Physiology and Biochemistry of Extremophiles

During the past 2 decades, the description of a diverse assortment of prokaryotic species that thrive under extreme environments that used to be considered inhospitable has broadened our understanding of the range of conditions under which life can persist. With the exception of heat-loving prokaryotes, however, the phylogenetic distribution of other extremophiles in molecular cladograms does not provide clues to their possible antiquity. Furthermore, given the huge gap existing in current descriptions of the evolutionary transition between the prebiotic synthesis of biochemical compounds and the last common ancestor (LCA) of all extant living beings, it is probably naïve to attempt to describe the origin of life and the nature of the first living systems from molecular phylogenies. It is unlikely that data on how life originated will be provided by the geological record. The remarkable coincidence between the monomeric constituents of living organisms and those synthesized in laboratory simulations of the prebiotic environment is too striking to be fortuitous, but at the same time the hiatus between the primitive soup and the RNA world, i.e., the evolutionary stage prior to the development of proteins and DNA genomes during which early life forms largely based on ribozymes may have existed, is discouragingly enormous. The diversity of environmental conditions under which prokaryotes can thrive should be understood as evidence of their adaptability and not as evidence that the origin of life took place under extreme conditions.

This chapter summarizes some of the thermal environments on Earth and describes the taxonomic, genetic, metabolic, and ecological diversity of these environments. Biogeology/biogeochemistry is especially of interest in thermal environments, where mineralization is active and the role of prokaryotes in mineralization is being examined. Water is readily available in circumneutral, freshwater hot springs, but there are thermal environments having low water potentials; e.g., in intraterrestrial environments because of high surface area-to-water ratios or in solar heated soils and sediments because of evaporation and high salinity. The authors believe that many environments that are classified as mesobiotic from their bulk temperature measurements contain temporary thermal microniches, created by localized biodegradation of organic material. Measuring the genetic diversity of 16S rRNA and functional genes, which has been an avenue for discovery of many enzymes for biotechnological applications and the isolation of novel microorganisms, provides only limited information about their in situ abundance and activity. In general, analysis of multiple approaches applied in single environments combined with that of similar approaches in different environments has enhanced the robustness of our understanding of the various high-temperature environments and the biodiversity they harbor. Considering how the initial discovery of life in shallow and deep-sea vents expanded our notion of global biodiversity, future approaches and discoveries, perhaps also in extraterrestrial thermal environments, will likely reveal additional information relevant to many fields of basic and applied science.

This chapter, using the rapidly expanding set of whole-genome sequences now available, examines the progress made in understanding life at elevated temperatures. Functional genomics uses high-throughput techniques like DNA microarrays, proteomics, metabolomics and mutation analysis to describe the function and interactions of genes. The first thermophilic methanogen to be analyzed comprehensively by microarray was Methanocaldococcus jannaschii. This study resulted in the discovery of a unique heat-shock-inducible prefoldin chaperone gene. In an early study, activities of several key metabolic enzymes were evaluated when Pyrococcus furiosus was grown on maltose and/or peptides, both with and without S0. This study revealed that Pyrococcus furiosus is able to utilize both peptides and maltose as sources of C and that it is able to grow well in the absence of S0, metabolic characteristics that set it apart from most other S0-reducing, heterotrophic hyperthermophiles. Comparative genomics also provides insights into the mobility of chromosomal sections and lateral gene transfer (LGT). Bacterial and archaeal thermophiles often share the same habitats, and there is abundant evidence from genomic analyses that LGT is common in the group. The application of microarray-based studies, already underway using the P. furiosus genome information, will be important to examine global stress regulation. Further studies of the growth physiology and molecular biology of model organisms such as hyperthermophiles and halophiles will be necessary to determine their potential for the production of gas fuels and the potential application of their extremely thermostable enzymes in biotechnology.

