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Microbiology Society logo Microbiology

Volume 170, Issue 5

A vector system for single and tandem expression of cloned genes and multi-colour fluorescent tagging in Open Access

Abstract

Archaeal cell biology is an emerging field expected to identify fundamental cellular processes, help resolve the deep evolutionary history of cellular life, and contribute new components and functions in biotechnology and synthetic biology. To facilitate these, we have developed plasmid vectors that allow convenient cloning and production of proteins and fusion proteins with flexible, rigid, or semi-rigid linkers in the model archaeon . For protein subcellular localization studies using fluorescent protein (FP) tags, we created vectors incorporating a range of codon-optimized fluorescent proteins for N- or C-terminal tagging, including GFP, mNeonGreen, mCherry, YPet, mTurquoise2 and mScarlet-I. Obtaining functional fusion proteins can be challenging with proteins involved in multiple interactions, mainly due to steric interference. We demonstrated the use of the new vector system to screen for improved function in cytoskeletal protein FP fusions, and identified FtsZ1–FPs that are functional in cell division and CetZ1–FPs that are functional in motility and rod cell development. Both the type of linker and the type of FP influenced the functionality of the resulting fusions. The vector design also facilitates convenient cloning and tandem expression of two genes or fusion genes, controlled by a modified tryptophan-inducible promoter, and we demonstrated its use for dual-colour imaging of tagged proteins in cells. These tools should promote further development and applications of archaeal molecular and cellular biology and biotechnology.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): archaea , CetZ , cloning vector , cytoskeleton , fluorescent protein and FtsZ
Funding
This study was supported by the:
  • Australian Research Council (Award FT160100010)
    • Principle Award Recipient: IainGeoffrey Duggin
  • Australian Research Council (Award DP160101076)
    • Principle Award Recipient: IainGeoffrey Duggin
© 2024 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2024-05-24
2025-05-21
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References

