1887

Abstract

Coevolution between bacteriophages (phages) and their prey is the result of mutualistic interactions. Here, we show that pseudolysogeny is a frequent outcome of infection by virulent phages of and that selection of resistant bacterial mutants is favoured by continuous production of phages. We investigated the frequency and characteristics of strain PAO1 variants resisting infection by different combinations of virulent phages belonging to four genera. The frequency of resistant bacteria was 10 for single phage infection and 10 for infections with combinations of two or four phages. The genome of 27 variants was sequenced and the comparison with the genome of the parental PAO1 strain allowed the identification of point mutations or small indels. Four additional variants were characterized by a candidate gene approach. In total, 27 independent mutations were observed affecting 14 genes and a regulatory region. The mutations affected genes involved in biosynthesis of type IV pilus, alginate, LPS and O-antigen. Half of the variants possessed changes in homopolymer tracts responsible for frameshift mutations and these phase variation mutants were shown to be unstable. Eleven double mutants were detected. The presence of free phage DNA was observed in association with exclusion of superinfection in half of the variants and no chromosomal mutation could be found in three of them. Upon further growth of these pseudolysogens, some variants with new chromosomal mutations were recovered, presumably due to continuous evolutionary pressure.

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2016-05-01
2024-12-09
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References

  1. Abedon S. T., Kuhl S. J., Blasdel B. G., Kutter E. M. 2011; Phage treatment of human infections. Bacteriophage 1:66–85 [View Article][PubMed]
    [Google Scholar]
  2. Augustin D. K., Song Y., Baek M. S., Sawa Y., Singh G., Taylor B., Rubio-Mills A., Flanagan J. L., Wiener-Kronish J. P., Lynch S. V. 2007; Presence or absence of lipopolysaccharide O antigens affects type III secretion by Pseudomonas aeruginosa . J Bacteriol 189:2203–2209 [View Article][PubMed]
    [Google Scholar]
  3. Baess I. 1971; Report on a pseudolysogenic mycobacterium and a review of the literature concerning pseudolysogeny. Acta Pathol Microbiol Scand B Microbiol Immunol 79:428–434[PubMed]
    [Google Scholar]
  4. Betts A., Kaltz O., Hochberg M. E. 2014; Contrasted coevolutionary dynamics between a bacterial pathogen and its bacteriophages. Proc Natl Acad Sci U S A 111:11109–11114 [View Article][PubMed]
    [Google Scholar]
  5. Bode W. 1967; Lysis inhibition in Escherichia coli infected with bacteriophage T4. J Virol 1:948–955[PubMed]
    [Google Scholar]
  6. Brockhurst M. A., Buckling A., Rainey P. B. 2005; The effect of a bacteriophage on diversification of the opportunistic bacterial pathogen, Pseudomonas aeruginosa . Proc Biol Sci 272:1385–1391 [View Article][PubMed]
    [Google Scholar]
  7. Bucior I., Pielage J. F., Engel J. N. 2012; Pseudomonas aeruginosa pili and flagella mediate distinct binding and signaling events at the apical and basolateral surface of airway epithelium. PLoS Pathog 8:e1002616 [View Article][PubMed]
    [Google Scholar]
  8. Buckling A., Rainey P. B. 2002; Antagonistic coevolution between a bacterium and a bacteriophage. Proc Biol Sci 269:931–936 [View Article][PubMed]
    [Google Scholar]
