Claire Le Marrec

1. Spontaneous vegetable fermentation is driven by the indigenous microbiota associated with the raw ingredients. The process often involves an initial heterofermentative phase followed by a predominantly homofermentative phase, both ensured by lactic acid bacteria (LAB). While microbial dynamics during fermentation are well characterized, phage communities remain poorly understood, despite evidence linking phage activity to LAB succession. We monitored viral and bacterial succession during sauerkraut and carrot fermentations using a metagenomics approach to characterize community dynamics. To further assess the impact of phage PLE001 on bacterial community dynamics, we developed a simplified carrot fermentation model comprising representative strains of Rahnella aquatilis, Leuconostoc mesenteroides and Lactiplantibacillus plantarum. Two consortia were assembled: one containing the PLE001-susceptible strain L. mesenteroides CIM3004, and the other containing also its phage-resistant derivative, CIM3004 BIM. We observed major shifts in the viral community during the first week of fermentation, coinciding with changes in bacterial structure during hetero- and homofermentative phases transition. In the simplified model, phage PLE001 reduced the population of the host during the heterofermentative phase but did not affect overall fermentation dynamics nor metabolite production. These results suggest that bacteriophages contribute to bacterial succession during fermentation, while communities remain resilient to phage infection, preserving fermentation stability.

Bacteriophages (phages) infecting Gram-positive bacteria must bind to host receptors across thick cell walls to initiate infection, yet the underlying structural mechanisms remain unclear. Here, we report cryo-electron microscopy structures of the Oenococcus oeni siphophage OE33PA, providing the first high-resolution view of a phage infecting this wine-industry bacterium. While the overall virion architecture is conserved, the adhesion device displays distinctive features. Its receptor-binding proteins adopt multiple orientations, revealing an intrinsically dynamic assembly. In situ cryo-electron tomography captures distinct conformations upon host attachment, providing rare structural insight into interactions with Gram-positive hosts. Functional assays further show that a mobile carbohydrate-binding module in the distal tail protein mediates host cell binding. Additionnally, the tape measure protein adopts a hexameric organization, updating the prevailing trimeric model in siphophages. Together, these findings reveal a dynamic adhesion device in a phage infecting Gram-positive bacteria and highlight the structural and functional diversity of phages.

This protocol describes an easy, quick, cheap, and effective method for the purification and concentration of bacteriophages (phages) produced in rich culture media, meeting the quality criteria required for structural analyses. It is based on a tube dialysis system that replaces the classical but expensive and tedious density gradient ultracentrifugation step. We developed this protocol for the Oenococcus oeni bacteriophage OE33PA from its amplification to imaging by negative stain electron microscopy (NS-EM). The host bacterium, O. oeni, is a lactic acid bacterium that lives in harsh oenological ecosystems and grows only in rich and complex media such as Man–Rogosa–Sharpe (MRS) or fruit juice-based media in laboratory conditions. This raises experimental challenges in pure and concentrated phage preparations for further uses such as structure-function studies.

Although the species is extensively studied, limited data are available on antiphage defense systems (APDSs) in Streptococcus mutans . The present study aimed to explore the diversity and the occurrence of APDSs and to search for prophages in the genomes of clinical isolates of S. mutans using bioinformatics tools. Forty‐four clinical isolates of S. mutans were obtained from saliva samples of people with Parkinson's disease. Genomic DNA was extracted, sequenced using Illumina MiSeq technology, and analyzed for the presence of defense systems using DefenseFinder and PADLOC. CRISPR‐Cas systems were characterized using CRISPRCasFinder, and prophages were detected by the PhiSpy pipeline from RAST. AcrFinder and AcrHub were used to identify anti‐CRISPR proteins. Each strain harbored between 6 and 12 APDS, with restriction‐modification systems being the most prevalent, followed by the MazEF toxin–antitoxin system and CRISPR‐Cas systems. Type II‐C CRISPR‐Cas systems were not identified here in S. mutans . Novel variations in type II‐A signature protein Cas9 were identified, allowing their classification into four distinct groups. Variability in direct repeat sequences within the same CRISPR array was also observed, and 80% of the spacers were classified as targeting “dark matter”. A unique prophage, phi_37bPJ2, was detected, showing high similarity with previously described phages. The AcrIIA5 protein encoded by phi_37bPJ2 was conserved and suggested to remain functionally active. This study reveals the diversity of APDSs in S. mutans and the limited presence of prophages. The findings provide a foundation for future research on the evolutionary dynamics of these systems and their role in S. mutans adaptation to phage pressure.

Oenococcus oeni is the predominant species of lactic acid bacteria in wine, where it carries out malolactic fermentation (MLF), which helps to ensure and preserve the quality of the wine. Today, existing combinations of grape varieties, soil composition, fluctuating climatic parameters, and specific technical processes implemented by wineries lead to incredibly varied wine compositions that pose challenges for spontaneous MLF. Commercial starter cultures have been developed for use as inoculants. However, their effectiveness in ensuring consistent and reliable MLF is also limited in modern wines. The selection process must therefore adapt to these new challenges, which means expanding current portfolios by selecting more robust bacteria from wines that are more varied in terms of grape varieties and chemical constraints. We have assembled a set of 21 wines produced in Europe from different grape varieties, with varying and, in some cases, extreme ethanol contents, total polyphenolic indices, and pH levels. The isolation and MLVA typing of 385 dominant colonies were combined with whole-genome sequencing of 48 representative strains, and we observed several strains with unique accessory genomic content. Different selective pressures led to the formation of groups of genetically related individuals, particularly in white and rosé wines with moderate ethanol content. However, cohabiting strains with contrasting genetic profiles were also observed in some red wines. Our data highlight the complexity of the factors involved in population heterogeneity and raise the possibility that this phenomenon may increase fitness through diversification of strategies or division of labor in specific production environments.

