Comparative genome analysis of a new Tequintavirus isolated on Salmonella enterica serotype Tennessee

Comparative genome analysis of a new Tequintavirus isolated on Salmonella enterica serotype Tennessee | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Comparative genome analysis of a new Tequintavirus isolated on Salmonella enterica serotype Tennessee Amandine Maurin, Carlos Zarate-Chaves, Cécile Breyton, Jacques Dainat, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6981461/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Nov, 2025 Read the published version in BMC Research Notes → Version 1 posted 10 You are reading this latest preprint version Abstract Objective : The 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. Results : We 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. Bacteriophage Salmonella enterica genome long tail fibre receptor binding protein Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Nontyphoidal Salmonella infections represent a major foodborne public health concern, ranking as the second leading cause of bacterial outbreaks in humans and frequently associated with direct animal contact. Salmonella enterica is an Enterobacteriaceae that is responsible for a substantial burden of gastroenteritis in both humans and animals, with more than 93.8 million cases and approximately 155,000 deaths reported per year [ 1 ]. The European Food Safety Authority (EFSA), the European Centre for Disease Prevention and Control (ECDC) and the U.S. Centers for Disease Control and Prevention (CDC) classified S. enterica as the second most reported-causing zoonoses [ 2 ] and foodborne disease [ 3 ]. Salmonella is a pathogen of global concern due to its ubiquity and persistence in diverse environments, including food processing facilities and agricultural settings. The extensive use of antibiotics as standard treatments in domains such as agriculture, livestock husbandry and food-processing further accelerates the emergence of resistant genotypes. The increasing prevalence of antimicrobial resistance (AMR) among pathogenic bacteria is a growing threat [ 4 ], compromising the efficacy of antibiotics or sanitary agents. Salmonella enterica is well known for its capacity to generate (multi)resistance genotypes, against both sanitary surface treatment and antibiotics [ 5 ]. As current sanitation and antimicrobial measures - particularly in the food industry - are becoming inadequate to fully eradicate Salmonella contaminations, it is an urgent necessity to consider alternative and sustainable methods for pathogen control. One promising strategy for bacterial biocontrol is to reconsider the use of bacteriophages (phages), viruses that exclusively replicate within bacterial cells. This strategy favors the use of virulent phages over temperate ones, because (i) of their inherent ability to lyse bacterial hosts and (ii) to avoid the risk of transferring virulent factor or antibiotic resistance genes, which is a known risk with temperate phages [ 6 ] [ 7 ] [ 8 ]). We thus aimed to isolate a virulent phage for the biocontrol of Salmonella enterica subsp. enterica serotype Tennessee ST5018, a contaminating pathogen isolated from a pet food-industry. We reported here the analysis of the complete genome of Salmonella phage Tennessee Salten, a member of a new Tequintavirus species. We made several genome comparisons between Salten’s genome and (i) it’s closest relative Escherichia phage HildyBeyeler, (ii) four representative Tequintavirus genomes and (iii) more globally to 184 published genomes from the Tequintavirus genus. Notably, the Long Tail Fibre - implicated in the reversible adsorption on bacterial surfaces - was highly divergent among all the representative Tequintaviruses. Materials & Methods Phage isolation The phage Salten was isolated from a bacterial strain of S. enterica enterica ( https://enterobase.warwick.ac.uk/ ; accession number SAL-QB8962AA – named Sten2) serotyped as Tennessee by Eurofins (Aix en Provence, France). Sten2 came from a collection of samples obtained by swabbing a food-processing factory in Poland, over a two-year period (2017–2019). The French National Reference Center for Escherichia coli , Shigella and Salmonella at the Institut Pasteur (Paris, France) attributed the Sequence Type (ST) ST5018 to Sten2. Bacteria were routinely cultured in Lysogeny Broth (LB Lennox, Athena Enzyme Systems; Baltimore, MD, USA) or LB agar (1.2%). A sample from Marseille’s wastewater (November 2017; 43°16′13″N, 5°24′00″E), filtered through a 0.22 µm Minisart polyethersulfone filter (#16541–K; Sartorius, Göttingen, Germany), and stored in glass bottles, was at the origin of the Salten isolate. Phage detection was done in a 96-deepwell plate, where each well contained 500 µL of LB inoculated with 2 µL of an overnight culture of Sten2 and 50 µL of filtered wastewater. The plate was incubated overnight at 37°C within a ventilated incubator (AL 265-5; Aqua-lytic) with 450 rpm shaking (1.5 mm orbital; Titramax 101 #544-11300-00; Heidolph Instruments, Schwabach, Germany). The following day, 50µL chloroform was added to each well and the plate was incubated at 4°C for at least four hours. From the supernatant, 2 µL were transferred to a new 96-well polystyrene plate (#82.1581001; Sarstedt, Nümbrecht, Germany), containing 200 µL of LB supplemented in 10mM CaCl2 (Sigma-Aldrich, #C3881) and inoculated with 2 µL of an overnight culture of Sten2. Bacterial growth was monitored by evaluating turbidity through the measure of Optical Density at 600 nm wave length (OD600nm) over 16h-20h at 37°C with 300 rpm shaking (spectrophotometer FLUOstar Omega, BMG Labtech, Ortenberg, Germany). The solution from the well showing the most delayed bacterial growth was transferred to a polypropylene 1,5 mL tube (Eppendorf SE, Hamburg, Germany). Residual bacteria were cleared by adding 10% chloroform and centrifugation (10 min at 15,871 Relative Centrifugal Force or rcf, Eppendorf 5415 R) to keep only phages. The phage strain was then purified using the double-layer method [ 9 ]. Briefly, 100 µL of the appropriate dilution of the phage solution was mixed with top LB agar (6g/L; #LF611001 Liofilchem, Italy) previously mixed with 100 µL of Sten2 overnight culture. After an overnight incubation at 35°C, one isolated lysis plaque was collected from the top agar, transferred into 200 µL SM buffer (100 mM NaCl, 10 mM MgSO 4 , 50 mM Tris-HCl, pH = 7.4) and incubated at 4°C for at least one hour. The phage was then purified through five consecutive rounds of the double-layer method, picking one isolated lysis plaque at each round. The last round, the entire top LB agar layer was collected in SM buffer, centrifuged (10 min, 3,000 rcf, Eppendorf Centrifuge 5702R), filtered through 0.22 µm filter, and stored at 4°C in polypropylen 15 mL tubes (#352096; Falcon, Corning, Mexico). The phage strain was named " Salmonella phage Tennessee Salten", and hereafter called "Salten". Transmission electron microscopy Salten solution (15 mL at 10 11 PFU/mL) was concentrated into 1.5 mL tubes by two rounds of centrifugation of one hour at 16,000 rcf, 4°C (Eppendorf Centrifuge 5415R). The pellet was resuspended in 600 µL ammonium acetate (100 mM; Sigma-Aldrich) and filtered through a 0.22 µm filter. Phages were then adsorbed onto a Formvar/carbon 300 grid (# CU 50/BX 9012.90.0000; Electron Microscopy Sciences, Hatfield, PA, USA), contrasted with 2% uranyl acetate, and visualized via transmission electronic microscopy (TEM, JEM-1400Plus, JEOL, Akishima, Tokyo, Japan). DNA extraction, preparation and sequencing Phage DNA extraction was done according to a protocol [ 10 ] adapted by Nicolas Ginet (Bacterial chemistry Laboratory, Marseille, France) after amplification of Salten in a more susceptible S. enterica Tennessee ST5018 isolate, i.e. Sten17 (Accession number SALQB8961AA in EnteroBase). Briefly, genetic material from bacterial origin potentially surrounding phages was eliminated by adding 10 µL of DNAse I (1 U/µL; #D5307; Sigma-Aldrich), 5 µL of RNAse A (10mg/mL; #EN0531; Thermo Fisher) and 2 µL of Dpn I (10 U/µL; #ER1702; Thermo Fisher). Phage DNA was extracted using phenol-chloroform-isoamyl acid 25/24/1 (#77617; Sigma-Aldrich). After DNA quantification with NanodropOne (Thermo Fischer) and Qubit 4 Fluoremeter (Invitrogen, Thermo Fisher), phage DNA was fragmented with a transposase enzyme. Fragmented end-prepared DNA was ligated to Illumina adaptors and then sorted with beads to select fragment sizes between 150–250 bp, for a final fragment size between 270–370 bp. Concentration of the final library was evaluated through Qubit 4 and fragment sizes were checked by migration using QIAxcel Advanced Instrument (QIAgen, Hilden, Germany). Fragments ready for sequencing sized between 280bp and 320bp. DNA libraries were sequenced in paired-ends in our in-lab sequencer (iSeq100; Illumina). Raw reads were deposited in the European Nucleotide Archive ( https://www.ebi.ac.uk/ena/browser/support ) with the following Accession Number: ERR13191102 ( Salmonella Phage Tennessee ). De novo phage assembly and annotation After Illumina sequencing, phage reads quality was controlled using FastQC v.0.12.1 [ 11 ] ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). Primers were trimmed with Fastp v.0.22.0 [ 12 ] using default parameters. Phage genome contigs were prepared following the workflow recommended in Turner et al.[ 13 ]. De novo phage genome assembly was carried out with SPAdes v.3.14.1 [ 14 ] [ 15 ] with default parameters. We obtained a large contig of 110,076 nt with high coverage (average 400 reads depth), and a number of short contigs (below 1200 nt) with low coverage (average 10–20 reads depth). We kept only the largest Salten contig and generated a complete genome with PhageTerm Virome v.4.3 [ 16 ]. After polishing with Pilon v.1.24[ 17 ], we obtained a new contig with a sequencing coverage ranged from 295× to 1171×. This contig corresponding to the complete genome was deposited in the European Nucleotide Archive with Accession Number OZ075147. Salten genome was annotated thanks to the Genome Annotation online tool from the Bacterial and Viral Bioinformatics Resource Center [ 18 ] (BV-BRC; https://www.bv-brc.org/ ) using VIGOR4 v.4.0. and based on Tequintavirus annotation [Taxonomy ID = 187218]). Pharokka v.1.7.5 [ 19 ] was used to complete the annotation. Structural genes were annotated manually based on Zivanovic et al [ 20 ] and Linares et al [ 21 ], using BLASTn or BLASTp from NCBI (Blast® services, available from: https://www.ncbi.nlm.nih.gov/Blast.cgi ). The number attributed to each Coding DNA Sequences (CDS) was provided by the BV-BRC annotation. Proksee web server [ 22 ] was used to generate Salten linear genome map, through CGView builder v.2.0.5 [ 23 ]. PhageScope web server 11 ( https://phagescope.deepomics.org/ ) tools were used for lifestyle prediction, virulent factor and antimicrobial resistance gene detection. Comparative genomics analysis The complete genome of the Salten was compared against the NCBI core nucleotide database using BLASTn. Hits with at least 30% query coverage were retained and, after genome comparison with VIRIDIC [ 24 ], a subset of 184 phage genomes showing ≥ 70% average nucleotide identity (ANI) was selected for a refined analysis ( Tequintavirus genus). Concerning