Chapter 21 : Molecular Genetics of

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Preview this chapter:
Zoom in

Molecular Genetics of , Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555815516/9781555813918_Chap21-1.gif /docserver/preview/fulltext/10.1128/9781555815516/9781555813918_Chap21-2.gif


This chapter discusses molecular genetics of . Gene transfer systems currently exist for species within all three physiological groups of archaea, halophiles, methanogens, and nonmethanogenic hyperthermophiles. The chapter reviews the development of these systems. Colonization of methanogenic and nonmethanogenic hyperthermophiles on solidified medium is equally problematic, as agar is rapidly dehydrated at high temperatures, especially at the concentrations required for it to remain solidified. Therefore, gellan gum (Gelrite) is used as the solidifying agent for growth of thermophiles and hyperthermophiles, which are incubated in a plastic bag or anaerobe jar to minimize dehydration. Haloarchaea are transformed via polyethylene glycol (PEG) mediated transformation, which was first described in . Gene disruption is required to identify and confirm the function of genes within the archaea. Random mutagenesis using chemical and UV radiation has been successfully used for , , , and . Progress in the development of methodologies for archaeal genetics has rapidly accelerated in the past decade. Additional methods are currently under development for all three archaeal phyla, including additional systems for markerless exchange, gene expression, topological mapping, protein tagging and expression, as well as others. The choice of archaeal genomes for sequencing is now largely driven by the availability of genetic systems, which at present include complete genomes of the halophiles and sp.

Citation: Sowers K, Anderson K. 2007. Molecular Genetics of , p 463-477. In Cavicchioli R (ed), Archaea. ASM Press, Washington, DC. doi: 10.1128/9781555815516.ch21
Highlighted Text: Show | Hide
Loading full text...

Full text loading...


Image of Figure 1.
Figure 1.

Recombinant plasmid showing construction typical for an archaeal shuttle vector. The construct includes the dependent R6K for replication of the plasmid in the gene for selection of transformants with ampicillin, pC2A and for replication in spp., and under transcriptional control of the archaeal gene for selection of methanosarcinal recombinants on puromycin. pC2A, autonomously replicating plasmid pC2A from puromycin -acetyltransferase flanked by the methylCoM reduc-tase archaeal promoter (pmcr) and terminator (tmcr); oriR6K, dependent R6K origin of replication; β-lactamase; mcs, multiple cloning site in the gene encoding β-galactosidase for blue-white screening of DNA insertion.

Citation: Sowers K, Anderson K. 2007. Molecular Genetics of , p 463-477. In Cavicchioli R (ed), Archaea. ASM Press, Washington, DC. doi: 10.1128/9781555815516.ch21
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 2.
Figure 2.

Gene disruption methods that are used in archaeal genetics. (a) Direct replacement of a gene with a selectable marker occurs by recombination between linear DNA flanked by regions of the target gene and the wild-type chromosomal gene. (b) The “pop-in pop-out” method uses circular DNA and selection for transformation to uracil prototrophy using a strain ( ). Recombinants that have lost the plasmid are counter-selected using 5-fluoroorotic acid (5-FOA), which inhibits growth of cells. Deletion mutants must be screened by Southern hybridization. (c) A variant of the “pop-in pop-out” method for gene deletion utilizes a genetic marker for gene disruption that allows direct selection ( ). (d) Another variant used for generating point mutations employs gene replacement with a marker and subsequent replacement of the disrupted target gene with a gene containing the desired point mutation ( ). Mutants with the desired point mutation are counter-selected with 5-FOA. Reprinted from ( ) with permission of the publisher.

Citation: Sowers K, Anderson K. 2007. Molecular Genetics of , p 463-477. In Cavicchioli R (ed), Archaea. ASM Press, Washington, DC. doi: 10.1128/9781555815516.ch21
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 3.
Figure 3.

Markerless disruption method that employs Flp recombinase. An artificial operon that expresses puromycin -acetyl-transferase and hypoxanthine phosphoribosyltransferase is flanked by Flp recombinase recognition sites (RP1 and RP2) and regions homologous to the target gene. The linear DNA is transformed into an Δhpt strain that is resistant to 8-aza-2,6-diamino-purine (8-ADP). The target gene is replaced by homologous recombination, and recombinants are selected by resistance to puromycin. The deletion mutant is subsequently transformed with the nonreplicating plasmid pMR55 encoding Flp recombinase, which removes the operon by site-specific recombination between RP1 and RP2. Reprinted from ( ) with permission of the publisher.

