Perspectives

Anecdotal, Historical and Critical Commentaries on Genetics Edited by James F. Crow and William F. Dove

What Archaea Have to Tell Biologists

William B. Whitman,* Felicitas Pfeifer,† Paul Blum‡ and Albrecht Klein§ *Department of Microbiology, University of Georgia, Athens Georgia 30602-2605, †Institut fuer Mikrobiologie und Genetik, Technischen Universitaet, D-64287 Darmstadt Germany, ‡School of Biological Sciences, University of Nebraska, Lincoln, Nebraska 68588-0666 and §Fachbereich Biologie-Genetik, Universitaet Marburg, D-35043 Marburg, Germany

E are excited to present the following review and While the study of fascinating microorganisms needs W research articles on archaeal research, and we no special justification, the archaea provide unique op- thank the Genetics Society of America for this opportu- portunities to gain insight into a number of fundamen- nity. In addition, we recognize the contributions of our tal problems in biology. As one of the most ancient colleagues, Charles Daniels (Ohio State University) and lineages of living organisms, the archaea set a boundary Michael Thomm (Universitaet Kiel), who along with for evolutionary diversity and have the potential to offer the authors served as coeditors of papers on archaea in key insights into the early evolution of life, including this volume. the origin of the eukaryotes. Many archaea are also More than two decades after the initial proposal, the extremophiles that flourish at high temperature, low or archaeal hypothesis remains the best explanation for high pH, or high salt and delineate another boundary the unexpected diversity of molecular and biochemical for life, the biochemical and geochemical boundary, properties found in the prokaryotes. This hypothesis which sets the physical limits of the biosphere. Finally, states simply that the prokaryotes are not a monophy- some archaea are fundamental components of the bio- letic group but contain two very ancient phylogenetic geochemical cycles on earth or dominate special ecosys- lineages (Woese and Fox 1977). In addition to the well- tems that are of great interest. characterized bacteria, there is a second prokaryotic Prokaryotes have been present if not abundant on lineage of very remote ancestry called the archaebacte- earth for more than 3.5 billion years, while evidence ria or, more recently, the archaea. Although originally for eukaryotes is limited to the last 2.1 billion years. controversial, the archaeal hypothesis has gained wide Thus, early life during the Archaean Eon was probably acceptance among prokaryotic biologists, in part due entirely prokaryotic. During this period, the major or- to a thorough examination of many aspects of archaeal ganizing principles of modern cells evolved and the biology, including lipid composition, cell envelope biosphere formed. In the absence of an informative structure, ribosomal structure, tRNA and rRNA struc- fossil record, comparative biology represents the major ture, elongation factors, DNA-dependent RNA polymer- approach to investigating life during this era. The ar- ases, and antibiotic sensitivity. These investigations, chaea, as representatives of one of the deepest lineages, which were conducted in many laboratories worldwide, offer special insights into the origin of cellular life and were summarized in Woese and Wolfe (1985). Perhaps, the ancestry of eukaryotes. For instance, most of the the most stunning confirmation of the archaeal hypoth- basic biosynthetic pathways for small molecules, such as esis was provided by the first archaeal genomic se- amino acids and nucleic acids, appear to be conserved quence, where even after extensive analyses less than between the bacteria and archaea, suggesting that these half of the open-reading frames (ORFs) discovered pathways were inherited from a common ancestor. Like- could be assigned a specific function based upon similar- wise, many aspects of the central paradigm of cellular ity to known bacterial and eukaryotic genes in the data- informatics including the genetic code, transcription, bases and analyses of motifs and other structural fea- and translation appear to be highly conserved, which tures (Bult et al. 1996; Koonin et al. 1997). also suggests that these features were established in the ancestors to modern cells. However, many mysteries remain, some of which are discussed in reviews and Corresponding author: William B. Whitman, Department of Microbiol- ogy, University of Georgia, Athens, GA 30602-2605. research articles in this volume. As described by Cann E-mail: [email protected] and Ishino (1999), the pathway of DNA replication in

