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.Evolution expérimentale en mileu extrême de la bactérie lactique Oenococcus oeni et applications à la sélection de levains malo-lactiques plus performant

Diversity of bacteriophages infecting Oenococcus oeni, the lactic acid bacteria responsible for the wine malolactic fermentation

Les bactériophages sont de puissants prédateurs bactériens. Leur développement est généralement redouté dans les industries agro-alimentaires mettant en oeuvre des fermentations, car les phages sont responsables d’accidents de fabrication affectant la qualité finale du produit. Leur impact lors de la vinification est moins bien défini. Le procédé comporte une étape de fermentation malolactique (FML), qui est assurée par la bactérie lactique Oenococcus oeni. La maîtrise de la FML est un moyen efficace pour protéger la qualité et la typicité des vins, vecteurs de commercialisation. Jusqu’à présent, cette étape fondamentale du processus de vinification n’est pas toujours maîtrisée. L’explication majeure réside dans l’insuffisance de la biomasse bactérienne endogène, liée aux conditions physico-chimiques difficiles du milieu. Des solutions visant à conduire rapidement la FML sont disponibles, comme l’inoculation de souches commerciales de O. oeni. Ces stratégies n’offrent toutefois pas une totale garantie de succès, et des retards ou non déclenchements de FML sont toujours observés. Ces situations inexpliquées amènent à s’interroger sur l’impact d’autres paramètres sur la fermentescibilité malolactique. L’objectif de cette thèse était d’évaluer la présence et la diversité des bactériophages antagonistes de la bactérie lactique O. oeni présents dans l’écosystème. La diversité des prophages présents dans le pangénome de O. oeni a été explorée. La lysogénie est fréquente dans l’espèce. Quatre groupes de prophages ont été identifiés sur la base de la séquence de l’intégrase, et du site de recombinaison site-spécifique utilisé. La pertinence de la classification établie a été vérifiée sur un panel de 40 phages isolés de moûts et de vins. Nos travaux suggèrent que la lysogénie est un moyen pour O. oeni de résister aux stress et aux phages, grâce à la présence de mécanismes de résistance sur les génomes prophagiques. La stabilité de la lysogénie lors de l’inoculation de souches lysogènes dans le vin et la possible libération de dérivés lytiques sont deux paramètres à prendre en compte lors des FML spontanées, et lors de l’inoculation de levains malolactiques. Ils sont susceptibles de moduler quantitativement et qualitativement la population.Bacteriophages are responsible for the predation of bacteria. They pose an ever-present threat to the food industries because they invade and destroy the starter and affect production. Their destructive potential is currently difficult to establish during spontaneous production of wine. Malolactic fermentation (MLF) represents a main stage in the winemaking process, and is essentially driven by the lactic acid bacterium (LAB) Oenococcus oeni. A rapid and efficient FML is essential to optimize wine quality and typicity, as sales rely on top-quality products. Up to now, this essential step is not controlled, and this results from the limited growth of MLF bacteria in wine, due to stressing conditions. Inoculation of commercial bacterial starter cultures is a strategy to improve MLF control, and will allow a rapid and complete fermentation. However, despite these evolutions, sluggish or complete failures of MLF are still reported by wine farmers, either during spontaneous or directed fermentations. Such cases suggest that additional factors need to be identified. The outline of this thesis was to demonstrate the occurrence of phages infecting O. oeni in the ecosystem and provide essential information regarding phage diversity. We analyzed prophage diversity through comparative genomics and demonstrated that lysogeny is widespread. Four prophage groups were identified according to the integrase gene sequence and attachment site used for site specific recombination. The relevance of the classification scheme was verified through the analysis of a panel of 40 phages isolated from wine and must. We suggest that lysogeny helps O. oeni to cope with stress and phage attack, through the presence of specific anti-phage mechanisms harbored by the prophages. The stability of lysogens during inoculation in wine, and the possible release of lytic particules have to be considered during spontaneous or directed MLF. They are expected to shape the population

Diversité des bactériophages infectant la bactérie lactique Oenococcus oeni, responsable de la fermentation malolactique des vins

