Extremophiles

Extremophiles are organisms that require extreme environments for growth. Although this is perhaps self-evident, what constitutes extreme? Extreme is a relative term, with the point of comparison being what is normal for humans. Extremophiles are therefore organisms that are “fond of ” or “love” (-phile) environments such as high temperature, pH, pressure, or salt concentration; or low temperature, pH, nutrient concentration, or water availability. Extremophiles are also organisms that can tolerate other extreme conditions, including high levels of radiation or toxic compounds, or living conditions that humans consider unusual, such as living in rocks 1.5 km below the surface of Earth. In addition, extremophiles may be found in environments with a combination of extreme conditions, such as high temperature and high acidity or high pressure and low temperature. 

I. INTRODUCTION 

Most extremophiles are microorganisms. For example, the upper temperature limits for archaeal, bacterial, and eukaryotic microorganisms are 113, 95, and 62 _C, respectively, in contrast to most metazoans (multicellular eukaryotes, e.g., animals and plants), which are unable to grow above temperatures of 50 _C. This example of thermal adaptation highlights an important distinction among the different classes of microorganisms (i.e., Archaea can grow at extremely high temperatures in comparison to their eukaryotic counterparts), and underscores the fundamentally different evolutionary origins of the members of the three domains of life, Archaea, Bacteria, and Eucarya. Although Archaea (formerly Archaebacteria) and Bacteria are both loosely defined as prokaryotes, they are by no means more similar to each other than Archaea are to eukaryotes. For example, archaea have a number of archaeal-specific traits (glycerol-1-phosphate– lipid backbones and methanogenesis), in addition to sharing many bacterial (metabolism, biosynthesis, energy generation, transport, and nitrogen fixation) and eukaryotic (transcription, translation, and replication) features. Due to the fact that Archaea are often found in extreme environments, the term “extremophile” is often used synonymously with “archaea”, and many of the extremophiles described here are members of the Archaea. It should be noted, however, that Archaea are also found in a broad range of nonextreme marine and soil environments. 

The first use of the term “extremophile” appeared in 1974 in a paper by MacElroy (Biosystems 6: 74–75) entitled, “Some Comments on the Evolution of Extremophiles.” In the 1990s, studies on extremophiles have progressed to the extent that the First International Congress on Extremophiles was convened in Portugal, June 2–6, 1996, and the scientific journal Extremophiles was established in February 1997. These developments in the field have arisen due to the isolation of extremophiles from environments previously considered impossible for sustaining biological life. As a result, the appreciation of microbial biodiversity has been reinvigorated and challenging new ideas about the origin and evolution of life on Earth have been generated. In addition, the novel cellular components and pathways identified in extremophiles have provided a burgeoning new biotechnology industry. In recognition of the importance of extremophiles to microbiology, the following sections describe the habitats, biochemistry, and physiology of a diverse range of extremophiles, followed by a concluding section on the biotechnological applications of extremophiles and their products. 

II. HYPERTHERMOPHILES 

A. Defining temperature classes of microorganisms Microorganisms can generally be separated into four groups with regard to their temperature optima for growth. In order of increasing temperature, they are psychrophiles (optimum of 15 _C, maximum _20 _C), mesophiles (optimum between 20 and 45 _C), thermophiles (optimum between 45 and 80 _C), and hyperthermophiles (optimum of 80 _C or higher). On the whole, these definitions are generally accepted terms; the reader should be aware, however, that some terms may have alternative meanings in specific fields. For example, the upper temperature for the growth of yeast is about 48 _C, and the majority of yeasts can grow below 20 _C. As a result, a thermophilic yeast is defined by its inability to grow below 20 _C, but with no restrictions placed on the maximum temperature for growth. B. Habitats and microorganisms Hyperthermophiles have been isolated from a range of natural geothermally heated environments (Table 37.1). The terrestrial environments tend to be acidic (as low as pH 0.5) with low salinity (0.1–0.5% salt), whereas the marine systems are saline (3% salt) and generally less acidic (pH 5–8.5). Artificial sources of isolates include coal refuse piles and geothermal power plants. The terrestrially based, volcanic systems include hot springs such as solfataras, which are sulfur-rich and generally acidic, or others that are boiling at a neutral pH, or that are iron-rich. Most of the heated soils and water contain elemental sulfur and sulfides, and most isolates metabolize sulfur. A by-product of oxidation tends to be sulfuric acid and as a result many of the hot springs are extremely acidic. Hyperthermophilic archaea have also been isolated from oil from geothermally heated oil reserves present in Jurassic sandstone and limestone 3500 m below the bed of the North Sea and below the Alaskan North Slope permafrost soil. As a by-product of their metabolism during oil extraction, they produce hydrogen sulfide; this condition is referred to as reservoir souring. Numerous hyperthermophiles have been isolated from submarine hydrothermal systems, including hot springs, sediments, sea mounts (submarine volcanoes), fumaroles (steam vents with temperatures up to 150–500 _C), and deep-sea vents. The hot deep-sea vents are often referred to as “black smokers” due to the thick plume of black material that forms from mineral-rich fluids at temperatures up to 400 _C, precipitating on mixing with the cold (1–5 _C) seawater. The submarine systems are a rich source of microbial and higher eukaryotic life that spans the temperature ranges suitable for hyperthermophiles through psychrophiles. Due to the extreme depth of some of the vents (3500 m), organisms are also exposed to extreme pressure, and as a result they tend to be barophilic or barotolerant (see Section V). The tube worms, giant clams, and mussels that inhabit the vents are dependent on the activity of the chemolithotrophic microorganisms to fix CO2 and use a broad range of inorganic energy sources emitted from the vents. The first eukaryotic organism living at very high temperatures was identified at the Axial Summit Caldera, west of Mexico. 

The Pompeii worm (Alvinella pompejana) was observed living in a hydrothermal system in which the posterior of the worm was in 80 _C water. Interestingly, it is speculated that the ability of the worm to survive the heat may be due to the presence of microorganisms that cover its exterior and secrete heat-stable enzymes. Most isolated hyperthermophiles are members of Archaea, and it is this characteristic that is often associated with this class of microorganism. However, it was the isolation of Thermus aquaticus, a bacterium, from a hot spring in Yellowstone National Park by Thomas Brock in 1969 that led to the discoveries of hyperthermophilic archaea. In 1972, Brock described Sulfolobus, an obligately aerobic archaeon that is able to oxidize H2S or S0 to H2SO4 and fix CO2 as a carbon source. The hot springs that Sulfolobus spp. thrive in are typically as hot as 90 _C, with an acidity of pH 1–5. Some hyperthermophiles have been found in unique locations, for example, Methanothermus spp., which have only been isolated from one solfataric area of Iceland. In contrast, Pyrococcus and Thermococcus spp. have been found in both submarine systems and subterranean oil reserves. Of all the isolates to date, Pyrolobus fumarii has the highest maximum temperature (113 _C). Furthermore, it is restricted to temperatures above 90 _C. Using a microscope slide suspended in the outflow from a black smoker at 125–140 _C, microbial films have been detected on the glass surfaces. Although organisms capable of growth at such high temperatures have not been isolated, this is good evidence that microorganisms are capable of growth in this extreme temperature range. C. Biochemistry and physiology of adaptation A broad diversity of hyperthermophiles with varied morphologies, metabolisms, and pH requirements have been isolated, and the characteristics of a number of these are shown in Table 37.2. Although organisms such as Sulfolobus spp. are aerobic chemoorganotrophs, most hyperthermophiles are anaerobic and many are chemolithotrophs. The extent of metabolic diversity in the hyperthermophiles is exemplified by the strict requirement of Methanococcus jannaschii for CO2 and H2 only, in comparison to the broad spectrum of substrates used as electron donors by the sulfate-reducing archaeon, Archaeoglobus fulgidus, including H2, formate, lactate, carbohydrates, starch, proteins, cell homogenates, and components of crude oil. This indicates that there are no particular carbonuse or energy-generation pathways that are exclusively linked to growth at high temperatures. For any microorganism, lipids, nucleic acids, and proteins are all generally susceptible to heat, and there is, in fact, no single factor that enables all hyperthermophiles to grow at high temperatures. 1. Membrane lipids and cell walls Archaeal lipids all contain ether-linkages (as opposed to the ester-linkages in most bacteria), which provide resistance to hydrolysis at high temperature. Some hyperthermophilic archaea contain membranespanning, tetra-ether lipids that provide a monolayer type of organization that gives the membranes a high degree of rigidity and may confer thermal stability. The bacterium Thermotoga maritima also contains a novel ester lipid, which may increase stability at high growth temperatures. However, the archaeon Methanopyrus kandleri, which can grow at up to 110 _C, contains unsaturated diether lipids that resemble terpenoids, and it is unclear how this may affect the thermal resistance of the cell. Lipid composition also varies with the growth temperature of individual organisms. For example, in Methanococcus jannaschii at 45 _C, 80% of the lipid content is one lipid (archaeol), whereas at 75 _C, two lipids (caldarchaeol and macrocyclic archaeol) account for 80% of the total core. Most archaeal species possess a paracrystalline surface layer (S-layer) consisting of protein or glycoprotein. It is likely that the S-layer functions as an external protective barrier. 

