Publications by authors named "Sylvain Moineau"

202 Publications

Phage Cocktail Development against subsp. Strains Is Compromised by a Prophage.

Viruses 2021 11 8;13(11). Epub 2021 Nov 8.

Institut de Biologie Intégrative et des Systèmes (IBIS), Pavillon Charles-Eugène-Marchand, Université Laval, Quebec City, QC G1V 0A6, Canada.

Aquaculture is a rapidly growing food production sector. Fish farmers are experiencing increasing problems with antibiotic resistance when fighting against pathogenic bacteria such as subsp. , the causative agent of furunculosis. Phage therapy may provide an alternative, but effective use must be determined. Here, we studied the inhibition of subsp. strains by five phages (HER98 [44RR2.8t.2], HER110 [65.2], SW69-9, L9-6 and Riv-10) used individually or as combinations of two to five phages. A particular combination of four phages (HER98 [44RR2.8t.2], SW69-9, Riv-10, and HER110 [65.2]) was found to be the most effective when used at an initial multiplicity of infection (MOI) of 1 against the subsp. strain 01-B526. The same phage cocktail is effective against other strains except those bearing a prophage (named Prophage 3), which is present in 2/3 of the strains from the province of Quebec. To confirm the impact of this prophage, we tested the effectiveness of the same cocktail on strains that were either cured or lysogenized with Prophage 3. While the parental strains were sensitive to the phage cocktail, the lysogenized ones were much less sensitive. These data indicate that the prophage content of subsp. can affect the efficacy of a cocktail of virulent phages for phage therapy purposes.
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http://dx.doi.org/10.3390/v13112241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8621227PMC
November 2021

Zebrafish: a big fish in the study of the gut microbiota.

Curr Opin Biotechnol 2021 Oct 12;73:308-313. Epub 2021 Oct 12.

Département de biochimie, de microbiologie et de bio-informatique, Faculté des sciences et de génie, Université Laval, Québec, QC, G1V 0A6, Canada; Groupe de recherche en écologie buccale, Faculté de médecine dentaire, Université Laval, Québec, QC, G1V 0A6, Canada; Félix d'Hérelle Reference Center for Bacterial Viruses, Université Laval, Québec, QC, G1V 0A6, Canada. Electronic address:

The importance of the gut microbiota in host health is now well established, but the underlying mechanisms remain poorly understood. Among the animal models used to investigate microbiota-host interactions, the zebrafish (Danio renio) is gaining attention. Several factors contribute to the recent interest in this model, including its low cost, the ability to assess large cohorts, the possibility to obtain germ-free larvae from non-axenic parents, and the availability of optical methodologies to probe the transparent larvae and adults from various genetic lines. We review recent findings on the zebrafish gut microbiota and its modulation by exogenous microbes, nutrition, and environmental factors. We also highlight the potential of this model for assessing the impact of the gut microbiota on brain development.
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http://dx.doi.org/10.1016/j.copbio.2021.09.007DOI Listing
October 2021

Induction and Elimination of Prophages Using CRISPR Interference.

CRISPR J 2021 08;4(4):549-557

Département de Biochimie, de Mmicrobiologie, et de Bio-informatique, Faculté des sciences et de Génie, Université Laval, Québec City, Canada; Université Laval, Québec City, Canada.

Prophages are widely spread among bacterial genomes, and they can have positive or negative effects on their hosts. A key aspect in the study of prophages is the discovery of their induction signals. Prophage induction can occur by inactivating a phage transcriptional repressor, which is responsible for maintaining the lysogenic state. This repressor can be inactivated through the bacterial SOS response. However, the induction signals for numerous prophages do not involve the SOS system, and therefore significant efforts are needed to identify these conditions. Similarly, curing bacterial strains of inducible prophages is a tedious process, requiring the screening of several colonies. Here, we investigated whether transcriptional silencing of a prophage repressor using CRISPR interference (CRISPRi) would lead to prophage induction. Using phages λ and P2 as models, we demonstrated the efficiency of CRISPRi for prophage induction and for curing lysogenic strains of their prophages.
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http://dx.doi.org/10.1089/crispr.2021.0026DOI Listing
August 2021

Primed CRISPR-Cas Adaptation and Impaired Phage Adsorption in Streptococcus mutans.

mSphere 2021 05 19;6(3). Epub 2021 May 19.