Bacteriophages are responsible for the predation of bacteria. They pose an ever-present threat to the food industries because they invade and destroy the starter and affect production. Their destructive potential is currently difficult to establish during spontaneous production of wine. Malolactic fermentation (MLF) represents a main stage in the winemaking process, and is essentially driven by the lactic acid bacterium (LAB) Oenococcus oeni. A rapid and efficient FML is essential to optimize wine quality and typicity, as sales rely on top-quality products. Up to now, this essential step is not controlled, and this results from the limited growth of MLF bacteria in wine, due to stressing conditions. Inoculation of commercial bacterial starter cultures is a strategy to improve MLF control, and will allow a rapid and complete fermentation. However, despite these evolutions, sluggish or complete failures of MLF are still reported by wine farmers, either during spontaneous or directed fermentations. Such cases suggest that additional factors need to be identified. The outline of this thesis was to demonstrate the occurrence of phages infecting O. oeni in the ecosystem and provide essential information regarding phage diversity. We analyzed prophage diversity through comparative genomics and demonstrated that lysogeny is widespread. Four prophage groups were identified according to the integrase gene sequence and attachment site used for site specific recombination. The relevance of the classification scheme was verified through the analysis of a panel of 40 phages isolated from wine and must. We suggest that lysogeny helps O. oeni to cope with stress and phage attack, through the presence of specific anti-phage mechanisms harbored by the prophages. The stability of lysogens during inoculation in wine, and the possible release of lytic particules have to be considered during spontaneous or directed MLF. They are expected to shape the population.Les bactériophages sont de puissants prédateurs bactériens. Leur développement est généralement redouté dans les industries agro-alimentaires mettant en oeuvre des fermentations, car les phages sont responsables d’accidents de fabrication affectant la qualité finale du produit. Leur impact lors de la vinification est moins bien défini. Le procédé comporte une étape de fermentation malolactique (FML), qui est assurée par la bactérie lactique Oenococcus oeni. La maîtrise de la FML est un moyen efficace pour protéger la qualité et la typicité des vins, vecteurs de commercialisation. Jusqu’à présent, cette étape fondamentale du processus de vinification n’est pas toujours maîtrisée. L’explication majeure réside dans l’insuffisance de la biomasse bactérienne endogène, liée aux conditions physico-chimiques difficiles du milieu. Des solutions visant à conduire rapidement la FML sont disponibles, comme l’inoculation de souches commerciales de O. oeni. Ces stratégies n’offrent toutefois pas une totale garantie de succès, et des retards ou non déclenchements de FML sont toujours observés. Ces situations inexpliquées amènent à s’interroger sur l’impact d’autres paramètres sur la fermentescibilité malolactique. L’objectif de cette thèse était d’évaluer la présence et la diversité des bactériophages antagonistes de la bactérie lactique O. oeni présents dans l’écosystème. La diversité des prophages présents dans le pangénome de O. oeni a été explorée. La lysogénie est fréquente dans l’espèce. Quatre groupes de prophages ont été identifiés sur la base de la séquence de l’intégrase, et du site de recombinaison site-spécifique utilisé. La pertinence de la classification établie a été vérifiée sur un panel de 40 phages isolés de moûts et de vins. Nos travaux suggèrent que la lysogénie est un moyen pour O. oeni de résister aux stress et aux phages, grâce à la présence de mécanismes de résistance sur les génomes prophagiques. La stabilité de la lysogénie lors de l’inoculation de souches lysogènes dans le vin et la possible libération de dérivés lytiques sont deux paramètres à prendre en compte lors des FML spontanées, et lors de l’inoculation de levains malolactiques. Ils sont susceptibles de moduler quantitativement et qualitativement la population

Diversity of bacteriophages infecting Oenococcus oeni, the lactic acid bacteria responsible for the wine malolactic fermentation

