Distinct ecophysiological characteristics of two genetically similar vibriophages

Abstract

Vibrio parahaemolyticus is a marine bacterium and a causative agent of gastroenteritis in humans. Phages targeting V. parahaemolyticus serve as natural antagonists to this pathogen, significantly controlling its population and, consequently, disease outbreaks. In this study, two myoviruses, vB_VpaM_R20L (R20L) and vB_VpaM_R19R (R19R), were isolated and characterized against V. parahaemolyticus ATCC 17802 ^T. Despite their genomic resemblance, phages R19R and R20L displayed notable differences in physiological characteristics, particularly in infection dynamics and thermal stability. Both phages had a latent period of approximately 10 min; however, R19R demonstrated significantly greater replication efficiency, with a burst size of 388 PFU/cell compared to 90 PFU/cell for R20L. Thermal stability assays showed that R20L maintained survival rates of 80%–100% at 5 °C–45 °C for over three days, while R19R’s viability declined to 50% within 12 h under the same conditions. Furthermore, molecular dynamics simulations identified the influence of temperature on the thermal stability of phages, primarily through impact on the structural proteins. These findings suggest that subtle genomic variations may drive differences in physiological characteristics, highlighting the complexity of vibriophage-host interactions and their selection in response to natural environmental pressures.

Keywords

Vibrio parahaemolyticus / Vibriophage / Ecophysiological characteristics / Genomic comparison

References

[1] Abedon ST. Harper DR, Abedon ST, Burrowes BH, McConville ML. Detection of bacteriophages: phage plaques. Bacteriophages: Biology, 2021, Technology, Therapy. Springer International Publishing507538

[2] Abedon ST, Yin J (2009) Bacteriophage plaques: theory and analysis. In Clokie MRJ, Kropinski AM (Eds.), Bacteriophages: Methods and Protocols, Volume 1: Isolation, Characterization, and Interactions. Humana Press pp 161–174

[3] Ahmadi H, Radford D, Kropinski AM, Lim L-T, Balamurugan S. Thermal-stability and reconstitution ability of Listeria phages P100 and A511. Front Microbiol, 2017, 8: 2375

[4] Ambalavanan L, Iehata S, Fletcher R, Stevens EH, Zainathan SC (2021) A review of marine viruses in coral ecosystem. J Mar Sci Eng 9：711

[5] Ashcroft AE, Lago H, Macedo J, Horn WT, Stonehouse NJ, Stockley PG. Engineering thermal stability in RNA phage capsids via disulphide bonds. J Nanosci Nanotechnol, 2005, 5: 2034-2041

[6] Bair CL, Black LW. A type IV modification dependent restriction nuclease that targets glucosylated hydroxymethyl cytosine modified DNAs. J Mol Biol, 2007, 366: 768-778

[7] Bair CL, Rifat D, Black LW. Exclusion of glucosyl-hydroxymethylcytosine DNA containing bacteriophages is overcome by the Injected Protein Inhibitor IPI*. J Mol Biol, 2007, 366: 779-789

[8] Baudoux AC, Brussaard CPD. Characterization of different viruses infecting the marine harmful algal bloom species Phaeocystis globosa. Virology, 2005, 341: 80-90

[9] Bochow S, Elliman J, Owens L. Bacteriophage adenine methyltransferase: a life cycle regulator modelled using Vibrio harveyi myovirus like. J Appl Microbiol, 2012, 113: 1001-1013

[10] Brister JR, Kropinski AM, Krupovic M, Lavigne R, Turner D, Sullivan MB. Taxonomic assignment of uncultivated prokaryotic virus genomes is enabled by gene-sharing networks. Nat Biotechnol, 2019, 37: 632-639

[11] Cai L, Ma R, Chen H, Yang Y, Jiao N, Zhang R. A newly isolated roseophage represents a distinct member of Siphoviridae family. Virol J, 2019, 16: 1-9

[12] Ceccarelli D, Hasan N, Huq A, Colwell R. Distribution and dynamics of epidemic and pandemic Vibrio parahaemolyticus virulence factors. Front Cell Infect Microbiol, 2013, 3 97

[13] Cequier-Sánchez E, Rodríguez C, Ravelo ÁG, Zárate R. Dichloromethane as a solvent for lipid extraction and assessment of lipid classes and fatty acids from samples of different natures. J Agric Food Chem, 2008, 56: 4297-4303

