Abstract
The methanogenic archaeon Methanosarcina mazei strain Gö1 has so far proven to be genetically intractable due to its low plating efficiency on solid medium and the lack of an effective transformation method. Here, we report the first significant improvement in plating efficiency (up to 10%), which was achieved by (1) selecting for a spontaneous mutant of M. mazei that shows significantly higher resistance to mechanical stress during spreading an agar plates, and (2) plating the cells in 0.5% top agar with trimethylamine as a carbon and energy source under a H2S-containing atmosphere (0.1%). Using this mutant we succeeded in establishing a liposome-mediated transformation protocol, which for the first time allowed genetic manipulation of the M. mazei Gö1 strain. We further report on the construction of the first chromosomal deletion mutant of M. mazei by means of homologous recombination. Characterization of this mutant strain revealed that M. mazei cells lacking a functional glnK1-gene exhibited a partial growth defect under nitrogen limitation when molecular nitrogen was used as the sole nitrogen source. Quantitative RT-PCR analysis, however, showed that genes involved in nitrogen assimilation or nitrogen fixation are transcribed in the glnK1 mutant as in the wild type. Thus, we propose that the archaeal GlnK1 protein is not directly involved in the transcriptional regulation of genes involved in nitrogen metabolism, but rather affects their protein products directly.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aravalli RN, Garrett RA (1997) Shuttle vectors for hyperthermophilic archaea. Extremophiles 1:183–191
Arcondeguy T, Jack R, Merrick M (2001) P(II) signal transduction proteins: pivotal players in microbial nitrogen control. Microbiol Mol Biol Rev 65:80–105
Argyle JL, Tumbula DL, Leigh JA (1996) Neomycin resistance as a selectable marker in Methanococcus maripaludis. Appl Environ Microbiol 62:4233–4237
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 182:2611–2618
Coutts G, Thomas G, Blakey D, Merrick M (2002) Membrane sequestration of the signal transduction protein GlnK by the ammonium transporter AmtB. EMBO J 21:536–545
Deppenmeier U et al (2002) The genome of Methanosarcina mazei: evidence for lateral gene transfer between bacteria and archaea. J Mol Microbiol Biotechnol 4:453–461
Deppenmeier U, Blaut M, Mahlmann A, Gottschalk G (1990) Reduced coenzyme F420: heterodisulfide oxidoreductase, a proton-translocating redox system in methanogenic bacteria. Proc Natl Acad Sci USA 87:9449–9453
Ehlers C, Grabbe R, Veit K, Schmitz RA (2002a) Characterization of GlnK1 from Methanosarcina mazei strain Gö1: complementation of an Escherichia coli glnK mutant strain by GlnK1. J Bacteriol 184:1028–1040
Ehlers C, Veit K, Gottschalk G, Schmitz RA (2002b) Functional organization of a single nif cluster in the mesophilic archaeon Methanosarcina mazei strain Gö1. Archaea 1:143–150
Ehlers C, Weidenbach K, Veit K, Forchhammer K, Schmitz RA (2004) Unique mechanistic features of posttranslational regulation of glutamine synthetase activity in Methanosarcina mazei strain Gö1 in response to nitrogen availability. Mol Microbiol 55:1841–1854
Gernhardt P, Possot O, Foglino M, Sibold L, Klein A (1990) Construction of an integration vector for use in the archaebacterium Methanococcus voltae and expression of a eubacterial resistance gene. Mol Gen Genet 221:273–279
Heinrich A, Maheswaran M, Ruppert U, Forchhammer K (2004) The Synechococcus elongatus P signal transduction protein controls arginine synthesis by complex formation with N-acetyl-L-glutamate kinase. Mol Microbiol 52:1303–1314
Hippe H, Caspari D, Fiebig K, Gottschalk G (1979) Utilization of trimethylamine and other N-methyl compounds for growth and methane formation by Methanosarcina barkeri. Proc Natl Acad Sci USA 76:494–498
Holmes ML, Dyall-Smith ML (1991) Mutations in DNA gyrase result in novobiocin resistance in halophilic archaeabacteria. J Bacteriol 173:642–648
Inoue H, Nojima H, Okayama H (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96:23–28