Citation: Sowers K, Anderson K. 2007. Molecular Genetics of , p 463-477. In Cavicchioli R (ed), Archaea. ASM Press, Washington, DC. doi: 10.1128/9781555815516.ch21
Permissions and Reprints Request Permissions
Download as Powerpoint


1. Aagaard, C.,, J. Z. Dalgaard, and, R. A. Garrett. 1995. Intercellular mobility and homing of an archaeal rDNA intron confers a selective advantage over intron - cells of Sulfolobus acidocalderius. Proc. Natl. Acad. Sci. USA 92: 1228512289.
2. Aagaard, C.,, I. Leviev,, R. N. Aravalli,, P. Forterre,, D. Prieur, and, R. A. Garrett. 1996. General vectors for archaeal hyper-thermophiles: strategies based on a mobile intron and a plasmid. FEMS Microbiol. Rev. 18: 93104.
3. Allers, T, and, M. Mevarech. 2005. Archaeal genetics—the third way. Nat. Rev. Genet. 6: 5873.
4. Allers, T,, H. P. Ngo,, M. Mevarech, and, R. G. Lloyd. 2004. Development of additional selectable markers for the halophilic archaeon Haloferax volcanii based on the leuB and trpA genes. Appl. Environ. Microbiol. 70: 943953.
5. Reference deleted.
6. Apolinario, E. A., and, K. R. Sowers. 1996. Plate colonization of Methanococcus maripaludis and Methanosarcina thermophila in a modified canning jar. FEMS Microbiol. Lett. 145: 131137.
7. Apolinario-Smith, E.,, K. M. Jackson, and, K. R. Sowers. 2005. Development of a plasmid-mediated reporter system for in vivo monitoring of gene expression in the archaeon Methanosarcina acetivorans. Appl. Envion. Microbiol. 71: 49144918.
8. Aravalli, R. N., and, R. A. Garrett. 1997. Shuttle vectors for hyperthermophilic archaea. Extremophiles 1: 183191.
9. Argyle, J. L.,, D. L. Tumbula, and, J. A. Leigh. 1996. Neomycin resistance as a selectable marker in Methanococcus maripaludis. Appl. Environ. Microbiol. 62: 42334237.
10. Balch, W. E.,, G. E. Fox,, L. J. Magrum,, C. R. Woese, and, R. S. Wolfe. 1979. Methanogens: reevaluation of a unique biological group. Microbiol. Rev. 43: 260296.
11. Balch, W. E., and, R. S. Wolfe. 1976. New approach to the cultivation of methanogenic bacteria: 2-mercaptoethanesul-fonic acid (HS-CoM)-dependent growth of Methanobacterium ruminantium in a pressurized atmosphere. Appl. Environ. Microbiol. 32: 781791.
12. Bardy, S. L., and, K. F. Jarrell. 2003. Cleavage of preflagellins by an aspartic acid signal peptidase is essential for flagellation in the archaeon Methanococcus voltae. Mol. Microbiol. 50: 13391347.
13. Bartolucci, S.,, M. Rossi, and, R. Cannio. 2003. Characterization and functional complementation of a nonlethal deletion in the chromosome of a beta-glycosidase mutant of Sulfolobus sol-fataricus. J. Bacterial. 185: 39483957.
14. Beckler, G. S.,, L. A. Hook, and, J. N. Reeve. 1984. Chloramphenicol acetyltransferase should not provide methanogens with resistance to chloramphenicol. Appl. Environ. Microbiol. 47: 868869.
15. Beneke, S.,, H. Bestgen, and, A. Klein. 1995. Use of the Escherichia coli uidA gene as a reporter in Methanococcus voltae for the analysis of the regulatory function of the inter-genic region between the operons encoding selenium-free hydrogenases. Mol. Gen. Genet. 248: 225228.
16. Bertani, G., and, L. Baresi. 1987. Genetic transformation in the methanogen Methanococcus voltae PS. J. Bacteriol. l69: 27302738.
17. Bitan-Banin,, R. O. G, and, M. Mevarech. 2003. Development of a gene knockout system for the halophilic archaeon Haloferax volcanii by use of the pyrE gene. J. Bacteriol. 185: 772778.
18. Blaseio, U., and, F. Pfeifer. 1990. Transformation of Halobacterium halobium: development of vectors and investigation of gas vesicle synthesis. Proc. Natl. Acad. Sci. USA 87: 67726776.