This chapter discusses the question of coping up of the nucleic acids with high temperature at the polynucleotide level—RNA, DNA, and their ribonucleoprotein derivatives (RNP/DNP). When nucleic acids are heated in aqueous solution, two types of phenomena take place: denaturation of their architecture and chemical degradation of their building blocks. In vivo, the half-lives of both RNA and DNA of thermophilic organisms are usually longer than that estimated in vitro, attesting to cellular strategies protecting the nucleic acids against the deleterious effects of heat. Despite the susceptibility of certain modified bases and of the ribonucleotide chain to thermal degradation, most naturally occurring tRNAs (especially those from hyperthermophilic organisms) appear fairly resistant to heat denaturation. Despite the intrinsic potentiality of nucleic acids to degrade at elevated temperatures, many hyperthermophiles can survive at very high temperatures approaching or even surpassing the boiling point of water. The majority of stable cellular RNAs, such as tRNA and rRNA molecules, contain a variety of modified nucleosides. Stabilizing strategies of RNAs and DNAs may be classified into three major categories: (i) those which are intrinsic to the chemical structures of the nucleic acids; (ii) those which are dependent on extrinsic interactions with other biomolecules; and (iii) those which are dependent on a battery of enzymes for detecting and repairing the DNA damage or to constantly renew functional RNA molecules. Genetic approach using mutant strains mutated in one or more biomolecules supposedly involved directly or indirectly in stabilization of nucleic acids should be more systematically used.

An intriguing question is how thermophilic organisms, in particular hyperthermophilic ones, cope with heat-sensitive thermolabile metabolites. Important phosphorylated metabolites, also used in glucose metabolism, are ATP and ADP. The comparison of the hyperthermophilic enzymes for tryptophan synthesis with the mesophilic homologs has suggested possible strategies by which hyperthermophiles cope with the thermolabile intermediates in tryptophan biosynthesis. In bacteria such as Escherichia coli and serovar Typhimurium, tryptophan synthase is an α2 β2 tetrameric enzyme complex that catalyzes the final two steps in the biosynthesis of tryptophan and involves the conversion of indole 3-glycerol phosphate (IGP) and serine to tryptophan. Important from the point of view of adaptation to thermophily is that interdomain signaling and channeling of NH3 in the enzyme of Aquifex aeolicus were found to be strongly temperature dependent. Hyperthermophiles appear to cope with the limitations posed by thermolabile metabolites and coenzymes by a range of mechanisms including rapid turnover or increased catalytic efficiency, local stabilization, substitution or bypassing, microenvironmental compartmentation, or metabolic channeling. Already some years ago, the enzymes of several metabolic pathways were suggested to be organized into structural and functional units. In this view, metabolic channeling of intermediates between physically associated enzymes that are sequential members of a metabolic pathway can be a major thermoprotective mechanism for thermolabile metabolites and therefore can play a critical role in the physiology of thermophiles.

This chapter compares and reviews the molecular adaptations shown by thermophilic and psychrophilic proteins. At both high and low temperatures, the proteins need to be active to maintain the cellular machinery in functional state. The proteins appear to achieve this by modulating their conformational stability/flexibility. The overall fold appears to remain conserved among the homologous thermophilic, mesophilic, and psychrophilic proteins, and only rather minor adjustments are required for adaptation of the protein to high and low temperatures. Thermophilic proteins have specific amino acid composition requirements. In general, thermophilic proteins favor charged residues (Glu, Arg, and Lys) capable of providing increased formation of the ion pairs and their networks. Surface loops in the thermophilic proteins may be undesirable due to the increased mobility at high temperatures. Psychrophilic proteins have high specific activities, yet their thermal stabilities are relatively low. Psychrophilic proteins are often more flexible, particularly, in the regions near the active sites. That proteins could adapt to the diverse living temperatures of their source organisms by modulating the electrostatic effects came to light from studies on citrate synthase.

Many thermophiles and hyperthermophiles (from now on designated (hyper)thermophiles) have been isolated from both fresh water and seawater sources. Compatible solutes must be highly soluble and they usually belong to one of the following groups of compounds: amino acids, sugars, polyols, betaines, and ectoines. In general, compatible solutes accumulate to high levels in the cytoplasm. The relative abundance combined with the low molecular mass of these compounds greatly facilitates the task of their molecular identification by resorting to two powerful analytical techniques: nuclear magnetic resonance (NMR) and mass spectrometry. The mannosyl-3-phosphoglycerate synthase (MPGS) characterized to date produce mannosyl-3-phosphoglycerate with the same anomeric configuration of the substrate and accordingly have been classified as members of glycosyltransferases family GT55, which comprises GDP-mannose: α-mannosyltransferases that retain the anomeric configuration of the substrate. The evolution of MG biosynthesis is a fascinating topic but a meaningful discussion would demand more ample data sets and reliable tools for genome analysis. Diglycerol phosphate (DGP) biosynthesis was investigated by the author and his team on Archaeoglobus fulgidus. In the case of solutes from hyperthermophiles, it was shown that the protecting effect was clearly dependent on the solute charge. The melting temperature of bovine ribonuclease A (RNase A) in the presence and absence of 2-a-O-mannosylglycerate (MG) depends on the ionization state of the solute. Due to the enhanced ability to stabilize biological materials, the application of hypersolutes in industrial applications was soon envisioned, and several industrial patents on their uses have been filed.

