Cas9 Allosteric Inhibition by the Anti-CRISPR Protein AcrIIA6.

In the arms race against bacteria, bacteriophages have evolved diverse anti-CRISPR proteins (Acrs) that block CRISPR-Cas immunity. Acrs play key roles in the molecular coevolution of bacteria with their predators, use a variety of mechanisms of action, and provide tools to regulate Cas-based genome manipulation. Here, we present structural and functional analyses of AcrIIA6, an Acr from virulent phages, exploring its unique anti-CRISPR action. Our cryo-EM structures and functional data of AcrIIA6 binding to Streptococcus thermophilus Cas9 (St1Cas9) show that AcrIIA6 acts as an allosteric inhibitor and induces St1Cas9 dimerization. AcrIIA6 reduces St1Cas9 binding affinity for DNA and prevents DNA binding within cells. The PAM and AcrIIA6 recognition sites are structurally close and allosterically linked. Mechanistically, AcrIIA6 affects the St1Cas9 conformational dynamics associated with PAM binding. Finally, we identify a natural St1Cas9 variant resistant to AcrIIA6 illustrating Acr-driven mutational escape and molecular diversification of Cas9 proteins.

Fermentation Practices Select for Thermostable Endolysins in Phages.

Endolysins are produced by (bacterio)phages and play a crucial role in degrading the bacterial cell wall and the subsequent release of new phage progeny. These lytic enzymes exhibit a remarkable diversity, often occurring in a multimodular form that combines different catalytic and cell wall-binding domains, even in phages infecting the same species. Yet, our current understanding lacks insight into how environmental factors and ecological niches may have influenced the evolution of these enzymes. In this study, we focused on phages infecting Streptococcus thermophilus, as this bacterial species has a well-defined and narrow ecological niche, namely, dairy fermentation. Among the endolysins found in phages targeting this species, we observed limited diversity, with a singular structural type dominating in most of identified S. thermophilus phages. Within this prevailing endolysin type, we discovered a novel and highly conserved calcium-binding motif. This motif proved to be crucial for the stability and activity of the enzyme at elevated temperatures. Ultimately, we demonstrated its positive selection within the host's environmental conditions, particularly under the temperature profiles encountered in the production of yogurt, mozzarella, and hard cheeses that rely on S. thermophilus.

Phage endolysins are adapted to specific hosts and are evolutionarily dynamic.

Endolysins are produced by (bacterio)phages to rapidly degrade the bacterial cell wall and release new viral particles. Despite sharing a common function, endolysins present in phages that infect a specific bacterial species can be highly diverse and vary in types, number, and organization of their catalytic and cell wall binding domains. While much is now known about the biochemistry of phage endolysins, far less is known about the implication of their diversity on phage-host adaptation and evolution. Using CRISPR-Cas9 genome editing, we could genetically exchange a subset of different endolysin genes into distinct lactococcal phage genomes. Regardless of the type and biochemical properties of these endolysins, fitness costs associated to their genetic exchange were marginal if both recipient and donor phages were infecting the same bacterial strain, but gradually increased when taking place between phage that infect different strains or bacterial species. From an evolutionary perspective, we observed that endolysins could be naturally exchanged by homologous recombination between phages coinfecting a same bacterial strain. Furthermore, phage endolysins could adapt to their new phage/host environment by acquiring adaptative mutations. These observations highlight the remarkable ability of phage lytic systems to recombine and adapt and, therefore, explain their large diversity and mosaicism. It also indicates that evolution should be considered to act on functional modules rather than on bacteriophages themselves. Furthermore, the extensive degree of evolvability observed for phage endolysins offers new perspectives for their engineering as antimicrobial agents.

Phagebook: The Social Network.

Much like social networks are used to connect with friends or relatives, bacteria communicate with relatives through quorum sensing. Viruses, though, were thought to be asocial-until now. Erez et al. (2017) reveal that viruses are also sharing information with relatives.

Building pyramids against the evolutionary emergence of pathogens.