In Pyrodictium spp., the highly irregularly shaped, flagellated cells are interconnected by extracellular glycoprotein tubules that remain stable at 140 _C. 2. Nucleic acids The thermal resistance of DNA could conceivably be improved by maintaining a high mol% G_C content; however, many hyperthermophiles have between 30 and 40% (Table 37.2), in comparison to, for example, the mesophile Escherichia coli, which has 50%. Acidianus infernus has a G_C content of 31%, which would rapidly lead to the melting of double-stranded DNA at its optimum growth temperature of 90 _C. Histone-like proteins that bind DNA have been identified in archaeal hyperthermophiles. It is likely that the DNA is protected by the histones and that this enables processes such as open-complex formation during transcription to occur without subsequent DNA melting. In addition, hyperthermophiles contain reverse gyrase, a type 1 DNA topoisomerase that causes positive supercoiling and therefore may stabilize the DNA. 3. Proteins and solutes Heat-shock proteins, including chaperones, are likely to be important for stabilizing and refolding proteins as they begin to denature. When Pyrodictium occultum is heat-stressed at 108 _C, 80% of total protein accumulated is a single chaperonin, termed the “thermosome,” the cells’ protein-folding machine. In addition to the differential expression of certain genes throughout the growth temperature range of the organism, proteins from hyperthermophiles are inherently more stable than those from thermophiles, mesophiles, or psychrophiles. Higher stability is a result of increased rigidity (decreased flexibility) of the protein. Certain structural properties favor a more rigid protein (see psychrophilic proteins in Section IV.B.2), including a higher degree of structure in hydrophobic cores, an increased number of hydrogen bonds and salt bridges, and a higher proportion of thermophilic amino acids (e.g., proline residues that have fewer degrees of freedom). As a result, the proteins from hyperthermophiles are extremely heat stable. For example, proteases from Pyrococcus furiosus have half-lives of _60 h at 95 _C and an amylase from Pyrococcus woesii is active at 130 _C. Protein stability may also be assisted by the accumulation of intracellular potassium and solutes, such as 2,3-diphosophoglycerate (cDPG). In Methanopyrus kandleri and Methanothermus fervidus, there is evidence that potassium is required for enzyme activity at high temperature and the potassium salt of cDPG acts as a thermal stabilizer. 

D. Evolution Earth is about 4.6 billion years old and life is believed to have evolved on Earth around 3.6–4 billion years ago. The atmosphere of early Earth was devoid of oxygen and contained gaseous H2O, CH4, CO2, N2, NH3, HCN, trace amounts of CO and H2, and large quantities of H2S and FeS. For the first 0.5 billion years, the surface of the planet was probably devoid of water because the temperature was higher than 100 _C. Subsequently, the planet cooled and water liquefied. The high temperatures imply that life evolving in these conditions must have possessed thermophilic properties. Due to the high temperature and the available carbon and energy substrates, a microorganism such as H2-oxidizing sulfur-reducing Methanopyrus kandleri would conceivably thrive. Studying the phylogenetic relationship of extant (living) microorganisms by the analysis of 16Sribosomal ribonucleic acid (16S-rRNA) sequences, reveals that the hyperthermophilic Archaea and Bacteria have short evolutionary branches that occur near the base of the tree of life. Short branches indicate a low rate of evolution and deep branches reflect a close relationship with primordial life forms. This suggests that hyperthermophiles living today may resemble some of the earliest forms of life on Earth. There is some evidence that microbial life may have existed (or still exists) on Mars and other terrestrial bodies (e.g., on Europa, one of Jupiter’s moons). In association with the characteristics of hyperthermophiles mentioned, this has led to the consideration that life on Earth may have originated from the introduction of extraterrestrial life. A possible scenario involves a meteor carrying microbial life similar to hyperthermophilic methanogens plunging into the ocean billions of years ago and initiating the use of inorganic matter to generate biological matter and the subsequent evolution of extant species. These possibilities are driving new research endeavors to discover extraterrestrial life (see Section IV.A on psychrophiles and lakes on Europa). III. EXTREME ACIDOPHILES A. Habitats and microorganisms An extreme acidophile has a pH optimum for growth at or below pH 3.0. This definition excludes microorganisms that are tolerant to pH below 3, but that have pH optima closer to neutrality, including many fungi, yeast, and bacteria (e.g., the ulcer- and gastric-cancer causing gut bacterium Helicobacter pylori). 

1. Natural environments Extremely acidic environments occur naturally and artificially. The hyperthermophilic extreme acidophiles Sulfolobus, Sulfurococcus, Desulfurolobus, and Acidianus produce sulfuric acid in solfataras in Yellowstone National Park from the oxidation of elemental sulfur or sulfidic ores. Other members of the Archaea found in these hot environments include species of Metallosphaera, which oxidize sulfidic ores, and Stygiolobus spp., which reduce elemental sulfur. The novel, cell-wall-less archaeon, Thermoplasma volcanium, which grows optimally at pH 2 and 55 _C, has also been isolated from sulfotaric fields around the world. The bacterium Thiobacillus caldus has been isolated in hot acidic soils. Bacillus acidocaldarius, Acidimicrobium ferrooxidans, and Sulfobacillus spp. have also been isolated from warm springs and hot spring runoff. The most extreme acidophiles known are species of Archaea, Picrophilus oshimae and Picrophilus torridus, which were isolated from two solfataric locations in northern Japan. One of the locations, which contained both organisms, is a dry soil, heated by solfataric gases to 55 _C and with a pH of less than 0.5. These remarkable species have aerobic heterotrophic growth with a temperature optimum of 60 _C and a pH optimum of 0.7 (i.e., growth in 1.2 M sulfuric acid). In addition to Archaea, the phototrophic red alga Galdieria sulphuraria (Cynadium caldarium), isolated in cooler streams and springs in Yellowstone National Park, has optimum growth at pH of 2–3 and 45 _C, and is able to grow at pH values around 0. The green algae Dunaliella acidophila is also adapted to a narrow pH range from 0 to 3. 2. Artificial environments The majority of extremely acid environments are associated with the mining of metals and coal. 

