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Abstract: Pyocins are high molecular weight bacteriocins produced by Pseudomonas aeruginosa that can be retargeted to new bacterial species by exchanging the pyocin tail fibers with bacteriophage receptor binding proteins (RBPs). Here, we develop retargeted pyocins called campycins as new antibacterials to precisely and effectively kill the major foodborne pathogen Campylobacter jejuni. We used two diverse RBPs (H-fibers) encoded by CJIE1 prophages found in the genomes of C. jejuni strains CAMSA2147 and RM1221 to construct campycin 1 and campycin 2, respectively. Campycins 1 and 2 could target all C. jejuni strains tested due to complementary antibacterial spectra. In addition, both campycins led to more than 3 log reductions in C. jejuni counts under microaerobic conditions at 42 °C, whereas the killing efficiency was less efficient under anaerobic conditions at 5 °C. Furthermore, we discovered that both H-fibers used to construct the campycins bind to the essential major outer membrane protein (MOMP) present in all C. jejuni in a strain-specific manner. Protein sequence alignment and structural modeling suggest that the highly variable extracellular loops of MOMP form the binding sites of the diverse H-fibers. Further in silico analyses of 5000 MOMP sequences indicated that the protein falls into three major clades predicted to be targeted by either campycin 1 or campycin 2. Thus, campycins are promising antibacterials against C. jejuni and are expected to broadly target numerous strains of this human pathogen in nature and agriculture. Key points: • Campycins are engineered R-type pyocins containing H-fibers from C. jejuni prophages • Campycins reduce C. jejuni counts by >3 logs at conditions promoting growth • Campycins bind to the essential outer membrane protein MOMP in a strain-dependent way

Abstract: Pyocins are high molecular weight bacteriocins produced by Pseudomonas aeruginosa that can be retargeted to new bacterial species by exchanging the pyocin tail fibers with bacteriophage receptor binding proteins (RBPs). Here, we develop retargeted pyocins called campycins as new antibacterials to precisely and effectively kill the major foodborne pathogen Campylobacter jejuni. We used two diverse RBPs (H-fibers) encoded by CJIE1 prophages found in the genomes of C. jejuni strains CAMSA2147 and RM1221 to construct campycin 1 and campycin 2, respectively. Campycins 1 and 2 could target all C. jejuni strains tested due to complementary antibacterial spectra. In addition, both campycins led to more than 3 log reductions in C. jejuni counts under microaerobic conditions at 42 °C, whereas the killing efficiency was less efficient under anaerobic conditions at 5 °C. Furthermore, we discovered that both H-fibers used to construct the campycins bind to the essential major outer membrane protein (MOMP) present in all C. jejuni in a strain-specific manner. Protein sequence alignment and structural modeling suggest that the highly variable extracellular loops of MOMP form the binding sites of the diverse H-fibers. Further in silico analyses of 5000 MOMP sequences indicated that the protein falls into three major clades predicted to be targeted by either campycin 1 or campycin 2. Thus, campycins are promising antibacterials against C. jejuni and are expected to broadly target numerous strains of this human pathogen in nature and agriculture. Key points: • Campycins are engineered R-type pyocins containing H-fibers from C. jejuni prophages • Campycins reduce C. jejuni counts by >3 logs at conditions promoting growth • Campycins bind to the essential outer membrane protein MOMP in a strain-dependent way

Viruses constitute the vast majority of all biological entities in the biosphere and represent one of the biggest reservoirs of undetected genetic diversity on Earth. Of all the viral particles inhabiting the ocean, phages are the most abundant and can affect the overall microbial composition of marine ecosystems and the dynamics of global biogeochemical cycles. The interaction between prokaryotic cells and their phages is among the oldest and most intertwined host-parasite relationships on the planet. It has been extensively studied by culture, molecular biology, and experimental evolution. However, due to the difficulties of culture with environmental samples, only a few studies have analyzed the mechanisms of phage-host interaction in the marine environment. Here, we have studied the genes involved in viral host recognition and their evolutionary dynamics by focusing on two species of the marine copiotrophic bacterium Alteromonas and several phages infecting them. We described the genomic and morphological characterization of the first Alteromonas phage belonging to the Myoviridae family (Alteromonas myovirus V22) that was isolated in coastal waters of the Mediterranean Sea, and we identified its receptor-binding protein (RBP) used for host recognition by combining fluorescence microscopy and spectrometry. In addition, using size-exclusion chromatography, we showed how this protein required co-expression with a downstream protein to be functional, which later was identified as a new type of intermolecular chaperone crucial for RBP maturation. We also identified a conserved host recognition module in V22 and other unrelated alterophages belonging to different viral families and with completely different morphologies, suggesting horizontal gene transfer between the ancestors of these phages. Furthermore, we described the first coevolution study of a host-parasite system performed with Alteromonas using a metagenomics-like approach. Finally, we analyzed the micro- a

