Xylella fastidiosa (Xf) is a plant pathogen causing significant losses in agriculture worldwide. Originating from America, this bacterium caused recent epidemics in southern Europe and is thus considered an emerging pathogen. As the European regulations do not authorize antibiotic treatment in plants, alternative treatments are urgently needed to control the spread of the pathogen and eventually to cure infected crops. One such alternative is the use of phage therapy, developed more than 100 years ago to cure human dysentery and nowadays adapted to agriculture. The first step towards phage therapy is the isolation of the appropriate bacteriophages. With this goal, we searched for phages able to infect Xf strains that are endemic in the Mediterranean area. However, as Xf is truly a fastidious organism, we chose the phylogenetically closest and relatively fast-growing organism X. albineans as a surrogate host for the isolation step. Our results showed the isolation from various sources and preliminary characterization of several phages active on different Xf strains, namely, from the fastidiosa (Xff), multiplex (Xfm), and pauca (Xfp) subspecies, as well as on X. albilineans. We sequenced their genomes, described their genomic features, and provided a phylogeny analysis that allowed us to propose new taxonomic elements. Among the 14 genomes sequenced, we could identify two new phage species, belonging to two new genera of the Caudoviricetes order, namely, Usmevirus (Podoviridae family) and Subavirus (Siphoviridae family). Interestingly, no specific phages could be isolated from infected plant samples, whereas one was isolated from vector insects captured in a contaminated area, and several from surface and sewage waters from the Marseille area.

Salmonella enterica, a Gram-negative zoonotic bacterium, is mainly a food-borne pathogen and the main cause of diarrhea in humans worldwide. The main reservoirs are found in poultry farms, but they are also found in wild birds. The development of antibiotic resistance in S. enterica species raises concerns about the future of efficient therapies against this pathogen and revives the interest in bacteriophages as a useful therapy against bacterial infections. Here, we aimed to decipher and functionally annotate 10 new Salmonella phage genomes isolated in Spain in the light of phage therapy. We designed a bioinformatic pipeline using available building blocks to de novo assemble genomes and perform syntaxic annotation. We then used genome-wide analyses for taxonomic annotation enabled by vContact2 and VICTOR. We were also particularly interested in improving functional annotation using remote homologies detection and comparisons with the recently published phage-specific PHROG protein database. Finally, we searched for useful functions for phage therapy, such as systems encoded by the phage to circumvent cellular defenses with a particular focus on anti-CRISPR proteins. We, thus, were able to genetically characterize nine virulent phages and one temperate phage and identify putative functions relevant to the formulation of phage cocktails for Salmonella biocontrol.

Bacterial genome diversity is influenced by prophages, which are viral genomes integrated into the bacterial chromosome. Most prophage genes are silent but those that are expressed can provide unexpected properties to their host. Using as a modelE.coliK-12 that carries 9 defective prophages in its genome, we aimed at highlighting the impact of genes encoded by prophages on host physiology. We focused our work on AppY, a transcriptional regulator encoded on the DLP12 prophage. By performing RNA-Seq experiments, we showed that AppY production modulates the expression of more than 200 genes. Among them, 11 were identified by ChIP-Seq as direct AppY targets. AppY directly and positively regulates several genes involved in the acid stress response including the master regulator genegadEbut alsonhaRandgadY, two genes important for biofilm formation. Moreover, AppY indirectly and negatively impacts bacterial motility by favoring the degradation of FlhDC, the master regulator of the flagella biosynthesis. As a consequence of these regulatory effects, AppY increases acid stress resistance and biofilm formation while also causing a strong defect in motility. Our research shed light on the importance to consider the genetic interactions occurring between prophages and bacteria to fully understand bacterial physiology. It also highlights how a prophage-encoded transcriptional regulator integrates in a complex manner into the host regulatory network and how it benefits its host, allowing it to cope with changing environmental conditions.

The study of bacteriophages (viruses of bacteria) includes a variety of approaches, such as structural biology, genetics, ecology, and evolution, with increasingly important implications for therapeutic and industrial uses. Researchers working with phages in France have recently established a network to facilitate the exchange on complementary approaches, but also to engage new collaborations. Here, we provide a summary of the topics presented during the second meeting of the French Phage Network that took place in Marseille in November 2016

I. Salmonella enterica - a Gram negative zoonotic bacterium – is mainly a food-borne pathogen and the main cause of diarrhea in humans worldwide. Main reservoirs are found in poultry farms but also in wild birds. The development of antibiotic resistance in S. enterica species raises concerns about the future of efficient therapies against this pathogen and revives the interest in bacteriophages as a useful therapy against bacterial infections. Here we aimed at deciphering and functionally annotate 10 new Salmonella phage genomes isolated in Spain in the light of phage therapy. We designed a bioinformatic pipeline using available building blocks to de novo assemble genomes and perform syntaxic annotation. We then used genome-wide analyses for taxonomic annotation enabled by vContact2 and VICTOR. We were also particularly interested in improving functional annotation using remote homologies detection and comparisons with the recently published phage-specific PHROG protein database. We finally searched for useful functions for phage therapy such as systems encoded by the phage to circumvent cellular defenses with a particular focus on anti-CRIPSR proteins. We thus were able to genetically characterized nine virulent phages and one temperate phage and identified putative functions relevant to the formulation of phage cocktails for Salmonella biocontrol.

Viral taxonomy is a challenging task due to the propensity of viruses for recombination and the lack of universal gene markers. As a result, recent ICTV updates increasingly rely on multiple tools for taxonomic ranking, with a growing emphasis on proteome-based clustering approaches. At the same time, the rapid expansion of viral datasets presents new challenges in organizing, analysing, and discovering phage relationships at scale. To address these challenges, we introduce hierarchical viruses, a framework for comparative genomics of bacteriophages that leverages protein Language Model (pLM) embeddings to generate proteome-wide vector representations of phages. Clustering the vector representations of 24 362 phages from the curated INPHARED dataset reveals a multi-scale hierarchical organization of phages. This hierarchy aligns with current ICTV taxonomic rankings at the genus and subfamily levels, with an adjusted mutual information score greater than 0.9 for both, in the Herelleviridae family, demonstrating that pLM-based proteome representations can effectively capture evolutionary relationships without relying on multiple sequence alignments. The framework builds the basics towards vectorial phage datasets that encode evolutionary information, thus allowing discovery of phage relationships at scale.