Mutations allowing pathogens to escape host immunity promote the spread of infectious diseases in heterogeneous host populations and can lead to major epidemics. Understanding the conditions that slow down this evolution is key for the development of durable control strategies against pathogens. Here, we use theory and experiments to compare the efficacy of three strategies for the deployment of resistance: (i) a mixing strategy where the host population contains two single-resistant genotypes, (ii) a pyramiding strategy where the host carries a double-resistant genotype, (iii) a combining strategy where the host population is a mix of a single-resistant genotype and a double-resistant genotype. First, we use evolutionary epidemiology theory to clarify the interplay between demographic stochasticity and evolutionary dynamics to show that the pyramiding strategy always yields lower probability of evolutionary emergence. Second, we test experimentally these predictions with the introduction of bacteriophages into bacterial populations where we manipulated the diversity and the depth of immunity using a Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated (CRISPR-Cas) system. These biological assays confirm that pyramiding multiple defences into the same host genotype and avoiding combination with single-defence genotypes is a robust way to reduce pathogen evolutionary emergence. The experimental validation of these theoretical recommendations has practical implications in various areas, including for the optimal deployment of resistance varieties in agriculture and for the design of durable vaccination strategies.

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9.

Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide a structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes.

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.

Detection of nucleotide modifications in bacteria and bacteriophages: Strengths and limitations of current technologies and software.

RNA and DNA modifications occur in eukaryotes and prokaryotes, as well as in their viruses, and serve a wide range of functions, from gene regulation to nucleic acid protection. Although the first nucleotide modification was discovered almost 100 years ago, new and unusual modifications are still being described. Nucleotide modifications have also received more attention lately because of their increased significance, but also because new sequencing approaches have eased their detection. Chiefly, third generation sequencing platforms PacBio and Nanopore offer direct detection of modified bases by measuring deviations of the signals. These unusual modifications are especially prevalent in bacteriophage genomes, the viruses of bacteria, where they mostly appear to protect DNA against degradation from host nucleases. In this Opinion article, we highlight and discuss current approaches to detect nucleotide modifications, including hardwares and softwares, and look onward to future applications, especially for studying unusual, rare, or complex genome modifications in bacteriophages. The ability to distinguish between several types of nucleotide modifications may even shed new light on metagenomic studies.

Streamlining CRISPR spacer-based bacterial host predictions to decipher the viral dark matter.

Thousands of new phages have recently been discovered thanks to viral metagenomics. These phages are extremely diverse and their genome sequences often do not resemble any known phages. To appreciate their ecological impact, it is important to determine their bacterial hosts. CRISPR spacers can be used to predict hosts of unknown phages, as spacers represent biological records of past phage-bacteria interactions. However, no guidelines have been established to standardize host prediction based on CRISPR spacers. Additionally, there are no tools that use spacers to perform host predictions on large viral datasets. Here, we developed a set of tools that includes all the necessary steps for predicting the hosts of uncharacterized phages. We created a database of >11 million spacers and a program to execute host predictions on large viral datasets. Our host prediction approach uses biological criteria inspired by how CRISPR-Cas naturally work as adaptive immune systems, which make the results easy to interpret. We evaluated the performance using 9484 phages with known hosts and obtained a recall of 49% and a precision of 69%. We also found that this host prediction method yielded higher performance for phages that infect gut-associated bacteria, suggesting it is well suited for gut-virome characterization.

Detection of preQ0 deazaguanine modifications in bacteriophage CAjan DNA using Nanopore sequencing reveals same hypermodification at two distinct DNA motifs.

In the constant evolutionary battle against mobile genetic elements (MGEs), bacteria have developed several defense mechanisms, some of which target the incoming, foreign nucleic acids e.g. restriction-modification (R-M) or CRISPR-Cas systems. Some of these MGEs, including bacteriophages, have in turn evolved different strategies to evade these hurdles. It was recently shown that the siphophage CAjan and 180 other viruses use 7-deazaguanine modifications in their DNA to evade bacterial R-M systems. Among others, phage CAjan genome contains a gene coding for a DNA-modifying homolog of a tRNA-deazapurine modification enzyme, together with four 7-cyano-7-deazaguanine synthesis genes. Using the CRISPR-Cas9 genome editing tool combined with the Nanopore Sequencing (ONT) we showed that the 7-deazaguanine modification in the CAjan genome is dependent on phage-encoded genes. The modification is also site-specific and is found mainly in two separate DNA sequence contexts: GA and GGC. Homology modeling of the modifying enzyme DpdA provides insight into its probable DNA binding surface and general mode of DNA recognition.