  9. Ceyssens P. J., Glonti T., Kropinski N. M., Lavigne R., Chanishvili N., Kulakov L., Lashkhi N., Tediashvili M., Merabishvili M. 2011; Phenotypic and genotypic variations within a single bacteriophage species. Virol J 8:134 [View Article][PubMed]
    [Google Scholar]
  10. Chaturongakul S., Ounjai P. 2014; Phage–host interplay: examples from tailed phages and Gram-negative bacterial pathogens. Front Microbiol 5:442 [View Article][PubMed]
    [Google Scholar]
  11. Chiang P., Burrows L. L. 2003; Biofilm formation by hyperpiliated mutants of Pseudomonas aeruginosa . J Bacteriol 185:2374–2378 [View Article][PubMed]
    [Google Scholar]
  12. Chibeu A., Ceyssens P. J., Hertveldt K., Volckaert G., Cornelis P., Matthijs S., Lavigne R. 2009; The adsorption of Pseudomonas aeruginosa bacteriophage ϕKMV is dependent on expression regulation of type IV pili genes. FEMS Microbiol Lett 296:210–218 [View Article][PubMed]
    [Google Scholar]
  13. Choi K. H., Kumar A., Schweizer H. P. 2006; A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J Microbiol Methods 64:391–397 [View Article][PubMed]
    [Google Scholar]
  14. Church G. M., Gilbert W. 1984; Genomic sequencing. Proc Natl Acad Sci U S A 81:1991–1995 [View Article][PubMed]
    [Google Scholar]
  15. de Siqueira R. S., Dodd C. E., Rees C. E. 2006; Evaluation of the natural virucidal activity of teas for use in the phage amplification assay. Int J Food Microbiol 111:259–262 [View Article][PubMed]
    [Google Scholar]
  16. Demerec M., Fano U. 1945; Bacteriophage-resistant mutants in Escherichia coli . Genetics 30:119–136[PubMed]
    [Google Scholar]
  17. Dennehy J. J. 2012; What can phages tell us about host-pathogen coevolution?. Int J Evol Biol 2012:396165 [View Article][PubMed]
    [Google Scholar]
  18. Essoh C., Blouin Y., Loukou G., Cablanmian A., Lathro S., Kutter E., Thien H. V., Vergnaud G., Pourcel C. 2013; The susceptibility of Pseudomonas aeruginosa strains from cystic fibrosis patients to bacteriophages. PLoS One 8:e60575 [View Article][PubMed]
    [Google Scholar]
  19. Essoh C., Latino L., Midoux C., Blouin Y., Loukou G., Nguetta S. P., Lathro S., Cablanmian A., Kouassi A. K., other authors. 2015; Investigation of a large collection of Pseudomonas aeruginosa bacteriophages collected from a single environmental source in Abidjan, Côte d'Ivoire. PLoS One 10:e0130548 [View Article][PubMed]
    [Google Scholar]
  20. Fomsgaard A., Freudenberg M. A., Galanos C. 1990; Modification of the silver staining technique to detect lipopolysaccharide in polyacrylamide gels. J Clin Microbiol 28:2627–2631[PubMed]
    [Google Scholar]
  21. Hahn H. P. 1997; The type-4 pilus is the major virulence-associated adhesin of Pseudomonas aeruginosa – a review. Gene 192:99–108 [View Article][PubMed]
    [Google Scholar]
  22. Hansen S. K., Haagensen J. A., Gjermansen M., Jørgensen T. M., Tolker-Nielsen T., Molin S. 2007; Characterization of a Pseudomonas putida rough variant evolved in a mixed-species biofilm with Acinetobacter sp. strain C6. J Bacteriol 189:4932–4943 [View Article][PubMed]
    [Google Scholar]
  23. Henderson I. R., Owen P., Nataro J. P. 1999; Molecular switches – the ON and OFF of bacterial phase variation. Mol Microbiol 33:919–932 [View Article][PubMed]
    [Google Scholar]
  24. Hitchcock P. J., Brown T. M. 1983; Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J Bacteriol 154:269–277[PubMed]
    [Google Scholar]