Les bactériophages sont de puissants prédateurs bactériens. Leur développement est généralement redouté dans les industries agro-alimentaires mettant en oeuvre des fermentations, car les phages sont responsables d’accidents de fabrication affectant la qualité finale du produit. Leur impact lors de la vinification est moins bien défini. Le procédé comporte une étape de fermentation malolactique (FML), qui est assurée par la bactérie lactique Oenococcus oeni. La maîtrise de la FML est un moyen efficace pour protéger la qualité et la typicité des vins, vecteurs de commercialisation. Jusqu’à présent, cette étape fondamentale du processus de vinification n’est pas toujours maîtrisée. L’explication majeure réside dans l’insuffisance de la biomasse bactérienne endogène, liée aux conditions physico-chimiques difficiles du milieu. Des solutions visant à conduire rapidement la FML sont disponibles, comme l’inoculation de souches commerciales de O. oeni. Ces stratégies n’offrent toutefois pas une totale garantie de succès, et des retards ou non déclenchements de FML sont toujours observés. Ces situations inexpliquées amènent à s’interroger sur l’impact d’autres paramètres sur la fermentescibilité malolactique. L’objectif de cette thèse était d’évaluer la présence et la diversité des bactériophages antagonistes de la bactérie lactique O. oeni présents dans l’écosystème. La diversité des prophages présents dans le pangénome de O. oeni a été explorée. La lysogénie est fréquente dans l’espèce. Quatre groupes de prophages ont été identifiés sur la base de la séquence de l’intégrase, et du site de recombinaison site-spécifique utilisé. La pertinence de la classification établie a été vérifiée sur un panel de 40 phages isolés de moûts et de vins. Nos travaux suggèrent que la lysogénie est un moyen pour O. oeni de résister aux stress et aux phages, grâce à la présence de mécanismes de résistance sur les génomes prophagiques. La stabilité de la lysogénie lors de l’inoculation de souches lysogènes dans le vin et la possible libération de dérivés lytiques sont deux paramètres à prendre en compte lors des FML spontanées, et lors de l’inoculation de levains malolactiques. Ils sont susceptibles de moduler quantitativement et qualitativement la population.Bacteriophages are responsible for the predation of bacteria. They pose an ever-present threat to the food industries because they invade and destroy the starter and affect production. Their destructive potential is currently difficult to establish during spontaneous production of wine. Malolactic fermentation (MLF) represents a main stage in the winemaking process, and is essentially driven by the lactic acid bacterium (LAB) Oenococcus oeni. A rapid and efficient FML is essential to optimize wine quality and typicity, as sales rely on top-quality products. Up to now, this essential step is not controlled, and this results from the limited growth of MLF bacteria in wine, due to stressing conditions. Inoculation of commercial bacterial starter cultures is a strategy to improve MLF control, and will allow a rapid and complete fermentation. However, despite these evolutions, sluggish or complete failures of MLF are still reported by wine farmers, either during spontaneous or directed fermentations. Such cases suggest that additional factors need to be identified. The outline of this thesis was to demonstrate the occurrence of phages infecting O. oeni in the ecosystem and provide essential information regarding phage diversity. We analyzed prophage diversity through comparative genomics and demonstrated that lysogeny is widespread. Four prophage groups were identified according to the integrase gene sequence and attachment site used for site specific recombination. The relevance of the classification scheme was verified through the analysis of a panel of 40 phages isolated from wine and must. We suggest that lysogeny helps O. oeni to cope with stress and phage attack, through the presence of specific anti-phage mechanisms harbored by the prophages. The stability of lysogens during inoculation in wine, and the possible release of lytic particules have to be considered during spontaneous or directed MLF. They are expected to shape the population

Complete genome sequence of lytic Oenococcus oeni bacteriophage OE33PA

Oenococcus oeni is the most common species of lactic acid bacteria associated with malolactic fermentation in wine. Here, we report the genome sequence of the lytic phage OE33PA (vB_OeS_OE33PA). It has a morphotype similar to that of members of the Siphoviridae family, a linear 39,866-bp double-stranded genome with cohesive ends, and 57 predicted open reading frames

Bacteriophage GC1, a novel tectivirus infecting gluconobacter cerinus, an acetic acid bacterium associated with wine-making

