Biochemistry and Molecular Biology Education 28 (2000) 186}191

The Archaea * a biochemical perspective

Clive Bullock
School of Life Sciences, University of Surrey, Roehampton Whitelands College. West Hill, London SW15 3SN, UK Received 16 September 1999; accepted 1 December 1999

Abstract The Archaea represent an unusual and instructive group of organisms for students of biochemistry. In this brief review some of their distinctive biochemical features are highlighted, along with appropriate topics and questions for student study. Archaea demonstrate unique structural features in the chemical makeup of their outer membranes and cell walls as well as in the components and design of their metabolic pathways, and the structure and function of their enzymes. Rationalising these biochemical features in relation to the chemically and physically harsh environments inhabited by these organisms and evaluating their potential (and limitations) for use in industrial biotechnology can form the basis of a range of pro"table student study topics. 2000 IUBMB. Published by Elsevier Science Ltd. All rights reserved.

1. Introduction The biochemical makeup of most living organisms is strikingly similar * common biomolecules, enzymes, metabolic pathways and so on. The Archaea are di!erent. This diverse group of microbes, possess some unusual and unique features which enable them to tolerate and thrive in some of the most inhospitable environments on earth. Studies of the biochemistry of these obscure but interesting organisms have led to a clearer understanding of cellular evolution and are revealing potential applications in biotechnology. From the student viewpoint, a study of the Archaea has the additional bene"t of breaking down the traditional divisions between biochemistry, microbiology and industrial biology.

anogens. This suggests that the Archaea may be descendants of the earliest forms of cellular life which would have had to evolve under such conditions. Despite outward appearances however, the Archaea are more closely biochemically related to the Eukarya than to the Bacteria.

3. Cell membranes Study topics: What are the unique chemical features of archeal cytoplasmic membranes, and how do they diwer from bacterial and eukaryotic membranes? To what extent do these diwerences account for the chemical and thermal tolerance of the Archaea? One of the most characteristic biochemical features of the group is the structure of their membrane lipids which are unique to the Archaea. The ester linkages (CH }OCOR) between fatty acids and glycerol usually  present in membrane glycerides are replaced by ether linkages (}CH OR), giving increased chemical stability  (resistance to hydrolysis, etc.) and the hydrocarbon chains (R) are essentially polymers based on the monomer isoprene (2-methylbut-1,3-diene). Isoprene is of course the monomer unit for natural rubber and the result is a hydrocarbon chain with regular methyl branches (Fig. 1). The major components of Archaean membranes are a glycerol diether with two C chains  (Fig. 1a) and a diglycerol tetraether with two C chains  (Fig. 1b), formed by covalently linking two diether molecules. Yet another unique feature is the presence of up to

2. The Archaea Around 20 years ago Carl Woese and others suggested that living organisms should be classi"ed into three primary groups, based on base-sequence studies of 16S and 18S ribosomal RNA molecules [1]. These groups, termed domains, are now referred to as the Bacteria, the Archaea and the Eukarya and it is generally accepted that the Archaea diverged earliest from the common ancestor and are therefore regarded as the most primitive group of organisms. Many are highly adapted to cope with extreme chemical and/or physical environments (temperature, pH, salinity, etc.) and the group can conveniently be divided into hyperthermophiles, halophiles and meth-

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Fig. 1. Lipids found in Archaean cell membranes (adapted from Refs. [2,3]).

Fig. 2. Comparison of Archaean and Eubacterial cell walls.

four cyclopentane rings in the hydrocarbon chains of thermoacidophiles such as Sulfolobus (Fig. 1c). Glycerol diethers assemble to form the usual bilayer membrane, while diglycerol tetraethers form an analagous monolayer (Fig. 1d), which can be thought of as a &fused' bilayer. Enzymes responsible for the biosynthesis of the ether linkages in glycerol diethers have been isolated [1], but routes for the biosynthesis of the tetraethers and the formation of cyclopentane units are still uncertain. All these features result in increased thermal stability and bilayers containing glycerol ethers retain a stable liquid-crystalline state over a wide temperature range [2]. It is likely that the presence of tetraethers in monolayers allows hyperthermophiles (optimum temperature '903C) to withstand extreme temperatures which would rapidly disrupt a bilayer structure. It should be noted however, that unlike many mesophilic organisms, the Archaea are unable to adapt to temperature changes by altering the degree of unsaturation or chain length of membrane lipids, though there is evidence that growth at higher temperatures increases the frequency of cyclopentane rings in Sulfolobus solfataricus and Thermoplasma acidophilum [1].

