Part 2 book “Physiology and biochemistry of extremophiles” has contents: Biodiversity in highly saline environments, molecular adaptation to high salt, physiology and ecology of acidophilic microorganisms, molecular adaptation to high salt, bioenergetic adaptations that support alkaliphily, environmental and taxonomic biodiversities of gram-positive alkaliphiles,… and other contents.
IV HALOPHILES This page intentionally left blank Physiology and Biochemistry of Extremophiles Edited by C Gerday and N Glansdorff © 2007 ASM Press, Washington, D.C Chapter 17 Biodiversity in Highly Saline Environments AHARON OREN NaCl from seawater, underground deposits of rock salt, as well as salted food products, highly saline soils, and others (Javor, 1989; Oren, 2002a) The two largest truly hypersaline inland salt lakes are the Great Salt Lake, Utah, and the Dead Sea The Great Salt Lake, a remnant of the ice-age saline Lake Bonneville that has largely dried out, has a salt composition that resembles that of seawater (“thalassohaline” brines) Owing to climatic changes and to human interference (division of the lake into a northern and a southern basin by a rockfill railroad causeway in the 1950s), the salinity of the lake has been subject to strong fluctuations in the past century The northern basin is nowadays saturated with respect to NaCl It is unfortunate that we know so little about the microbiology of the Great Salt Lake: after the pioneering studies by Fred Post in the 1970s (Post, 1977), the study of the microbial communities in the lake has been sadly neglected However, a recent renewed interest in the biology of the lake is expected to change the picture, so that we soon may expect to get a much better picture of the diversity of microorganisms in the largest of all hypersaline lakes, their properties, and their dynamics (Baxter et al., 2005) The Dead Sea, with its present-day salt concentration of over 340 g/liter, is an example of an “athalassohaline” brine, which has an ionic composition greatly different from that of seawater Magnesium, not sodium, is the dominant cation, calcium is present as well in very high concentrations, and the pH is relatively low: around 6, as compared with 7.5 to in thalassohaline brines Indeed, the present-day Dead Sea is a remnant of the Pleistocene Lake Lisan, whose salts were of marine origin, but massive precipitation of halite and other geological phenomena have greatly changed the chemical properties of the brine Yet, a few types of microorganisms can survive even in the INTRODUCTION About 70% of the surface of planet Earth is covered by seawater: a salty environment that contains approximately 35 g of total dissolved salts per liter, 78% of which is NaCl Although many microorganisms are unable to cope with life at seawater salinity, the marine environment cannot be considered “extreme”: the seas are populated by a tremendous diversity of micro- and macroorganisms, at least as diverse as the world of freshwater organisms However, there are also environments with salt concentrations much higher than those found in the sea When salt concentrations increase, the biological diversity decreases, and at concentrations about 150 to 200 g/liter, macroorganisms no longer survive On the other hand, highly salt-tolerant and often even highly salt-requiring microorganisms can be found up to the highest salt concentrations: NaCl-saturated brines that contain salt concentrations of over 300 g/liter Halophilic Archaea, Bacteria, and eukaryotic unicellular algae live in the Dead Sea, in the Great Salt Lake, in saltern crystallizer ponds, and in other salt-saturated environments, and they often reach high densities in such environments This chapter explores the world of high salt environments worldwide and the diversity of microorganisms that inhabit these environments DIVERSITY OF HYPERSALINE ENVIRONMENTS Highly saline environments can be encountered on all continents They include natural salt lakes with highly diverse chemical compositions, artificial salt lakes such as solar salterns for the production of A Oren • The Institute of Life Sciences and the Moshe Shilo Minerva Center for Marine Biogeochemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel 223 224———OREN waters of the Dead Sea However, the increase in salt concentration and relative increase in divalent cation concentrations in the past decades have made the Dead Sea environment too extreme for massive development of even the most salt-adapted microorganisms Only when the upper water layers become diluted as a result of winter rain floods dense microbial communities develop in the lake A 10 to 15% dilution is sufficient to trigger massive blooms of the green alga Dunaliella and different types of red halophilic Archaea (Oren, 1988, 1999a) Other natural hypersaline lakes are highly alkaline Mono Lake, California (total salt concentration of around 90 g/liter; pH about 9.7 to 10), is an example of such a soda lake Even more extreme are some of the soda lakes of the East African Rift Valley such as Lake Magadi, Kenya, as well as the lakes of Wadi Natrun, Egypt, and some soda lakes in China: here dense communities of halophilic Archaea and other prokaryotes are found in salt-saturated brines at pH values above 10 This illustrates that some halophilic microorganisms are true “polyextremophiles” (Rothschild and Mancinelli, 2001), organisms that can simultaneously cope with more than one type of environmental stress The discovery of a truly thermophilic halophile, Halothermothrix orenii, isolated from a salt lake in Tunisia, shows that also life at high temperatures is compatible with life at high salt This anaerobic fermentative bacterium grows up to salt concentrations of 200 g/ liter (optimum: 100 g/liter) at temperatures up to 68°C (optimum 60°C) (Cayol et al., 1994) Coastal solar salterns, found worldwide in dry tropical and subtropical climates, are man-made, thalassohaline hypersaline environments in which seawater is evaporated for the production of salt Such saltern systems are operated as a series of ponds of increasing salinity, enabling controlled sequential precipitation of different minerals (calcite, gypsum, and halite) As a result, these saltern ecosystems present us with a more or less stable gradient of salt concentrations, from seawater salinity to NaCl precipitation and beyond, with each pond enabling the growth of those microbial communities adapted to the specific salinity of its brines Dense and varied microbial communities generally develop both in the water and in the surface sediments of the saltern ponds (Oren, 2005) It is therefore not surprising that these saltern ecosystems have become popular objects for the study of microbial biodiversity and community dynamics at high salt concentrations, and much of our understanding of the biology of halophilic microorganisms is based on studies of the saltern environment and in-depth studies of microorganisms isolated from such salterns Another hypersaline aquatic habitat that appears to harbor interesting communities of halophilic microorganisms is the highly saline anoxic brines found in several sites near the bottom of the sea Owing to the fact that these deep-sea anoxic hypersaline basins are not easily accessible for sampling, little is known thus far on their microbiology However, a preliminary exploration of such brines from the bottom of the Red Sea, using culture-independent techniques, yielded evidence for the presence of a wealth of novel types of halophiles (Eder et al., 1999) A comprehensive multidisciplinary research program was recently