[14] Chan PP, Lin BY, Mak AJ, Lowe TM. tRNAscan-SE 2.0: improved detection and functional classification of transfer RNA genes. Nucleic Acids Res, 2021, 49: 9077-9096

[15] Chen LX, Jaffe AL, Borges AL, Penev PI, Nelson TC, Warren LA, Banfield JF. Phage-encoded ribosomal protein S21 expression is linked to late-stage phage replication. ISME Commun, 2022, 2 31

[16] Chen Y, Li W, Shi K, Fang Z, Yang Y, Zhang R. Isolation and characterization of a novel phage belonging to a new genus against Vibrio parahaemolyticus. Virol J, 2023, 20: 81

[17] Clokie MRJ, Kropinski AM, Lavigne R (2009) Bacteriophages: Methods Mol Biol, Volume 1: Isolation, characterization, and interactions. Humana Press, Totowa pp 113–126

[18] Cochran PK, Paul JH. Seasonal abundance of lysogenic bacteria in a subtropical estuary. Appl Environ Microbiol, 1998, 64: 2308-2312

[19] De Schryver P, Defoirdt T, Sorgeloos P. Early mortality syndrome outbreaks: a microbial management issue in shrimp farming. PLoS Pathog, 2014, 10 e1003919

[20] de Souza Valente C, Wan AHL. Vibrio and major commercially important vibriosis diseases in decapod crustaceans. J Invertebr Pathol, 2021, 181 107527

[21] Elena SF, Patricia AR, Jasna L (2009) The evolution of viruses in multi-host fitness landscapes. Open Virol J 3：1–6

[22] Evseev P, Shneider M, Miroshnikov K. Evolution of phage tail sheath protein. Viruses, 2022, 14: 1148

[23] Faruque SM, Mekalanos JJ. Phage-bacterial interactions in the evolution of toxigenic Vibrio cholerae. Virulence, 2012, 3: 556-565

[24] Fibriansah G, Ng TS, Kostyuchenko VA, Lee J, Lee S, Wang J, Lok SM. Structural changes in dengue virus when exposed to a temperature of 37°C. J Virol, 2013, 87: 7585-7592

[25] Fujino T, Okuno Y, Nakada D, Aoyama A, Fukai K, Mukai T, Ueho T. On the bacteriological examination of shirasu-food poisoning. Med J Osaka Univ, 1953, 4: 299-304

[26] Gabig M, Obuchowski M, Srutkowska S, Wegrzyn G (1998) Regulation of replication of λ phage and λ plasmid DNAs at low temperature. Mol Genet Genomics 258：494–502

[27] Gallet R, Kannoly S, Wang IN. Effects of bacteriophage traits on plaque formation. BMC Microbiol, 2011, 11: 181

[28] Garneau JR, Depardieu F, Fortier LC, 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

[29] Geagea H, Labrie SJ, Subirade M, Moineau S. The tape measure protein is involved in the heat stability of Lactococcus lactis phages. Appl Environ Microbiol, 2018, 84 e02082-02017

[30] Gilbert JA, Steele JA, Caporaso JG, Steinbrück L, Reeder J, Temperton B, Huse S, McHardy AC, Knight R, Joint I, Somerfield P, Fuhrman JA, Field D. Defining seasonal marine microbial community dynamics. ISME J, 2012, 6: 298-308

[31] Gilchrist CL, Chooi YH. Clinker & clustermap. js: automatic generation of gene cluster comparison figures. Bioinformatics, 2021, 37: 2473-2475

[32] González-Gómez JP, Soto-Rodriguez SA, Gomez-Gil B, Serrano-Hernández JM, Lozano-Olvera R, López-Cuevas O, Castro-del Campo N, Chaidez C. Effect of phage therapy on survival, histopathology, and water microbiota of Penaeus vannamei challenged with Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (AHPND). Aquaculture, 2023, 576 739851

[33] Gregory MA, Till R, Smith MC. Integration site for Streptomyces phage φBT1 and development of site-specific integrating vectors. J Bacteriol, 2003, 185: 5320-5323

[34] Hazem A. Effects of temperatures, pH values, ultraviolet light, ethanol and chloroform on the growth of isolated thermophilic Bacillus phages. New Microbiol, 2002, 25: 469-476

[35] Hazen TH, Pan L, Gu JD, Sobecky PA. The contribution of mobile genetic elements to the evolution and ecology of Vibrios. FEMS Microbiol Ecol, 2010, 74: 485-499

