1887

Abstract

As the smallest and most abundant primary producer in the oceans, the cyanobacterium is of interest to diverse branches of science. For the past 30 years, research on this minimal phototroph has led to a growing understanding of biological organization across multiple scales, from the genome to the global ocean ecosystem. Progress in understanding drivers of its diversity and ecology, as well as molecular mechanisms underpinning its streamlined simplicity, has been hampered by the inability to manipulate these cells genetically. Multiple attempts have been made to develop an efficient genetic transformation method for over the years; all have been unsuccessful to date, despite some success with their close relative, . To avoid the pursuit of unproductive paths, we report here what has not worked in our hands, as well as our progress developing a method to screen the most efficient electroporation parameters for optimal DNA delivery into cells. We also report a novel protocol for obtaining axenic colonies and a new method for differentiating live and dead cells. The electroporation method can be used to optimize DNA delivery into any bacterium, making it a useful tool for advancing transformation systems in other genetically recalcitrant microorganisms.

Funding
This study was supported by the:
  • Simons Foundation (Award 337262, 509034SCFY17, 329108)
    • Principle Award Recipient: Sallie W. Chisholm
  • National Science Foundation (Award 1645061)
    • Principle Award Recipient: Sallie W. Chisholm
  • This is an open-access article distributed under the terms of the Creative Commons.
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2020-02-19
2024-04-16
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References

  1. Dick GJ, Lam P. Omic approaches to microbial geochemistry. Elements 2015; 11:403–408 [View Article]
    [Google Scholar]
  2. Castelle CJ, Banfield JF. Major new microbial groups expand diversity and alter our understanding of the tree of life. Cell 2018; 172:1181–1197 [View Article]
    [Google Scholar]
  3. Godzik A. Metagenomics and the protein universe. Curr Opin Struct Biol 2011; 21:398–403 [View Article]
    [Google Scholar]
  4. Liu H, Deutschbauer AM. Rapidly moving new bacteria to model-organism status. Curr Opin Biotechnol 2018; 51:116–122 [View Article]
    [Google Scholar]
  5. Price MN, Wetmore KM, Waters RJ, Callaghan M, Ray J et al. Mutant phenotypes for thousands of bacterial genes of unknown function. Nature 2018; 557:503–509 [View Article]
    [Google Scholar]
  6. Brahamsha B. A genetic manipulation system for oceanic cyanobacteria of the genus Synechococcus . Appl Environ Microbiol 1996; 62:1747–1751 [View Article]
    [Google Scholar]
  7. Drecktrah D, Samuels DS. Genetic manipulation of Borrelia spp. Curr Top Microbiol Immunol 2017; 6:23–27
    [Google Scholar]
  8. Kohler PRA, Metcalf WW. Genetic manipulation of Methanosarcina spp. Front Microbiol 2012; 3: [View Article]
    [Google Scholar]
  9. Kananavičiūtė R, Čitavičius D. Genetic engineering of Geobacillus spp. J Microbiol Methods 2015; 111:31–39 [View Article]
    [Google Scholar]
  10. Szvetnik A, Bihari Z, Szabó Z, Kelemen O, Kiss I. Genetic manipulation tools for Dietzia spp. J Appl Microbiol 2010; 109:1845–1852 [View Article]
    [Google Scholar]
  11. Salyers AA, Bonheyo G, Shoemaker NB. Starting a new genetic system: lessons from bacteroides . Methods 2000; 20:35–46 [View Article]
    [Google Scholar]