The endless battle between phages and CRISPR-Cas systems in Streptococcus thermophilus.

This review describes the contribution of basic research on phage-bacteria interactions to the understanding of CRISPR-Cas systems and their various applications. It focuses on the natural function of CRISPR-Cas systems as adaptive defense mechanisms against mobile genetic elements such as bacteriophage genomes and plasmids. Some of the advances in the characterization of the type II-A CRISPR-Cas system of Streptococcus thermophilus and Streptococcus pyogenes led to the development of the CRISPR-Cas9 genome-editing technology. We mostly discuss the 3 stages of the CRISPR-Cas system in S. thermophilus, namely the adaptation stage, which is unique to this resistance mechanism; the CRISPR RNA biogenesis; and the DNA-cutting activity in the interference stage to protect bacteria against phages. Finally, we look into applications of CRISPR-Cas in microbiology, including overcoming limitations in genome editing.

Genomic diversity and CRISPR-Cas systems in the cyanobacterium Nostoc in the High Arctic.

Nostoc (Nostocales, Cyanobacteria) has a global distribution in the Polar Regions. However, the genomic diversity of Nostoc is little known and there are no genomes available for polar Nostoc. Here we carried out the first genomic analysis of the Nostoc commune morphotype with a recent sample from the High Arctic and a herbarium specimen collected during the British Arctic Expedition (1875-76). Comparisons of the polar genomes with 26 present-day non-polar members of the Nostocales family highlighted that there are pronounced genetic variations among Nostoc strains and species. Osmoprotection and other stress genes were found in all Nostoc strains, but the two Arctic strains had markedly higher numbers of biosynthetic gene clusters for uncharacterised non-ribosomal peptide synthetases, suggesting a high diversity of secondary metabolites. Since viral-host interactions contribute to microbial diversity, we analysed the CRISPR-Cas systems in the Arctic and two temperate Nostoc species. There were a large number of unique repeat-spacer arrays in each genome, indicating diverse histories of viral attack. All Nostoc strains had a subtype I-D system, but the polar specimens also showed evidence of a subtype I-B system that has not been previously reported in cyanobacteria, suggesting diverse cyanobacteria-virus interactions in the Arctic.

Evolutionary emergence of infectious diseases in heterogeneous host populations.

The emergence and re-emergence of pathogens remains a major public health concern. Unfortunately, when and where pathogens will (re-)emerge is notoriously difficult to predict, as the erratic nature of those events is reinforced by the stochastic nature of pathogen evolution during the early phase of an epidemic. For instance, mutations allowing pathogens to escape host resistance may boost pathogen spread and promote emergence. Yet, the ecological factors that govern such evolutionary emergence remain elusive because of the lack of ecological realism of current theoretical frameworks and the difficulty of experimentally testing their predictions. Here, we develop a theoretical model to explore the effects of the heterogeneity of the host population on the probability of pathogen emergence, with or without pathogen evolution. We show that evolutionary emergence and the spread of escape mutations in the pathogen population is more likely to occur when the host population contains an intermediate proportion of resistant hosts. We also show that the probability of pathogen emergence rapidly declines with the diversity of resistance in the host population. Experimental tests using lytic bacteriophages infecting their bacterial hosts containing Clustered Regularly Interspaced Short Palindromic Repeat and CRISPR-associated (CRISPR-Cas) immune defenses confirm these theoretical predictions. These results suggest effective strategies for cross-species spillover and for the management of emerging infectious diseases.

Investigating Lactococcus lactis MG1363 Response to Phage p2 Infection at the Proteome Level.