[36] Hendrix RW, Smith MC, Burns RN, Ford ME, Hatfull GF. Evolutionary relationships among diverse bacteriophages and prophages: all the world's a phage. Proc Natl Acad Sci USA, 1999, 96: 2192-2197

[37] Hyman P, Abedon ST. Practical methods for determining phage growth parameters. Bacteriophages: Methods and Protocols, Volume 1: Isolation, characterization, and interactions, 2009, Totowa, NJ, Humana Press175202

[38] Jang HB, Bolduc B, Zablocki O, Kuhn JH, Roux S, Adriaenssens EM, Brister JR, Kropinski AM, Krupovic M, Lavigne R, Turner D, Sullivan MB. Taxonomic assignment of uncultivated prokaryotic virus genomes is enabled by gene-sharing networks. Nat Biotechnol, 2019, 37: 632-639

[39] Jayasree L, Janakiram P, Madhavi R. Characterization of Vibrio spp. associated with diseased shrimp from culture ponds of Andhra Pradesh (India). J World Aquacult Soc, 2006, 37: 523-532

[40] Jończyk E, Kłak M, Międzybrodzki R, Górski A. The influence of external factors on bacteriophages—review. Folia Microbiol, 2011, 56: 191-200

[41] Jurczak-Kurek A, Gąsior T, Nejman-Faleńczyk B. Biodiversity of bacteriophages: morphological and biological properties of a large group of phages isolated from urban sewage. Sci Rep, 2016, 6: 34338

[42] Kalatzis PG, Castillo D, Katharios P, Middelboe M. Bacteriophage interactions with marine pathogenic Vibrios: implications for phage therapy. Antibiotics, 2018, 7: 15

[43] Kita K, Tsuda J, Kato T, Okamoto K, Yanase H, Tanaka M. Evidence of horizontal transfer of the EcoO109I restriction-modification gene to Escherichia coli chromosomal DNA. J Bacteriol, 1999, 181: 6822-6827

[44] Kita K, Kawakami H, Tanaka H. Evidence for horizontal transfer of the EcoT38I restriction-modification gene to chromosomal DNA by the P2 phage and diversity of defective P2 prophages in Escherichia coli TH38 strains. J Bacteriol, 2003, 185: 2296-2305

[45] Kovall R, Matthews BW. Toroidal structure of λ-exonuclease. Science, 1997, 277: 1824-1827

[46] Kutter E (2009) Phage host range and efficiency of plating. In MRJ Clokie, AM Kropinski (Eds.) Bacteriophages: methods and protocols, Volume 1: Isolation, characterization, and interactions, pp141–149

[47] Kuzmanic A, Zagrovic B. Determination of ensemble-average pairwise root mean-square deviation from experimental B-factors. Biophys J, 2010, 98: 861-871

[48] Kuzminov A. Recombinational repair of DNA damage in Escherichia coli and bacteriophage λ. Microbiol Mol Biol Rev, 1999, 63: 751-813

[49] Labrie SJ, Samson JE, Moineau S. Bacteriophage resistance mechanisms. Nat Rev Microbiol, 2010, 8: 317-327

[50] Letchumanan V, Chan KG, Lee LH. Vibrio parahaemolyticus: a review on the pathogenesis, prevalence, and advance molecular identification techniques. Front Microbiol, 2014, 5: 705

[51] Li S, Fan H, An X, Fan H, Jiang H, Chen Y, Tong Y. Scrutinizing virus genome termini by high-throughput sequencing. PLoS ONE, 2014, 9 e85806

[52] Li P, Yong S, Zhou X, Shen J. Characterization of a new temperate Escherichia coli phage vB_EcoP_ZX5 and its regulatory protein. Pathogens, 2022, 11: 1445

[53] Li Y, Yun H, Chen R, Jiao N, Zheng Q, Yang Y, Zhang R. Characterization of a vibriophage infecting pathogenic Vibrio harveyi. Int J Mol Sci, 2023, 24: 16202

[54] Liang J, Li X, Zha T, Chen Y, Hao H, Liu C, Duan R, Xiao Y, Su M, Wang X, Jing H. DTDP-rhamnosyl transferase RfbF, is a newfound receptor-related regulatory protein for phage phiYe-F10 specific for Yersinia enterocolitica serotype O:3. Sci Rep, 2016, 6: 22905

