Genetic technologies for Archaea Michael Rother 1 and William W Metcalf 2

1 Genetic technologies for Archaea Michael Rother 1 and William W Metcalf 2 Members of the third domain of life, the Archaea, possess structural, phys...

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Genetic technologies for Archaea Michael Rother1 and William W Metcalf2 Members of the third domain of life, the Archaea, possess structural, physiological, biochemical and genetic features distinct from Bacteria and Eukarya and, therefore, have drawn considerable scientific interest. Physiological, biochemical and molecular analyses have revealed many novel biological processes in these important prokaryotes. However, assessment of the function of genes in vivo through genetic analysis has lagged behind because suitable systems for the creation of mutants in most Archaea were established only in the past decade. Among the Archaea, sufficiently sophisticated genetic systems now exist for some thermophilic sulfur-metabolizing Archaea, halophilic Archaea and methanogenic Archaea. Recently, there have been developments in genetic analysis of thermophilic and methanogenic Archaea and in the use of genetics to study the physiology, metabolism and regulatory mechanisms that direct gene expression in response to changes of environmental conditions in these important microorganisms. Addresses 1 Institut fu¨r Mikrobiologie, Johann Wolfgang Goethe-Universita¨t, Marie-Curie-Strasse 9, D-60439 Frankfurt (Main), Germany 2 Department of Microbiology, University of Illinois at UrbanaChampaign, 601 South Goodwin Avenue, Urbana, Il 61801, USA Corresponding author: Metcalf, William W ([email protected])

Current Opinion in Microbiology 2005, 8:745–751 This review comes from a themed issue on Growth and development Edited by John N Reeve and Ruth Schmitz Available online 28th October 2005 1369-5274/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2005.10.010

Introduction The domains Archaea, Eukarya and Bacteria represent three distinct phylogenetic lineages that encompass all known life on earth. Archaea were only recognized as a distinct phylogenetic group less than 30 years ago [1,2] and were initially renowned for being strictly anaerobic and/or inhabiting inhospitable environments such as solfataric hot springs, soda lakes and submarine volcanic vents. However, it is now apparent that Archaea are ubiquitous and constitute a significant portion of the global biomass [3]. Like the environments that they inhabit, the variety of Archaea is vast. Significantly, they share unique features not typically found in the other two domains: characteristic rRNAs and tRNAs [4];

linked isoprenoid lipids ([5] and references therein); the absence of peptidoglycan cell walls [6]; and a 11– 12 subunit DNA-dependent RNA polymerase [7,8]. Based on 16S rRNA analysis, the archaeal domain is split between four kingdom-level phyla: the Euryarchaeota, which include the methanogenic and the halophilic Archaea as well as the Archaeoglobus and the Pyrococcus groups (Thermococcales); the Crenarchaeota, which are dominated by thermophilic organisms such as Sulfolobus and Thermoproteus; the Korarchaeota [9], for which only DNA sequence data exist to date; and the Nanoarchaeota, which are represented to date by only one species, Nanoarchaeum equitans [10]. As can be expected from the variety of organisms known, the Archaea have drawn considerable scientific interest. Physiological, biochemical, molecular and phylogenetic analyses have produced a wealth of information about many aspects of this important group of prokaryotes. Thanks to those efforts, a scaffold of understanding about archaeal metabolism — sometimes densely meshed, sometimes thinly — is now in place. However, much of this knowledge is still fragmentary, partly because many members of the Archaea are difficult to handle and partly because many questions cannot be answered with these methods. For example, large portions of all known archaeal genome sequences encode proteins of unknown function that have no homologs in the other two domains. Clearly, the means to conduct in vivo characterization of these, and other, genes through genetic manipulation was pressingly needed. Much of the recent effort in this area has therefore gone into the development of facile genetic techniques for Archaea. As a result, in vivo genetic techniques including highly efficient transformation, targeted and random gene disruption, plasmid-based genotypic complementation and expression analysis using reporter gene fusions have been established over the years for some members of the Archaea. Currently, the main principle species for which sufficiently sophisticated genetic methodologies are available fall into three main groups: the methanogenic Archaea, the halophilic Archaea and thermophilic sulfur-metabolizing Archaea. In this review, we will highlight some of the latest developments in genetic techniques applicable to thermophilic and methanogenic Archaea, and will discuss recent studies in which genetic analysis was crucial to address the respective problems. Detailed reviews on the history of archaeal genetics, the basic requirements of genetic systems, the problems encountered in their development and how they were solved, as well as the Current Opinion in Microbiology 2005, 8:745–751

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state of the art in genetic analysis of halophilic Archaea are published elsewhere [11,12,13].

analysis of methanogenic Archaea readily possible. The model genetic organisms studied to date are members of the two genera, Methanococcus and Methanosarcina.

