What Archaea Have to Tell Biologists

Corresponding author: William B. Whitman, Department of Microbiology, University of Georgia, Athens, GA 30602-2605., whitman{at}arches.uga.edu (E-mail)

WE are excited to present the following review and research articles on archaeal research, and we thank the Genetics Society of America for this opportunity. In addition, we recognize the contributions of our colleagues, Charles Daniels (Ohio State University) and Michael Thomm (Universitaet Kiel), who along with the authors served as coeditors of papers on archaea in this volume.

More than two decades after the initial proposal, the archaeal hypothesis remains the best explanation for the unexpected diversity of molecular and biochemical properties found in the prokaryotes. This hypothesis states simply that the prokaryotes are not a monophyletic group but contain two very ancient phylogenetic lineages (WOESE and FOX 1977 ). In addition to the well-characterized bacteria, there is a second prokaryotic lineage of very remote ancestry called the archaebacteria or, more recently, the archaea. Although originally controversial, the archaeal hypothesis has gained wide acceptance among prokaryotic biologists, in part due to a thorough examination of many aspects of archaeal biology, including lipid composition, cell envelope structure, ribosomal structure, tRNA and rRNA structure, elongation factors, DNA-dependent RNA polymerases, and antibiotic sensitivity. These investigations, which were conducted in many laboratories worldwide, were summarized in WOESE and WOLFE 1985 . Perhaps, the most stunning confirmation of the archaeal hypothesis was provided by the first archaeal genomic sequence, where even after extensive analyses less than half of the open-reading frames (ORFs) discovered could be assigned a specific function based upon similarity to known bacterial and eukaryotic genes in the databases and analyses of motifs and other structural features (BULT et al. 1996 ; KOONIN et al. 1997 ).

While the study of fascinating microorganisms needs no special justification, the archaea provide unique opportunities to gain insight into a number of fundamental problems in biology. As one of the most ancient lineages of living organisms, the archaea set a boundary for evolutionary diversity and have the potential to offer key insights into the early evolution of life, including the origin of the eukaryotes. Many archaea are also extremophiles that flourish at high temperature, low or high pH, or high salt and delineate another boundary for life, the biochemical and geochemical boundary, which sets the physical limits of the biosphere. Finally, some archaea are fundamental components of the biogeochemical cycles on earth or dominate special ecosystems that are of great interest.

Prokaryotes have been present if not abundant on earth for more than 3.5 billion years, while evidence for eukaryotes is limited to the last 2.1 billion years. Thus, early life during the Archaean Eon was probably entirely prokaryotic. During this period, the major organizing principles of modern cells evolved and the biosphere formed. In the absence of an informative fossil record, comparative biology represents the major approach to investigating life during this era. The archaea, as representatives of one of the deepest lineages, offer special insights into the origin of cellular life and the ancestry of eukaryotes. For instance, most of the basic biosynthetic pathways for small molecules, such as amino acids and nucleic acids, appear to be conserved between the bacteria and archaea, suggesting that these pathways were inherited from a common ancestor. Likewise, many aspects of the central paradigm of cellular informatics including the genetic code, transcription, and translation appear to be highly conserved, which 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 research articles in this volume. As described by CANN and ISHINO 1999 , the pathway of DNA replication in archaea is still not known. However, the components of replication identified by genomic sequencing appear more similar to the eukaryotic system than the bacterial system. If this initial observation is confirmed upon further biochemical characterization, it may imply that archaea and bacteria diverged during the establishment of DNA as a component of the modern cell. In archaea, transcription shares many similarities with that found in eukaryotes, including structural and functional similarities of the RNA polymerase, transcription factors, and promoter sequence (THOMM 1996 ; REEVE et al. 1997 ). Similarities in transcription and translation between the archaea and eukaryotes provide one of the strongest arguments that these two ancient lineages share a common ancestor to the exclusion of the bacteria. From this perspective, transcriptional regulation in archaea is of special interest because it may provide insight into the natures of the last common ancestors with the bacteria as well as the eukaryotes. If the ancestor to the archaea and eukaryotes was functionally similar to modern organisms it should have had complex regulatory systems, some of which might remain in descendents in both lineages. Alternatively, if the ancestor was a more primitive organism, regulation may have developed independently in each lineage, and modern regulatory systems should not be homologous. At present, transcriptional regulation in archaea is not well described (LEIGH 1999 ). Articles in this volume present stories of two operons in Methanococcus. In one case, the major regulatory element for transcription of the selenium-independent hydrogenases of Methanococcus voltae resembles a silencer, which is common in eukaryotes (NOLL et al. 1999 ). In the other case, transcriptional regulation of the genes encoding the nitrogenase of Methanococcus maripaludis occurs at a repressor-binding site and is more bacterial like (KESSLER and LEIGH 1999 ). A third article in this volume by HASELTINE et al. 1999 uses a classical genetic approach to examine catabolite repression in the hyperthermophile Sulfolobus solfataricus. A novel group of pleiotropic extragenic regulatory mutations were recovered. Their analysis reveals the presence of a trans-acting transcriptional regulatory mechanism for glycosyl hydrolase expression and is suggestive of a positively regulated system. As described by MACARIO and CONWAY DE MACARIO 1999 , the protein folding apparatus of archaea contains both eukaryotic and bacterial components. Because the bacterial chaperone represented by DnaK is found in only a few archaeal lineages, it may have been acquired late in evolution by lateral gene transfer. Similarly, work on the functional genomics of Pyrococcus species by MAEDER et al. 1999 provides one of the first insights into hyperthermophilic diversity at the whole genome level and suggests that horizontal gene exchange may be an important evolutionary process in this group of archaea. Although speculative, these observations argue that the common ancestor of the bacteria and archaea was not a fully modern cell but one whose exact nature is yet to be determined (for a discussion of this idea see WOESE 1998 ). Finally, given the lack of detailed understanding of early evolution, the most important point is that these and other hypotheses concerning the nature of early life are testable by careful examination of archaeal biology.

