Archaea: Molecular and Cellular Biology

This chapter describes the recent advances that have been made in understanding the biochemical players that facilitate the complex macromolecular process that mediates faithful replication of archaeal chromosomes. The current state of knowledge of the machineries that drive the archaeal cell cycle is discussed. In bacteria, the functional single-stranded DNA-binding proteins (SSBs) is a homotetramer that wraps 65 nucleotides. Pol α, Pol δ, and Pol ε are the major replicative polymerases, with Pol δ acting on the lagging strand and both acting on the leading strand of DNA replication. Sliding clamps are well known for their role in DNA replication, but they also interact with factors involved in other cellular processes, such as DNA repair and recombination, and cell cycle regulators. While an ever-growing body of data has yielded considerable insight into the form and function of the archaeal DNA replication machinery, much less is known about the details of the archaeal cell cycle and its control. Indeed, what little is known appears to be suggesting that diverse mechanisms may be employed to regulate chromosome copy number, to coordinate DNA replication and cell division, and even to mediate the process of cell division itself. Researchers examined nucleoid distribution during the cell cycle, and the results suggested that chromosome segregation was concomitant with DNA replication, as was proposed for M. thermautotrophicus, in a mode akin to that employed by bacteria.

This chapter examines the ‘’natural genetics’’ of methanogenic, halophilic, and thermophilic archaea, progressing from the molecular scale to cells and finally to populations. The simplest reconciliation of observations would seem to be that the thermophilic and hyperthermophilic archaea have alternative molecular strategies that assume the function of classical MMR and NER systems but do not involve homologous proteins. In its most general sense, genetic recombination means the creation of new DNA sequences from existing sequences by processes involving strand exchange rather than error-prone synthesis. ‘’Illegitimate’’ recombination breaks and joins DNA sequences with negligible influence of the sequences involved. Limited migration elevates the importance of mutation and recombination in the evolution of natural populations. Testing hypothesis that archaea, or major archaeal groups will involve measuring molecular processes central to the survival, reproduction, and evolution of archaea and to the development of experimental tools for establishing gene function at the molecular level. Compared with bacteria and unicellular eucarya, archaea that have been analyzed in genetic terms have rather low rates of neutral mutation and high rates of recombination. This combination of properties would seem ideal for evolutionary adaptation. Combining computational genetic analyses of natural populations with experimental analyses of cultured archaea will help clarify how molecular mechanisms of archaea determine genetic properties and how genetic properties of archaea affect genome stability and evolution.

Most organisms contain between 40 and 50 different transfer RNA (tRNA) molecules that read one or more of the 61 or 62 different sense codons through specific codon-anticodon interactions and position the appropriate amino acid for insertion into the growing polypeptide chain. Additional complexity in the tRNA processing and modification pathway occurs in cases where archaeal tRNA genes contain an intron. The chapter describes introns in archaeal transcripts. The genes for ribosomal RNA (rrn) are located in operons in the archaeal genome and are transcribed to produce multicistronic precursor RNAs. Two early studies used nuclease protection and primer extension assays to define the intermediates generated during processing of the primary rRNA transcript from two canonical rrn operons: the single-rrn operon in Halobacterium salinarum and the canonical rrnA operon in Haloarcula marismortui. The proportion of modified nucleotides in tRNA can approach 50% or more. The two most frequent modifications in RNA are ribose methylation and the pseudouridylation. In an attempt to distinguish between these two alternatives, actinomycin D was used to inhibit transcription and Northern hybridization was used to follow the decay of the various mRNA fragments. There was a strong correlation between fragment length and stability: the shorter the fragment, the longer the half-life. This correlation was used to argue that the transcript fragments are generated by endonucleotic cleavage rather than premature transcription termination, and that the distal sequences released following cleavage are selectively degraded.

This chapter discusses four unique aspects of archaeal aa-tRNA formation that led to a much deeper understanding of this process not only in the Archaea, but in all domains of life. The topics are: (i) processing of half-tRNA genes to mature tRNA in Nanoarchaeum equitans, (ii) RNA-dependent cysteine synthesis in methanogens, (iii) pyrrolysyl-tRNA formation in the Methanosarcinaceae, and (iv) glutaminyl- tRNA synthesis in archaea. The bulge-helix-bulge (BHB) motifs postulated to form at the intron-exon junctions of archaeal tRNAs show divergence from the canonical structure. Once mature tRNA has been generated each tRNA species needs to be acylated (charged) with the correct amino acid. This is primarily achieved by the direct attachment of an amino acid to the corresponding tRNA by an aminoacyl-tRNA synthetase. However, since many organisms lack the complete set of 20 aminoacyl-tRNA synthetases (aaRSs), many biochemical, genetic, and genomic studies revealed the existence of an essential indirect two-step pathway that also provides correctly charged aa-tRNA. The aminoacyl-tRNA synthetases are an ancient family of enzymes that esterify an amino acid to the 3’ end of the cognate tRNA species. The current understanding of the Methanothermobacter thermautotrophicus Glu-tRNAGln amidotransferase GatDE is discussed. Discovery of the tRNA-dependent cysteine biosynthetic route in M. jannaschii may have implications that reach far beyond the only problem of the formation of Cys-tRNACys in three methanogenic archaea. The Methanosarcinaceae are an exception among the methanogens, as they are able to use compounds like methanol, methylated thiols, and methylamines as energy sources.