The microbial processes that produce the environments are a result of dissimilatory oxidation of sulfide minerals, including iron, copper, lead, and zinc sulfides. This process can be written as Me2_S2_ (insoluble metal complex) → Me2__SO2 4 _; where Me represents a cationic metal. As a result of the extremely low pH in these environments, and due to the geochemistry of the mining sites, cationic metals (e.g., Fe2_, Zn2_, Cu2_, and Al2_) and metaloid elements (e.g., arsenic) are solubilized; this process is referred to as microbial ore leaching. Most mining sites tend to have low levels of organic compounds, and as a result chemolithoautotrophs, such as the bacteria Thiobacillus ferrooxidans, Thiobacillus thiooxidans, Leptospirillum ferrooxidans, and Leptospirillum thermoferrooxidans, are prolific. In addition, mixotrophic Thiobacillus cuprinus and heterotrophic Acidiphilium spp. have been isolated from acidic coal refuse and mine drainage. Thermophilic acidophilic bacterial species include Thiobacillus caldus from coal refuse, Acidimicrobium ferrooxidans from copperleaching dumps, and Sulfobacillus spp. from coal refuse and mine water. The archaeal microorganism Thermoplasma acidophilum is frequently isolated from coal refuse piles. Coal refuse contains coal, pyrite (an iron sulfide), and organic material extracted from coal. As a result of spontaneous combustion, the refuse piles are self-heating and provide the thermophilic environment necessary for sustaining Thermoplasma and other thermophilic microorganisms. In illuminated regions (e.g., mining outflows and tailings dams), phototrophic algae, including Euglena, Chlorella, Chlamydomonas, Ulothrix, and Klebsormidium species, have been isolated. Other eukaryotes include species of yeast (Rhodotorula, Candida, and Cryptococcus), filamentous fungi (Acontium, Trichosporon, and Caphalosporium) and protozoa (Eutreptial, Bodo, Cinetochilium, and Vahlkampfia). B. Biochemistry and physiology of adaptation Acidophiles (and alkaliphiles; see Section VII) keep their internal pH close to neutral. 

Most extreme acidophiles maintain an intracellular pH above 6, and even Picrophilus maintains an internal pH of 4.6 when the outside pH is 0.5–4. As a result, extreme acidophiles have a large chemical proton gradient across the membrane. Proton movement into the cell is minimized by an intracellular net positive charge and as a result cells have a positive inside-membrane potential. This is caused by amino acid side chains of proteins and phosphorylated groups of nucleic acids and metabolic intermediates, acting as titratable groups. In effect, the low intracellular pH leads to protonation of titratable groups and produces a net intracellular positive charge. In addition to this passive effect, some acidophiles (e.g., Bacillus coagulans) produce an active proton-diffusion potential that is sensitive to agents that disrupt the membrane potential, such as ionophores. The ability of lipids from the archaeon Picrophilus oshimae to form vesicles is lost when the pH is neutral, thus indicating that the membrane lipids are adapted for activity at low pH to minimize proton permeability. In Dunaliella acidophila, the surface charge and inside membrane potential are both positive, which is expected to reduce influx of protons into the cell. In addition, it overexpresses a potent cytoplasmic membrane H_-ATPase to facilitate proton efflux from the cell. The pH to which a protein is exposed affects the dissociation of functional groups in the protein and may be affected by salt and solute concentrations. Few periplasmic surface-exposed or -secreted proteins from extreme acidophiles have been studied to identify the structural features important for activity and stability. In Thiobacillus ferrooxidans, the acid stability of rusticyanin (acid-stable electron carrier) has been attributed to a high degree of inherent secondary structure and the hydrophobic environment in which it is located in the cell. Arelatively low number of positive charges have been linked to the acid stability of secreted proteins (thermopsin, a protease from Sulfolobus acidocaldarius, and an _-amylase from Alicyclobacillus acidocaldarius), by minimizing electrostatic repulsion and protein unfolding. 

IV. PSYCHROPHILES 

A. Habitats and microorganisms Over 80% of the total biosphere of Earth is at a temperature permanently below 5 _C, and it is therefore not surprising that a large number and variety of organisms have adapted to cold environments. These natural environments include cold soils; water (fresh and saline, still and flowing); in and on ice in polar or alpine regions; polar and alpine lakes, and sediments; caves; plants, and cold-blooded animals (e.g. Antarctic fish). Artificial sources include many refrigerated appliances and equipment. Organisms thriving in low-temperature environments (0 _C or close to 0 _C) are commonly classified as psychrophilic or psychrotolerant. Psychrophiles (cold-loving) grow fastest at a temperature of 15_C or lower, and are unable to grow over 20 _C. Psychrotolerant (also termed psychrotrophic) organisms grow well at temperatures close to the freezing point of water; however, their fastest rates of growth are above 20 _C. Psychrophilic and psychrotolerant microorganisms include bacteria, archaea, yeast, fungi, protozoa, and microalgae. It is generally found that psychrophiles predominate in permanently cold, stable environments that have good sources of nutrition (e.g., old, consolidated forms of sea ice exposed to algal blooms). It is likely that the permanency of the cold in stable environments obviates the need for cells to be able to grow at higher temperatures. In addition, at low temperature the affinity of uptake and transport systems decrease, and as a result, psychrophiles tend to be found in environments that are rich in organic substrates, thus providing compensation for less effective uptake and transport systems. 1. Natural environments Psychrophiles have been isolated from permanently cold, deep-ocean waters, as well as from ocean sediments as deep as 500 m below the ocean floor. As a consequence of the pressure in these environments, many of these psychrophiles are also barotolerant or barophiles (see Section V). A unique source of cold-adapted microorganisms are lakes in the Vestfold Hills region in Antarctica. The Vestfold Hills lakes are only about 10,000 years old and differ in their salinity and ionic strength, oxygen content, depth, and surface ice coverage. As a result, they have proven to be a rich resource of diverse and unusual microorganisms, including a cell-wall-less Spirochaete, a coiled or “C-shape” bacterium, and the only known free-living, psychrophilic archaeal species. The archaeal species include the methanogens Methanococcoides burtonii and Methangenium frigidum, and the extreme halophile Halobacterium lacusprofundi. The only other low-temperature-adapted archaeal isolate that has been studied is the symbiont Cenarchaeum symbiosum, which was isolated from a marine sponge off the Californian coast. It is perhaps surprising that more low-temperature-adapted Archaea have not been isolated throughout the world, as 16S-rRNA analysis of numerous aquatic and soil samples have indicated the prevalence of Archaea in these cold habitats. Gram-negative bacteria, including members of the genera Pseudomonas, Achromobacter, Flavobacterium, Alcaligenes, Cytophaga, Aeromonas, Vibrio, Serratia, Escherichia, Proteus, and Psychrobacter are more frequently found in cold environments than are gram-positive bacteria (e.g. Arthrobacter, Bacillus, and Micrococcus). Psychrophilic yeast are of the genera Candida and Torulopsis, and psychrotolerant members are mostly of the genera Candida, Cryptococcus, Rhodotorula, Torulopsis, Hanseniaspora, and Saccharomyces. Lowtemperature- adapted fungi and molds include isolates of the genera Penicillium, Cladosporium, Phoma, and Aspergillus. The most common snow alga is Chlamydomonas nivalis, which produces bright red spores, marking its location clearly against the white snow background. A remarkable, and as yet unstudied, lowtemperature environment has been discovered in Antarctica. Lake Vostok is a subglacial lake found about one kilometer below Vostok Station, 