Département de Biochimie, de Microbiologie, et de Bio-informatique, Faculté des Sciences et de Génie, Université Laval, Québec City, Québec, Canada

strain P42S possesses a type II-A CRISPR-Cas system that protects against phage infection and plasmid transformation. The analysis of 293 bacteriophage-insensitive mutants (BIMs) obtained upon exposure to the virulent phage M102AD revealed the acquisition of 399 unique spacers, including several ectopic spacer acquisitions and a few cases of native spacer deletions. The acquisition of multiple spacers was also observed and appears to be mostly due to priming, which has been rarely reported for type II-A systems. Analyses of the acquired spacers indicated that 88% of them are identical to a region of the phage M102AD genome. The remaining 12% of spacers had mismatches with the phage genome, primarily at the 5' end of the spacer, leaving the seed sequence at the 3' end largely intact. When a high multiplicity of infection (MOI) was used in the phage challenge assays, we also observed the emergence of CRISPR BIMs that, in addition to the acquisition of new spacers, displayed a reduced phage adsorption phenotype. While CRISPR-Cas and adsorption resistance work in tandem to protect P42S against phage M102AD, nonidentified antiviral mechanisms are also likely at play in this strain. Bacteria are under the constant threat of viral predation and have therefore developed several defense mechanisms, including CRISPR-Cas systems. While studies on the mode of action of CRISPR-Cas systems have already provided great insights into phage-bacterium interactions, still more information is needed on the biology of these systems. The additional characterization of the type II-A CRISPR-Cas system of P42S in this study provides novel information on the spacer acquisition step, especially regarding protospacer-adjacent motif (PAM) recognition, multiple-spacer acquisition, and priming.
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http://dx.doi.org/10.1128/mSphere.00185-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8265633PMC
May 2021

Functional Study of the Type II-A CRISPR-Cas System of Hypervirulent Strains.

CRISPR J 2021 04;4(2):233-242

ISP, Université de Tours, INRAE, Tours, France; Dangé-Saint-Romain, France.

Nearly all strains of , the leading cause of invasive infections in neonates, encode a type II-A clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system. Interestingly, strains belonging to the hypervirulent Sequence Type 17 (ST17) contain significantly fewer spacers in their CRISPR locus than other lineages, which could be the result of a less functional CRISPR-Cas system. Here, we revealed one large deletion in the ST17 promoter region and we evaluated its impact on the transcription of genes as well as the functionalities of the CRISPR-Cas system. We demonstrated that Cas9 interference is functional and that the CRISPR-Cas system of ST17 strains can still acquire new spacers, despite the absence of a regular promoter. We demonstrated that a promoter sequence upstream of a small RNA partially overlapping the antisense tracrRNA, is responsible for the ST17 CRISPR-Cas adaptation and interference activities.
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http://dx.doi.org/10.1089/crispr.2020.0145DOI Listing
April 2021

Complete Genome Sequences of 10 Lactococcal Phages Isolated from Cheddar Cheese Whey Samples in Canada.

Microbiol Resour Announc 2021 Apr 15;10(15). Epub 2021 Apr 15.

Département de Biochimie, de Microbiologie et de Bio-informatique, Faculté des Sciences et de Génie, Université Laval, Québec, QC, Canada

We report the complete genome sequences of 10 virulent phages of the genus () that infect strains used for cheddar cheese production in Canada. Their linear genomes range from 28,969 bp to 31,042 bp with GC contents of 34.1 to 35.1% and 55 to 60 predicted open reading frames (ORFs).
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http://dx.doi.org/10.1128/MRA.00098-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8050962PMC
April 2021

Ectopic Spacer Acquisition in CRISPR3 Array.

Microorganisms 2021 Mar 1;9(3). Epub 2021 Mar 1.

