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Genetic Methods and Construction of Chromosomal Mutations in Methanogenic Archaea

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2522))

Abstract

Genetic manipulation through markerless exchange enables the modification of several genomic regions without leaving a selection marker in the genome. Here, a method using hpt coding for hypoxanthine phosphoribosyltransferase as a counter selectable marker is described. For Methanosarcina species a chromosomal deletion of the hpt gene is firstly generated, which confers resistance to the purine analogue 8-aza-2,6-diaminopurine (8-ADP). In a second step, the reintroduction of the hpt gene on a plasmid leads to a selectable loss of 8-ADP resistance after a homologous recombination event (pop-in). A subsequent pop-out event restores the 8-ADP resistance and can generate chromosomal mutants with frequencies of about 50%.

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References

  1. Pfeifer K, Ergal İ, Koller M et al (2020) Archaea biotechnology. Biotechnol Adv 47:107668. https://doi.org/10.1016/j.biotechadv.2020.107668

    Article  CAS  PubMed  Google Scholar 

  2. Enzmann F, Mayer F, Rother M et al (2018) Methanogens: biochemical background and biotechnological applications. AMB Express 8(1):1. https://doi.org/10.1186/s13568-017-0531-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Balch WE, Fox GE, Magrum LJ et al (1979) Methanogens: reevaluation of a unique biological group. Microbiol Rev 43(2):260–296

    Article  CAS  Google Scholar 

  4. Liu Y, Whitman WB (2008) Metabolic, phylogenetic, and ecological diversity of the methanogenic archaea. Ann N Y Acad Sci 1125:171–189. https://doi.org/10.1196/annals.1419.019

    Article  CAS  PubMed  Google Scholar 

  5. Leigh JA, Albers S-V, Atomi H et al (2011) Model organisms for genetics in the domain archaea: methanogens, halophiles, Thermococcales and Sulfolobales. FEMS Microbiol Rev 35(4):577–608. https://doi.org/10.1111/j.1574-6976.2011.00265.x

    Article  CAS  PubMed  Google Scholar 

  6. de Vrieze J, Hennebel T, Boon N et al (2012) Methanosarcina: the rediscovered methanogen for heavy duty biomethanation. Bioresour Technol 112:1–9. https://doi.org/10.1016/j.biortech.2012.02.079

    Article  CAS  PubMed  Google Scholar 

  7. Rother M, Metcalf WW (2005) Genetic technologies for archaea. Curr Opin Microbiol 8(6):745–751. https://doi.org/10.1016/j.mib.2005.10.010

    Article  CAS  PubMed  Google Scholar 

  8. Deppenmeier U, Johann A, Hartsch T et al (2002) The genome of Methanosarcina mazei: evidence for lateral gene transfer between bacteria and archaea. J Mol Microbiol Biotechnol 4(4):453–461

    CAS  PubMed  Google Scholar 

  9. Kohler PRA, Metcalf WW (2012) Genetic manipulation of Methanosarcina spp. Front Microbiol 3:259. https://doi.org/10.3389/fmicb.2012.00259

    Article  PubMed  PubMed Central  Google Scholar 

  10. Ehlers C, Jäger D, Schmitz RA (2011) Establishing a markerless genetic exchange system for Methanosarcina mazei strain Gö1 for constructing chromosomal mutants of small RNA genes. Archaea 2011:439608. https://doi.org/10.1155/2011/439608

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Tumbula DL, Makula RA, Whitman WB (1994) Transformation of Methanococcus maripaludis and identification of a PstI-like restriction system. FEMS Microbiol Lett 121(3):309–314. https://doi.org/10.1111/j.1574-6968.1994.tb07118.x

    Article  CAS  Google Scholar 

  12. Bertani G, Baresi L (1987) Genetic transformation in the methanogen Methanococcus voltae PS. J Bacteriol 169(6):2730–2738. https://doi.org/10.1128/jb.169.6.2730-2738.1987

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Metcalf WW, Zhang JK, Apolinario E et al (1997) A genetic system for archaea of the genus Methanosarcina: liposome-mediated transformation and construction of shuttle vectors. Proc Natl Acad Sci U S A 94(6):2626–2631. https://doi.org/10.1073/pnas.94.6.2626

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Gernhardt P, Possot O, Foglino M et al (1990) Construction of an integration vector for use in the archaebacterium Methanococcus voltae and expression of a eubacterial resistance gene. Mol Gen Genet 221(2):273–279. https://doi.org/10.1007/BF00261731

    Article  CAS  PubMed  Google Scholar 

  15. Tumbula DL, Bowen TL, Whitman WB (1997) Characterization of pURB500 from the archaeon Methanococcus maripaludis and construction of a shuttle vector. J Bacteriol 179(9):2976–2986. https://doi.org/10.1128/jb.179.9.2976-2986.1997

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Walters AD, Smith SE, Chong JPJ (2011) Shuttle vector system for Methanococcus maripaludis with improved transformation efficiency. Appl Environ Microbiol 77(7):2549–2551. https://doi.org/10.1128/AEM.02919-10