  1. Charles-Orszag A, Lord SJ, Mullins RD. High-temperature live-cell imaging of cytokinesis, cell motility, and cell-cell interactions in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Front Microbiol 2021; 12:707124 [View Article] [PubMed]
     [Google Scholar]
  2. Duggin IG, McCallum SA, Bell SD. Chromosome replication dynamics in the archaeon Sulfolobus acidocaldarius. Proc Natl Acad Sci U S A 2008; 105:16737–16742 [View Article] [PubMed]
     [Google Scholar]
  3. Samson RY, Obita T, Hodgson B, Shaw MK, Chong PL-G et al. Molecular and structural basis of ESCRT-III recruitment to membranes during archaeal cell division. Mol Cell 2011; 41:186–196 [View Article] [PubMed]
     [Google Scholar]
  4. Pulschen AA, Mutavchiev DR, Culley S, Sebastian KN, Roubinet J et al. Live imaging of a hyperthermophilic archaeon reveals distinct roles for two ESCRT-III homologs in ensuring a robust and symmetric division. Curr Biol 2020; 30:2852–2859 [View Article] [PubMed]
     [Google Scholar]
  5. Mullakhanbhai MF, Larsen H. Halobacterium volcanii spec. nov., a Dead Sea halobacterium with a moderate salt requirement. Arch Microbiol 1975; 104:207–214 [View Article] [PubMed]
     [Google Scholar]
  6. Allers T, Barak S, Liddell S, Wardell K, Mevarech M. Improved strains and plasmid vectors for conditional overexpression of His-tagged proteins in Haloferax volcanii. Appl Environ Microbiol 2010; 76:1759–1769 [View Article] [PubMed]
     [Google Scholar]
  7. Pérez-Arnaiz P, Dattani A, Smith V, Allers T. Haloferax volcanii-a model archaeon for studying DNA replication and repair. Open Biol 2020; 10:200293 [View Article] [PubMed]
     [Google Scholar]
  8. Bisson-Filho AW, Zheng J, Garner E. Archaeal imaging: leading the hunt for new discoveries. Mol Biol Cell 2018; 29:1675–1681 [View Article] [PubMed]
     [Google Scholar]
  9. de Silva RT, Abdul-Halim MF, Pittrich DA, Brown HJ, Pohlschroder M et al. Improved growth and morphological plasticity of Haloferax volcanii. Microbiology 2021; 167:001012 [View Article] [PubMed]
     [Google Scholar]
  10. Delmas S, Duggin IG, Allers T. DNA damage induces nucleoid compaction via the Mre11-Rad50 complex in the archaeon Haloferax volcanii. Mol Microbiol 2013; 87:168–179 [View Article] [PubMed]
     [Google Scholar]
  11. Duggin IG, Aylett CHS, Walsh JC, Michie KA, Wang Q et al. CetZ tubulin-like proteins control archaeal cell shape. Nature 2015; 519:362–365 [View Article] [PubMed]
     [Google Scholar]
  12. Turkowyd B, Schreiber S, Wörtz J, Segal ES, Mevarech M et al. Establishing live-cell single-molecule localization microscopy imaging and single-particle tracking in the archaeon Haloferax volcanii. Front Microbiol 2020; 11:583010 [View Article] [PubMed]
     [Google Scholar]
  13. Lippincott-Schwartz J, Snapp E, Kenworthy A. Studying protein dynamics in living cells. Nat Rev Mol Cell Biol 2001; 2:444–456 [View Article] [PubMed]
     [Google Scholar]
  14. Landgraf D, Okumus B, Chien P, Baker TA, Paulsson J. Segregation of molecules at cell division reveals native protein localization. Nat Methods 2012; 9:480–482 [View Article] [PubMed]
     [Google Scholar]
  15. Margolin W. The price of tags in protein localization studies. J Bacteriol 2012; 194:6369–6371 [View Article] [PubMed]
     [Google Scholar]
  16. Ma X, Ehrhardt DW, Margolin W. Colocalization of cell division proteins FtsZ and FtsA to cytoskeletal structures in living Escherichia coli cells by using green fluorescent protein. Proc Natl Acad Sci U S A 1996; 93:12998–13003 [View Article] [PubMed]
     [Google Scholar]
  17. McQuillen R, Xiao J. Insights into the structure, function, and dynamics of the bacterial Cytokinetic FtsZ-Ring. Annu Rev Biophys 2020; 49:309–341 [View Article] [PubMed]
     [Google Scholar]
  18. Moore DA, Whatley ZN, Joshi CP, Osawa M, Erickson HP. Probing for binding regions of the FtsZ protein surface through site-directed insertions: discovery of fully functional FtsZ-fluorescent proteins. J Bacteriol 2017; 199:e00553-16 [View Article] [PubMed]
     [Google Scholar]
  19. Crivat G, Taraska JW. Imaging proteins inside cells with fluorescent tags. Trends Biotechnol 2012; 30:8–16 [View Article] [PubMed]
     [Google Scholar]
  20. Abdul-Halim MF, Schulze S, DiLucido A, Pfeiffer F, Bisson Filho AW et al. Lipid anchoring of archaeosortase substrates and midcell growth in haloarchaea. mBio 2020; 11:e00349-20 [View Article] [PubMed]
     [Google Scholar]