launched, aimed at the elucidation of the biology of the deep-sea hypersaline anoxic basins in the Eastern Mediterranean Sea The first published data that emerged from this program (van der Wielen et al., 2005) prove that we may expect many surprises from this previously unexplored type of hypersaline environment An overview of the biology of natural and manmade hypersaline lakes, as related to their chemical and physical properties, can be found in a recent monograph (Oren, 2002a) Halophilic and halotolerant microorganisms are not only found in aquatic habitats They can be recovered from many other environments in which high salt concentrations and/or low water activities occur Halophilic and highly halotolerant bacteria can easily be recovered from saline soils Some plants that grow on saline soils in arid areas actively excrete salt from their leaves, and the phylloplane of these plants thus appeared to be an interesting novel environment for halophiles (Simon et al., 1994), an environment that deserves to be investigated in further depth Salted food products—especially when crude solar salt is used for salting—can be an excellent growth substrate for halophilic or halotolerant microorganisms In fact, the production of some traditionally fermented food products in the Far East is based on the activity of halophilic bacteria Maybe the most surprising environment in which halophilic microorganisms have been found is the rock salt deposits found in many places worldwide Live bacteria (endospore-forming organisms of the genus Bacillus) have even been recovered from rock salt crystals that had been buried for 250 million years (Vreeland et al., 2000), while viable Archaea of the family Halobacteriaceae or their 16S ribosomal RNA genes were recovered from ancient salt deposits as well (Fish et al., 2002; Leuko et al., 2005) These microorganisms appear to survive within small liquid inclusions within the solid rock salt Although the claim that these organisms indeed had survived within the crystals for millions of years is not uncontested, it is now well established that indeed halophilic Bacteria and Archaea can retain their viability for long times in such brine inclusions within salt crystals CHAPTER 17 PHYLOGENETIC DIVERSITY OF HALOPHILIC MICROORGANISMS The ability to grow at salt concentrations exceeding those of seawater is widespread in the tree of life (Oren, 2000, 2002a, 2002b) Figure presents the three-domain Archaea–Bacteria–Eukarya phylogenetic tree, based on small subunit rRNA gene comparisons, indicating those branches that contain representatives able to grow at salt concentrations above 100 g/liter Halophiles are thus found in all three domains of life Among the Eukarya, we find relatively few representatives Halophilic macroorganisms are rare; one of the few existing ones is the brine shrimp (genus Artemia) found in many salt lakes worldwide at salt concentrations up to 330 g/liter (Javor, 1989) The most widespread representative of the Eukarya in hypersaline ecosystems is the algal genus Dunaliella Dunaliella is a unicellular green alga that is present as the major or sole primary producer in the Great Salt Lake, the Dead Sea, and salterns Some species can accumulate massive amounts of `-carotene, and their cells are therefore orange-red rather than green Some Dunaliella species prefer the low-salt marine habitat, but others, notably D salina, can still grow in the NaCl-saturated brines of saltern crystallizer ponds Also among the protozoa, we find halophilic and halotolerant types Different ciliate, flagellate, and amoeboid protozoa can be observed in the biota of saltern evaporation ponds of intermediate salinity (Oren, • BIODIVERSITY IN HIGHLY SALINE ENVIRONMENTS———225 2005) Although predation of the halophilic microbial communities is possible up to the highest salt concentrations (Hauer and Rogerson, 2005; Park et al., 2003), protozoa not appear to be very abundant in most hypersaline ecosystems Another, often neglected, group of eukaryal halophiles is that of the fungi Fungi are generally not abundantly found in environments of high salt concentrations However, it was recently ascertained that certain fungi, notably the halophilic black yeasts, find their natural ecological niches in the hypersaline waters of solar salterns (Gunde-Cimerman et al., 2000) More recent surveys have shown that the role that fungi may play in high salt environments has been grossly underestimated thus far (GundeCimerman et al., 2004; Butinar et al., 2005) Within the domain Archaea, we find halophiles in two major branches of Euryarchaeota: the Halobacteriales and the methanogens The branch of extremely halophilic, generally red pigmented, aerobic Archaea of the order Halobacteriales consists entirely of halophiles (Oren, 2001a) These are the organisms that dominate the heterotrophic communities in the Dead Sea, in the northern basin of the Great Salt Lake, in the crystallizer ponds of solar salterns, and also in many soda lakes Their massive presence is generally obvious by the red coloration of the brines, caused mainly by 50-carbon carotenoids (_-bacteroruberin and derivatives), but retinal based protein pigments (the lightdriven proton pump bacteriorhodopsin and the light-driven primary chloride pump halorhodopsin) may also contribute to the coloration of the cells Figure 1.–The small subunit rRNA sequence-based tree of life Branches that harbor organisms able to grow at salt concentrations above 100 g/liter are highlighted Based in part on Fig 11.13 in Madigan et al (2003) 226———OREN There are also obligatory anaerobic halophilic methanogenic Archaea Here, the halophiles not form a separate phylogenetic branch, but they appear interspersed between non-halophilic relatives Most known halophilic and halotolerant prokaryote species belong to the domain Bacteria Microscopic examination of water and sediment samples of saltern evaporation ponds of intermediate salinity shows an abundance of forms of bacteria Diverse communities of cyanobacteria, unicellular as well as filamentous, are conspicuously found in the microbial mats that cover the bottom sediments of salterns at salt concentrations up to 200 to 250 g/liter Below the cyanobacterial layer, massive development of photosynthetic purple sulfur bacteria (Halochromomatium, Halorhodospira, and related organisms, belonging to the Proteobacteria branch of the domain Bacteria) is often seen as well (Oren, 2005) The domain Bacteria contains many aerobic heterotrophic organisms of widely varying phylogenetic affiliation (Ventosa et al., 1998) The recent discovery of the genus Salinibacter (Bacteroidetes phylum), a genus abundant in saltern crystallizer ponds (see below), shows that the domain Bacteria contains some microorganisms that are no less salt tolerant and salt dependent than the most halophilic among the archaeal order Halobacteriales, which was thus far considered to contain the best salt adapted of all microorganisms There is one lineage within the Bacteria that appears to consist entirely of halophiles: the group of obligatory anaerobic bacteria of the order Halanaerobiales (families Halanaerobiaceae and Halobacteroidaceae) (Oren, 2001b) These fermentative organisms, which typically grow optimally at salt concentrations between 50 and 200 g/liter, may well be responsible for much of the anaerobic degradation of carbohydrates and other compounds in the anaerobic sediments of hypersaline lakes Last but not least, this survey of microbial diversity at high salt concentrations should also mention the occurrence of viruses Many halophilic Bacteria and Archaea