The Gluconobacter phage GC1 is a novel member of the Tectiviridae family isolated from a juice sample collected during dry white wine making. The bacteriophage infects Gluconobacter cerinus, an acetic acid bacterium which represents a spoilage microorganism during wine making, mainly because it is able to produce ethyl alcohol and transform it into acetic acid. Transmission electron microscopy revealed tail-less icosahedral particles with a diameter of similar to 78 nm. The linear double-stranded DNA genome of GC1 (16,523 base pairs) contains terminal inverted repeats and carries 36 open reading frames, only a handful of which could be functionally annotated. These encode for the key proteins involved in DNA replication (protein-primed family B DNA polymerase) as well as in virion structure and assembly (major capsid protein, genome packaging ATPase (adenosine triphosphatase) and several minor capsid proteins). GC1 is the first tectivirus infecting an alphaproteobacterial host and is thus far the only temperate tectivirus of gram-negative bacteria. Based on distinctive sequence and life-style features, we propose that GC1 represents a new genus within the Tectiviridae, which we tentatively named Gammatectivirus. Furthermore, GC1 helps to bridge the gap in the sequence space between alphatectiviruses and betatectiviruses

Int J Food Microbiol.

Oenophages have so far been mostly isolated from red wines under malolactic fermentation (MLF), and correspond to temperate or ex-temperate phages of Oenococcus oeni. Their genomes are clustered into 4 integrase gene sequence groups, which are also related to the chromosomal integration site. Our aims were to survey the occurrence of oenophages in a broader and more diverse collection of samples than those previously explored. Active phages were isolated from 33 out of 166 samples, which mostly originated from must and MLF. Seventy one phage lysates were produced and 30% were assigned to a novel group with unusual genomic characteristics, called unk. All unk members produced similar RAPD and DNA restriction patterns, were negative by PCR for the signature sequences previously identified in the integrase and endolysin genes of oenophages, and lacked any BamHI restriction site in their genome. The data support that development of additional and novel signature genes for assessing oenophage diversity is now required

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

Limosilactobacillus reuteri BIO7251 administration improves metabolic phenotype in obese mice fed a high fat diet: an inter-organ crosstalk between gut, adipose tissue and nervous system

Gut microbiota is implicated in the control of host physiology by releasing bioactive actors that could exert a direct or indirect effect on tissue. A dysfunction of the gut microbiota to tissue axis could participate in the development of pathological states such as obesity and diabetes. The aim of this study was to identify the metabolic effect of Limosilactobacillus reuteri (known as Lactobacillus reuteri) BIO7251 (L. reuteri BIO7251) isolated from Corsican clementine orange. Body weight gain, adiposity, glucose tolerance, glucose absorption and food intake were measured in mice fed a high-fat diet in response to a preventive oral administration of L. reuteri BIO7251. This strain of bacteria exerts a beneficial effect on body weight gain by decreasing the subcutaneous adipose tissue mass. The treatment with L. reuteri BIO7251 decreases glucose absorption and food intake in obese/diabetic mice. L. reuteri BIO7251 could be tested as new probiotic strain that could manage body weight during obesity

Characterization of the first virulent phage infecting Oenococcus oeni, the queen of the cellars

International audienceThere has been little exploration of how phages contribute to the diversity of the bacterial community associated with winemaking and may impact fermentations and product quality. Prophages of Oenococcus oeni, the most common species of lactic acid bacteria (LAB) associated with malolactic fermentation of wine, have been described, but no data is available regarding phages of O. oeni with true virulent lifestyles. The current study reports on the incidence and characterization of the first group of virulent oenophages named Vinitor, isolated from the enological environment. Vinitor phages are morphologically very similar to siphoviruses infecting other LAB. Although widespread during winemaking, they are more abundant in musts than temperate oenophages. We obtained the complete genomic sequences of phages Vinitor162 and Vinitor27, isolated from white and red wines, respectively. The assembled genomes shared 97.6% nucleotide identity and belong to the same species. Coupled with phylogenetic analysis, our study revealed that the genomes of Vinitor phages are architecturally mosaics and represent unique combinations of modules amongst LAB infecting-phages. Our data also provide some clues to possible evolutionary connections between Vinitor and (pro)phages associated to epiphytic and insect-related bacteria