The rise in multidrug-resistant pathogenic bacteria presents a major current challenge, highlighting the urgent need for alternatives and sustainable biocontrol strategies. Here, we report the genome analysis of a bacteriophage called Salmonella phage Tennessee Salten and attributed to a new Tequintavirus species. Salten was isolated following infection of Salmonella enterica subsp. enterica serotype Tennessee (sequence type ST5018). Its genome is 109,999 bp in length and contains 220 predicted proteins on which 197 are CDS and 23 tRNAs. Compared to its closest known relative phage Escherichia phage HildyBeyeler - sharing 84.6% identity - Salten harbours 16 unique or highly divergent genes. Of these, 13 encode proteins with unknown function, one encodes for a putative adenine methyltransferase and two encode HNH homing endonucleases. Moreover, the Long Tail Fibre protein, whose structure was predicted based on that of phage T5, was highly divergent among the Tequintavirus genus.

Siphophages are tailed bacteriophages characterised by their long noncontractile tails. In this review, we compare the recent electron cryo-microscopy structures of eight siphophage tails. We confirm and extend common building block organisation within the siphophage tails, particularly within the tail tip. Moreover, the description of the structure of siphophages T5 and λ tail after receptor binding, showing conformational changes only in the tail tip, explains how the siphophage tail opens, leading to DNA ejection. Conserved structural elements point to a general mechanism of infection for Gram-negative-infecting siphophages and allow considerations regarding the classification of the receptor-binding proteins into two different categories: host recognition receptors and membrane sensing receptors that trigger DNA ejection.

This article presents an in-depth analysis of selected CASP16 targets, with a focus on their biological and functional significance. The authors highlight the most relevant features of the target proteins and discuss how well these were reproduced in the submitted predictions. While the overall performance of structure prediction methods remains impressive, challenges persist, particularly in modeling rare structural motifs, flexible regions, small molecule interactions, posttranslational modifications, and biologically important interfaces. Addressing these limitations can strengthen the role of structure prediction in complementing experimental efforts and advancing both basic research and biomedical applications.

ObjectiveThe rise in multidrug-resistant pathogenic bacteria presents a major current challenge, highlighting the urgent need for alternatives and sustainable biocontrol strategies. We aimed to isolate a bacteriophage against Salmonella enterica serotype Tennessee sequence type 5018, a food-processing pathogen.ResultsWe report the genome analysis of Salmonella phage Tennessee Salten, a new species from the Tequintavirus genus. Its genome is 109,999 bp in length and contains 197 predicted coding sequences and 23 tRNAs. Compared to its closest known relative phage Escherichia phage HildyBeyeler-sharing 84.6% identity-Salten harbours 16 unique or highly divergent genes. Of these, 13 encode proteins with unknown function, one encodes for a putative adenine methyltransferase and two encode HNH homing endonucleases. Moreover, the Long Tail fibre protein, whose structure was predicted based on that of phage T5, was highly divergent among the Tequintavirus genus.

Amide-proton-detected magic-angle-spinning NMR of deuterated proteins has become a main technique in NMR-based structural biology. In standard deuteration protocols that rely on D 2 O-based culture media, non-exchangeable amide sites remain deuterated, making these sites unobservable. Here we demonstrate that proteins produced with a H 2 O-based culture medium doped with deuterated cell lysate allow scientists to overcome this "reprotonation bottleneck" while retaining a high level of deuteration (ca. 80 %) and narrow linewidths. We quantified coherence lifetimes of several proteins prepared with this labeling pattern over a range of magic-angle-spinning (MAS) frequencies (40-100 kHz). We demonstrate that under commonly used conditions (50-60 kHz MAS), the amide 1 H linewidths with our labeling approach are comparable to those of perdeuterated proteins and better than those of protonated samples at 100 kHz. For three proteins in the 33-50 kDa size range, many previously unobserved amides become visible. We report how to prepare the deuterated cell lysate for our approach from fractions of perdeuterated cultures which are usually discarded, and we show that such media can be used identically to commercial media. The residual protonation of H α sites allows for well-resolved H α -detected spectra and H α resonance assignment, exemplified by the de novo assignment of 168 H α sites in a 39 kDa protein. The approach based on this H 2 O/cell-lysate deuteration and MAS frequencies compatible with 1.3 or 1.9 mm rotors presents a strong sensitivity benefit over 0.7 mm 100 kHz MAS experiments.