the comparison of Salten to four other Tequintaviruses, gene coordinates were adjusted to standardize the starting point at the deoxynucleoside-5'-monophosphatase (dmp) gene for all genomes. VICTOR web platform was used to infer the phylogeny from nucleotides and using the D0 formula [ 25 ]. EasyFig [ 26 ] was used to generate detailed synteny diagrams. PanExplorer web server [ 27 ] was used to determine the core genome and Salten specific-genes (PanACoTA v.1.4.0 option with > 80% blast identity [ 28 ]) by comparing to the 184 Tequintavirus genomes selected after VIRIDIC analysis. A phylogenetic tree based on the aminoacid sequences of the Long Tail fibre proteins was performed based on a MUSCLE v.5.1 [ 29 ] alignment followed by a tree construction through the Jukes-Cantor genetic distance model with neighbor-joining and 1000 bootstrap replicates, in Geneious Prime v.2025.1.3 [ 30 ]. Results and discussion Phage morphology Transmission electron microscopy (TEM) of Salten revealed a T5-like Siphophage morphology, with a long non-contractile flexible tail of about 180 nm, attached to an icosahedral head of 60 nm of diameter (Fig. 1 ). Phage genome Salten harboured a complete genome of 109,999 base pairs with a ~ 39% GC content, varying across the genome (Fig. 2 ). A total of 220 Open Reading Frames (ORF) were predicted, including 197 CDS (Table S1 ) and 23 tRNAs. According to PhageScope, Salten was predicted to be virulent (lytic cycle) and did not encode any genes associated with virulence or antimicrobial resistance. CDS annotation and associated known function are provided in Table S1 . Thanks to the well modeled structure of T5 tail [ 21 ], we manually annotated the CDSs encoding for Salten’s tail proteins (Fig. 1 B). The Tail Tube Protein pb6 (TTPpb6; CDS175) is surrounded by the Tape Measure Protein pb2 (TMPpb2; CDS171), which determines the length of the tube. Three lateral Long Tail fibres (each fiber being formed by the LTFpb1; CDS165) are connected by their amino-terminal (N-terminal) part to the TTPpb6 protein through three lateral fibres p132 (CDS166). Anchoring of the LTFpb1 onto the central fibre is followed by the sequential positioning of pb9 (CDS169), pb4 (CDS167), pb3 (CDS168), and finally the receptor-binding protein pb5 (RBPpb5; CDS189) at the tip. RBPpb5 binds to outer membrane transporter, such as FhuA, FepA or BtuB, to trigger infection [ 31 ]. Small proteins p140 and p142 are encoded by CDS174 and CDS176, respectively. Comparative genomics with other Tequintaviruses After complete genome comparison (VIRIDIC and BLASTn), Escherichia phage HildyBeyeler (MZ501074.1) was found to be the most similar to Salten, harbouring 84.6% average nucleotide identity (ANI). Based on the criterion that strains of the same species share at least 95% genome sequence identity [ 32 ], Salten can be considered as a member from a new species within the Tequintavirus genus. This statement was confirmed by the phylogenetic tree generated by VICTOR that classified HildyBeyeler and Salten in two different species (Fig. S1 ). Salten and HildyBeyeler shared 97.2% nucleotide identity within the core genome (i.e. CDS having > 80% blast identity). Salten harboured 16 CDS (CDS7, 8, 9, 10, 11, 13, 28, 33, 51, 62, 67, 84, 85, 101, 116 and 127) absent in HildyBeyeler, of which 11 were annotated as "hypothetical proteins" and two as "Phage protein" (CDS33 and CDS127). CDS13 was annotated as a DNA N-6-adenine methyltransferase ( Salmonella phage vB_SalS-SIY1lw [WVH10139.1]; 93.9% identity on 89% coverage), while the two last CDS (CDS51 and CDS62) were annotated as "Phage HNH homing endonuclease". VIRIDIC was used to delimit the Tequintavirus genus (i.e. >70% ANI) among NCBI phage accessions. To compare Salten to the diversity of Tequintaviruses, we selected four "representative" genomes out of the 184 genomes from the Tequintavirus genus. Selection was based on an interval of approximately 5% of ANI divergence, relative to Salten (since this threshold has been proposed as a species level of diversification [ 32 ]. We thus selected Escherichia phage HildyBeyeler, Salmonella phage 8sent1748 (MT653146.1), Escherichia phage T5 (AY543070.1) and Salmonella phage S147 (NC_048012.1) with 84.6%, 80.4%, 76% and 70.1% ANI values, respectively. Genome comparisons performed with MAUVE [ 33 ] showed a high degree of synteny (Fig. 3 A), with three large collinear blocks, shared by all the genomes and kept in the same order. One of the HNH homing endonucleases (CDS62) - found in Salten and absent in HildyBeyeler - was also present in Salmonella phage S147, which was seen as a small collinear block in a different position of the genome (in dark green; Fig. 3 A). Knowing that HNH homing endonuclease are mobile genetic elements able to move by their own [ 34 ] [ 35 ] [ 36 ], it is not surprising to retrieve this CDS and its flanking sequences in different parts of the genomes. The pangenome of the five phages corresponded to 374 CDS (PanACoTA analysis). Among these pangenomic CDS, 64 of them were homologs between the five genomes - including Salten (> 80% BLASTp identity), 121 CDS were shared by two or more genomes (and Salten harboured 80 of them) and 190 CDS were strain-specific (Salten harboured 54 of them). More globally, the pangenome of the 185 Tequintaviruses (including Salten) corresponded to 1472 CDS (PanACoTA analysis). Among these pangenomic CDS, only eight of them were homologs CDS (> 80% BLASTp identity; pink on Fig. 3 B; Table 1 ). These proteins can be considered essential for phage multiplication, such as DNA replication (dihydrofolate reductase and NAD-dependant DNA ligase) and protein degradation (metallopeptidase). The two genes annotated as "Phage proteins" (CDS113 and CDS140) have been compared to NCBI dataset (BLASTn) and CDS113 corresponded to a hypothetical protein or virion-structural protein ( Escherichia phage phiLLS [YP_009790087.1]; 97.7% identity with 100% coverage) and CDS140 corresponded to a helicase ( Escherichia phage DT57C [YP_009149867.1]; 99.6% identity with 100% coverage). When comparing Salten to all other 184 Tequintaviruses, 17 CDS were considered as unique to Salten (or highly divergent; represented in blue arrows in Fig. 3 B) and were all annotated as "hypthetical proteins" or "Phage protein" (with no ability to improve annotation). To conclude, Salten as well as Tequintavirus genomes showed a high degree of variation and strain-specific CDS (accessory genome). Table 1 List of the core genes (> 80% blastp identity) among the 185 Tequintavirus genomes and named according to Salten BV-BRC annotation. CDS number Protein name 56 ATP-dependent Clp protease proteolytic subunit 111 Metallopeptidase phage-associated 113 Phage protein / structural protein 120 Ribonucleotide reductase of class Ia (aerobic) 124 Phage phosphate starvation-inducible protein 140 Phage protein / helicase 148 DNA ligase phage-associated 175 Phage major Tail Tube Protein TTPpb6 Host recognition Tequintaviruses recognize their host(s) through a two step process, that is first reversibly adsorbing to the LPS via the LTFpb1 and then irreversibly adsorbing to an outer-membrane transporter - such as FhuA, BtuB or FepA - via the receptor binding protein [ 37 ] (RBPpb5). To infer the potential second receptor of Salten, we compared its RBPpb5 with those of the four representative genomes. Unlike the core-genome phylogeny, Salten’s RBPpb5 showed the highest similarity to those of HildyBeyeler and S147 (Fig. S3 ). These two phages are known to target BtuB [ 38 ] [ 39 ], which suggests that Salten could also adsorb on this outer-membrane transporter. Future experiments are needed to test if BtuB is indeed Salten’s receptor, for exemple by generating knockouts of each of the potential receptor gene and assessing phage adsorption [ 38 ]. Concerning the reversible adsorption to the LPS, EasyFig detected a highly variability (< 64% of ANI) in Salten’s LTFpb1 compared to the one harbored by the four representative genomes (Fig. 3 B; blue arrow annotated "LTFpb1" on Salten genome map). Indeed, in line with Skutel et al. [ 40 ] findings that compared 15 LTFpb1 of Tequintavirus , the LTFpb1 aminoacid sequences of T5, HildyBeyeler, 8sent1748 and S147 respectively shared with Salten 85.4%, 85%, 51.1% and 41.7% identity (coverage 78%, 48%, 31% and 31%). The N-terminal part of the protein (phage central tail binding domain; residues 1 to 41 on consensus sequence; Fig. S2 ) which is known to attach LTFpb1 to the phage central tail tube by the collar with p132 [ 20 ] was well conserved. Moreover, the coiled-coil domain (residues 42 to 225; Fig. S2 ), responsible for projecting the lateral fibre away from the central tube, was also relatively well conserved. Notably, in Salten LTFpb1, the coiled-coil region contains insertions (repeats), leading to an extended coiled-coil. On the C-terminal part of LTFpb1, the chaperone encoding domain was also well conserved. However, the lateral fibre encoding domain (downstream the coiled-coil region; residues 226 to 874) showed a high level of variation (Fig. S2 and Fig. 4 B-C). The LTFpb1 phylogenetic analysis was not congruent with the analysis made with the complete genomes of the phages. Indeed, Salten’s LTFpb1 was phylogeneticaly closer to T5 than the one of HildyBeyeler, and these three proteins were similarly distinct to the ones of 8sent1748 and S147 (Fig. 4 A; Jukes-Cantor genetic distance model with neighbor-joining). This phylogenetic analysis was consistent with the structural analysis. Indeed, the structure of the lateral fibres of both T5 - crystallized and well modeled by Garcia-Doval et al. [ 41 ] - and Salten were similar, but distinct from S147 (Fig. 4 B). We therefore superimposed the predicted lateral fibres structure of Salten with the ones of S147 to visualise structure divergence between the two clusters (Fig. 4 C). As proposed by sequence alignments (Fig. S2 ), their C-terminal domains did not overlapped. Given that this domain is known to reversibly bind to the bacterial polysaccharide moiety of lipopolysaccharides (LPS) [ 41 ] [ 42 ], it is likely involved in host specificity and may play a critical role in determining the phage host range. Conclusion The Salten bacteriophage was isolated from wastewater and exhibits a lytic activity against Salmonella enterica serotype Tennessee ST5018. It belongs to the Tequintavirus genus but remains the first member of a new species. As it does not harbour any bacterial virulence genes nor antimicrobial resistance genes, Salten is a promising candidate for the biocontrol of Salmonella enterica serotype Tennessee ST5018. Notably, the LTFpb1 appeared to be highly diverse among the Tequintavirus genus. Given that this protein directly interacts with LPS on the host cell surface, such LTFpb1 diversity -particularly in the C-terminal domain - may explain Tequintaviruses host specificity. Indeed, O-antigen is known to be variable among Gram-negative bacteria [ 43 ] [ 44 ]. Tequintavirus adaptation to a new bacterial host may thus first reside in modification of their LTFpb1. Further studies would be interesting to determine which Salten’s aminoacids are involved in its interaction with its bacterial host. We could also experimentally investigate the capacity of Salten to increase its host range on other sequence types of