Genetic analysis in thermophilic sulfurmetabolizing Archaea

Genetics of Methanococcus species

Within the Crenarchaeota, the thermophilic sulfur-metabolizing Sulfolobales represent a major branch. They are aerobic or microaerophilic and are capable of growth on various organic substrates such as yeast extract, peptone, tryptone, amino acids and various mono-, oligo- and even poly-meric sugars. Unlike the situation in other Archaea, many of the genetic tools in Sulfolobus emerged from studies of their genetic elements, such as mobile introns [14], cryptic and conjugative plasmids [15–17], and viruses [18]. A facile genetic system is available for Sulfolobus, which includes highly efficient transformation [19], multiple resistance markers [20,21], shuttle vectors [20–23] and a reporter gene system [21]. Few aspects of the physiology of these organisms have been addressed by genetic analysis; however, the mechanism of mercury resistance [24], the mode of regulation of a catabolic gene [25] and the role of threonyl-tRNA synthetase [26] were recently studied using in vivo genetic techniques.

Methanococcus species are known to use only H2/CO2 or formate as carbon and energy sources. The first targeted chromosomal mutant ever created in a methanogen was in Methanococcus voltae [31]. The genetic methodology developed for this organism includes liposome- or polyethylene glycol-mediated transformation and targeted gene disruption by homologous recombination based on non-replicating plasmids [31,32]. A system for insertion of fusions of promoters that have the reporter genes uidA (encodes b-glucuronidase from E. coli) or lacZ (encodes b-galactosidase from E. coli) into the chromosome, and use of the hisA gene (encodes N-[50 -phosphoribosyl-formimino]-5-amino1-[50 -phosphoribosyl]4-imidazolecarboxamide isomerase) both as a permissive site for chromosomal integration and as a selectable marker through complementation of histidine auxotrophy in hisA strains, have also been developed for use in Methanococcus species [33,34].

Other species of thermophilic sulfur-metabolizing Archaea for which genetic systems are just beginning to emerge include members of the euryarchaeal order Thermococcales — Pyrococcus and Thermococcus. Accordingly, selectable plasmid shuttle vectors have now been developed for some hyperthermophilic species [23]. An especially promising approach has been the recent development of a method for targeted gene disruption in Thermococcus [27,28], which allowed for the identification of a key enzyme in glucose biosynthesis [29].

Using these genetic tools, the mechanisms that underlie flagellum biosynthesis, excretion of flagellin subunits and their assembly outside of the cell have been studied ([35– 37] and references therein). Deletion of the gene for structural maintenance of chromosomes (smc) demonstrated the importance of the gene product in the cell cycle and in the ordered DNA partitioning during division, based on the gross defects in chromosome segregation and cell morphology seen in smc deletion mutants [38]. Also, the role of several chromatin genes (genes that encode histones and histone-like proteins) was assessed by mutational analysis [39]. However, because only a single locus could be deleted in a particular strain, possible compensatory effects that result from expression of highly similar chromosomal genes have hindered interpretation of the resultant mutant phenotypes.

Genetic analysis in methanogenic Archaea Methanogenic Archaea mediate all significant biological methane production on earth. Methanogenesis proceeds by conversion of simple C1 and C2 compounds to methane by way of coenzyme-bound intermediates (see review by RK Thauer in this issue). During this stepwise conversion, an electrochemical ion gradient is generated that allows energy conservation by a chemiosmotic mechanism [30]. Studies on methanogenesis in Archaea have produced a wealth of novel physiological and biochemical knowledge because of the unique nature of methanogenic metabolism. Although the basic biochemistry of methane production has been elucidated, relatively little is known about other aspects of the methanoarchaea, many of which have direct relevance to the process of methanogenesis. One reason for this lack of understanding is the extreme sensitivity of methanogens towards oxygen, which necessitates elaborate methods and equipment for their cultivation and manipulation. Another significant factor is the dearth of genetic methods that can be applied to solve this problem. However, this has recently changed and new methods now make genetic Current Opinion in Microbiology 2005, 8:745–751