Comprehending the full extent of prokaryotic diversity remains one of the great challenges of modern biology. The number of prokaryotes is enormous, on the order of 5 x 10³⁰ cells, and even given the disparity in cell size the total biomass of prokaryotes is comparable to that of eukaryotes (WHITMAN et al. 1998 ). How can we discover the limits of the genetic diversity in this enormous population? One approach is to use comparisons of the most distantly related organisms to delineate the outermost boundary. If these organisms are well chosen, the diversity of other organisms will fall within this boundary. Thus, comparisons of familar bacteria to the archaea, which represents the deepest known phylogenetic lineage of prokaryotes, might describe much of the diversity within prokaryotes.

This approach has many successes, some of which are described in this volume. Aminoacyl tRNA synthetases have long been thought to be among the most conserved biomolecules. Because of their fundamental role in protein biosynthesis, they may well have been one of the earliest systems to evolve, and their essential nature may have limited their variability. However, as described in the review by TUMBULA et al. 1999 , some archaea contain a novel class I lysyl-tRNA synthetase. Further examination revealed that the novel synthetase was also present in spirochetes as well as in a variety of other bacteria. Thus, discovery of a novel form of aminoacyl tRNA synthetase in archaea led to recognition of an unanticipated diversity in other prokaryotes. Likewise, KESSLER and LEIGH 1999 present evidence of a novel function of GlnB-like proteins. In proteobacteria such as Escherichia coli, GlnB functions in the regulation of adenylylation of glutamine synthetase, a key enzyme in ammonia assimilation. In the archaeon M. maripaludis, a homologous protein is involved in the post-transcriptional regulation of nitrogenase, a key enzyme in the fixation of N₂ to ammonia. However, it remains to be determined whether this novel function of GlnB is found in other prokarotes.

Although archaea represent a phylogenetic extreme of modern life, many but not all archaea are also extremophiles in terms of the habitats in which they are found. Extremophiles are organisms that thrive under conditions normally considered inhospitable, such as high temperature, high salt concentrations, low pH, or high pH. Extremophiles are important not only because they determine the boundaries of the biosphere but also because they determine the physical and chemical limits of the basic biological processes. For instance, most of the known hyperthermophiles, or organisms that grow optimally above 80°, are archaea. Many of the archaeal hyperthermophiles are also acidophiles and flourish at pH 1.5–3. Another group of archaeal extremophiles are the halobacteria, which grow at moderate temperatures but only at high salt concentrations. Some of the halophilic archaea are also alkaliphilic and grow in alkaline salt lakes at pH 9.5–11. While halophily is not uniquely archaeal, the halobacteria are very successful in this habitat.

