Biology subjects

Oechslin, F.

Publications and source records attributed to Oechslin, F..

5 recordsLinked to original sources

bioRxiv2026

Isolation and characterization of novel filamentous phages from Swiss-type cheeses infecting the Gram-positive bacterium Propionibacterium freudenreichii

Filamentous phages infecting Gram-positive bacteria remain largely unexplored. Notably, only two filamentous phages, B5 and Philemon infecting Propionibacterium freudenreichii, have been described to date in the phage-rich dairy ecosystem. Although both were genomically characterized, only B5 was confirmed to be an infective filamentous single-stranded DNA phage. The aim of this study was to isolate and characterize new filamentous phages from Swiss-type cheese to investigate their diversity, structural features, host specificity, and potential adaptation to the dairy environment. Thirty raw and pasteurized milk cheeses from France were screened for phages infecting P. freudenreichii strains. Eleven phages were isolated, nine of which displayed a filamentous morphology. Named MINOG1 to MINOG9, these filamentous phages exhibited genomic features typical of this morphotype, including small single-stranded DNA genomes with collinear genes organized into functional modules. Comparison with B5 and Philemon revealed sequence divergence ranging from 0.1% to 7%. These phages also exhibited a diverse host range. To further explore phage-P. freudenreichii interactions, we screened the genomes of the strains used in this study, as well as additional genomes retrieved from the NCBI database, for CRISPR spacers predicted to target these filamentous phages. Numerous strains contained CRISPR spacers showing 79 to 100% identity to genomic regions of these phages. Two P. freudenreichii strains displayed markedly different phage resistance levels despite exact spacer-protospacer matches with phages B5, MINOG1, MINOG2, and MINOG8. Conversely, several strains were resistant to nearly all tested phages despite lacking CRISPR spacers targeting them suggesting the presence of additional defense systems in P. freudenreichii. IMPORTANCEFilamentous phages can play important roles in bacterial ecology by modulating host physiology, population dynamics, and bacterial adaptation to specific environments. However, filamentous phages infecting Gram-positive bacteria remain among the least explored bacterial viruses, and their diversity, ecology, and interactions with their hosts are still poorly understood. This knowledge gap is particularly relevant in dairy ecosystems, where phages are abundant and can influence microbial communities and fermentation processes. In characterizing nine new filamentous phages infecting Propionibacterium freudenreichii from Swiss-type cheeses, this study expands the known diversity of filamentous phages associated with Gram-positive bacteria and provides new insights into phage-host interactions and bacterial defense strategies in dairy-associated bacteria.

microbiology↗

bioRxiv2026

Predictable adaptive evolution of a phage endolysin through substrate recognition optimization

Bacteriophages release progeny by producing endolysins that degrade the bacterial cell wall. While the frequent horizontal transfer of endolysins suggests substantial evolutionary plasticity, the mechanisms by which these enzymes adapt to new phage-host contexts remain poorly understood. Here, we investigated the evolutionary dynamics and structural mechanisms governing endolysin adaptation following experimental evolution of a chimeric phage generated via heterologous endolysin exchange between phages infecting different hosts. Using replicate experimental evolution and time-resolved PacBio sequencing, we identified a dominant, highly reproducible adaptive trajectory characterized by the stepwise fixation of three key mutations. This constrained mutational order correlated with incremental gains in enzymatic activity, reflecting a rugged yet predictable fitness landscape. High-resolution structural analyses revealed that these substitutions lie exclusively outside the catalytic site; instead, they enhance substrate recognition through electrostatic tuning, optimized hydrophobic packing, and local conformational refinement, resulting in significantly higher binding affinity. While the adaptive trajectory was largely conserved, one replicate followed an alternative path, highlighting the interplay between selection and historical contingency. Adaptation was further shaped by a functional trade-off, whereby increased lytic activity on the novel host was accompanied by reduced activity on the ancestral host, consistent with antagonistic pleiotropy. Genome-wide sequencing additionally identified a compensatory mutation in a lytic transglycosylase, suggesting coordinated evolution of the broader lysis machinery. Together, these results demonstrate that endolysins evolve through reproducible adaptive walks constrained by structure, selection, and trade-offs, providing a mechanistic framework for understanding enzyme evolution and informing rational protein engineering.