Département de Biochimie, De Microbiologie et de Bio-Informatique, Faculté des Sciences et de Génie, Université Laval, Québec, QC G1V 0A6, Canada.

relies heavily on two type II-A CRISPR-Cas systems, CRISPR1 and CRISPR3, to resist siphophage infections. One hallmark of these systems is the integration of a new spacer at the 5' end of the CRISPR arrays following phage infection. However, we have previously shown that ectopic acquisition of spacers can occur within the CRISPR1 array. Here, we present evidence of the acquisition of new spacers within the array of CRISPR3 of . The analysis of randomly selected bacteriophage-insensitive mutants of the strain Uy01 obtained after phage infection, as well as the comparison with other strains with similar CRISPR3 content, showed that a specific spacer within the array could be responsible for misguiding the adaptation complex. These results also indicate that while the vast majority of new spacers are added at the 5' end of the CRISPR array, ectopic spacer acquisition is a common feature of both CRISPR1 and CRISPR3 systems in , and it can still provide phage resistance. Ectopic spacer acquisition also appears to have occurred naturally in some strains of , suggesting that it is a general phenomenon, at least in type II-A systems.
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http://dx.doi.org/10.3390/microorganisms9030512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7999890PMC
March 2021

Cooperation between Different CRISPR-Cas Types Enables Adaptation in an RNA-Targeting System.

mBio 2021 03 30;12(2). Epub 2021 Mar 30.

University of Jyväskylä Department of Biological and Environmental Science, Nanoscience Center, Jyväskylä, Finland

CRISPR-Cas immune systems adapt to new threats by acquiring new spacers from invading nucleic acids such as phage genomes. However, some CRISPR-Cas loci lack genes necessary for spacer acquisition despite variation in spacer content between microbial strains. It has been suggested that such loci may use acquisition machinery from cooccurring CRISPR-Cas systems within the same strain. Here, following infection by a virulent phage with a double-stranded DNA (dsDNA) genome, we observed spacer acquisition in the native host that carries an acquisition-deficient CRISPR-Cas subtype VI-B system and a complete subtype II-C system. We show that the VI-B locus acquires spacers from both the bacterial and phage genomes, while the newly acquired II-C spacers mainly target the viral genome. Both loci preferably target the terminal end of the phage genome, with priming-like patterns around a preexisting II-C protospacer. Through gene deletion, we show that the RNA-cleaving VI-B system acquires spacers in using acquisition machinery from the DNA-cleaving II-C system. Our observations support the concept of cross talk between CRISPR-Cas systems and raise further questions regarding the plasticity of adaptation modules. CRISPR-Cas systems are immune systems that protect bacteria and archaea against their viruses, bacteriophages. Immunity is achieved through the acquisition of short DNA fragments from the viral invader's genome. These fragments, called spacers, are integrated into a memory bank on the bacterial genome called the CRISPR array. The spacers allow for the recognition of the same invader upon subsequent infection. Most CRISPR-Cas systems target DNA, but recently, systems that exclusively target RNA have been discovered. RNA-targeting CRISPR-Cas systems often lack genes necessary for spacer acquisition, and it is thus unknown how new spacers are acquired and if they can be acquired from DNA phages. Here, we show that an RNA-targeting system "borrows" acquisition machinery from another CRISPR-Cas locus in the genome. Most new spacers in this locus are unable to target phage mRNA and are therefore likely redundant. Our results reveal collaboration between distinct CRISPR-Cas types and raise further questions on how other CRISPR-Cas loci may cooperate.
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http://dx.doi.org/10.1128/mBio.03338-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092290PMC
March 2021

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

Environ Microbiol 2021 06 21;23(6):2955-2968. Epub 2021 Apr 21.

Centre for Northern Studies (CEN), Université Laval, Quebec City, QC, G1V 0A6, Canada.

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.
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http://dx.doi.org/10.1111/1462-2920.15481DOI Listing
June 2021

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

Nucleic Acids Res 2021 04;49(6):3127-3138

Département de biochimie, de microbiologie et de bio-informatique, Faculté des sciences et de génie, Université Laval, Québec City, Québec G1V 0A6, Canada.

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.
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http://dx.doi.org/10.1093/nar/gkab133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034630PMC
April 2021

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

Biochem Cell Biol 2021 08 3;99(4):397-402. Epub 2021 Feb 3.

Département de biochimie, de microbiologie, et de bio-informatique, Faculté des sciences et de génie, Université Laval, Québec, QC G1V 0A6, Canada.

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 and led to the development of the CRISPR-Cas9 genome-editing technology. We mostly discuss the 3 stages of the CRISPR-Cas system in , 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.
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http://dx.doi.org/10.1139/bcb-2020-0593DOI Listing
August 2021

Delivery of CRISPR-Cas systems using phage-based vectors.