This unfortunate limitation (i.e. the inability to construct strains that have multiple mutations) was overcome in Methanococcus maripaludis by the development of a neomycin resistance marker (to go along with the previously developed putomycin-resistance marker) [40] and by adaptation of markerless deletion and transposon-mutagenesis strategies that had been previously developed for Methanosarcina (see below, [41]). With these improvements, elegant genetic studies that examine the regulation and structure/function relationship of components involved in nitrogen metabolism of M. maripaludis were conducted [42–46], whereas other studies have used genetic analysis to demonstrate the existence of two biosynthetic routes for aromatic amino acids in this organism [47].

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The recently published complete genome sequence of M. maripaludis [48] has helped to answer a fundamental question in the process of translation, namely how this organism is able to insert cysteine into growing polypeptides independent of cysteinyl-tRNA synthetase (the enzyme that attaches cysteine to its cognate tRNA [49]). In this newly discovered method, free cysteine is not synthesized. Instead, cysteine is synthesized from Ophosphoserine only after this biosynthetic intermediate is charged onto tRNAcys. This mechanism is reminiscent of that in selenocysteine biosynthesis [50,51] and, thus, could indicate how one system might have evolved from the other. Genetics of Methanosarcina species

Methanosarcina species are more metabolically versatile than other methanogenic Archaea and can use H2/CO2, carbon monoxide, methanol, methylamines, methylsulfides and acetate as substrates in substantially different methanogenic pathways [52–54]. The capacity to utilize alternative routes for methanogenesis makes Methanosarcina particularly attractive for genetic analysis of the methanogenic process itself because mutants of one methanogenic pathway remain viable as one of the other pathways can be used instead [55,56,57]. By contrast, other methanogens typically possess only a single methanogenic pathway. For example, Methanococcus and Methanobacterium species can only grow via reduction of CO2 to CH4, Methanosaeta species can only grow on acetate, and Methanococcoides species can only grow on C1 compounds such as methanol. Therefore, because all known methanogens are obligate methanogens, mutations that block the ability to utilize these sole substrates are expected to be lethal mutations. Thus, due to their metabolic diversity, Methanosarcina species are the only methanogenic organisms in which genetic analysis of the methanogenic process is possible. As such, they are currently the only organisms in which genetic analysis of methanogenes is itself is readily possible. Genetic techniques available for use in Methanosarcina include plasmid shuttle vectors, highly efficient liposome-mediated transformation, in vivo transposon mutagenesis [58], multiple selectable markers [59–61] and homologous recombinationmediated gene replacement [61]. Recently, a method was developed [62] that makes it possible to generate strains that carry multiple mutations using only one antibiotic resistance marker. This method, analogous to numerous selection/counterselection strategies used in Bacteria [63,64], is based on a Methanosarcina acetivorans mutant that carries a defective hpt gene (encodes hypoxanthine phosphoribosyl transferase) to make them resistant to the toxic base analog 8-aza-2,6diamino-purine (8-ADP). (Strains with the wild-type hpt allele are sensitive to this compound.) After insertion of a non-replicating plasmid that carries the desired mutation as well as the pac gene (confers resistance to the antibiotic

puromycin) and the wild-type hpt gene, partial diploids are selected as puromycin-resistant 8-ADP-sensitive recombinants. Subsequently, clones that have resolved the partial diploid state are selected as 8-ADP-resistant recombinants. As the resolution of the partial diploid state results in two possible mutant types that have identical phenotypes regarding puromycin and 8-ADP resistance — one that carries the desired mutation and one in which the first recombination event is reverted (and thus is wildtype) — this method requires (sometimes tedious) screening for the desired mutation. To obviate the requirement for this screening, a revised method for markerless disruption, again modeled on similar bacterial systems [65], was developed (Zhang, Rother and Metcalf, unpublished; Figure 1). Similar to the method described above, this method is based on an M. acetivorans mutant that carries a defective hpt gene. In the first step, this strain is transformed with a linear DNA construct in which the gene of interest is disrupted by a gene cassette that encodes an artificial pac–hpt operon flanked by two flp recombinase recognition sites. Insertion of this construct into the chromosome by a double recombination event results in the deletion and/or disruption of the target gene, which confers both puromycin-resistance and 8-ADPsensitivity. In the second step, the mutant is transiently transfected with a non-replicating plasmid that carries the gene for flp recombinase under the control of a strong Methanosarcina promoter. Transient expression of flp recombinase leads to deletion of the region between its recognition sites and thus the generation of puromycinsensitive/8-ADP-resistant mutants, which can be selected for in the presence of the toxic base analog. A system for testing essential genes by conditional gene inactivation has also recently been developed [66]. By fusing the gene of interest to a highly regulated promoter and testing growth, or lack thereof, under permissive (expressing) and non-permissive (non-expressing) conditions, evidence can be obtained to show if a gene is essential. This system can also be employed for the identification of genes that are controlled by a common regulatory mechanism by making regulatory mutations phenotypically selectable. As expression of an essential gene, and thus growth of the organism, is prevented under non-permissive conditions, suppressed mutants can readily be obtained by selection for growth under these nonpermissive conditions. If trans-active factors are affected by the adaptive mutation, other genes under the same regulatory control will also be deregulated. Novel insights into the different pathways of methanogenesis have been obtained by employing the genetic tools developed for Methanosarcina in conjunction with the published genome sequences of three species ([67,68]; metba.home.html) as a resource. For example, genetic Current Opinion in Microbiology 2005, 8:745–751