[55] Ma R, Chen X, Li Y, Jiao N, Zhang R. Diversity, evolution and life strategies of CbK-like phages. Environ Microbiol, 2023, 25: 1250-1264

[56] Mäntynen S, Sundberg LR, Oksanen HM, Poranen MM. Half a century of research on membrane-containing bacteriophages: bringing new concepts to modern virology. Viruses, 2019, 11 1

[57] Martínez L. Automatic identification of mobile and rigid substructures in molecular dynamics simulations and fractional structural fluctuation analysis. PLoS ONE, 2015, 10 e0119264

[58] Mateus L, Costa L, Silva Y, Pereira C, Cunha A, Almeida A. Efficiency of phage cocktails in the inactivation of Vibrio in aquaculture. Aquaculture, 2014, 424: 167-173

[59] Medhekar B, Miller JF. Diversity-generating retroelements. Curr Opin Microbiol 10: 388–395Meier-Kolthoff JP, Göker M (2017) VICTOR: genome-based phylogeny and classification of prokaryotic viruses. Bioinformatics, 2007, 33: 3396-3404

[60] Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M (2022) ColabFold: making protein folding accessible to all. Nat Methods 19：679–682

[61] Mizusawa S, Gottesman S. Protein degradation in Escherichia coli: the lon gene controls the stability of sulA protein. Proc Natl Acad Sci USA, 1983, 80: 358-362

[62] Moldovan R, Chapman-McQuiston E, Wu X (2007) On kinetics of phage adsorption. Biophys J 93:303–315

[63] Moolenaar GF, Moorman C, Goosen N (2000) Role of the Escherichia coli nucleotide excision repair proteins in DNA replication. J Bacteriol 182:5706–5714

[64] Moraru C, Varsani A, Kropinski AM. VIRIDIC—a novel tool to calculate the intergenomic similarities of prokaryote-infecting viruses. Viruses, 2020, 12: 1268

[65] Murphy J, Mahony J, Ainsworth S, Nauta A, van Sinderen D. Bacteriophage orphan DNA methyltransferases: insights from their bacterial origin, function, and occurrence. Appl Environ Microbiol, 2013, 79: 7547-7555

[66] Mushegian AR. Are there 10³¹ virus particles on Earth, or more, or fewer?. J Bacteriol, 2020, 202: 101-128

[67] Nelapati S, Nelapati K, Chinnam BK. Vibrio parahaemolyticus—an emerging foodborne pathogen—a review. Vet World, 2012, 5: 48-63

[68] Osbourn AE, Field B. Operons. Cell Mol Life Sci, 2009, 66: 3755-3775

[69] Paepe MD, Taddei F. Viruses' life history: towards a mechanistic basis of a trade-off between survival and reproduction among phages. PLoS Biol, 2006, 4 e193

[70] Paul JH. Prophages in marine bacteria: dangerous molecular time bombs or the key to survival in the seas?. ISME J, 2008, 2: 579-589

[71] Paul JH, Sullivan MB, Segall AM, Rohwer F. Marine phage genomics. Comp Biochem Physiol B Biochem Mol Biol, 2002, 133: 463-476

[72] Pickard DJJ (2009) Preparation of bacteriophage lysates and pure DNA. Bacteriophages: Methods and Protocols, Volume 2: Molecular and Applied Aspects, pp3–9

[73] Pronk S, Páll S, Schulz R, Larsson P, Bjelkmar P, Apostolov R, Shirts MR, Smith JC, Kasson PM, Van Der Spoel D. GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics, 2013, 29: 845-854

[74] Rifat D, Wright NT, Varney KM, Weber DJ, Black LW. Restriction endonuclease inhibitor IPI* of bacteriophage T4: a novel structure for a dedicated target. J Mol Biol, 2008, 375: 720-734

[75] Sancar A. DNA excision repair. Annu Rev Biochem, 1996, 65: 43-81

[76] Santini S, Jeudy S, Bartoli J, Poirot O, Lescot M, Abergel C, Barbe V, Wommack KE, Noordeloos AAM, Brussaard CPD, Claverie J-M. Genome of Phaeocystis globosa virus PgV-16T highlights the common ancestry of the largest known DNA viruses infecting eukaryotes. Proc Natl Acad Sci, 2013, 110: 10800-10805