have bacteriophages that attack them and may cause their lysis Free virus-like particles as well as lysing cells releasing large number of mature bacteriophages have been observed during electron microscopic examination of the biomass of saltern crystallizer ponds (Guixa-Boixareu et al., 1996) and the Dead Sea (Oren et al., 1997), and the viral assemblage in Spanish saltern pond has been partially characterized by pulsed-field gel electrophoresis (Diez et al., 2000) It was calculated that lysis by viruses is quantitatively far more important than bacterivory by protozoa in regulating the prokaryotic community densities of saltern ponds at the highest salinities (Guixa-Boixareu et al., 1996) METABOLIC DIVERSITY OF HALOPHILIC MICROORGANISMS As salt concentrations increase, the number of physiological types of microorganisms encountered in hypersaline lakes and other high salt ecosystems decreases To give a few examples: we not know any methanogenic Archaea growing at salt concentrations above 100 g/liter and using hydrogen plus carbon dioxide or acetate as their substrates Methanogenesis at higher salt concentrations does occur, but it is mainly based on degradation of methylated amines No truly halophilic dissimilatory sulfate-reducing bacteria are known to oxidize acetate, while sulfate reduction with lactate as electron donor can proceed up to salt concentrations of 200 to 250 g/liter at least Other metabolic activities that are notably absent at the highest salt concentrations are the two stages of autotrophic nitrification: oxidation of ammonium ions to nitrite and oxidization of nitrite to nitrate Microbial activities that are possible up to the highest salt concentrations are aerobic respiration and oxygenic photosynthesis Denitrification, anoxygenic photosynthesis with sulfide as electron donor and fermentations are processes that have been documented to proceed in environments at or close to salt saturation, as well as in cultures of isolated microorganisms grown at salt concentrations of 200 g/liter and higher (Oren, 1999b, 2000, 2002a) A possible explanation has been brought forward for the apparent absence of certain metabolic types of microorganisms at the highest salt concentrations This explanation was based on the balance between the energetic cost of osmotic adaptation and the amount of energy made available to the organisms in the course of their dissimilatory metabolism (Oren, 1999b) Life at high salt concentrations is energetically costly as the cells have to accumulate high concentrations of solutes to provide osmotic balance between their cytoplasm and the brines in which they live No microorganism uses NaCl to balance the NaCl outside, and therefore osmotic balance is always accompanied by the establishment of concentration gradients across the cell membrane, and this can only be done at the expense of energy Two fundamentally different modes of osmotic adaptation are known in the microbial world: accumulation of KCl, i.e., inorganic ions, to provide the osmotic equilibrium, or synthesis of accumulation of organic osmotic solutes The “high salt-in” strategy, based on the accumulation of potassium and chloride ions up to molar concentrations in the cytoplasm, is used by a few groups of microorganisms only The aerobic halophilic Archaea of the order Halobacteriales use this mode of osmotic adaptation CHAPTER 17 Not all halophilic Archaea use this strategy: the halophilic members of the methanogens accumulate organic osmotic solutes Within the domain Bacteria, we thus far know only two groups of halophiles that use the “high salt-in” strategy One is the fermentative anaerobes of the order Halanaerobiales (low GϩC branch of the Firmicutes) (Oren, 2002a) The second is the only recently discovered red aerobic Salinibacter (Bacteroidetes branch) (Oren et al., 2002) It is interesting to note that both the halophilic Archaea and Salinibacter possess halorhodopsin, a light-driven primary chloride pump, to facilitate the uptake of chloride into the cells Calculations have shown that the “high salt-in” strategy of osmotic adaptation is energetically favorable (Oren, 1999b) However, this mode of life depends on the complete adaptation of the intracellular enzymatic machinery to function in the presence of high ionic concentration Special adaptations of the protein structure are necessary to achieve this, and as a result, those microorganisms that use KCl as their osmotic solute have become strictly dependent on the presence of high salt concentrations Such organisms are generally restricted to life at a narrow range of extremely high salt concentrations They lack the flexibility to adapt to a wide range of salt concentrations and to changes in the salt concentration of their medium, a flexibility that is so characteristic of many microorganisms that use the second strategy of osmotic adaptation That second strategy is based on the exclusion of inorganic ions from the cytoplasm to a large extent while balancing the osmotic pressure exerted by the salts in the environment with simple uncharged or zwitterionic organic solutes A tremendous variety of such organic solutes have been detected in different halophilic and halotolerant microorganisms Thus, algae of the genus Dunaliella produce and accumulate molar concentrations of glycerol while regulating the intracellular glycerol in accordance with the outside salinity Glycerol is never found as an osmotic solute in the prokaryote world Osmotic, “compatible” solutes produced by different groups of prokaryotes include simple sugars (sucrose and trehalose), amino acid derivatives [glycine betaine, ectoine (1,4,5,6-tetrahydro2-methyl-4-pyrimidine carboxylic acid), and others], and other classes of compounds (Oren, 2002a) In many cases, more than one solute may be produced by a single organism For example, photosynthetic sulfur bacteria of the genus Halorhodospira (a-Proteobacteria) typically contain cocktails of glycine betaine, ectoine, and trehalose De novo biosynthesis of such organic osmotic solutes is energetically expensive However, most “low salt-in” organisms are also able to accumulate suitable • BIODIVERSITY IN HIGHLY SALINE ENVIRONMENTS———227 organic solutes when such compounds are present in the medium, thus enabling the cells to save considerable amounts of energy The great advantage of the “low salt-in” strategy of life at high salt concentration is that no or little adaptation of the intracellular enzymatic machinery is necessary Cells that use organic osmotic solutes to provide osmotic balance generally display a large extent of adaptability to a wide range of salinities and can rapidly adjust to changes in medium salinity Integration of the available information on the energetic cost of osmotic adaptation and information on the amount of energy generated by the different types of dissimilatory metabolism has enabled the establishment of a coherent model that may explain which types of metabolism can occur at the highest salt concentrations and which cannot (Oren, 1999b) Processes that provide plenty of energy (e.g., aerobic respiration and denitrification) can function at high salt concentrations, independent of the mode of osmotic adaptation of the organisms that perform them On the other hand, dissimilatory processes that yield little energy only (e.g., autotrophic nitrification and production of methane from acetate) are problematic at the highest salt concentrations unless the cells can economize on the amount of energy required to produce or accumulate osmotic solutes There, the “high salt-in” strategy appears to be advantageous, and