Curr Opin Biotechnol 2021 04 23;68:174-180. Epub 2020 Dec 23.

Département de biochimie, de microbiologie, et de bio-informatique, Faculté des sciences et de génie, Université Laval, Québec City, QC, Canada; Groupe de recherche en écologie buccale, Faculté de médecine dentaire, Université Laval, Québec City, QC, Canada; Félix d'Hérelle Reference Center for Bacterial Viruses, Faculté de médecine dentaire, Université Laval, Québec City, QC, Canada. Electronic address:

Antimicrobial resistance has spread quickly on a worldwide scale, reducing therapeutic options for bacterial infections. CRISPR-Cas is an adaptive immune system found in many prokaryotes that can be designed to target bacterial genomes, leading to cell death. Repurposing the CRISPR-Cas system as a therapeutic strategy offers an attractive way to overcome antimicrobial resistance. However, this strategy requires efficient vectors for the CRISPR-Cas system to reach the bacterial genomes. Engineered phages offer an attractive option as cargo delivery vectors. In this review, we discuss the production of phage-based vectors and the relevance of using repurposed CRISPR-Cas systems as antimicrobials. We also discuss recent progress in phage engineering that can potentially overcome the limitations and increase the efficiency of CRISPR-Cas delivery.
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http://dx.doi.org/10.1016/j.copbio.2020.11.012DOI Listing
April 2021

Symposium on Lactic Acid Bacteria-reading while waiting for a meeting.

FEMS Microbiol Rev 2021 03;45(2)

Département de biochimie, de microbiologie, et de bio-informatique, Faculté des sciences et de génie1045, avenue de la MédecineUniversité Laval, Québec, Canada, G1V 0A6.

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http://dx.doi.org/10.1093/femsre/fuaa049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968516PMC
March 2021

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

Nucleic Acids Res 2020 10;48(18):10383-10396

Department of Plant and Environmental Science, University of Copenhagen, Denmark.

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.
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http://dx.doi.org/10.1093/nar/gkaa735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544227PMC
October 2020

Structural Insights into Lactococcal Siphophage p2 Baseplate Activation Mechanism.

Viruses 2020 08 11;12(8). Epub 2020 Aug 11.

Architecture et Fonction des Macromolécules Biologiques, Aix-Marseille Université, Campus de Luminy, 13288 Marseille CEDEX 09, France.

Virulent phages infecting , an industry-relevant bacterium, pose a significant risk to the quality of the fermented milk products. Phages of the Skunavirus genus are by far the most isolated lactococcal phages in the cheese environments and phage p2 is the model siphophage for this viral genus. The baseplate of phage p2, which is used to recognize its host, was previously shown to display two conformations by X-ray crystallography, a rested state and an activated state ready to bind to the host. The baseplate became only activated and opened in the presence of Ca. However, such an activated state was not previously observed in the virion. Here, using nanobodies binding to the baseplate, we report on the negative staining electron microscopy structure of the activated form of the baseplate directly observed in the p2 virion, that is compatible with the activated baseplate crystal structure. Analyses of this new structure also established the presence of a second distal tail (Dit) hexamer as a component of the baseplate, the topology of which differs largely from the first one. We also observed an uncoupling between the baseplate activation and the tail tip protein (Tal) opening, suggesting an infection mechanism more complex than previously expected.
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http://dx.doi.org/10.3390/v12080878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472080PMC
August 2020

A Lactococcal Phage Protein Promotes Viral Propagation and Alters the Host Proteomic Response During Infection.

Viruses 2020 07 24;12(8). Epub 2020 Jul 24.

Département de biochimie, de microbiologie, et de bio-informatique, Faculté des sciences et de génie and PROTEO, Québec City, QC G1V 0A6, Canada.