19. Boccazzi, P.,, K. J. Zhang, and, W. W. Metcalf. 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: 26112618.
20. Bock, A., and, O. Kandler. 1985. Antibiotic sensitivity of archaebacteria. In C. R. Woese and, R. S. Wolfe (ed.), The Bacteria. A Treatise on Structure and Function, vol. 8. Academic Press, Inc., New York, N.Y.
21. Bowen, T. L., and, W. B. Whitman. 1987. Incorporation of exogenous purines and pyrimidines by Methanococcus voltae and isolation of analog-resistant mutants. Appl. Environ. Microbiol. 53: 18221826.
22. Cannio, R.,, P. Contursi,, M. Rossi, and, S. Bartolucci. 1998. An autonomously replicating transforming vector for Sulfolobus sol-fataricus. J. Bacteriol. 180: 32373240.
23. Cannio, R.,, P. Contursi,, M. Rossi, and, S. Bartolucci. 2001. Thermoadaptation of a mesophilic hygromycin B phosphotransferase by directed evolution in hyperthermophilic Ar-chaea: selection of a stable genetic marker for DNA transfer into Sulfolobus sol-fataricus. Extremophiles 5: 153159.
24. Charlebois, R. L.,, W. L. Lam,, S. W. Cline, and, W. F. Doolittle. 1987. Characterization of pHV2 from Halobacterium volcanii and its use in demonstrating transformation of an ar-chaebacterium. Proc. Natl. Acad. Sci. USA 84: 85308534.
25. Cline, S., and, W. F. Doolittle. 1992. Transformation of members of the genus Haloarcula with shuttle vectors based on Halobacterium halobium and Haloferax volcanii plasmid replicons. J. Bacteriol. 174: 10761080.
26. Cline, S. W., and, W. F. Doolittle. 1987. Efficient transfection of the archaebacterium Halobacterium halobium. J. Bacteriol. 169: 13411344.
27. Cohen-Kupiec, R.,, C. Blank, and, J. A. Leigh. 1997. Transcriptional regulation in Archaea: In vivo demonstration of a repressor binding site in a methanogen. Proc. Natl. Acad. Sci. USA 94: 13161320.
28. Conover, R. K., and, W. F. Doolittle. 1990. Characterization of a gene involved in histidine biosynthesis in Halobacterium (Haloferax) volcanii: isolation and rapid mapping by transformation of an auxotroph with cosmid DNA. J. Bacteriol. 172: 32443249.
29. Contursi, P.,, R. Cannio,, S. Prato,, G. Fiorentino,, M. Rossi, and, S. Bartolucci. 2003. Development of a genetic system for hyperthermophilic Archaea: expression of a moderate thermophilic bacterial alcohol dehydrogenase gene in Sulfolobus sol-fataricus. FEMS Microbiol. Lett. 218: 115120.
30. DasSarma, S., and, E. M. Fleischmann (ed.). 1995. Halophiles. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
31. Ehlers, C.,, K. Weidenbach,, K. Veit,, U. Deppenmeier,, W. W. Metcalf, and, R. A. Schmitz. 2005. Development of genetic methods and construction of a chromosomal glnK1 mutant in Methanosarcina mazei strain Go1. Mol. Genet. Genomics 273: 290298.
32. Elferink, M. G.,, C. Schleper, and, W. Zillig. 1996. Transformation of the extremely thermoacidophilic archaeon Sulfolobus sol-fataricus via a self-spreading vector. FEMS Microbiol. Lett. 137: 3135.
33. Erauso, G.,, S. Marsin,, N. Benbouzid-Rollet,, M. F. Baucher,, T. Barbeyron,, Y. Zivanovic,, D. Prieur, and, P. Forterre. 1996. Sequence of plasmid pGT5 from the archaeon Pyrococcus abyssi: evidence for rolling-circle replication in a hyperthermophile. J. Bacteriol. 178: 32323237.
34. Erauso, G.,, D. Prieur,, A. Godfroy, and, G. Raguenes. 1995. Plate cultivation techniques for strictly anaerobic, ther-mophilic, sulfur-metabolizing Archaea, p. 2529. In F. T. Robb,, A. R. Place,, K. R. Sowers,, H. J. Schreier,, S. DasSarma, and, E. M. Fleischmann (ed.), ArchaeaA Laboratory Manual. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
35. Firtel, M.,, G. B. Patel, and, T. J. Beveridge. 1995. S layer regeneration in Methanococcus voltae protoplasts. Microbiology 141: 817824.