this is therefore the strategy adopted by the Halanaerobiales, the specialized group of halophilic fermentative Bacteria The model explains why for example autotrophic nitrification is not likely to occur at high salt concentrations: only very little energy is gained in the process and (most) nitrifying bacteria belong to the Proteobacteria, a group that uses organic osmotic solutes rather than KCl to provide osmotic balance Also, the apparent lack of certain types of methanogens and sulfatereducing bacteria becomes understandable: those reactions that yield little energy not occur at the highest salinities and those reactions that are energetically more favorable Both groups depend on organic osmotic solutes for growth at high salt concentrations (Oren, 1999b, 2002a) SALINIBACTER RUBER, AN EXTREMELY HALOPHILIC MEMBER OF THE BACTERIA The recently discovered Salinibacter ruber, a species of red, extremely halophilic Bacteria isolated from saltern crystallizer ponds, presents us with an interesting model for the study of the adaptation of microorganisms to life at the highest salt concentrations (Oren, 2004; Oren et al., 2004) 228———OREN In the past, Archaea of the order Halobacteriales, family Halobacteriaceae, were always considered to be the extreme halophiles par excellence, being the sole heterotrophs active at the highest salinities such as those that occur in saltern crystallizer ponds and other NaCl-saturated environments All known heterotrophs representatives of the domain Bacteria could be classified as moderate halophiles Those few that were still able to grow at salt concentrations above 300 g/liter did so at very slow rates only and had their optimum growth at far lower salt concentrations (Ventosa et al., 1998) However, evidence for the presence of significant number of extremely halophilic representatives of the domain Bacteria in saltern crystallizer ponds, sometimes representing up to 15 to 20% and more of the prokaryotic community, was first obtained in the late 1990s on the basis of molecular ecological, culture-independent studies (Antón et al., 2000) When soon afterward the organism, a rod-shaped red aerobic bacterium, was brought into culture (Antón et al., 2002), the organism appeared to be extremely interesting, and its study has deepened our understanding of phylogenetic as well as physiological and metabolic diversity in the world of halophiles Salinibacter ruber, as the organism was named, belongs phylogenetically to the Salinibacter Bacteroidetes branch of the Bacteria Its closest relative as based on 16S rRNA sequence comparison is the genus Rhodothermus, red, aerobic thermophiles isolated from marine hot springs Salinibacter is no less halophilic than the most salt-requiring and salttolerant organisms within the Halobacteriaceae: it is unable to grow at salt concentrations below 150 g/ liter, it thrives optimally at 200 to 250 g/liter, and it grows in media saturated with NaCl as well Examination of the mode of osmotic adaptation and the properties of the intracellular enzymes showed a great similarity between Salinibacter and the Halobacteriaceae: in contrast to all earlier examined aerobic halophilic or halotolerant members of the Bacteria, Salinibacter did not contain organic osmotic solutes but was found to use KCl to provide osmotic balance (Oren et al., 2002) Accordingly, the intracellular enzymatic systems were found to be salt tolerant, and in many cases salt dependent The finding of a gene coding for halorhodopsin, the lightdriven inward chloride pump known thus far from halophilic Archaea only, made the similarity between the two even greater We may here have an example of convergent evolution, in which two, phylogenetically disparate organisms have obtained highly similar adaptations that have enabled them to grow at the highest salt concentrations but have also restricted their possibility to survive at lower salinities (Mongodin et al., 2005; Oren, 2004) We still know little about the interrelationships between Salinibacter and halophilic Archaea in the habitat they share: the brines of saltern crystallizer ponds and probably other salt lakes as well Being very similar in their physiological properties, Salinibacter should be expected to compete with the Halobacteriaceae for the same substrates and other resources What selective advantages either group has to ensure its coexistence with the other remains to be determined THE MICROBIAL COMMUNITY STRUCTURE IN HYPERSALINE ENVIRONMENTS— CULTURE-DEPENDENT AND CULTURE-INDEPENDENT APPROACHES As described in the previous section, it was the application of culture-independent studies of the microbial diversity, using small subunit rRNA gene sequence-based techniques that presented the first evidence of the existence of Salinibacter (Antón et al., 2000), an organism that was until that time completely overlooked, even when it probably had been present as colonies on agar plates inoculated with saltern brines in the past Microbiologists working with halophiles silently assumed that red colonies that developed on plates with salt concentrations of 200 to 250 g/liter can only belong to members of the Halobacteriaceae After the molecular approach had indicated what to look for, the isolation of the organism harboring the novel 16S rRNA gene sequence followed rapidly (Antón et al., 2002) The application of molecular biological techniques to the study of the microbial diversity in hypersaline ecosystems started in the mid-1990s with the studies by Benlloch et al (1995) in the salterns of Santa Pola, Alicante, Spain Sequencing of 16S rRNA genes amplified from DNA extracted from the biomass showed that the dominant phylotype in this environment indeed belonged to a member of the Halobacteriaceae, but differed from all thus far isolated members of the family at the genus level Fluorescence in situ hybridization experiments then showed that this phylotype belongs to a highly unusually shaped prokaryote: extremely thin, flat, perfectly square, or rectangular cells that contain gas vesicles (Antón et al., 1999) This type of cell was first detected during microscopic examination of water from a coastal brine pool on the Sinai Peninsula (Walsby, 1980) The abundance of such cells in the salterns had become well known in subsequent years (Guixa-Boixareu et al., 1996; Oren et al., 1996) However, until recently, this intriguing microorganism defied all attempts toward its isolation The elusive flat square halophilic Archaea were brought into culture in 2004, independently by two CHAPTER 17 groups of investigators, working in salterns in Spain (Bolhuis et al., 2004) and in Australia (Burns et al., 2004a) Using appropriate growth media (preferentially low in nutrients) and in addition a large amount of patience (incubation times of to 12 weeks), Burns et al (2004b) showed that in fact the majority of prokaryotes that can be detected in the saltern crystallizer ponds using 16S rRNA gene sequencebased, culture-independent techniques can also be cultured In most non-extreme ecosystems, there still is a tremendous difference, generally of many orders of magnitude, between the numbers of prokaryotes observed microscopically and the numbers that can be grown as colonies on plates Thanks to the recently developed new approaches, the saltern crystallizer environment is probably the first ecosystem for which the “great plate count anomaly,” as the phenomenon is often designated, has ceased to exist More extensive molecular ecological studies have been made in the Alicante salterns along the salt gradient, to obtain a more complete picture of the