  25. Hosseinidoust Z., Tufenkji N., van de Ven T. G. 2013a; Predation in homogeneous and heterogeneous phage environments affects virulence determinants of Pseudomonas aeruginosa . Appl Environ Microbiol 79:2862–2871 [View Article][PubMed]
    [Google Scholar]
  26. Hosseinidoust Z., van de Ven T. G., Tufenkji N. 2013b; Evolution of Pseudomonas aeruginosa virulence as a result of phage predation. Appl Environ Microbiol 79:6110–6116 [View Article][PubMed]
    [Google Scholar]
  27. Hyman P., Abedon S. T. 2010; Bacteriophage host range and bacterial resistance. Adv Appl Microbiol 70:217–248 [View Article][PubMed]
    [Google Scholar]
  28. Islam S. T., Lam J. S. 2014; Synthesis of bacterial polysaccharides via the Wzx/Wzy-dependent pathway. Can J Microbiol 60:697–716 [View Article][PubMed]
    [Google Scholar]
  29. Islam S. T., Huszczynski S. M., Nugent T., Gold A. C., Lam J. S. 2013; Conserved-residue mutations in Wzy affect O-antigen polymerization and Wzz-mediated chain-length regulation in Pseudomonas aeruginosa PAO1. Sci Rep 3:3441 [View Article][PubMed]
    [Google Scholar]
  30. Kim K., Oh J., Han D., Kim E. E., Lee B., Kim Y. 2006; Crystal structure of PilF: functional implication in the type 4 pilus biogenesis in Pseudomonas aeruginosa . Biochem Biophys Res Commun 340:1028–1038 [View Article][PubMed]
    [Google Scholar]
  31. King J. D., Kocíncová D., Westman E. L., Lam J. S. 2009; Review: Lipopolysaccharide biosynthesis in Pseudomonas aeruginosa . Innate Immun 15:261–312 [View Article][PubMed]
    [Google Scholar]
  32. Klausen M., Heydorn A., Ragas P., Lambertsen L., Aaes-Jørgensen A., Molin S., Tolker-Nielsen T. 2003; Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants. Mol Microbiol 48:1511–1524 [View Article][PubMed]
    [Google Scholar]
  33. Klockgether J., Munder A., Neugebauer J., Davenport C. F., Stanke F., Larbig K. D., Heeb S., Schöck U., Pohl T. M., other authors. 2010; Genome diversity of Pseudomonas aeruginosa PAO1 laboratory strains. J Bacteriol 192:1113–1121 [View Article][PubMed]
    [Google Scholar]
  34. Labrie S. J., Samson J. E., Moineau S. 2010; Bacteriophage resistance mechanisms. Nat Rev Microbiol 8:317–327 [View Article][PubMed]
    [Google Scholar]
  35. Lam J. S., Taylor V. L., Islam S. T., Hao Y., Kocíncová D. 2011; Genetic and functional diversity of Pseudomonas aeruginosa lipopolysaccharide. Front Microbiol 2:118 [View Article][PubMed]
    [Google Scholar]
  36. Latino L., Essoh C., Blouin Y., Vu Thien H., Pourcel C. 2014; A novel Pseudomonas aeruginosa bacteriophage, Ab31, a chimera formed from temperate phage PAJU2 and P. putida lytic phage AF: characteristics and mechanism of bacterial resistance. PLoS One 9:e93777 [View Article][PubMed]
    [Google Scholar]
  37. Le S., Yao X., Lu S., Tan Y., Rao X., Li M., Jin X., Wang J., Zhao Y., other authors. 2014; Chromosomal DNA deletion confers phage resistance to Pseudomonas aeruginosa . Sci Rep 4:4738 [View Article][PubMed]
    [Google Scholar]
  38. León M., Bastías R. 2015; Virulence reduction in bacteriophage resistant bacteria. Front Microbiol 6:343 [View Article][PubMed]
    [Google Scholar]
  39. Liebens V., Defraine V., Van der Leyden A., De Groote V. N., Fierro C., Beullens S., Verstraeten N., Kint C., Jans A., other authors. 2014; A putative de-N-acetylase of the PIG-L superfamily affects fluoroquinolone tolerance in Pseudomonas aeruginosa . Pathog Dis 71:39–54 [View Article][PubMed]
    [Google Scholar]