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Figure 1

Schematic of the markerless disruption method. Linear DNA that contains an artificial pac–hpt operon flanked by Flp recombinase recognition sites (RP1 and RP2) and regions homologous to the target gene (green boxes) is transformed into an M. acetivorans Dhpt strain. Strains that replace the target gene by homologous recombination (shown by dotted lines) are selected as puromycin-resistant recombinants. Subsequently, the pac–hpt operon is removed by site-specific recombination between RP1 and RP2, which is catalyzed by Flp recombinase encoded on the non-replicating plasmid pMR55. See text for details. 8-ADP, 8-aza-2,6-diamino-purine; flp, gene for Flp recombinase; Hpt, gene for hypoxanthine phosphoribosyl transferase; pac, gene for puromycin N-acetyl transferase; Pur, puromycin.

analysis of mutants that lack the Ech hydrogenase led to the identification of the long-sought electron donor for the first step in hydrogenotrophic methanogenesis and revealed a central role for Ech hydrogenase in catabolism and anabolism in Methanosarcina barkeri [69]. Other mutational studies have answered long-standing questions regarding the potential to bypass energy-consuming steps (i.e. the Na+-pumping methyl coenzyme M:tetrahydromethanopterin methyltransferase) and at the same time revealed a novel methanogenic pathway [57]. Comparative genetics led to clues as to why M. acetivorans is unable to utilize the H2 as a reductant for methanogenesis; these clues were subsequently verified by a combined genetic and biochemical approach [55]. Furthermore, M. acetivorans was shown to produce substantial amounts of acetate during growth when using Current Opinion in Microbiology 2005, 8:745–751

carbon monoxide as the sole energy source. Mutational analysis suggests that the organism can grow acetogenically in some circumstances. This conclusion was strongly supported by genetic experiments that showed the pta–ack operon (key genes known to be involved in acetate production [70,71]) is required for growth on carbon monoxide [72]. A phenomenon often encountered in Methanosarcina species is the presence of multiple genes that encode homologous proteins [67,68]. Genetic analysis of the methanolspecific methyltransferase system indicates that this is not mere ‘redundancy’, but that these isoforms serve distinct metabolic roles as they possess different biochemical properties and are differentially regulated ([56]; A Bose and WW Metcalf, unpublished).

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Finally, genetic analysis has been used to characterize factors that participate in the biogenesis of pyrrolysine — the 22nd co-translationally inserted amino acid found in enzymes involved in the conversion of methylamines to methane (see review by JA Krzycki in this issue) — and in the regulation of nitrogen metabolism of M. mazei [73].

Conclusions Genetic analysis of Archaea has proven extremely fruitful over the past decade. Once basic obstacles, such as the development of efficient plating and transformation procedures, had been overcome, many questions about these important prokaryotes, which could not be addressed by molecular or biochemical approaches, have been solved. Resourceful genetic systems are now in place for thermophilic sulfur-metabolizing, halophilic and methanogenic Archaea. Development of these is ongoing and more sophisticated tools are continuously being developed, expanding the number of approaches that can be used to address specific questions in these organisms. As many techniques developed for genetic analysis in Bacteria can be adapted to Archaea, the repertoire of tools is expected to increase even further in the future to allow their genetic manipulation on a routine basis, which will encourage more researchers to study these fascinating, and not so extreme, organisms.