development of the microbial diversity as the salinity increases during the gradual evaporation of seawater (Benlloch et al., 2001, 2002; Casamajor et al., 2002; see also Oren, 2002c) Benthic cyanobacterial mats that develop on the bottom of saltern ponds of intermediate salinity have been the subject of molecular ecological studies as well (Mouné et al., 2002) Similar techniques have been used to characterize the microbial diversity in the athalassohaline alkaline Mono Lake, California (Humayoun et al., 2003) These studies make it clear that many of the microorganisms that dominate the communities before NaCl saturation is reached still await isolation and characterization EPILOGUE Although only few groups of macroorganisms have learned to live at salt concentrations much higher than those of seawater, many types of microorganisms have developed the adaptations necessary for life in hypersaline environments Many can even live at the salinity of saturated solutions of NaCl, the salt concentration encountered in some natural salt lakes as well as in saltern crystallizer ponds It has been suggested that the ability to live at high salt concentrations may have appeared very early in prokaryote evolution and that life may even have emerged in a hypersaline environment—a concentrated solution of organic compounds in tidal pools of partially evaporated seawater (Dundas, 1998) The theory of a hypersaline origin of life is, however, not supported by phylogenetic evidence: most halophiles are located on distant, • BIODIVERSITY IN HIGHLY SALINE ENVIRONMENTS———229 relatively “recent” branches of the small subunit rRNA gene sequence-based phylogenetic tree Moreover, the great variety in strategies used by the present-day halophiles to cope with the high salinity in their environment shows that adaptation to life at high salt concentrations has probably arisen many times during the evolution of the three domains of life (Oren, 2002a) The world of the halophilic microorganisms is highly diverse We find halophiles dispersed all over the phylogenetic tree of life Metabolically, they are almost as diverse as the “non-extremophilic” microbial world: we know halophilic autotrophs as well as heterotrophs, aerobes as well as anaerobes, phototrophs as well as chemoautotrophs Thus, hypersaline ecosystems can function to a large extent in the same way as “conventional” freshwater and marine ecosystems Owing to the absence of macroorganisms and the generally low levels of predation by protozoa, the microbial community densities of halophiles in hypersaline environments may be extremely high: counts of 107 to 108 red halophilic Archaea per ml of brine are not exceptionally high in Great Salt Lake, the Dead Sea, and in saltern crystallizer ponds, and they often impart a bright red color to the brines The presence of such dense communities makes such environments ideal model systems for the study of the functioning of microorganisms in nature While osmotic equilibrium of the cell’s cytoplasm with the salinity of the environment is essential for any halophilic or halotolerant microorganism to function, there are multiple ways in which this osmotic equilibrium can be achieved There is therefore a considerable diversity within the world of the halophilic microorganisms with respect to the way the cells cope with the salt outside Notably, there are two basically different approaches toward the solution of the problem: keeping the salt out or allowing massive amounts of salt (KCl rather than NaCl) to enter the cytoplasm There is no clear correlation between the phylogenetic position of a halophilic microorganism and the strategy it uses to obtain osmotic balance As the case of Salinibacter clearly shows, similar solutions have turned up in completely unrelated microorganisms Culture-independent techniques have taught us how diverse the microbial communities in salt lakes really are A few recent breakthroughs have enabled the cultivation of a number of halophiles (the flat square gas-vacuolated Archaea and Salinibacter) that are among the dominant forms of life in many hypersaline environments An in-depth study of such ecologically relevant organisms will undoubtedly deepen our understanding of the functioning of the highly saline ecosystems, as well as shed more light on the nature of the adaptation of life to function at the highest salt concentrations 230———OREN REFERENCES Antón, J., E Llobet-Brossa, F Rodríguez-Valera, and R Amann 1999 Fluorescence in situ hybridization analysis of the prokaryotic community inhabiting crystallizer ponds Environ Microbiol 1:517–523 Antón, J., R Rosselló-Mora, F Rodríguez-Valera, and R Amann 2000 Extremely 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Comp Biochem Physiol A 133:677–688 Xu, Y., Y Nogi, C Kato, Z Liang, H.-J Ruger, D De Kegel, and N Glansdorff 2003c Psychromonas profunda sp nov., a psychropiezophilic bacterium from deep Atlantic sediments Int J Syst Evol Microbiol 53:527–532 Xu, Y., Y Nogi, C Kato, Z Liang, H.-J Ruger, D De Kegel, and N Glansdorff 2003d Moritella profunda sp nov and Moritella abyssi sp nov., two psychropiezophilic organisms isolated from deep Atlantic sediments Int J Syst Evol Microbiol 53:533–538 Yayanos, A A 1995 Microbiology to 10,500 meters in the deep sea Annu Rev Microbiol 49:1356–1361 Zillig, W., P Palm, and H P Klenk 1992 The nature of the common ancestor of the three domains of life and the origin of the Eucarya, p 181–193 In J Tran Thanh Van, J C Mounolou, J Schneider, and C McKay (ed.), Frontiers of Life Editions Frontiers, Gif-sur-Yvette This page intentionally left blank INDEX Accretion ice, 146 Acidimicrobium/Ferrimicrobium spp., 264–265 Acidiphilium spp., 265–266, 274 Acidithiobacillus ferrooxidans, 260, 262, 263–264, 271, 279 Acidithiobacillus spp., 262–263 Acidocella organivorus, 261, 266 Acidocella spp., 265–266 Acidophiles adaptations of living at low pH, 259–260 arsenic resistance mechanisms, 273, 274 copper resistance, 274–275 energy transformations and carbon assimilation, 260–261 extremely acidic environments, 257–259 mercury resistance mechanisms, 275 phylogenetic diversity and physiological characteristics, 259–261 protection from metal toxicity, 272–273 Acidophiles, genomics of, 279–280 acidophily and acid resistance of pathogens, 281–284 chaperone genes, 287–289 evolution of thermoacidophiles and concept of “lifestyle genes,” 289–290 genes for DNA superstructure formation, repair, and restriction, 287 genome sequences of acidophilic archaea, 280–281 genome-based reconstruction of major metabolic pathways, 284–286 transport, 286–287 Acidophilic communities macroscopic growths and streamer communities, 267–268 relationships and interactions, 266–267 Acidophilic prokaryotes dissimilatory oxidoreduction of iron, 263–265 iron- and sulfur-oxidizing aerobes, 261–263 iron-reducing heterotrophs, 265–266 sulfur-oxidizing archaea, 263 sulfur-reducing acidophiles, 266 Aeromonas hydrophila, 186 Alicyclobacillus spp And other acidophilic Firmicutes, 264 Alkaliphiles, 295–296 aerobic mesophiles other than Bacillus spp and thermophiles, 304 anaerobic and lactic acid bacterial groups, 303 features in the ATP synthase, 318 gram-positive high GϩC bacteria, 307 gram-positive low GϩC bacteria, 302–306 gut of higher termites, 298 indigo fermentation, 300–301 MotPS and NavBP in motility chemotaxis, 320–322 Naϩ/solute symporters, 319–320 other environments, 301–302 sea and related samples, 299–300 segments of membrane proteins, 324 soda lakes, 298–299 soil samples, 296–298 thermophiles, 307 Alkaliphilic Bacillus spp., 321–325 B pseudofirmus, 313, 320 characteristics, 