East Antarctica. More than sixty smaller subglacial lakes also exist in central regions of the Antarctic Ice Sheet. Due to the isolation of the lakes, they are likely to contain many novel microorganisms, some of which could be expected to have developed along a separate evolutionary path from that of currently known life. The exploration of Lake Vostoc is being considered using specialized robots based on thermal-probe technology for ice penetration, and submersible technology for lake exploration. Interestingly, the ocean on Europa (a moon of Jupiter) is also located below a kilometer-thick covering of ice. The technology developed for Lake Vostok will be a model for future exploration (in 2003–2018) of the ocean on Europa and provides the unprecedented potential to identify extraterrestrial life in an aquatic environment. 2. Artificial environments The abundance of microorganisms in cold environments was realized as early as 1887, when the first low-temperature-adapted bacterium was probably isolated from a preserved fish stored at 0 _C. Since then, numerous psychrotolerant organisms have been found in artificial habitats and are frequently responsible for the spoilage of food. Members of the genera Pseudomonas, Acinetobacter, Alcaligenes, Chromobacterium, and Flavobacterium are often associated with spoilage of dairy products, whereas Lactobacillus viridescens and Brochothrix thermospacta contaminate meat products. Psychrotolerant bacterial pathogens include Yersinia enterocolitica, Clostridium botulinum, and certain Aeromonas strains. B. Biochemistry and physiology of adaptation Unicellular organisms are unable to insulate themselves against low temperatures and, as a consequence, psychrophilic and psychrotolerant microorganisms need to adapt the structures of their cellular components. 1. Membranes It is well documented that microorganisms adjust the fatty acid composition of their membrane phospholipids in response to changes in the growth temperature. Normal cell function requires membrane lipids that are largely fluid. As temperature is decreased, the fatty acids chains in membrane bilayers undergo a change of state from a fluid disordered state to a more ordered crystalline array of fatty acid chains. In order to adapt to low temperature, microorganisms decrease the transition temperature of the disordered-to-ordered state by altering the fatty acyl composition. This alteration consists of one or a combination of the following changes to the membrane lipids: an increase in unsaturation, a decrease in average chain length, an increase in methyl branching, an increase in the ratio of anteiso-branching compared to iso-branching, and an isomeric alteration of acyl chains in sn-1 and sn-2 positions. The most common alterations occur in fatty acid saturation and chain length. 

Changes in the amount and type of methylbranching occur mostly in gram-positive bacteria. Most bacteria only have monosaturated fatty acids; however a notable exception was found in some marine psychrophilic bacteria that increase membrane fluidity at low temperatures by incorporating polyunsaturated fatty acids into their membranes. Alterations in the membrane lipid composition can be mediated in a rapid fashion through the increase of unsaturation catalyzed by desaturases. Changes in fatty acyl chain length and the amount and type of methyl branching, however, require de novo fatty acid synthesis and are therefore much slower processes. 2. Enzymes The thermodynamic problems associated with enzymes in an environment of low kinetic energy (i.e., low temperature) relate to the lack of sufficient energy to achieve the activation state for catalysis. Lowtemperature adaptation of proteins therefore lowers the free energy of the activated state by decreasing the enthalpy-driven interactions necessary for activation. The necessity for weaker interactions leads to a less rigid, more flexible protein structure, or parts thereof. Consequently, factors that confer a more “loose” or flexible structure are expected to be important for coldadapted proteins. Based on the comparison of proteins from low-temperature-adapted Bacteria, Archaea, and Eukarya with those from mesophiles to hyperthermophiles, including comparisons of three-dimensional structures, a number of structural differences have been identified; including the reduction of the number of salt bridges, the reduction of aromatic interactions, the reduction of hydrophobic clustering, the reduction of proline content, the addition of loop structures, and an increase in solvent interaction. It should be noted that no cold-adapted protein has been studied that exhibits all of these features. This highlights the importance of the molecular context of the changes to stability, activity, or both. 

As a general rule, however, enzymes from psychrophilic organisms have reduced thermostability and a lower apparent temperature optima for their activity when compared to their mesophilic or thermophilic counterparts. 3. Cold shock and cold acclimation The response to a rapid decrease in temperature (cold shock) has been well studied in mesophilic laboratory microorganisms, including Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae. In response to cold shock, the pattern of gene expression is altered (cold-shock response). In bacteria, a class of small (7–8 kDa) acidic proteins are transiently induced. These cold-shock proteins (CSPs) have been well characterized and shown, in some cases, to function as transcriptional enhancers and RNA-binding proteins. In S. cerevisiae, TIP1 (temperature-shockinducible protein 1) is a major CSP; it is targeted to the outside of the plasma membrane and appears to be heavily glycosylated with O-mannose, therefore invoking a role for TIP1 in membrane protection during lowtemperature adaptation. Following a cold shock, cells resume growth, albeit at a lower growth rate, indicating that the cold-shock response is an adaptive response aimed at maintaining growth, rather than a stress response aimed only at cell survival. In general, the process of protein synthesis is temperature sensitive. As a result, psychrophilic microorganisms must have specially adapted ribosomes and accessory factors (e.g., initiation and elongation factors). In addition, psychrophilic and psychrotolerant microorganisnms (particularly those that are food-borne) may be exposed to sudden changes in their environmental temperature. A cold-shock response has been demonstrated in a number of psychrophilic and psychrotolerant organisms, including Trichosporon pullulans, Bacillus psychrophilus, Aquaspirrillum arcticum, and Arthrobacter globiformis; and homologs of a major E. coli cold-shock protein (CspA) have been identified in a number of these. The extent of the response (i.e., the number of proteins induced) in these microorganisms is dependent on the magnitude of the temperature shift. Some of the CSPs are also cold-acclimation proteins (proteins expressed continuously during growth at low temperature), suggesting that this class of CSP is important for cold-shock adaptation and growth maintenance at low temperatures. V. BAROPHILES A. Habitats and microorganisms Barophiles (“weight lovers”) are organisms that thrive in high-pressure habitats. An etymologically more accurate term is “piezophiles” (“pressure lovers”); however, it is less frequently used in the literature. There is apparent confusion and contradiction in the literature concerning the defining traits of pressure-adapted organisms. This mainly stems from the complicating effects of temperature on growth rate. The effect of both temperature (T) and pressure (P) on the growth rate (k) of different organisms has been thoroughly investigated using “PTk-diagrams.” With all other conditions held constant, there is a unique pressure, Pkmax, and temperature, Tkmax, at which the growth rate of an individual microorganism is maximal (kmax). 