basis of lysozyme resistance in the Archaea. A detailed study of the basic stereochemistry of cell wall components (interesting because some unusual D and L sugar and amino-acid residues are present) Like the Bacteria, almost all Archaea have a cell wall to maintain cellular integrity but again there are subtle but signi"cant di!erences. In bacteria, a rigid peptidoglycan (murein) sheet is present composed of polysaccharide chains (N-acetylglucosamine {1-4 N-acetylmuramic acid {1-4) connected by peptide cross bridges which di!er slightly between gram positive and gram negative organisms (Fig. 2). Pseudopeptidoglycan is found in the methanogenic Archaea (Fig. 2) and its polysaccharide backbone (N-acetylglucosamine {1-3 N-acetyltalosaminuronic acid {1-3) is resistant to the antibacterial enzyme lysozyme as a result of the 1}3 linkages. The other Archaea exhibit a wide variety of complex cell wall structures composed of polysaccharide, glycoprotein or protein as well as inorganic components. In common with many bacteria, Archaeans possess a paracrystalline surface layer (S-layer), a crystalline protein surface sheet which probably acts as a selective barrier to solute entry. In halophilic Archaea such as Halobacterium which thrive in salt lakes and the Dead Sea where salinities exceed ten times that of coastal sea water (35%) the glycoprotein of the cell wall has a high proportion of the acidic amino acids aspartate and glutamate as sodium salts. Interestingly, this sodium binding is essential to

4. Cell walls Study topics: Chemical similarities and diwerences between archaeal and bacterial cell walls. The chemical

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maintain the cell wall and dilution of the medium leads to repulsion between the free carboxylate groups leading to cell wall disintegration and cell lysis. Halococcus, on the other hand, incorporates regular uronic acid residues, bearing charged sulphate groups [3].

5. Biochemical pathways Study topics: How do the unique archaeal coenzymes compare in structure and function with more familiar bacterial and eukaryotic coenzymes? How is carbon xxation achieved (from carbon dioxide) achieved in methanogenic Archaea? The Archaea are predominantly anaerobic organisms re#ecting the absence of oxygen from the early atmosphere and the decline in oxygen solubility at higher environmental temperatures. The Sulfolobales, the most recently evolved group, are the exception and Sulfolobus can grow aerobically by oxidising sulphur or anaerobically by reducing sulphur. Some Archaeans are chemoorganotrophic using organic sources such as glucose or acetate as energy sources whereas others are chemolithoautotrophic using carbon dioxide as a carbon source. Fermentation is rare but is exhibited by Hyperthermus butylicus which converts peptides to carbon dioxide, alcohols and organic acids. Other hyperthermic Archaea such as Pyrococcus and Pyrodictium may also become fermentative in the absence of sulphur and/or hydrogen [1]. Many metabolic pathways show similarities to those found in bacteria and eukaryotes; the citric acid cycle operates in the usual oxidative mode in aerobic Archaea and extreme halophiles show autotropic carbon dioxide "xation, for example, but some of the more unusual features are noted here (Fig. 3). Glycolysis appears to be absent in the Archaea and glucose is catabolised by a simple oxidation pathway in some organisms (Sulfolobus, Thermoplasma) (Fig. 3a). It has been proposed that glucose synthesis takes place by the reverse of this pathway in aerobic Archaea since the enzymes normally associated with gluconeogenesis are lacking. There is evidence for an unusual carbon monoxide dehydrogenase pathway in the hyperthermophile Archaeoglobus fulgidus which grows on lactate and sulphate. This route is e!ectively an alternative to the citric acid cycle for the conversion of lactate to carbon dioxide (Fig. 3b) [1]. An interesting reversal of the citric acid cycle occurs in Sulfolobus sp. and Thermoplasma acidophilum, where the pathway is run in reductive mode with carbon dioxide "xation (Fig. 3c). Methane production is a unique feature of the energy metabolism of one group of obligate anaerobes, the methanogenic Archaea. Three major routes have been identi"ed [3]:

Fig. 3. Metabolic pathways in the Archaea: (a) glucose catabolism in Sulfolobus; (b) lactate oxidation in Archaeglobus fulgidus; reductive citrate cycle (Sulfolobus and Thermoproteus neutrophilus) (adapted from Ref. [1]).

