Genomic characterization of the novel bacteriophage PfAn1 from Lake Baikal against Pseudomonas fluorescens

Genomic characterization of the novel bacteriophage PfAn1 from Lake Baikal against Pseudomonas fluorescens | 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 Research Article Genomic characterization of the novel bacteriophage PfAn1 from Lake Baikal against Pseudomonas fluorescens Anna Gorshkova, Olga Belykh, Irina Tikhonova, Li Xi, Maria Siniagina, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5526281/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 May, 2025 Read the published version in Archives of Virology → Version 1 posted 5 You are reading this latest preprint version Abstract We isolated a novel bacteriophage from Lake Baikal. Transmission electron microscopy revealed that phage PfAn1 has a head with a diameter of 50 nm and a short tail. Its genome is 39,156 bp in length with a GC content of 57%. It is predicted to contain 53 open reading frames (ORFs). The results of evolutionary analysis suggest that phage PfAn1 should be considered a new member of the class Caudoviricetes . Figures Figure 1 Figure 2 Figure 3 Full Text With the development of high-throughput sequencing technology, numerous novel phages have been discovered from metagenomes and viromes. However, the analysis of these phage sequences is a great challenge because the reference genomes of phages are very limited due to the fact that most phages cannot be cultured independently [ 1 ] . Here, we isolated a novel Pseudomonas fluorescens phage, PfAn1, from the water of Lake Baikal (Russia). Evolutionary and phylogenetic analysis suggested that phage PfAn1 should be considered a new member of the class Caudoviricetes . Bacteriophage PfAn1 was isolated from liquid enrichment culture obtained by adding a concentrated R2A medium [ 2 ] to a natural sample. The enrichment was based on the hypothesis that actively multiplying bacteria would trigger the accumulation of viruses, according to the “kill the winner” theory. Strains of bacteria and bacteriophages were simultaneously isolated from the enrichment cultures. PfAn1 was enriched from a sample taken at the center of the Maloye More Strait at a depth of 5 m. The host strain was isolated from a water sample collected at the depth of 5 m from the central site of the Ukhan Cape-Tonky Cape transect in the central basin of Lake Baikal. The enrichment culture was centrifuged at 8,000 g for 10 min, and the supernatant was filtered through a 0.22 μm filter. Then, the supernatant was tested for specific phages by the double-layer agar method. It resulted in lysis on cells that were identified as Pseudomonas fluorescens by 16S sequencing. Phages were concentrated by PEG precipitation. The phages were further purified by CsCl density gradient centrifugation (100,000 × g , 2 h) and dialyzed against SM buffer (100 mM NaCl, 10 mM MgSO 4 , 50 mM Tris-HCl; pH 7.5). Purified phages were used for further experiments. For electron microscopy, a 20 uL aliquot of phage suspension was placed on a formvar-coated copper grid for 15 min and then dried using filter paper. The preparation was then stained with 2% (w/v) uranyl acetate for 15 min. Finally, phage morphology was examined using a LEO906E transmission electron microscope (Carl Zeiss, Germany). Genomic DNA was extracted using the phenol-chloroform protocol [ 3 ] . Genome sequencing was performed using a FASTAseq 300 (GeneMind, China) in Kazan Federal University. The phage genome assembly was carried out according to the recommendations of A. Shen and A. Millard [ 4 ] . The raw reads were analyzed in FastQC v. 0.12.1 [ 5 ] . Then reads were filtered to remove low-quality reads and adapter regions, using Trimmomatic v. 0.36 [ 6 ] . The genome sequence was de novo assembled using the SPAdes v. 4.0.0 software [ 7 ] . The coverage depth was calculated using Bowtie2 v. 2.4.4 [ 8 ] and SAMtools v. 1.13 [ 9 ] . The assembled contig was checked via Bandage v. 0.8.1 [ 10 ] . Identification, quality assessment, and completeness of the virus genome were estimated with CheckV v. 1.0.3 [ 11 ] . Genome termini were analyzed through inspection using read mapping and PhageTerm [ 12 ] . Open reading frames were determined with Prodigal v. 2.6.3 [ 13 ] . Taxonomic identification of viral genome was performed using geNomad v. 1.8.0 [ 14 ] , Diamond v. 2.1.8.162 [15] with e-value parameters (10 -5 , bit score ≥ 50, more sensitive) and BLASTn v. 2.12.0+ (e-value 10 -5 ) using the amino acid and nucleotide database of RefSeq v. 226. Functional analysis of translated ORFs was performed applying PHROG v.4 [ 16 ] , the Virus Orthologous Groups Database (VOGDB) v. 219 [17] and HHMER v. 3.2.1 [ 18 ] . The IMG/VR v.4 database was used to identify uncultivated relatives [ 19 ] , and Phyre2 tool was used for predicting and analyzing protein structure and function [20]. VipTree v. 4.0 [ 21 ] and VirClust v. 2.0 [ 22 ] were used for comparative genomic analysis. tRNAs were searched using tRNAscan-SE 2.0 [ 23 ]. For the phylogenetic tree based on the terminase large subunit proteins, the sequences were aligned using the MAFFT v. 7.407 [24] program with the -L-INS-i parameter. TrimAl v. 1.2 (-gappyout) [25] was used to remove ambiguous regions. Trees were computed with IQ-TREE software v. 1.6.9 [26]; model was selected with ModelFinder [27], and branch supports were determined using the approximate likelihood ratio test (1000 repetitions) [28] and the ultrafast bootstrap (1000 repetitions) [29]. The resulting trees were visualized and edited in iTOL [30]. Phage PfAn1 formed small plaques that became visible six days after the cell lawn has already grown (Supplementary Fig. S1). Transmission electron microscopy revealed that this phage possesses a head ~50 nm in diameter and a short tail (Fig. 1) and, thus, exhibits "podovirus" morphology. Phage PfAn1 has a double-stranded DNA genome of 39,156 bp with an average GC content of 57%, which is similar to that of its host DNA (60%). Its genome contains 53 putative protein-coding genes. One tRNA gene was detected in the PfAn1 genome. Only 30 PfAn1 gene products exhibit similarity to proteins of known function from diverse organisms (Fig. 2). For proteins having related proteins, the average coverage was 87%, and the average identity was 44% (26% to 66%) compared to proteins from the RefSeq, VOG, PHROG, and Uniref90 databases. Proteins of uncultivated bacteriophages from the IMG/VR database accounted for an average of 55% identity (32% to 90%) (Supplementary Table S1). We predicted the function of two proteins only based on tertiary structure because the function of proteins with similar amino acid composition is unknown. These are OFR-10 gene product presumably encoding the tail fiber protein and OFR-13 gene product encoding an oxidoreductase enzyme. The identified assembly genes include a small, large terminase and portal protein. Virion particle genes include a portal protein, a major capsid protein, a scaffolding protein, a head-tail adaptor protein, a head-closure protein, and three tail proteins, including a major tail