These values have been used to define barophiles with Pkmax_0.1 MPa and extreme barophiles with Pkmax b 0.1 MPa. In addition, the values for Tkmax have been used to delineate psychro- , meso-, or thermo- (extreme) barophiles. Other terms used are obligate (extreme) barophiles, for organisms that cannot grow at atmospheric pressure (0.1 MPa), irrespective of temperature, and barotolerants, for those that grow best under atmospheric pressure but that can also grow at up to 40 MPa. Barophiles were first isolated from the deep sea (deeper than 1000 m), and this environment still represents the most thoroughly studied habitat. Other high-pressure habitats include the deep-Earth and the deep-sediment layers below the ocean floor. The deep sea is a cold (_5 _C), dark, oligotrophic (low in nutrients) environment with pressure as high as 110 MPa, as is found in the Mariana Trench (almost 11,000 m). In contrast, fumarole and black-smoker hydrothermal vents, produced by the extrusion of hot subsurface water, represent a hyperthermal environment with high metabolic activity (see hyperthermophiles in Section II). The microbiota of Atlantic and Pacific vents are remarkably similar, indicating that the (hyper-) thermobarophilic microorganisms are able to survive for long periods in cold waters, thus facilitating their effective dispersal throughout the oceans. Supporting this, these microorganisms have been isolated from ocean waters far removed from hydrothermal vents. Psychrophilic deep-sea (extreme) barophilic isolates predominantly belong to five genera of the _-Proteobacteria—Photobacterium, Shewanella, Colwellia, Moritella, and a new group containing the strain CNPT3. One Bacillus species (strain DSK25) has also been isolated. Hyperthermobarophilic archaeal isolates include Pyrococcus spp. It is noteworthy that the difficulty in isolating microorganisms from the deep sea, and the specific enrichment and culturing techniques that are used, are likely to produce a bias in the types of barophiles that are isolated; a complete analysis of the phylogenetic distribution of barophilic microorganisms is yet to be attempted. B. Biochemistry and physiology of adaptation 1. General physiological adaptations Membrane lipids from barophilic bacteria have been well studied. In response to high pressure, the relative amount of monounsaturation and polyunsaturation in the membrane is increased. The increase in unsaturation produces a more fluid membrane and counteracts the effects of the increase in viscosity caused by high pressure. This response is analogous to adaptations caused by temperature reduction (see Section IV). Note, however, that an increase in unsaturation was not observed in two extreme barophiles that have been studied, thus indicating that alternative mechanisms of adaptation exist. An interesting observation for many barophiles is that they are extremely sensitive to UV light and need to be grown in dark or light-reduced environments. This adaptation is not unexpected, considering the darkness that prevails in the deep sea. 

There are presently no studies on barophilic microorganisms investigating the adaptation of molecular processes such as chromosomal replication, cell division, transcription, or translation. Studies of E. coli, however, have revealed that when cells are grown at their upper pressure limit, DNA synthesis is completely inhibited, protein synthesis is slowed down, and mRNA synthesis and decay appears to be unaffected. This demonstrates that specific cellular processes are affected by pressure, and therefore barophiles are likely to have adapted mechanisms to compensate. Pressure may stabilize proteins and retard thermal denaturation. In support of this, thermal inactivation of DNA polymerases from the hyperthermophilic microorganisms Pyrococcus furiosus, Pyrococcus strain ES4, and Thermus aquaticus is reduced by hydrostatic pressure. 2. Gene and protein expression Pressure-regulated gene and protein expression has been observed and investigated in deep-sea bacteria. Photobacterium sp. SS9 expresses two outer-membrane proteins (porins) under different pressures, the OmpH protein at 28MPa and OmpL at 0.1 MPa. The genes are regulated by a homolog of the toxRS system from Vibrio cholerae. ToxR and ToxS are cytoplasmic membrane proteins that are thought to be pressure sensors controlled by membrane fluidity. A pressure-regulated operon has been identified in the barophilic bacterium DB6705. A complex promoter region, which is controlled by a variety of regulatory proteins expressed under different pressure conditions, was identified upstream of three open reading frames (ORFs). The function of the first two ORFs is unknown, while the third ORF encodes the CydD protein. CydD is required for the assembly of the cytochrome-bd complex in the aerobic respiratory chain. The membrane location of this protein highlights the apparent importance of membrane components in high-pressure adaptation. VI. HALOPHILES A. Habitats and microorganisms The first recorded observation of organisms adapted to high salt concentrations (halophiles and halotolerants) probably dates to 2500 BC, when the Chinese noted a red coloration of saturated salterns. What they detected was most likely a bloom of extreme halophilic Archaea that possess red or orange C50 carotenoides. The definition of a hypersaline environment is one that possesses a salt concentration greater than that of seawater (3.5% w/v). For water-containing environments, the salt composition depends greatly on the historical development of the habitat, and the environments are normally described as thalassohaline or athalassohaline. Thalassohaline waters are marine derived and therefore contain, at least initially, a seawater composition; however, with increasing evaporation the concentration of various salts alters depending on the thresholds for the crystallization and precipitation of different minerals (see Table 37.3). Athalassohaline water may also be influenced by the influx of seawater; however, the chemical composition is mainly determined by geological, geographical, and topographical parameters. Examples of athalassohaline environments include the Great Salt Lake in Utah and the Dead Sea. 

In addition to lakes formed by evaporation in moderate climate conditions, hypersaline Antarctic lakes (e.g., Vestfold Hills; see Section IV) have been formed from the effects of frost and dryness in this environment. Antarctic and moderate temperature soils also contain salinities between 10 and 20% (w/v) and efforts have been directed at characterizing these comparatively poorly studied ecosystems. Less obvious saline habitats known to be colonized by microorganisms are animals skins, plant surfaces, and building surfaces. In addition, interest is also being focused on subterranean salt deposits as a habitat for extreme halophilic Archaea, and as a possible source of ancient prokaryotic lineages preserved in fluid inclusion bodies of salt crystals. Organic compounds in hypersaline lakes are mostly produced by cyanobacteria, anoxygenic phototrophic bacteria, and by species of the green algae Dunaliella spp. They are found in most natural and artificial hypersaline lakes around the world. With seasonal and transient contributions from animals or plants, these environments can contain dissolved organic carbon levels up to 1 g/l. Due to the low solubility of oxygen in saline solutions, however, many habitats become anaerobic and as a result aerobic growth is often restricted to the upper layers. Organisms living in saline habitats exhibit different levels of adaptation to salt. To account for the variety of tolerances, an extensive set of definitions exist and are summarized in Table 37.4. A salt concentration of about 1.5 M is the upper limit for vertebrates, although some eukaryotes such as the brine shrimp (Artemia salina) and the brine fly (Ephydra) can be found in habitats with higher salinity. 

For salinities above 1.5 M, prokaryotes become the predominant group with moderate halophilic and haloversatile bacteria at salt concentrations between 1.5 and 3.0 M, and extreme halophilic Archaea (halobacteria) at salinities around the point of sodium chloride precipitation. Aerobic gram-negative chemoorganotrophic bacteria are abundant in brines of medium salinity, and many strains have been isolated, including members of the genera Vibrio, Alteromonas, Acinetobacter, Deleya, Marinomonas, Pseudomonas, Flavobacterium, Halomonas, and Halovibrio. Gram-positive aerobic bacteria of the Marinococcus, Sporosarcina, Salinococcus and Bacillus species have been found in saline soils, salterns, and occasionally in solar salterns. Members of the genera Halomonas and Flavobacterium have been isolated from Antarctic lakes. Two of the most remarkable isolates from Antarctica are the extreme halophilic archaeal species Halobacterium lacusprofundi and a Dunaliella spp., which are found in association in Deep Lake. These are the only two microorganisms growing in this lake, which has 4.8 M salt and whose temperature for 8 months of the year is less than 0 _C. With the extremes of temperature, salt, and ionic strength, and with primary production rates of 10_g C/m2/year, Deep Lake has been described as one of the most inhospitable environments on Earth. Wherever light reaches the anoxic layer of hypersaline brines, anoxygenic phototrophs such as Chromatium salexigens, Thiocapsa halophila, Rhodospirillum salinarum, and Ectothiorhodospira are commonly found and represent the primary producers in these environments. Sulfate reducers Desulfovibrio halophilus and Desulfohalobium retbaense have been isolated from anaerobic sediments and are thought to perform dissimilatory sulfate reduction to H2S. H2S is subsequently used for growth by most anoxygenic phototrophs (except for members of the Rhodospirillaceae family). Anaerobic fermentative halophiles from the bacterial lineage of Haloanaerobiaceae have also been described. Most of the bacterial species mentioned here are predominant in environments up to 2M salt. At higher concentrations, extremely halophilic Archaea (including the confusingly named genus, Halobacteria) are more abundant. Most of these Archaea require at least 1.5 M salt for growth and for retaining their structural integrity. 