(1) From carbon dioxide or carbon monoxide or methanoate CO #4H PCH #2H O     4CO#2H OPCH #3CO    G"!131 kJ mol\ G"!210 kJ mol\

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Fig. 5. Methanogenesis from carbon dioxide (adapted from Ref. [3]). Key: MF"methofuran, MP"tetrahydromethanopterin, F "co enzyme F , CoM"coenzyme M, HS-HTP "7-mercaptohep tanoyl-threonine phosphate.

central carbon ring and the lack of methyl substitutents found in #avins. HS-HTP shows clear similarities to the pantothenic acid component found in acetyl coenzyme A, while coenzyme M is remarkable for its sheer simplicity. It acts as a methyl carrier in the "nal stage of methanogenesis: Coenzyme M!SCH #2HPCoenzyme M#CH .   A detailed description of all the methanogenic pathways is beyond the scope of this review, but an excellent summary can be found in Ref. [3]. A summary of the methanogenesis from carbon dioxide is shown as an example (Fig. 5). Electron transport leads to the generation of a proton gradient across the cell membrane (protons are pumped outwards) and this is linked to the phosphorylation of ADP to ATP via a proton-translocating ATPase in conventional fashion. The methanogens have speci"c requirements for only three trace metals, iron colbalt and nickel, largely related to their unusual enzymes and coenzymes. Nickel is required for Factor430, carbon monoxide dehydrogenase and hydrogenase. Ammonium ion (NH>) is their major nitrogen  source and several are capable of nitrogen "xation. Halophytic Archaea such as Halobacterium maintain an osmotic balance with the environment by actively accumulating high concentrations of potassium ions from the medium. This is an unusual feature since halophytic bacteria tend to produce organic solutes (glycine betaine, glutamate, etc.) for this purpose. A wellknown feature of Halobacterium and some other halophiles is its purple membrane resulting from the presence of the light-mediated proton pump, bacteriorhodopsin which is produced under low oxygen conditions. This pigment is associated with a carotene derivative, retinal. Light triggers the conversion of trans retinal to the cis form and its subsequent reversion to the trans isomer is coupled to the pumping of hydrogen ions to the outside of the membrane. The proton gradient is then used to drive other process such as ATP synthesis (photosynthetic phosphorylation) and K> uptake (Fig. 6).

Fig. 4. The coenzymes of Archaea.

4HCOO\#4H>PCH #3CO #2H O    G"!145 kJ mol\ (2) From methanol or methyl amines or methyl sulphides CH OHPCH #CO #2H O     G"!319 kJ mol\ CH NH ) HCl#2H OP3CH #CO #4NH Cl       G"!230 kJ mol\ (3) From acetate (Methanosarcina sp. and Methanothrix sp. only) CH COO\#H OPCH #HCO\     G"!31 kJ mol\ The methanogenic pathways incorporate a number of coenzymes unique to this group of Archaea (Fig. 4) which largely replace the common coenzymes NAD, FMN and FAD. Interesting and unusual chemical features include the presence of a furan ring (C H O) in methanofuran,   the presence of a pteridine rather than a purine ring system in methanopterin and the use of nickel in a porphyrin-type ring system in coenzyme F430. Coenzyme F420 shows obvious resemblances to the common #avin coenzyme FMN, but note the &missing' nitrogen in the

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Fig. 7. The tetrabrachion-STABLE complex in Staphylococcus marinus (redrawn from Ref. [6]).

Fig. 6. Biochemical functions associated with the membrane of Halobacterium.