Viruses constitute the vast majority of all biological entities in the biosphere and represent one of the biggest reservoirs of undetected genetic diversity on Earth. Of all the viral particles inhabiting the ocean, phages are the most abundant and can affect the overall microbial composition of marine ecosystems and the dynamics of global biogeochemical cycles. The interaction between prokaryotic cells and their phages is among the oldest and most intertwined host-parasite relationships on the planet. It has been extensively studied by culture, molecular biology, and experimental evolution. However, due to the difficulties of culture with environmental samples, only a few studies have analyzed the mechanisms of phage-host interaction in the marine environment. Here, we have studied the genes involved in viral host recognition and their evolutionary dynamics by focusing on two species of the marine copiotrophic bacterium Alteromonas and several phages infecting them. We described the genomic and morphological characterization of the first Alteromonas phage belonging to the Myoviridae family (Alteromonas myovirus V22) that was isolated in coastal waters of the Mediterranean Sea, and we identified its receptor-binding protein (RBP) used for host recognition by combining fluorescence microscopy and spectrometry. In addition, using size-exclusion chromatography, we showed how this protein required co-expression with a downstream protein to be functional, which later was identified as a new type of intermolecular chaperone crucial for RBP maturation. We also identified a conserved host recognition module in V22 and other unrelated alterophages belonging to different viral families and with completely different morphologies, suggesting horizontal gene transfer between the ancestors of these phages. Furthermore, we described the first coevolution study of a host-parasite system performed with Alteromonas using a metagenomics-like approach. Finally, we analyzed the micro- a

This experimental study investigated a novel strategy to reduce phage titer during milk acidification using phage adsorption to non-permissive host strains. For this purpose, two 936 species phages and six Lactococcus lactis strains were selected based on phage adsorption abilities. Challenge tests were conducted in two separate systems each containing the two phages and four strains. In the first system, phages were both able to propagate on their permissive host strain and to adsorb to one non-permissive host each. The second system contained the same permissive host strains and two other strains without phage adsorption. In the challenge tests, whey was transferred between successive cycles of milk acidifications to investigate the development of phage titer. Challenges tests were initiated with two different phage concentrations. Furthermore, a comparative sequence analysis was conducted on the C-terminal part of receptor-binding proteins (RBP) belonging to phages from the challenge test and selected phages propagating on the non-permissive hosts. A reduction of 1 log (PFU/mL) was observed for one of the phages adsorbing to the non-permissive host. However, this reduction became significantly smaller by adding a 100-fold lower phage concentration from the beginning. Finally, the comparison of RBPs indicated that the non-permissive host (responsible for reduction) may have contain two different receptors.

This experimental study investigated a novel strategy to reduce phage titer during milk acidification using phage adsorption to non-permissive host strains. For this purpose, two 936 species phages and six Lactococcus lactis strains were selected based on phage adsorption abilities. Challenge tests were conducted in two separate systems each containing the two phages and four strains. In the first system, phages were both able to propagate on their permissive host strain and to adsorb to one non-permissive host each. The second system contained the same permissive host strains and two other strains without phage adsorption. In the challenge tests, whey was transferred between successive cycles of milk acidifications to investigate the development of phage titer. Challenges tests were initiated with two different phage concentrations. Furthermore, a comparative sequence analysis was conducted on the C-terminal part of receptor-binding proteins (RBP) belonging to phages from the challenge test and selected phages propagating on the non-permissive hosts. A reduction of 1 log (PFU/mL) was observed for one of the phages adsorbing to the non-permissive host. However, this reduction became significantly smaller by adding a 100-fold lower phage concentration from the beginning. Finally, the comparison of RBPs indicated that the non-permissive host (responsible for reduction) may have contain two different receptors.