protein, tail protein and putative tail fibers. The proteins encoded by ORF-15 and ORF-16 likely encode internal virion proteins involved in DNA ejection. The proteins associated with lysis of the host cell include the following gene cassette: endolysin, spanin and holin. Moreover, we identified a similarity of ORF-17 with peptidoglycan transglycosylase. ORF-17 is located in the morphogenic module. A recent study indicates that lytic transglycosylases are morphological components of the phage particle, which contact with the bacterial cell wall from the outside and might facilitate the passage of the viral genome through the cell wall by introducing small, localized gaps into the peptidoglycan layer [31]. We identified a gene encoding a repressor protein homologous to the phage λ C1 repressor that allows virus to establish and maintain latency. Phyre2 modeled gene product of ORF-38 with bacteriophage λ repressor-like DNA-binding domains with 95.2% confidence and 82% coverage. However, we did not identify other proteins involved in the integration and excision of viral DNA characteristic of temperate λ-like phages like an integrase. The module of genes responsible for DNA metabolism and regulation of transcription and translation contained proteins homologous to various nucleases that are involved in numerous nucleic acid cleavage events (ORF-25, ORF-30, ORF-31, and ORF-52), to transcription and translation repressor proteins (ORF-27, ORF-38 and ORF-44) and DNA recombination enzymes (ORF-29 and OFR-49). BLASTn analysis revealed that the genome of phage PfAn1 showed the highest similarity at DNA level to Ralstonia phage Firinga (NC_054961) and Ralstonia phage RSK1 (NC_022915) (68% identity and 9% query coverage). In bacterial genomes, we also detected short fragments similar to the PfAn1 genome, which amounted to <10% of the phage genome. The proteome analysis was performed using the genome sequence of phage PfAn1 and those of 54778 other phages, followed by the construction of a proteome tree. Phage PfAn1 formed a separate branch in the proteome tree with Ralstonia phages from the genus Firingavirus . Ralstonia phage Firinga infecting the plant pathogen R. solanacearum has a podovirus morphology [32]. Bacteriophage PfAn1 is similar to these phages, mainly in structural proteins; other proteins did not show a significant similarity (Fig.3). As Pseudomonas is the host genus of phage PfAn1; therefore, we attempted to search for related phages among viruses known for this group. The OrthoANIu [33] values were obtained by comparing phage PfAn1 with 1375 other Pseudomonas phage genomes, deposited in the GenBank database. Only eight bacteriophages had the OrthoANIu values > 0 (Supplementary Fig. S2). Bacteriophage PfAn1 formed a cluster with lytic cold-active phage HU1 infecting P. lactis [34]. Other phage infect P. aeruginosa and, like bacteriophage HU1, belong to unclassified Caudoviricetes . The OrthoANIu values were obtained by comparing phage PfAn1 with 5153 Caudoviricetes phages genomes, deposited in the RefSeq database. Only twenty-one bacteriophages had the OrthoANIu values > 0. The Firinga, phi297, and YMC11/07/P54_PAE_BP phages showed highest similarity with phage PfAn1. Combining the results of the search in both databases, we can conclude that the highest nucleotide similarity to phage PfAn1 was found for unclassified HU1 and phi297-like phages, as well as phages of the genus Firingavirus . Notably, the intergenomic similarity of PfAn1 and the OrthoANIu selected phages was very low. The gene for the terminase large subunit, an enzyme that packages DNA into a capsid, is one of the most conservative genes. As shown in Supplementary Figure S3, phylogenetic analysis was performed based on multiple alignments of the amino acid sequences of the DNA terminase large subunit. In the terminase large subunit-based tree, it was most closely related to phages Firinga and RKS1 belonging to the genus Firingavirus . Unclassified Pseudomonas phages and P22-like bacteriophages also appeared to be closely related phages. Phyre2 modeled the protein structure of the PfAn1 terminase large subunit that turned out to be the most similar to large terminase bacteriophage P22, with 100% confidence and 60% identity. Interestingly, the DNA packaging strategy is well studied for the temperate transducing phage P22, which, apparently, is also inherent in bacteriophage PfAn1. Thus, we can assume that phage PfAn1 can be classified as a P22-like phages based on the similarity of the structural module of the genome. However, the PfAn1 genome did not show homologous genes typical of phages of this group, which are responsible for the integration into the host genome. Fig. 3 demonstrates the intergenomic alignments of phages PfAn1, RSK1, P22, and HU1, which were performed using DiGAlign [35]. As shown in this figure, phage PfAn1 has some similarity in the structural module of the genome with phage RSK1 and phage P22 (56% identity and 12% query coverage), while the second half of the genome is more similar to the unclassified phage HU1 (87% identity and 28% query coverage). In conclusion, we isolated a novel phage against P. fluorescens , PfAn1, from Lake Baikal water. Many of the putative proteins show sequence relatedness to proteins from a great variety of other phages (Supplementary Fig. S4), supporting the hypothesis that this phage has evolved through the recombinational exchange of genetic information with other viruses. Genomic and proteomic analyses, as well as phage PfAn1 genome organization with known bacteriophages, did not allow us to assign this virus to any taxonomic group of the class Caudoviricetes . Declarations Author contributions All authors contributed to the study conception and design. Experimental work, data collection, and analysis were performed by AG, SP, AK, IT, and LX. This study was conceived and designed by AG, SP, OB, and VD. The first draft of the manuscript was written by AG, and all authors commented on previous versions of the manuscript. All authors have read and approved the final manuscript. Funding This study was supported by the National Government and was carried out within the framework of State Project No. 0279-2021-0015 “Viral and bacterial communities as the basis for the stable functioning of freshwater ecosystems”. Acknowledgments Density gradient ultracentrifugation was carried out on the experimental basis of the Center of Collective Use “Bioanalytic SIFIBR SB RAS”. TEM was performed at the Ultramicroanalysis Research Center at the Limnological Institute Siberian Branch of the Russian Academy of Sciences, Irkutsk. Olga Markina assisted in experiments. Conflict of interest. All authors declare that they have no competing interests. Ethical approval. This research does not contain any studies with human participants or animals performed by any of the authors. References Wu S., Fang Z., Tan J., Li M., Wang C., Guo Q., Xu C., Jiang X., Zhu H. (2021) DeePhage: distinguishing virulent and temperate phage-derived sequences in metavirome data with a deep learning approach. Giga Science 