This chapter focuses on other modalities of signal transduction, including intracellular second messengers, feedback regulation, and posttranslational modifications such as the phosphorylation-dephosphorylation of proteins. Empirical studies of archaeal-archaeal and archaeal-bacterial communication have been few in number and preliminary in nature. Inspection of archaeal genomes has revealed them to be devoid of homologs of the prototypic bacterial quorum-sensing proteins LuxS and LuxR. Two-component systems differ in several fundamental respects from protein-serine/threonine/tyrosine phosphorylation cascades. First, autophosohorylation is the predominant mechanism of phosphorylation in the two-component system, whereas protein-serine/threonine/tyrosine phosphorylation cascades rely primarily on phosphotransfer reactions catalyzed by protein kinases that are distinct from the phosphoacceptor protein. Second, the chemical nature of the phosphoryl moieties formed during two-component signaling differs significantly from that of protein-serine/threonine/tyrosine phosphorylation. Posttranslational modifications have discussed and demonstrated, at least in some instances, to modulate the function of one or more target proteins from the Archaea or other organisms. It is widely presumed that (poly)ADP-ribosylation regulates the functional properties of proteins, as is the case with other covalent modifications such as protein phosphorylation-dephosphorylation. Given that the members of the bacterial domain have been the subject of decades of intensive study, it appears highly likely that the Archaea will not only be found to contain new sensor-response mechanisms and molecules, but that they will provide new insights into this vital process in other organisms as well.

Recent genomic sequencing, proteomic analyses, and development of genetic systems continue to expand one's understanding of methanogenesis and the Archaea. The conversion of the methyl group of acetate to methane (acetate fermentation pathway) produces about two-thirds of the annual production, whereas one-third derives from the reduction of carbon dioxide with electrons supplied from the oxidation of formate or hydrogen (carbon dioxide reduction pathway). Thus, the methanogens rely on the first two groups to supply substrates for growth and methanogenesis. Methanogens are the main constituency of the Euryarchaeota and are subdivided into five orders, including such as Methanobacteriales, Methanococcales and Methanomicrobiales; each with distinctive characteristics. Methanofuran (MF) and tetrahydromethanopterin (THMPT) function as one-carbon carriers, the latter coenzyme also functioning in methylotrophic microbes from the Bacteria domain. A proteomic and transcriptional analysis of cold adaptation has revealed the thermal regulation of several genes essential for methanogenesis by dismutation of the methyl group of trimethylamine. The extent of regulation of genes essential for methanogenesis and other fundamental processes in response to temperature is consistent with a role in providing the cell with an ecological advantage in cold environments. The genome sequences of M. acetivorans, M. mazei, and M. thermophila harbor two homologs of mtaA, three homologs of mtaB, and three homologs of mtaC encoding enzymes specific for methanogenesis from methanol.

This chapter summarizes the different biosynthetic steps of isoprenoid ether lipid biosynthesis in archaea, describing the underlying enzymatic reactions that have been characterized. The evolution of this lipid biosynthesis apparatus in a variety of archaea is discussed. The effect of the environment on the nature of the lipids present in archaeal cell membranes is scrutinized in an attempt to link structure and function. Similar to other isoprenoids, archaeal lipid side chains are assembled from two universal precursors: isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In archaea, geranylgeranyl diphosphate (GGPP) synthase can elongate DMAPP to obtain both farnesyl diphosphate (FPP) and GGPP, the latter being the isoprenyl forming the side chains of C20-C20 diether lipids. However, similar to the studies on partially saturated side chains, there is little experimental evidence on when these structures are formed during the process of archaeal lipid biosynthesis. The detection of CDP-archaeol synthase and archaetidylserine synthase activities in Methanothermobacter thermautotrophicus suggests that the biochemical steps for the addition of polar head groups on archaeal lipid precursors, might proceed in a manner analogous to fatty acid biosynthesis in bacteria. High-performance liquid chromotography (HPLC), in combination with electrospray mass spectrometry (ES-MS) was used to characterize the membrane phospholipids and glycolipids of halophilic archaea and the cold-adapted methanogen M. burtonii.