ABSTRACT Bacteriophages are viruses infecting bacteria. The vast majority of them bear a tail, allowing host recognition, cell wall perforation, and DNA injection into the host cytoplasm. Using electron cryo-microscopy (cryo-EM) and single particle analysis, we determined the organization of the tail proximal extremity of siphophage T5 that possesses a long flexible tail and solved the structure of its tail terminator protein p142 (TrP 142 ). It allowed us to confirm the common evolutionary origin between T5 TrP p142 and other known or putative TrPs from siphophages, myophages, and bacterial tail-like machines, despite very poor sequence conservation. By also determining the structure of the T5 tail proximal extremity after interaction with T5 bacterial receptor FhuA, we showed that no conformational changes occur in TrP p142 and confirmed that the infection signal transduction is not carried by the tube itself. We also investigated the location of T5 Neck1 or tail completion protein p143 (TCP p143 ) and showed, thanks to a combination of cryo-EM and structure prediction using Alphafold2, that it is not located at the capsid-to-tail interface as suggested by its position in the genome, but instead, very unexpectedly, on the side of T5 tail tip, and that it appears to be monomeric. Based on structure comparison with other putative TCPs predicted structures, this feature could not be shared by other TCPs and questions the affiliation of p143 to this family of protein. IMPORTANCE Bacteriophages, viruses infecting bacteria, are the most abundant living entities on Earth. They are present in all ecosystems where bacteria develop and are instrumental in the regulation, diversity, evolution, and pathogeny of microbial populations. Moreover, with the increasing number of pathogenic strains resistant to antibiotics, virulent phages are considered a serious alternative or complement to classical treatments. 96% of all phages present a tail that allows host recognition and safe channeling of the DNA to the host cytoplasm. We present the atomic model of the proximal extremity of the siphophage T5 tail, confirming structural similarities with other phages. This structure, combined with results previously published and further explored, also allowed a review and a discussion on the role and localization of a mysterious tail protein, the tail completion protein, which is known to be present in the phage tails, but that was never identified in a phage structure.

We report herein the synthesis of three detergents bearing a perfluorinated cyclohexyl group connected through a short, hydrogenated spacer (i.e., propyl, butyl, or pentyl) to a β-maltoside polar head that are, respectively, called FCymal-3, FCymal-4, and FCymal-5. Increasing the length of the spacer decreased the critical micellar concentration (CMC), as demonstrated by surface tension (SFT) and isothermal titration calorimetry (ITC), from 5 mM for FCymal-3 to 0.7 mM for FCymal-5. The morphology of the micelles was studied by dynamic light scattering (DLS), analytical ultracentrifugation (AUC), and small-angle X-ray scattering (SAXS), indicating heterogeneous rod-like shapes. While micelles of FCymal-3 and -4 have similar hydrodynamic diameters of ∼10 nm, those of FCymal-5 were twice as large. We also investigated the ability of the detergents to solubilize lipid membranes made of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC). Molecular modeling indicated that the FCymal detergents generate disorder in lipid bilayers, with FCymal-3 being inserted more deeply into bilayers than FCymal-4 and -5. This was experimentally confirmed using POPC vesicles that were completely solubilized within 2 h with FCymal-3, whereas FCymal-5 required >8 h. A similar trend was noticed for the direct extraction of membrane proteins from E. coli membranes, with FCymal-3 being more potent than FCymal-5. An opposite trend was observed in terms of stabilization of the two model membrane proteins bacteriorhodopsin (bR) and SpNOX. In all three FCymal detergents, bR was stable for at least 2 months with no signs of aggregation. However, while the structural integrity of bR was fully preserved in FCymal-4 and -5, minor bleaching was observed in FCymal-3. Similarly, SpNOX exhibited the least activity in FCymal-3 and the highest activity in FCymal-5. By combining solubilizing and stabilizing potency, FCymal detergents push forward our expectations of the usefulness of fluorinated detergents for handling and investigating membrane proteins.

Graphene is an excellent support film for high-resolution transmission electron microscopy (TEM) but its use with biological samples, notably in cryo-TEM, is hindered by its inherent hydrophobicity. Whereas surface treatments have been proposed to render graphene hydrophilic, they are often difficult to reproduce due to a lack of information on the structural changes that modify the wetting properties of graphene. This study aims to correlate the atomic structure of graphene with its wetting properties to allow a reproducible protocol to advance its application in cryo-TEM. We follow the change in the atomic structure of graphene as a function of low-energy hydrogen plasma treatment duration on monolayer graphene transferred onto TEM grids. With finely controlled plasma exposure, partial hydrogenation, monoatomic vacancies, and pores of a few nanometers are realized in the graphene. The introduction of defects (vacancies and pores) enables the formation of continuous layers of vitreous ice on TEM grids. Grids with defect-integrated graphene are reproduced and used in the vitrification of the mouse serotonin 5-HT3 receptor, a membrane protein. Single particle analysis of the membrane protein on graphene compared to conventional holey carbon film give insight into the strengths and discretions in using graphene membrane for protein structural studies.