The lactococcal virulent phage p2 is a model for studying the genus, the most prevalent group of phages causing milk fermentation failures in cheese factories worldwide. This siphophage infects MG1363, a model strain used to study Gram-positive lactic acid bacteria. The structural proteins of phage p2 have been thoroughly described, while most of its non-structural proteins remain uncharacterized. Here, we developed an integrative approach, making use of structural biology, genomics, physiology, and proteomics to provide insights into the function of ORF47, the most conserved non-structural protein of unknown function among the genus. This small phage protein, which is composed of three α-helices, was found to have a major impact on the bacterial proteome during phage infection and to significantly reduce the emergence of bacteriophage-insensitive mutants.
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http://dx.doi.org/10.3390/v12080797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472136PMC
July 2020

Source Tracking Based on Core Genome SNV and CRISPR Typing of Serovar Heidelberg Isolates Involved in Foodborne Outbreaks in Québec, 2012.

Front Microbiol 2020 17;11:1317. Epub 2020 Jun 17.

Laboratoire de Santé Publique du Québec, Institut National de Santé Publique du Québec, Sainte-Anne-de-Bellevue, QC, Canada.

Whole-genome sequencing (WGS) is the method of choice for bacterial subtyping and it is rapidly replacing the more traditional methods such as pulsed-field gel electrophoresis (PFGE). Here we used the high-resolution core genome single nucleotide variant (cgSNV) typing method to characterize clinical and food from serovar Heidelberg isolates in the context of source attribution. Additionally, clustered regularly interspaced short palindromic repeats (CRISPR) analysis was included to further support this method. Our results revealed that cgSNV was highly discriminatory and separated the outbreak isolates into distinct clusters (0-4 SNVs). CRISPR analysis was also able to distinguish outbreak strains from epidemiologically unrelated isolates. Specifically, our data clearly demonstrated the strength of these two methods to determine the probable source(s) of a 2012 epidemiologically characterized outbreak of Heidelberg. Using molecular cut-off of 0-10 SNVs, the cgSNV analysis of 246 clinical and food isolates of Heidelberg collected in Québec, in the same year of the outbreak event, revealed that retail and abattoir chicken isolates likely represent an important source of human infection to . Heidelberg. Interestingly, the isolates genetically related by cgSNV also harbored the same CRISPR as outbreak isolates and clusters. This indicates that CRISPR profiles can be useful as a complementary approach to determine source attribution in foodborne outbreaks. Use of the genomic analysis also allowed to identify a large number of cases that were missed by PFGE, indicating that most outbreaks are probably underestimated. Although epidemiological information must still support WGS-based results, cgSNV method is a highly discriminatory method for the resolution of outbreak events and the attribution of these events to their respective sources. CRISPR typing can serve as a complimentary tool to this analysis during source tracking.
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http://dx.doi.org/10.3389/fmicb.2020.01317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7311582PMC
June 2020

Characterization of a Type II-A CRISPR-Cas System in .

mSphere 2020 06 24;5(3). Epub 2020 Jun 24.

Département de Biochimie, de Microbiologie, et de Bio-Informatique, Faculté des Sciences et de Génie, Université Laval, Québec City, Quebec, Canada

and its virulent phages are important members of the human oral microbiota. is also the primary causal agent of dental caries. To survive in this ecological niche, must encode phage defense mechanisms, which include CRISPR-Cas systems. Here, we describe the CRISPR-Cas type II-A system of strain P42S, which was found to display natural adaptation and interference activity in response to phage infection and plasmid transformation. Newly acquired spacers were integrated both at the 5' end of the CRISPR locus and ectopically. In comparisons of the genes of P42S to those of other strains of , , , and appear to be highly conserved within the species. However, more diversity was observed with While the nuclease domains of Cas9 (SmCas9) are conserved, the C terminus of the protein, including the protospacer adjacent motif (PAM) recognition domain, is less conserved. In support of these findings, we experimentally demonstrated that the PAMs associated with SmCas9 of strain P42S are NAA and NGAA. These PAMs are different from those previously reported for the CRISPR-Cas system of the model strain UA159. This study illustrates the diversity of CRISPR-Cas type II-A systems that can be found within the same bacterial species. CRISPR-Cas is one of the mechanisms used by bacteria to defend against viral predation. Increasing our knowledge of the biology and diversity of CRISPR-Cas systems will also improve our understanding of virus-bacterium interactions. As CRISPR-Cas systems acquiring novel immunities under laboratory conditions are rare, strain P42S provides an alternative model to study the adaptation step, which is still the least understood step in CRISPR-Cas biology. Furthermore, the availability of a natural Cas9 protein recognizing an AT-rich PAM opens up new avenues for genome editing purposes.
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http://dx.doi.org/10.1128/mSphere.00235-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316486PMC
June 2020

A short overview of the CRISPR-Cas adaptation stage.

