Biology subjects

Breyton, C.

Publications and source records attributed to Breyton, C..

7 recordsLinked to original sources

bioRxiv2026

Superinfection exclusion strategy of siphophage T5: analysis of the FhuA:Llp complex

Superinfection exclusion is a widespread viral mechanism that protects the infected cell from over infection by other, identical or closely related viruses. Shortly after infection by bac-teriophage T5, the bacterium E. coli produces Llp coded by the just injected viral genome. Llp is a small lipoprotein targeted to the inner-leaflet of the host outer-membrane. It binds to FhuA, an outer-membrane iron-ferrichrome transporter, which is T5 receptor at the E. coli cell surface. The interaction between Llp and FhuA prevents any further bacteriophage binding and infection. Here, we determined the RMN structure of Llp and analyse the formation of the FhuA:Llp complex using a wide range of techniques and mutants, both in vivo and in vitro. Interaction of Llp to FhuA is governed by a two-step equilibrium with a strong contribution of induced fit: Llp binding requires remodelling of FhuA periplasmic plug surface, allowing the further large conformational reorganisation of the extracellular loops. Analysis of FhuA mu-tants show the importance of the intertwined interactions between the extracellular loops and the plug in the communication between the periplasmic and the extracellular faces of FhuA.

biophysics↗

bioRxiv2025

The complete genome and comparative analysis of a new Tequintavirus: Salmonella phage Tennessee Salten

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.

microbiology↗

bioRxiv2024

About bacteriophage tail terminator and tail completion proteins: structure of the proximal extremity of siphophage T5 tail

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 organisation of the tail proximal extremity of siphophage T5 that possess a long flexible tail, and solved the structure of its tail terminator protein (TrP) p142 (TrP142). It allowed to confirm the common evolutionary origin between T5 TrPp142 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 T5 tail proximal extremity after interaction with T5 bacterial receptor FhuA, we showed that no conformational changes occur in TrPp142 and confirmed that the infection signal transduction is not carried by the tube itself. We also investigated the location of T5 tail completion protein (TCP) p143 (TCPp143) 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. The stoichiometry of the Tape Measure Protein is also discussed. ImportanceBacteriophages, 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 as a serious alternative or complement to classical treatments. 96% of all phages present a tail that allows host recognition and safe channelling of the DNA to the host cytoplasm. We present the atomic model of the proximal extremity of siphophage T5 tail, confirming structural similarities with other phages. This structure, combined to results previously published further explored, also allowed a review and a discussion on the role and localisation 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.

microbiology↗

bioRxiv2024

Deuteration of proteins boosted by cell lysates: high-resolution amide and Hα MAS NMR without re-protonation bottleneck

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 H2O-based culture medium doped with deuterated cell lysate allow to overcome this "reprotonation bottleneck", while retaining a high level of deuteration (ca. 80 %) and narrow line widths. We quantified coherence life times of several proteins prepared with this labelling pattern over a range of MAS frequencies (40-100 kHz). We demonstrate that under commonly used conditions (50-60 kHz MAS), amide 1H line widths with our labelling 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 show that such media can be used identically to commercial media. The residual protonation of H sites allows for well-resolved H-detected spectra and H resonance assignment, exemplified by the de novo assignment of 168 H 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.

biochemistry↗

bioRxiv2022

Deciphering bacteriophage T5 host recognition mechanism and infection trigger

Bacteriophages, viruses infecting bacteria, recognise their host with high specificity, either binding to saccharide motifs or proteins of the cell wall of their host. In the majority of bacteriophages, this host recognition is performed by Receptor Binding Proteins (RBPs) located at the extremity of a tail. Interaction between the RBPs and the host is the trigger for bacteriophage infection, but the molecular details of the mechanisms are unknown for the majority of bacteriophages. Here, we present the electron cryo-microscopy structure of bacteriophage T5 RBPpb5 in complex with its E. coli receptor, the iron ferrichrome transporter FhuA. Monomeric RBPpb5 is located at the extremity of T5 long flexible tail, and its irreversible binding to FhuA commits T5 to infection. Analysis of RBPpb5 structure within the complex, comparison with its AlphaFold2 predicted structure, and its fit into a previously determined map of T5 tail tip in complex with FhuA allow us to propose a mechanism of transmission of RBPpb5 receptor binding to the straight fibre, initiating the cascade of events that commits T5 to DNA ejection.

biophysics↗

bioRxiv2022

Structural basis of bacteriophage T5 infection trigger and E. coli cell wall perforation

The vast majority of bacteriophages (phages) - bacterial viruses - present a tail that allows host recognition, cell wall perforation and safe channelling of the viral DNA from the capsid to the cytoplasm of the infected bacterium. The majority of tailed phages bears a long flexible tail (Siphoviridae) at the distal end of which a tip complex, often called baseplate, harbours one or more Receptor Binding Protein{middle dot}s (RBPs). Interaction between the RBPs and the host surface triggers cell wall perforation and DNA ejection, but little is known on these mechanisms for Siphoviridae. Here, we present the structure of siphophage T5 tip at high resolution, determined by electron cryo-microscopy, allowing to trace most of its constituting proteins, including 35 C-terminal residues of the Tape Measure Protein. We also present the structure of T5 tip after interaction with its E. coli receptor FhuA reconstituted into nanodisc. It brings out the dramatic conformational changes underwent by T5 tip upon infection, i.e. bending of the central fibre on the side, opening of the tail tube and its anchoring to the membrane, and formation of a transmembrane channel. These new structures shed light on the mechanisms of host recognition and activation of the viral entry for Siphoviridae.

microbiology↗

bioRxiv2018

DET1-mediated degradation of a SAGA-like deubiquitination module controls H2Bub homeostasis

DE-ETIOLATED 1 (DET1) is an evolutionarily conserved component of the ubiquitination machinery that mediates the destabilization of key regulators of cell differentiation and proliferation in multicellular organisms. In this study, we provide evidence from Arabidopsis that DET1 is essential for the regulation of histone H2B monoubiquitination (H2Bub) over most genes by controlling the stability of a plant deubiquitination module (DUBm). In contrast with yeast and metazoan DUB modules that are associated with the large SAGA complex, the Arabidopsis DUBm only comprises three proteins (hereafter named SGF11, ENY2 and UBP22) and appears to act independently as a major H2Bub deubiquitinase activity. Our study further unveils that DET1-DDB1-Associated-1 (DDA1) protein interacts with SGF11 in vivo, linking the DET1 complex to light-dependent ubiquitin-mediated proteolytic degradation of the DUBm. Collectively, these findings uncover a signaling path controlling DUBm availability, potentially adjusting H2Bub turnover capacity to the cell transcriptional status.

plant biology↗