297 phylogenetic tree derived from 16S rRNA gene sequences, 300 Alkaliphily, bioenergetic adaptations, 311–312 alkaliphiles versus neutrophiles, 312–313 contribution to cytoplasmic pH homeostasis, 313–317 cycles supporting alkaliphile pH homeostasis, 317–322 _-Amylases, 362–365 `-Amylases, 365 Amylomaltases, 369–370 Anthropogenic environments, 17 Archaea, 104, 409, 411 See also Hyperthermophiles archaeal versus bacterial membrane lipids, 107–110 cellulolytic, hemicellulolytic, chitinolytic, and proteolytic enzymes, 371–372 classification according to optimal growth temperatures, 14 Crenarchaeota, 105 ether linkages in archaeal lipids, 110 Euryarchaeota, 105 membrane adaptations to heat stress, 111 Archaea–Bacteria–Eukarya phylogenetic tree, 225 Archaeal genomes density of genes encoding transporter proteins, 286 size and coding density distribution of selected, 281 Arctic winter permafrost, 134, 135 Arctic winter sea ice concentration of EPS scaled to brine volume of ice sections, 138 microscopic images, 135 viral and bacterial dynamics in section of, 140 Arthrobacter globiformis, 186 423 424 INDEX Astrobiology habitats for life, 354–355 life requirements, 351–354 perspectives in, 152 plausibility categories, 356 searching for life in the universe, 355–356 ATP synthase (alkaliphile-specific features), 318 Autonomous IS elements, 36 Bacteria, 409, 411 archaeal versus bacterial membrane lipids, 107–110 cellulolytic, hemicellulolytic, chitinolytic, pectinolytic and proteolytic enzymes, 374–376 classification according to optimal growth temperatures, 14 membrane adaptations to heat stress, 111 membranes of, 107, 110 Bacterial membrane fluidity, low-temperature-induced changes, 194–195 changes in fatty acid desaturation, 195 changes in proportion of cis and trans fatty acids, 197–199 perception of temperature and signal transduction, 200–201 role of carotenoids, 199 role of cold-inducible genes, 199–200 sensing of temperature, 202–203 sensors and transducers of low temperature, 201–202 temperature-dependent changes, 195–197 Bacterial piezophiles, phylogenetic tree of, 335 Barophilicity, 113 Biocatalysts from extremophiles alcohol dehydrogenases, 388 C–C bond-forming enzymes, 389 cellulose-degrading enzymes, 370–373 chitin-degrading enzymes, 377–378 DNA-processing enzymes, 380–384 extremozymes of industrial relevance, 390 glucose and arabinose isomerases, 388–389 lipases and esterases, 384–388 nitrile-degrading enzymes, 389 pectin-degrading enzymes, 378–379 proteolytic enzymes, 379–380 starch-processing enzymes, 362–370 xylan-degrading enzymes, 373–377 Biodiversity, 13 Bioenergetics function of cytoplasmic membrane, 105–107 Biogeochemistry and thermal environments, 15–16 Biogeography and thermal environments, 15–16 Biomining, 390–391 Biopolymers, 393 Biotechnological applications (extremophiles) alcohol dehydrogenases, 388 _-amylases, 362–365 `-amylases, 365 amylomaltases, 369–370 branching enzymes, 369 C–C bond-forming enzymes, 389 cellulose-degrading enzymes, 370–373 CGTases, 369 chitin-degrading enzymes, 377–378 DNA-processing enzymes, 380–384 extremophilic archaea and bacteria, 362 glucoamylases, 365–368 glucose and arabinose isomerases, 388–389 _-glucosidases, 368 `-glucosidases, 370, 373 lipases and esterases, 384–388 nitrile-degrading enzymes, 389 pectin-degrading enzymes, 378–379 proteolytic enzymes, 379–380 pullulanases, 368–369 starch-processing enzymes, 362 xylan-degrading enzymes, 373–377 Biotechnological applications (psychrophiles) bioremediation and natural cycle processes, 125 biotechnologically important properties, 125 cleaning additives, 128 food industry, 125–128 molecular biology, 128 textile industry, 128 Branching enzymes, 369 Caloramator fervidus, 112–113 Carbamoyl phosphate metabolism in hyperthermophiles, 65–69 Carbamoyl phosphate, 66 Cellulose-degrading enzymes, 370–373 Cenarchaeum symbiosum, 105 CGTases, 369 Chasmoendolithic organisms, 123 Chemical evolution, 6–7 Chemolithotrophy, 21–22, 136 Chitin-degrading enzymes, 377–378 Cold environments man-made psychrophilic environments, 124–125 marine environments, 120–122 soil environments, 122–124 Cold-adapted enzymes catalysis at low temperatures, 167–170 cold denaturation, 174 importance of flexibility, 174–176 inactivation and conformational stability, 171–172 mutagenesis studies, 173–174 properties, 165–167 structural modifications, 172–173 thermodynamics of activity, 170–171 Cold-adapted extracellular protease activity, frequency of detection, 139 Cold-adapted microorganisms See Psychrophiles Cold-growing bacteria with complete genomes, 211 Cold-shock response adenylate metabolism, 186 Aeromonas hydrophila, 186 Arthrobacter globiformis, 186 of cold-adapted bacteria, 184–185 cold-adapted enzymes, 185 cold-shock proteins of psychrotrophic bacteria, 186 of CspA family of E coli, 181–184 INDEX cyanobacteria, 187–188 E coli versus psychrotrophs, 185 global transcript profiling, 188–189 membrane-associated changes, 185 of mesophilic bacteria, 180–181 protective effect of sugars during cold shock, 184 protein folding at low temperature, 184 Pseudomonas fragi, 186 thermophilic bacteria, 187 translation, 185–186 Compact tertiary structures, generation of, 48–49 Compatible solutes, 86, 88, 393–396 distribution in hyperthermophilic microorganisms, 88 hyperthermophiles preference, 96–97 mannosylglycerate biosynthesis, 91–95 molecular basis of protein stabilization, 97–101 occurrence in hyperthermophiles, 87–91 physiological relevance of compatible solute accumulation, 96 polyol-phosphodiesters biosynthesis, 95–96 Covalent modification of nucleosides, 44–48 Crenarchaeota, 105 Cryoprotection, 137–138 Cryptoendolithic organisms, 123, 147 Cytophaga-Flavobacterium-Bacteroides (CFB) group, 138 Cytoplasmic membrane adaptations to heat stress, 111 in bioenergetics, 105–107 permeability under physiological conditions, 111–113 De novo purine nucleotide biosynthesis, 63–65 Decontamination and hydrogen production, 391 DNA repair, controlling DNA damages by efficient, 50 DnaJ cochaperone, 237, 238, 240 DnaK chaperone, 235, 236, 237 DNA-processing enzymes DNA sequencing, 383 ligase chain reaction, 383–384 PCR, 380–383 DNAs, stabilizing strategies of, 51–52 E coli genes influencing high-pressure (HP) resistance or high-pressure growth in, 342 morphology at low and high pressure, 338 Earth environmental requirements and limitations for active life on, 351 life on, primitive, as an extreme environment, Embden–Meyerhof (EM) glucose-degrading pathway, 58 Endoliths, 123–124 Entner–Doudoroff (ED) glucose-degrading pathway, 58 non-phosphorylative and semi-phosphorylative, 59 EPS concentration scaled to brine volume of sections from Arctic winter sea ice, 138 Cytophaga-Flavobacterium-Bacteroides (CFB) group, 138 potential roles in winter sea-ice brine pores, 141 425 Eukarya, 44, 45, 50, 91, 104, 148, 200, 210, 216, 225, 240, 287, 333, 334, 338, 342, 409, 410, 411, 413, 414, 415 Euryarchaeota, 105 Exobiology See Astrobiology Extracellular enzyme activity, temperature-dependent, 139 Extreme environment primitive Earth, chemical evolution and, 6–7 Extremophiles advantages and limitations of using, 396 applications, xii biocatalysts from See Biocatalysts from extremophiles DNA-modifying enzymes from, 381–382 definition, xii early history, xi and “intelligent design,” 418 limits of life and combined, 415–418 Extremophilic archaea and bacteria, 362 Extremophilic microorganisms, applications of, 394 Facultative anaerobic acidophiles catalyzing dissimilatory iron oxidoreduction Acidimicrobium/Ferrimicrobium spp., 264–265 Acidithiobacillus ferrooxidans, 263–264 