10(9), giab056. https://doi.org/10.1093/gigascience/giab056 Reasoner D. J., Geldreich E. E. (1985) A new medium for the enumeration and subculture of bacteria from potable water. Applied and Environmental Microbiology 49(1). https://doi.org/10.1128/aem.49.1.1-7.1985 Maniatis T. Fritsch E, Sambrook J. (1984) Methods of Genetic Engineering. Molecular Cloning. Moscow: Mir. Shen A., Millard A. (2021) Phage Genome Annotation: Where to Begin and End. PHAGE: Therapy, Applications, and Research 2(4). https://doi.org/10.1089/phage.2021.0015 Andrews S. (2010) FastQC - A quality control tool for high throughput sequence data. Babraham Bioinformatics. http://www.bioinformatics.babraham.ac.uk/projects/fastqc/. Bolger A. M., Lohse M., Usadel B. (2014) Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30(15). https://doi.org/10.1093/bioinformatics/btu170 Prjibelski A., Antipov D., Meleshko D., Lapidus A., Korobeynikov A. (2020) Using SPAdes De Novo Assembler. Current Protocols in Bioinformatics 70(1), e102. https://doi.org/https://doi.org/10.1002/cpbi.102 Langmead B., Salzberg S. L. (2012) Fast gapped-read alignment with Bowtie 2. Nature Methods 9(4). https://doi.org/10.1038/nmeth.1923 Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., Marth G., Abecasis G., Durbin R. (2009). The Sequence Alignment/Map format and SAMtools. Bioinformatics 25(16). https://doi.org/10.1093/bioinformatics/btp352 Wick R. R., Schultz M. B., Zobel J., Holt K. E. (2015) Bandage: Interactive visualization of de novo genome assemblies. Bioinformatics 31(20). https://doi.org/10.1093/bioinformatics/btv383 Nayfach S., Camargo A. P., Schulz F., Eloe-Fadrosh E., Roux S., & Kyrpides N. C. (2021) CheckV assesses the quality and completeness of metagenome-assembled viral genomes. Nature Biotechnology 39(5). https://doi.org/10.1038/s41587-020-00774-7 Garneau J. R., Depardieu F., Fortier L.-C., Bikard D., Monot M. (2017) PhageTerm: a tool for fast and accurate determination of phage termini and packaging mechanism using next-generation sequencing data. Scientific Reports 7(1):8292. https://doi.org/10.1038/s41598-017-07910-5 Hyatt D., Chen G. L., LoCascio P. F., Land M. L., Larimer F. W., Hauser, L. J. (2010) Prodigal: Prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11. https://doi.org/10.1186/1471-2105-11-119 Camargo A. P., Roux S., Schulz F., Babinski M., Xu Y., Hu B., Chain P. S. G., Nayfach S., Kyrpides, N. C. (2024) Identification of mobile genetic elements with geNomad. Nature Biotechnology 42(8). https://doi.org/10.1038/s41587-023-01953-y Buchfink B., Reuter K., Drost HG. (2021) Sensitive protein alignments at tree-of-life scale using DIAMOND. Nat Methods 18(4):366-368. DOI: 10.1038/s41592-021-01101-x Terzian P., Olo Ndela E., Galiez C., Lossouarn J., Pérez Bucio R. E., Mom R., Toussaint A., Petit M. A., Enault F. (2021) PHROG: Families of prokaryotic virus proteins clustered using remote homology. NAR Genomics and Bioinformatics 3(3). https://doi.org/10.1093/nargab/lqab067 Trgovec-Greif L., Hellinger H.-J., Mainguy J., Pfundner A., Frishman D., Kiening M., Webster N.S., Laffy P.W., Feichtinger M., Rattei T. (2024) VOGDB – Database of Virus Orthologous Groups. Viruses 16, 1191. https://doi.org/10.3390/v16081191 Eddy S. R. (2011) Accelerated profile HMM searches. PLoS Computational Biology 7(10). https://doi.org/10.1371/journal.pcbi.1002195 Camargo A. P., Nayfach S., Chen I. M. A., Palaniappan K., Ratner A., Chu K., Ritter S. J., Reddy T. B. K., Mukherjee S., Schulz F., Call L., Neche, R. Y., Woyke T., Ivanova N. N., Eloe-Fadrosh E. A., Kyrpides N. C., Roux S. (2023) IMG/VR v4: an expanded database of uncultivated virus genomes within a framework of extensive functional, taxonomic, and ecological metadata. Nucleic Acids Research 51(D1). https://doi.org/10.1093/nar/gkac1037 Kelley L. A., Mezulis S., Yates C. M., Wass M. N., Sternberg M. J. E. (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols 10(6):845–858. https://doi.org/10.1038/nprot.2015.053 Nishimura Y., Yoshida T., Kuronishi M., Uehara H., Ogata H., Goto S. (2017) ViPTree: The viral proteomic tree server. Bioinformatics 33(15). https://doi.org/10.1093/bioinformatics/btx157 Moraru, C. (2023) VirClust—A Tool for Hierarchical Clustering, Core Protein Detection and Annotation of (Prokaryotic) Viruses. Viruses 15(4). https://doi.org/10.3390/v15041007 Chan P. P., Lin B. Y., Mak A. J., Lowe T. M. (2021). tRNAscan-SE 2.0: improved detection and functional classification of transfer RNA genes. Nucleic Acids Research 49(16):9077–9096. https://doi.org/10.1093/nar/gkab688 Katoh K., Standley D.M. (2013) MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Mol Biol Evol 30:772–780. doi: 10.1093/molbev/mst010. Capella-Gutiérrez S., Silla-Martínez J.M., Gabaldón T. (2009) TrimAl: A Tool for Automated Alignment Trimming in Large-Scale Phylogenetic Analyses. Bioinformatics 25:1972–1973. doi: 10.1093/bioinformatics/btp348 Nguyen L.-T., Schmidt H.A., von Haeseler A., Minh B.Q. (2015) IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. Mol Biol Evol 32:268–274. doi: 10.1093/molbev/msu300 Kalyaanamoorthy S., Minh B.Q., Wong T.K.F., Haeseler A., Jermiin L.S. (2017) ModelFinder: Fast Model Selection for Accurate Phylogenetic Estimates. Nat Methods 14:587–589. doi: 10.1038/nmeth.4285 Guindon S., Dufayard J.-F., Lefort V., Anisimova M., Hordijk W., Gascuel O. (2010) New Algorithms and Methods to Estimate Maximum-Likelihood Phylogenies: Assessing the Performance of PhyML 3.0. Syst. Biol. 59:307–321. doi: 10.1093/sysbio/syq010 Hoang D.T., Chernomor O., von Haeseler A., and Minh B.Q., Vinh L.S. (2018) UFBoot2: Improving the Ultrafast Bootstrap Approximation. Mol Biol Evol 35:18–522. doi: 10.1093/molbev/msx281 Letunic I., Bork P. (2007) Interactive Tree Of Life (ITOL): An Online Tool for Phylogenetic Tree Display and Annotation. Bioinformatics 23:127–128. doi: 10.1093/bioinformatics/btl529 Lehnherr H., Hansen A.-M., Ilyina T. (1998) Penetration of the bacterial cell wall: a family of lytic transglycosylases in bacteriophages and conjugative plasmids. Molecular Microbiology 30(2):454–457. https://doi.org/https://doi.org/10.1046/j.1365-2958.1998.01069.x Trotereau A., Boyer C., Bornard I., Pécheur M. J. B., Schouler C., Torres-Barceló C. (2021) High genomic diversity of novel phages infecting the plant pathogen Ralstonia solanacearum , isolated in Mauritius and Reunion islands. Scientific Reports 11(1):5382. https://doi.org/10.1038/s41598-021-84305-7 Lee I., Ouk Kim Y., Park S.C., Chun J. (2016) OrthoANI: An improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol 66(2):1100-1103. doi: 10.1099/ijsem.0.000760. Tanaka C., Nakayama T., Toba T., Kashiwagi A. (2020) Complete genomic sequence of Pseudomonas lactis bacteriophage HU1 isolated from raw cow’s milk. Archives of Virology 165(1):215–217. https://doi.org/10.1007/s00705-019-04423-6 Nishimura Y., Yamada K., Okazaki Y., Ogata H. (2024) DiGAlign: Versatile and Interactive Visualization of Sequence Alignment for Comparative Genomics. Microbes and Environments 39(1). https://doi.org/10.1264/jsme2.ME23061 Supplementary Files SupplementaryFigures.docx TableS1.xlsx PfAn1completegenome.fasta Cite Share Download PDF Status: Published Journal Publication published 16 May, 2025 Read the published version in Archives of Virology → Version 1 posted Editorial decision: Accept 26 Apr, 2025 Reviewers agreed at journal 31 Mar, 2025 Reviewers invited by journal 31 Mar, 2025 Editor assigned by journal 29 Mar, 2025 First submitted to journal 27 Mar, 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. 