36. Gardner, W. L., and, W. B. Whitman. 1999. Expression vectors for Methanococcus maripaludis: overexpression of acetohydroxyacid synthase and beta-galactosidase. Genetics 152: 14391447.
37. Gernhardt, P.,, O. Possot,, M. Foglino,, L. Sibold, and, A. Klein. 1990. Construction of an integration vector for use in the ar-chaebacterium Methanococcus voltae and expression of a eubacterial resistance gene. Mol. Gen. Genet. 22: 273279.
38. Gregor, D., and, F. Pfeifer. 2001. Use of a halobacterial bgaH reporter gene to analyse the regulation of gene expression in halophilic archaea. Microbiology 147: 17451754.
39. Grogan, D. W. 1996. Exchange of genetic markers at extremely high temperatures in the archaeon Sulfolobus acidocaldarius. J. Bacteriol. 178: 32073211.
40. Grogan, D. W. 1991. Selectable mutant phenotypes of the extremely thermophilic archaebacterium Sulfolobus acidocaldarius. J. Bacterial. 173: 77257727.
41. Guss, A. M.,, B. Mukhopadhyay,, J. K. Zhang, and, W. W. Metcalf. 2005. Genetic analysis of mch mutants in two Methanosarcina species demonstrates multiple roles for the methanopterin-dependent C-1 oxidation/reduction pathway and differences in H-2 metabolism between closely related species. Mol. Microbiol. 55: 16711680.
42. Hackett, N. R., and, S. DasSarma. 1989. Characterization of the small endogenous plasmid of Halobacterium strain SB3 and its use in transformation. Can. J. Microbiol. 35: 8691.
43. Harris, J. E. 1985. Gelrite as an agar substitute for the cultivation of mesophilic Methanobacterium and Methanobre-vibacter species. Appl. Environ. Microbiol. 50: 11071109.
44. Harris, J. E., and, P. A. Pinn. 1985. Bacitracin-resistant mutants of a mesophilic Methanobacterium species. Arch. Microbiol. 143: 151153.
45. Haydock, A. K.,, I. Porat,, W. B. Whitman, and, J. A. Leigh. 2004. Continuous culture of Methanococcus maripaludis under defined nutrient conditions. FEMS Microbiol. Lett. 238: 8591.
46. Heinicke, I.,, J. Muller,, M. Pittelkow, and, A. Klein. 2004. Mutational analysis of genes encoding chromatin proteins in the archaeon Methanococcus voltae indicates their involvement in the regulation of gene expression. Mol. Genet. Genomics 272: 7687.
47. Holmes, M.,, F. Pfeifer, and, M. Dyall-Smith. 1994. Improved shuttle vectors for Haloferax volcanii including a dual-resistance plasmid. Gene 146: 117121.
48. Holmes, M. L., and, M. L. Dyall-Smith. 1990. A plasmid vector with a selectable marker for halophilic archaebacteria. J. Bacteriol. 172: 756761.
49. Holmes, M. L., and, M. L. Dyall-Smith. 2000. Sequence and expression of a halobacterial β-galactosidase gene. Mol. Microbiol. 36: 114122.
50. Holmes, M. L.,, S. D. Nuttall, and, M. L. Dyall-Smith. 1991. Construction and use of halobacterial shuttle vectors and further studies on Haloferax DNA gyrase. J. Bacteriol. 173: 38073813.
51. Holmes, M. L.,, R. K. Scopes,, R. L. Moritz,, R. J. Simpson,, C. Englert,, F. Pfeifer, and, M. L. Dyall-Smith. 1997. Purification and analysis of an extremely halophilic β-galactosidase from Haloferax alicantei. BBA Prot. Struct. Mol. Enzymol. 1337: 276286.
52. Hook, L. A.,, R. E. Corder,, P. T. Hamilton,, J. I. Frea, and, J. N. Reeve (ed.). 1984. Development of a Plating System for Genetic Exchange Studies in Methanogens Using a Modified Ultra-Low Oxygen Chamber. The Ohio State University Press, Columbus, Ohio.
53. Jenal, U.,, T. Rechsteiner,, P. Y. Tan,, E. Buhlmann,, L. Meile, and, T. Leisinger. 1991. Isoleucyl-tRNA synthetase of Methanobacterium thermoautotrophicum Marburg. Cloning of the gene, nucleotide sequence, and localization of a base change conferring resistance to pseudomonic acid. J. Biol. Chem. 266: 1057010577.