Alicyclobacillus spp and other acidophilic Firmicutes, 264 Ferroplasma spp., 265 Sulfobacillus spp., 264 Fatty acid composition glycerolipids from wild type and mutant cells of Synechocystis sp PCC 6803 and Synechococcus sp PCC 7002, 198 mesophilic versus psychrophile, 196 Fatty acyl chain length of lipids, 160 Fatty acyl unsaturation, 157–159, 160 Ferroplasma spp., 265 Flagellum, components of, 337 Functional genomics adaptations required for thermophily and structural genomics, 31–32 definition, 30 future trends, 36 LGT, genome plasticity, and phylogeny, 34–36 transcriptional analysis of stress and responses and metabolic regulation, 32–34 GϩC content in RNA, high, 41–42 Genomic DNA of hyperthermophiles, 41, 47, 48, 49, 50, 149 Glacial ice, 146 Glucoamylases, 365–368 Glucose catabolism, 57–61 _-glucosidases, 368 `-glucosidases, 370, 373 Haloarchaea, 232–233 adaptation to osmotic stress, 235–238 osmoregulation in, 233–235 Haloferax volcanii genome, transcriptional map of, 234 426 INDEX Halophiles diversity of hypersaline environments, 223–224 metabolic diversity, 226–227 microbial community structure in hypersaline environments, 228–229 phylogenetic diversity, 225–226 Salinibacter ruber, 227–228 Halophilic archaea See Haloarchaea Halophilic genome sequences, 240 archaea, 241 eubacteria, 241 Halophilic proteins from nonextreme halophile with available high-resolution structures, 245 ions detected and net charge of (X-ray structure), 243 with available high-resolution structures, 242 Homeo-proton permeability adaptation, 112, 162 Hydrogen production, 391 Hydrogenobaculum acidophilum, 263 Hypersaline environments, 223–224 See also Halophiles Hypersolutes, 87, 96, 101 Hyperthermophiles, 104 See also Cytoplasmic membrane classification according to optimal growth temperatures, 14 compatible solutes, 87–91 compatible solutes restricted to, 90 function of cytoplasmic membrane in bioenergetics, 105–107 identifying heat-shock proteins, 33 importance of cell-specific polyamines, 44 membrane adaptations to heat stress, 111 membrane lipids, 107–110 membrane permeability under physiological conditions, 111–113 physiochemical properties of membranes, 110–111 preference for negatively charged compatible solutes, 96–97 tree of life, 91 Hypolithic microbial communities, 123 Ice accretion, 146 benefits of exopolymers in very cold ice, 138–140 comparison of very cold glacial ice, permafrost, and sea ice, 135–137 formations, 134 freeze-concentration effect in very cold ice, 137–138 glacial, 146 life in Vostok ice core, 148–151 potential for discovery, 141–142 transition, 146 Insertion sequence (IS) elements, 34–36, 234 Iron- and sulfur-oxidizing aerobes Acidithiobacillus spp., 262–263 Hydrogenobaculum acidophilum, 263 Leptospirillum spp., 261–262 Thiobacillus spp., 263 Iron oxidation and CO2 fixation, 260 Iron-reducing heterotrophs Acidiphilium spp., 265 Acidocella organivorus, 266 Acidocella spp., 265–266 Last universal common ancestor (LUCA), 48, 409–410, 411 Leptospirillum spp., 261–262, 266, 267 phenotypic characteristics of, 262 Life at high temperature, 30–31 high temperatures and, 13–16 in ice formations at very cold temperatures, 133–135 in subglacial environments, 147–148 in Vostok ice core, 148–151 molecular cladistics and origin and early evolution, 4–6 on Earth, timescale for origin and early evolution, Lipids, 391–392 LUCA See Last universal common ancestor Man-made psychrophilic environments, 124–125 Mannosyl-3-phosphoglycerate synthase genes biochemical properties of recombinant, 93 unrooted phylogenetic tree based on known or putative sequences, 94 Mannosylglycerate, 87 biosynthesis, 91 distribution, 91 genomic organization, 95 pathways for synthesis, 92 Mannosylglycerate biosynthesis, 91–94 genes for, 94–95 synthesis, 95 Marine environments, 16–17 Antarctic marine environments, 121 deep marine environments, 120–121 glacial ice, 122 sea ice, 121–122 Mesobiotic environments, 17–18 Mesophilic versus psychrophile enzymes, 170 fatty acid composition, 196 Methanopyrus kandleri, 109–110 Methyl branched fatty acids, 159–160 Microbial adaptation to high pressure cell division, 338 culture-based studies, 334–337 culture-independent studies, 333–334 membranes and transport, 341–342 motility, 337 pressure stress responses, 338–339 protein/extrinsic factors, 342 signaling changes in gene expression, 340–341 translation, 339–340 Microecology, 23 Molecular adaptation by psychrophilic proteins, 80–82 by thermophilic/hyperthermophilic proteins, 76–80 Molecular adaptation to high salt crystallographic studies of halophilic proteins, 242–245 evolutionary mechanisms, 242 INDEX halophilic genome sequences, 240–242 protein–DNA interactions in a halophilic context, 245–246 salt and halophilic protein stability, 247 solvation, 246–247 Molecular cladistics and origin and early evolution of life, 4–6 Mrp Naϩ/Hϩ antiporter system, 316 Naϩ and proton cycles supporting alkaline pH homeostasis, 315 Nonautonomous IS elements, 36 Nucleic acids—effects on heat in vitro studies, 39–40 stabilization of, by thermophiles, 41–50 strategies of thermostabilization, 42 thermoresistance (in vivo versus in vitro), 40–41 Oligotrophic environments, 17, 138, 150, 155 Pectin-degrading enzymes, 378–379 Phospholipids core structures in bacteria and tetraether lipids, 108 freeze-fracture and freeze-etch, 109 mobility of acyl chains in fluid membrane phase, 107 Photobacterium profundum SS9, 211, 213, 214, 216, 217, 338 genes influencing high-pressure (HP) resistance or high-pressure growth in, 342 Piezophile genomics and functional genomics, 342–343 Piezophiles See Microbial adaptation to high pressure Piezophilic Bacteria and Archaea, 336 Planctomycetes, 123, 410, 411, 415 Polar glacial ice, 134 Polyol-phosphodiesters biosynthesis DGP biosynthesis, 96 di-myo-inositol phosphate, 95–96 Primeval cell, xv, 410 Prokaryotes, modern, 410 Protein stabilization by solutes probing effects of solutes on protein dynamics, 100–101 probing effects of solutes on unfolding pathway of proteins, 99–100 types of molecular interactions, 97–99 Proteins molecular adaptation by psychrophilic, 80–82 molecular adaptation by thermophilic/ hyperthermophilic, 76–80 and peptides, 392 Proteolytic enzymes, 379–380 Protoeukaryote, 410, 411 Protoeukaryotic LUCA, domains of life from (alternative evolutionary scenarios), 414 Proton motive force (PMF), 105–106, 111, 113 Pseudomonas fragi, 186 Psychrophiles See also Cold environments biotechnical applications, 125–128 cardinal temperatures, 119 genome sequencing projects, 126 427 genomics, 125 habitats, 119 Psychrophiles, membrane adaptations future trends, 162–163 genomics and, 161–162 membrane stability and temperature, 156–157 regulation of adaptive changes in lipid composition, 160–161 solute transport across membranes, 162 thermal changes in lipid fatty acyl composition, 157–160 Psychrophilic habitats, microbial genera isolated from, 120 Psychrophilic prokaryotes genomes, 208–209 bacteria under cold conditions, 213 bacterial genome organization, 213–214 early studies, 210–211 genome programs, 211–213 psychrophily, 209–210 RNAs, 214–217 Psychrophilic versus mesophilic enzymes, 170 fatty acid composition, 196 Psychrophily, 128, 168, 209, 213, 412, 415, 417 Psychrotrophs, 119, 128, 185, 186 Pullulanases, 368–369 Pyrite oxidation by consortia of acidophilic prokaryotes, 267 Pyrolobus fumarii, 14, 105 Radiation dose giving ~37% survival for UV and ionizing radiation, 353 Reverse gyrase, 8, 48, 49, 50, 287, 411, 412 Ribosome, components of, 340 RNA damages by RNA turnover, eliminating, 50 RNAs, 214–215 factors allowing tRNA molecules to function