This chapter discusses the recent insights into the mechanisms of membrane adaptation of Archaea and Bacteria to high temperatures, with an emphasis on the structure and function of the lipids that constitute the membrane of hyperthermophiles. The cytoplasmic membrane plays an essential role in many metabolic processes, energy transduction, and signaling. Membranes of bacteria mainly contain phospholipids with a core structure consisting of a glycerol, a three-carbon alcohol, to which two fatty acid acyl chains are linked via ester bonds. The archaeal membrane lipids differ in composition from those of bacteria in three important ways. First, the lipid acyl chains are joined to a glycerol backbone by ether rather than ester linkages. High temperatures impose a burden on the cellular metabolism and require a higher stability of enzymes and other macromolecules. Second, the acyl chains are branched rather than linear. Finally, the stereochemistry of the central glycerol is inverted as compared with the ester-based phospholipids. Ether links are far more resistant to oxidation and high temperatures than ester links. Consequently, liposomes prepared from archaeal tetraether lipids are more thermostable. Bacteria respond to changes in ambient temperature through adaptations of the lipid composition of their cytoplasmic membranes. The thermoresistance and tolerance of the membranes of hyperthermophiles is likely a result of an interplay between lipids and proteins.

Cold-adapted organisms are generally classed in two overlapping groups: psychrophiles and psychrotrophs (or psychrotolerants). This chapter reviews the diversity of cold-adapted microorganisms known to exist in each of the major cold environments. Dissimilatory sulfate reduction is one of the most important bacterial reactions in anoxic marine sediments, and it is thought to account for approximately half of the total organic carbon remineralization. The Dry Valleys of Eastern Antarctica are the most extreme example of polar soils and are arguably the coldest and driest deserts on Earth. The most important lithic characteristics are porosity (providing interstitial spaces for microbial colonization) and translucence (facilitating photosynthetic activity). Alternatively, antifreeze proteins produced by psychrophiles could have applications in the food industry for products where low-temperature storage is critical but where ice formation would damage texture or structure. Habitats for cold-adapted microorganisms are widespread on Earth. Both the steady growth of new methods for metagenomic gene recovery and the continued expansion of the industrial enzyme market suggest that psychrophiles, even if relatively unexploited at present, will play an increasingly important role in the future of biotechnology.

The most vibrant and extensive of within-ice microbial ecosystems are those that develop in seasonal ice formed from seawater. Permanently frozen soil, or permafrost, represents an endmember to glacial ice in being primarily lithogenic by definition, with only a limited volume of either frozen or liquid water held within the soil matrix. The fraction of liquid inclusions is relatively large as a result of the non-linear process of ice-formation from seawater, which promotes the retention of pockets of seawater as the ice grows. With shrinking pore space, the encased liquid becomes increasingly salty. Temperature thus determines the salinity of the brine, though not in linear fashion, in part because the subzero precipitation points of individual sea salts differ. Enzymes may have been present in the initial seawater prior to freezing, and thus derived from an unknown myriad of possible organisms, only to partition and eventually concentrate in the brine inclusions during the freezing process and onset of winter. Tracers to measure the synthesis must enter the cell at lower temperatures permissive of solute diffusion, but incorporation into protein appears possible via conformational changes of enzymes that do not require diffusion. The wonders of life in the extreme cold continue to beckon.

The study of subglacial lakes requires a multidisciplinary approach, because basic questions are addressed to life, Earth, atmosphere, and climate sciences. In view of the large geological time span, paleosciences are at the base of the study. The scientific objectives comprise (i) understanding formation and evolution of subglacial processes and environments; (ii) determining the existence, origins, evolution, and maintenance of life; and (iii) understanding limnology and paleoclimate history recorded in lake sediments. Novel responses to the environment are expected to be found in the lake systems, important end members for biodiversity and polar-community dynamics. The only information on living organisms in the Lake Vostok area is coming from the ice core. The findings are reported in several publications in issues of top journals of the past decade. Methane clathrates are known to exist at great marine depths, but their microbiological characterization is scanty; clathrates of other gases are even less known. Evidence from at least four independent laboratories indicates that accretion ice contains bacteria. The danger of irreversibly contaminating extraterrestrial bodies with biological material coming from Earth is an even major concern for space missions, and again, this calls for extreme care.

