Enterococcus faecalis Bacteriophage 156 Is an Effective Biotechnological Tool for Reducing the Presence of Tyramine and Putrescine in an Experimental Cheese Model.

Biogenic amines (BA) - nitrogenous compounds of low molecular weight - are the result of metabolism of certain amino acids. They are biologically present in all living organisms and play essential physiological roles. However, their accumulation in foodstuffs due to the metabolic activity of certain microorganisms represents a toxicological risk. Containing such microorganisms, and with an abundance of precursor substrate amino acids, fermented foods in general, and cheeses in particular, provide an ideal matrix for the accumulation of these toxic compounds. Unfortunately, the main microorganisms responsible for BA accumulation are members of the lactic acid bacteria (LAB) group, which are also essential for the development of the organoleptic characteristics of the final product. The methods used to reduce the BA content of cheese, such as milk pasteurization, commonly fail to do so, and affect desirable non-BA-producing LAB as well. Bacteriophages have been proposed as biotechnological tools for diminishing the presence of undesirable microorganisms in dairy products. Given their specificity, they could be used to target the population of BA-producing bacteria. In this work, we aimed to explore the use of Enterococcus faecalis infecting phages as a tool to reduce the content of BA in dairy products. For this, we proceeded to the isolation and characterization of E. faecalis bacteriophage 156, a member of the family Myoviridae. Its genome was sequenced and compared with that of E. faecalis family Myoviridae phages available in public databases. Its capacity to decrease the accumulation of the BA tyramine and putrescine in an experimental laboratory-scale cheese model was proven.

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.

Phage-host interactions in Streptococcus thermophilus: Genome analysis of phages isolated in Uruguay and ectopic spacer acquisition in CRISPR array.

Three cos-type virulent Streptococcus thermophilus phages were isolated from failed mozzarella production in Uruguay. Genome analyses showed that these phages are similar to those isolated elsewhere around the world. The CRISPR1 and CRISPR3 arrays of the three S. thermophilus host strains from Uruguay were also characterized and similarities were noted with previously described model strains SMQ-301, LMD-9 and DGCC7710. Spontaneous bacteriophage-insensitive S. thermophilus mutants (BIMs) were obtained after challenging the phage-sensitive wild-type strain Uy02 with the phage 128 and their CRISPR content was analyzed. Analysis of 23 BIMs indicated that all of them had acquired at least one new spacer in their CRISPR1 array. While 14 BIMs had acquired spacer at the 5'-end of the array, 9 other BIMs acquired a spacer within the array. Comparison of the leader sequence in strains Uy02 and DGCC7710 showed a nucleotide deletion at position -1 in Uy02, which may be responsible for the observed ectopic spacer acquisition. Analysis of the spacer sequences upstream the newly acquired ectopic spacer indicated presence of a conserved adenine residue at position -2. This study indicates that natural strains of S. thermophilus can also acquire spacers within a CRISPR array.

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.

CRISPR-Cas and restriction-modification systems are compatible and increase phage resistance.

Bacteria have developed a set of barriers to protect themselves against invaders such as phage and plasmid nucleic acids. Different prokaryotic defence systems exist and at least two of them directly target the incoming DNA: restriction-modification (R-M) and CRISPR-Cas systems. On their own, they are imperfect barriers to invasion by foreign DNA. Here, we show that R-M and CRISPR-Cas systems are compatible and act together to increase the overall phage resistance of a bacterial cell by cleaving their respective target sites. Furthermore, we show that the specific methylation of phage DNA does not impair CRISPR-Cas acquisition or interference activities. Taken altogether, both mechanisms can be leveraged to decrease phage contaminations in processes relying on bacterial growth and/or fermentation.

Cleavage of phage DNA by the Streptococcus thermophilus CRISPR3-Cas system.

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.

Involvement of the major capsid protein and two early-expressed phage genes in the activity of the lactococcal abortive infection mechanism AbiT.

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 ORF6(p2) and ORF5(p2), a protease, led to the formation of procapsids. To our knowledge, AbiT is the first Abi system involving distinct phage genes.

The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA.

Bacteria and Archaea have developed several defence strategies against foreign nucleic acids such as viral genomes and plasmids. Among them, clustered regularly interspaced short palindromic repeats (CRISPR) loci together with cas (CRISPR-associated) genes form the CRISPR/Cas immune system, which involves partially palindromic repeats separated by short stretches of DNA called spacers, acquired from extrachromosomal elements. It was recently demonstrated that these variable loci can incorporate spacers from infecting bacteriophages and then provide immunity against subsequent bacteriophage infections in a sequence-specific manner. Here we show that the Streptococcus thermophilus CRISPR1/Cas system can also naturally acquire spacers from a self-replicating plasmid containing an antibiotic-resistance gene, leading to plasmid loss. Acquired spacers that match antibiotic-resistance genes provide a novel means to naturally select bacteria that cannot uptake and disseminate such genes. We also provide in vivo evidence that the CRISPR1/Cas system specifically cleaves plasmid and bacteriophage double-stranded DNA within the proto-spacer, at specific sites. Our data show that the CRISPR/Cas immune system is remarkably adapted to cleave invading DNA rapidly and has the potential for exploitation to generate safer microbial strains.

Structure and function of phage p2 ORF34(p2), a new type of single-stranded DNA binding protein.

Lactococcus lactis, a Gram-positive bacterium widely used by the dairy industry, is subject to infection by a diverse population of virulent phages, predominantly by those of the 936 group, including the siphovirus phage p2. Confronted with the negative impact of phage infection on milk fermentation, the study of the biology of lactococcal provides insight from applied and fundamental perspectives. We decided to characterize the product of the orf34 gene from lactococcus phage p2, which was considered as a candidate single-stranded DNA binding protein (SSB) due to its localization downstream of a gene coding for a single-strand annealing protein. Two-dimensional gel electrophoresis showed that ORF34(p2) is expressed in large amounts during the early phases of phage infection, suggesting an important role in this process. Gel-shift assays, surface plasmon resonance and atomic force microscopy demonstrated that ORF34(p2) interacts with single-strand DNA with nanomolar affinity. We also determined the crystal structure of ORF34(p2) and showed that it bears a variation of the typical oligonucleotide/oligosaccharide binding-fold of SSBs. Finally, we found that ORF34(p2) is able to stimulate Escherichia coli RecA-mediated homologous recombination. The specific structural and biochemical properties that distinguish ORF34(p2) from other SSB proteins are discussed.

Fast real-time polymerase chain reaction for quantitative detection of Lactobacillus delbrueckii bacteriophages in milk.

One of the main microbiological problems of the dairy industry is the susceptibility of starter bacteria to virus infections. Lactobacillus delbrueckii, a component of thermophilic starter cultures used in the manufacture of several fermented dairy products, including yogurt, is also sensitive to bacteriophage attacks. To avoid the problems associated with these viruses, quick and sensitive detection methods are necessary. In the present study, a fast real-time quantitative polymerase chain reaction assay for the direct detection and quantification of L. delbrueckii phages in milk was developed. A set of primers and a TaqMan MGB probe was designed, based on the lysin gene sequence of different L. delbrueckii phages. The results show the proposed method to be a rapid (total processing time 30 min), specific and highly sensitive technique for detecting L. delbrueckii phages in milk.

Multiplex fast real-time PCR for quantitative detection and identification of cos- and pac-type Streptococcus thermophilus bacteriophages.

The fermentation of milk by Streptococcus thermophilus is a widespread industrial process that is susceptible to bacteriophage attack. In this work, a preventive fast real-time PCR method for the detection, quantification, and identification of types of S. thermophilus phages in 30 min is described.