Discovery and characterization of the first bacterioviruses amongst the order of thermotogales

Notre connaissance de la diversité virale associée aux microorganismes procaryotiques issus des sources hydrothermales océaniques profondes demeure encore limitée. Peu d’études se sont intéressées à l’abondance virale ou à l’impact des virus sur la mortalité microbienne au sein de ces écosystèmes. Le nombre de virus caractérisés, issus de ces environnements, reste faible. Deux virus, PAV1 et TPV1, associés à des archées hyperthermophiles anaérobies appartenant à l’ordre des Thermococcales ont été décrits dans notre laboratoire. Afin de poursuivre nos recherches sur la diversité virale infectant les microorganismes hydrothermaux marins, l’ordre bactérien des Thermotogales a été ciblé. Cet ordre est composé de bactéries chimioorganotrophes anaérobies pour la plupart hyper/thermophiles. Elles partagent la même niche écologique que les Thermococcales et sont métaboliquement proches. De nombreux transferts latéraux de gènes ont, par ailleurs, contribué à l’histoire évolutive des Thermotogales, subodorant l’implication potentielle de virus. La présence de séquences CRISPR a également été rapportée au sein de plusieurs génomes de Thermotogales, suggérant que les Thermotogales sont ou ont certainement déjà été exposées à des infections virales. Pour autant, à ce jour, les seuls éléments génétiques mobiles à avoir réellement été décrits chez les Thermotogales sont 3 miniplasmides et aucun virus. Une cinquantaine de souches de Thermotogales provenant majoritairement de la collection de notre laboratoire (Souchothèque de Bretagne et Collection Ifremer) a été passée au crible quant à la présence d’éventuels bactériovirus associés. A l’issue de ce criblage, des éléments à ADN extra-chromosomiques, incluant 2 plasmides et 7 bactériovirus (du type Siphoviridae) ont été découverts au sein de souches appartenant aux genres Thermosipho et Marnitoga. Des analyses préliminaires ont été réalisées sur ces différents éléments et l’un des nouveaux systèmes hôte/virus a été caractérisé en détail. MPV1 (Marinitoga piezophila virus 1) est un siphovirus-like tempéré isolé d’une bactérie piezophile, il constitue le premier bactériovirus associé à l’ordre des Thermotogales. La souche hôte est piezophile mais aisément cultivable à pression atmosphérique au terme de plusieurs repiquages. Si l’essentiel des analyses a été mené à pression atmosphérique, la production virale s’est avérée tout à fait effective à pression hydrostatique. Nous avons réalisé les analyses de la séquence complète du génome MPV1 (43,7 kb, extrait des capsides virales purifiées) et sa comparaison avec le provirus présent au sein du génome séquencé de Marinitoga piezophila KA3. Les analyses de ce génome viral ont suggéré une proximité évolutive de MPV1 avec les bactériovirus de Firmicutes. Nous avons également mis en évidence que le bactériovirus partage son hôte avec un élément génétique extra chromosomique circulaire de 13,3 kb (pMP1). Ce « ménage à 3 » est surprenant dans le sens où l’élément de 13,3 kb, contenant 13 ORF de fonctions majoritairement inconnues, utilise les capsides virales afin de se propager. Ceci pourrait, ainsi, illustrer un nouvel exemple de piratage moléculaire.Our knowledge of the viral diversity associated to procaryotic microorganisms inhabiting the deep sea hydrothermal vents is still limited. Only few studies have focused on viral abundance and impact on microbial mortality within these ecosystems. A limited number of viruses from these environments were isolated and characterized. Two viruses, PAV1 and TPV1, associated to hyperthermophilic anaerobic Archaea, Thermococcales order, have ever been described in our laboratory. The topic of this phD thesis was to extend our investigation to other deep sea vent microorganisms in order to deepen our knowledge on the marine hydrothermal virosphere. We decided to focus more precisely on the bacterial order of Thermotogales. This order is composed of anaerobic chemoorganotrophic bacteria that are, for almost, hyper/thermophilic. They share the same ecological niche as the Thermococcales and are metabolically close. Numerous lateral gene transfers have contributed to the evolutionary history of the Thermotogales, implying the potential involvement of viruses. The presence of CRISPRs has also been reported in many genomes, suggesting that Thermotogales certainly are or have been exposed to viral infections. However, up till now, only 3 miniplasmids have been described within Thermotogales and no viruses. Fifty strains of Thermotogales, mostly from the LM2E culture collection (Ifremer and “UBOCC”), were screened for the presence of potential bacteriovirus. Extrachromosomal DNA elements, including 2 plasmids and 7 bacterioviruses (siphovirus-like), were discovered amongst strains belonging to both Thermosipho and Marinitoga genera. Preliminary studies were performed on these elements and one of the new virus-host systems was characterized in details. MPV1 (Marinitoga piezophila virus 1) is a temperate siphovirus-like isolated from a piezophilic bacterium, it is the first bacteriovirus associated to the Thermotogales order. This host strain is piezophilic but easily cultivable at atmospheric pressure after several subcultures. Whether most experiments were performed at atmospheric pressure, the viral production appeared to be effective at hydrostatic pressure. We reported the analyses of the complete sequence of the MPV1 genome (43.7 kb, extracted from purified virions) and its comparison to the provirus present in the sequenced genome of Marinitoga piezophila KA3. Analyses of the viral genome suggested a close evolutionary relationship of MPV1 to Firmicutes bacterioviruses .We also reported that this bacteriovirus shares its host with a circular extrachromosomal genetic element of 13.3 kb (pMP1). This ‘ménage à trois’ is surprising in the sense where the 13.3kb element, that contains 13 ORFs of mostly unknown function, uses the viral capsid to propagate. Therefore, it would likely correspond to a new example of molecular piracy