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Sowers KR, Gunsalus RP (1988) Plasmid DNA from the acetotrophic methanogen Methanosarcina acetivorans. J Bacteriol 170(10):4979–4982. https://doi.org/10.1128/jb.170.10.4979-4982.1988

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Wood AG, Whitman WB, Konisky J (1985) A newly-isolated marine methanogen harbors a small cryptic plasmid. Arch Microbiol 142(3):259–261. https://doi.org/10.1007/BF00693400

    Article  CAS  PubMed  Google Scholar 

  19. Guss AM, Rother M, Zhang JK et al (2008) New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species. Archaea 2(3):193–203. https://doi.org/10.1155/2008/534081

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Mondorf S, Deppenmeier U, Welte C (2012) A novel inducible protein production system and neomycin resistance as selection marker for Methanosarcina mazei. Archaea 2012:973743. https://doi.org/10.1155/2012/973743

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Argyle JL, Tumbula DL, Leigh JA (1996) Neomycin resistance as a selectable marker in Methanococcus maripaludis. Appl Environ Microbiol 62(11):4233–4237. https://doi.org/10.1128/AEM.62.11.4233-4237.1996

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Farley KR, Metcalf WW (2019) The streptothricin acetyltransferase (sat) gene as a positive selectable marker for methanogenic archaea. FEMS Microbiol Lett 366(17):fnz216. https://doi.org/10.1093/femsle/fnz216

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Boccazzi P, Zhang JK, Metcalf WW (2000) Generation of dominant selectable markers for resistance to pseudomonic acid by cloning and mutagenesis of the ileS gene from the archaeon Methanosarcina barkeri Fusaro. J Bacteriol 182(9):2611–2618. https://doi.org/10.1128/jb.182.9.2611-2618.2000

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Zhang JK, White AK, Kuettner HC et al (2002) Directed mutagenesis and plasmid-based complementation in the methanogenic archaeon Methanosarcina acetivorans C2A demonstrated by genetic analysis of proline biosynthesis. J Bacteriol 184(5):1449–1454. https://doi.org/10.1128/jb.184.5.1449-1454.2002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Pritchett MA, Zhang JK, Metcalf WW (2004) Development of a markerless genetic exchange method for Methanosarcina acetivorans C2A and its use in construction of new genetic tools for methanogenic archaea. Appl Environ Microbiol 70(3):1425–1433. https://doi.org/10.1128/aem.70.3.1425-1433.2004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Moore BC, Leigh JA (2005) Markerless mutagenesis in Methanococcus maripaludis demonstrates roles for alanine dehydrogenase, alanine racemase, and alanine permease. J Bacteriol 187(3):972–979. https://doi.org/10.1128/JB.187.3.972-979.2005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Wolin EA, Wolin MJ, Wolfe RS (1963) Formation of methane by bacterial extracts. J Biol Chem 238:2882–2886

    Article  CAS  Google Scholar 

  28. Pfennig N, Lippert KD (1966) Über das Vitamin B12-Bedürfnis phototropher Schwefelbakterien. Archiv Mikrobiol 55(3):245–256. https://doi.org/10.1007/BF00410246

    Article  CAS  Google Scholar 

  29. Hippe H, Caspari D, Fiebig K et al (1979) Utilization of trimethylamine and other N-methyl compounds for growth and methane formation by Methanosarcina barkeri. Proc Natl Acad Sci U S A 76(1):494–498. https://doi.org/10.1073/pnas.76.1.494

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ehlers C, Weidenbach K, Veit K et al (2005) Development of genetic methods and construction of a chromosomal glnK1 mutant in Methanosarcina mazei strain Gö1. Mol Gen Genomics 273(4):290–298. https://doi.org/10.1007/s00438-005-1128-7

    Article  CAS  Google Scholar 

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Acknowledgments

The project was financially supported by Bundesministerium für Bildung und Forschung (BMBF) (031B0851B to RAS, within the MethanoPEP consortium). The authors thank Britta Jordan for providing the colony pictures and for helpful suggestions.

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Authors and Affiliations

  1. Institute For General Microbiology, Christian-Albrechts-University, Kiel, Germany

    Johanna Thomsen, Katrin Weidenbach & Ruth A. Schmitz

  2. Department of Microbiology, University of Illinois, Urbana, IL, USA

    William W. Metcalf

Authors
  1. Johanna Thomsen
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  2. Katrin Weidenbach
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  3. William W. Metcalf
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  4. Ruth A. Schmitz
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Corresponding author

Correspondence to Ruth A. Schmitz .

Editor information

Editors and Affiliations

  1. Cellular Biochemistry of Microorganisms - Biochemie III, Universität Regensburg, Regensburg, Bayern, Germany

    Sébastien Ferreira-Cerca

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Thomsen, J., Weidenbach, K., Metcalf, W.W., Schmitz, R.A. (2022). Genetic Methods and Construction of Chromosomal Mutations in Methanogenic Archaea. In: Ferreira-Cerca, S. (eds) Archaea. Methods in Molecular Biology, vol 2522. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2445-6_6

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  • DOI: https://doi.org/10.1007/978-1-0716-2445-6_6

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2444-9

  • Online ISBN: 978-1-0716-2445-6

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