S. enterica and related bacteria, as it was done for T3 phage and its gp17 fibre in Yehl and al. [ 45 ]. Abbreviations AMR : antimicrobial resistance ANI : Average nucleotide identity BV-BRC : Bacterial and Viral Bioinformatics Resource Center CDC : Centers for Disease Control and Prevention CDS : Coding DNA sequence dmp : Deoxynucleoside-5'-monophosphatase ECDC : European Centre for Disease Prevention and Control EFSA : European Food Safety Authority LB : Lysogeny Broth LPS : Lipopolysaccharides LTF : Long tail fibre O-Ag : O-antigen OD : Optical Density ORF : Open Reading Frame Phage : Bacteriophage RBP : Receptor-Binding Protein Salten : Salmonella Phage Tennessee Salten S. enterica : Salmonella enterica SM buffer : ST : Sequence type Sten : Salmonella enterica serotype Tennessee TTP : Tail Tube Protein TMP : Tape Measure Protein TEM : Transmission electronic microscopy deoxynucleoside-5'-monophosphatase (dmp) Declarations Acknowledgments The authors thank M. Ansaldi (LCB, Marseille) for providing wastewater from which we isolated our phage, F-X. Weill for the bacterial sequencing, C. Mariac (DIADE, Montpellier) for the library quality check before sequencing and for QiAxcel use, A. Talman (MIVEGEC, Montpellier) for the help on the iSeq use, J. Garneau for PhageTerm help, M. Monot (I. Pasteur, Paris), A. Dereeper (PHIM, Montpellier), S. Bouzidi (MIVEGEC, Montpellier) and J. Hayer (MIVEGEC, Montpellier) for bioinformatics advices. We thank UMR MIVEGEC for providing an efficient working environment and acknowledge the ISO 9001 certified IRD itrop HPC (member of the South Green Platform) and the whole bioinformatics team support in Montpellier for providing HPC resources that have contributed to the research results reported within this paper. URL: https://bioinfo.ird.fr/ - http://www.southgreen.fr. Data availability The complete genome sequence generated and analysed during the current study is available in the European Nucleotide Archive repository with Accession Number OZ075147 (https://www.ebi.ac.uk/ena/browser/view/OZ075147). The supplemental data that support the findings of this study are openly available at https://src.koda.cnrs.fr/MAURINAmandine/salten_report Author contribution Conceptualisation, R.F. and A.M.; Methodology & Investigation, R.F. and A.M. with the help of C.Z.-C. for the genomic comparison; Results analysis, R.F., A.M., C.Z.-C., C.B., and J.D.; Writing, R.F. and A.M.; Revision, R.F., A.M., C.Z.-C., C.B., and J.D.. Conflict of interest The authors have no conflicts of interest to declare. Funder Information This work was funded by Royal Canin through the support of the CNRS (contract 245420). Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Note References [46], [47], [48], [49], [50], [51], [52], [53] and [54] are cited in the supplementary material document, in Table S1. References Eng S-K, Pusparajah P, Ab Mutalib N-S, Ser H-L, Chan K-G, Lee L-H. Salmonella: A review on pathogenesis, epidemiology and antibiotic resistance. Frontiers in Life Science. 2015;8:284–93. The European Union One. Health 2023 Zoonoses report. EFSA J. 2024;22:e9106. Tack DM. Preliminary Incidence and Trends of Infections with Pathogens Transmitted Commonly Through Food — Foodborne Diseases Active Surveillance Network, 10 U.S. Sites, 2015–2018. MMWR Morb Mortal Wkly Rep. 2019;68. Murray CJL, Ikuta KS, Sharara F, Swetschinski L, Aguilar GR, Gray A, et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet. 2022;399:629–55. Curiao T, Marchi E, Grandgirard D, León-Sampedro R, Viti C, Leib SL, et al. Multiple adaptive routes of Salmonella enterica Typhimurium to biocide and antibiotic exposure. BMC Genomics. 2016;17:491. Kuhl S, Abedon ST, Hyman P. Diseases caused by phages. In: Bacteriophages in health and disease. 2012. pp. 21–32. Balcazar JL. Bacteriophages as Vehicles for Antibiotic Resistance Genes in the Environment. PLoS Pathog. 2014;10:e1004219. Brown-Jaque M, Calero-Cáceres W, Muniesa M. Transfer of antibiotic-resistance genes via phage-related mobile elements. Plasmid. 2015;79:1–7. Kropinski AM, Mazzocco A, Waddell TE, Lingohr E, Johnson RP. Enumeration of Bacteriophages by Double Agar Overlay Plaque Assay. In: Clokie MRJ, Kropinski AM, editors. Bacteriophages. Totowa, NJ: Humana; 2009. pp. 69–76. Gendre J, Ansaldi M, Olivenza DR, Denis Y, Casadesús J, Ginet N. Genetic Mining of Newly Isolated Salmophages for Phage Therapy. Int J Mol Sci. 2022;23:8917. Babraham Bioinformatics -. FastQC A Quality Control tool for High Throughput Sequence Data. https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ . Accessed 17 Apr 2024. Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34:i884–90. Turner D, Adriaenssens EM, Tolstoy I, Kropinski AM. Phage Annotation Guide: Guidelines for Assembly and High-Quality Annotation. PHAGE. 2021;2:170–82. Antipov D, Raiko M, Lapidus A, Pevzner PA. MetaviralSPAdes: assembly of viruses from metagenomic data. Bioinformatics. 2020;36:4126–9. Prjibelski A, Antipov D, Meleshko D, Lapidus A, Korobeynikov A. Using SPAdes De Novo Assembler. Curr Protocols Bioinf. 2020;70:e102. Garneau JR, Depardieu F, Fortier L-C, Bikard D, Monot M. PhageTerm: a tool for fast and accurate determination of phage termini and packaging mechanism using next-generation sequencing data. Sci Rep. 2017;7:8292. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, et al. Pilon: An Integrated Tool for Comprehensive Microbial Variant Detection and Genome Assembly Improvement. PLoS ONE. 2014;9:e112963. Olson RD, Assaf R, Brettin T, Conrad N, Cucinell C, Davis JJ, et al. Introducing the Bacterial and Viral Bioinformatics Resource Center (BV-BRC): a resource combining PATRIC, IRD and ViPR. Nucleic Acids Res. 2023;51:D678–89. Bouras G, Nepal R, Houtak G, Psaltis AJ, Wormald P-J, Vreugde S. Pharokka: a fast scalable bacteriophage annotation tool. Bioinformatics. 2023;39:btac776. Zivanovic Y, Confalonieri F, Ponchon L, Lurz R, Chami M, Flayhan A, et al. Insights into Bacteriophage T5 Structure from Analysis of Its Morphogenesis Genes and Protein Components. J Virol. 2014;88:1162–74. Linares R, Arnaud C-A, Effantin G, Darnault C, Epalle NH, Boeri Erba E, et al. Structural basis of bacteriophage T5 infection trigger and E. coli cell wall perforation. Sci Adv. 2023;9:eade9674. Grant JR, Enns E, Marinier E, Mandal A, Herman EK, Chen C-Y, et al. Proksee: in-depth characterization and visualization of bacterial genomes. Nucleic Acids Res. 2023;51:W484–92. Stothard P, Wishart DS. Circular genome visualization and exploration using CGView. Bioinformatics. 2005;21:537–9. Moraru C, Varsani A, Kropinski AM. VIRIDIC—A Novel Tool to Calculate the Intergenomic Similarities of Prokaryote-Infecting Viruses. Viruses. 2020;12:1268. Meier-Kolthoff JP, Göker M. VICTOR: genome-based phylogeny and classification of prokaryotic viruses. Bioinformatics. 2017;33:3396–404. Sullivan MJ, Petty NK, Beatson SA. Easyfig: a genome comparison visualizer. Bioinformatics. 2011;27:1009–10. Dereeper A, Summo M, Meyer DF. PanExplorer: a web-based tool for exploratory analysis and visualization of bacterial pan-genomes. Bioinformatics. 2022;38:4412–4. Perrin A, Rocha EPC. PanACoTA: a modular tool for massive microbial comparative genomics. NAR Genom Bioinform. 2021;3:lqaa106. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32:1792–7. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, et al. Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;28:1647–9. Degroux S, Effantin G, Linares R, Schoehn G, Breyton C. Deciphering Bacteriophage T5 Host Recognition Mechanism and Infection Trigger. J Virol. 2023;97:e01584–22. Adriaenssens E, Brister JR. How to Name and Classify Your Phage: An Informal Guide. Viruses. 2017;9:70. Darling ACE, Mau B, Blattner FR, Perna NT. Mauve: Multiple Alignment of Conserved Genomic Sequence With Rearrangements. Genome Res. 2004;14:1394–403. Hafez M, Hausner G. Homing endonucleases: DNA scissors on a mission. Genome. 2012;55:553–69. Stoddard BL. Homing endonucleases from mobile group I introns: discovery to genome engineering. Mob DNA. 2014;5:7. Barth ZK, Dunham DT, Seed KD. Nuclease genes occupy boundaries of genetic exchange between bacteriophages. NAR Genomics Bioinf. 2023;5:lqad076. Bertozzi Silva J, Storms Z, Sauvageau D. Host receptors for bacteriophage adsorption. FEMS Microbiol Lett. 2016;363:fnw002. Maffei E, Shaidullina A, Burkolter M, Heyer Y, Estermann F, Druelle V, et al. Systematic exploration of Escherichia coli phage–host interactions with the BASEL phage collection. PLoS Biol. 2021;19:e3001424. Gencay YE, Gambino M, Prüssing TF, Brøndsted L. The genera of bacteriophages and their receptors are the major determinants of host range. Environ Microbiol. 2019;21:2095–111. Skutel M, Andriianov A, Zavialova M, Kirsanova M, Shodunke O, Zorin E, et al. T5-like phage BF23 evades host-mediated DNA restriction and methylation. microLife. 2023;4:uqad044. Garcia-Doval C, Castón JR, Luque D, Granell M, Otero JM, Llamas-Saiz AL, et al. Structure of the Receptor-Binding Carboxy-Terminal Domain of the Bacteriophage T5 L-Shaped Tail Fibre with and without Its Intra-Molecular Chaperone. Viruses. 2015;7:6424–40. Heller KJ. Identification of the phage gene for host receptor specificity by analyzing hybrid phages of T5 and BF23. Virology. 1984;139:11–21. Goldman RC, Leive L. Heterogeneity of Antigenic-Side-Chain Length in Lipopolysaccharide from Escherichia coli 0111 and Salmonella typhimurium LT2. Eur J Biochem. 1980;107:145–53. Reeves PR, Cunneen MM. Chapter 18 - Biosynthesis of O-antigen chains and assembly. In: Holst O, Brennan PJ, von Itzstein M, Moran AP, editors. Microbial Glycobiology. San Diego: Academic; 2010. pp. 319–35. Yehl K, Lemire S, Yang AC, Ando H, Mimee M, Torres MDT, et al. Engineering Phage Host-Range and Suppressing Bacterial Resistance through Phage Tail Fiber Mutagenesis. Cell. 2019;179:459–e4699. Davison J. Pre-early functions of bacteriophage T5 and its relatives. Bacteriophage. 2015;5:e1086500. Shi L, Potts M, Kennelly PJ. The serine, threonine, and/or tyrosine-specific protein kinases and protein phosphatases of prokaryotic organisms: a family portrait. FEMS Microbiol Rev. 1998;22:229–53. Kim Y-I, Levchenko I, Fraczkowska K, Woodruff RV, Sauer RT, Baker TA. Molecular determinants of complex formation between Clp/Hsp100 ATPases and the ClpP peptidase. Nat Struct Mol Biol. 2001;8:230–3. Young R. Bacteriophage lysis: mechanism and regulation. Microbiol Rev. 1992;56:430–81. Wang J, Jiang Y, Vincent M, Sun Y, Yu H, Wang J, et al. Complete genome sequence of bacteriophage T5. Virology. 2005;332:45–65. Hogenkamp HPC. Nature and properties of the bacterial ribonucleotide reductases. Pharmacol Ther. 1983;23:393–405. Rojowska AM. Structural and functional analysis of DNA binding by the Rad50 catalytic head from Thermotoga maritima. PhD. Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München; 2013. Warner HR, Thompson RB, Mozer TJ, Duncan BK. The properties of a bacteriophage T5 mutant unable to induce deoxyuridine 5’-triphosphate nucleotidohydrolase. Synthesis of uracil-containing T5 deoxyribonucleic acid. J Biol Chem. 1979;254:7534–9. Casjens SR, Gilcrease EB. Determining DNA Packaging Strategy by Analysis of the Termini of the Chromosomes in Tailed-Bacteriophage Virions. In: Clokie MRJ, Kropinski AM, editors. Bacteriophages: Methods and Protocols. Molecular and Applied Aspects. Volume 2. Totowa, NJ: Humana; 2009. pp. 91–111. Additional Declarations No competing interests reported. Supplementary Files FigureS1.pdf FigureS2.pdf FigureS3.pdf TableS1.pdf Cite Share Download PDF Status: Published Journal Publication published 21 Nov, 2025 Read the published version in BMC Research Notes → Version 1 posted Editorial decision: Revision requested 08 Sep, 2025 Reviews received at journal 06 Sep, 2025 Reviews received at journal 02 Sep, 2025 Reviewers agreed at journal 27 Aug, 2025 Reviewers agreed at journal 25 Aug, 2025 Reviewers invited by journal 13 Aug, 2025 Editor assigned by journal 13 Aug, 2025 Editor invited by journal 13 Aug, 2025 Submission checks completed at journal 11 Aug, 2025 First submitted to journal 11 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6981461","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":503114259,"identity":"fdde4e29-7dc9-454b-952d-aef0caf5abc7","order_by":0,"name":"Amandine Maurin","email":"","orcid":"","institution":"MIVEGEC (Univ. Montpellier, CNRS, IRD)","correspondingAuthor":false,"prefix":"","firstName":"Amandine","middleName":"","lastName":"Maurin","suffix":""},{"id":503114261,"identity":"749b08d8-ae04-40e2-835f-b77ec575a6a9","order_by":1,"name":"Carlos Zarate-Chaves","email":"","orcid":"","institution":"LIPME (Univ. Toulouse, INRAE, CNRS)","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"","lastName":"Zarate-Chaves","suffix":""},{"id":503114262,"identity":"9353cbf1-e623-4825-b2f4-772ff852ed25","order_by":2,"name":"Cécile Breyton","email":"","orcid":"","institution":"IBS (Univ. Grenoble Alpes, CNRS, CEA)","correspondingAuthor":false,"prefix":"","firstName":"Cécile","middleName":"","lastName":"Breyton","suffix":""},{"id":503114263,"identity":"ffd4a68d-430b-45e7-aba7-23a48a1af275","order_by":3,"name":"Jacques Dainat","email":"","orcid":"","institution":"MIVEGEC (Univ. Montpellier, CNRS, IRD)","correspondingAuthor":false,"prefix":"","firstName":"Jacques","middleName":"","lastName":"Dainat","suffix":""},{"id":503114264,"identity":"693245b5-47bb-42b6-94fa-dbc40de8ec0f","order_by":4,"name":"Alexandre Feugier","email":"","orcid":"","institution":"Natural Intelligence Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Alexandre","middleName":"","lastName":"Feugier","suffix":""},{"id":503114265,"identity":"6167639e-a25c-4ce9-bcd5-deab526153c3","order_by":5,"name":"Rémy Froissart","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYNACGwYGNjgLBCoIakmDaUlIgwicIUYLBCQcJqzFvL07TYIh4Z4cH//xh58rf5yXN5c+/oDh4B7cWmTOnN0G1FJszCaRYyx5JuG24c6+HAOGA89wa5GQyN0mwfgjIbFNgodBsiHhdoLBGR4G5g8H8GiRfwuyBaiF//jjnw0J54Ba2B8wHMCnRYIXqoUhwQxoywGgFgYD/Fp4cjdbJCQkgPxiZtmQlmy44QyPwQG8WtjPbrzxISFBTr7/+OObDTZ28kCHPXyATwsQsEgkoAvh18AADB4CCkbBKBgFo2CkAwBhUE/b41VNFgAAAABJRU5ErkJggg==","orcid":"","institution":"MIVEGEC (Univ. Montpellier, CNRS, IRD)","correspondingAuthor":true,"prefix":"","firstName":"Rémy","middleName":"","lastName":"Froissart","suffix":""}],"badges":[],"createdAt":"2025-06-26 08:53:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6981461/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6981461/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13104-025-07533-4","type":"published","date":"2025-11-21T15:57:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89565443,"identity":"dbed78f3-253e-460f-a666-08c5976ee3bd","added_by":"auto","created_at":"2025-08-21 10:45:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":322570,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphology of the Tequintavirus Salten. A-\u003c/strong\u003e Transmission electron microscopy (TEM). The scale bar represents 100 nm.\u003cstrong\u003e B- \u003c/strong\u003eSchematic structure of T5 adapted from Zivanovic et al. [20] with TTPpb6: cyan; TMPpb2: red; p140 \u0026amp; p142: blue; pb9: orange; pb3: yellow; pb4: green; p132 \u0026amp; LTFpb1: pink; RBPpb5: dark green.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6981461/v1/8de87930b37cdb39b0341af7.png"},{"id":89565444,"identity":"4183bae2-6c96-4762-8dc2-de504c613d1d","added_by":"auto","created_at":"2025-08-21 10:45:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":100508,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenome linear map of Salten. \u003c/strong\u003eGC content is indicated in black between the genome length and the predicted ORFs represented in arrows, where the direction indicates the forward (left to right) or reverse (right to left) strand of transcription. ORFs in light blue are associated to DNA transformation, in orange to structural proteins, in green to proteins modification, in yellow to bacterial cell-wall perforation and in purple to tRNA. Structural tail proteins names are colored according to the Figure 1.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6981461/v1/d291777d6f771412bca57221.png"},{"id":89565452,"identity":"839e71d6-c21f-4c9a-85d7-651b20321835","added_by":"auto","created_at":"2025-08-21 10:45:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":984169,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative genomics analysis between Salten and the four representative Tequintaviruses. A- \u003c/strong\u003eMAUVE alignment of five \u003cem\u003eTequintavirus\u003c/em\u003e genomes. The phylogenetic tree was built by VICTOR using the d0 formula and then re-arranged to have Salten on the upper part.\u003cstrong\u003e B-\u003c/strong\u003e EasyFig was used to obtain a more detailed synteny diagram. Lines connecting the genome maps indicate gene-level identity, ranging from 64% to 100%, represented by varying shades of grey. According to PanExplorer, genes in pink are homologs (\u0026gt;80% blastp identity) in the 185 \u003cem\u003eTequintavirus \u003c/em\u003egenomes\u003cem\u003e, \u003c/em\u003ewhereas genes in blue are unique to Salten or highly divergent. The percentage of ANI between each representative \u003cem\u003eTequintavirus \u003c/em\u003egenome and Salten is indicated in the right.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6981461/v1/4206aefaa10c0ed4d3e3db6d.png"},{"id":89565794,"identity":"77fac9c6-f1ee-4a43-86e1-5c0f0b63c4dc","added_by":"auto","created_at":"2025-08-21 10:53:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":526590,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative analysis of the LTFpb1 among the four representative \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTequintavirus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand Salten. A-\u003c/strong\u003e LTFpb1 phylogenetic relationship reconstruction. LTFpb1 sequences were aligned with MUSCLE v3.8.425 in Geneious Prime 2025 and phylogenetic tree was constructed with Geneious Tree builder with Jukes-Cantor genetic distance model and neighbor-joining build method with bootstrap 1000. \u003cstrong\u003eB-\u003c/strong\u003e AlphaFold3 predicted structure of the C-terminal fibre domain of the lateral fibers from Salten, HildyBayeler, 8Sent and S147, and structure of that of T5 (d'Acapito and Breyton, in preparation). AlphaFold predictions are coloured by confidence level according to the scale (red: very poor, blue: maximum prediction confidence). Structures have been placed according to their position on the LTFpb1 phylogenetic tree and aligned vertically on the pb1 structure.\u003cstrong\u003e C- \u003c/strong\u003eTop: superimposition of the fiber domaine of T5 LTFpb1 (pink) and S147 LTFpb1 (green). Bottom: Superimposition of a single monomer to better visualise the overlay region between the fiber domain structure of LTFpb1 of T5 (pink) and S147 (green). C- and N-termini of the proteins are indicated.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6981461/v1/f267e9d536ba0015b0c40900.png"},{"id":96650254,"identity":"6a205f30-9c16-4088-ac70-6e146193b698","added_by":"auto","created_at":"2025-11-24 16:10:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2624034,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6981461/v1/6b93234a-a988-4eb2-a58d-d8f6b411c9a6.pdf"},{"id":89565446,"identity":"f7798288-9bfc-491f-bf3c-32bf2e3ee732","added_by":"auto","created_at":"2025-08-21 10:45:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":67365,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6981461/v1/f1f2983a851debe7a4cb3ef9.pdf"},{"id":89565793,"identity":"f4ec0e56-459f-4c9b-8c03-6560b0305627","added_by":"auto","created_at":"2025-08-21 10:53:02","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":638042,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6981461/v1/7aa8678fac6310d4c481403b.pdf"},{"id":89565796,"identity":"d5bf1ad8-01e1-4b6f-a2e5-ef5190d45d14","added_by":"auto","created_at":"2025-08-21 10:53:02","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":136254,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6981461/v1/38d1417a899ba0a55ef8ac27.pdf"},{"id":89566427,"identity":"4bf81934-3240-4ede-a8a2-31fe70a78334","added_by":"auto","created_at":"2025-08-21 11:01:02","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":44289,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6981461/v1/ebd4da47d16a6b27b468a7b7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative genome analysis of a new Tequintavirus isolated on Salmonella enterica serotype Tennessee","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNontyphoidal \u003cem\u003eSalmonella\u003c/em\u003e infections represent a major foodborne public health concern, ranking as the second leading cause of bacterial outbreaks in humans and frequently associated with direct animal contact. \u003cem\u003eSalmonella enterica\u003c/em\u003e is an \u003cem\u003eEnterobacteriaceae\u003c/em\u003e that is responsible for a substantial burden of gastroenteritis in both humans and animals, with more than 93.8\u0026nbsp;million cases and approximately 155,000 deaths reported per year [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The European Food Safety Authority (EFSA), the European Centre for Disease Prevention and Control (ECDC) and the U.S. Centers for Disease Control and Prevention (CDC) classified \u003cem\u003eS. enterica\u003c/em\u003e as the second most reported-causing zoonoses [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and foodborne disease [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. \u003cem\u003eSalmonella\u003c/em\u003e is a pathogen of global concern due to its ubiquity and persistence in diverse environments, including food processing facilities and agricultural settings. The extensive use of antibiotics as standard treatments in domains such as agriculture, livestock husbandry and food-processing further accelerates the emergence of resistant genotypes. The increasing prevalence of antimicrobial resistance (AMR) among pathogenic bacteria is a growing threat [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], compromising the efficacy of antibiotics or sanitary agents. \u003cem\u003eSalmonella enterica\u003c/em\u003e is well known for its capacity to generate (multi)resistance genotypes, against both sanitary surface treatment and antibiotics [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. As current sanitation and antimicrobial measures - particularly in the food industry - are becoming inadequate to fully eradicate \u003cem\u003eSalmonella\u003c/em\u003e contaminations, it is an urgent necessity to consider alternative and sustainable methods for pathogen control.