COPYRIGHTED MATERIAL Extremophiles Have Evolved to Adapt to Severe Geological Conditions

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MOLECULAR EVOLUTION OF EXTREMOPHILES

Debamitra Chakravorty Department of Biotechnology, Indian Institute of Technology, Guwahati, Assam, India

Ashwinee Kumar Shreshtha Kathmandu University, Dhulikhel, Nepal

V. R. Sarath Babu Max Biogen Max Fermentek Pvt. Ltd., Hyderabad, Andhra Pradesh, India

Sanjukta Patra Department of Biotechnology, Indian Institute of Technology, Guwahati, Assam, India

1.1. INTRODUCTIONCOPYRIGHTED MATERIAL Extremophiles have evolved to adapt to severe geological conditions. Their adaptation cannot be justiﬁed merely as stress responses, as they not only survive but thrive in such milieus. The term extreme is used anthropocentrically to designate optimal growth for extremophiles under such conditions. This can be proved through the time-evolved complexity pertaining to their molecular mechanism of adaptation. Their evolution can be mapped on the geological time scale of life, in which a thermophile is argued to be the last common ancestor from which life has arisen. The early existence of methanogens has been proved

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by isotopic records about 2.7 Gya (gigayears ago). Extremophiles span the three domains of life and not only thrive on Earth but also occupy extraterrestrial space. One of the most im- pressive eukaryotic polyextremophiles is the tardigrade, a microscopic invertebrate found in all Earth habitats (Romano, 2003). Extremophiles such as archaea have evolved through the phenomenon of lateral gene transfer by orthologous replacement or incorporation of paralogous genes (Allers and Mevarech, 2005). It has been suggested that the switch from an anaerobic to an aerobic lifestyle by the methanogenic ancestor of haloarchaea was facil- itated by the phenomenon of lateral gene transfer of respiratory chain genes from bacteria. They also possess coregulated genes in operons, leading to coinheritance by lateral gene transfer (Allers and Mevarech, 2005). According to the 16S rRNA classiﬁcation domain, archaea can be divided into four kingdoms: Crenarchaeota, Euryarchaeota, Korarchaeota, and the recently discovered Nanoarchaeota (Grant and Larsen, 1989; Huber et al., 2002). Crenarchaeota includes thermophiles and psychrophiles, Euryarchaeota comprises hyperthermophiles and halophiles, hyperthermophilic archaea represent Korarchaeota, and to date, Nanoarcheota is represented by only a single species, Nanoarchaeum equitans. Recently, sequencing of extremophilic genomes and their global analysis has shed light on their genetic evolution. An appropriate example of such an evolutionary process is that of the radioactive damage-resistant microorganism Deinococcus radiodurans. Deinococcus possesses a robust DNA repair system that performs interchromosomal DNA recombination to cope up with radiation damage of its genome. The natural selection pressure that led to this novel evolution is known as desiccation (Cavicchioli, 2002). These organisms educate us on exactly what extremophilic evolution means. The evolutionary history of extremophiles will aid us in understanding the adaptation of microbes to extreme conditions and environments. Research on extremophiles speeded up only recently after the ﬁrst genome sequence of the methanogenic archaeon Methanococcus jannaschii was published (Bult et al., 273). Success in extremophilic research is limited, due to the lack of proper in vitro conditions and genetic systems for extremophile cloning, library creation, and expression (Allers and Mevarech, 2005). Knowing the evolutionary route of extremophiles can lead to novel discoveries, adding to industrial economy. In-depth understanding of extremophilic evolution will help in engineering extremophiles and hence add to the multibillion-dollar biotechnology industry. In this chapter we discuss the natural routes followed by extremophiles in their evolution and in ways that they can be engineered in vitro.

