Evolution of Lactococcus lactis phages within a cheese factory

We have sequenced the double-stranded DNA genomes of six lactococcal phages (SL4, CB13, CB14, CB19, CB20, and GR7) from the 936 group that were isolated over a 9-year period from whey samples obtained from a Canadian cheese factory. These six phages infected the same two industrial Lactococcus lactis strains out of 30 tested. The CB14 and GR7 genomes were found to be 100% identical even though they were isolated 14 months apart, indicating that a phage can survive in a cheese plant for more than a year. The other four genomes were related but notably different. The length of the genomes varied from 28,144 to 32,182 bp, and they coded for 51 to 55 open reading frames. All five genomes possessed a 3 overhang cos site that was 11 nucleotides long. Several structural proteins were also identified by nano-high-performance liquid chromatography–tandem mass spectrometry, confirming bioinformatic analyses. Comparative analyses suggested that the most recently isolated phages (CB19 and CB20) were derived, in part, from older phage isolates (CB13 and CB14/GR7). The organization of the five distinct genomes was similar to the previously sequenced lactococcal phage genomes of the 936 group, and from these sequences, a core genome was determined for lactococcal phages of the 936 group

Characterization of two polyvalent phages infecting Enterobacteriaceae

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

Characterization of CRISPR-Cas systems in the Ralstonia solanacearum species complex

Clustered regularly interspaced short palindromic repeats (CRISPRs) are composed of an array of short DNA repeat sequences separated by unique spacer sequences that are flanked by associated (Cas) genes. CRISPR‐Cas systems are found in the genomes of several microbes and can act as an adaptive immune mechanism against invading foreign nucleic acids, such as phage genomes. Here, we studied the CRISPR‐Cas systems in plant‐pathogenic bacteria of the Ralstonia solanacearum species complex (RSSC). A CRISPR‐Cas system was found in 31% of RSSC genomes present in public databases. Specifically, CRISPR‐Cas types I‐E and II‐C were found, with I‐E being the most common. The presence of the same CRISPR‐Cas types in distinct Ralstonia phylotypes and species suggests the acquisition of the system by a common ancestor before Ralstonia species segregation. In addition, a Cas1 phylogeny (I‐E type) showed a perfect geographical segregation of phylotypes, supporting an ancient acquisition. Ralstoniasolanacearum strains CFBP2957 and K60T were challenged with a virulent phage, and the CRISPR arrays of bacteriophage‐insensitive mutants (BIMs) were analysed. No new spacer acquisition was detected in the analysed BIMs. The functionality of the CRISPR‐Cas interference step was also tested in R. solanacearum CFBP2957 using a spacer‐protospacer adjacent motif (PAM) delivery system, and no resistance was observed against phage phiAP1. Our results show that the CRISPR‐Cas system in R. solanacearum CFBP2957 is not its primary antiviral strategy

Complete genome sequence of Escherichia coli Siphophage BRET

The lytic Escherichia coli siphophage BRET was isolated from a chicken obtained at a local market in Abidjan, Côte d’Ivoire. Its linear genome sequence consists of 59,550 bp (43.4% GC content) and contains 88 predicted genes, including 4 involved in archaeosine biosynthesis. Phage BRET is related (95% nucleotide identity) to Enterobacteria phage JenK

Étude de l'évolution des phages de l'espèce 936 de Lactococcus lactis

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2007-2008

Phage resistance in lactic acid bacteria

Lactic Acid Bacteria (LAB) are used in a variety of industrial fermentation processes because of their ability to convert a variety of substrates into complex products. Any technological process that relies on bacterial fermentation is vulnerable to bacteriophage infections. This chapter describes the relationship between bacteriophages and their LAB hosts in the context of the food fermentation industry. Specifically, we highlight the most significant antiviral mechanisms in LAB. The primary focus will be given to LAB used by the dairy industry because it has openly acknowledged the problem of phage infections and has teamed up with academia and starter cultures companies to develop natural and engineered phage resistance systems to curtail the propagation of diverse phages

Le système CRISPR-Cas

CRISPR-Cas est un système immunitaire adaptatif utilisé par de nombreux microbes pour se défendre contre l’invasion d’acides nucléiques tels que les génomes viraux et autres éléments génétiques mobiles. Le système microbien utilise son locus CRISPR pour stocker de l’information génétique afin de produire des ARN guides. Ces derniers, de concert avec des endonucléases (Cas), empêchent des invasions futures. Des parties de ce système microbien ont été exploitées pour développer un puissant outil d’édition des génomes dans une panoplie d’organismes. La capacité de CRISPR-Cas9 à couper efficacement et à des endroits très précis de l’ADN pourrait peut-être permettre un jour de guérir certaines maladies génétiques humaines. La malléabilité de cet outil d’édition rend possible une variété d’applications allant de la modulation de l’expression de gènes à des modifications épigénétiques. Les locus CRISPR représentent également une mine d’informations pouvant servir de méthode de typage de souches microbiennes ou encore une façon d’étudier les interactions entre les bactéries et leurs habitats

Identification and Characterization of the Phage Gene sav, Involved in Sensitivity to the Lactococcal Abortive Infection Mechanism AbiV▿

Lactococcus lactis phage mutants that are insensitive to the recently characterized abortive infection mechanism AbiV were isolated and analyzed in an effort to elucidate factors involved in the sensitivity to AbiV. Whole-genome sequencing of the phage mutants p2.1 and p2.2 revealed mutations in an orf that is transcribed early, indicating that this orf was responsible for AbiV sensitivity. Sequencing of the homologous regions in the genomes of other AbiV-insensitive mutants derived from p2 and six other lactococcal wild-type phages revealed point mutations in the homologous orf sequences. The orf was named sav (for sensitivity to AbiV), and the encoded polypeptide was named SaV. The purification of a His-tagged SaV polypeptide by gel filtration suggested that the polypeptide formed a dimer in its native form. The overexpression of SaV in L. lactis and Escherichia coli led to a rapid toxic effect. Conserved, evolutionarily related regions in SaV polypeptides of different phage groups are likely to be responsible for the AbiV-sensitive phenotype and the toxicity

Targeted genome editing of virulent phages using CRISPR-Cas9

This protocol describes a straightforward method to generate specific mutations in the genome of strictly lytic phages. Briefly, a targeting CRISPR-Cas9 system and a repair template suited for homologous recombination are provided inside a bacterial host, here the Gram-positive model Lactococcus lactis MG1363. The CRISPR-Cas9 system is programmed to cleave a specific region present on the genome of the invading phage, but absent from the recombination template. The system either triggers the recombination event or exerts the selective pressure required to isolate recombinant phages. With this methodology, we generated multiple gene knockouts, a point mutation and an insertion in the genome of the virulent lactococcal phage p2. Considering the broad host range of the plasmids used in this protocol, the latter can be extrapolated to other phage-host pairs