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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-5526281","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":436198254,"identity":"29a074ce-8c31-419a-b677-6668729f17e4","order_by":0,"name":"Anna Gorshkova","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYBACCRDBU8AgB+cQqcWAwZh0LYkNRGuRbO8x/PDGwCZ9w/GzB298bGPINzhAQIs0zxljyTkGabkbzuQlW844w2C5gZAWOYm0BGkeg8O5Gw7kmEnzVDAYELQFqCX5N1BLusH5N2bSfwyI0CItkXwMZEuCwQ2gLQzE2CLZc/iYJdAvhjNvvDG27DkjYSBJSIvE8cbmG28qbOT5zucY3vjZZmPAR0gLHChAVBIXNRAg30CC4lEwCkbBKBhZAADt0T5++6PeGQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-2408-0837","institution":"Limnological Institute of Siberian Branch of Russian Academy of Sciences: FGBUN Limnologiceskij institut Sibirskogo otdelenia Rossijskoj akademii nauk","correspondingAuthor":true,"prefix":"","firstName":"Anna","middleName":"","lastName":"Gorshkova","suffix":""},{"id":436198255,"identity":"fcc31add-b18c-4172-9d22-74493acace71","order_by":1,"name":"Olga Belykh","email":"","orcid":"","institution":"Limnological Institute of Siberian Branch of Russian Academy of Sciences: FGBUN Limnologiceskij institut Sibirskogo otdelenia Rossijskoj akademii nauk","correspondingAuthor":false,"prefix":"","firstName":"Olga","middleName":"","lastName":"Belykh","suffix":""},{"id":436198256,"identity":"5e474cfd-e305-40a9-b135-1b445cc6482e","order_by":2,"name":"Irina Tikhonova","email":"","orcid":"","institution":"Limnological Institute of Siberian Branch of Russian Academy of Sciences: FGBUN Limnologiceskij institut Sibirskogo otdelenia Rossijskoj akademii nauk","correspondingAuthor":false,"prefix":"","firstName":"Irina","middleName":"","lastName":"Tikhonova","suffix":""},{"id":436198257,"identity":"a9b70e35-51e9-4254-b84c-f3af0cfaaa69","order_by":3,"name":"Li Xi","email":"","orcid":"","institution":"Institute of Urban Environment Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Xi","suffix":""},{"id":436198258,"identity":"815c1bcc-dd3f-468e-af8e-1c7c9d18e1f8","order_by":4,"name":"Maria Siniagina","email":"","orcid":"","institution":"Kazan Federal University: Kazanskij Privolzskij federal'nyj universitet","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Siniagina","suffix":""},{"id":436198259,"identity":"61527bc8-bec1-4209-a670-5d11b67bfd3f","order_by":5,"name":"Valentin Drucker","email":"","orcid":"","institution":"Limnological Institute of Siberian Branch of Russian Academy of Sciences: FGBUN Limnologiceskij institut Sibirskogo otdelenia Rossijskoj akademii nauk","correspondingAuthor":false,"prefix":"","firstName":"Valentin","middleName":"","lastName":"Drucker","suffix":""},{"id":436198260,"identity":"579e4b04-51d9-4e0e-99aa-de5830d260c7","order_by":6,"name":"Sergey Potapov","email":"","orcid":"","institution":"Limnological Institute SB RAS: FGBUN Limnologiceskij institut Sibirskogo otdelenia Rossijskoj akademii nauk","correspondingAuthor":false,"prefix":"","firstName":"Sergey","middleName":"","lastName":"Potapov","suffix":""}],"badges":[],"createdAt":"2024-11-26 09:15:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5526281/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5526281/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00705-025-06315-4","type":"published","date":"2025-05-16T15:57:45+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79667635,"identity":"12a4f25e-8c8a-439e-bcdd-244a0ff2ca19","added_by":"auto","created_at":"2025-04-01 10:35:16","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":430958,"visible":true,"origin":"","legend":"\u003cp\u003eA, B) Negative staining transmission electron microscopic images of phages after density gradient ultracentrifugation. The scale bar corresponds to 100 nm. A) Black arrow indicates bacteriophage band.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5526281/v1/2a68c64477ea24dd6657efd9.jpg"},{"id":79667636,"identity":"2b4eb58c-59e7-4de6-a1ef-dc81d7ba3362","added_by":"auto","created_at":"2025-04-01 10:35:16","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":995555,"visible":true,"origin":"","legend":"\u003cp\u003eGenome organization of PfAn1 phage. Arrows indicate transcriptional orientation of genes. Arrows colored according to the legend indicate selected predicted protein functions. Numbers on arrows refer to the ORF number in Table S1. Circular permutation of the PfAn1 genome is shown.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5526281/v1/a32cba0ea4ccefb0e30ed368.jpg"},{"id":79667196,"identity":"3f6631b0-f4a9-451a-bf0a-39836c3e3784","added_by":"auto","created_at":"2025-04-01 10:27:16","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1315775,"visible":true,"origin":"","legend":"\u003cp\u003eComparative analysis between the PfAn1 genome with those of related phage belonging to the genus \u003cem\u003eFiringavirus\u003c/em\u003e (\u003cem\u003eRalstonia\u003c/em\u003ephage RSK1), phage belonging to the genus \u003cem\u003eLederbergvirus\u003c/em\u003e(\u003cem\u003eSalmonella\u003c/em\u003e phage P22) and the unclassified phage (\u003cem\u003ePseudomonas\u003c/em\u003e phage HU1).\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5526281/v1/d948064ade2110fbdb179a3d.jpg"},{"id":83067815,"identity":"107435c2-1b53-4558-95d1-db40dad60b4f","added_by":"auto","created_at":"2025-05-19 16:06:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3086315,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5526281/v1/5671c482-f38d-4af2-b741-a112802ce5af.pdf"},{"id":79667198,"identity":"5f184b7e-06fc-426d-b0da-7723411db680","added_by":"auto","created_at":"2025-04-01 10:27:16","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3260028,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-5526281/v1/683242b6dffc4cb8db7801d4.docx"},{"id":79667200,"identity":"6855f4b7-8afa-46e8-9b3c-8d2ab50b789a","added_by":"auto","created_at":"2025-04-01 10:27:16","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":24071,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5526281/v1/db91f12adf250ec4724a5726.xlsx"},{"id":79667637,"identity":"f522bb30-2c00-435f-89cf-6efea6b7742c","added_by":"auto","created_at":"2025-04-01 10:35:17","extension":"fasta","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":39192,"visible":true,"origin":"","legend":"","description":"","filename":"PfAn1completegenome.fasta","url":"https://assets-eu.researchsquare.com/files/rs-5526281/v1/a4221273085be0afc42fde79.fasta"}],"financialInterests":"","formattedTitle":"Genomic characterization of the novel bacteriophage PfAn1 from Lake Baikal against Pseudomonas fluorescens","fulltext":[{"header":"Full Text","content":"\u003cp\u003eWith the development of high-throughput sequencing technology, numerous novel phages have been discovered from metagenomes and viromes. However, the analysis of these phage sequences is a great challenge because the reference genomes of phages are very limited due to the fact that most phages cannot be cultured independently \u003cspan lang=\"EN-US\"\u003e[\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e1\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e]\u003c/span\u003e. Here, we isolated a novel \u003cem\u003ePseudomonas fluorescens\u0026nbsp;\u003c/em\u003ephage, PfAn1, from the water of Lake Baikal (Russia). Evolutionary and phylogenetic analysis suggested that phage PfAn1 should be considered a new member of the class \u003cem\u003eCaudoviricetes\u003c/em\u003e. Bacteriophage PfAn1 was isolated from liquid enrichment culture obtained by adding a concentrated R2A medium\u0026nbsp;\u003cspan lang=\"EN-US\"\u003e[\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e2\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e]\u003c/span\u003e to a natural sample.