 5 PFU/infected cell. Moreover, phage SH6 was sensitive to acidic conditions (pH < 5) while phage SH7 was stable from pH 3 to 11 for 1 hour. Of the 35 bacterial strains tested, SH6 infected its S. flexneri host strain and 8 strains of E. coli. Phage SH7 lysed additionally strains of E. coli O157:H7, Salmonella Paratyphi, and Shigella dysenteriae. The broader host ranges of these two phages as well as their microbiological properties suggest that they may be useful for controlling bacterial populations.

Lactococcal Abortive Infection Protein AbiV Interacts Directly with the Phage Protein SaV and Prevents Translation of Phage Proteins

ABSTRACT 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.

Inactivation of dairy bacteriophages by commercial sanitizers and disinfectants.

Many commercial sanitizers and disinfectants have been used over the years to control microbial contamination but their efficacy on phages is often unknown. Here, 23 commercial chemical products, including 21 food-grade sanitizers were tested against virulent dairy phages. These food-grade chemicals included oxidizing agents, halogenated agents, alcohols, quaternary ammonium compounds, anionic acids, iodine-based acids, and an amphoteric chemical. Phage P008 was first exposed to each sanitizer for 2 and 15min at room temperature and at two different concentrations, namely the lowest and highest no-rinse sanitizing concentrations. Organic matter (whey or milk) was also added to the testing solutions. At the end of the exposure period, the test solution was neutralized and the number of infectious phages was determined by plaque assays. The five most efficient sanitizers against phage P008 (<4 log of inactivation) were then tested against virulent lactococcal phages P008, CB13, AF6, P1532 of the 936 group, P001 (c2), Q54, and 1358 as well as Lactobacillus plantarum phage B1 and Streptococcus thermophilus phage 2972 using the same protocol. The oxidizing agents and the quaternary ammonium compounds were the most efficient against all phages although phages CB13 and P1532 were less sensitive to these chemicals than the other phages. This study may help in the selection of appropriate chemicals for controlling phage contamination in industrial factories and research laboratories.

Involvement of the Major Capsid Protein and Two Early-Expressed Phage Genes in the Activity of the Lactococcal Abortive Infection Mechanism AbiT

ABSTRACT 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 [e14 bIL170]), orf41 (P008 [orf41 P008]), and orf6 (p2 [orf6 p2] and P008 [orf6 P008]). The genes e14 bIL170 and orf41 P008 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.

Programming Native CRISPR Arrays for the Generation of Targeted Immunity

ABSTRACT The adaptive immune system of prokaryotes, called CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes), results in specific cleavage of invading nucleic acid sequences recognized by the cell’s “memory” of past encounters. Here, we exploited the properties of native CRISPR-Cas systems to program the natural “memorization” process, efficiently generating immunity not only to a bacteriophage or plasmid but to any specifically chosen DNA sequence. IMPORTANCE CRISPR-Cas systems have entered the public consciousness as genome editing tools due to their readily programmable nature. In industrial settings, natural CRISPR-Cas immunity is already exploited to generate strains resistant to potentially disruptive viruses. However, the natural process by which bacteria acquire new target specificities (adaptation) is difficult to study and manipulate. The target against which immunity is conferred is selected stochastically. By biasing the immunization process, we offer a means to generate customized immunity, as well as provide a new tool to study adaptation. CRISPR-Cas systems have entered the public consciousness as genome editing tools due to their readily programmable nature. In industrial settings, natural CRISPR-Cas immunity is already exploited to generate strains resistant to potentially disruptive viruses. However, the natural process by which bacteria acquire new target specificities (adaptation) is difficult to study and manipulate. The target against which immunity is conferred is selected stochastically. By biasing the immunization process, we offer a means to generate customized immunity, as well as provide a new tool to study adaptation.

Investigating the requirement for calcium during lactococcal phage infection.

Calcium is widely used in the study and successful propagation of virulent bacteriophages infecting lactic acid bacteria; however, it has not been assessed if and why this divalent cation is required for the infection process. Lactococcal phages are a persistent problem in the calcium-rich dairy environment and therefore were used as the model for this study. Using representative members of nine of the currently recognized ten lactococcal phage groups, encompassing phages of the Podoviridae and Siphoviridae families, we present data to suggest that calcium is not an explicit requirement for many of these phages. However, calcium expedited the pace of the lytic cycle for certain phages. Additionally, for calcium-dependent phages belonging to the 936 siphogroup, we could substitute this cation with magnesium or manganese, indicating that these phages are more adaptable than lactococcal phages of other groups. We postulate that the ability of phages to adapt to their environment and to harness the available mineral content may ultimately decide the success of a given phage infection. This may explain, in part, why 936 phages are one of the most frequently isolated phages in the dairy industry.