\u003c/p\u003e\u003cp\u003eOne promising strategy for bacterial biocontrol is to reconsider the use of bacteriophages (phages), viruses that exclusively replicate within bacterial cells. This strategy favors the use of virulent phages over temperate ones, because (i) of their inherent ability to lyse bacterial hosts and (ii) to avoid the risk of transferring virulent factor or antibiotic resistance genes, which is a known risk with temperate phages [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]).\u003c/p\u003e\u003cp\u003eWe thus aimed to isolate a virulent phage for the biocontrol of \u003cem\u003eSalmonella enterica\u003c/em\u003e subsp. \u003cem\u003eenterica\u003c/em\u003e serotype Tennessee ST5018, a contaminating pathogen isolated from a pet food-industry. We reported here the analysis of the complete genome of \u003cem\u003eSalmonella phage Tennessee\u003c/em\u003e Salten, a member of a new \u003cem\u003eTequintavirus\u003c/em\u003e species. We made several genome comparisons between Salten\u0026rsquo;s genome and (i) it\u0026rsquo;s closest relative \u003cem\u003eEscherichia phage\u003c/em\u003e HildyBeyeler, (ii) four representative \u003cem\u003eTequintavirus\u003c/em\u003e genomes and (iii) more globally to 184 published genomes from the \u003cem\u003eTequintavirus\u003c/em\u003e genus. Notably, the Long Tail Fibre - implicated in the reversible adsorption on bacterial surfaces - was highly divergent among all the representative Tequintaviruses.\u003c/p\u003e"},{"header":"Materials \u0026 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePhage isolation\u003c/h2\u003e\u003cp\u003eThe phage Salten was isolated from a bacterial strain of \u003cem\u003eS. enterica enterica\u003c/em\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://enterobase.warwick.ac.uk/\u003c/span\u003e\u003cspan address=\"https://enterobase.warwick.ac.uk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accession number SAL-QB8962AA \u0026ndash; named Sten2) serotyped as Tennessee by Eurofins (Aix en Provence, France). Sten2 came from a collection of samples obtained by swabbing a food-processing factory in Poland, over a two-year period (2017\u0026ndash;2019). The French National Reference Center for \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eShigella\u003c/em\u003e and \u003cem\u003eSalmonella\u003c/em\u003e at the Institut Pasteur (Paris, France) attributed the Sequence Type (ST) ST5018 to Sten2. Bacteria were routinely cultured in Lysogeny Broth (LB Lennox, Athena Enzyme Systems; Baltimore, MD, USA) or LB agar (1.2%).\u003c/p\u003e\u003cp\u003eA sample from Marseille\u0026rsquo;s wastewater (November 2017; 43\u0026deg;16\u0026prime;13\u0026Prime;N, 5\u0026deg;24\u0026prime;00\u0026Prime;E), filtered through a 0.22 \u0026micro;m Minisart polyethersulfone filter (#16541\u0026ndash;K; Sartorius, G\u0026ouml;ttingen, Germany), and stored in glass bottles, was at the origin of the Salten isolate. Phage detection was done in a 96-deepwell plate, where each well contained 500 \u0026micro;L of LB inoculated with 2 \u0026micro;L of an overnight culture of Sten2 and 50 \u0026micro;L of filtered wastewater. The plate was incubated overnight at 37\u0026deg;C within a ventilated incubator (AL 265-5; Aqua-lytic) with 450 rpm shaking (1.5 mm orbital; Titramax 101 #544-11300-00; Heidolph Instruments, Schwabach, Germany). The following day, 50\u0026micro;L chloroform was added to each well and the plate was incubated at 4\u0026deg;C for at least four hours. From the supernatant, 2 \u0026micro;L were transferred to a new 96-well polystyrene plate (#82.1581001; Sarstedt, N\u0026uuml;mbrecht, Germany), containing 200 \u0026micro;L of LB supplemented in 10mM CaCl2 (Sigma-Aldrich, #C3881) and inoculated with 2 \u0026micro;L of an overnight culture of Sten2. Bacterial growth was monitored by evaluating turbidity through the measure of Optical Density at 600 nm wave length (OD600nm) over 16h-20h at 37\u0026deg;C with 300 rpm shaking (spectrophotometer FLUOstar Omega, BMG Labtech, Ortenberg, Germany).\u003c/p\u003e\u003cp\u003eThe solution from the well showing the most delayed bacterial growth was transferred to a polypropylene 1,5 mL tube (Eppendorf SE, Hamburg, Germany). Residual bacteria were cleared by adding 10% chloroform and centrifugation (10 min at 15,871 Relative Centrifugal Force or rcf, Eppendorf 5415 R) to keep only phages. The phage strain was then purified using the double-layer method [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Briefly, 100 \u0026micro;L of the appropriate dilution of the phage solution was mixed with top LB agar (6g/L; #LF611001 Liofilchem, Italy) previously mixed with 100 \u0026micro;L of Sten2 overnight culture. After an overnight incubation at 35\u0026deg;C, one isolated lysis plaque was collected from the top agar, transferred into 200 \u0026micro;L SM buffer (100 mM NaCl, 10 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 50 mM Tris-HCl, pH\u0026thinsp;=\u0026thinsp;7.4) and incubated at 4\u0026deg;C for at least one hour. The phage was then purified through five consecutive rounds of the double-layer method, picking one isolated lysis plaque at each round. The last round, the entire top LB agar layer was collected in SM buffer, centrifuged (10 min, 3,000 rcf, Eppendorf Centrifuge 5702R), filtered through 0.22 \u0026micro;m filter, and stored at 4\u0026deg;C in polypropylen 15 mL tubes (#352096; Falcon, Corning, Mexico). The phage strain was named \"\u003cem\u003eSalmonella phage Tennessee\u003c/em\u003e Salten\", and hereafter called \"Salten\".\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTransmission electron microscopy\u003c/h3\u003e\n\u003cp\u003eSalten solution (15 mL at 10\u003csup\u003e11\u003c/sup\u003e PFU/mL) was concentrated into 1.5 mL tubes by two rounds of centrifugation of one hour at 16,000 rcf, 4\u0026deg;C (Eppendorf Centrifuge 5415R). The pellet was resuspended in 600 \u0026micro;L ammonium acetate (100 mM; Sigma-Aldrich) and filtered through a 0.22 \u0026micro;m filter. Phages were then adsorbed onto a Formvar/carbon 300 grid (# CU 50/BX 9012.90.0000; Electron Microscopy Sciences, Hatfield, PA, USA), contrasted with 2% uranyl acetate, and visualized via transmission electronic microscopy (TEM, JEM-1400Plus, JEOL, Akishima, Tokyo, Japan).\u003c/p\u003e\n\u003ch3\u003eDNA extraction, preparation and sequencing\u003c/h3\u003e\n\u003cp\u003ePhage DNA extraction was done according to a protocol [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] adapted by Nicolas Ginet (Bacterial chemistry Laboratory, Marseille, France) after amplification of Salten in a more susceptible \u003cem\u003eS. enterica\u003c/em\u003e Tennessee ST5018 isolate, \u003cem\u003ei.e.\u003c/em\u003e Sten17 (Accession number SALQB8961AA in EnteroBase). Briefly, genetic material from bacterial origin potentially surrounding phages was eliminated by adding 10 \u0026micro;L of DNAse I (1 U/\u0026micro;L; #D5307; Sigma-Aldrich), 5 \u0026micro;L of RNAse A (10mg/mL; #EN0531; Thermo Fisher) and 2 \u0026micro;L of Dpn I (10 U/\u0026micro;L; #ER1702; Thermo Fisher). Phage DNA was extracted using phenol-chloroform-isoamyl acid 25/24/1 (#77617; Sigma-Aldrich). After DNA quantification with NanodropOne (Thermo Fischer) and Qubit 4 Fluoremeter (Invitrogen, Thermo Fisher), phage DNA was fragmented with a transposase enzyme. Fragmented end-prepared DNA was ligated to Illumina adaptors and then sorted with beads to select fragment sizes between 150\u0026ndash;250 bp, for a final fragment size between 270\u0026ndash;370 bp. Concentration of the final library was evaluated through Qubit 4 and fragment sizes were checked by migration using QIAxcel Advanced Instrument (QIAgen, Hilden, Germany). Fragments ready for sequencing sized between 280bp and 320bp. DNA libraries were sequenced in paired-ends in our in-lab sequencer (iSeq100; Illumina). Raw reads were deposited in the European Nucleotide Archive (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/ena/browser/support\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/ena/browser/support\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e with the following Accession Number: ERR13191102 (\u003cem\u003eSalmonella Phage Tennessee\u003c/em\u003e).\u003c/p\u003e\n\u003ch3\u003eDe novo phage assembly and annotation\u003c/h3\u003e\n\u003cp\u003eAfter Illumina sequencing, phage reads quality was controlled using FastQC v.0.12.1 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.babraham.ac.uk/projects/fastqc/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e Primers were trimmed with Fastp v.0.22.0 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] using default parameters. Phage genome contigs were prepared following the workflow recommended in Turner et al.[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. \u003cem\u003eDe novo\u003c/em\u003e phage genome assembly was carried out with SPAdes v.3.14.1 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] with default parameters. We obtained a large contig of 110,076 nt with high coverage (average 400 reads depth), and a number of short contigs (below 1200 nt) with low coverage (average 10\u0026ndash;20 reads depth). We kept only the largest Salten contig and generated a complete genome with PhageTerm Virome v.4.3 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. After polishing with Pilon v.1.24[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], we obtained a new contig with a sequencing coverage ranged from 295\u0026times; to 1171\u0026times;. This contig corresponding to the complete genome was deposited in the European Nucleotide Archive with Accession Number OZ075147.\u003c/p\u003e\u003cp\u003eSalten genome was annotated thanks to the Genome Annotation online tool from the Bacterial and Viral Bioinformatics Resource Center [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] (BV-BRC; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bv-brc.org/\u003c/span\u003e\u003cspan address=\"https://www.bv-brc.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e using VIGOR4 v.4.0. and based on \u003cem\u003eTequintavirus\u003c/em\u003e annotation [Taxonomy ID\u0026thinsp;=\u0026thinsp;187218]). Pharokka v.1.7.5 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] was used to complete the annotation. Structural genes were annotated manually based on Zivanovic et al [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and Linares et al [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], using BLASTn or BLASTp from NCBI (Blast\u0026reg; services, available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e The number attributed to each Coding DNA Sequences (CDS) was provided by the BV-BRC annotation. Proksee web server [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] was used to generate Salten linear genome map, through CGView builder v.2.0.5 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. PhageScope web server 11 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phagescope.deepomics.org/\u003c/span\u003e\u003cspan address=\"https://phagescope.deepomics.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e tools were used for lifestyle prediction, virulent factor and antimicrobial resistance gene detection.