  12. Dhakal D, Jha AK, Pokhrel A, Shrestha A, Sohng JK. Genetic manipulation of Nocardia species. Curr Protoc Microbiol 2016; 2016:10F.2.1–10.210
    [Google Scholar]
  13. Picardeau M. Toolbox of molecular techniques for studying leptospira Spp. Current Topics in Microbiology and Immunology 2017
    [Google Scholar]
  14. Freed E, Fenster J, Smolinski SL, Walker J, Henard CA et al. Building a genome engineering toolbox in nonmodel prokaryotic microbes. Biotechnol Bioeng 2018; 115:2120–2138 [View Article]
    [Google Scholar]
  15. Johnston CD, Cotton SL, Rittling SR, Starr JR, Borisy GG et al. Systematic evasion of the restriction-modification barrier in bacteria. Proc Natl Acad Sci U S A 2019; 116:11454–11459 [View Article]
    [Google Scholar]
  16. van Opijnen T, Bodi KL, Camilli A. Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods 2009; 6:767–772 [View Article]
    [Google Scholar]
  17. van Opijnen T, Camilli A. Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms. Nat Rev Microbiol 2013; 11:435–442 [View Article]
    [Google Scholar]
  18. Wetmore KM, Price MN, Waters RJ, Lamson JS, He J et al. Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly Bar-Coded transposons. MBio 2015; 6:1–15 [View Article]
    [Google Scholar]
  19. Flombaum P, Gallegos JL, Gordillo RA, Rincón J, Zabala LL et al. Present and future global distributions of the marine Cyanobacteria Prochlorococcus and Synechococcus . Proc Natl Acad Sci USA 2013; 110:9824–9829 [View Article]
    [Google Scholar]
  20. Partensky F, Hess WR, Vaulot D. Prochlorococcus, a marine photosynthetic prokaryote of global significance. MicrobiolMol BiolRev 1999; 63:106–127 [View Article]
    [Google Scholar]
  21. Partensky F, Garczarek L. Microbiology: Arms race in a drop of sea water. Nature 2011; 474:582–583 [View Article]
    [Google Scholar]
  22. Sunagawa S, Coelho LP, Chaffron S, Kultima JR, Labadie K et al. Structure and function of the global ocean microbiome. Science 2015; 348:1261359 [View Article]
    [Google Scholar]
  23. Mende DR, Bryant JA, Aylward FO, Eppley JM, Nielsen T et al. Environmental drivers of a microbial genomic transition zone in the ocean’s interior. Nat Microbiol 2017; 2:1367–1373 [View Article]
    [Google Scholar]
  24. Saito MA, McIlvin MR, Moran DM, Goepfert TJ, DiTullio GR et al. Multiple nutrient stresses at intersecting Pacific Ocean biomes detected by protein biomarkers. Science 2014; 345:1173–1177 [View Article]
    [Google Scholar]
  25. Shi Y, Tyson GW, Eppley JM, DeLong EF. Integrated metatranscriptomic and metagenomic analyses of stratified microbial assemblages in the open ocean. Isme J 2011; 5:9991013 [View Article]
    [Google Scholar]
  26. Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D et al. Environmental genome shotgun sequencing of the Sargasso Sea. Science 2004; 304:6674 [View Article]
    [Google Scholar]
  27. Biller SJ, Berube PM, Dooley K, Williams M, Satinsky BM et al. Marine microbial metagenomes sampled across space and time. Sci Data 2018; 5: [View Article]
    [Google Scholar]
  28. Biller SJ, Berube PM, Lindell D, Chisholm SW. Prochlorococcus: the structure and function of collective diversity. Nat Rev Microbiol 2015; 13:13–27 [View Article]
    [Google Scholar]
  29. Biller SJ, Berube PM, Berta-Thompson JW, Kelly L, Roggensack SE et al. Genomes of diverse isolates of the marine cyanobacterium Prochlorococcus . Sci Data 2014; 1:140034 [View Article]
    [Google Scholar]
  30. Berube PM, Biller SJ, Hackl T, Hogle SL, Satinsky BM et al. Single cell genomes of Prochlorococcus, Synechococcus, and sympatric microbes from diverse marine environments. Sci Data 2018; 5:180154 [View Article]
