Most of studied bacteriophages (phages) are terrestrial viruses. However, marine phages are shown to be highly involved in all levels of oceanic regulation. They are, however, still largely overlooked by the scientific community. By inducing cell lysis on half of the bacterial population daily, their role and influence on the bacterial biomass and evolution, as well as their impact in the global biogeochemical cycles, is undeniable. Cobetia marina virus 1 (Carin-1) is a member of the Podoviridae family infecting the γ-protoabacteria C. marina. Here, we present the almost complete, nearly-atomic resolution structure of Carin-1 comprising capsid, portal, and tail machineries at 3.5 Å, 3.8 Å and 3.9 Å, respectively, determined by cryo-electron microscopy (cryo-EM). Our experimental results, combined with AlphaFold2 (AF), allowed us to obtain the nearly-atomic structure of Carin-1 by fitting and refining the AF atomic models in the high resolution cryo-EM map, skipping the bottleneck of de-novo manual building and speeding up the structure determination process. Our structural results highlighted the T7-like nature of Carin1, as well as several novel structural features like the presence of short spikes on the capsid, reminiscent those described for Rhodobacter capsulatus gene transfer agent (RcGTA). This is, to our knowledge, the first time such assembly is described for a bacteriophage, shedding light into the common evolution and shared mechanisms between gene transfer agents and phages. This first full structure determined for a marine podophage allowed to propose an infection mechanism different than the one proposed for the archetypal podophage T7. IMPORTANCE Oceans play a central role in the carbon cycle on Earth and on the climate regulation (half of the planet\'s CO2 is absorbed by phytoplankton photosynthesis in the oceans and just as much O2 is liberated). The understanding of the biochemical equilibriums of marine biology represents a major goal for our future. By lysing half of the bacterial population every day, marine bacteriophages are key actors of these equilibriums. Despite their importance, these marine phages have, so far, only been studied a little and, in particular, structural insights are currently lacking, even though they are fundamental for the understanding of the molecular mechanisms of their mode of infection. The structures described in our manuscript allow us to propose an infection mechanism that differs from the one proposed for the terrestrial T7 virus, and might also allow us to, in the future, better understand the way bacteriophages shape the global ecosystem.

Understanding interactions of bacterial surface polysaccharides with receptor protein scaffolds is important for the development of antibiotic therapies. The corresponding protein recognition domains frequently form low-affinity complexes with polysaccharides that are difficult to address with experimental techniques due to the conformational flexibility of the polysaccharide. In this work, we studied the tailspike protein (TSP) of the bacteriophage Sf6. Sf6TSP binds and hydrolyzes the high-rhamnose, serotype Y O-antigen polysaccharide of the Gram-negative bacterium Shigella flexneri (S. flexneri) as a first step of bacteriophage infection. Spectroscopic analyses and enzymatic cleavage assays confirmed that Sf6TSP binds long stretches of this polysaccharide. Crystal structure analysis and saturation transfer difference (STD) NMR spectroscopy using an enhanced method to interpret the data permitted the detailed description of affinity contributions and flexibility in an Sf6TSP-octasaccharide complex. Dodecasaccharide fragments corresponding to three repeating units of the O-antigen in complex with Sf6TSP were studied computationally by molecular dynamics simulations. They showed that distortion away from the low-energy solution conformation found in the octasaccharide complex is necessary for ligand binding. This is in agreement with a weak-affinity functional polysaccharide-protein contact that facilitates correct placement and thus hydrolysis of the polysaccharide close to the catalytic residues. Our simulations stress that the flexibility of glycan epitopes together with a small number of specific protein contacts provide the driving force for Sf6TSP-polysaccharide complex formation in an overall weak-affinity interaction system.

The cell surface receptor for bacteriophage Lambda is LamB (maltoporin). Responsible for phage binding to LamB is the C-terminal part, gpJ, of phage tail protein J. To study the interaction between LamB and gpJ, a chimera protein composed of maltose binding protein (MBP or MalE) connected to the C-terminal part of J (gpJ, amino acids 684-1131) of phage tail protein J of bacteriophage Lambda was expressed in Escherichia coli and purified to homogeneity. The interaction of the MBP-gpJ chimera protein with reconstituted LamB and its mutants LamB Y118G and the loop deletion mutant LamB Delta4+Delta6+Delta9v was studied using planar lipid bilayer membranes on a single-channel and multichannel level. Titration with the MBP-gpJ chimera blocked completely the ion current through reconstituted LamB when it was added to the cis side, the extracellular side of LamB with a half-saturation constant of approximately 6 nM in 1 M KCl. Control experiments with LamB Delta4+Delta6+Delta9v from which all major external loops had been removed showed similar blocking, whereas MBP alone caused no visible effect. Direct conductance measurement with His(6)-gpJ that contained a hexahistidyl tag (His(6) tag) at the N-terminal end of the protein for easy purification revealed no blocking of the ion current, requiring other measurements for the binding constant. However, when maltoporin was preincubated with His-gpJ, MBP-gpJ could not block the channel, which indicated that also His(6)-gpJ bound to the channel. High-molecular mass bands on SDS-PAGE and Western blots, confirming the planar lipid bilayer experiment results, also demonstrated stable complex formation between His(6)-gpJ and LamB or LamB mutants. The results revealed that phage Lambda binding includes not only the extracellular loops.