6. Enzymes Study topics: What chemical features are associated with thermal and chemical stability in archaeal enzymes? To what extent does thermal stability inyuence enzyme activity? Much recent research on the Archaea centres on the remarkable thermal and chemical stability (towards detergents and organic solvents for example) of their enzymes, in the search for industrially useful biocatalysts. For these applications enzymes which are both thermophilic (active at higher temperatures, typically above 503C) and thermostable(resistant to irreversible denaturation at these temperatures) are desirable. However, comparative studies across microbial metabolism reveal that enzymes with similar functions often have similar catalytic activities at their optimum temperatures. This suggests that the structural features which enhance thermostability are likely to involve changes (such as reduced conformational #exibility) which will reduce overall activity [1]. Features associated with thermostability are reviewed in Ref. [4] and include: (a) Substitution of amino acids associated with conformational #exibility (glycine, serine, alanine) with those associated with rigidity (threonine, valine, proline); (b) substitution of amino acids with chemically active groups (cys (}SH), met, gln and asn (}CONH )); (c) a higher ratio of arg : lys  which promotes stronger polar interactions; (d) immobilisation onto an insoluble matrix (as in the example of STABLE below). Thermostable enzymes are common in all thermophilic organisms but hyperthermophilic Archaea which grow at 70}1153C are likely to be the source of the most stable examples. Over 30 enzymes have been isolated and puri"ed from Pyrococcus furiosus which grows optimally at 983 and even more thermophilic organisms such as Pyrodictium brockii (optimum 1053C) are under investigation [5]. One hyperthermostable enzyme isolated from Staphylothermus marinus, &stalk-associated archaebacterial endoprotease' (STABLE) has been extensively investigated [6]. S. marinus, a peptide-fermenting organ-

ism, inhabits marine geothermal vents and grows optimally at 923C. Its S layer contains an unusual glycoprotein complex, tetrabrachion, made up of repeating stalked units linked to form a cage-like cell wall (Fig. 7). Each stalk each stalk bears two globular protein molecules of the enzyme. STABLE is structurally related to the subtilisin family but shows remarkable stability. It is stable over a pH range of 3.2}12.7 (optimum pH 9.0), resistant to irreversible denaturation up to 1253C (regarded as the maximum temperature for primary structure stability) and retains residual activity even after exposure to 1353C. Analysis of STABLE has not revealed obvious structural features which would account for its extreme properties, but immobilisation within such a rigid cell wall framework undoubtedly makes a signi"cant contribution.

7. Applications in biotechnology Study topics: What are the potential applications of the Archaea and their enzymes in industrial systems? What are the limitations and problems in the economic exploitation of Archaea? Methanogenic Archaea make a major contribution to the controlled anaerobic digestion of human and animal wastes to biogas (predominantly methane). Intensive farming in the US allows the collection of virtually all animal dung and slurry with energy conversion rates approaching 60% and in the UK over half the sewage sludge produced undergoes anaerobic digestion [7]. In developing countries too, small scale sewage and animal dung digestion to biogas is a signi"cant energy technology. Land"ll gas is the biggest source of methane production however, and one US site alone (Staten Island, NY) produces 300,000 m/day and is a popular tourist attraction! Bacterial thermostable enzymes are already well established in biotechnology, in the processing of starch to liquid sugar syrups, for example and the 1980s saw biocatalysts double their share of the global catalyst market (from 10 to 20%) [8]. It seems unlikely that hyperstable enzymes will o!er signi"cant advantages in well-established, large-scale industrial processes, but could "nd applications in the production of specialist low-volume, high-value products and processes [5,8]. A typical example is development of the polymerase