54. Jones, W. J.,, W. B. Whitman,, F. D. Fields, and, R. S. Wolfe. 1983. Growth and plating efficiency of methanococci on agar media. Appl. Environ. Microbiol. 46: 220226.
55. Jonuscheit, M.,, E. Martusewitsch,, K. M. Stedman, and, C. Schleper. 2003. A reporter gene system for the hyperthermophilic archaeon Sulfolobus sol-fataricus based on a selectable and integrative shuttle vector. Mol. Microbiol. 48: 12411252.
56. Kandler, O., and, H. Konig. 1998. Cell wall polymers in Archaea (Archaebacteria). Cell Mol. Life Sci. 54: 305308.
57. Kiener, A.,, H. Konig,, J. Winter, and, T. Leisinger. 1987. Purification and use of Methanobacterium wolfei pseudomurein endopeptidase for lysis of Methanobacterium thermoautotrophicum. J. Bacteriol. 169: 10101016.
58. Kiener, A., and, T. Leisinger. 1983. Oxygen sensitivity of methanogenic bacteria. Syst. Appl. Microbiol. 4: 305312.
59. Krebs, M. P.,, T. Hauss,, M. P. Heyn, and, U. L. RajBhandary. 1991. Expression of the bacterioopsin gene in Halobacterium halobium using a multicopy plasmid. Proc. Natl. Acad. Sci. USA 88: 859863.
60. Krebs, M. P.,, R. Mollaaghababa, and, H. G. Khorana. 1993. Gene replacement in Halobacterium halobium and expression of bacteriorhodopsin mutants. Proc. Natl. Acad. Sci. USA 90: 19871991.
61. Ladapo, J., and, W. B. Whitman. 1990. Method for isolation of auxotrophs in the methanogenic archaebacteria: role of the acetyl-CoA pathway of autotrophic CO 2 fixation in Methanococcus mari-paludis. Proc. Natl. Acad. Sci. USA 87: 55985602.
62. Lam, W. L., and, W. F. Doolittle. 1989. Shuttle vectors for the archaebacterium Halobacterium volcanii. Proc. Natl. Acad. Sci. USA 86: 54785482.
63. Lie, T, and, J. Leigh. 2003. A novel repressor of ra/and glnA expression in the methanogenic archaeon Methanococcus maripaludis. Mol. Microbiol. 47: 235246.
64. Lie, T. J.,, G. E. Wood, and, J. A. Leigh. 2005. Regulation of nif expression in Methanococcus maripaludis: roles of the euryarchaeal repressor NrpR, 2-oxoglutarate, and two operators. J. Biol. Chem. 280: 52365241.
65. Lindstrom, E. B., and, H. M. Sehlin. 1989. High efficiency of plating of the thermophilic sulfur-dependent archaebacterium Sulfolobus acidocaldarius. Appl. Environ. Microbiol. 55: 30203021.
66. Long, S. W., and, D. M. Faguy. 2004. Anucleate and titan cell phenotypes caused by insertional inactivation of the structural maintenance of chromosomes (smc) gene in the archaeon Methanococcus voltae. Mol. Microbiol. 52: 15671577.
67. Lucas, S.,, L. Toffin,, Y. Zivanovic,, D. Charlier,, H. Moussard,, P. Forterre,, D. Prieur, and, G. Erauso. 2002. Construction of a shuttle vector for, and spheroplast transformation of, the hyperthermophilic archaeon Pyrococcus abyssi. Appl. Environ. Microbiol. 68: 55285536.
68. Mahapatra, A.,, A. Patel,, J. A. Soares,, R. C. Larue,, J. K. Zhang,, W. W. Metcalf, and, J. A. Krzycki. 2006. Characterization of a Methanosarcina acetivorans mutant unable to translate UAG as pyrrolysine. Mol. Microbiol. 59: 5666.
69. Mankin, A. S.,, I. M. Zyrianova,, V. K. Kagramanova, and, R. A. Garrett. 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: 65356539.
70. Martusewitsch, E.,, C. W. Sensen, and, C. Schleper. 2000. High spontaneous mutation rate in the hyperthermophilic archaeon Sulfolobus sol-fataricus is mediated by transposable elements. J. Bacteriol. 182: 25742581.
71. Meile, L.,, P. Abendschein, and, T. Leisinger. 1990. Transduction in the archaebacterium Methanobacterium thermau-totrophicum Marburg. J. Bacteriol. 172: 35073508.