  21. Delpech F, Collien Y, Mahou P, Beaurepaire E, Myllykallio H et al. Snapshots of archaeal DNA replication and repair in living cells using super-resolution imaging. Nucleic Acids Res 2018; 46:10757–10770 [View Article] [PubMed]
     [Google Scholar]
  22. Liao Y, Ithurbide S, Evenhuis C, Löwe J, Duggin IG. Cell division in the archaeon Haloferax volcanii relies on two FtsZ proteins with distinct functions in division ring assembly and constriction. Nat Microbiol 2021; 6:594–605 [View Article] [PubMed]
     [Google Scholar]
  23. Kamekura M, Oesterhelt D, Wallace R, Anderson P, Kushner DJ. Lysis of halobacteria in bacto-peptone by bile acids. Appl Environ Microbiol 1988; 54:990–995 [View Article] [PubMed]
     [Google Scholar]
  24. Allers T, Ngo HP, Mevarech M, Lloyd RG. Development of additional selectable markers for the halophilic archaeon Haloferax volcanii based on the leuB and trpA genes. Appl Environ Microbiol 2004; 70:943–953 [View Article] [PubMed]
     [Google Scholar]
  25. Cline SW, Schalkwyk LC, Doolittle WF. Transformation of the archaebacterium Halobacterium volcanii with genomic DNA. J Bacteriol 1989; 171:4987–4991 [View Article] [PubMed]
     [Google Scholar]
  26. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods 2012; 9:671–675 [View Article]
     [Google Scholar]
  27. Ducret A, Quardokus EM, Brun YV. MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis. Nat Microbiol 2016; 1:16077 [View Article] [PubMed]
     [Google Scholar]
  28. Arai R, Ueda H, Kitayama A, Kamiya N, Nagamune T. Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Eng 2001; 14:529–532 [View Article] [PubMed]
     [Google Scholar]
  29. Chen X, Zaro JL, Shen WC. Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev 2013; 65:1357–1369 [View Article] [PubMed]
     [Google Scholar]
  30. Li G, Huang Z, Zhang C, Dong B-J, Guo R-H et al. Construction of a linker library with widely controllable flexibility for fusion protein design. Appl Microbiol Biotechnol 2016; 100:215–225 [View Article] [PubMed]
     [Google Scholar]
  31. Shaner NC, Lambert GG, Chammas A, Ni Y, Cranfill PJ et al. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods 2013; 10:407–409 [View Article] [PubMed]
     [Google Scholar]
  32. Nguyen AW, Daugherty PS. Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat Biotechnol 2005; 23:355–360 [View Article] [PubMed]
     [Google Scholar]
  33. Goedhart J, von Stetten D, Noirclerc-Savoye M, Lelimousin M, Joosen L et al. Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%. Nat Commun 2012; 3:751 [View Article] [PubMed]
     [Google Scholar]
  34. Bindels DS, Haarbosch L, van Weeren L, Postma M, Wiese KE et al. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nat Methods 2017; 14:53–56 [View Article] [PubMed]
     [Google Scholar]
  35. Reuter CJ, Maupin-Furlow JA. Analysis of proteasome-dependent proteolysis in Haloferax volcanii cells, using short-lived green fluorescent proteins. Appl Environ Microbiol 2004; 70:7530–7538 [View Article] [PubMed]
     [Google Scholar]
  36. Nussbaum P, Ithurbide S, Walsh JC, Patro M, Delpech F et al. An oscillating MinD protein determines the cellular positioning of the motility machinery in archaea. Curr Biol 2020; 30:4956–4972 [View Article] [PubMed]
     [Google Scholar]
  37. Snapp E. Design and use of fluorescent fusion proteins in cell biology. Curr Protoc Cell Biol 2005; Chapter 21:21 [View Article] [PubMed]
     [Google Scholar]
  38. Large A, Stamme C, Lange C, Duan Z, Allers T et al. Characterization of a tightly controlled promoter of the halophilic archaeon Haloferax volcanii and its use in the analysis of the essential cct1 gene. Mol Microbiol 2007; 66:1092–1106 [View Article] [PubMed]
     [Google Scholar]
  39. Sheridan DL, Berlot CH, Robert A, Inglis FM, Jakobsdottir KB et al. A new way to rapidly create functional, fluorescent fusion proteins: random insertion of GFP with an in vitro transposition reaction. BMC Neurosci 2002; 3:7 [View Article] [PubMed]
     [Google Scholar]
/content/journal/micro/10.1099/mic.0.001461
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A vector system for single and tandem expression of cloned genes and multi-colour fluorescent tagging in Haloferax volcanii
Microbiology 170, 001461 (2024); https://doi.org/10.1099/mic.0.001461
/content/journal/micro/10.1099/mic.0.001461
/content/journal/micro/10.1099/mic.0.001461
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