at high temperatures, 51 membrane fluidity, protein export, and secretion, 217 metabolic constraints, 216–217 phylogenetic distribution of modified nucleosides, 45 proteome, 215–216 reactive oxygen species, 216 schematic representation of tertiary interactions in tRNA structure, 46 stabilizing strategies of, 51–52 RNP particles with thermostable proteins, generation of, 49 16S rRNA gene sequences phylogenetic and genetic diversity, 20 restriction enzyme phylotypes, 20–21 16S rRNA gene sequences, phylogenetic tree derived from alkaliphilic Bacillus spp., 300 alkaliphiles that are high GϩC and low GϩC gram-positive bacteria, 305 Salt and halophilic protein stability electrostatic contributions, 247 neutron-scattering studies of molecular dynamics in extreme halophiles, 250–251 salt bridges, ion binding, and stabilization of active dime and tetramer of Hm MalDH by salt, 247–248 428 INDEX salts and stabilization of halophilic proteins, 248–249 solubility, 249–250 Small ligand binding, 42–44 Sodium motive force (SMF), 106, 111, 112–113 Soil environments fellfield communities, 124 lithic communities, 123–124 montane environments, 123 ornithogenic environments, 124 polar soils, 122–123 Starch-processing enzymes, 362 _-amylases, 362–365 `-amylases, 365 amylomaltases, 369–370 branching enzymes, 369 CGTases, 369 extremophilic archaea, 363–364 extremophilic bacteria, 366 glucoamylases, 365–368 _-glucosidases, 368 `-glucosidases, 370, 373 pullulanases, 368–369 Subglacial environments, 147–148 Subglacial lakes of Antarctica See also Vostok, Lake life in subglacial environments, 147–148 scientific objectives, 146–147 Subsurface environments, 17, 354 Sulfobacillus spp., 264 Sulfolobus acidocaldarius, 105, 110 Sulfolobus solfataricus, 109 Sulfur-oxidizing archaea, 263 Sulfur-reducing acidophiles, 266 Synthetic membrane spanning lipids, 110 Temperature profiles (generic), 134 Temperature, high, and life, 13–16 Temporary environments, 17 Terrestrial, nonanthropogenic environments, 16 Tetraether liposomes, 110–111, 113 Thermal adaptation and macromolecular organization of metabolism, 69–70 Thermal environments cultural diversity, 13–16, 18–20 ecological diversity, 22–23 high temperatures and life, 13–14 metabolic diversity, 21–22 phylogenetic and genetic diversity, 20–21 types, 16–18 Thermoacidophilic archaea, E coli acid resistance genes among, 284 Thermoactive enzymes of industrial relevance, 385–386 Thermolabile metabolites CP metabolism in hyperthermophiles, 65–69 de novo purine nucleotide biosynthesis, 63–65 glucose catabolism, 57–61 thermal adaptation and macromolecular organization of metabolism, 69–70 tryptophan biosynthesis, 61–63 Thermophile genomes, sequenced, 31 Thermophiles, stabilization of their nucleic acids controlling DNA damages by efficient DNA repair, 50 eliminating RNA damages by RNA turnover, 50 generation of compact tertiary structures, 48–49 generation of RNP particles with thermostable proteins, 49 high temperatures and life, 13–16 stabilization of nucleic acid structures, 42–48 use of high GϩC content in RNA, 41–42 Thermophiles and thermolabile metabolites CP metabolism in hyperthermophiles, 65–69 de novo purine nucleotide biosynthesis, 63–65 glucose catabolism, 57–61 thermal adaptation and macromolecular organization of metabolism, 69–70 tryptophan biosynthesis, 61–63 Thermophilic archaea, 19, 20, 22 analysis of genomic data, 77 branched polyamines in, 44 changes in lipid composition, 111 and chitinolytic enzymes, 377 Di-myo-inositol-1,1v-phosphate, 393 DnaK system, 236 genomes, 287 glucose catabolism, 57 heat-stable proteases, 379 heat-stress response, 96 recombinational DNA-repair system, 50 reverse gyrase, 48 salt concentration in cytosol, 245 tetraether lipids, 113 UAG codons and, 36 and xylanolytic enzymes, 377 Thermophilic bacteria, 19 amino acid positions, 236 anaerobic, 22 cellular machinery, 180 chemolithoautotrophic, 22 chitin-hydrolyzing enzymes, 378 cold-shock response, 187 composition, 107 CspA homologs, 181 genomes, 280 glucose isomerases, 388 growth temperature membranes, 112 heat-stable lipases, 384 life at high temperature, 30 maximal growth rate temperatures, 411 metabolic channeling, 65 nature of membrane lipids, 411 phylogenetic and genetic diversity, 20 polyamines in, 43–44 proton permeability of membranes, 112 pullulanase type I, 369 reverse gyrase, 48 thermoactive enzymes, 389 thermostable serine proteases, 379 Thermophilicity, 78, 104 Thermophily adaptation, 412 before LUCA, 412–413 INDEX extreme, 410–412 LUCA, 413 Thiobacillus ferrooxidans See Acidithiobacillus ferrooxidans Thiobacillus spp., 263 Transition ice, 146 Tryptophan biosynthesis, 61–63 Tryptophan biosynthetic pathway, 62 Vostok, Lake, 145–146 life in subglacial environments, 147–148 scientific objectives, 146–147 Universal tree of life, 409–410 Xylan-degrading enzymes, 373–377 Whole-cell biocatalysis biomining, 390–391 decontamination and hydrogen production, 391 429 Color Plate (Chapter 6) Stereo diagram depicting superimposition of highly homologous cold-shock proteins from Bacillus caldolyticus (red, PDB entry: 1C9O chain A, BcCSP) and Bacillus subtilis (green, PDB entry: 1CSP, BsCSP) The backbones of the two proteins are shown as thin lines and the nonconserved residues between the two protein structures are shown as balls and sticks (see text for details) For the sake of clarity, the conserved residues between the two proteins are not shown The N- and C-termini are indicated by the first (Met 1) and the last residues (Leu 66) in BcCSP Arg 3, which along with Leu 66 mostly confers thermostability on BcCSP (Perl et al., 2000), is also shown Cold-shock proteins have emerged as a model system to understand molecular factors involved in protein thermostability Modulation of protein electrostatics appears to be responsible for the greater stability of BcCSP However, this is not achieved by incorporation of additional salt bridges or ion pairs in BcCSP In this sense, the case of the cold-shock proteins appears exceptional Color Plate (Chapter 19) Crystallographic structures of halophilic proteins PYMOL was used to represent the electrostatic potential at the surface of the proteins The names of halophilic proteins are defined in Table Pf LDH, the lactate dehydrogenase from the nonhalophilic Plasmodium falciparum, is shown for comparison Pw TBP is the TATA-binding box protein from the hyperthermophile Pyrococcus woesei Color Plate (Chapter 19) Crystallographic structures of R207S, R292S mutant of Hm MalDH Two orthogonal views are presented, with the four polypeptide chains of the tetramer shown in ribbons of different colours; NADH is shown in a stick representation, chloride ions as grey balls, and water as small red dots Color Plate (Chapter 20) Macroscopic growths of acidophilic microbial communities at two mine sites in north Wales: (a) stalactite-like (“pipe”) growths and (b) surface slimes within a disused pyrite mine and (c) streamer growths in acid waters draining an abandoned copper mine ... chargesa Naϩ; ClϪ Kϩ PO3Ϫ SO4Ϫ, 16 ClϪ, Kϩ 12 Mg2ϩ, ClϪ, Naϩ SO4Ϫ, Mg2ϩ, Naϩ Ca2ϩ, Mg2ϩ 1 ,21 2 128 1 62 1,4 62 804 2, 184 960 1 12 12 1 52 28 14 150 147 368 1 32 At pH All numbers are given per biologically... MalDH 3 .2 2.95 2. 6 1.95 1.9 NMR NMR 2. 6 1.7 1.6 2. 35 2. 2 Dym et al., 1995 Richard et al., 20 00 Richard et al., 20 00 Irimia et al., 20 03 Frolow et al., 1996 Marg et al., 20 05 Marg et al., 20 05 Pieper... Res 14 :22 21 22 34 Charlebois, R L., L C Schalkwyk, J D Hofman, and W F Doolittle 1991 Detailed physical map and set of overlapping clones covering the genome of the archaebacterium Haloferax volcanii