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Chapter 33 : Genetics of Archaea

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Abstract:

In recent years several laboratories have developed effective plating techniques, identifying genetic markers that do not target cell wall synthesis, fusing archaeal promoters with recombinant genes, and isolating native vectors and promiscuous nonnative vectors. This chapter focuses on tractable systems that are currently available for the . Due to fundamental differences between gene transfer systems for each archaeal branch, the chapter is divided into three inclusive sections covering the halophilic and methanogenic Euryarchaeota and the hyperthermophilic Crenarchaeota. Despite varying degrees of difficulty growing , all three systems are routinely used by laboratories conducting research on archaeal genetics and can be mastered by anyone with a fundamental knowledge of microbial genetic techniques. Under low oxygen tension, sp. NRC-1 induces purple membrane patches in the cell membrane and buoyant gas vesicles intracellularly, which increases the availability of light and oxygen and allows a period of light-driven proton pumping and phototrophic growth. Targeted manipulation of the chromosome by directed recombination was recently added to the growing list of approaches for the genetic analysis of . Plasmids that do not replicate in can be used to introduce DNA into the genome.

Citation: Sowers K, Blum P, Dassarma S. 2007. Genetics of Archaea, p 800-824. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch33

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Bacteria and Archaea
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Methyl Coenzyme M Reductase
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Image of FIGURE 1
FIGURE 1

Haloarchaeal shuttle vector pNG168. This plasmid contains the pTZ19r replicon and the NRC-1 pNRC100 minimal replication region. The gene provides selection with ampicillin in , and the gene provides selection with mevinolin in haloarchaea. The multiple cloning site is located in the α fragment gene and permits blue-white screening in . The plasmid is available from ATCC (catalog no. MBA-77) and the sequence is available in GenBank (accession no. AY291460).

Citation: Sowers K, Blum P, Dassarma S. 2007. Genetics of Archaea, p 800-824. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch33
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Image of FIGURE 2
FIGURE 2

Gene knockout and replacement in the halophilic Euryarchaeota. The example shown is for selection and counterselection with 3. A cloned haloarchaeal target gene (geneX) in a plasmid vector, which does not replicate in haloarchaea, is used for PCR amplification (primers designated by arrowheads) and recircularization to provide for a precisely deleted gene. The plasmid is introduced into a ∆haloarchaeon by transformation. Integrants are selected by uracil prototrophy using uracil dropout plates. Excisants are selected for by plating on plates containing 5- FOA. Depending on the site of the recombination (1 or 2), different outcomes are possible. Alternatively, mevinolin selection can also be used for integration and the gene can be used for selection and counterselection.

Citation: Sowers K, Blum P, Dassarma S. 2007. Genetics of Archaea, p 800-824. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch33
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Image of FIGURE 3
FIGURE 3

Recombinant plasmid showing construction typical for an -shuttle vector. The construct includes the dependent R6K for replication of the plasmid in and the gene for selection of transformants with ampicillin; pC2A and for replication in spp.; and under transcriptional control of the archaeal methyl-coenzyme M reductase gene for selection of methansarcinal recombinants on puromycin. Filled and open elements represent genes from the Bacteria and Archaea, respectively.

Citation: Sowers K, Blum P, Dassarma S. 2007. Genetics of Archaea, p 800-824. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch33
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Image of FIGURE 4
FIGURE 4

Random gene mutagenesis in with an -shuttle vector containing a transposable element. pJK60 is a modified -shuttle vector that contains the puromycin resistance gene, the R6K plasmid origin of replication, and the kanamycin resistance gene flanked by the transposable elements of the insect family transposon. The transposase is expressed in the methanogen from the methyl-coenzyme M reductase gene () from . The vector is transformed into spp. and transposed into random sites in the genome, and then puromycin-resistant colonies with the desired phenotype are selected. The transposed DNA is purified and digested with EcoRI, and the fragments are closed by treatment with T4 ligase. The circular DNA is transformed into the DNA is repurified from kanamycin-resistant clones, and then DNA flanking the transposable element is sequenced to identify the disrupted gene.

Citation: Sowers K, Blum P, Dassarma S. 2007. Genetics of Archaea, p 800-824. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch33
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Image of FIGURE 5
FIGURE 5

Reporter vector pWLG30+for detecting archaeal promoter strength based on β-galactosidase activity. This shuttle vector includes a bacterial for replication of the plasmid in and the gene for selection of transformants with ampicillin; methanococcal pURB500 for replication in ; and under transcriptional control of the archaeal methyl-coenzyme M reductase gene for selection of methanococcal recombinants on puromycin. The methanococcal hydrogenase promoter is fused upstream of for measuring expression of hydrogenase by β-galactosidase activity. Filled and open elements represent genes from the Bacteria and Archaea, respectively.

Citation: Sowers K, Blum P, Dassarma S. 2007. Genetics of Archaea, p 800-824. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch33
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Image of FIGURE 6
FIGURE 6

Specialized apparatus for plating methanogenic Euryarchaeota. (A) Anaerobic glove box used for plating methanogenic Archaea. The gas phase is composed of a mixture of N, CO, and Hin a volume ratio of 75:20:5. The COmaintains the equilibrium of the carbonate buffer at a neutral pH, the Hcombined with palladium catalyst pellets located in the glove box reduces any oxygen that may diffuse into the chamber, and the Nis inert. Lower figures show anaerobic jars for incubation of colony clones on solidified medium. Examples include a modified glass canning jar (B), a commercial jar manufactured by TORBAL (C), and a modified paint pressure tank (D). Photo in panel D is courtesy of W. B. Whitman.

Citation: Sowers K, Blum P, Dassarma S. 2007. Genetics of Archaea, p 800-824. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch33
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Image of FIGURE 7
FIGURE 7

Transformation enrichment culture time courses. PBL2025 (circles) and PBL2030 (triangles and squares) were transformed with plasmids pLacS (filled circles) or pMalA (filled triangles and squares) to growth on lactose (circles), glycogen (triangles), or starch (squares). Untransformed control cultures were PBL2025 (open circles) and PBL2030 (open triangles and open squares).

Citation: Sowers K, Blum P, Dassarma S. 2007. Genetics of Archaea, p 800-824. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch33
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Tables

Generic image for table
TABLE 1

Archaeal genomic sequences

Citation: Sowers K, Blum P, Dassarma S. 2007. Genetics of Archaea, p 800-824. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch33
Generic image for table
TABLE 2

Archaeal strain characteristics and sources

ATCC, American Type Culture Collection (http://www.atcc.org/); DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (http://www.dsmz.de/); JCM, Japan Collection of Microorganisms (http://www.jcm.riken.go.jp/); OCM, Oregon Collection of Methanogens (http://methanogens.pdx.edu/).

Citation: Sowers K, Blum P, Dassarma S. 2007. Genetics of Archaea, p 800-824. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch33
Generic image for table
TABLE 3

Archaeal genetic vectors

Citation: Sowers K, Blum P, Dassarma S. 2007. Genetics of Archaea, p 800-824. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch33
Generic image for table
TABLE 4

Selectable genetic markers for Archaea

SCES, sole carbon and energy source.

Citation: Sowers K, Blum P, Dassarma S. 2007. Genetics of Archaea, p 800-824. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch33

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