    [Google Scholar]
  31. Braakman R, Follows MJ, Chisholm SW. Metabolic evolution and the self-organization of ecosystems. Proc Natl Acad Sci USA 2017; 114:E3091–E3100 [View Article]
    [Google Scholar]
  32. Rocap G, Larimer FW, Lamerdin J, Malfatti S, Chain P et al. Genome divergence in two Prochlorococcus ecotypes reflects oceanic niche differentiation. Nature 2003; 424:1042–1047 [View Article]
    [Google Scholar]
  33. Kettler GC, Martiny AC, Huang K, Zucker J, Coleman ML et al. Patterns and implications of gene gain and loss in the evolution of Prochlorococcus . PLoS Genet 2007; 3:e231–2528 [View Article]
    [Google Scholar]
  34. Rusch DB, Martiny AC, Dupont CL, Halpern AL, Venter JC. Characterization of Prochlorococcus clades from iron-depleted oceanic regions. Proc Natl Acad Sci USA 2010; 107:16184–16189 [View Article]
    [Google Scholar]
  35. Malmstrom RR, Rodrigue S, Huang KH, Kelly L, Kern SE et al. Ecology of uncultured Prochlorococcus clades revealed through single-cell genomics and biogeographic analysis. Isme J 2013; 7:184–198 [View Article]
    [Google Scholar]
  36. Kashtan N, Roggensack SE, Berta-Thompson JW, Grinberg M, Stepanauskas R et al. Fundamental differences in diversity and genomic population structure between Atlantic and Pacific Prochlorococcus . Isme J 2017; 11:1997–2011 [View Article]
    [Google Scholar]
  37. Kashtan N, Roggensack SE, Rodrigue S, Thompson JW, Biller SJ et al. Single-Cell genomics reveals hundreds of coexisting subpopulations in wild Prochlorococcus . Science 2014; 344:416–420 [View Article]
    [Google Scholar]
  38. Coleman ML, Chisholm SW. Code and context: Prochlorococcus as a model for cross-scale biology. Trends Microbiol 2007; 15:398–407 [View Article]
    [Google Scholar]
  39. Partensky F, Garczarek L. Prochlorococcus : Advantages and Limits of Minimalism. Ann Rev Mar Sci 2010; 2:305–331 [View Article]
    [Google Scholar]
  40. Martiny AC, Coleman ML, Chisholm SW. Phosphate acquisition genes in Prochlorococcus ecotypes: evidence for genome-wide adaptation. Proc Natl Acad Sci USA 2006; 103:12552–12557 [View Article]
    [Google Scholar]
  41. Tagwerker C, Dupont CL, Karas BJ, Ma L, Chuang R-Y et al. Sequence analysis of a complete 1.66 Mb Prochlorococcus marinus MED4 genome cloned in yeast. Nucleic Acids Res 2012; 40:10375–10383 [View Article]
    [Google Scholar]
  42. Hutchison CA, Chuang R-Y, Noskov VN, Assad-Garcia N, Deerinck TJ et al. Design and synthesis of a minimal bacterial genome. Science 2016; 351:aad6253 [View Article]
    [Google Scholar]
  43. Noack S, Baumgart M. Communities of Niche-optimized strains: small-genome organism consortia in bioproduction. Trends Biotechnol 20181–14
    [Google Scholar]
  44. Calero P, Nikel PI. Chasing bacterial chassis for metabolic engineering: a perspective review from classical to non-traditional microorganisms. Microb Biotechnol 2019; 12:98-124 [View Article]
    [Google Scholar]
  45. Hopkinson BM, Young JN, Tansik AL, Binder BJ. The minimal CO2-concentrating mechanism of Prochlorococcus spp. MED4 is effective and efficient. Plant Physiol 2014; 166:2205–2217 [View Article]
    [Google Scholar]
  46. Motwalli O, Essack M, Jankovic BR, Ji B, Liu X et al. In silico screening for candidate chassis strains of free fatty acid-producing cyanobacteria. BMC Genomics 2017; 18:33 [View Article]
    [Google Scholar]
  47. Moore LR, Coe A, Zinser ER, Saito MA, Sullivan MB et al. Culturing the marine cyanobacterium Prochlorococcus . Limnol Oceanogr 2007; 5:353–362 [View Article]