Genetics 152: 1245–1248 (August 1999) 1246 W. B. Whitman et al. archaea is still not known. However, the components a fully modern cell but one whose exact nature is yet of replication identified by genomic sequencing appear to be determined (for a discussion of this idea see Woese more similar to the eukaryotic system than the bacterial 1998). Finally, given the lack of detailed understanding system. If this initial observation is confirmed upon fur- of early evolution, the most important point is that these ther biochemical characterization, it may imply that and other hypotheses concerning the nature of early life archaea and bacteria diverged during the establishment are testable by careful examination of archaeal biology. of DNA as a component of the modern cell. In archaea, Comprehending the full extent of prokaryotic diver- transcription shares many similarities with that found sity remains one of the great challenges of modern in eukaryotes, including structural and functional simi- biology. The number of prokaryotes is enormous, on larities of the RNA polymerase, transcription factors, the order of 5 ϫ 1030 cells, and even given the disparity and promoter sequence (Thomm 1996; Reeve et al. in cell size the total biomass of prokaryotes is compara- 1997). Similarities in transcription and translation be- ble to that of eukaryotes (Whitman et al. 1998). How tween the archaea and eukaryotes provide one of the can we discover the limits of the genetic diversity in this strongest arguments that these two ancient lineages enormous population? One approach is to use compari- share a common ancestor to the exclusion of the bacte- sons of the most distantly related organisms to delineate ria. From this perspective, transcriptional regulation in the outermost boundary. If these organisms are well archaea is of special interest because it may provide chosen, the diversity of other organisms will fall within insight into the natures of the last common ancestors this boundary. Thus, comparisons of familar bacteria with the bacteria as well as the eukaryotes. If the ancestor to the archaea, which represents the deepest known to the archaea and eukaryotes was functionally similar phylogenetic lineage of prokaryotes, might describe to modern organisms it should have had complex regu- much of the diversity within prokaryotes. latory systems, some of which might remain in descend- This approach has many successes, some of which are ents in both lineages. Alternatively, if the ancestor was described in this volume. Aminoacyl tRNA synthetases a more primitive organism, regulation may have devel- have long been thought to be among the most con- oped independently in each lineage, and modern regu- served biomolecules. Because of their fundamental role latory systems should not be homologous. At present, in protein biosynthesis, they may well have been one of transcriptional regulation in archaea is not well de- the earliest systems to evolve, and their essential nature scribed (Leigh 1999). Articles in this volume present may have limited their variability. However, as described stories of two operons in Methanococcus. In one case, in the review by Tumbula et al. (1999), some archaea the major regulatory element for transcription of the contain a novel class I lysyl-tRNA synthetase. Further selenium-independent hydrogenases of Methanococcus examination revealed that the novel synthetase was also voltae resembles a silencer, which is common in eukary- present in spirochetes as well as in a variety of other otes (Noll et al. 1999). In the other case, transcriptional bacteria. Thus, discovery of a novel form of aminoacyl regulation of the genes encoding the nitrogenase of tRNA synthetase in archaea led to recognition of an Methanococcus maripaludis occurs at a repressor-binding unanticipated diversity in other prokaryotes. Likewise, site and is more bacterial like (Kessler and Leigh Kessler and Leigh (1999) present evidence of a novel 1999). A third article in this volume by Haseltine et function of GlnB-like proteins. In proteobacteria such al. (1999) uses a classical genetic approach to examine as Escherichia coli, GlnB functions in the regulation of catabolite repression in the hyperthermophile Sulfolobus adenylylation of glutamine synthetase, a key enzyme in solfataricus. A novel group of pleiotropic extragenic reg- ammonia assimilation. In the archaeon M. maripaludis, ulatory mutations were recovered. Their analysis reveals a homologous protein is involved in the post-transcrip- the presence of a trans-acting transcriptional regulatory tional regulation of nitrogenase, a key enzyme in the mechanism for glycosyl hydrolase expression and is sug- fixation of N2 to ammonia. However, it remains to be gestive of a positively regulated system. As described by determined whether this novel function of GlnB is Macario and Conway de Macario (1999), the protein found in other prokarotes. folding apparatus of archaea contains both eukaryotic Although archaea represent a phylogenetic extreme and bacterial components. Because the bacterial chap- of modern life, many but not all archaea are also extrem- erone represented by DnaK is found in only a few arch- ophiles in terms of the habitats in which they are found. aeal lineages, it may have been acquired late in evolution Extremophiles are organisms that thrive under condi- by lateral gene transfer. Similarly, work on the functions normally considered inhospitable, such as high tional genomics of Pyrococcus species by Maeder et al. temperature, high salt concentrations, low pH, or high (1999) provides one of the first insights into hyperther- pH. Extremophiles are important not only because they mophilic diversity at the whole genome level and sug- determine the boundaries of the biosphere but also gests that horizontal gene exchange may be an impor- because they determine the physical and chemical limits tant evolutionary process in this group of archaea. of the basic biological processes. For instance, most of Although speculative, these observations argue that the the known hyperthermophiles, or organisms that grow common ancestor of the