\u003c/p\u003e\n\u003ch3\u003eComparative genomics analysis\u003c/h3\u003e\n\u003cp\u003eThe complete genome of the Salten was compared against the NCBI core nucleotide database using BLASTn. Hits with at least 30% query coverage were retained and, after genome comparison with VIRIDIC [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], a subset of 184 phage genomes showing\u0026thinsp;\u0026ge;\u0026thinsp;70% average nucleotide identity (ANI) was selected for a refined analysis (\u003cem\u003eTequintavirus\u003c/em\u003e genus). Concerning the comparison of Salten to four other Tequintaviruses, gene coordinates were adjusted to standardize the starting point at the deoxynucleoside-5'-monophosphatase (dmp) gene for all genomes. VICTOR web platform was used to infer the phylogeny from nucleotides and using the D0 formula [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. EasyFig [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] was used to generate detailed synteny diagrams. PanExplorer web server [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] was used to determine the core genome and Salten specific-genes (PanACoTA v.1.4.0 option with \u0026gt;\u0026thinsp;80% blast identity [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]) by comparing to the 184 \u003cem\u003eTequintavirus\u003c/em\u003e genomes selected after VIRIDIC analysis.\u003c/p\u003e\u003cp\u003eA phylogenetic tree based on the aminoacid sequences of the Long Tail fibre proteins was performed based on a MUSCLE v.5.1 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] alignment followed by a tree construction through the Jukes-Cantor genetic distance model with neighbor-joining and 1000 bootstrap replicates, in Geneious Prime v.2025.1.3 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003ePhage morphology\u003c/h2\u003e\u003cp\u003eTransmission electron microscopy (TEM) of Salten revealed a T5-like Siphophage morphology, with a long non-contractile flexible tail of about 180 nm, attached to an icosahedral head of 60 nm of diameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePhage genome\u003c/h3\u003e\n\u003cp\u003eSalten harboured a complete genome of 109,999 base pairs with a\u0026thinsp;~\u0026thinsp;39% GC content, varying across the genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A total of 220 Open Reading Frames (ORF) were predicted, including 197 CDS (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and 23 tRNAs. According to PhageScope, Salten was predicted to be virulent (lytic cycle) and did not encode any genes associated with virulence or antimicrobial resistance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCDS annotation and associated known function are provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Thanks to the well modeled structure of T5 tail [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], we manually annotated the CDSs encoding for Salten\u0026rsquo;s tail proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The Tail Tube Protein pb6 (TTPpb6; CDS175) is surrounded by the Tape Measure Protein pb2 (TMPpb2; CDS171), which determines the length of the tube. Three lateral Long Tail fibres (each fiber being formed by the LTFpb1; CDS165) are connected by their amino-terminal (N-terminal) part to the TTPpb6 protein through three lateral fibres p132 (CDS166). Anchoring of the LTFpb1 onto the central fibre is followed by the sequential positioning of pb9 (CDS169), pb4 (CDS167), pb3 (CDS168), and finally the receptor-binding protein pb5 (RBPpb5; CDS189) at the tip. RBPpb5 binds to outer membrane transporter, such as FhuA, FepA or BtuB, to trigger infection [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Small proteins p140 and p142 are encoded by CDS174 and CDS176, respectively.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003eComparative genomics with other\u003c/em\u003e Tequintaviruses\u003c/h2\u003e\u003cp\u003eAfter complete genome comparison (VIRIDIC and BLASTn), \u003cem\u003eEscherichia phage\u003c/em\u003e HildyBeyeler (MZ501074.1) was found to be the most similar to Salten, harbouring 84.6% average nucleotide identity (ANI). Based on the criterion that strains of the same species share at least 95% genome sequence identity [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], Salten can be considered as a member from a new species within the \u003cem\u003eTequintavirus\u003c/em\u003e genus. This statement was confirmed by the phylogenetic tree generated by VICTOR that classified HildyBeyeler and Salten in two different species (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Salten and HildyBeyeler shared 97.2% nucleotide identity within the core genome (i.e. CDS having\u0026thinsp;\u0026gt;\u0026thinsp;80% blast identity). Salten harboured 16 CDS (CDS7, 8, 9, 10, 11, 13, 28, 33, 51, 62, 67, 84, 85, 101, 116 and 127) absent in HildyBeyeler, of which 11 were annotated as \"hypothetical proteins\" and two as \"Phage protein\" (CDS33 and CDS127). CDS13 was annotated as a DNA N-6-adenine methyltransferase (\u003cem\u003eSalmonella phage\u003c/em\u003e vB_SalS-SIY1lw [WVH10139.1]; 93.9% identity on 89% coverage), while the two last CDS (CDS51 and CDS62) were annotated as \"Phage HNH homing endonuclease\".\u003c/p\u003e\u003cp\u003eVIRIDIC was used to delimit the \u003cem\u003eTequintavirus\u003c/em\u003e genus (i.e. \u0026gt;70% ANI) among NCBI phage accessions. To compare Salten to the diversity of Tequintaviruses, we selected four \"representative\" genomes out of the 184 genomes from the \u003cem\u003eTequintavirus\u003c/em\u003e genus. Selection was based on an interval of approximately 5% of ANI divergence, relative to Salten (since this threshold has been proposed as a species level of diversification [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. We thus selected \u003cem\u003eEscherichia phage\u003c/em\u003e HildyBeyeler, \u003cem\u003eSalmonella phage\u003c/em\u003e 8sent1748 (MT653146.1), \u003cem\u003eEscherichia phage\u003c/em\u003e T5 (AY543070.1) and \u003cem\u003eSalmonella phage\u003c/em\u003e S147 (NC_048012.1) with 84.6%, 80.4%, 76% and 70.1% ANI values, respectively. Genome comparisons performed with MAUVE [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] showed a high degree of synteny (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), with three large collinear blocks, shared by all the genomes and kept in the same order. One of the HNH homing endonucleases (CDS62) - found in Salten and absent in HildyBeyeler - was also present in \u003cem\u003eSalmonella\u003c/em\u003e phage S147, which was seen as a small collinear block in a different position of the genome (in dark green; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Knowing that HNH homing endonuclease are mobile genetic elements able to move by their own [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], it is not surprising to retrieve this CDS and its flanking sequences in different parts of the genomes.\u003c/p\u003e\u003cp\u003eThe pangenome of the five phages corresponded to 374 CDS (PanACoTA analysis). Among these pangenomic CDS, 64 of them were homologs between the five genomes - including Salten (\u0026gt;\u0026thinsp;80% BLASTp identity), 121 CDS were shared by two or more genomes (and Salten harboured 80 of them) and 190 CDS were strain-specific (Salten harboured 54 of them). More globally, the pangenome of the 185 Tequintaviruses (including Salten) corresponded to 1472 CDS (PanACoTA analysis). Among these pangenomic CDS, only eight of them were homologs CDS (\u0026gt;\u0026thinsp;80% BLASTp identity; pink on Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These proteins can be considered essential for phage multiplication, such as DNA replication (dihydrofolate reductase and NAD-dependant DNA ligase) and protein degradation (metallopeptidase). The two genes annotated as \"Phage proteins\" (CDS113 and CDS140) have been compared to NCBI dataset (BLASTn) and CDS113 corresponded to a hypothetical protein or virion-structural protein (\u003cem\u003eEscherichia\u003c/em\u003e phage phiLLS [YP_009790087.1]; 97.7% identity with 100% coverage) and CDS140 corresponded to a helicase (\u003cem\u003eEscherichia\u003c/em\u003e phage DT57C [YP_009149867.1]; 99.6% identity with 100% coverage). When comparing Salten to all other 184 Tequintaviruses, 17 CDS were considered as unique to Salten (or highly divergent; represented in blue arrows in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and were all annotated as \"hypthetical proteins\" or \"Phage protein\" (with no ability to improve annotation). To conclude, Salten as well as \u003cem\u003eTequintavirus\u003c/em\u003e genomes showed a high degree of variation and strain-specific CDS (accessory genome).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eList of the core genes (\u0026gt;\u0026thinsp;80% blastp identity) among the 185 \u003cem\u003eTequintavirus\u003c/em\u003e genomes and named according to Salten BV-BRC annotation.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCDS number\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProtein name\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eATP-dependent Clp protease proteolytic subunit\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e111\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMetallopeptidase phage-associated\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e113\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePhage protein / structural protein\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e120\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRibonucleotide reductase of class Ia (aerobic)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e124\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePhage phosphate starvation-inducible protein\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e140\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePhage protein / helicase\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e148\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDNA ligase phage-associated\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e175\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePhage major Tail Tube Protein TTPpb6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eHost recognition\u003c/h2\u003e\u003cp\u003eTequintaviruses recognize their host(s) through a two step process, that is first reversibly adsorbing to the LPS via the LTFpb1 and then irreversibly adsorbing to an outer-membrane transporter - such as FhuA, BtuB or FepA - via the receptor binding protein [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] (RBPpb5). To infer the potential second receptor of Salten, we compared its RBPpb5 with those of the four representative genomes. Unlike the core-genome phylogeny, Salten\u0026rsquo;s RBPpb5 showed the highest similarity to those of HildyBeyeler and S147 (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). These two phages are known to target BtuB [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], which suggests that Salten could also adsorb on this outer-membrane transporter. Future experiments are needed to test if BtuB is indeed Salten\u0026rsquo;s receptor, for exemple by generating knockouts of each of the potential receptor gene and assessing phage adsorption [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eConcerning the reversible adsorption to the LPS, EasyFig detected a highly variability (\u0026lt;\u0026thinsp;64% of ANI) in Salten\u0026rsquo;s LTFpb1 compared to the one harbored by the four representative genomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB; blue arrow annotated \"LTFpb1\" on Salten genome map). Indeed, in line with Skutel et al. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] findings that compared 15 LTFpb1 of \u003cem\u003eTequintavirus\u003c/em\u003e, the LTFpb1 aminoacid sequences of T5, HildyBeyeler, 8sent1748 and S147 respectively shared with Salten 85.4%, 85%, 51.1% and 41.7% identity (coverage 78%, 48%, 31% and 31%). The N-terminal part of the protein (phage central tail binding domain; residues 1 to 41 on consensus sequence; Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) which is known to attach LTFpb1 to the phage central tail tube by the collar with p132 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] was well conserved. Moreover, the coiled-coil domain (residues 42 to 225; Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), responsible for projecting the lateral fibre away from the central tube, was also relatively well conserved. Notably, in Salten LTFpb1, the coiled-coil region contains insertions (repeats), leading to an extended coiled-coil. On the C-terminal part of LTFpb1, the chaperone encoding domain was also well conserved. However, the lateral fibre encoding domain (downstream the coiled-coil region; residues 226 to 874) showed a high level of variation (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C). The LTFpb1 phylogenetic analysis was not congruent with the analysis made with the complete genomes of the phages. Indeed, Salten\u0026rsquo;s LTFpb1 was phylogeneticaly closer to T5 than the one of HildyBeyeler, and these three proteins were similarly distinct to the ones of 8sent1748 and S147 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA; Jukes-Cantor genetic distance model with neighbor-joining). This phylogenetic analysis was consistent with the structural analysis. Indeed, the structure of the lateral fibres of both T5 - crystallized and well modeled by Garcia-Doval et al. [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] - and Salten were similar, but distinct from S147 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). We therefore superimposed the predicted lateral fibres structure of Salten with the ones of S147 to visualise structure divergence between the two clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). As proposed by sequence alignments (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), their C-terminal domains did not overlapped. Given that this domain is known to reversibly bind to the bacterial polysaccharide moiety of lipopolysaccharides (LPS) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], it is likely involved in host specificity and may play a critical role in determining the phage host range.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe Salten bacteriophage was isolated from wastewater and exhibits a lytic activity against \u003cem\u003eSalmonella enterica\u003c/em\u003e serotype Tennessee ST5018. It belongs to the \u003cem\u003eTequintavirus\u003c/em\u003e genus but remains the first member of a new species. As it does not harbour any bacterial virulence genes nor antimicrobial resistance genes, Salten is a promising candidate for the biocontrol of \u003cem\u003eSalmonella enterica\u003c/em\u003e serotype Tennessee ST5018.\u003c/p\u003e\u003cp\u003eNotably, the LTFpb1 appeared to be highly diverse among the \u003cem\u003eTequintavirus\u003c/em\u003e genus. Given that this protein directly interacts with LPS on the host cell surface, such LTFpb1 diversity -particularly in the C-terminal domain - may explain Tequintaviruses host specificity. Indeed, O-antigen is known to be variable among Gram-negative bacteria [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. \u003cem\u003eTequintavirus\u003c/em\u003e adaptation to a new bacterial host may thus first reside in modification of their LTFpb1. Further studies would be interesting to determine which Salten\u0026rsquo;s aminoacids are involved in its interaction with its bacterial host. We could also experimentally investigate the capacity of Salten to increase its host range on other sequence types of \u003cem\u003eS. enterica\u003c/em\u003e and related bacteria, as it was done for T3 phage and its gp17 fibre in Yehl and al. [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAMR : antimicrobial resistance\u003c/p\u003e\n\u003cp\u003eANI : Average nucleotide identity\u003c/p\u003e\n\u003cp\u003eBV-BRC : Bacterial and Viral Bioinformatics Resource Center\u003c/p\u003e\n\u003cp\u003eCDC : Centers for Disease Control and Prevention\u003c/p\u003e\n\u003cp\u003eCDS : Coding DNA sequence\u003c/p\u003e\n\u003cp\u003edmp : Deoxynucleoside-5\u0026apos;-monophosphatase\u003c/p\u003e\n\u003cp\u003eECDC : European Centre for Disease Prevention and Control\u003c/p\u003e\n\u003cp\u003eEFSA : European Food Safety Authority\u003c/p\u003e\n\u003cp\u003eLB : Lysogeny Broth\u003c/p\u003e\n\u003cp\u003eLPS : Lipopolysaccharides\u003c/p\u003e\n\u003cp\u003eLTF : Long tail fibre\u003c/p\u003e\n\u003cp\u003eO-Ag : O-antigen\u003c/p\u003e\n\u003cp\u003eOD : Optical Density\u003c/p\u003e\n\u003cp\u003eORF : Open Reading Frame\u003c/p\u003e\n\u003cp\u003ePhage : Bacteriophage\u003c/p\u003e\n\u003cp\u003eRBP : Receptor-Binding Protein\u003c/p\u003e\n\u003cp\u003eSalten : \u003cem\u003eSalmonella Phage Tennessee\u003c/em\u003e Salten\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS. enterica\u003c/em\u003e : Salmonella enterica\u003c/p\u003e\n\u003cp\u003eSM buffer :\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eST : Sequence type\u003c/p\u003e\n\u003cp\u003eSten : \u003cem\u003eSalmonella enterica\u003c/em\u003e serotype Tennessee\u003c/p\u003e\n\u003cp\u003eTTP : Tail Tube Protein\u003c/p\u003e\n\u003cp\u003eTMP : Tape Measure Protein\u003c/p\u003e\n\u003cp\u003eTEM : Transmission electronic microscopy\u003c/p\u003e\n\u003cp\u003edeoxynucleoside-5\u0026apos;-monophosphatase (dmp)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eThe authors thank M. Ansaldi (LCB, Marseille) for providing wastewater from which we isolated our phage, F-X. Weill for the bacterial sequencing, C. Mariac (DIADE, Montpellier) for the library quality check before sequencing and for QiAxcel use, A. Talman (MIVEGEC, Montpellier) for the help on the iSeq use, J. Garneau for PhageTerm help, M. Monot (I. Pasteur, Paris), A. Dereeper (PHIM, Montpellier), S. Bouzidi (MIVEGEC, Montpellier) and J. Hayer (MIVEGEC, Montpellier) for bioinformatics advices. We thank UMR MIVEGEC for providing an efficient working environment and acknowledge the ISO 9001 certified \u0026nbsp;IRD itrop HPC (member of the South Green Platform) and the whole bioinformatics team support in Montpellier for providing HPC resources that have contributed to the research results reported within this paper. URL: https://bioinfo.ird.fr/ - http://www.southgreen.fr.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eThe complete genome sequence generated and analysed during the current study is available in the European Nucleotide Archive repository with Accession Number OZ075147 (https://www.ebi.ac.uk/ena/browser/view/OZ075147). The supplemental data that support the findings of this study are openly available at\u0026nbsp;https://src.koda.cnrs.fr/MAURINAmandine/salten_report\u003c/p\u003e\n\u003cp\u003eAuthor contribution\u003c/p\u003e\n\u003cp\u003eConceptualisation, R.F. and A.M.; Methodology \u0026amp; Investigation, R.F. and A.M. with the help of C.Z.-C. for the genomic comparison; Results analysis, R.F., A.M., C.Z.-C., C.B., and J.D.; Writing, R.F. and A.M.; Revision, R.F., A.M., C.Z.-C., C.B., and J.D..\u003c/p\u003e\n\u003cp\u003eConflict of interest\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003eFunder\u0026nbsp;Information\u003c/p\u003e\n\u003cp\u003eThis work was funded by Royal Canin through the support of the CNRS (contract 245420).\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNote\u003c/p\u003e\n\u003cp\u003eReferences [46], [47], [48], [49], [50], [51], [52], [53] and [54] are cited in the supplementary material document, in Table S1.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEng S-K, Pusparajah P, Ab Mutalib N-S, Ser H-L, Chan K-G, Lee L-H. Salmonella: A review on pathogenesis, epidemiology and antibiotic resistance. Frontiers in Life Science. 2015;8:284\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThe European Union One. Health 2023 Zoonoses report. EFSA J. 2024;22:e9106.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTack DM. Preliminary Incidence and Trends of Infections with Pathogens Transmitted Commonly Through Food \u0026mdash; Foodborne Diseases Active Surveillance Network, 10 U.S. Sites, 2015\u0026ndash;2018. MMWR Morb Mortal Wkly Rep. 2019;68.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMurray CJL, Ikuta KS, Sharara F, Swetschinski L, Aguilar GR, Gray A, et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet. 2022;399:629\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCuriao T, Marchi E, Grandgirard D, Le\u0026oacute;n-Sampedro R, Viti C, Leib SL, et al. Multiple adaptive routes of Salmonella enterica Typhimurium to biocide and antibiotic exposure. BMC Genomics. 2016;17:491.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKuhl S, Abedon ST, Hyman P. Diseases caused by phages. In: Bacteriophages in health and disease. 2012. pp. 21\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBalcazar JL. Bacteriophages as Vehicles for Antibiotic Resistance Genes in the Environment. PLoS Pathog. 2014;10:e1004219.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrown-Jaque M, Calero-C\u0026aacute;ceres W, Muniesa M. Transfer of antibiotic-resistance genes via phage-related mobile elements. Plasmid. 2015;79:1\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKropinski AM, Mazzocco A, Waddell TE, Lingohr E, Johnson RP. Enumeration of Bacteriophages by Double Agar Overlay Plaque Assay. In: Clokie MRJ, Kropinski AM, editors. Bacteriophages. Totowa, NJ: Humana; 2009. pp. 69\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGendre J, Ansaldi M, Olivenza DR, Denis Y, Casades\u0026uacute;s J, Ginet N. Genetic Mining of Newly Isolated Salmophages for Phage Therapy. Int J Mol Sci. 2022;23:8917.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBabraham Bioinformatics -. FastQC A Quality Control tool for High Throughput Sequence Data. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.babraham.ac.uk/projects/fastqc/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Accessed 17 Apr 2024.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34:i884\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTurner D, Adriaenssens EM, Tolstoy I, Kropinski AM. Phage Annotation Guide: Guidelines for Assembly and High-Quality Annotation. PHAGE. 2021;2:170\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAntipov D, Raiko M, Lapidus A, Pevzner PA. MetaviralSPAdes: assembly of viruses from metagenomic data. Bioinformatics. 2020;36:4126\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePrjibelski A, Antipov D, Meleshko D, Lapidus A, Korobeynikov A. Using SPAdes De Novo Assembler. Curr Protocols Bioinf. 