  40. Loś M., Węgrzyn G. 2012; Pseudolysogeny. Adv Virus Res 82:339–349 [View Article][PubMed]
    [Google Scholar]
  41. Loś M., Węgrzyn G., Neubauer P. 2003; A role for bacteriophage T4 rI gene function in the control of phage development during pseudolysogeny and in slowly growing host cells. Res Microbiol 154:547–552 [View Article][PubMed]
    [Google Scholar]
  42. Lu M. J., Henning U. 1989; The immunity (imm) gene of Escherichia coli bacteriophage T4. J Virol 63:3472–3478[PubMed]
    [Google Scholar]
  43. Lyczak J. B., Cannon C. L., Pier G. B. 2000; Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect 2:1051–1060 [View Article][PubMed]
    [Google Scholar]
  44. Maillou J., Dreiseikelmann B. 1990; The sim gene of Escherichia coli phage P1: nucleotide sequence and purification of the processed protein. Virology 175:500–507 [View Article][PubMed]
    [Google Scholar]
  45. Maura D., Debarbieux L. 2012; On the interactions between virulent bacteriophages and bacteria in the gut. Bacteriophage 2:229–233 [View Article][PubMed]
    [Google Scholar]
  46. Maura D., Galtier M., Le Bouguénec C., Debarbieux L. 2012; Virulent bacteriophages can target O104 : H4 enteroaggregative Escherichia coli in the mouse intestine. Antimicrob Agents Chemother 56:6235–6242 [View Article][PubMed]
    [Google Scholar]
  47. Murphy K., Park A. J., Hao Y., Brewer D., Lam J. S., Khursigara C. M. 2014; Influence of O polysaccharides on biofilm development and outer membrane vesicle biogenesis in Pseudomonas aeruginosa PAO1. J Bacteriol 196:1306–1317 [View Article][PubMed]
    [Google Scholar]
  48. O'Toole G. A., Kolter R. 1998; Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol 30:295–304 [View Article][PubMed]
    [Google Scholar]
  49. Olszak T., Zarnowiec P., Kaca W., Danis-Wlodarczyk K., Augustyniak D., Drevinek P., de Soyza A., McClean S., Drulis-Kawa Z. 2015; In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas aeruginosa strains from cystic fibrosis patients. Appl Microbiol Biotechnol 99:6021–6033 [View Article][PubMed]
    [Google Scholar]
  50. Olvera C., Goldberg J. B., Sánchez R., Soberón-Chávez G. 1999; The Pseudomonas aeruginosa algC gene product participates in rhamnolipid biosynthesis. FEMS Microbiol Lett 179:85–90 [View Article][PubMed]
    [Google Scholar]
  51. Poon K. K., Westman E. L., Vinogradov E., Jin S., Lam J. S. 2008; Functional characterization of MigA and WapR: putative rhamnosyltransferases involved in outer core oligosaccharide biosynthesis of Pseudomonas aeruginosa . J Bacteriol 190:1857–1865 [View Article][PubMed]
    [Google Scholar]
  52. Pritt B., O'Brien L., Winn W. 2007; Mucoid Pseudomonas in cystic fibrosis. Am J Clin Pathol 128:32–34 [View Article][PubMed]
    [Google Scholar]
  53. Pulcrano G., Iula D. V., Raia V., Rossano F., Catania M. R. 2012; Different mutations in mucA gene of Pseudomonas aeruginosa mucoid strains in cystic fibrosis patients and their effect on algU gene expression. New Microbiol 35:295–305[PubMed]
    [Google Scholar]
  54. Ripp S., Miller R. V. 1997; The role of pseudolysogeny in bacteriophage–host interactions in a natural freshwater environment. Virology 143:2065–2070
    [Google Scholar]
  55. Ripp S., Miller R. V. 1998; Dynamics of the pseudolysogenic response in slowly growing cells of Pseudomonas aeruginosa . Microbiology 144:2225–2232 [View Article][PubMed]
    [Google Scholar]