\u0026nbsp;The\u0026nbsp;enrichment was based on the hypothesis that actively multiplying bacteria would trigger the accumulation of viruses, according to the \u0026ldquo;kill the winner\u0026rdquo; theory. Strains of bacteria and bacteriophages were simultaneously isolated from the enrichment cultures. PfAn1 was enriched from a sample taken at the center of the Maloye More Strait at a depth of 5 m. The host strain was isolated from a water sample collected at the depth of 5 m from the central site of the Ukhan Cape-Tonky Cape transect in the central basin of Lake Baikal. The enrichment culture was centrifuged at 8,000 \u003cem\u003eg\u0026nbsp;\u003c/em\u003efor 10 min, and the supernatant was filtered through a 0.22\u0026nbsp;\u0026mu;m filter. Then, the supernatant was tested for specific phages by the double-layer agar method. It resulted in lysis on cells that were identified as \u003cem\u003ePseudomonas fluorescens\u0026nbsp;\u003c/em\u003eby 16S sequencing. Phages were concentrated by PEG precipitation. The phages were further purified by CsCl density gradient centrifugation (100,000 \u003cem\u003e\u0026times; g\u003c/em\u003e, 2 h) and dialyzed against SM buffer (100 mM NaCl, 10 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 50 mM Tris-HCl; pH 7.5). Purified phages were used for further experiments. For electron microscopy, a 20 uL aliquot of phage suspension was placed on a formvar-coated copper grid for 15 min and then dried using filter paper. The preparation was then stained with 2% (w/v) uranyl acetate for 15 min. Finally, phage morphology was examined using a LEO906E transmission electron microscope (Carl Zeiss, Germany).\u0026nbsp;Genomic DNA was extracted using the phenol-chloroform protocol \u003cspan lang=\"EN-US\"\u003e[\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e3\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e]\u003c/span\u003e. Genome sequencing was performed using a FASTAseq 300 (GeneMind, China) in Kazan Federal University.\u003c/p\u003e\n\u003cp\u003eThe phage genome assembly was carried out according to the recommendations of A. Shen and A. Millard\u0026nbsp;\u003cspan lang=\"EN-US\"\u003e[\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e4\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e]\u003c/span\u003e. The raw reads were analyzed in FastQC v. 0.12.1\u0026nbsp;\u003cspan lang=\"EN-US\"\u003e[\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e5\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e]\u003c/span\u003e. Then reads were filtered to remove low-quality reads and adapter regions, using Trimmomatic v. 0.36\u0026nbsp;\u003cspan lang=\"EN-US\"\u003e[\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e6\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e]\u003c/span\u003e. The genome sequence was de novo assembled using the SPAdes v. 4.0.0 software\u0026nbsp;\u003cspan lang=\"EN-US\"\u003e[\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e7\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e]\u003c/span\u003e. The coverage depth was calculated using Bowtie2 v. 2.4.4\u0026nbsp;\u003cspan lang=\"EN-US\"\u003e[\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e8\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e]\u003c/span\u003e and SAMtools v. 1.13\u0026nbsp;\u003cspan lang=\"EN-US\"\u003e[\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e9\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e]\u003c/span\u003e. The assembled contig was checked via Bandage v. 0.8.1\u0026nbsp;\u003cspan lang=\"EN-US\"\u003e[\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e10\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e]\u003c/span\u003e. Identification, quality assessment, and completeness of the virus genome were estimated with CheckV v. 1.0.3\u0026nbsp;\u003cspan lang=\"EN-US\"\u003e[\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e11\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e]\u003c/span\u003e. Genome termini were analyzed through inspection using read mapping and PhageTerm\u0026nbsp;\u003cspan lang=\"EN-US\"\u003e[\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e12\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e]\u003c/span\u003e. Open reading frames were determined with Prodigal v. 2.6.3\u0026nbsp;\u003cspan lang=\"EN-US\"\u003e[\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e13\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e]\u003c/span\u003e. Taxonomic identification of viral genome was performed using geNomad v. 1.8.0\u0026nbsp;\u003cspan lang=\"EN-US\"\u003e[\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e14\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e]\u003c/span\u003e, Diamond v. 2.1.8.162 [15]\u0026nbsp;with e-value parameters (10\u003csup\u003e-5\u003c/sup\u003e, bit score \u0026ge; 50, more sensitive) and BLASTn v. 2.12.0+ (e-value 10\u003csup\u003e-5\u003c/sup\u003e) using the amino acid and nucleotide database of RefSeq v. 226. Functional analysis of translated ORFs was performed applying PHROG v.4\u0026nbsp;\u003cspan lang=\"EN-US\"\u003e[\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e16\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e]\u003c/span\u003e, the Virus Orthologous Groups Database (VOGDB) v. 219 [17] and HHMER v. 3.2.1\u0026nbsp;\u003cspan lang=\"EN-US\"\u003e[\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e18\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e]\u003c/span\u003e. The\u0026nbsp;IMG/VR v.4 database was used to identify uncultivated relatives\u0026nbsp;\u003cspan lang=\"EN-US\"\u003e[\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e19\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e]\u003c/span\u003e, and Phyre2 tool was used for predicting and analyzing protein structure and function [20]. VipTree v. 4.0\u0026nbsp;\u003cspan lang=\"EN-US\"\u003e[\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e21\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e]\u003c/span\u003e and\u0026nbsp;VirClust v. 2.0\u0026nbsp;\u003cspan lang=\"EN-US\"\u003e[\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e22\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e]\u003c/span\u003e were used for comparative genomic analysis. tRNAs were searched using tRNAscan-SE 2.0\u0026nbsp;\u003cspan lang=\"EN-US\"\u003e[\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e23\u003c/span\u003e\u003cspan lang=\"EN-US\"\u003e].