Recent analyses of the archaeal Sec and Tat pathways have revealed novel and crucial information about archaeal protein translocation, as well as protein translocation in general. This chapter provides an overview on protein translocation into and across archaeal cytoplasmic membranes. The Sec pathway is the only known universally conserved protein translocation pathway. Protein translocation may be driven by one or several extracytoplasmic activities that provide directionality by preventing movement of the polypeptide chain back into the cytoplasm. In vitro studies suggest that the proton motive force (PMF), in concert with the action of SecA, facilitates bacterial secretion via the Sec pore. Furthermore, the PMF is apparently sufficient to drive translocation of proteins via the twin-arginine translocation (Tat) pore. Thus, it is possible that an ion gradient across the archaeal membrane is the sole source of energy for protein translocation. Many bacteria and archaea possess an additional general secretion pathway, described as the Tat pathway. The presence of the twin-arginine motif in the Tat signal sequence provided a means of identifying novel Tat substrates by computational pattern-matching techniques. Recent in vivo, in vitro, and in silico studies have led to a better understanding of archaeal protein translocation. Moreover, the elucidation of an archaeal Sec-pore X-ray crystal structure strikingly demonstrates how analysis of this pathway in archaea can significantly advance the field of protein translocation as a whole. In addition to standard molecular and biochemical approaches, it is now crucial to develop in vitro Sec and Tat protein translocation systems that will more clearly define the mechanisms of these pathways and reveal the energetics of these cellular processes in archaea.

The existence of many informational proteins that are common to Archaea and Eucarya and absent from Bacteria is not only true for DNA replication, but also for translation, transcription, and RNA and protein processing. Many reviews have summarized insight from newly sequenced archaeal genomes and have usually focused on aspects of comparative genomics. This chapter focuses on the description of archaeal genomes and what can be learned from archaeal genomics about the mechanisms of genome evolution, and the history of the Archaea domain itself. Compared with Bacteria, the number of completely sequenced archaeal genomes is much smaller. The larger genomes are from mesophilic archaea and contain a high proportion of genes recruited from bacteria by horizontal gene transfer (HGT). The chromosome terminus appears to be a hot spot of recombination in archaea, as it is in bacteria. This was clearly shown from a genome comparison of the two closely related species, Pyrococcus abyssi and Pyrococcus horikoshii. Comparative genomics has shown that gene loss, gene duplication, and integration of foreign DNA are major forces shaping genome evolution. The continuous evolution of archaeal genomes by gene loss and acquisition is obvious from the comparative analyses of the proteomes of closely related species. In a recent study using unfolding simulation experiments to determine amino acid composition, the number of charged residues in hyperthermophiles was reported to be much greater than it would need to be for the stabilization. Ongoing metagenomics projects will continue to broaden the understanding of archaeal diversity and evolution.

This chapter discusses molecular genetics of Archaea. Gene transfer systems currently exist for species within all three physiological groups of archaea, halophiles, methanogens, and nonmethanogenic hyperthermophiles. The chapter reviews the development of these systems. Colonization of methanogenic and nonmethanogenic hyperthermophiles on solidified medium is equally problematic, as agar is rapidly dehydrated at high temperatures, especially at the concentrations required for it to remain solidified. Therefore, gellan gum (Gelrite) is used as the solidifying agent for growth of thermophiles and hyperthermophiles, which are incubated in a plastic bag or anaerobe jar to minimize dehydration. Haloarchaea are transformed via polyethylene glycol (PEG) mediated transformation, which was first described in Haloferax volcanii. Gene disruption is required to identify and confirm the function of genes within the archaea. Random mutagenesis using chemical and UV radiation has been successfully used for H. volcanii, Methanococcus voltae, Methanococcus maripaludis, and Pyrococcus abyssi. Progress in the development of methodologies for archaeal genetics has rapidly accelerated in the past decade. Additional methods are currently under development for all three archaeal phyla, including additional systems for markerless exchange, gene expression, topological mapping, protein tagging and expression, as well as others. The choice of archaeal genomes for sequencing is now largely driven by the availability of genetic systems, which at present include complete genomes of the halophiles Haloarcula marismortui and Halobacterium sp.

Archaeosomes have been developed from various archaea for use as drug delivery systems and vaccine applications that utilize their adjuvant properties. This chapter describes the mechanism of archaeosome adjuvants as self-adjuvanting antigen carrier systems that are taken up by specific receptor-mediated endocytosis to promote both CD4+ and CD8+ T-cell responses. Effects of lipid structure on the immune response are also reviewed in this chapter. Archaeal lipids possess several features that make them ideal for the preparation of archaeosomes. The first is the inherent stability of the polar lipids. Second, archaeal lipids form archaeosomes over physiological temperature ranges, allowing preparation of vaccines at ambient temperatures. Third, once formed, archaeosomes of 50 to 250-nm diameters remain suspended indefinitely and resist fusion or aggregation over long storage periods. Two signals are required to activate T cells. The first is antigen presentation in the context of MHC, and the second is costimulation of the specific T cell recognizing the presented antigen. Several lines of evidence support a phosphatidylserine (PS) receptormediated endocytic mechanism. Data using test antigens indicate the strong potential for archaeosome vaccines to achieve immunity against intracellular bacteria. Because Listeria monocytogenes is an intracellular pathogen that is typically cleared by the host CD8+ T-cell immunity, this model has been used as a test for potency of archaeosome vaccines.