Can J Microbiol 2021 Jan 19;67(1):1-12. Epub 2020 Jun 19.

Département de biochimie, de microbiologie et de bio-informatique, Faculté des sciences et de génie, Université Laval, QC G1V 0A6, Canada.

CRISPR research began over 30 years ago with the incidental discovery of an unusual nucleotide arrangement in the 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 . 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.
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http://dx.doi.org/10.1139/cjm-2020-0212DOI Listing
January 2021

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

Environ Microbiol 2020 08 7;22(8):3413-3428. Epub 2020 Jul 7.

Département de Biochimie, de Microbiologie et de Bio-Informatique, Faculté des Sciences et de Génie, Université Laval, Québec, Québec City, Canada.

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.
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http://dx.doi.org/10.1111/1462-2920.15113DOI Listing
August 2020

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

BMC Genomics 2020 May 26;21(1):374. Epub 2020 May 26.

Ottawa Laboratory Fallowfield, Canadian Food Inspection Agency, Ottawa, Ontario, Canada.

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.
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http://dx.doi.org/10.1186/s12864-020-6765-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7251866PMC
May 2020

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

Appl Environ Microbiol 2020 06 17;86(13). Epub 2020 Jun 17.

Département de Biochimie, de Microbiologie, et de Bio-informatique, Faculté des Sciences et de Génie, Université Laval, Québec City, Quebec, Canada

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 have been described. Here, we describe a fifth group. Phages P738 and D4446 are virulent siphophages that infect a few industrial strains of 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 phages. In fact, these two novel phages share similarities with streptococcal phages of nondairy origin, suggesting that they emerged recently in the dairy environment. 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 These two related viruses represent a fifth group of phages, as they are significantly distinct from other known 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.
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http://dx.doi.org/10.1128/AEM.00227-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7301855PMC
June 2020

A Jumbo Formation in the Viral Game Plan.

CRISPR J 2020 02;3(1):14-17

Département de biochimie, de microbiologie et de bio-informatique, Faculté des sciences et de génie, Université Laval, Québec, Canada; Université Laval, Québec, Canada.

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http://dx.doi.org/10.1089/crispr.2020.29082.jcoDOI Listing
February 2020

Author Correction: A mutation in the methionine aminopeptidase gene provides phage resistance in Streptococcus thermophilus.

Sci Rep 2020 Feb 11;10(1):2682. Epub 2020 Feb 11.

Département de biochimie, de microbiologie, et de bio-informatique, Faculté des sciences et de genie, Université Laval, Québec City, QC, G1V 0A6, Canada.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-59767-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010727PMC
February 2020

Phage diversity, genomics and phylogeny.

Nat Rev Microbiol 2020 03 3;18(3):125-138. Epub 2020 Feb 3.

Département de biochimie, de microbiologie et de bio-informatique, Faculté des sciences et de génie, Université Laval, Québec City, Québec, Canada.

Recent advances in viral metagenomics have enabled the rapid discovery of an unprecedented catalogue of phages in numerous environments, from the human gut to the deep ocean. Although these advances have expanded our understanding of phage genomic diversity, they also revealed that we have only scratched the surface in the discovery of novel viruses. Yet, despite the remarkable diversity of phages at the nucleotide sequence level, the structural proteins that form viral particles show strong similarities and conservation. Phages are uniquely interconnected from an evolutionary perspective and undergo multiple events of genetic exchange in response to the selective pressure of their hosts, which drives their diversity. In this Review, we explore phage diversity at the structural, genomic and community levels as well as the complex evolutionary relationships between phages, moulded by the mosaicity of their genomes.
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http://dx.doi.org/10.1038/s41579-019-0311-5DOI Listing
March 2020

Virulent coliphages in 1-year-old children fecal samples are fewer, but more infectious than temperate coliphages.

Nat Commun 2020 01 17;11(1):378. Epub 2020 Jan 17.

Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78350, Jouy-en-Josas, France.