bacteria and archaea was not optimally above 80Њ, are archaea. Many of the archaeal Perspectives 1247 hyperthermophiles are also acidophiles and flourish at nasch and Mottl 1985). In this volume, Takai and pH 1.5–3. Another group of archaeal extremophiles are Horikoshi (1999) describe the archaeal components the halobacteria, which grow at moderate temperatures of five hydrothermal vent communities in the Pacific but only at high salt concentrations. Some of the halo- Ocean. A large diversity was observed not only within philic archaea are also alkaliphilic and grow in alkaline certain vent communities but also between vent commu- salt lakes at pH 9.5–11. While halophily is not uniquely nities, and evidence for many novel archaeal taxa was archaeal, the halobacteria are very successful in this obtained. Likewise, in saline lakes, the halophilic arch- habitat. aea are important heterotrophs and reoxidize photosyn- Many of the articles in this volume describe basic mo- thate produced by algae and photosynthetic bacteria. lecular processes in extremophilic archaea. Hethke et In spite of the availability of six complete genomic al. (1999) examine the thermostability of transcription sequences for archaea, the development of classical ge- at 95Њ in Pyrococcus. Russell et al. (1999) add to the netics in archaea has proven to be a slow business. Two growing understanding of mechanisms controlling RNA main difficulties have been encountered. Most of the processing in hyperthermophiles, which must necessar- archaea are extremophiles and require special growth ily overcome inherent constraints imposed by nucleic conditions to manipulate them. Even the more temper- acid chemical instability. Kletzin et al. (1999) describe ate methanogens are strictly anaerobic, and plating re- plasmid replication in the hyperthermophilic acido- quires an anaerobic chamber and other cumbersome phile Acidianus. This plasmid seems to be a member of techniques. The requirement for the development of a larger family of plasmids found in the chrenarchaeotes the methodologies to handle these organisms has cer- Acidianus and Sulfolobus. Finally, a novel family of vi- tainly hindered progress. More fundamentally, because ruses discovered by Prangishvili et al. (1999) indicates of the differences in molecular properties, many of the that additional biodiversity occurs at the temperature convenient tools developed for bacteria are ineffective extremes of life. in archaea. Even though our recognition of archaea as a distinct In spite of these difficulties, great progress has been phylogenetic group is relatively recent, the participation made in some archaeal groups, especially the halobact- of these prokaryotes in important biogeochemical pro- eria. In these organisms, a halobacterial transformation cesses or key ecosystems has been long known. The system is frequently used to investigate gene functions methanogenic archaea are the major source of atmo- and promoter activities. Different types of shuttle vec- spheric methane, which is an important greenhouse tors containing compatible halobacterial origins of rep- gas. Total biogenic methane production on earth is lication and conferring resistance to antibiotics, such as about 0.77 Pg of C yrϪ1 (Reeburgh et al. 1993). During novobiocin and mevinolin, are available. Thus, comple- methane production, one CO2 is formed for every CH4. mentation studies are possible and were successfully Therefore, the total amount of carbon processed is used to investigate, for example, the formation of gas about 1.54 Pg of C or 1.6% of the total primary produc- vesicles (Offner et al. 1996, 1998). Also, vectors that tion on earth. This calculation emphasizes the impor- enable the expression of halobacterial ORFs in halobact- tance of this archaea-catalyzed process, and, given its erial transformants are available (Pfeifer et al. 1994). magnitude, it is not surprising that methanogenic arch- In this volume, a method based upon a suicide vector aea are common in a wide variety of anaerobic habitats, was devised to identify genes involved in nitrate respira- from the gastrointestinal tract of mammals and insects tion of Haloferax volcanii (Wanner and Soppa 1999). to wetlands and hyperthermophilic vents at the bottoms Articles in this volume also describe recent progress in of oceans. In fact, the methanogens are the most cosmo- the development of genetic systems in thermophilic and politan of the archaea presently cultured, and they are methanogenic archaea. Stedman et al. (1999) report abundant in a wide variety of moderate as well as ex- on an efficient shuttle vector for the thermoacidophile treme habitats. On the basis of this abundance, other S. solfataricus based upon the virus SSV1. Despite the archaeal groups may have important global biogeo- extremes of high temperature, Schmidt et al. (1999) chemical roles. For instance, direct cloning of rRNA demonstrate that the horizontal transmission of genetic genes from the environment indicates that archaea may information can also occur in hyperthermophiles via represent up to 30% of the prokaryotes in Antarctic sea a conjugation-like system requiring cell-to-cell contact. water (Delong et al. 1994). Because these archaea have Gardner and Whitman (1999) describe an expression so far eluded cultivation, we can only speculate as to system in the temperate methanogen M. maripaludis. their properties. Finally, Kim and Whitman (1999) report on a method Archaea are also important participants in some spe- of random insertional mutagenesis using a suicide plas- cialized, but very interesting, ecosystems. Hydrothermal mid in the same methanogen. With the recent develop- vent communities are found in the deep sea along geo- ment of genetic systems in archaea, the investigation of logical faults. These communities are of special interest genes and gene function is possible and allows further because the primary producers are chemolithotrophic comparative studies in this fascinating group of organ- prokaryotes rather than photosynthetic organisms (Jan- isms. 1248 W. B. Whitman et al.

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