Genetics of Archaea

Kevin R. Sowers; Paul H. Blum; Shiladitya Dassarma

doi:10.1128/9781555817497.ch33

Chapter 33 : Genetics of Archaea

Authors: Kevin R. Sowers, Paul H. Blum, Shiladitya Dassarma

Content Type: Reference

Publication Year: 2007

Category: Microbial Genetics and Molecular Biology

Book DOI: 10.1128/9781555817497

Chapter DOI: 10.1128/9781555817497.ch33

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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 Archaea. 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 Archaea, 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, Halobacterium 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 Sulfolobus solfataricus. Plasmids that do not replicate in S. solfataricus 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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Genetics of Archaea

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FIGURE 1

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

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FIGURE 1

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FIGURE 2

Gene knockout and replacement in the halophilic Euryarchaeota. The example shown is for selection and counterselection with ura3. 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 ∆ura3 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 pyrE2 gene can be used for selection and counterselection.

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FIGURE 2

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FIGURE 3

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

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FIGURE 3

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FIGURE 4

Random gene mutagenesis in Methanosarcina with an E. coli-Methanosarcina shuttle vector containing a transposable element. pJK60 is a modified E. coli-Methanosarcina shuttle vector that contains the puromycin resistance pac gene, the R6K E. coli plasmid origin of replication, and the kanamycin resistance aph gene flanked by the transposable elements of the insect marinerfamily Himar1 transposon. The transposase is expressed in the methanogen from the methyl-coenzyme M reductase gene (mcrB) from Methanosarcina barkeri. The vector is transformed into Methanosarcina 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 E. coli, the DNA is repurified from kanamycin-resistant clones, and then DNA flanking the transposable element is sequenced to identify the disrupted gene.

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FIGURE 4

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FIGURE 5

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

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FIGURE 5

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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 N2, CO2, and H2 in a volume ratio of 75:20:5. The CO2 maintains the equilibrium of the carbonate buffer at a neutral pH, the H2 combined with palladium catalyst pellets located in the glove box reduces any oxygen that may diffuse into the chamber, and the N2 is 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.

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FIGURE 6

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FIGURE 7

Transformation enrichment culture time courses. S. solfataricus 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).

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FIGURE 7

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Tables

Genetics of Archaea

/content/10.1128/9781555817497.chap33.tab33-1

TABLE 1

Archaeal genomic sequences

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TABLE 1

Archaeal genomic sequences

/content/10.1128/9781555817497.chap33.tab33-2

TABLE 2

Archaeal strain characteristics and sources a

a 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/).

10.1128/9781555817497/tab33-2_thmb.gif

10.1128/9781555817497/tab33-2.gif

TABLE 2

Archaeal strain characteristics and sources a

/content/10.1128/9781555817497.chap33.tab33-3

TABLE 3

Archaeal genetic vectors

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TABLE 3

Archaeal genetic vectors

/content/10.1128/9781555817497.chap33.tab33-4

TABLE 4

Selectable genetic markers for Archaea

a SCES, sole carbon and energy source.

10.1128/9781555817497/tab33-4_thmb.gif

10.1128/9781555817497/tab33-4.gif

TABLE 4

Selectable genetic markers for Archaea

a SCES, sole carbon and energy source.