\u003c/span\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the phylogenetic tree based on the terminase large subunit proteins, the sequences were aligned using the MAFFT v. 7.407 [24] program with the -L-INS-i parameter. TrimAl v. 1.2 (-gappyout) [25] was used to remove ambiguous regions. Trees were computed with IQ-TREE software v. 1.6.9 [26]; model was selected with ModelFinder [27], and branch supports were determined using the approximate likelihood ratio test (1000 repetitions) [28] and the ultrafast bootstrap (1000 repetitions) [29]. The resulting trees were visualized and edited in iTOL [30].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePhage PfAn1 formed small plaques that became visible six days after the cell lawn has already grown (Supplementary Fig. S1). Transmission electron microscopy revealed that this phage possesses a head ~50 nm in diameter and a short tail (Fig. 1) and, thus, exhibits \u0026quot;podovirus\u0026quot; morphology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePhage PfAn1 has a double-stranded DNA genome of 39,156 bp with an average GC content of 57%, which is similar to that of its host DNA (60%). Its genome contains 53 putative protein-coding\u0026nbsp;genes. \u0026nbsp;One tRNA gene was detected in the PfAn1 genome.\u0026nbsp;Only 30 PfAn1 gene products exhibit similarity to proteins of known function from diverse organisms (Fig. 2). For proteins having related proteins, the average coverage was 87%, and the average identity was 44% (26% to 66%) compared to proteins from the RefSeq, VOG, PHROG, and Uniref90 databases. Proteins of uncultivated bacteriophages from the IMG/VR database accounted for an average of 55% identity (32% to 90%) (Supplementary Table S1). We predicted the function of two proteins only based on tertiary structure because the function of proteins with similar amino acid composition is unknown. These are OFR-10 gene product presumably encoding the tail fiber protein and OFR-13 gene product encoding an oxidoreductase enzyme.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe identified assembly genes include a small, large terminase and portal protein. Virion particle genes include a portal protein, a major capsid protein, a scaffolding protein, a head-tail adaptor protein, a head-closure protein, and three tail proteins, including a major tail protein, tail protein and putative tail fibers. The proteins encoded by ORF-15 and ORF-16 likely encode internal virion proteins involved in DNA ejection. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe proteins associated with lysis of the host cell include the following gene cassette: endolysin, spanin and holin. Moreover, we identified a similarity of ORF-17 with peptidoglycan transglycosylase. ORF-17 is located in the morphogenic module. A recent study indicates that lytic transglycosylases are morphological components of the phage particle, which contact with the bacterial cell wall from the outside and might facilitate the passage of the viral genome through the cell wall by introducing small, localized gaps into the peptidoglycan layer [31].\u003c/p\u003e\n\u003cp\u003eWe identified a gene encoding a repressor protein homologous to the phage \u0026lambda; C1 repressor that allows virus to establish and maintain latency. Phyre2 modeled gene product of ORF-38 with bacteriophage \u0026lambda; repressor-like DNA-binding domains with 95.2% confidence and 82% coverage. However, we did not identify other proteins involved in the integration and excision of viral DNA characteristic of temperate \u0026lambda;-like phages like an integrase.\u003c/p\u003e\n\u003cp\u003eThe module of genes responsible for DNA metabolism and regulation of transcription and translation contained proteins homologous to various nucleases that are involved in numerous nucleic acid cleavage events (ORF-25, ORF-30, ORF-31, and ORF-52), to transcription and translation repressor proteins (ORF-27, ORF-38 and ORF-44) and DNA recombination enzymes (ORF-29 and OFR-49).\u003c/p\u003e\n\u003cp\u003eBLASTn analysis revealed that the genome of phage PfAn1 showed the highest similarity at DNA level to \u003cem\u003eRalstonia\u003c/em\u003e phage Firinga (NC_054961) and \u003cem\u003eRalstonia\u0026nbsp;\u003c/em\u003ephage RSK1 (NC_022915) (68% identity and 9% query coverage). In bacterial genomes, we also detected short fragments similar to the PfAn1 genome, which amounted to \u0026lt;10% of the phage genome. The proteome analysis was performed using the genome sequence of phage PfAn1 and those of 54778 \u0026nbsp;other phages, followed by the construction of a proteome tree. Phage PfAn1 formed a separate branch in the proteome tree with \u003cem\u003eRalstonia\u003c/em\u003e phages from the genus \u003cem\u003eFiringavirus\u003c/em\u003e. \u003cem\u003eRalstonia\u003c/em\u003e phage Firinga infecting the plant pathogen \u003cem\u003eR. solanacearum\u003c/em\u003e has a podovirus morphology\u003cem\u003e\u0026nbsp;\u003c/em\u003e[32]. Bacteriophage PfAn1 is similar to these phages, mainly in structural proteins; other proteins did not show a significant similarity (Fig.3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs \u003cem\u003ePseudomonas\u003c/em\u003e is the host genus of phage\u0026nbsp;PfAn1; therefore, we attempted to search for related phages among viruses known for this group. \u0026nbsp;The OrthoANIu [33] values were obtained by comparing phage PfAn1 with 1375 other \u003cem\u003ePseudomonas\u003c/em\u003e phage genomes, deposited in the GenBank database. Only eight bacteriophages had the OrthoANIu values \u0026gt; 0 (Supplementary Fig. S2). Bacteriophage PfAn1 formed a cluster with lytic cold-active phage HU1 infecting \u003cem\u003eP.\u003c/em\u003e \u003cem\u003elactis\u0026nbsp;\u003c/em\u003e[34]. Other phage infect \u003cem\u003eP. aeruginosa\u0026nbsp;\u003c/em\u003eand, like bacteriophage HU1, belong to unclassified \u003cem\u003eCaudoviricetes\u003c/em\u003e. The OrthoANIu values were obtained by comparing phage PfAn1 with 5153 \u003cem\u003eCaudoviricetes\u003c/em\u003e phages genomes, deposited in the RefSeq database. \u0026nbsp; Only twenty-one bacteriophages had the OrthoANIu values \u0026gt; 0. The Firinga, phi297, and YMC11/07/P54_PAE_BP phages showed highest similarity with phage PfAn1. \u0026nbsp;Combining the results of the search in both databases, we can conclude that the highest nucleotide similarity to phage PfAn1 was found for unclassified HU1 and phi297-like phages, as well as phages of the genus \u003cem\u003eFiringavirus\u003c/em\u003e. Notably, the intergenomic similarity of PfAn1 and the OrthoANIu selected phages was very low.