Oenococcus oeni is the predominant lactic acid bacteria species in wine, where it performs the malolactic fermentation, which helps to secure and preserve wine quality. Here, we describe the morphological, biological, and genomic characterization of siphophage Krappator X ₂₇ , a strictly lytic phage that was previously isolated from Merlot wines. Several aspects of the life cycle of the phage were investigated using the sensitive strain IOEBS277 under optimal growth conditions. X ₂₇ has a large burst size (149 Plaque Forming unit [PFU] per infected cell) and targets industrially relevant strains of O. oeni used for production of wines. X ₂₇ also shows lytic activity against its host in red and white grape juice media. The phage genome consists of 41,633 nucleotides. Structure prediction of the viral adhesion devices reveals elongated multi-domain machinery containing carbohydrate-binding modules likely involved in host recognition and binding. Genome-based phylogeny revealed that X ₂₇ has less than 30% intergenomic similarities with its most similar phages and represents a new species in a yet unassigned genus, for which we propose the name « Krappavirus » . Aiming to expand the available collection of genomes in this genus, we sequenced two independently isolated phage homologs from Merlot wines. Modular protein evolution may play a role in the diversification of this specific phage lineage, and naturally occurring domain combinations were found in three crucial proteins of known function: the small terminase subunit, the endolysin, and the replisome organizer. IMPORTANCE: Oenococcus oeni is commonly used for wine and cider production. Characterizing strictly lytic oenophages, understanding their genetic relationships, and studying their interactions with various hosts are the necessary steps for preventing and controlling phage attacks that occur along the fermentation process.

Background/Objectives: Bacteriophages infecting the genus Streptococcus play a crucial role in microbial ecology and have potential applications in biotechnology and medicine. Despite their importance, significant gaps remain in our understanding of their lysis modules. This study aims to address these deficiencies by analyzing the genomic diversity and lysis module organization in Streptococcus phages. Methods: A search was conducted in the NCBI RefSeq database to identify phage genomes infecting Streptococcus. A representative panel was selected based on taxonomic diversity. Lysis modules were annotated and visualized, functional domains in endolysins were identified, and holins were characterized. Results: A total of 205 phage genomes were retrieved from the NCBI RefSeq database, of which 185 complete genomes were analyzed. A subset of 34 phages was selected for in-depth analysis, ensuring the representation of taxonomic diversity. The lysis modules were annotated and visualized, revealing five distinct organizations. Among the 256 identified endolysins, 25 distinct architectural organizations were observed, with amidase activity being the most prevalent. Holins were classified into 9 of the 74 families listed in the Transporter Classification Database, exhibiting one to three transmembrane domains. Conclusions: This study provides insights into the structural diversity of lysis modules in Streptococcus phages, paving the way for future research and potential biotechnological applications.

Bacteriophages infecting the genus Streptococcus play a crucial role in microbial ecology and have potential applications in biotechnology and medicine. Despite their importance, significant gaps remain in our understanding of their lysis modules. This study aims to address these deficiencies by analyzing the genomic diversity and lysis module organization of phages infecting the Streptococcus genus. A search was conducted in the NCBI RefSeq database to identify phage genomes infecting the Streptococcus genus. A representative panel was selected based on taxonomic diversity. Lysis modules were annotated and visualized, functional domains in endolysins were identified, and holins were characterized. A total of 205 phage genomes were retrieved from the NCBI RefSeq database, of which 185 complete genomes were analyzed. A subset of 34 phages was selected for in-depth analysis, ensuring representation of taxonomic diversity. The lysis modules were annotated and visualized, revealing five distinct organizations. Among the 256 identified endolysins, 25 distinct architectural organizations were observed, with amidase activity being the most prevalent. Holins were classified into 9 of the 74 families listed in the Transporter Classification Database, exhibiting 1 to 3 transmembrane domains. This study provides insights into the structural diversity of lysis modules in Streptococcus phages, paving the way for future research and potential biotechnological applications.

Les virus de bactéries, ou bactériophages, sont les virus les plus abondants sur Terre, et leurs hôtes sont les organismes vivants les plus répandus dans la biosphère. Ils sont retrouvés à l’état libre, sous forme de virions, et aussi très abondants dans les génomes de bactéries, à l’état de prophages. Les bactériophages sont présents dans tous les biotopes colonisés par les bactéries, eaux, sols, environnements extrêmes, microbiotes humains, animaux et végétaux, où ils participent aux échanges génétiques. Tous les facteurs qui impactent ces environnements ont donc des conséquences importantes sur la dynamique des populations de bactéries et de leurs virus.