Découverte et caractérisation des premiers virus de Thermotogales (bactéries thermophiles et anaérobies) issus de sources hydrothermales océaniques profondes

Our knowledge of the viral diversity associated to procaryotic microorganisms inhabiting the deep sea hydrothermal vents is still limited. Only few studies have focused on viral abundance and impact on microbial mortality within these ecosystems. A limited number of viruses from these environments were isolated and characterized. Two viruses, PAV1 and TPV1, associated to hyperthermophilic anaerobic Archaea, Thermococcales order, have ever been described in our laboratory. The topic of this phD thesis was to extend our investigation to other deep sea vent microorganisms in order to deepen our knowledge on the marine hydrothermal virosphere. We decided to focus more precisely on the bacterial order of Thermotogales. This order is composed of anaerobic chemoorganotrophic bacteria that are, for almost, hyper/thermophilic. They share the same ecological niche as the Thermococcales and are metabolically close. Numerous lateral gene transfers have contributed to the evolutionary history of the Thermotogales, implying the potential involvement of viruses. The presence of CRISPRs has also been reported in many genomes, suggesting that Thermotogales certainly are or have been exposed to viral infections. However, up till now, only 3 miniplasmids have been described within Thermotogales and no viruses. Fifty strains of Thermotogales, mostly from the LM2E culture collection (Ifremer and “UBOCC”), were screened for the presence of potential bacteriovirus. Extrachromosomal DNA elements, including 2 plasmids and 7 bacterioviruses (siphovirus-like), were discovered amongst strains belonging to both Thermosipho and Marinitoga genera. Preliminary studies were performed on these elements and one of the new virus-host systems was characterized in details. MPV1 (Marinitoga piezophila virus 1) is a temperate siphovirus-like isolated from a piezophilic bacterium, it is the first bacteriovirus associated to the Thermotogales order. This host strain is piezophilic but easily cultivable at atmospheric pressure after several subcultures. Whether most experiments were performed at atmospheric pressure, the viral production appeared to be effective at hydrostatic pressure. We reported the analyses of the complete sequence of the MPV1 genome (43.7 kb, extracted from purified virions) and its comparison to the provirus present in the sequenced genome of Marinitoga piezophila KA3. Analyses of the viral genome suggested a close evolutionary relationship of MPV1 to Firmicutes bacterioviruses .We also reported that this bacteriovirus shares its host with a circular extrachromosomal genetic element of 13.3 kb (pMP1). This ‘ménage à trois’ is surprising in the sense where the 13.3kb element, that contains 13 ORFs of mostly unknown function, uses the viral capsid to propagate. Therefore, it would likely correspond to a new example of molecular piracy.Notre connaissance de la diversité virale associée aux microorganismes procaryotiques issus des sources hydrothermales océaniques profondes demeure encore limitée. Peu d’études se sont intéressées à l’abondance virale ou à l’impact des virus sur la mortalité microbienne au sein de ces écosystèmes. Le nombre de virus caractérisés, issus de ces environnements, reste faible. Deux virus, PAV1 et TPV1, associés à des archées hyperthermophiles anaérobies appartenant à l’ordre des Thermococcales ont été décrits dans notre laboratoire. Afin de poursuivre nos recherches sur la diversité virale infectant les microorganismes hydrothermaux marins, l’ordre bactérien des Thermotogales a été ciblé. Cet ordre est composé de bactéries chimioorganotrophes anaérobies pour la plupart hyper/thermophiles. Elles partagent la même niche écologique que les Thermococcales et sont métaboliquement proches. De nombreux transferts latéraux de gènes ont, par ailleurs, contribué à l’histoire évolutive des Thermotogales, subodorant l’implication potentielle de virus. La présence de séquences CRISPR a également été rapportée au sein de plusieurs génomes de Thermotogales, suggérant que les Thermotogales sont ou ont certainement déjà été exposées à des infections virales. Pour autant, à ce jour, les seuls éléments génétiques mobiles à avoir réellement été décrits chez les Thermotogales sont 3 miniplasmides et aucun virus. Une cinquantaine de souches de Thermotogales provenant majoritairement de la collection de notre laboratoire (Souchothèque de Bretagne et Collection Ifremer) a été passée au crible quant à la présence d’éventuels bactériovirus associés. A l’issue de ce criblage, des éléments à ADN extra-chromosomiques, incluant 2 plasmides et 7 bactériovirus (du type Siphoviridae) ont été découverts au sein de souches appartenant aux genres Thermosipho et Marnitoga. Des analyses préliminaires ont été réalisées sur ces différents éléments et l’un des nouveaux systèmes hôte/virus a été caractérisé en détail. MPV1 (Marinitoga piezophila virus 1) est un siphovirus-like tempéré isolé d’une bactérie piezophile, il constitue le premier bactériovirus associé à l’ordre des Thermotogales. La souche hôte est piezophile mais aisément cultivable à pression atmosphérique au terme de plusieurs repiquages. Si l’essentiel des analyses a été mené à pression atmosphérique, la production virale s’est avérée tout à fait effective à pression hydrostatique. Nous avons réalisé les analyses de la séquence complète du génome MPV1 (43,7 kb, extrait des capsides virales purifiées) et sa comparaison avec le provirus présent au sein du génome séquencé de Marinitoga piezophila KA3. Les analyses de ce génome viral ont suggéré une proximité évolutive de MPV1 avec les bactériovirus de Firmicutes. Nous avons également mis en évidence que le bactériovirus partage son hôte avec un élément génétique extra chromosomique circulaire de 13,3 kb (pMP1). Ce « ménage à 3 » est surprenant dans le sens où l’élément de 13,3 kb, contenant 13 ORF de fonctions majoritairement inconnues, utilise les capsides virales afin de se propager. Ceci pourrait, ainsi, illustrer un nouvel exemple de piratage moléculaire