Phage host-range expansion is predicted to be at the cost of lower mean fitness. We aimed at following the adaptive walks of a virulent phage ( Tequintavirus ) evolving in a spatially variable environment composed of four susceptible and four resistant strains ( Salmonella enterica enterica pv Tennessee, sequence types ST5018 and ST319 respectively). We evolved a single ancestor through serial passages on the non-coevolving bacterial strains following Appelmans’ protocol and obtained several evolved phage populations with expanded host-range and increased virulence. Phage populations sequencing revealed multiple mutations appearing at the same loci (parallel mutations), notably on exo- and endo-nuclease, dUTPase and caudal proteins. Two parallel mutations present on the long tail fiber gene showed to become fixed within the population before the other parallel mutations. Introduction by reverse-genetics of these two mutations into the ancestral genome expanded host-range but not virulence.Highlights In Brief Despite that generalism is predicted to evolve at the cost of lower mean fitness, we experimentally selected phage populations with expanded host-range and increased virulence, demonstrating that generalist phages could be easily and usefully generated for phage therapy efforts. - 39 words - 278 characters### Competing Interest StatementThe authors have declared no competing interest.

Amide-proton-detected magic-angle-spinning NMR of deuterated proteins has become a main technique in NMR-based structural biology. In standard deuteration protocols that rely on D2O-based culture media, non-exchangeable amide sites remain deuterated, making these sites unobservable. Here we demonstrate that proteins produced with a H2O-based culture medium doped with deuterated cell lysate allow scientists to overcome this “reprotonation bottleneck” while retaining a high level of deuteration (ca. 80 %) and narrow linewidths. We quantified coherence lifetimes of several proteins prepared with this labeling pattern over a range of magic-angle-spinning (MAS) frequencies (40–100 kHz). We demonstrate that under commonly used conditions (50–60 kHz MAS), the amide 1H linewidths with our labeling approach are comparable to those of perdeuterated proteins and better than those of protonated samples at 100 kHz. For three proteins in the 33–50 kDa size range, many previously unobserved amides become visible. We report how to prepare the deuterated cell lysate for our approach from fractions of perdeuterated cultures which are usually discarded, and we show that such media can be used identically to commercial media. The residual protonation of Hα sites allows for well-resolved Hα-detected spectra and Hα resonance assignment, exemplified by the de novo assignment of 168 Hα sites in a 39 kDa protein. The approach based on this H2O/cell-lysate deuteration and MAS frequencies compatible with 1.3 or 1.9 mm rotors presents a strong sensitivity benefit over 0.7 mm 100 kHz MAS experiments.

We report herein the synthesis of zwitterionic sulfobetaine (SB) and dimethylamine oxide (AO) de-tergents whose alkyl chain is made of either a perfluorohexyl (F6H3) or a perfluoropentyl (F5H5) group linked to a hydrogenated spacer arm. In aqueous solution, the critical micellar concentrations (CMCs) measured by surface tensiometry (SFT) and isothermal titration calorimetry (ITC) were found in the millimolar range (1.3-2.4 mM). The morphologies of the aggregates were evaluated by dynamic light scattering (DLS), analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), and trans-mission electron microscopy (TEM), demonstrating that the two perfluoropentyl derivatives formed small micelles less than 10 nm in diameter, whereas the perfluorohexyl derivatives formed larger and more heterogeneous micelles. The two SB detergents were able to solubilize synthetic lipid vesicles in a few hours; by contrast, the perfluoropentyl AO induced much faster solubilization, whereas the per-fluorohexyl AO did not show any solubilization. All detergents were tested for their abilities to stabilize three membrane proteins, namely, bacteriorhodopsin (bR), the Bacillus subtilis ABC transporter BmrA, and the Streptococcus pneumoniae enzyme SpNOX. The SB detergents outperformed the AO derivatives as well as their hydrogenated analogs in stabilizing these proteins. Among the four new compounds, F5H5SB combines many desirable properties for membrane-protein study, as it is a powerful yet gentle detergent.(c) 2022 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.