    [Google Scholar]
  48. Lindell D. The genus prochlorococcus, phylum cyanobacteria. Prokaryotes Other Major Lineages Bact Archaea 9783642389 2014 pp 829–845
    [Google Scholar]
  49. Saito MA, Sigman DM, Morel FMM. The bioinorganic chemistry of the ancient ocean: the co-evolution of cyanobacterial metal requirements and biogeochemical cycles at the Archean–Proterozoic boundary?. Inorganica Chim Acta 2003; 356:308–318 [View Article]
    [Google Scholar]
  50. Hawco NJ, Saito MA. Competitive inhibition of cobalt uptake by zinc and manganese in a pacific Prochlorococcus strain: Insights into metal homeostasis in a streamlined oligotrophic cyanobacterium. Limnol Oceanogr 2018; 63:2229–2249 [View Article]
    [Google Scholar]
  51. Morris JJ, Johnson ZI, Szul MJ, Keller M, Zinser ER. Dependence of the cyanobacterium Prochlorococcus on hydrogen peroxide scavenging microbes for growth at the ocean's surface. PLoS One 2011; 6:e16805 [View Article]
    [Google Scholar]
  52. Van Mooy BAS, Rocap G, Fredricks HF, Evans CT, Devol AH. Sulfolipids dramatically decrease phosphorus demand by picocyanobacteria in oligotrophic marine environments. Proc Natl Acad Sci USA 2006; 103:8607–8612 [View Article]
    [Google Scholar]
  53. Osburne MS, Holmbeck BM, Frias-Lopez J, Steen R, Huang K et al. UV hyper-resistance in Prochlorococcus MED4 results from a single base pair deletion just upstream of an operon encoding nudix hydrolase and photolyase. Environ Microbiol 2010; 12:1978–1988 [View Article]
    [Google Scholar]
  54. Tolonen AC, Liszt GB, Hess WR. Genetic manipulation of Prochlorococcus strain MIT9313: green fluorescent protein expression from an RSF1010 plasmid and Tn5 transposition. Appl Environ Microbiol 2006; 72:7607–7613 [View Article]
    [Google Scholar]
  55. Stucken K, Koch R, Dagan T. Cyanobacterial defense mechanisms against foreign DNA transfer and their impact on genetic engineering. Biol Res 2013; 46:373–382 [View Article]
    [Google Scholar]
  56. Taton A, Unglaub F, Wright NE, Zeng WY, Paz-Yepes J et al. Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria. Nucleic Acids Res 2014; 42:gku673 [View Article]
    [Google Scholar]
  57. Stuart RK, Brahamsha B, Busby K, Palenik B. Genomic island genes in a coastal marine Synechococcus strain confer enhanced tolerance to copper and oxidative stress. ISME J 2013
    [Google Scholar]
  58. Chen H, Lin H, Jiang P, Li F, Qin S. Genetic transformation of marine cyanobacterium Synechococcus sp. CC9311 (Cyanophyceae) by electroporation. Chin J Ocean Limnol 2013; 31:416–420 [View Article]
    [Google Scholar]
  59. Martínez-García E, Aparicio T, de Lorenzo V, Nikel PI. New transposon tools tailored for metabolic engineering of gram-negative microbial cell factories. Front Bioeng Biotechnol 2014; 2:46 [View Article]
    [Google Scholar]
  60. McCarren J, Brahamsha B. Transposon mutagenesis in a marine Synechococcus strain: isolation of swimming motility mutants. J Bacteriol 2005; 187:4457–4462 [View Article]
    [Google Scholar]
  61. Coe A, Ghizzoni J, LeGault K, Biller S, Roggensack SE et al. Survival of Prochlorococcus in extended darkness. Limnol Oceanogr 2016; 61:1375–1388 [View Article]
    [Google Scholar]
  62. Yoshida N, Sato M. Plasmid uptake by bacteria: a comparison of methods and efficiencies. Appl Microbiol Biotechnol 2009; 83:791–798 [View Article]
    [Google Scholar]
  63. Kato J, Amie J, Murata Y, Kuroda A, Mitsutani A et al. Development of a genetic transformation system for an alga-lysing bacterium. Appl Environ Microbiol 1998; 64:2061–2064 [View Article]