Many of the articles in this volume describe basic molecular processes in extremophilic archaea. HETHKE et al. 1999 examine the thermostability of transcription at 95° in Pyrococcus. RUSSELL et al. 1999 add to the growing understanding of mechanisms controlling RNA processing in hyperthermophiles, which must necessarily overcome inherent constraints imposed by nucleic acid chemical instability. KLETZIN et al. 1999 describe plasmid replication in the hyperthermophilic acidophile Acidianus. This plasmid seems to be a member of a larger family of plasmids found in the chrenarchaeotes Acidianus and Sulfolobus. Finally, a novel family of viruses discovered by PRANGISHVILI et al. 1999 indicates that additional biodiversity occurs at the temperature extremes of life.

Even though our recognition of archaea as a distinct phylogenetic group is relatively recent, the participation of these prokaryotes in important biogeochemical processes or key ecosystems has been long known. The methanogenic archaea are the major source of atmospheric methane, which is an important greenhouse gas. Total biogenic methane production on earth is about 0.77 Pg of C yr^-1 (REEBURGH et al. 1993 ). During methane production, one CO₂ is formed for every CH₄. Therefore, the total amount of carbon processed is about 1.54 Pg of C or 1.6% of the total primary production on earth. This calculation emphasizes the importance of this archaea-catalyzed process, and, given its magnitude, it is not surprising that methanogenic archaea are common in a wide variety of anaerobic habitats, from the gastrointestinal tract of mammals and insects to wetlands and hyperthermophilic vents at the bottoms of oceans. In fact, the methanogens are the most cosmopolitan of the archaea presently cultured, and they are abundant in a wide variety of moderate as well as extreme habitats. On the basis of this abundance, other archaeal groups may have important global biogeochemical roles. For instance, direct cloning of rRNA genes from the environment indicates that archaea may represent up to 30% of the prokaryotes in Antarctic sea water (DELONG et al. 1994 ). Because these archaea have so far eluded cultivation, we can only speculate as to their properties.

Archaea are also important participants in some specialized, but very interesting, ecosystems. Hydrothermal vent communities are found in the deep sea along geological faults. These communities are of special interest because the primary producers are chemolithotrophic prokaryotes rather than photosynthetic organisms (JANNASCH and MOTTL 1985 ). In this volume, TAKAI and HORIKOSHI 1999 describe the archaeal components of five hydrothermal vent communities in the Pacific Ocean. A large diversity was observed not only within certain vent communities but also between vent communities, and evidence for many novel archaeal taxa was obtained. Likewise, in saline lakes, the halophilic archaea are important heterotrophs and reoxidize photosynthate produced by algae and photosynthetic bacteria.

In spite of the availability of six complete genomic sequences for archaea, the development of classical genetics in archaea has proven to be a slow business. Two main difficulties have been encountered. Most of the archaea are extremophiles and require special growth conditions to manipulate them. Even the more temperate methanogens are strictly anaerobic, and plating requires an anaerobic chamber and other cumbersome techniques. The requirement for the development of the methodologies to handle these organisms has certainly hindered progress. More fundamentally, because of the differences in molecular properties, many of the convenient tools developed for bacteria are ineffective in archaea.

In spite of these difficulties, great progress has been made in some archaeal groups, especially the halobacteria. In these organisms, a halobacterial transformation system is frequently used to investigate gene functions and promoter activities. Different types of shuttle vectors containing compatible halobacterial origins of replication and conferring resistance to antibiotics, such as novobiocin and mevinolin, are available. Thus, complementation studies are possible and were successfully used to investigate, for example, the formation of gas vesicles (OFFNER et al. 1996 , OFFNER et al. 1998 ). Also, vectors that enable the expression of halobacterial ORFs in halobacterial transformants are available (PFEIFER et al. 1994 ). In this volume, a method based upon a suicide vector was devised to identify genes involved in nitrate respiration of Haloferax volcanii (WANNER and SOPPA 1999 ). Articles in this volume also describe recent progress in the development of genetic systems in thermophilic and methanogenic archaea. STEDMAN et al. 1999 report on an efficient shuttle vector for the thermoacidophile S. solfataricus based upon the virus SSV1. Despite the extremes of high temperature, SCHMIDT et al. 1999 demonstrate that the horizontal transmission of genetic information can also occur in hyperthermophiles via a conjugation-like system requiring cell-to-cell contact. GARDNER and WHITMAN 1999 describe an expression system in the temperate methanogen M. maripaludis. Finally, KIM and WHITMAN 1999 report on a method of random insertional mutagenesis using a suicide plasmid in the same methanogen. With the recent development of genetic systems in archaea, the investigation of genes and gene function is possible and allows further comparative studies in this fascinating group of organisms.

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