This chapter talks about membrane adaptations, with emphasis on the adaptive changes occurring in prokaryotes; where relevant, the distinctive changes in eukaryotes will be compared. Genotypic adaptation refers to adaptive changes on an evolutionary time scale (usually longer than that for phenotypic adaptation), which involve an alteration in genetic structure, i.e., mutations occur and are positively selected if favorable to become established as part of the genome. Of particular relevance to the membrane adaptations of psychrophiles are the phenotypic and genotypic adaptations in lipid composition (the cellular “lipiome”), for which there is much information. Like the membranes of higher organisms, those of microorganisms are comprised mainly of proteins and lipids, together with a smaller amount of carbohydrate in the form of glycoproteins, glycolipids, or other molecules, organized as described originally in the Fluid-Mosaic Model of membrane structure. The presence of lipids that have a tendency to form non-bilayer phases gives a certain tension to the membrane and may be important in helping to drive processes such as sporulation and cell division that involve segregation of membranes. Microorganisms modify their membrane lipid fatty acyl composition in response to thermal changes by altering unsaturation, (methyl) branching, or chain length. The trans-unsaturated fatty acids are synthesized by direct and non-reversible isomerization of cisunsaturated fatty acyl chains without a saturated intermediate. The gene for the cis/trans isomerase enzyme has been cloned and the enzyme purified. Anteiso-branched fatty acids seem to be particularly associated with growth at low temperatures.

Cold-adapted enzymes are produced by microorganisms living at permanently low temperature, which constitutes the major environment on planet Earth and includes deep sea, polar, and mountain regions. This chapter deals with those enzymes that are significantly adapted to low temperatures, that is, displaying a high specific activity at low temperatures. Many enzymes produced by cold-adapted microorganisms have now been fully characterized in terms of their physical, chemical, and kinetic properties but still only 11 structures have been solved by X-ray crystallography: α-amylase, citrate synthase, malate dehydrogenase, triosephosphate isomerase, Ca2+-Zn2+ protease, xylanase, adenylate kinase, cellulase, subtilisin-like protease, tyrosine phosphatase, and β-galactosidase. The in vitro growth temperature of these psychrophilic microorganisms is very important for enzyme production, especially for extracellular enzymes, since the production is highly dependent on temperature. In another systematic investigation, the production of various extracellular enzymes such as cellulases, pectate lyases, chitinases, and chitobiases by several strains permanently or seasonally exposed to cold temperatures was followed as a function of growth temperature. The structural modifications believed to be involved in cold-adaptation have been examined in some limited cases using site-directed mutagenesis and directed evolution approaches. The two main properties of cold-adaptation enzymes—a high specific activity at low and moderate temperatures and a low thermostability enabling their rapid inactivation in a complex mixture—render these enzymes particularly suitable for various low to moderate temperature biotechnological processes.

When a bacterial culture growing exponentially at a temperature optimum for its growth is shifted to low temperature, it exhibits cold-shock response. This is irrespective of the preferred optimum growth temperature; thus all types of bacteria such as psychrotrophic, psychrophilic, mesophilic, and thermophilic bacteria possess cellular machinery to elicit this response. Recent global transcript profiling of Escherichia coli cells undergoing cold shock showed that several genes encoding proteins involved in sugar transport and metabolism were induced by cold shock. Cold-shock response of cold-adapted bacteria is similar to that of mesophiles in aspects such as in many cases a lag phase of growth precedes acclimation to low temperature, specific proteins are induced by temperature downshift, membranes undergo adaptive changes, and enzymes are adapted to function at low temperature. One of the main differences in the cold-shock response of these two types of bacteria is the presence of cold acclimation proteins (Caps) in cold-adapted bacteria. The cold-shock response machinery of cyanobacteria is different from that of E. coli. The two main differences are: (i) the absence of CspA homologs and (ii) the presence of desaturases. Desaturases play an important role in cold-shock response of cyanobacteria. With the advent of DNA microarray technology, several groups have carried out global transcript profiling of cold-shock response of different bacteria. Cellular events occurring during cold-shock response are used in applications such as in food and agricultural industry and in research.