Phages are viruses that specifically infect and eventually kill their bacterial hosts. Bacterial fermentation and biotechnology industries see them as enemies, however, they are also investigated as antibacterial agents for the treatment or prevention of bacterial infections in various sectors. They also play key ecological roles in all ecosystems. Despite decades of research some aspects of phage biology are still poorly understood. In this study, we used label-free quantitative proteomics to reveal the proteotypes of Lactococcus lactis MG1363 during infection by the virulent phage p2, a model for studying the biology of phages infecting Gram-positive bacteria. Our approach resulted in the high-confidence detection and quantification of 59% of the theoretical bacterial proteome, including 226 bacterial proteins detected only during phage infection and 6 proteins unique to uninfected bacteria. We also identified many bacterial proteins of differing abundance during the infection. Using this high-throughput proteomic datasets, we selected specific bacterial genes for inactivation using CRISPR-Cas9 to investigate their involvement in phage replication. One knockout mutant lacking gene llmg_0219 showed resistance to phage p2 because of a deficiency in phage adsorption. Furthermore, we detected and quantified 78% of the theoretical phage proteome and identified many proteins of phage p2 that had not been previously detected. Among others, we uncovered a conserved small phage protein (pORFN1) coded by an unannotated gene. We also applied a targeted approach to achieve greater sensitivity and identify undetected phage proteins that were expected to be present. This allowed us to follow the fate of pORF46, a small phage protein of low abundance. In summary, this work offers a unique view of the virulent phages' takeover of bacterial cells and provides novel information on phage-host interactions.

A short overview of the CRISPR-Cas adaptation stage.

CRISPR research began over 30 years ago with the incidental discovery of an unusual nucleotide arrangement in the Escherichia coli genome. It took 20 years to find the main function of CRISPR-Cas systems as an adaptive defence mechanism against invading nucleic acids, and our knowledge of their biology has steadily increased ever since. In parallel, the number of applications derived from CRISPR-Cas systems has risen spectacularly. The CRISPR-based genome editing tool is arguably the most exciting application in both basic and applied research. Lately, CRISPR-Cas research has partially shifted to the least understood aspect of its biology: the ability of CRISPR-Cas systems to acquire new immunities during the so-called adaptation step. To date, the most efficient natural system to readily acquire new spacers is the type II-A system of the gram-positive dairy bacterium Streptococcus thermophilus. The discovery of additional systems able to acquire new spacers will hopefully draw more attention to this step of CRISPR-Cas biology. This review focuses on the breakthroughs that have helped to unravel the adaptation phase and on questions that remain to be answered.

Comparative genomic analysis of 142 bacteriophages infecting Salmonella enterica subsp. enterica.

BACKGROUND: Bacteriophages are bacterial parasites and are considered the most abundant and diverse biological entities on the planet. Previously we identified 154 prophages from 151 serovars of Salmonella enterica subsp. enterica. A detailed analysis of Salmonella prophage genomics is required given the influence of phages on their bacterial hosts and should provide a broader understanding of Salmonella biology and virulence and contribute to the practical applications of phages as vectors and antibacterial agents. RESULTS: Here we provide a comparative analysis of the full genome sequences of 142 prophages of Salmonella enterica subsp. enterica which is the full complement of the prophages that could be retrieved from public databases. We discovered extensive variation in genome sizes (ranging from 6.4 to 358.7 kb) and guanine plus cytosine (GC) content (ranging from 35.5 to 65.4%) and observed a linear correlation between the genome size and the number of open reading frames (ORFs). We used three approaches to compare the phage genomes. The NUCmer/MUMmer genome alignment tool was used to evaluate linkages and correlations based on nucleotide identity between genomes. Multiple sequence alignment was performed to calculate genome average nucleotide identity using the Kalgin program. Finally, genome synteny was explored using dot plot analysis. We found that 90 phage genome sequences grouped into 17 distinct clusters while the remaining 52 genomes showed no close relationships with the other phage genomes and are identified as singletons. We generated genome maps using nucleotide and amino acid sequences which allowed protein-coding genes to be sorted into phamilies (phams) using the Phamerator software. Out of 5796 total assigned phamilies, one phamily was observed to be dominant and was found in 49 prophages, or 34.5% of the 142 phages in our collection. A majority of the phamilies, 4330 out of 5796 (74.7%), occurred in just one prophage underscoring the high degree of diversity among Salmonella bacteriophages. CONCLUSIONS: Based on nucleotide and amino acid sequences, a high diversity was found among Salmonella bacteriophages which validate the use of prophage sequence analysis as a highly discriminatory subtyping tool for Salmonella. Thorough understanding of the conservation and variation of prophage genomic characteristics will facilitate their rational design and use as tools for bacterial strain construction, vector development and as anti-bacterial agents.