Plasmid pMO1 from Marinitoga okinawensis, first non-cryptic plasmid reported within Thermotogota

Mobile genetic elements (MGEs), such as viruses and plasmids, drive the evolution and adaptation of their cellular hosts from all three domains of life. This includes microorganisms thriving in the most extreme environments, like deep-sea hydrothermal vents. However, our knowledge about MGEs still remains relatively sparse in these abyssal ecosystems. Here we report the isolation, sequencing, assembly, and functional annotation of pMO1, a 28.2 kbp plasmid associated with the reference strain Marinitoga okinawensis. Carrying restriction/modification and chemotaxis protein-encoding genes, pMO1 likely affects its host’s phenotype and represents the first non-cryptic plasmid described among the phylum Thermotogota

Caractérisation et modélisation des populations de bactériophages de la croûte de fromages (type époisses) sous différentes conditions environnementales d’affinage

National audienc

Caractérisation et modélisation des populations de bactériophages de la croûte de fromages (type époisses) sous différentes conditions environnementales d’affinage

National audienc

An abyssal mobilome: Viruses, plasmids and vesicles from deep-sea hydrothermal vents

Mobile genetic elements (MGEs) such as viruses, plasmids, vesicles, gene transfer agents (GTAs), transposons and transpovirions, which collectively represent the mobilome, interact with cellular organisms from all three domains of life, including those thriving in the most extreme environments. While efforts have been made to better understand deep-sea vent microbial ecology, our knowledge of the mobilome associated with prokaryotes inhabiting deep-sea hydrothermal vents remains limited. Here we focus on the abyssal mobilome by reviewing accumulating data on viruses, plasmids and vesicles associated with thermophilic and hyperthermophilic Bacteria and Archaea present in deep-sea hydrothermal vents