    [Google Scholar]
  64. Lee HH, Ostrov N, Wong BG, Gold MA, Khalil AS et al. Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi. Nat Microbiol 2019; 4:1105–1113 [View Article]
    [Google Scholar]
  65. Harris JR, Lundgren BR, Grzeskowiak BR, Mizuno K, Nomura CT. A rapid and efficient electroporation method for transformation of Halomonas sp. O-1. J Microbiol Methods 2016; 129:127–132 [View Article]
    [Google Scholar]
  66. van Ooijen G, Knox K, Kis K, Bouget F-Y, Millar AJ. Genomic Transformation of the Picoeukaryote Ostreococcus tauri . JoVE 2012; 65: [View Article]
    [Google Scholar]
  67. Dower WJ, Miller JF, Ragsdale CW. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 1988; 16:6127–6145 [View Article]
    [Google Scholar]
  68. Moore LR, Chisholm SW. Photophysiology of the marine cyanobacterium Prochlorococcus: Ecotypic differences among cultured isolates. Limnol Oceanogr 1999; 44:628–638 [View Article]
    [Google Scholar]
  69. Steglich C, Mullineaux CW, Teuchner K, Hess WR, Lokstein H. Photophysical properties of Prochlorococcus marinus SS120 divinyl chlorophylls and phycoerythrin in vitro and in vivo. FEBS Lett 2003; 553:7984 [View Article]
    [Google Scholar]
  70. Ohse M, Takahashi K, Kadowaki Y, Kusaoke H. Effects of Plasmid DNA Sizes and Several Other Factors on Transformation of Bacillus subtilis ISW1214 with Plasmid DNA by Electroporation. Biosci Biotechnol Biochem 1995
    [Google Scholar]
  71. Chassy B, Mercenier A, Flickinger J. Transformation of bacteria by electroporation. Trends Biotechnol 1988; 6:303–309 [View Article]
    [Google Scholar]
  72. Zang X, Liu B, Liu S, Arunakumara KKIU, Zhang X. Optimum conditions for transformation of Synechocystis sp. PCC 6803. J Microbiol 2007; 45:241–245
    [Google Scholar]
  73. Garcia PA, Ge Z, Moran JL, Buie CR. Microfluidic screening of electric fields for electroporation. Sci Rep 2016; 6: [View Article]
    [Google Scholar]
  74. Morris JJ, Kirkegaard R, Szul MJ, Johnson ZI, Zinser ER. Facilitation of Robust Growth of Prochlorococcus Colonies and Dilute Liquid Cultures by "Helper" Heterotrophic Bacteria. Appl Environ Microbiol 2008; 74:4530–4534 [View Article]
    [Google Scholar]
  75. Morris JJ, Johnson ZI, Szul MJ, Keller M, Zinser ER. Dependence of the cyanobacterium Prochlorococcus on hydrogen peroxide scavenging microbes for growth at the ocean's surface. PLoS One 2011; 6:e16805 [View Article]
    [Google Scholar]
  76. Tanaka T, Kawasaki K, Daimon S, Kitagawa W, Yamamoto K et al. A hidden pitfall in the preparation of agar media undermines microorganism Cultivability. Appl Environ Microbiol 2014; 80:7659–7666 [View Article]
    [Google Scholar]
  77. Giandomenico AR, Cerniglia GE, Biaglow JE, Stevens CW, Koch CJ. The importance of sodium pyruvate in assessing damage produced by hydrogen peroxide. Free Radical Biology and Medicine 1997; 23:426–434 [View Article]
    [Google Scholar]
  78. Berube PM, Biller SJ, Kent AG, Berta-Thompson JW, Roggensack SE et al. Physiology and evolution of nitrate acquisition in Prochlorococcus . Isme J 2015; 9:1195–1207 [View Article]
    [Google Scholar]
  79. Godbey WT. “Chapter 12 - Genetic Engineering”. An Introduction to Biotechnology 2014
    [Google Scholar]
  80. Goryshin IY, Jendrisak J, Hoffman LM, Meis R, Reznikoff WS. Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes. Nat Biotechnol 2000; 18:97–100 [View Article]
    [Google Scholar]