Members of the genera Haloarcula, Halobacterium, and Halorubrum are frequently found in hypersaline waters reaching the sodium chloride saturation point, and the proteolytic species Halobacterium salinarium is often associated with salted food. Other Archaea are also found in saline lakes, marine stromatolites, and solar ponds, and these tend to be methanogens (see Section X). In addition, the eukaryote Dunaliella can adapt to a wide range of salt concentrations, from less than 100 mM to saturation (5.5M). B. Biochemistry and physiology of adaptation Cells exposed to an environment with a higher salinity than the one inside the cytoplasm inevitably experience a loss of water and undergo plasmolysis (Fig. 37.1). Microorganisms living in high-salt environments generally adopt one of two strategies (either salt-in-cytoplasm or compatible-solute adaptation) to prevent the loss of cytoplasmic water and to establish osmotic equilibrium across their cell membranes. 1. Salt-in-cytoplasm adaptation Extremely halophilic archaea and anaerobic halophilic bacteria use a salt-in-cytoplasm strategy. This involves cations flowing through the membrane into the cytoplasm (Fig. 37.1A). Archaea accumulate intracellular potassium and exclude sodium, whereas Bacteria accumulate sodium rather than potassium. As a consequence of the high salt, intracellular components (e.g., proteins, nucleic acid and cofactors) require protection from the denaturing effects of salt. The most common protective mechanism is the presence of excess negative charges on their exterior surfaces. Malate dehydrogenase from Haloarcula marismortui has a 20 mol% excess of acidic residues over basic amino acid residues, compared to only 6 mol% excess for a nonhalophilic counterpart. Structural analysis shows that the acidic residues are mainly located on the surface of the protein and are either involved in the formation of stabilizing salt bridges or in attracting water and salt to form a strong hydration shell. This malate dehydrogenase is able to bind extraordinary amounts of water and salt (0.8–1.0 g water and 0.3 g salt/g protein) compared to a nonhalophilic malate dehydrogenase (0.2–0.3g water and 0.01 g salt/g protein). Due to the adaptation mechanism in these cells, they have an obligate requirement for high concentrations of salt. In low-salt environments, the lack of salt removes the shielding effect of cations from the proteins and leads to the rapid denaturation of the three-dimensional structure. 2. Compatible-solute adaptation Most halotolerant and moderately halophilic organisms, including the anoxygenic phototrophic bacteria, aerobic heterotrophic bacteria, cyanobacteria, and methanogens, maintain a salt-minimized cytoplasm. They accumulate small organic and osmotically active molecules, referred to as compatible solutes. These compounds can be synthesized de novo or imported from the surrounding medium (Fig. 37.1b). The latter mechanism is also used by nonhalophilic organisms; they adapt to increased salt concentrations by importing extracellular compatible solutes using transporters involved in amino acid or sugar uptake. A large range of compatible solutes has been identified in a broad range of halophiles (Table 37.5). All these molecules are polar, highly soluble, and uncharged or zwitterionic at physiological pH values. They are strong water-structure formers and as such are probably excluded from the hydration shell of proteins (preferential exclusion), and therefore exert a stabilizing effect without interfering directly with the structure of the protein. In addition to being stabilizers against salt stress, they have also been shown to prevent the denaturation of proteins caused by heating, freezing, and drying. 

3. Membranes Even though the cytoplasmic interior is protected from the effects of external salt by cytoplasmic-compatible solutes, the outer surface of the cytoplasmic membrane is permanently exposed to high salt concentrations. To protect the membrane in halophilic bacteria, the proportion of anionic phospholipids (often phosphatidylglycerol and glycolipids) increases with increasing salinity at the expense of neutral zwitterionic phospholipids. These alterations produce additional surface charges to the membrane and help to maintain the hydration state of the membrane. Most halophilic Archaea posses an S-layer consisting of sulfated glycoproteins, which surrounds the cytoplasmic membrane. The sulfate groups confer a negative charge to the S-layer and possibly provides structural integrity at high ionic concentrations. In addition, archaeal ether lipids have been shown to be more stable at high salt concentrations (up to 5M) compared to the ester lipids found in the membranes of bacteria. VII. ALKALIPHILES A. Habitats and microorganisms Alkaliphiles are defined by optimal growth at pH values above 10, whereas alkalitolerant microorganisms may grow well up to pH 10, but exhibit more rapid growth below pH 9.5. Alkaliphiles are further subdivided into facultative alkaliphiles (that grow well at neutral pH) and obligate alkaliphiles (that grow only above pH 9). In addition, due to their natural habitats, many alkaliphilic organisms are adapted to high salt concentrations and are referred to as haloalkaliphiles. Alkali environments include soils where the pH has been increased by microbial ammonification and sulfate reduction, and water derived from leached silicate minerals. These environments tend to have only a limited buffering capacity and the pH of the environments fluctuates. As a result, alkalitolerant microorganisms are more abundant in these habitats than are alkaliphiles. Artificial environments include locations of cement manufacture, mining, and paper and pulp production. Probably the best studied and most stable alkaline environments are soda lakes and soda deserts (e.g., in the East African Rift Valley or central Asia). The formation of soda lakes and deserts is similar to the formation of athalassohaline salt lakes (see Section V), with the exception that carbonate is the major anion in solution, due to the lack of divalent cations (Mg2_, Ca2_) in the surrounding environment. A typical soda lake composition is shown in Table 37.2. Bacteria, Archaea, yeast, and fungi have been isolated from alkali environments. Archaeal alkaliphiles include members of the Halobacteriaceae (Halorubrum, Natronbacterium, and Natronococcus spp.) and Methanosarcinaceae (Methanohalophilus spp.). Cyanobacterial genera Spirulina and Synechococcus represent the dominant primary producers in the aerobic layers of soda lakes. Other alkaliphilic bacteria are members of the Actinomyces, Bacillaceae, Clostridiaceae, Haloanaerobiales, and _-Proteobacteria (Ectothiorodospira, Halomonadaceae, and Pseudomonas). Anaerobic thermophilic alkaliphiles (alkalithermophiles) include Clostridium and Thermoanaerobacter spp., and the only representative of a new taxon, Thermopallium natronophilum. B. Biochemistry and physiology of adaptation 1. pH homeostasis Studies of alkaliphiles (particularly Bacillus spp.) have demonstrated that they maintain a neutral or slightly alkaline cytoplasm. This is reflected by a neutral pH optimum for intracellular enzymes compared to a high pH optimum for extracellular enzymes. The intracellular pH regulation has been shown to be dependent on the presence of sodium. The Na_ ions are exchanged from the cytoplasm into the medium by H_/Na_ antiporters (Fig. 37.2). Electrogenic proton extrusion is mediated in aerobic cells by respiratory chain activity and protons are transported back into the cell via antiporters that are efficient at transporting H_ into the cell at the expense of Na_ export from the cell. The resulting net production and influx of protons creates a more acidic cytoplasm. In addition to controlling protons, Na_-dependent pH homeostasis requires the reentry of Na_ into the cell. 

