lysogeny in Oenococcus oeni species : Impact on malolactic starters performance

Oenococcus oeni est la bactérie lactique majoritairement responsable de la fermentation malolactique (FML) des vins. Elle est la cible de phages qui sont des virus spécifiques des bactéries. La vaste majorité des oenophages isolés sont tempérés selon la littérature scientifique. Ces phages sont capables d’entrer en latence en intégrant leur génome au sein de celui de leur hôte bactérien pour former un prophage. La lysogénie est instable puisque les prophages peuvent s’induire dans certaines conditions et conduire à la mort de la bactérie pour se répliquer. Les données de séquençage indiquent que la lysogénie est fréquente dans l’espèce O. oeni, y compris chez les levains commerciaux. L’impact de la lysogénie est très peu abordé dans la filière vinicole, elle est pourtant redoutée dans l’élaboration d’autres produits fermentés en raison des risques d’induction. Néanmoins, la prévalence de la lysogénie laisse supposer qu’elle confère des bénéfices, a fortiori car les prophages peuvent encoder des fonctions utiles aux bactéries les hébergeant. Durant cette thèse nous nous sommes intéressés à la fréquence et à l’incidence de la lysogénie au sein de flores indigènes d’O. oeni et dans un panel de souches commerciales. Elle a débuté par un inventaire des séquences prophagiques présentes dans les génomes accessibles publiquement pour développer un outil PCR de criblage de la lysogénie. Nos travaux ont confirmé un fort taux de la lysogénie in vino, dans les souches responsables de FML spontanées de vins rouges de la région bordelaise et ont montré que les prophages apportent de la diversité génétique. Une méthode de criblage de la lysogénie basée sur des morphotypes de colonie a également été développée et exploitée dans des expérimentations de compétition entre une souche lysogène et une non-lysogène. La lysogène s'est montrée la plus compétitive dans toutes les conditions testées. Enfin, une étude des phénotypes conférés par les prophages d’un panel de souches commerciales a montré que la lysogénie a une influence importante sur la physiologie bactérienne. Dans leur ensemble ces travaux confirment la prévalence de la lysogénie dans l’espèce O. oeni et indiquent qu’elle confère un avantage sélectif aux lysogènes. Ceci au sein des flores indigènes responsables des FML spontanées et parmi les souches développées comme levains commerciaux.Oenococcus oeni is the lactic acid bacterium mainly responsible for malolactic fermentation (MLF) of wines. It is the prey of phages which are viruses specific to bacteria. The vast majority of isolated oenophages are temperate according to the scientific literature. These phages are able to enter into latency by integrating their genome within that of their bacterial host to form a prophage. Lysogeny is unstable since the prophages can be induced under certain conditions and lead to the bacterial death in order to replicate. Sequencing data indicate that lysogeny is common in O. oeni species, including in commercial strains. The impact of lysogeny is little discussed in the wine industry, yet it is feared in the development of other fermented products because of the risks of induction. Nevertheless, the prevalence of lysogeny suggests that it confers benefits, all the more because the prophages can encode useful functions to the bacteria hosting them. During this thesis we investigated the frequency and incidence of lysogeny in indigenous O. oeni flora and in a panel of commercial strains. It started with an inventory of the prophagic sequences present in the publicly available genomes in order to develop a PCR tool to screen lysogeny. Our work confirmed a high rate of lysogeny in vino, in strains responsible for spontaneous MLF in red wines from the Bordeaux region and showed that prophages bring genetic diversity. A lysogeny screening method based on colony morphotypes was also developed and exploited in competition experiments between a lysogenic and a non-lysogenic strain. The lysogen was the most competitive under all conditions tested. Finally, a study of the phenotypes conferred by the prophages of a panel of commercial strains showed that lysogeny has an important influence on bacterial physiology.Taken as a whole, this work confirms the prevalence of lysogeny in O. oeni species and indicates that it confers a selective advantage to lysogens. This within the native flora responsible for spontaneous MLF and among strains developed as commercial starters

"French Phage Network" Annual Conference-Fifth Meeting Report

Attracting about 100 participants, the fifth edition of our French Phages.fr annual conference was once more a success. This year's conference took place at the Institute for Structural Biology on the European Electron and Photon Campus in Grenoble, October 8 and 9, 2019. Similar to previous years, our meeting gathered scientists mainly working in France, from academic labs and hospitals as well as from industry. We also had the pleasure of welcoming attendees from different European countries and even beyond. The conference was divided into four sessions: Ecology and Evolution, Phage Therapy and Biotechnology, Structure and Assembly and Phage-Host Interaction, each opened by a keynote lecture. The talks, selected from abstracts, gave the opportunity for young scientists (especially students and post-docs) to present their project and results in a friendly atmosphere. Poster sessions also favoured interactions and discussions between young researchers and more senior scientists. Here, we provide a summary of the topics developed during the conference

Phage Encounters Recorded in CRISPR Arrays in the Genus <i>Oenococcus</i>

The Oenococcus genus comprises four recognized species, and members have been found in different types of beverages, including wine, kefir, cider and kombucha. In this work, we implemented two complementary strategies to assess whether oenococcal hosts of different species and habitats were connected through their bacteriophages. First, we investigated the diversity of CRISPR-Cas systems using a genome-mining approach, and CRISPR-endowed strains were identified in three species. A census of the spacers from the four identified CRISPR-Cas loci showed that each spacer space was mostly dominated by species-specific sequences. Yet, we characterized a limited records of potentially recent and also ancient infections between O. kitaharae and O. sicerae and phages of O. oeni, suggesting that some related phages have interacted in diverse ways with their Oenococcus hosts over evolutionary time. Second, phage-host interaction analyses were performed experimentally with a diversified panel of phages and strains. None of the tested phages could infect strains across the species barrier. Yet, some infections occurred between phages and hosts from distinct beverages in the O. oeni species

Distribution of Prophages in the Oenococcus oeni Species

Oenococcus oeni is the most exploited lactic acid bacterium in the wine industry and drives the malolactic fermentation of wines. Although prophage-like sequences have been identified in the species, many are not characterized, and a global view of their integration and distribution amongst strains is currently lacking. In this work, we analyzed the complete genomes of 231 strains for the occurrence of prophages, and analyzed their size and positions of insertion. Our data show the limited variation in the number of prophages in O. oeni genomes, and that six sites of insertion within the bacterial genome are being used for site-specific recombination. Prophage diversity patterns varied significantly for different host lineages, and environmental niches. Overall, the findings highlight the pervasive presence of prophages in the O. oeni species, their role as a major source of within-species bacterial diversity and drivers of horizontal gene transfer. Our data also have implications for enhanced understanding of the prophage recombination events which occurred during evolution of O. oeni, as well as the potential of prophages in influencing the fitness of these bacteria in their distinct niches