  81. Fels SR, Zane GM, Blake SM, Wall JD. Rapid transposon liquid enrichment sequencing (TnLE-seq) for gene fitness evaluation in underdeveloped bacterial systems. Appl Environ Microbiol 2013; 79:7510–7517 [View Article]
    [Google Scholar]
  82. Vidal JE, Chen J, Li J, McClane BA. Use of an EZ-Tn5-based random mutagenesis system to identify a novel toxin regulatory locus in Clostridium perfringens strain 13. PLoS One 2009; 4:e6232 [View Article]
    [Google Scholar]
  83. Gallagher LA, Ramage E, Weiss EJ, Radey M, Hayden HS et al. Resources for genetic and genomic analysis of emerging pathogen Acinetobacter baumannii . J Bacteriol 2015; 197:2027–2035 [View Article]
    [Google Scholar]
  84. Dawoud TM, Jiang T, Mandal RK, Ricke SC, Kwon YM. Improving the efficiency of transposon mutagenesis in Salmonella enteritidis by overcoming Host-Restriction barriers. Mol Biotechnol 2014; 56:1004–1010 [View Article]
    [Google Scholar]
  85. Vogel J, Axmann IM, Herzel H, Hess WR. Experimental and computational analysis of transcriptional start sites in the cyanobacterium Prochlorococcus MED4. Nucleic Acids Res 2003; 31:2890–2899 [View Article]
    [Google Scholar]
  86. Trubitsyna M, Michlewski G, Finnegan DJ, Elfick A, Rosser SJ et al. Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection. Nucleic Acids Res 2017; 45:e89 [View Article]
    [Google Scholar]
  87. Liang X, Potter J, Kumar S, Zou Y, Quintanilla R et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol 2015; 208:44–53 [View Article]
    [Google Scholar]
  88. Ungerer J, Pakrasi HB. Cpf1 is a versatile tool for CRISPR genome editing across diverse species of cyanobacteria. Sci Rep 2016; 6: [View Article]
    [Google Scholar]
  89. Doron S, Melamed S, Ofir G, Leavitt A, Lopatina A et al. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 2018; 359:eaar4120 [View Article]
    [Google Scholar]
  90. Martinez A, Kolvek SJ, Yip CLT, Hopke J, Brown KA et al. Genetically modified bacterial strains and novel bacterial artificial chromosome shuttle vectors for constructing environmental libraries and detecting heterologous natural products in multiple expression hosts. Appl Environ Microbiol 2004; 70:2452–2463 [View Article]
    [Google Scholar]
  91. Waldbauer JR, Rodrigue S, Coleman ML, Chisholm SW. Transcriptome and proteome dynamics of a light-dark synchronized bacterial cell cycle. PLoS One 2012; 7:e43432 [View Article]
    [Google Scholar]
  92. Jeamton W, Dulsawat S, Tanticharoen M, Vonshak A, Cheevadhanarak S. Overcoming intrinsic restriction enzyme barriers enhances transformation efficiency in Arthrospira platensis C1. Plant Cell Physiol 2017; 58:822–830 [View Article]
    [Google Scholar]
  93. Kawata Y, Yano S, Kojima H, Toyomizu M. Transformation of Spirulina platensis strain C1 (Arthrospira sp. PCC9438) with Tn5 transposase-transposon DNA-cation liposome complex. Mar Biotechnol 2004
    [Google Scholar]
  94. Puigbò P, Guzmán E, Romeu A, Garcia-Vallvé S. OPTIMIZER: a web server for optimizing the codon usage of DNA sequences. Nucleic Acids Res 2007; 35:W126–W131 [View Article]
    [Google Scholar]
  95. Osburne MS, Holmbeck BM, Coe A, Chisholm SW. The spontaneous mutation frequencies of Prochlorococcus strains are commensurate with those of other bacteria. Environ Microbiol Rep 2011; 3:744–749 [View Article]
    [Google Scholar]
  96. Larsen RA, Wilson MM, Guss AM, Metcalf WW. Genetic analysis of pigment biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria. Arch Microbiol 2002; 178:193–201 [View Article]
    [Google Scholar]
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