72. Metcalf, W. W. 1999. Genetic analysis in the domain Ar-chaea, p. 277326. In M. C. Smith and, R. E. Sockett (ed.), Genetic Methods for Diverse Prokaryotes, vol. 29. Academic Press, New York, N.Y.
73. Metcalf, W. W.,, J. K. Zhang,, E. Apolinario,, K. R. Sowers, and, R. S. Wolfe. 1997. A genetic system for Archaea of the genus Methanosarcina: Liposome-mediated transformation and construction of shuttle vectors. Proc. Natl. Acad. Sci. USA 94: 26262631.
74. Metcalf, W. W.,, J. K. Zhang, and, R. S. Wolfe. 1998. An anaerobic, intrachamber incubator for growth of Methanosarcina spp. on methanol-containing solid media. Appl. Environ. Microbiol. 64: 768770.
75. Mevarech, M., and, R. Werczberger. 1985. Genetic transfer in Halobacterium volcanii. J. Bacteriol. 162: 461462.
76. Moore, B. C., and, J. A. Leigh. 2005. Markerless mutagenesis in Methanococcus maripaludis demonstrates roles for alanine dehydrogenase, alanine racemase, and alanine permease. J. Bacteriol. 187: 972979.
77. Morii, H., and, Y. Koga. 1992. An improved assay method for a pseudomurien-degrading enzyme of Methanobacterium wolfei and the protoplast formation of Methanobacterium thermoautotrophicum by the enzyme. J. Ferment. Bioeng. 73: 610.
78. Ng, W.-L.,, P. Arora, and, S. DasSarma. 1994. Large deletions in class III gas-vesicles deficient mutants of Halobacterium halobium. Syst. Appl. Microbiol. 16: 560568.
79. Ng, W.-L., and, S. DasSarma. 1993. Minimal replication origin of the 200-kilobase Halobacterium plasmid pNRC100. J. Bacteriol. 175: 45844596.
80. Ng, W. L., and, S. DasSarma. 1993. Minimal replication origin of the 200-kilobase Halobacterium plasmid pNRC100. J. Bacteriol. 175: 45844596.
81. Nieuwlandt, D. T., and, C. J. Daniels. 1990. An expression vector for the archaebacterium Haloferax volcanii. J. Bacteriol. 172: 71047110.
82. Ortenberg, R.,, O. Rozenblatt-Rosen, and, M. Mevarech. 2000. The extremely halophilic archaeon Haloferax volcanii has two very different dihydrofolate reductases. Mol. Microbiol. 35: 14931505.
83. Patel, G. B.,, J. H. E. Nash,, B. J. Agnew, and, G. D. Sprott. 1994. Natural and electroporation-mediated transformation of Methanococcus voltae protoplasts. Appl. Environ. Microbiol. 60: 903907.
84. Patenge, N.,, A. Haase,, H. Bolhuis, and, D. Oesterhelt. 2000. The gene for a halophilic β-galactosidase (bgaH) of Haloferax alicantei as a reporter gene for promoter analysis in Halobacterium salinarium. Mol. Microbiol. 36: 105113.
85. Peck, R. E,, S. DasSarma, and , M. P. Krebs. 2000. Homologous gene knockout in the archaeon Halobacterium with ura3 as a counterselectable marker. Mol. Microbiol. 35: 667676.
86. Pfeiffer, M.,, H. Bestgen,, A. Burger, and , A. Klein. 1998. The vhuU gene encoding a small subunit of a selenium-containing [NiFe]-hydrogenase in Methanococcus voltae appears to be essential for the cell. Arch. Microbiol. 170: 418426.
87. Porat, I.,, B. W. Waters,, Q. Teng, and, W. B. Whitman. 2004. Two biosynthetic pathways for aromatic amino acids in the archaeon Methanococcus maripaludis. J. Bacterial. 186: 49404950.
88. Possot, O.,, P. Gernhardt,, A. Klein, and, L. Sibold. 1988. Analysis of drug resistance in the archaebacterium Methanococcus voltae with respect to potential use in genetic engineering. Appl. Environ. Microbiol. 54: 734740.
89. Pritchett, M. A.,, J. K. Zhang, and , W. W Metcalf. 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: 14251433.
90. Reuter, C. J., and, J. A. Maupin-Furlow. 2004. Analysis of proteasome-dependent proteolysis in Haloferax volcanii cells, using short-lived green fluorescent proteins. Appl. Environ. Microbiol. 70: 75307538.