Na_-coupled solute symporter and sodium-driven flagella rotation ensure a net sodium balance. The combined action of the antiporters coupled with respiration provides the cell with a means to control its internal pH while maintaining sufficient Na_ levels through symport and flagella rotation.  The exterior surfaces of the cell are also important for maintaining a pH differential. This is supported by evidence that the protoplasts of alkaliphilic Bacillus spp. are unstable in alkaline conditions. The peptidoglycan in these strains has a higher cross-linking rate at higher pH values, which may provide a shielding effect by “tightening” the cell wall. Large amounts of acidic compounds, including teichuronic acid, teichoic acid, uronic acids, and acidic amino acids, are evident in alkaliphilic cell walls compared to the cell walls of nonalkaliphilic microorganisms. The negative charge of these acidic substances may create a more neutral layer close to the outer surface of the cell. 2. Bioenergetics Alkaliphiles have unique bioenergetic properties. Nonalkaliphilic respiring bacteria energize their cytoplasmic membrane with a chemiosmotic driving force (p) by generating an electrochemical gradient of ions that has two parameters—acidic conditions outside (caused by the extrusion of protons and described by the term pH) and a positive charge outside (described by the transmembrane electrical potential, _). The p is used for proton-coupled symport of solutes, protondriven mobility (flagella) and ATP synthesis. In alkaliphilic environments, the contribution of pH to p becomes smaller with increasing extracellular pH. However, with increasing extracellular pH, sodium ion export is increased (see Section VII.B.1) and may contribute to an increased _. This partially compensates for the decrease of p by the reduction of pH. Interestingly, the sodium gradient is used to energize solute transport and flagella movement, but not for ATP synthesis. No sodium-dependent ATP-synthases have been identified in alkaliphiles. In addition, ATPsynthases in alkaliphilic Bacillus spp. have been shown to be exclusively proton translocating. VIII. OLIGOTROPHS A. Habitats and microorganisms A most important environmental factor in microbial ecology is the availability of energy, and virtually all microbial cells in nature are limited in their growth by the availability of one or more essential growth nutrients. For example, in the intestinal tract, the number of E. coli doubles about twice per day, whereas in ideal laboratory conditions it doubles in 20 min. Similarly, soil bacteria are estimated to grow in soil at about 1% of the maximal rate of growth observed in the laboratory. This highlights that in natural ecosystems nutrient limitation is the rule, rather than the exception. In addition to the overall nutrient status, in aquatic and soil environments, nutrient levels are often transient; for example, a fallen leaf provides nutrients to soil microorganisms, or a dead fish provides nutrients in an aquatic environment. As a result of this apparent “feast or famine,” microorganisms have adopted two main strategies for surviving in nutrient-depleted (oligotrophic) environments. Eutrophic microorganisms (also referred to as copiotrophs, saphrophytes, and heterotrophs) grow in bursts when nutrients are available and produce resting-stage cells when nutrients are in short supply (referred to as r-strategy). In contrast, oligotrophic microorganisms (also referred to as oligocarbophiles; low-nutrient, LN, bacteria; low-Ks bacteria; and dilute-nutrient-broth, DNB, organisms) grow slowly, using low concentrations of nutrients (referred to as K-strategy). It is important to note that whereas oligotrophs grow slowly in oligotrophic environments, eutrophs are in a resting stage. In contrast, when members of these two classes are subcultured from oligotrophic environments into rich media, the eutrophs resume rapid growth, whereas the oligotrophs do not grow at all. The cellular responses that prevent growth of the oligotrophs in rich media is not understood; however, the response highlights the physiological differences between these two classes of microorganisms. In addition, the difficulty in growing oligotrophs in the laboratory has proven problematic for examining their physiology. Fortunately, some isolates have adapted (faculative oligotrophs) to growth in rich media and are amenable to laboratory studies. Although the definition of an oligotroph and an oligotrophic environment remain the subject of debate, it is generally accepted that an oligotroph is able to grow in a medium containing 0.2–16.8mg dissolved organic carbon/liter. 

The terms “obligate oligotroph” (implying the inability to grow in high concentrations of nutrients) and “facultative oligotroph” (indicating the ability to grow in low and high concentrations of nutrients) are also used to further clarify the nutritional requirements for growth. In natural ecosystems, oligotrophs and eutrophs coexist, and the proportion of each varies depending on their individual abilities to dominate in the particular environment. For example, when the marine oligotrophic ultramicrobacterium Sphingomonas sp. strain RB2256 was isolated from Resurrection Bay, Alaska, it was a numerically dominant species in a place where the total bacterioplankton population was 0.2–1.07_106 cells/ml. In contrast, significantly lower numbers (_1%) of larger, faster-growing cells (typical of eutrophs) were able to be immediately cultured in rich media and on plates.  B. Biochemistry and physiology of adaptation A characteristic that is often associated with oligotrophic bacteria is their ultramicro size (_0.1_m3). Ultramicrobacteria (or dwarf cells) are commonly found in aquatic and soil environments. Oligotrophic ultramicrobacteria, such as Sphingomonas RB2256, retain their ultramicro size irrespective of growth phase, carbon source, or carbon concentration. In contrast, eutrophic microorganisms, such as Vibrio angustum S14 and Vibrio ANT-300, undergo reductive cell division during starvation, but resume their normal size (_1_m3) when grown in rich media. The characteristics that are thought to be important for oligotrophic microorganisms include a substrate uptake system that is able to acquire nutrients from its surroundings, and the capacity to use the nutrients in order to maintain its integrity and growth. As a result, oligotrophs would ideally have large surface-area-tovolume ratios, and high-affinity uptake systems with broad substrate specificities. Consistent with this, a number of microorganisms that are adapted to lownutrient environments produce appendages to enhance their surface area. These include members of the bacterial genera, Caulobacter, Hyphomicrobium, Prosthecomicrobium, Ancalomicrobium, Labrys, and Stella. Other bacteria have tiny cell volumes (ultramicrobacteria:_0.1 _m3) to maximize their surface-area-to-volume ratio. The most comprehensive physiological studies of oligotrophic marine isolates have been performed on Sphingomonas sp. strain RB2256. The characteristics that distinguish it from typical marine eutrophs include constant ultramicro size irrespective of growth or starvation conditions, a mechanism for avoiding predation (ultramicro size), a relatively slow maximum specific growth rate (_0.2 h_1), a single copy of the rRNA operon compared to 8–11 copies for Vibrio spp., a relatively small genome size compared to faster-growing heterotrophs, an ability to use low concentrations of nutrients, high-affinity broadspecificity uptake systems, an ability to simultaneously take up mixed substrates, an ability to immediately respond to nutrient addition without a lag in growth, and an inherent resistance to environmental stresses (e.g., heat, hydrogen peroxide, and ethanol). 