This chapter focuses on the various mechanisms by which membrane fluidity is modulated in bacteria vis-à-vis its importance in cold adaptation. A detailed update on the perception and transduction of low-temperature signals in bacteria is also included. Subsequently, it was found that trans-monounsaturated are predominant in gram-negative bacteria and are synthesized by direct isomerization of cis-unsaturated fatty acids to trans-unsaturated fatty acids without shifting of a double bond. One of the predominant signal transduction mechanisms employed by bacteria is the phosphotransfer pathway commonly referred to as the two-component signal transduction system, which consists of a sensor kinase (histidine kinase) and a response regulator, found in bacteria, Archaea, and Eukarya. The first direct evidence for the two-component signal transduction mechanism involved in sensing cold has come from studies on Bacillus subtilis. Modulation in membrane fluidity appears to be crucial for low-temperature sensing in bacteria, and this is normally achieved by the conversion of saturated fatty acids to unsaturated fatty acids. Yet we are far from understanding many key aspects of bacterial signal transduction in response to low temperature.

This chapter summarizes the knowledge about the genomes of psychrophilic bacteria, a subclass of the cold-adapted bacteria, with emphasis on the specific selective features relevant to cold adaptation. A detailed analysis of the general features of genomes and proteomes from psychrophilic bacteria is presented. All investigators involved in sequencing the genomes of psychrophilic Bacteria looked for common features which would account for cold-adaptation. The genomes of psychrophilic bacteria also have the counterpart of major chaperonins such as the essential GroES GroEL complex. A remarkable observation poses interesting questions about the role of this complex. In the presence of molecular oxygen (dioxygen), this has the consequence that reactive oxygen species (ROS) are more frequent and stable for a longer time. Membrane fluidity can be increased in two ways: either by incorporating unsaturated fatty acids or by including branched-chain fatty acids in the diglycerides. Photobacterium profundum SS9 was found to exhibit enhanced proportions of both monounsaturated and polyunsaturated fatty acids when grown at a decreased temperature or elevated pressure. Three main features can be observed in the genomes and proteomes of these organisms: a variety of means to cope with ROS, a multiplicity of nucleic acid folding and unfolding devices, and, finally, a bias in the amino acid composition of their proteome.

This chapter explores the world of high salt environments worldwide and the diversity of microorganisms that inhabit these environments. Highly saline environments can be encountered on all continents. Coastal solar salterns, found worldwide in dry tropical and subtropical climates, are man-made, thalassohaline hypersaline environments in which sea-water is evaporated for the production of salt. 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 one's 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. When soon afterward the organism, a rod-shaped red aerobic bacterium, was brought into culture, the organism appeared to be extremely interesting, and its study has deepened the understanding of phylogenetic as well as physiological and metabolic diversity in the world of halophiles. 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.

Haloarchaea are highly specialized for life under extreme conditions. They can grow in saturated sodium chloride concentrations, and most of them require a minimum of 1.5 to 3M NaCl and 0.005 to 0.04M magnesium salts for growth. The description of different haloarchaeal genomes has created new means of understanding the biology of this group of organisms. The transcriptional response to different osmotic conditions appears to be quite widespread over the Haloferax volcanii genome. The authors have distinguished specific high-salt and low-salt responses, as well as more general stress behaviors such as responses to both low and high salt and to both osmotic stress and heat shock, which may help to understand the osmoadaptation processes and the connection between different networks of adaptation to environmental conditions. Organization of genes in gene clusters, not necessarily cotranscribed nor organized in operons, may allow global regulatory mechanisms such as DNA topology to play an effective role in adaptation to the environment. A general stress behavior, with response to heat shock, has also been observed for certain sequences responding to low-salt conditions, while it has not been observed for specific high-salt responses. Furthermore, the overlap of responses to heat shock and osmotic stress, particularly hypoosmotic stress, seems to be a frequent feature within the haloarchaeal genome. In fact, in haloarchaea, both hypoosmotic stress and heat shock would promote haloarchaeal protein destabilization and aggregation.