Thermosipho spp. Immune System Differences Affect Variation in Genome Size and Geographical Distributions

Thermosipho species inhabit thermal environments such as marine hydrothermal vents, petroleum reservoirs, and terrestrial hot springs. A 16S rRNA phylogeny of available Thermosipho spp. sequences suggested habitat specialists adapted to living in hydrothermal vents only, and habitat generalists inhabiting oil reservoirs, hydrothermal vents, and hotsprings. Comparative genomics of 15 Thermosipho genomes separated them into three distinct species with different habitat distributions: The widely distributed T. africanus and the more specialized, T. melanesiensis and T. affectus. Moreover, the species can be differentiated on the basis of genome size (GS), genome content, and immune system composition. For instance, the T. africanus genomes are largest and contained the most carbohydrate metabolism genes, which could explain why these isolates were obtained from ecologically more divergent habitats. Nonetheless, all the Thermosipho genomes, like other Thermotogae genomes, show evidence of genome streamlining. GS differences between the species could further be correlated to differences in defense capacities against foreign DNA, which influence recombination via HGT. The smallest genomes are found in T. affectus that contain both CRISPR-cas Type I and III systems, but no RM system genes. We suggest that this has caused these genomes to be almost devoid of mobile elements, contrasting the two other species genomes that contain a higher abundance of mobile elements combined with different immune system configurations. Taken together, the comparative genomic analyses of Thermosipho spp. revealed genetic variation allowing habitat differentiation within the genus as well as differentiation with respect to invading mobile DNA

Virulent Phages Isolated from a Smear-Ripened Cheese Are Also Detected in Reservoirs of the Cheese Factory

International audienceSmear-ripened cheeses host complex microbial communities that play a crucial role in the ripening process. Although bacteriophages have been frequently isolated from dairy products, their diversity and ecological role in such this type of cheese remain underexplored. In order to fill this gap, the main objective of this study was to isolate and characterize bacteriophages from the rind of a smear-ripened cheese. Thus, viral particles extracted from the cheese rind were tested through a spot assay against a collection of bacteria isolated from the same cheese and identified by sequencing the full-length small subunit ribosomal RNA gene. In total, five virulent bacteriophages infecting Brevibacterium aurantiacum, Glutamicibacter arilaitensis, Leuconostoc falkenbergense and Psychrobacter aquimaris species were obtained. All exhibit a narrow host range, being only able to infect a few cheese-rind isolates within the same species. The complete genome of each phage was sequenced using both Nanopore and Illumina technologies, assembled and annotated. A sequence comparison with known phages revealed that four of them may represent at least new genera. The distribution of the five virulent phages into the dairy-plant environment was also investigated by PCR, and three potential reservoirs were identified. This work provides new knowledge on the cheese rind viral community and an overview of the distribution of phages within a cheese factory

Newly identified proviruses in Thermotogota suggest that viruses are the vehicles on the highways of interphylum gene sharing

Phylogenomic analyses of bacteria from the phylum Thermotogota have shown extensive lateral gene transfer with distantly related organisms, particularly with Firmicutes. One likely mechanism of such DNA transfer is viruses. However, to date, only three temperate viruses have been characterized in this phylum, all infecting bacteria from the Marinitoga genus. Here we report 17 proviruses integrated into genomes of bacteria belonging to eight Thermotogota genera and induce viral particle production from one of the proviruses. All except an incomplete provirus from Mesotoga fall into two groups based on sequence similarity, gene synteny and taxonomic classification. Proviruses of Group 1 are found in the genera Geotoga, Kosmotoga, Marinitoga, Thermosipho and Mesoaciditoga and are similar to the previously characterized Marinitoga viruses, while proviruses from Group 2 are distantly related to the Group 1 proviruses, have different genome organization and are found in Petrotoga and Defluviitoga. Genes carried by both groups are closely related to Firmicutes and Firmicutes (pro)viruses in phylogenetic analyses. Moreover, one of the groups show evidence of recent gene exchange and may be capable of infecting cells from both phyla. We hypothesize that viruses are responsible for a large portion of the observed gene flow between Firmicutes and Thermotogota

Draft Genome Sequences of Two Marinitoga camini Isolates Producing Bacterioviruses

Here, we present the draft genome sequences of two thermophilic Marinitoga strain members of the Thermotogales order, Marinitoga camini DV1155 and Marinitoga camini DV1197. These strains were isolated from deep-sea hydrothermal vents of the Mid-Atlantic Ridge