The small (0.2 _m3) oligotrophic bacterium Cycloclasticus oligotrophicus RB1, which was also isolated from Resurrection Bay, shares some properties similar to Sphingomonas RB2256 (e.g., single copy of the rRNA operon, and relatively small cell size and genome size). Interestingly, while this chemoorganotroph is unable to grow using glucose and amino acids, it can use acetate and a few aromatic hydrocarbons such as toluene. IX. RADIATION-RESISTANT MICROORGANISMS A. Habitats and microorganisms Radiation from the sun drives photosynthetic reactions, thus ensuring primary production throughout the global ecosystem. Although the visible spectrum leads to biomass production, visible light and other portions of the electromagnetic spectrum (particularly short-wavelength) also cause cellular damage. Damage to cells primarily occurs directly to nucleic acids (e.g., UV-induced thymine-dimer formation and strand breakage) or indirectly through the production of reactive oxygen species (e.g., H2O2, O2 _ 0OH, and 1O2), which cause damage to lipids, proteins, and nucleic acids. Due to the prevalence of natural radiation, most cells have developed a range of DNA-repair and other protective mechanisms to facilitate their survival. In contrast to natural forms of relatively low-level radiation, microorganisms may be exposed to intense sources of radiation in the form of _-irradiation (60Co and 137Cs) as a means of sterilization or by being in close proximity to nuclear reactors. It is mainly these forms of radiation that have been the sources of highly radiation-resistant extremophiles. The most wellstudied of these is the bacterium Deinococcus radiodurans, which was first isolated in 1956 from tins of meat that had been irradiated with _-rays. Although some microorganisms escape radiation damage by forming spores (e.g., Clostridium botulinum), D. radiodurans is resistant while in the exponential growth phase. The degree of resistance of these cells is illustrated by their ability to survive 3,000,000 rad, a dose that is sufficient to kill most spores (a lethal dose for humans is about 500 rad). As a consequence of the extreme radiation resistance of D. radiodurans and other microorganisms, biocides are routinely added to the cooling waters of nuclear reactors to prevent the proliferation of microorganisms. By virtue of their resistant properties, this class of extremophile can be readily isolated by exposing samples to intense UV or _-irradiation and then plating them out on a rich medium. Although Deinococcus spp. have been found in dust, processed meats, medical instruments, textiles, dried food, animal feces, and sewage, their natural habitats have not been clearly defined. Thermophilic species have, however, been isolated from hot springs in Italy, and deinococci have been identified in many soil environments. It therefore appears that this class of radiation resistant extremophiles exists in a broad range of environmental niches. In addition to Deinococcus spp., some hyperthermophilic Archaea (e.g., Thermococcus stetteri and Pyrococcus furiosus) are able to survive high levels of _-irradiation. B. Biochemistry and physiology of adaptation Resistance to radiation could conceivably occur by two main mechanisms, prevention of damage or efficient repair of damage. In D. radiodurans, it has been clearly shown that the DNA is severely damaged during _-irradiation (e.g., ~110 double-strand breaks per cell when exposed to 300,000 rad); however, within 3h of recovery, the fragmented DNA is replaced by essentially intact, chromosomal DNA. Furthermore, even though the DNA within the cells has been extensively degraded, viability is unaffected. P. furiosus is also able to repair fragmented DNA after exposure to 250,000 rad when the cells are grown at 95 _C. As a result, it has been suggested that active DNA-repair mechanisms may be important determinants of survival of hyperthermophiles in their natural environments. In addition to _-radiation resistance, D. radiodurans is resistant to highly mutagenic chemicals, with the exception of those that cause DNA deletions (e.g., nitrosoguanadine, NTG). D. radiodurans is also extremely resistant to UV irradiation, surviving doses as high as 1000J/m2. 

The dose that is required to inactivate a single colony-forming unit of an irradiated population is for D. radiodurans 550–600J/m2 compared to just 30J/m2 for E. coli. The DNA-repair systems in D. radiodurans are so effective that they have proven to be a hindrance for genetic studies, that is, it is difficult to isolate stable mutants. However, through the combined use of chemical mutagenesis and screens for mitomycin C-, UV radiation-, and ionizing radiation-sensitive strains, genes involved in nucleotide-excision repair, base-excision repair, and recombinational repair have been identified. In addition, D. radiodurans is multigenomic (e.g., there are 2.5–10 copies of the chromosome depending on growth rate) and a novel mechanism of interchromosomal recombination has been proposed. This process would help to circumvent problems of reassembling a complete and contiguous chromosome from the chromosome fragments that have been generated as a result of irradiation. It appears that D. radiodurans’s ability to use its genome multiplicity to repair DNA damage is the most fundamental reason for this species’s extraordinary radioresistance. Almost all Deinococcus spp. that have been isolated (even those isolated without selection for radiation resistance) are radiation resistant, thus demonstrating that the extreme resistance is not a result of selection by irradiation, but a normal characteristic of the genus. Desiccation leads to DNA damage in all cells and prevents DNA repair from occurring. For a cell to be viable when it is rehydrated, it needs to be able to repair the damaged DNA. It is therefore likely that the evolutionary process that has led to the inherent radiation resistance in Deinococcus is natural selection for resistance to desiccation. This is supported by evidence that D. radiodurans is also exceptionally resistant to desiccation. X. OTHER EXTREMOPHILES As a group, methanogenic Archaea (methanogens) are often considered extremophiles. They are the most thermally diverse organisms known, inhabiting environments from close to freezing in Antarctic lakes to 110_C in hydrothermal vents. Those isolated from Antarctica include Methanococcoides burtonii and Methanogenium frigidum, and those from hydrothermal vents include Methanopyrus kandleri, Methanothermus fervidus, Methanothermus sociabilis, Methanococcus jannaschii, and Methanococcus igneus. Methanogens can also be isolated from a diverse range of salinities, from fresh water to saturated brines. Methanohalobium evestigatum was isolated from a microbial mat in Sivash Lake and grows in pH neutral, hypersaline conditions from 2.6–5.1M. Alkaliphilic (Methanosalsus zhilinaeae, pH 8.2–10.3) and acidophilic (Methanosarcina sp., pH 4–5) methanogens have also been isolated. Other microorganisms that may be considered to be extremophiles include toxitolerants (those tolerant to organic solvents, hydrocarbons, and heavy metals, such as Rhodococcus sp., which can grow on benzene as a sole carbon source), and xerophiles and xerotolerant microbes that survive very low-water activity (e.g., extremely halophilic Archaea, fungi such as Xeromyces bisporus, and endolithic microorganisms that live in rocks). XI. BIOTECHNOLOGY OF EXTREMOPHILES A major impetus driving research on extremophiles is the biotechnological potential associated with the microorganisms and their cellular products. In 1992, of the patents related to Archaea, about 60% were for methanogens, 20% for halophiles, and 20% thermophiles. Examples of “extremozymes” that are presently used commercially include alkaline proteases for detergents. This is a huge market, with 30% of the total worldwide enzyme production for detergents. In 1994, the total market for alkaline proteases in Japan alone was ~15,000 million yen. DNA polymerases have been isolated from the hyperthermophiles Thermus aquaticus, Thermotoga maritima, Thermococcus litoralis, Pyrococcus woesii, and Pyrococcus furiosus for use in the polymerase chain reaction (PCR). Aeukaryotic homolog of the myc oncogene product from halophilic Archaea has been used to screen the sera of cancer patients. Its utility is demonstrated by the fact that the archaeal homolog produced a higher number of positive reactions than the recombinant protein expressed in E. coli. -carotene is commercially produced from the green algae Dunaliella bardawil. The applications in industry are still limited; however, the potential applications are extensive. Some examples of their uses and potential applications are listed in Table 37.6. The biotechnology potential is increasing exponentially with the isolation of new organisms, the identification of novel compounds and pathways, and the molecular and biochemical characterization of cellular components. Major advances are likely in the area of protein engineering. For example, the identification of the structural properties important for thermal activity and stability will enable the construction of proteins with required catalytic and thermal properties. Recently, a metalloprotease from the moderately thermophilic bacterium Bacillus stearothermophilus was mutated using a rational design process in an effort to increase its thermostability. The mutant protein was 340 times more stable than the wild-type protein and was able to function at 100 _C in the presence of denaturing agents, while retaining wild-type activity at 37 _C. Advances are also likely to arise from the construction of recombinant microorganisms for specific purposes. A recombinant strain of Deinococcus radiodurans has been engineered to degrade organopollutants in radioactive mixed-waste environments. The recombinant Deinococcus expresses toluene dioxygenase, enabling it to oxidize toluene, chlorobenzene, 2,3-dichloro-1-butene, and indole in a highly irradiating environment (6000rad/h), while remaining tolerant to the solvent effects of toluene and trichloroethylene at levels exceeding those of many radioactive waste sites. In recognition of the number of waste sites contaminated with organopollutants plus radionuclides and heavy metals around the world, and the safety hazards and cost involved in clean up using physicochemical means, the potential use of genetically engineered extremophilic microorganisms is an important and exciting prospect.