This chapter presents the insights into adaptation provided by the study of the four genomes of extreme halophiles sequenced to date. The focus then shifts to molecular adaptation of halophilic proteins, defined as proteins isolated from extreme halophiles. Different aspects concerning solvation, stabilization of the folded and associated assemblies of proteins, and salt effect is presented. Molecular adaptation of the iron translocation function to high salt would be obtained by the replacement of acidic side chains by the more basic His residues. A number of hyperthermophilic Archaea accumulates moderate salt concentration in their cytosol. The three-dimensional structures of their proteins share some of the features emphasized for halophilic proteins. The dynamics of soluble and membrane proteins from extreme halophiles has been studied extensively with the aim of understanding the molecular mechanisms leading to stability, solubility, and activity in highly concentrated salt environments. Studies reported in other sections of this chapter have shown that soluble proteins from extremely halophilic Archaea are active and soluble in a wide range of solvent salt conditions with varying stability. Extreme halophilic organisms require high salt concentrations for growth. They accumulate multimolar salt concentrations in their cytosol to counterbalance the high osmotic pressure of their environment.

Acidophilic microorganisms are distributed throughout the three domains of living organisms. Within the archaeal domain, both extremely acidophilic Euryarchaeota and Crenarchaeota are known, and a number of different bacterial phyla (Firmicutes, Actinobacteria, and Proteobacteria [α, β, and γ subphyla], Nitrospira, and Aquifex) also contain extreme acidophiles. Acidophilic microorganisms exhibit a range of energy-transforming reactions and means of assimilating carbon as neutrophiles. Entire genomes have been sequenced of the iron/sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans and of the archaea Thermoplasma acidophilum, Picrophilus torridus, Sulfolobus tokodaii, and Ferroplasma acidarmanus, and more genome sequences of acidophiles are due to be completed in the near future. Currently, there are four recognized species of this genus that grow autotrophically on sulfur, sulfide, and reduced inorganic sulfur compounds (RISCs). Acidithiobacillus ferrooxidans is the most well studied of all acidophilic microorganisms and has often, though erroneously, been regarded as an obligate aerobe. In both natural and anthropogenic environments, acidophilic microorganisms live in communities that range from relatively simple (two to three dominant members) to highly complex, and within these, acidophiles interact positively or negatively with each other. In a study which examined slime biofilms and snotites that had developed on the exposed surface of a pyrite ore within the abandoned Richmond mine at Iron Mountain, the major microorganisms identified were Leptospirillum spp. (L. ferriphilum and smaller numbers of L. ferrodiazotrophum) and Fp. acidarmanus, Sulfobacillus, and Acidimicrobium/Ferrimicrobium-related species. Using a modified plating technique, the uncharacterized β-proteobacterium can be isolated in pure culture and shown to be a novel iron-oxidizing acidophile.

Acidophiles are microorganisms belonging to eubacteria, archaea, and eukaryotes that need to grow in environments of low pH (<3). Other microorganisms are highly tolerant to extremely acidic conditions but can also grow at neutral pH. Ecological success in any given environment is termed fitness, a measure of the ability of one genotype to reproduce in comparison with another. Hot, acidic, and metal-rich environments are often dominated by archaeal members of the Thermoplasmales family. Volcanic activities contributed to a constant supply of arsenic that was mainly in the form of arsenite under the prevailing reducing conditions. Acidophiles are not intrinsically arsenic or metal resistant but often achieve high levels of resistance through plasmids and/or transposons. Mercury resistance in acidophiles has been studied extensively in Acidithiobacillus ferrooxidans. In various strains of A. ferrooxidans, an additional mercury volatilization system that is dependent on iron oxidation can be found. The mechanisms characterized to date are very similar to those found in organisms that grow at neutral pH, and their genes are often found to be located on either plasmids or transposons that would facilitate their spread by interspecies gene transfer.