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chain reaction (PCR) for the ampli"cation of DNA samples. The thermostable Taq DNA polymerase from Thermus aquaticus (a bacterium) enabled the automation of PCR because the Taq enzyme can withstand the temperatures required to separate the chains of duplex DNA. More recent developments involve the use of robust, hyperstable DNA polymerases from Archaeans such as Pyrococcus furiosus which o!er the twin advantages of greater accuracy of DNA copying in addition to enhanced thermostability [5]. The chemical stability which accompanies thermostability in archaean enzymes makes them ideal candidates for catalysing chiral syntheses where attack by organic solvents on enzymes is a frequent problem. The stereospeci"c reduction of ketones to chiral alcohols by alcohol dehydrogenase from Sulfolobus solfataricus is a promising example [5]. The enzymes of halophilic Archaea have not been exploited commercially because the requirement for high solute concentrations in many cases (up to 4.5 mol/l) makes isolation di$cult, though their action in enhancing #avour (and sometimes deterioration) in salted meats and "sh is of historical importance. Attempts to harness the light-trapping properties of the membrane of Halobacterium halobium have a long history [9] and there have been numerous attempts to create a solar cell by incorporating bacteriorhodopsin into arti"cial lipid bilayers or lipid-impregnated "lter sytems. These are assembled to promote unidirectional proton pumping across the membrane thus generating an electrical potential. Other potential applications include desalination employing the H>/Na> antiport or halorhodopsin (light-driven Cl\ pump) in the purple membrane, the use of bacteriorhodopsin for the synthesis of ATP on a commercial scale and most recently a possible role in the development of &biochips'. Purple membranes and their components have proved to be robust and resistant to irreversible denaturation. Halophytic Archaea also produce useful chemical products including a stable exopolysaccharide used as an emulsi"er and mobility controller in oil extraction (Halobacterium mediterranei), membrane surfactants for bitumen extraction from tar sands and the biodegradable polymer polyhydroxybutyrate (PHB) (Haloferax mediterranei) [8]. Gene transfer from halophiles to increase salt tolerance in crop plants is a further potential development. Microbiological mining of metals such as copper, uranium and gold dates back to the eighteenth century with the Rio Tinto copper mines in Spain and currently it is estimated that these processes account for at least 10% of copper production in the US. Although bacteria such as Thiobacillus sp. are involved, the Archaean Sulfolobus plays an important role in the oxidation of sulphur compounds and iron (Fe>) leading to the breakdown of the ore chalcopyrite (CuFeS2) and can also break down molybdenite (MoS ), a remarkable property since molyb denum is acutely toxic to most bacteria. Sulfolobus can grow in acidic conditions in the presence or absence of

oxygen at temperatures up to 853C and could speed up copper extraction at these temperatures. Unfortunately the absence of peptidoglycan in its cell wall renders it sensitive to abrasion and no industrial scale bioleaching currently utilises Sulfolobus [10]. This sensitivity has also hindered the use of S. brierleyi in the desulphurisation of coal where its unusual ability to metabolise either organic or inorganic sulphur could be exploited. Most biomining employs indigenous populations of acidophiles, but it is conceivable that seeding mineral leaching sites with Archaea such as Sulfolobus, Acidianus and Metallosphaera would enhance metal extraction since mineral oxidation is an exothermic process and therefore the use of thermophiles should signi"cantly increase reaction rates [8]. 8. Conclusions From any perspective, the Archaea are a truly unique group of organisms but our knowledge of all aspects of their way of life is limited, not least because of di$culties in discovering and extracting them from their obscure, natural habitats. Increased understanding of the biochemical features which have enabled them to cope with the harshest environments on earth will undoubtedly enhance their current status as key subjects in the search for new &industrial microorganisms' and biocatalysts, and is likely to push back the accepted environmental limits for the survival of living organisms still further. All the references cited in this brief review are recommended as accessible and informative sources for students researching the study topics suggested. References
[1] D.A. Cowan, Biochemistry and molecular biology of the extremely thermophilic archaeobacteria, in: R.A. Herbert (Ed.), Molecular Biology and Biotechnology of Extremophiles, Blackie, New York, 1992. [2] C. Edwards, (Ed.), Microbiology of Extreme Environments, Open University Press, Milton Keynes, 1990. [3] M.T. Madigan, J.M. Martinko, J. Parker, J. Brock, Biology of Microorganisms, 8th Edition, Prentice Hall, Englewood cli!s, NJ, 1997. [4] C. Bullock, Thermostable enzymes * new catalysts?, Educ. Chem. 36 (3) (1999) 68}71. [5] D.A. Cowan, Biotechnology of the Archaea, Trends Biotechnol. 10 (1992) 315}323. [6] J. Mayr, K.A.J. Lupas, C. Eckerskorn, W. Baumeister, J. Peters, A hyperstable protease of the subtilisin family bound to the surface layer of the Arachaeon Staphylothermus marinus, Curr. Biol. 6 (6) (1996) 739}749. [7] S. Pirt, Renewable sources of energy and materials, Biotechnol. Educ. 3 (1) (1992) 33}36. [8] R.A. Herbert, A perspective on the biotechnological potential of extremophiles Trends Biotechnol. 10 (1992) 395}401. [9] S. Prentis, Microbes that capture the sun, New Scientist 101 (1981) 159}163. [10] D.E. Rawlings, S. Silver, Mining with microbes, Biotechnology 13 (1995) 773}778.