\u003c/p\u003e\n\u003cp\u003eThe gene for the terminase large subunit, an enzyme that packages DNA into a capsid, is one of the most conservative genes. As shown in Supplementary Figure S3, phylogenetic analysis was performed based on multiple alignments of the amino acid sequences of the DNA terminase large subunit. In the terminase large subunit-based tree, it was most closely related to phages Firinga and RKS1 belonging to the genus \u003cem\u003eFiringavirus\u003c/em\u003e. Unclassified \u003cem\u003ePseudomonas\u003c/em\u003e phages and P22-like bacteriophages also appeared to be closely related phages. Phyre2 modeled the protein structure of the PfAn1 terminase large subunit that turned out to be the most similar to large terminase bacteriophage P22, with 100% confidence and 60% identity. Interestingly, the DNA packaging strategy is well studied for the temperate transducing phage P22, which, apparently, is also inherent in bacteriophage PfAn1. Thus, we can assume that phage PfAn1 can be classified as a P22-like phages based on the similarity of the structural module of the genome. However, the PfAn1 genome did not show homologous genes typical of phages of this group, which are responsible for the integration into the host genome. Fig. 3 demonstrates the intergenomic alignments of phages PfAn1, RSK1, P22, and HU1, which were performed using DiGAlign [35]. As shown in this figure, phage PfAn1 has some similarity in the structural module of the genome with phage RSK1 and phage P22 (56% identity and 12% query coverage), while the second half of the genome is more similar to the unclassified phage HU1 (87% identity and 28% query coverage).\u003c/p\u003e\n\u003cp\u003eIn conclusion, we isolated a novel phage against\u003cem\u003e\u0026nbsp;P. fluorescens\u003c/em\u003e, PfAn1, from Lake Baikal water. Many of the putative proteins show sequence relatedness to proteins from a great variety of other phages (Supplementary Fig. S4), supporting the hypothesis that this phage has evolved through the recombinational exchange of genetic information with other viruses. Genomic and proteomic analyses, as well as phage PfAn1 genome organization with known bacteriophages, did not allow us to assign this virus to any taxonomic group of the class \u003cem\u003eCaudoviricetes\u003c/em\u003e.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e All authors contributed to the study conception and design. Experimental work, data collection, and analysis were performed by AG, SP, AK, IT, and LX. This study was conceived and designed by AG, SP, OB, and VD. The first draft of the manuscript was written by AG, and all authors commented on previous versions of the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This study was supported by the National Government and was carried out within the framework of State Project No. 0279-2021-0015 \u0026ldquo;Viral and bacterial communities as the basis for the stable functioning of freshwater ecosystems\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e Density gradient ultracentrifugation was carried out on the experimental basis of the Center of Collective Use \u0026ldquo;Bioanalytic SIFIBR SB RAS\u0026rdquo;. TEM was performed at the Ultramicroanalysis Research Center at the Limnological Institute Siberian Branch of the Russian Academy of Sciences, Irkutsk. Olga Markina assisted in experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003c/strong\u003eConflict of interest. All authors declare that they have no competing interests. Ethical approval. This research does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWu S., Fang Z., Tan J., Li M., Wang C., Guo Q., Xu C., Jiang X., Zhu H. (2021) DeePhage: distinguishing virulent and temperate phage-derived sequences in metavirome data with a deep learning approach. Giga Science 10(9), giab056. https://doi.org/10.1093/gigascience/giab056\u003c/li\u003e\n\u003cli\u003eReasoner D. J., Geldreich E. E. (1985) A new medium for the enumeration and subculture of bacteria from potable water. Applied and Environmental Microbiology 49(1). https://doi.org/10.1128/aem.49.1.1-7.1985\u003c/li\u003e\n\u003cli\u003eManiatis T. Fritsch E, Sambrook J. (1984) Methods of Genetic Engineering. Molecular Cloning. Moscow: Mir.\u003c/li\u003e\n\u003cli\u003eShen A., Millard A. (2021) Phage Genome Annotation: Where to Begin and End. PHAGE: Therapy, Applications, and Research 2(4). https://doi.org/10.1089/phage.2021.0015\u003c/li\u003e\n\u003cli\u003eAndrews S. (2010) FastQC - A quality control tool for high throughput sequence data. Babraham Bioinformatics. http://www.bioinformatics.babraham.ac.uk/projects/fastqc/. \u003c/li\u003e\n\u003cli\u003eBolger A. M., Lohse M., Usadel B. (2014) Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30(15). https://doi.org/10.1093/bioinformatics/btu170\u003c/li\u003e\n\u003cli\u003ePrjibelski A., Antipov D., Meleshko D., Lapidus A., Korobeynikov A. (2020) Using SPAdes De Novo Assembler. Current Protocols in Bioinformatics 70(1), e102. https://doi.org/https://doi.org/10.1002/cpbi.102\u003c/li\u003e\n\u003cli\u003eLangmead B., Salzberg S. L. (2012) Fast gapped-read alignment with Bowtie 2. Nature Methods 9(4). https://doi.org/10.1038/nmeth.1923\u003c/li\u003e\n\u003cli\u003eLi H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., Marth G., Abecasis G., Durbin R. (2009). The Sequence Alignment/Map format and SAMtools. Bioinformatics 25(16). https://doi.org/10.1093/bioinformatics/btp352\u003c/li\u003e\n\u003cli\u003eWick R. R., Schultz M. B., Zobel J., Holt K. E. (2015) Bandage: Interactive visualization of de novo genome assemblies. Bioinformatics 31(20). https://doi.org/10.1093/bioinformatics/btv383\u003c/li\u003e\n\u003cli\u003eNayfach S., Camargo A. P., Schulz F., Eloe-Fadrosh E., Roux S., \u0026amp; Kyrpides N. C. (2021) CheckV assesses the quality and completeness of metagenome-assembled viral genomes. Nature Biotechnology 39(5). https://doi.org/10.1038/s41587-020-00774-7\u003c/li\u003e\n\u003cli\u003eGarneau J. R., Depardieu F., Fortier L.-C., Bikard D., Monot M. (2017) PhageTerm: a tool for fast and accurate determination of phage termini and packaging mechanism using next-generation sequencing data. Scientific Reports 7(1):8292. https://doi.org/10.1038/s41598-017-07910-5\u003c/li\u003e\n\u003cli\u003eHyatt D., Chen G. L., LoCascio P. F., Land M. L., Larimer F. W., Hauser, L. J. (2010) Prodigal: Prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11. https://doi.org/10.1186/1471-2105-11-119\u003c/li\u003e\n\u003cli\u003eCamargo A. P., Roux S., Schulz F., Babinski M., Xu Y., Hu B., Chain P. S. G., Nayfach S., Kyrpides, N. C. (2024) Identification of mobile genetic elements with geNomad. Nature Biotechnology 42(8). https://doi.org/10.1038/s41587-023-01953-y\u003c/li\u003e\n\u003cli\u003eBuchfink B., Reuter K., Drost HG. (2021) Sensitive protein alignments at tree-of-life scale using DIAMOND. Nat Methods 18(4):366-368. DOI: 10.1038/s41592-021-01101-x\u003c/li\u003e\n\u003cli\u003eTerzian P., Olo Ndela E., Galiez C., Lossouarn J., P\u0026eacute;rez Bucio R. E., Mom R., Toussaint A., Petit M. A., Enault F. (2021) PHROG: Families of prokaryotic virus proteins clustered using remote homology. NAR Genomics and Bioinformatics 3(3). https://doi.org/10.1093/nargab/lqab067\u003c/li\u003e\n\u003cli\u003eTrgovec-Greif L., Hellinger H.