Acidophily is a trait of organisms referring to the ability to survive and preferentially multiply at low pH (<3). Extremely acidic environments are found in natural geothermal areas as well as in man-made habitats such as mining wastes. Acidophiles that thrive in these habitats are found among the archaeal, bacterial, and eukaryotic microorganisms. Genome sequence analysis of prokaryotic microorganisms is proceeding at an enormous pace and in the past few years has been applied to several acid-adapted microbes. Since among acidophiles most genomic information is available on representatives of the thermoacidophilic archaeal lineages Sulfolobales and Thermoplasmatales, this chapter focuses mainly on these organisms. A more comprehensive understanding of the mechanisms that underlie protein stability at harsh conditions may be accessible by a comparative analysis of protein structures. A recent study of the structure of the maltose-maltodextrin-binding protein (MBP) from Alicyclobacillus acidocaldarius gives some insights into the molecular basis for protein acidostability. The majority of acidophilic archaea, especially the extreme acidophiles of the Thermoplasmatales, have a scavenging life style—they rely on the decomposition of organic matter for their nutrition and usually require yeast, meat, or bacterial extracts to grow in culture. An important metabolic feature for acidophiles, related to their life style, is the ability to efficiently metabolize weak organic acids. The secondary transporters of thermoacidophiles are believed to utilize protons and not Na+ as a motive force. Thermophilic and acidophilic archaea form two distinct phylogenetic groups, one belonging to the euryarchaeal and the second one to the crenarchaeal lineage.

This chapter focuses on the environmental and taxonomic distributions of gram-positive alkaliphiles. Garbeva et al. developed a polymerase chain reaction (PCR) system for studying the diversity of the species of Bacillus and related taxa using DNA directly obtained from soil. Detection of Bacillus halodurans by this procedure indicated that although the soil samples were slightly acidic, Bacillus halodurans might be one of the major Bacillus species in the soil samples used in that study. Microbial diversities of soda lakes in Africa, Europe, and North America have been detected on the basis of the analysis of DNA clone libraries produced by amplification of obtained DNA as well as from the isolation of microorganisms from the environments. The major gram-negative isolates are members of the gamma subdivision of Proteobacteria. Indigo-reducing bacteria have been isolated by Takahara and Tanabe and identified as Bacillus sp. they have been named Bacillus alcalophilus. This is the only species that can grow at 5°C among the currently known alkaliphilic Bacillus spp. The chapter provides facts that suggest that niches of Bacillus patagoniensis are in soil and in rhizosphere of certain plants. Some of the strains in this group were formally classified as Bacillus. In the next decade, the understanding of the distribution in the environment and of the taxonomic diversities of alkaliphiles will proceed further not only by isolation of novel species of alkaliphiles but also from results of analyses of DNA directly obtained from various environments.

Two themes that run through this chapter are the whole-cell, systems biology aspects of alkaliphile bioenergetics and the diverse ion transporters, pumps, and channels that participate in this system, many of which were first discovered in alkaliphiles and many of which have alkaliphile-specific roles or adaptations. All alkaliphiles examined to date, including both anaerobes and aerobes, do indeed maintain a cytoplasmic pH much lower than the external pH. The growing amount of comparative genomic data between alkaliphiles and neutrophiles has made it much easier to identify putative alkaliphile-specific deviations in conserved and functionally important residues or motifs in proteins of bioenergetic interest. Compelling genomic and biochemical evidence attest to the fact that extreme alkaliphiles experience a low proton motive force (PMF) at high pH. Alkaliphily in bacteria depends upon one or more Na+/H+ antiporters that catalyze proton uptake in exchange for cytoplasmic Na+. The specific properties of the antiporters of alkaliphilic Bacillus that support its functions are not yet clear, but antiporter properties of interest in relation to alkaliphily have emerged for a different alkaliphile. The proton transfer might involve direct protein–protein interactions with a respiratory chain complex, as suggested by for mitochondria, and/or involve the abundant cardiolipin of the alkaliphile membrane.

Piezomicrobiology is one of the lesser studied areas in extremophilic microbiology, although it constitutes a significant field of research, considering that piezophilic microorganisms reside in the largest habitat on Earth—the deep sea. High-pressure microbial habitats include the abyssal and hadal deep-sea environments, which are typified by low temperatures, darkness, sporadic nutrient inputs, and high diversity (low biomass) of invertebrate and vertebrate life. The abyssal plain is commonly thought of as a barren desert, punctuated by the presence of reducing environments such as hydrothermal vents, cold seeps, and whale falls. Culture-independent analyses of microbial diversity in low-temperature deep-ocean habitats have indicated the presence of particular groups of Eukarya, Archaea, and Bacteria. One of the classic responses of mesophilic microbial cells to growth-permissive elevated pressure is the impairment of cell division. The SOS regulon includes genes whose products repair DNA damage as well as prevent cell division. In order to gain further insight into the nature of elevated pressure as a stress, the response of Escherichia coli to pressure has been examined. Many DNA-binding proteins display pressure-sensitive binding properties, and in many instances, this is due to hydration effects. Translation is another pressure-sensitive cellular process involving nucleic acid-protein interactions. Among the ribosome structures present throughout the elongation cycle the most pressure-sensitive one appears to be the posttranslocational complex. The description of genes required for high-pressure growth is now remarkably small but is likely to be greatly expanded as a result of ongoing genetic and genomic studies.