Bacteriophages constitute an important part of the human gut microbiota, but their impact on this community is largely unknown. Here, we cultivate temperate phages produced by 900 E. coli strains isolated from 648 fecal samples from 1-year-old children and obtain coliphages directly from the viral fraction of the same fecal samples. We find that 63% of strains hosted phages, while 24% of the viromes contain phages targeting E. coli. 150 of these phages, half recovered from strain supernatants, half from virome (73% temperate and 27% virulent) were tested for their host range on 75 E. coli strains isolated from the same cohort. Temperate phages barely infected the gut strains, whereas virulent phages killed up to 68% of them. We conclude that in fecal samples from children, temperate coliphages dominate, while virulent ones have greater infectivity and broader host range, likely playing a role in gut microbiota dynamics.
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http://dx.doi.org/10.1038/s41467-019-14042-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6969025PMC
January 2020

Versatile and robust genome editing with CRISPR1-Cas9.

Genome Res 2020 01 3;30(1):107-117. Epub 2020 Jan 3.

Centre Hospitalier Universitaire de Québec Research Center-Université Laval, Québec, Québec G1V 4G2, Canada.

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 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.
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http://dx.doi.org/10.1101/gr.255414.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6961573PMC
January 2020

Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants.

Nat Rev Microbiol 2020 02 19;18(2):67-83. Epub 2019 Dec 19.

National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD, USA.

The number and diversity of known CRISPR-Cas systems have substantially increased in recent years. Here, we provide an updated evolutionary classification of CRISPR-Cas systems and cas genes, with an emphasis on the major developments that have occurred since the publication of the latest classification, in 2015. The new classification includes 2 classes, 6 types and 33 subtypes, compared with 5 types and 16 subtypes in 2015. A key development is the ongoing discovery of multiple, novel class 2 CRISPR-Cas systems, which now include 3 types and 17 subtypes. A second major novelty is the discovery of numerous derived CRISPR-Cas variants, often associated with mobile genetic elements that lack the nucleases required for interference. Some of these variants are involved in RNA-guided transposition, whereas others are predicted to perform functions distinct from adaptive immunity that remain to be characterized experimentally. The third highlight is the discovery of numerous families of ancillary CRISPR-linked genes, often implicated in signal transduction. Together, these findings substantially clarify the functional diversity and evolutionary history of CRISPR-Cas.
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http://dx.doi.org/10.1038/s41579-019-0299-xDOI Listing
February 2020

7-Deazaguanine modifications protect phage DNA from host restriction systems.

Nat Commun 2019 11 29;10(1):5442. Epub 2019 Nov 29.

Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, 32611, USA.

Genome modifications are central components of the continuous arms race between viruses and their hosts. The archaeosine base (G), which was thought to be found only in archaeal tRNAs, was recently detected in genomic DNA of Enterobacteria phage 9g and was proposed to protect phage DNA from a wide variety of restriction enzymes. In this study, we identify three additional 2'-deoxy-7-deazaguanine modifications, which are all intermediates of the same pathway, in viruses: 2'-deoxy-7-amido-7-deazaguanine (dADG), 2'-deoxy-7-cyano-7-deazaguanine (dPreQ) and 2'-deoxy-7- aminomethyl-7-deazaguanine (dPreQ). We identify 180 phages or archaeal viruses that encode at least one of the enzymes of this pathway with an overrepresentation (60%) of viruses potentially infecting pathogenic microbial hosts. Genetic studies with the Escherichia phage CAjan show that DpdA is essential to insert the 7-deazaguanine base in phage genomic DNA and that 2'-deoxy-7-deazaguanine modifications protect phage DNA from host restriction enzymes.
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http://dx.doi.org/10.1038/s41467-019-13384-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6884629PMC
November 2019

Cas9 Allosteric Inhibition by the Anti-CRISPR Protein AcrIIA6.

Mol Cell 2019 12 8;76(6):922-937.e7. Epub 2019 Oct 8.

Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique (CNRS), Campus de Luminy, Case 932, 13288 Marseille Cedex 09, France; Architecture et Fonction des Macromolécules Biologiques, Aix-Marseille Université, Campus de Luminy, Case 932, 13288 Marseille Cedex 09, France. Electronic address:

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.
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http://dx.doi.org/10.1016/j.molcel.2019.09.012DOI Listing
December 2019
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