Beyond the A-layer: adsorption of lipopolysaccharides and characterization of bacteriophage-insensitive mutants of Aeromonas salmonicida subsp. salmonicida.

Aeromonas salmonicida subsp. salmonicida is a fish pathogen that causes furunculosis. Antibiotherapy used to treat furunculosis in fish has led to resistance. Virulent phages are increasingly seen as alternatives or complementary treatments against furunculosis in aquaculture environments. For phage therapy to be successful, it is essential to study the natural mechanisms of phage resistance in A. salmonicida subsp. salmonicida. Here, we generated bacteriophage-insensitive mutants (BIMs) of A. salmonicida subsp. salmonicida, using a myophage with broad host range and characterized them. Phage plaques were different depending on whether the A-layer surface array protein was expressed or not. The genome analysis of the BIMs helped to identify mutations in genes involved in the biogenesis of lipopolysaccharides (LPS) and on an uncharacterized gene (ASA_1998). The characterization of the LPS profile and gene complementation assays identified LPS as a phage receptor and confirmed the involvement of the uncharacterized protein ASA_1998 in phage infection. In addition, we confirmed that the presence of an A-layer at the bacterial surface could act as protection against phages. This study brings new elements into our understanding of the phage adsorption to A. salmonicida subsp. salmonicida cells.

DNA tandem repeats contribute to the genetic diversity of Brevibacterium aurantiacum phages.

This report presents the characterization of the first virulent phages infecting Brevibacterium aurantiacum, a bacterial species used during the manufacture of surface-ripened cheeses. These phages were also responsible for flavour and colour defects in surface-ripened cheeses. Sixteen phages (out of 62 isolates) were selected for genome sequencing and comparative analyses. These cos-type phages with a long non-contractile tail currently belong to the Siphoviridae family (Caudovirales order). Their genome sizes vary from 35,637 to 36,825 bp and, similar to their host, have a high GC content (~61%). Genes encoding for an immunity repressor, an excisionase and a truncated integrase were found, suggesting that these virulent phages may be derived from a prophage. Their genomic organization is highly conserved, with most of the diversity coming from the presence of long (198 bp) DNA tandem repeats (TRs) within an open reading frame coding for a protein of unknown function. We categorized these phages into seven genomic groups according to their number of TR, which ranged from two to eight. Moreover, we showed that TRs are widespread in phage genomes, found in more than 85% of the genomes available in public databases.

Novel Genus of Phages Infecting Streptococcus thermophilus: Genomic and Morphological Characterization.

Streptococcus thermophilus is a lactic acid bacterium commonly used for the manufacture of yogurt and specialty cheeses. Virulent phages represent a major risk for milk fermentation processes worldwide, as they can inactivate the added starter bacterial cells, leading to low-quality fermented dairy products. To date, four genetically distinct groups of phages infecting S. thermophilus have been described. Here, we describe a fifth group. Phages P738 and D4446 are virulent siphophages that infect a few industrial strains of S. thermophilus The genomes of phages P738 and D4446 were sequenced and found to contain 34,037 and 33,656 bp as well as 48 and 46 open reading frames, respectively. Comparative genomic analyses revealed that the two phages are closely related to each other but display very limited similarities to other S. thermophilus phages. In fact, these two novel S. thermophilus phages share similarities with streptococcal phages of nondairy origin, suggesting that they emerged recently in the dairy environment.IMPORTANCE Despite decades of research and adapted antiphage strategies such as CRISPR-Cas systems, virulent phages are still a persistent risk for the milk fermentation industry worldwide, as they can cause manufacturing failures and alter product quality. Phages P738 and D4446 are novel virulent phages that infect the food-grade Gram-positive bacterial species Streptococcus thermophilus These two related viruses represent a fifth group of S. thermophilus phages, as they are significantly distinct from other known S. thermophilus phages. Both phages share similarities with phages infecting nondairy streptococci, suggesting their recent emergence and probable coexistence in dairy environments. These findings highlight the necessity of phage surveillance programs as the phage population evolves in response to the application of antiphage strategies.

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.