evolutionary biology↗

bioRxiv2023

Dairy practices select for thermostable endolysins in phages

Endolysins are phage lytic enzymes that degrade the bacterial cell wall to release phage progeny. We found that the diversity of endolysins is unsuspectedly low and dominated by a single structural type in distinct phages that infect Streptococcus thermophilus. Through X-ray crystallography, we discovered that this type of endolysin contains a highly conserved calcium-binding motif in its cell wall binding domain. Inactivation of the motif in the purified endolysin or in the phage genome revealed its key role in stabilizing the enzyme under conditions that mimic the cheesemaking process, including at elevated temperatures. Fermented dairy products such as yogurt and cheeses are incubated or cooked at higher temperatures and require the addition of thermophilic lactic acid bacteria such as S. thermophilus to drive milk fermentation. It appears that dairy practices have influenced the genetic signatures of streptococcal phages by selecting for thermostable endolysins, which in turn have limited their diversity.

microbiology↗

bioRxiv2020

Intramolecular synergism and bacterial lysis regulation can explain the complex multi-domain architecture of the bacteriophage endolysin PlySK1249

Endolysins are peptidoglycan hydrolases produced at the end of the bacteriophage (phage) replication cycle to lyse the host cell. Gram-positive phages endolysins come in a variety of multi-modular forms that combine different catalytic domains and may have evolved to adapt to their bacterial hosts. However, the reason why phage can adopt endolysin with such complex multidomain architecture is for the moment not well understood. We used the Streptococcus dysgalactiae phage endolysin PlySK1249 as a model to study the implication of multi-domain architecture in phage-induced bacterial lysis and lysis regulation. The activity of the enzyme relied on a bacteriolytic amidase (Ami), a non-bacteriolytic L-Ala-D-Ala endopeptidase (CHAP) acting as a de-chaining enzyme and central LysM cell wall binding domain (CBD). Ami and CHAP synergized for peptidoglycan digestion and bacteriolysis in the native enzyme or when expressed individually and reunified in vitro. This cooperation could be modulated by bacterial cell wall-associated proteases, which specifically cleaved the two linkers connecting the different domains. While both catalytic domains were observed to act coordinately to optimize bacterial lysis, the CBD is expected to delay diffusion of the enzyme until proteolytic inactivation is achieved. As for certain autolysins, PlySK1249 cleavage by bacterial cell wall associated proteases might be an example of dual phage-bacterial regulation and mutual coevolution. In addition, understanding more thoroughly the multidomain interplay of PlySK1249 opens new perspectives on the ideal architecture of therapeutic antibacterial endolysins.

microbiology↗

bioRxiv2019

Phage efficacy in infecting dual-strain biofilms of Pseudomonas aeruginosa

Bacterial viruses, or phage, play a key role in shaping natural microbial communities. Yet much research on bacterial-phage interactions has been conducted in liquid cultures involving single bacterial strains. Critically, phage often have a very narrow host range meaning they can only ever target a subset of strains in a community. Here we explore how strain diversity affects the success of lytic phage in structured communities. In particular, we infect a susceptible Pseudomonas aeruginosa strain PAO1 with lytic phage Pseudomonas 352 in the presence versus absence of an insensitive P. aeruginosa strain PA14, in liquid culture versus colonies growing on agar. We find that competition between the two bacterial strains reduces the likelihood of the susceptible strain evolving resistance to the phage. This result holds in liquid culture and in colonies. However, while in liquid the phage eliminate the whole sensitive population, colonies contain refuges wherein bacteria can remain sensitive yet escape phage infection. These refuges form mainly due to reduced growth in colony centers. We find little evidence that the presence of the insensitive strain provides any additional protection against phage. Our study reveals that living in a spatially structured population can protect bacteria against phage infection, while the presence of competing strains may instead reduce the likelihood of evolving resistance to phage, if encountered.

microbiology↗