[77] Shaburova OV, Krylov SV, Veiko VP, Pleteneva EA, Burkal'Tseva MV, Miroshnikov KA, Cornelissen A, Lavigne R, Sykilinda NN, Kadykov VA. Search for destruction factors of bacterial biofilms: comparison of phage properties in a group of Pseudomonas putida bacteriophages and specificity of their halo-formation products. Russ J Genet, 2009, 45: 161-170

[78] Sheikh HI, Najiah M, Fadhlina A, Laith AA, Nor MM, Jalal KCA, Kasan NA. Temperature upshift mostly but not always enhances the growth of Vibrio species: a systematic review. Front Mar Sci, 2022, 9 959830

[79] Shenvi SS, Isloor AM, Ismail AF. A review on RO membrane technology: developments and challenges. Desalination, 2015, 368: 10-26

[80] Sinha RP, Häder DP. UV-induced DNA damage and repair: a review. Photochem Photobiol Sci, 2002, 1: 225-236

[81] Steczynska JP, Kerr SJ, Kelly ML, Whittle MJ, Bilverstone TW, Minton NP. A tale of two phage tails: engineering the host range of bacteriophages infecting Clostridioides difficile. bioRxiv, 2023, 10562632

[82] Stone E, Campbell K, Grant I, McAuliffe O. Understanding and exploiting phage-host interactions. Viruses, 2019, 11: 567

[83] Storms ZJ, Arsenault E, Sauvageau D. Bacteriophage adsorption efficiency and its effect on amplification. Bioprocess Biosyst Eng, 2010, 33: 823-831

[84] Suttle CA. Viruses in the sea. Nature, 2005, 437: 356-361

[85] Suttle CA. Marine viruses—major players in the global ecosystem. Nat Rev Microbiol, 2007, 5: 801-812

[86] Takemura AF, Chien DM, Polz MF. Associations and dynamics of vibrionaceae in the environment, from the genus to the population level. Front Microbiol, 2014, 5 38

[87] Thammatinna K, Sinprasertporn A, Naknaen A, Samernate T, Nuanpirom J, Chanwong P, Somboonwiwat K, Pogliano J, Sathapondecha P, Thawonsuwan J, Nonejuie P, Chaikeeratisak V. Nucleus-forming vibriophage cocktail reduces shrimp mortality in the presence of pathogenic bacteria. Sci Rep, 2023, 13: 17844

[88] Tokman JI, Kent DJ, Wiedmann M, Denes T. Temperature significantly affects the plaquing and adsorption efficiencies of listeria phages. Front Microbiol, 2016, 7 631

[89] Vezzulli L, Previati M, Pruzzo C, Marchese A, Bourne DG, Cerrano C, Consortium TV. Vibrio infections triggering mass mortality events in a warming Mediterranean Sea. Environ Microbiol., 2010, 12(72007-19

[90] Vidaver AK, Koski RK, Van Etten JL. Bacteriophage φ6: a lipid-containing virus of Pseudomonas phaseolicola. J Virol, 1973, 11: 799-805

[91] Wang Z, Zheng P, Ji W, Fu Q, Wang H, Yan Y, Sun J. SLPW: a virulent bacteriophage targeting methicillin-resistant Staphylococcus aureus in vitro and in vivo. Front Microbiol, 2016, 7934

[92] Wei N, Lu L, Li Y, Ding B, Cai L, Yang Y. A novel roseosiphovirus infecting Dinoroseobacter shibae DFL12T represents a new genus. BMC Genomics, 2025, 26: 121

[93] Wu H, Zhang Y, Jiang Y, Wu H, Huang Y. Characterization and genomic analysis of SHP3, a new transposable bacteriophage infecting Stenotrophomonas maltophilia. J Virol, 2021, 95: 1128

[94] Yan J, Mao J, Xie J. Bacteriophage polysaccharide depolymerases and biomedical applications. BioDrugs, 2014, 28: 265-274

[95] Yang M, Chen H, Guo S, Tan S, Xie Z, Zhang J, Wu Q, Tan Z. Characterization and genome analysis of a novel Vibrio parahaemolyticus phage vB_VpP_DE17. Virus Res, 2022, 307 198580

[96] Yu Y, Gong T, Jost G, Liu W, Ye D, Luo Z. Isolation and characterization of five lytic bacteriophages infecting a Vibrio strain closely related to Vibrio owensii. FEMS Microbiol Lett, 2013, 348: 112-119

[97] Zhang X, Lin H, Wang X, Austin B. Significance of Vibrio species in the marine organic carbon cycle—a review. Sci China, 2018, 61: 1357-1368

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