2020;70:e102.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGarneau JR, Depardieu F, Fortier L-C, Bikard D, Monot M. PhageTerm: a tool for fast and accurate determination of phage termini and packaging mechanism using next-generation sequencing data. Sci Rep. 2017;7:8292.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWalker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, et al. Pilon: An Integrated Tool for Comprehensive Microbial Variant Detection and Genome Assembly Improvement. PLoS ONE. 2014;9:e112963.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOlson RD, Assaf R, Brettin T, Conrad N, Cucinell C, Davis JJ, et al. Introducing the Bacterial and Viral Bioinformatics Resource Center (BV-BRC): a resource combining PATRIC, IRD and ViPR. Nucleic Acids Res. 2023;51:D678\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBouras G, Nepal R, Houtak G, Psaltis AJ, Wormald P-J, Vreugde S. Pharokka: a fast scalable bacteriophage annotation tool. Bioinformatics. 2023;39:btac776.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZivanovic Y, Confalonieri F, Ponchon L, Lurz R, Chami M, Flayhan A, et al. Insights into Bacteriophage T5 Structure from Analysis of Its Morphogenesis Genes and Protein Components. J Virol. 2014;88:1162\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLinares R, Arnaud C-A, Effantin G, Darnault C, Epalle NH, Boeri Erba E, et al. Structural basis of bacteriophage T5 infection trigger and E. coli cell wall perforation. Sci Adv. 2023;9:eade9674.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGrant JR, Enns E, Marinier E, Mandal A, Herman EK, Chen C-Y, et al. Proksee: in-depth characterization and visualization of bacterial genomes. Nucleic Acids Res. 2023;51:W484\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStothard P, Wishart DS. Circular genome visualization and exploration using CGView. Bioinformatics. 2005;21:537\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoraru C, Varsani A, Kropinski AM. VIRIDIC\u0026mdash;A Novel Tool to Calculate the Intergenomic Similarities of Prokaryote-Infecting Viruses. Viruses. 2020;12:1268.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeier-Kolthoff JP, G\u0026ouml;ker M. VICTOR: genome-based phylogeny and classification of prokaryotic viruses. Bioinformatics. 2017;33:3396\u0026ndash;404.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSullivan MJ, Petty NK, Beatson SA. Easyfig: a genome comparison visualizer. Bioinformatics. 2011;27:1009\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDereeper A, Summo M, Meyer DF. PanExplorer: a web-based tool for exploratory analysis and visualization of bacterial pan-genomes. Bioinformatics. 2022;38:4412\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePerrin A, Rocha EPC. PanACoTA: a modular tool for massive microbial comparative genomics. NAR Genom Bioinform. 2021;3:lqaa106.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEdgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32:1792\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, et al. Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;28:1647\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDegroux S, Effantin G, Linares R, Schoehn G, Breyton C. Deciphering Bacteriophage T5 Host Recognition Mechanism and Infection Trigger. J Virol. 2023;97:e01584\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAdriaenssens E, Brister JR. How to Name and Classify Your Phage: An Informal Guide. Viruses. 2017;9:70.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDarling ACE, Mau B, Blattner FR, Perna NT. Mauve: Multiple Alignment of Conserved Genomic Sequence With Rearrangements. Genome Res. 2004;14:1394\u0026ndash;403.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHafez M, Hausner G. Homing endonucleases: DNA scissors on a mission. Genome. 2012;55:553\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStoddard BL. Homing endonucleases from mobile group I introns: discovery to genome engineering. Mob DNA. 2014;5:7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBarth ZK, Dunham DT, Seed KD. Nuclease genes occupy boundaries of genetic exchange between bacteriophages. NAR Genomics Bioinf. 2023;5:lqad076.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBertozzi Silva J, Storms Z, Sauvageau D. Host receptors for bacteriophage adsorption. FEMS Microbiol Lett. 2016;363:fnw002.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMaffei E, Shaidullina A, Burkolter M, Heyer Y, Estermann F, Druelle V, et al. Systematic exploration of Escherichia coli phage\u0026ndash;host interactions with the BASEL phage collection. PLoS Biol. 2021;19:e3001424.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGencay YE, Gambino M, Pr\u0026uuml;ssing TF, Br\u0026oslash;ndsted L. The genera of bacteriophages and their receptors are the major determinants of host range. Environ Microbiol. 2019;21:2095\u0026ndash;111.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSkutel M, Andriianov A, Zavialova M, Kirsanova M, Shodunke O, Zorin E, et al. T5-like phage BF23 evades host-mediated DNA restriction and methylation. microLife. 2023;4:uqad044.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGarcia-Doval C, Cast\u0026oacute;n JR, Luque D, Granell M, Otero JM, Llamas-Saiz AL, et al. Structure of the Receptor-Binding Carboxy-Terminal Domain of the Bacteriophage T5 L-Shaped Tail Fibre with and without Its Intra-Molecular Chaperone. Viruses. 2015;7:6424\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHeller KJ. Identification of the phage gene for host receptor specificity by analyzing hybrid phages of T5 and BF23. Virology. 1984;139:11\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGoldman RC, Leive L. Heterogeneity of Antigenic-Side-Chain Length in Lipopolysaccharide from Escherichia coli 0111 and Salmonella typhimurium LT2. Eur J Biochem. 1980;107:145\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eReeves PR, Cunneen MM. Chapter 18 - Biosynthesis of O-antigen chains and assembly. In: Holst O, Brennan PJ, von Itzstein M, Moran AP, editors. Microbial Glycobiology. San Diego: Academic; 2010. pp. 319\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYehl K, Lemire S, Yang AC, Ando H, Mimee M, Torres MDT, et al. Engineering Phage Host-Range and Suppressing Bacterial Resistance through Phage Tail Fiber Mutagenesis. Cell. 2019;179:459\u0026ndash;e4699.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDavison J. Pre-early functions of bacteriophage T5 and its relatives. Bacteriophage. 2015;5:e1086500.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShi L, Potts M, Kennelly PJ. The serine, threonine, and/or tyrosine-specific protein kinases and protein phosphatases of prokaryotic organisms: a family portrait. FEMS Microbiol Rev. 1998;22:229\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim Y-I, Levchenko I, Fraczkowska K, Woodruff RV, Sauer RT, Baker TA. Molecular determinants of complex formation between Clp/Hsp100 ATPases and the ClpP peptidase. Nat Struct Mol Biol. 2001;8:230\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYoung R. Bacteriophage lysis: mechanism and regulation. Microbiol Rev. 1992;56:430\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang J, Jiang Y, Vincent M, Sun Y, Yu H, Wang J, et al. Complete genome sequence of bacteriophage T5. Virology. 2005;332:45\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHogenkamp HPC. Nature and properties of the bacterial ribonucleotide reductases. Pharmacol Ther. 1983;23:393\u0026ndash;405.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRojowska AM. Structural and functional analysis of DNA binding by the Rad50 catalytic head from Thermotoga maritima. PhD. Dissertation zur Erlangung des Doktorgrades der Fakult\u0026auml;t f\u0026uuml;r Chemie und Pharmazie der Ludwig-Maximilians-Universit\u0026auml;t M\u0026uuml;nchen; 2013.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWarner HR, Thompson RB, Mozer TJ, Duncan BK. The properties of a bacteriophage T5 mutant unable to induce deoxyuridine 5\u0026rsquo;-triphosphate nucleotidohydrolase. Synthesis of uracil-containing T5 deoxyribonucleic acid. J Biol Chem. 1979;254:7534\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCasjens SR, Gilcrease EB. Determining DNA Packaging Strategy by Analysis of the Termini of the Chromosomes in Tailed-Bacteriophage Virions. In: Clokie MRJ, Kropinski AM, editors. Bacteriophages: Methods and Protocols. Molecular and Applied Aspects. Volume 2. Totowa, NJ: Humana; 2009. pp. 91\u0026ndash;111.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bacteriophage, Salmonella enterica, genome, long tail fibre, receptor binding protein","lastPublishedDoi":"10.21203/rs.3.rs-6981461/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6981461/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e: The 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 \u003cem\u003eSalmonella enterica\u003c/em\u003eserotype Tennessee sequence type 5018, a food-processing pathogen.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: We report the genome analysis of \u003cem\u003eSalmonella phage Tennessee \u003c/em\u003eSalten, a new species from the \u003cem\u003eTequintavirus \u003c/em\u003egenus. Its genome is 109,999 bp in length and contains 197 predicted coding sequences and 23 tRNAs. Compared to its closest known relative phage \u003cem\u003eEscherichia phage\u003c/em\u003e 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 \u003cem\u003eTequintavirus \u003c/em\u003egenus.\u003c/p\u003e","manuscriptTitle":"Comparative genome analysis of a new Tequintavirus isolated on Salmonella enterica serotype Tennessee","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-21 10:44:57","doi":"10.21203/rs.3.rs-6981461/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-08T10:33:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-07T02:48:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-02T11:43:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"10524537220897155490907905392047463032","date":"2025-08-27T13:49:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62170554265087584668902815676180585727","date":"2025-08-25T23:17:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-13T15:07:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-13T14:31:23+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-13T14:16:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-11T18:02:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Research Notes","date":"2025-08-11T17:59:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d1417691-9086-431b-919f-689cd02689ec","owner":[],"postedDate":"August 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-24T16:04:56+00:00","versionOfRecord":{"articleIdentity":"rs-6981461","link":"https://doi.org/10.1186/s13104-025-07533-4","journal":{"identity":"bmc-research-notes","isVorOnly":false,"title":"BMC Research Notes"},"publishedOn":"2025-11-21 15:57:08","publishedOnDateReadable":"November 21st, 2025"},"versionCreatedAt":"2025-08-21 10:44:57","video":"","vorDoi":"10.1186/s13104-025-07533-4","vorDoiUrl":"https://doi.org/10.1186/s13104-025-07533-4","workflowStages":[]},"version":"v1","identity":"rs-6981461","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6981461","identity":"rs-6981461","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}