-J., Mainguy J., Pfundner A., Frishman D., Kiening M., Webster N.S., Laffy P.W., Feichtinger M., Rattei T. (2024) VOGDB \u0026ndash; Database of Virus Orthologous Groups. Viruses \u003cem\u003e \u003c/em\u003e16, 1191. https://doi.org/10.3390/v16081191 \u003c/li\u003e\n\u003cli\u003eEddy S. R. (2011) Accelerated profile HMM searches. PLoS Computational Biology 7(10). https://doi.org/10.1371/journal.pcbi.1002195\u003c/li\u003e\n\u003cli\u003eCamargo A. P., Nayfach S., Chen I. M. A., Palaniappan K., Ratner A., Chu K., Ritter S. J., Reddy T. B. K., Mukherjee S., Schulz F., Call L., Neche, R. Y., Woyke T., Ivanova N. N., Eloe-Fadrosh E. A., Kyrpides N. C., Roux S. (2023) IMG/VR v4: an expanded database of uncultivated virus genomes within a framework of extensive functional, taxonomic, and ecological metadata. Nucleic Acids Research 51(D1). https://doi.org/10.1093/nar/gkac1037\u003c/li\u003e\n\u003cli\u003eKelley L. A., Mezulis S., Yates C. M., Wass M. N., Sternberg M. J. E. (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols 10(6):845\u0026ndash;858. https://doi.org/10.1038/nprot.2015.053\u003c/li\u003e\n\u003cli\u003eNishimura Y., Yoshida T., Kuronishi M., Uehara H., Ogata H., Goto S. (2017) ViPTree: The viral proteomic tree server. Bioinformatics 33(15). https://doi.org/10.1093/bioinformatics/btx157\u003c/li\u003e\n\u003cli\u003eMoraru, C. (2023) VirClust\u0026mdash;A Tool for Hierarchical Clustering, Core Protein Detection and Annotation of (Prokaryotic) Viruses. Viruses 15(4). https://doi.org/10.3390/v15041007\u003c/li\u003e\n\u003cli\u003eChan P. P., Lin B. Y., Mak A. J., Lowe T. M. (2021). tRNAscan-SE 2.0: improved detection and functional classification of transfer RNA genes. Nucleic Acids Research 49(16):9077\u0026ndash;9096. https://doi.org/10.1093/nar/gkab688\u003c/li\u003e\n\u003cli\u003eKatoh K., Standley D.M. (2013) MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Mol Biol Evol 30:772\u0026ndash;780. doi: 10.1093/molbev/mst010.\u003c/li\u003e\n\u003cli\u003eCapella-Guti\u0026eacute;rrez S., Silla-Mart\u0026iacute;nez J.M., Gabald\u0026oacute;n T. (2009) TrimAl: A Tool for Automated Alignment Trimming in Large-Scale Phylogenetic Analyses. Bioinformatics 25:1972\u0026ndash;1973. doi: 10.1093/bioinformatics/btp348\u003c/li\u003e\n\u003cli\u003eNguyen L.-T., Schmidt H.A., von Haeseler A., Minh B.Q. (2015) IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. Mol Biol Evol 32:268\u0026ndash;274. doi: 10.1093/molbev/msu300\u003c/li\u003e\n\u003cli\u003eKalyaanamoorthy S., Minh B.Q., Wong T.K.F., Haeseler A., Jermiin L.S. (2017) ModelFinder: Fast Model Selection for Accurate Phylogenetic Estimates. Nat Methods 14:587\u0026ndash;589. doi: 10.1038/nmeth.4285\u003c/li\u003e\n\u003cli\u003eGuindon S., Dufayard J.-F., Lefort V., Anisimova M., Hordijk W., Gascuel O. (2010) New Algorithms and Methods to Estimate Maximum-Likelihood Phylogenies: Assessing the Performance of PhyML 3.0. Syst. Biol. 59:307\u0026ndash;321. doi: 10.1093/sysbio/syq010\u003c/li\u003e\n\u003cli\u003eHoang D.T., Chernomor O., von Haeseler A., and Minh B.Q., Vinh L.S. (2018) UFBoot2: Improving the Ultrafast Bootstrap Approximation. Mol Biol Evol 35:18\u0026ndash;522. doi: 10.1093/molbev/msx281\u003c/li\u003e\n\u003cli\u003eLetunic I., Bork P. (2007) Interactive Tree Of Life (ITOL): An Online Tool for Phylogenetic Tree Display and Annotation. Bioinformatics 23:127\u0026ndash;128. doi: 10.1093/bioinformatics/btl529\u003c/li\u003e\n\u003cli\u003eLehnherr H., Hansen A.-M., Ilyina T. (1998) Penetration of the bacterial cell wall: a family of lytic transglycosylases in bacteriophages and conjugative plasmids. Molecular Microbiology 30(2):454\u0026ndash;457. https://doi.org/https://doi.org/10.1046/j.1365-2958.1998.01069.x\u003c/li\u003e\n\u003cli\u003eTrotereau A., Boyer C., Bornard I., P\u0026eacute;cheur M. J. B., Schouler C., Torres-Barcel\u0026oacute; C. (2021) High genomic diversity of novel phages infecting the plant pathogen \u003cem\u003eRalstonia solanacearum\u003c/em\u003e, isolated in Mauritius and Reunion islands. Scientific Reports 11(1):5382. https://doi.org/10.1038/s41598-021-84305-7\u003c/li\u003e\n\u003cli\u003eLee I., Ouk Kim Y., Park S.C., Chun J. (2016) OrthoANI: An improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol 66(2):1100-1103. doi: 10.1099/ijsem.0.000760.\u003c/li\u003e\n\u003cli\u003eTanaka C., Nakayama T., Toba T., Kashiwagi A. (2020) Complete genomic sequence of \u003cem\u003ePseudomonas\u003c/em\u003e lactis bacteriophage HU1 isolated from raw cow\u0026rsquo;s milk. Archives of Virology 165(1):215\u0026ndash;217. https://doi.org/10.1007/s00705-019-04423-6\u003c/li\u003e\n\u003cli\u003eNishimura Y., Yamada K., Okazaki Y., Ogata H. (2024) DiGAlign: Versatile and Interactive Visualization of Sequence Alignment for Comparative Genomics. Microbes and Environments 39(1). https://doi.org/10.1264/jsme2.ME23061\u003c/li\u003e\n\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":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5526281/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5526281/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe isolated a novel bacteriophage from Lake Baikal. Transmission electron microscopy revealed that phage PfAn1 has a head with a diameter of 50 nm and a short tail. Its genome is 39,156 bp in length with a GC content of 57%. It is predicted to contain 53 open reading frames (ORFs). The results of evolutionary analysis suggest that phage PfAn1 should be considered a new member of the class \u003cem\u003eCaudoviricetes\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Genomic characterization of the novel bacteriophage PfAn1 from Lake Baikal against Pseudomonas fluorescens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-01 10:27:12","doi":"10.21203/rs.3.rs-5526281/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2025-04-26T09:56:24+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-03-31T08:29:31+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-31T08:22:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-29T06:29:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Virology","date":"2025-03-28T03:37:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"dbb752d6-7293-4112-9eea-84be3353bb60","owner":[],"postedDate":"April 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-19T16:01:32+00:00","versionOfRecord":{"articleIdentity":"rs-5526281","link":"https://doi.org/10.1007/s00705-025-06315-4","journal":{"identity":"archives-of-virology","isVorOnly":false,"title":"Archives of Virology"},"publishedOn":"2025-05-16 15:57:45","publishedOnDateReadable":"May 16th, 2025"},"versionCreatedAt":"2025-04-01 10:27:12","video":"","vorDoi":"10.1007/s00705-025-06315-4","vorDoiUrl":"https://doi.org/10.1007/s00705-025-06315-4","workflowStages":[]},"version":"v1","identity":"rs-5526281","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5526281","identity":"rs-5526281","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}