91. Rodriguez-Valera, F. 1995. Cultivation of halophilic Archaea, p. 1316. In F. T. Robb,, A. R. Place,, K. R. Sowers,, H. J. Schreier,, S. DasSarma, and, E. M. Fleischmann (ed.), ArchaeaA Laboratory Manual. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
92. Rosenshine, I.,, R. Tchelet, and, M. Mevarech. 1989. The mechanism of DNA transfer in the mating system of an archaebacterium. Science 245: 13871389.
93. Rother, M.,, P. Boccazzi,, A. Bose,, M. A. Pritchett, and, W. W. Metcalf. 2005. Methanol-dependent gene expression demonstrates that methyl-coenzyme M reductase is essential in Methanosarcina acetivorans C2A and allows isolation of mutants with defects in regulation of the methanol utilization pathway. J. Bacteriol. 187: 55525559.
94. Rother, M., and, W. W. Metcalf. 2005. Genetic technologies for Archaea. Curr. Opin. Microbiol. 8: 745751.
95. Sato, T,, T. Fukui,, H. Atomi, and, T. Imanaka. 2005. Improved and versatile transformation system allowing multiple genetic manipulations of the hyperthermophilic archaeon Thermococcus kodakaraensis. Appl. Environ. Microbiol. 71: 38893899.
96. Sato, X,, X. Fukui,, H. Atomi, and, X. Imanaka. 2003. Targeted gene disruption by homologous recombination in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. J. Bacteriol. 185: 210220.
97. Schleper, C,, I. Holz,, D. Janekovic,, J. Murphy, and, W. Zillig. 1995. A multicopy plasmid of the extremely thermophilic archaeon Sulfolobus effects its transfer to recipients by mating. J. Bacteriol. 177: 44174426.
98. Schleper, C,, K. Kubo, and, W. Zillig. 1992. The particle SSV1 from the extremely thermophilic archaeon Sulfolobus is a virus: demonstration of infectivity and of transfection with viral DNA. Proc. Natl. Acad. Sci. USA 89: 76457649.
99. She, Q.,, H. Phan,, R. A. Garrett,, S.-V. Albers,, K. M. Stedman, and, W. Zillig. 1998. Genetic profile of pNOB8 from Sulfolobus: the first conjugative plasmid form an archaeon. Extremophiles 2: 417425.
100. Sniezko, I.,, C. Dobson-Stone, and, A. Klein. 1998. The treA gene of Bacillus subtilis is a suitable reporter gene for the archaeon Methanococcus voltae. FEMS Microbiol. Lett. 164: 237242.
101. Sowers, K. R.,, J. E. Boone, and, R. P. Gunsalus. 1993. Disag-gregation of Methanosarcina spp. and growth as single cells at elevated osmolarity. Appl. Environ. Microbiol. 59: 38323839.
102. Sowers, K. R., and, R. P. Gunsalus. 1988. Plasmid DNA from the Acetotrophic Methanogen Methanosarcina acetivorans. J. Bacteriol. 170: 49794982.
103. Sowers, K. R., and, H. J. Schreier. 1999. Gene transfer systems for the archaea. Trends Microbiol. 7: 212219.
104. Sowers, K. R., and, H. J. Schreier. 1995. Techniques for anaerobic growth, p. 1555. In F. T. Robb,, K. R. Sowers,, S. DasSharma,, A. R. Place,, H. J. Schreier, and, E. M. Fleischmann (ed.), ArchaeaA Laboratory Manual. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
105. Tan, G. T.,, A. DeBlasio, and, A. S. Mankin. 1996. Mutations in the peptidyl transferase center of 23 S rRNA reveal the site of action of sparsomycin, a universal inhibitor of translation. J. Mol. Biol. 261: 222230.
106. Thomas, N. A.,, S. Mueller,, A. Klein, and, K. F. Jarrell. 2002. Mutants in flaI and flaJ of the archaeon Methanococcus voltae are deficient in flagellum assembly. Mol. Microbiol. 46: 879887.
107. Tumbula, D. L.,, T. L. Bowen, and, W. B. Whitman. 1997. Characterization of pURB500 from the archaeon Methanococcus maripaludis and construction of a shuttle vector. J. Bacteriol. 179: 29762986.
108. Tumbula, D. L.,, T. L. Bowen, and, W. B. Whitman. 1995. Growth of methanogens on solidified medium, p. 4955. In F. T. Robb,, A. R. Place,, K. R. Sowers,, H. J. Schreier,, S. Das-Sarma, and, E. M. Fleischmann (ed.), ArchaeaA Laboratory Manual. Cold Spring Harbor Laboratory Press, Plainview, N.Y.