Astrobiology is the search for the origin, evolution, distribution, and future of life in the universe. This chapter examines the requirements for life as we know it. With these constraints as guidelines, the chapter then examines potential habitats for life in our solar system and, finally, elaborates on a strategy to search for life in the universe. Environmental requirements and limitations for active life on Earth have been discussed in the chapter. Most life forms on Earth (including most bacteria, all fungi, and all animals) are heterotrophs that live off energy captured by autotrophic organisms. Salinity affects biological activity, in part, because it controls water availability. The two solar system bodies beyond Earth that have elicited the most interest as potential habitats for life are Mars and Europa because both have clearly been subjected to aqueous processes. The chapter examines these two solar-system bodies in detail. In principle, there are three potential habitats for active life: the surface of planetary bodies, its subsurface, and its atmosphere. Advantages for subsurface life include stable temperatures and vapor pressures and protection from damaging radiation and meteoritic impacts. In the search for life in our solar system, important resources for life (liquid water, readily available energy, and organic compounds), constraints for life, biosignatures, and geosignatures are appropriate criteria. In the search for life beyond our solar system, attention should focus on (i) energy (especially solar) sources, (ii) liquid water, (iii) complex organic chemistry, (iv) the presence of an atmosphere, and (v) O2 (O3) disequilibrium.

This chapter focuses on extremophilic archaea and extremophilic bacteria and their relevance for industrial biotechnology. The majority of extremophiles identified to date belong to the archaeal domain (nearly 300 species), which consists of four kingdoms: Crenarchaeota, Euryarchaeota, Korarchaeota, and Nanoarchaeota. Other hemicellulases (glucoronidase, β-mannanase, β-mannosidase, galactosidase, acetyl xylan esterase, feruloyl esterase and α-arabinofuranosidase), isolated from extremophiles, are efficient enzymes for the complete saccharification of plant cell wall. In the field of industrial biotechnology, esterases too are gaining increasing attention because of their application in organic biosynthesis. The use of high pressure leads to better flavor and color preservation. On the other hand, a number of limitations did not allow so far the broad application of extremophiles. These limitations include difficulties associated with large-scale cultivation of extremophiles, non efficient systems for the overexpression of archaeal genes and unknown factors that confer enzyme stability under extremes of temperature, pH, and pressure. The growing demand for more robust biocatalysts has shifted the trend toward improving the properties of existing proteins for established industrial processes and producing new enzymes tailor-made for entirely new areas of application. The new technologies such as genomics, metanogenomics, gene shuffling, and mutagenesis provide valuable tools for improving or adapting enzyme properties to the desired requirements. Thus, the modern methods of genetic engineering combined with an increasing knowledge of structure and function and process engineering will allow further adaptation of biocatalysts to industrial needs, the exploration of novel applications and protection of the environment.

Modern prokaryotes are the only forms of life featuring organisms capable of growth above 62°C, and inside each domain, the first phylogenetic analyses singled out the most extreme of these thermophiles as the earliest lines of descent: Aquificales and Thermotogales among Bacteria, different Euryarchaeota and Crenarchaeota among Archaea. A recent research study used a new algorithm automatically picking up “representative” proteins, including both ubiquitous and non-ubiquitous but rather well-conserved proteins; here Thermotoga and Aquifex remained together in a basal position with a weak bootstrap support. Modern thermophiles are the result of more than 3 billion years evolution, during which further adaptation has certainly occurred, and molecular adaptations to thermophily look rather elaborated in the only living organisms we can investigate. Temperature is an all-pervasive factor with straightforward effects on the physical state of the universal life solvent, which has to remain in the liquid state to allow suitably adapted organisms to grow. The world of extremophiles offers similar test cases; some of the most obvious ones concern extreme halophily and thermophily, conditions that impose adaptation to the whole proteome.