Acknowledgements Portions of the work described here were supported by grants to WWM from The National Science Foundation (MCB0212466) and the Department of Energy (DE-FG02-02ER15296). MR was supported through a fellowship from the Deutsche Forschungsgemeinschaft (RO 2445/1-1).

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60. Boccazzi P, Zhang JK, Metcalf WW: 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 2000, 182:2611-2618.

67. Galagan JE, Nusbaum C, Roy A, Endrizzi MG, Macdonald P, FitzHugh W, Calvo S, Engels R, Smirnov S, Atnoor D et al.: The genome of M. acetivorans reveals extensive metabolic and physiological diversity. Genome Res 2002, 12:532-542.

61. Zhang JK, White AK, Kuettner HC, Boccazzi P, Metcalf WW: Directed mutagenesis and plasmid-based complementation in the methanogenic archaeon Methanosarcina acetivorans C2A demonstrated by genetic analysis of proline biosynthesis. J Bacteriol 2002, 184:1449-1454.

68. Deppenmeier U, Johann A, Hartsch T, Merkl R, Schmitz RA, Martinez-Arias R, Henne A, Wiezer A, Baumer S, Jacobi C et al.: The genome of Methanosarcina mazei: evidence for lateral gene transfer between bacteria and archaea. J Mol Microbiol Biotechnol 2002, 4:453-461.

62. Pritchett MA, Zhang JK, Metcalf WW: 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 2004, 70:1425-1433. The method to introduce unmarked mutations is applied for the first time to a member of the methanogenic Archaea.

69. Meuer J, Kuettner HC, Zhang JK, Hedderich R, Metcalf WW: Genetic analysis of the archaeon Methanosarcina barkeri Fusaro reveals a central role for Ech hydrogenase and ferredoxin in methanogenesis and carbon fixation. Proc Natl Acad Sci USA 2002, 99:5632-5637.

63. Ried JL, Collmer A: An nptI-sacB-sacR cartridge for constructing directed, unmarked mutations in gram-negative bacteria by marker exchange-eviction mutagenesis. Gene 1987, 57:239-246. 64. Russell CB, Dahlquist FW: Exchange of chromosomal and plasmid alleles in Escherichia coli by selection for loss of a dominant antibiotic sensitivity marker. J Bacteriol 1989, 171:2614-2618. 65. Fabret C, Ehrlich SD, Noirot P: A new mutation delivery system for genome-scale approaches in Bacillus subtilis. Mol Microbiol 2002, 46:25-36. 66. Rother M, Boccazzi P, Bose A, Pritchett MA, Metcalf WW:  Methanol-dependent gene expression demonstrates that methyl-CoM reductase is essential in Methanosarcina acetivorans C2A and allows isolation of mutants with defects in regulation of the methanol utilization pathway. J Bacteriol 2005, 187:5552-5559. Development of a method to assess the essentiality of a gene in M. acetivorans; the same system can be used to identify genes under a common regulatory control.

70. Schaupp A, Ljungdahl LG: Purification and properties of acetate kinase from Clostridium thermoaceticum. Arch Microbiol 1974, 100:121-129. 71. Drake HL, Hu SI, Wood HG: Purification of five components from Clostridium thermoaceticum which catalyze synthesis of acetate from pyruvate and methyltetrahydrofolate: properties of phosphotransacetylase. J Biol Chem 1981, 256:11137-11144. 72. Rother M, Metcalf WW: Anaerobic growth of Methanosarcina  acetivorans C2A on carbon monoxide: An unusual way of life for a methanogenic archaeon. Proc Natl Acad Sci USA 2004, 101:16929-16934. Production of acetate and formate in addition to methane during growth of M. acetivorans on carbon monoxide exemplifies the catabolic flexibility of this organism; mutational analysis suggests that acetate is produced via the Wood–Ljungdahl pathway. 73. Ehlers C, Weidenbach K, Veit K, Deppenmeier U, Metcalf WW,  Schmitz RA: Development of genetic methods and construction of a chromosomal glnK(1) mutant in Methanosarcina mazei strain Go¨1. Mol Genet Genomics 2005, 273:290-298. Characterization of the first M. mazei strain obtained through targeted mutagenesis.

Current Opinion in Microbiology 2005, 8:745–751

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