Javelle A, Severi E, Thornton J, Merrick M (2004) Ammonium sensing in Escherichia coli. Role of the ammonium transporter AmtB and AmtB-GlnK complex formation. J Biol Chem 279:8530–8538
Jiang P, Peliska JA, Ninfa AJ (1998) Reconstitution of the signal-transduction bicyclic cascade responsible for the regulation of Ntr gene transcription in Escherichia coli. Biochemistry 37:12795–12801
Kessler PS, Blank C, Leigh JA (1998) The nif gene operon of the methanogenic archaeon Methanococcus maripaludis. J Bacteriol 180:1504–1511
Kessler PS, Daniel C, Leigh JA (2001) Ammonia switch-off of nitrogen fixation in the methanogenic archaeon Methanococcus maripaludis: mechanistic features and requirement for the novel GlnB homologues, NifI(1) and NifI(2). J Bacteriol 183:882–889
Kiener A, Rechsteiner T, Leisinger T (1986) Mutation to pseudomonic acid resistance of Methanobacterium thermoautotrophicum leads to an altered isoleucyl-tRNA synthetase. FEMS Microbiol Lett 33:15–18
Krebs MP, Mollaaghababa, Khorana HG (1993) Gene replacement in Halobacterium halobium and expression of bacteriorhodopsin mutants. Proc Natl Acad Sci USA 90:1987–1991
Mankin AS, Zyrianova IM, Kagramanova VK, Garrett RA (1992) Introducing mutations into the single-copy chromosomal 23S rRNA gene of the archaeon Halobacterium halobium by using an rRNA operon-based transformation system. Proc Natl Acad Sci USA 89:6535–6539
Metcalf WW, Zhang JK, Apolinario E, Sowers KR, Wolfe RS (1997) A genetic system for Archaea of the genus Methanosarcina: liposome-mediated transformation and construction of shuttle vectors. Proc Natl Acad Sci USA 94:2626–2631
Metcalf WW, Zhang JK, Wolfe RS (1998) An anaerobic, intrachamber incubator for growth of Methanosarcina spp. on methanol-containing solid media. Appl Environ Microbiol 64:768–770
Miller VL, Mekalanos JJ (1988) A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol 170:2575–2583
Possot O, Gernhardt P, Klein A, Sibold L (1988) Analysis of drug resistance in the archaebacterium Methanococcus voltae with respect to potential use in genetic engineering. Appl Environ Microbiol 54:734–740
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 70:1425–1433
Reitzer L (2003) Nitrogen assimilation and global regulation in Escherichia coli. Annu Rev Microbiol 57:155–176
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY
Sowers KR, Gunsalus RP (1988) Plasmid DNA from the acetotrophic methanogen Methanosarcina acetivorans. J Bacteriol 170:4979–4982
Talaat AM, Lyons R, Howard ST, Johnston SA (2004) The temporal expression profile of Mycobacterium tuberculosis infection in mice. Proc Natl Acad Sci USA 101:4602–4607
Tumbula DL, Makula RA, Whitman WB (1994) Transformation of Methanococcus maripaludis and identification of a PstI-like restriction system. FEMS Microbiol Lett 121:309–314
Zhang JK, Pritchett MA, Lampe DJ, Robertson HM, Metcalf WW (2000) In vivo transposon mutagenesis of the methanogenic archaeon Methanosarcina acetivorans C2A using a modified version of the insect mariner-family transposable element Himar1. Proc Natl Acad Sci USA 97:9665–9670
Zhang JK, White AK, Kuettner HC, Boccazzi P, Metcalf WW (2002) Directed mutagenesis and plasmid-based complementation in the methanogenic archaeon Methanosarcina acetivorans C2A demonstrated by genetic analysis of proline biosynthesis. J Bacteriol 184:1449–1454
Acknowledgements
We thank Gerhard Gottschalk for continuous support and helpful discussions. This work was carried out in compliance with the current laws governing genetic experimentation in the country concerned. This work was supported by the Deutsche Forschungsgemeinschaft (SCHM1052/6-1 and 6-2) and by a Ph.D. fellowship awarded to C.E. by the Fonds der Chemischen Industrie.
Author information
Authors and Affiliations
Corresponding author
Additional information
A. Hirsch
Rights and permissions
About this article
Cite this article
Ehlers, C., Weidenbach, K., Veit, K. et al. Development of genetic methods and construction of a chromosomal glnK1 mutant in Methanosarcina mazei strain Gö1. Mol Genet Genomics 273, 290–298 (2005). https://doi.org/10.1007/s00438-005-1128-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00438-005-1128-7