  56. Rocchetta H. L., Burrows L. L., Lam J. S. 1999; Genetics of O-antigen biosynthesis in Pseudomonas aeruginosa . Microbiol Mol Biol Rev 63:523–553[PubMed]
    [Google Scholar]
  57. Scanlan P. D., Hall A. R., Blackshields G., Friman V. P., Davis M. R. Jr, Goldberg J. B., Buckling A. 2015; Coevolution with bacteriophages drives genome-wide host evolution and constrains the acquisition of abiotic-beneficial mutations. Mol Biol Evol 32:1425–1435 [View Article][PubMed]
    [Google Scholar]
  58. Segura A., Hurtado A., Duque E., Ramos J. L. 2004; Transcriptional phase variation at the flhB gene of Pseudomonas putida DOT-T1E is involved in response to environmental changes and suggests the participation of the flagellar export system in solvent tolerance. J Bacteriol 186:1905–1909 [View Article][PubMed]
    [Google Scholar]
  59. Siringan P., Connerton P. L., Cummings N. J., Connerton I. F. 2014; Alternative bacteriophage life cycles: the carrier state of Campylobacter jejuni . Open Biol 4:130200 [View Article][PubMed]
    [Google Scholar]
  60. Sistrom M., Park D., O'Brien H. E., Wang Z., Guttman D. S., Townsend J. P., Turner P. E. 2015; Genomic and gene-expression comparisons among phage-resistant type-IV pilus mutants of Pseudomonas syringae pathovar phaseolicola . PLoS One 10:e0144514 [View Article][PubMed]
    [Google Scholar]
  61. Spencer D. H., Kas A., Smith E. E., Raymond C. K., Sims E. H., Hastings M., Burns J. L., Kaul R., Olson M. V. 2003; Whole-genome sequence variation among multiple isolates of Pseudomonas aeruginosa . J Bacteriol 185:1316–1325 [View Article][PubMed]
    [Google Scholar]
  62. Stover C. K., Pham X. Q., Erwin A. L., Mizoguchi S. D., Warrener P., Hickey M. J., Brinkman F. S., Hufnagle W. O., Kowalik D. J., other authors. 2000; Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406:959–964 [View Article][PubMed]
    [Google Scholar]
  63. Tanji Y., Hattori K., Suzuki K., Miyanaga K. 2008; Spontaneous deletion of a 209-kilobase-pair fragment from the Escherichia coli genome occurs with acquisition of resistance to an assortment of infectious phages. Appl Environ Microbiol 74:4256–4263 [View Article][PubMed]
    [Google Scholar]
  64. Taylor V. L., Udaskin M. L., Islam S. T., Lam J. S. 2013; The D3 bacteriophage α-polymerase inhibitor (Iap) peptide disrupts O-antigen biosynthesis through mimicry of the chain length regulator Wzz in Pseudomonas aeruginosa . J Bacteriol 195:4735–4741 [View Article][PubMed]
    [Google Scholar]
  65. Vu-Thien H., Corbineau G., Hormigos K., Fauroux B., Corvol H., Clément A., Vergnaud G., Pourcel C. 2007; Multiple-locus variable-number tandem-repeat analysis for longitudinal survey of sources of Pseudomonas aeruginosa infection in cystic fibrosis patients. J Clin Microbiol 45:3175–3183 [View Article][PubMed]
    [Google Scholar]
  66. West S. E., Schweizer H. P., Dall C., Sample A. K., Runyen-Janecky L. J. 1994; Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa . Gene 148:81–86 [View Article][PubMed]
    [Google Scholar]
  67. Williams H. T. 2013; Phage-induced diversification improves host evolvability. BMC Evol Biol 13:17 [View Article][PubMed]
    [Google Scholar]
  68. Wommack K. E., Colwell R. R. 2000; Virioplankton: viruses in aquatic ecosystems. Microbiol Mol Biol Rev 64:69–114 [View Article][PubMed]
    [Google Scholar]
  69. Zierdt C. H., Schmidt P. J. 1964; Dissociation in Pseudomonas aeruginosa . J Bacteriol 87:1003–1010[PubMed]
    [Google Scholar]
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