109. Tumbula, D. L.,, R. A. Makula, and, W. B. Whitman. 1994. Transformation of Methanococcus maripaludis and identification of a PstI-like restriction system. FEMS Microbiol. Lett. 121: 309314.
110. Völkl, P.,, R. Huber,, E. Drobner,, R. Rachel,, S. Burggraf,, A. Trincone, and, K. Stetter. 1993. Pyrobaculum aerophilum sp. nov., a novel nitrate-reducing hyperthermophilic archaeum. Appl. Environ. Microbiol. 59: 29182926.
111. Wang, G.,, S. P. Kennedy,, S. Fasiludeen,, C. Rensing, and, S. DasSarma. 2004. Arsenic resistance in Halobacterium sp. strain NRC-1 examined by using an improved gene knockout system. J. Bacteriol. 186: 31873194.
112. Watrin, L.,, S. Lucas,, C. Purcarea,, C. Legrain, and, D. Prieur. 1999. Isolation and characterization of pyrimidine auxotrophs, and molecular cloning of the pyrE gene from the hyperthermophilic archaeon Pyrococcus abyssi. Mol. Gen. Genet. 262: 378381.
113. Watrin, L., and, D. Prieur. 1996. UV and ethyl methanesulfonate effects in hyperthermophilic archaea and isolation of auxotrophic mutants of Pyrococcus strains. Curr. Microbiol. 33: 377382.
114. Welander, P. V., and, W. W. Metcalf. 2005. Loss of the mtr operon in Methanosarcina blocks growth on methanol, but not methanogenesis, and reveals an unknown methanogenic pathway. Proc. Natl. Acad. Sci. USA 102: 1066410669.
115. Wendoloski, D.,, C. Ferrer, and, M. L. Dyall-Smith. 2001. A new simvastin (mevinolin)-resistant marker from Haloarchula hispanica and a new Haloferax volcanii strain cured of plas-mid pHV2. Microbiology 147: 959964.
116. Wood, G. E.,, A. K. Haydock, and, J. A. Leigh. 2003. Function and regulation of the formate dehydrogenase genes of the methanogenic archaeon Methanococcus maripaludis. J. Bacteriol. 185: 25482554.
117. Worthington, P.,, V. Hoang,, P. Perez-Pomares, and, P. Blum. 2003. Targeted disruption of the a-amylase gene in the hyperthermophilic archaeon Sulfolobus sol-fataricus. J. Bacterial. 185: 482488.
118. Zhang, J. K.,, M. A. Pritchett,, D. J. Lampe,, H. M. Robertson, and, W. W. Metcalf. 2000. In vivo transposon mutagenesis of the methanogenic archaeon Methanosarcina acetivorans C2A using a modified version of the insect mariner-family transposable element Himarl. Proc. Natl. Acad. Sci. USA 97: 96659670.
119. Zhang, J. K.,, A. K. White,, H. C. Kuettner,, P. Boccazzi, and, W. W. Metcalf. 2002. Directed mutagenesis and plasmid-based complementation in the methanogenic archaeon Methanosarcina acetivorans C2A demonstrated by genetic analysis of proline biosynthesis. J. Bacteriol. 184: 14491454.
120. Zhou, M.,, H. Xiang,, C. Sun, and, H. Tan. 2004. Construction of a novel shuttle vector based on an RCR-plasmid from a haloalkaliphilic archaeon and transformation into other haloarchaea. Biotechnol. Lett. 26: 11071113.


Generic image for table
Table 1.

Physiological characteristics and growth efficiency of archaea on solidified medium

Citation: Sowers K, Anderson K. 2007. Molecular Genetics of , p 463-477. In Cavicchioli R (ed), Archaea. ASM Press, Washington, DC. doi: 10.1128/9781555815516.ch21
Generic image for table
Table 2.

Gene transfer methods for archaea

Citation: Sowers K, Anderson K. 2007. Molecular Genetics of , p 463-477. In Cavicchioli R (ed), Archaea. ASM Press, Washington, DC. doi: 10.1128/9781555815516.ch21
Generic image for table
Table 3.

Shuttle vectors for use in archaea and

Citation: Sowers K, Anderson K. 2007. Molecular Genetics of , p 463-477. In Cavicchioli R (ed), Archaea. ASM Press, Washington, DC. doi: 10.1128/9781555815516.ch21

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error