Longitudinal Study of Lactococcus Phages in a Canadian Cheese Factory.

The presence of virulent phages is closely monitored during cheese manufacturing, as these bacterial viruses can significantly slow down the milk fermentation process and lead to low-quality cheeses. From 2001 to 2020, whey samples from cheddar cheese production in a Canadian factory were monitored for the presence of virulent phages capable of infecting proprietary strains of Lactococcus cremoris and Lactococcus lactis used in starter cultures. Phages were successfully isolated from 932 whey samples using standard plaque assays and several industrial Lactococcus strains as hosts. A multiplex PCR assay assigned 97% of these phage isolates to the Skunavirus genus, 2% to the P335 group, and 1% to the Ceduovirus genus. DNA restriction profiles and a multilocus sequence typing (MLST) scheme distinguished at least 241 unique lactococcal phages from these isolates. While most phages were isolated only once, 93 of them (out of 241, 39%) were isolated multiple times. Phage GL7 was isolated 132 times from 2006 to 2020, demonstrating that phages can persist in a cheese factory for long periods of time. Phylogenetic analysis of MLST sequences showed that phages could be clustered based on their bacterial hosts rather than their year of isolation. Host range analysis showed that Skunavirus phages exhibited a very narrow host range, whereas some Ceduovirus and P335 phages had a broader host range. Overall, the host range information was useful in improving the starter culture rotation by identifying phage-unrelated strains and helped mitigating the risk of fermentation failure due to virulent phages. IMPORTANCE Although lactococcal phages have been observed in cheese production settings for almost a century, few longitudinal studies have been performed. This 20-year study describes the close monitoring of dairy lactococcal phages in a cheddar cheese factory. Routine monitoring was conducted by factory staff, and when whey samples were found to inhibit industrial starter cultures under laboratory conditions, they were sent to an academic research laboratory for phage isolation and characterization. This led to a collection of at least 241 unique lactococcal phages, which were characterized through PCR typing and MLST profiling. Phages of the Skunavirus genus were by far the most dominant. Most phages lysed a small subset of the Lactococcus strains. These findings guided the industrial partner in adapting the starter culture schedule by using phage-unrelated strains in starter cultures and removing some strains from the starter rotation. This phage control strategy could be adapted for other large-scale bacterial fermentation processes.

Lactococcus lactis type III-A CRISPR-Cas system cleaves bacteriophage RNA.

CRISPR-Cas defends microbial cells against invading nucleic acids including viral genomes. Recent studies have shown that type III-A CRISPR-Cas systems target both RNA and DNA in a transcription-dependent manner. We previously found a type III-A system on a conjugative plasmid in Lactococcus lactis which provided resistance against virulent phages of the Siphoviridae family. Its naturally occurring spacers are oriented to generate crRNAs complementary to target phage mRNA, suggesting transcription-dependent targeting. Here, we show that only constructs whose spacers produce crRNAs complementary to the phage mRNA confer phage resistance in L. lactis. In vivo nucleic acid cleavage assays showed that cleavage of phage dsDNA genome was not detected within phage-infected L. lactis cells. On the other hand, Northern blots indicated that the lactococcal CRISPR-Cas cleaves phage mRNA in vivo. These results cannot exclude that single-stranded phage DNA is not being targeted, but phage DNA replication has been shown to be impaired.

The CRISPR-Cas Immune System and Genetic Transfers: Reaching an Equilibrium.

Horizontal gene transfer drives the evolution of bacterial genomes, including the adaptation to changing environmental conditions. Exogenous DNA can enter a bacterial cell through transformation (free DNA or plasmids) or through the transfer of mobile genetic elements by conjugation (plasmids) and transduction (bacteriophages). Favorable genes can be acquired, but undesirable traits can also be inadvertently acquired through these processes. Bacteria have systems, such as clustered regularly interspaced short palindromic repeat CRISPR-associated genes (CRISPR-Cas), that can cleave foreign nucleic acid molecules. In this review, we discuss recent advances in understanding CRISPR-Cas system activity against mobile genetic element transfer through transformation and conjugation. We also highlight how CRISPR-Cas systems influence bacterial evolution and how CRISPR-Cas components affect plasmid replication.

Revenge of the phages: defeating bacterial defences.

Bacteria and their viral predators (bacteriophages) are locked in a constant battle. In order to proliferate in phage-rich environments, bacteria have an impressive arsenal of defence mechanisms, and in response, phages have evolved counter-strategies to evade these antiviral systems. In this Review, we describe the various tactics that are used by phages to overcome bacterial resistance mechanisms, including adsorption inhibition, restriction-modification, CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems and abortive infection. Furthermore, we consider how these observations have enhanced our knowledge of phage biology, evolution and phage-host interactions.

Effect of the abortive infection mechanism and type III toxin/antitoxin system AbiQ on the lytic cycle of Lactococcus lactis phages.

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.

Structure and activity of AbiQ, a lactococcal endoribonuclease belonging to the type III toxin-antitoxin system.

AbiQ is a phage resistance mechanism found on a native plasmid of Lactococcus lactis that abort virulent phage infections. In this study, we experimentally demonstrate that AbiQ belongs to the recently described type III toxin-antitoxin systems. When overexpressed, the AbiQ protein (ABIQ) is toxic and causes bacterial death in a bacteriostatic manner. Northern and Western blot experiments revealed that the abiQ gene is transcribed and translated constitutively, and its expression is not activated by a phage product. ABIQ is an endoribonuclease that specifically cleaves its cognate antitoxin RNA molecule in vivo. The crystal structure of ABIQ was solved and site-directed mutagenesis identified key amino acids for its anti-phage and/or its RNase function. The AbiQ system is the first lactococcal abortive infection system characterized to date at a structural level.

Bacteriophages in food fermentations: new frontiers in a continuous arms race.

Phage contamination represents an important risk to any process requiring bacterial growth, particularly in the biotechnology and food industries. The presence of unwanted phages may lead to manufacturing delays, lower quality product, or, in the worst cases, total production loss. Thus, constant phage monitoring and stringent application of the appropriate control measures are indispensable. In fact, a systematic preventive approach to phage contamination [phage analysis and critical control points (PACCP)] should be put in place. In this review, sources of phage contamination and novel phage detection methods are described, with an emphasis on bacterial viruses that infect lactic acid bacteria used in food fermentations. Recent discoveries related to antiphage systems that are changing our views on phage-host interactions are highlighted. Finally, future directions are also discussed.

Lactococcal abortive infection protein AbiV interacts directly with the phage protein SaV and prevents translation of phage proteins.

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.

Characterization of Lactococcus lactis phage 949 and comparison with other lactococcal phages.

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.

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.