1.2. MOLECULAR EVOLUTION OF THERMOPHILES

1.2.1. Habitat Thermophilic environments are widespread throughout the Earth, encompassing hot springs, volcanic areas, geothermal vents and mud holes, and solfataric fields (Huber et al., 2000). They are found in several parts of the world, the largest being Yellowstone Na- tional Park in the United States. These ecosystems have high salt concentrations (3%) and slightly acidic-to-alkaline pH values (pH 5 to 8.5) (Bock, 1996). In volcanic regions large amounts of steam are formed which contain such gases as carbon dioxide, hydrogen sulfide, methane, nitrogen, carbon monoxide, and traces of ammonia or nitrate. Coal refuse piles and hot outflows from geothermal power plants constitute artificial high-temperature milieus (Huber et al., 2000). Domestic and industrial hot-water systems are the anthropogenic habitats for thermophiles. JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

MOLECULAR EVOLUTION OF THERMOPHILES 3

1.2.2. Cellular Organization In the cell membranes of thermophiles, the lipid side-chain branching and ether linkages of the phosholipid bilayer contribute to their thermoadaptation (van de Vossenberg et al., 1995; Mathai et al., 2001; Futterer et al., 2004). A few extreme thermophiles such as Pyrobolus and Thermoplasma acidophilum use a modiﬁed lipid that forms a monolayer instead of a bilayer, thus making it immune to the tendency of high temperature to pull bilayers apart. In thermophiles growing above 60◦C, modiﬁcations are also observed in the metabolic pathways. Synthesis of heme, acetyl-CoA, acyl-CoA, and folic acid are either reduced or absent in thermophiles (Kawashima et al., 2000).

1.2.3. Genome Thermophilies have adapted to temperature maxima by the evolution of a wealth of structural and functional features. Recently, genomes of hyperthermophilic archaea, Nanoarchaeum equitans and Thermococcus kodakaraensis, Sulfolobus acidocaldarius, and Carboxydothermus hydrogenoformans were sequenced, providing insight into high- temperature evolution of their genome and metabolic versatility in speciﬁc thermophilic environments (Podar and Reysenbach, 2006). It has been observed that the primary structure of DNA is prone to denaturation at increased temperature by hydrolysis of the N-glycosyl bond and due to deamination of the cytosine bases (Kampmann, 2004). The genomes of such thermophiles as T. kodakaraensis show a high guanineÐcytosine (GC) content (Bao et al., 2002; Saunders et al., 2003). They also show a preference for purine-rich codons, favoring charged amino acids (Paz et al., 2004). DNA shows positive supercoils in thermophiles, hence greater stability. The positive supercoils are catalyzed by the enzyme reverse gyrase, which is present only in hyperthermophiles, in contrast to the negative supercoils in mesophiles (Madigan, 2000). Additionally, monovalent and divalent salts enhance the stability of nucleic acids by screening the negative charges of the phosphate groups, protecting DNA from depurination and hydrolysis (Rothschild and Mancinelli, 2001). In some hyperthermophiles, heat-resistant protein (histone-like protein) binds and stabilizes the DNA by lowering its melting point (Tm) (Madigan, 2000). Recently, work carried out by Zeldovich et al. (2007) concludes that an increase in purine (A + G) of thermophilic bacterial genomes due to the preference for isoleucine, valine, tyrosine, tryptophan, arginine, glutamine, and leucine, which have purine-rich codon patterns, is responsible for the possible primary adaptation mechanism for thermophilicity. These amino acid residues increase the content of hydrophobic and charged amino acids, enhancing thermostability.

1.2.4. Proteome Thermophiles are under constant threat of high temperature on their proteins. Thus, thermophilic intracellular protein and enzymes, compatible solutes, molecular chaperones, and translational modiﬁcations lead synergistically to their dramatic stability at high temperature (England et al., 2003). Hyperthermophilic proteins are more resistant to denaturation due to restriction on the ﬂexibility of these proteins (Scandurra et al., 1998). Factors such as increased van der Waals interactions, higher core hydrophobicity, hydrogen bonds, ionic interactions, coordination with metal ions, and compactness of proteins contribute to the- mostability (Berezovsky et al., 2007). The presence of increased salt bridges in proteins of JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

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thermophiles has been a unanimous observation claimed by many researchers (Scandurra et al., 1998). In thermophiles we also observe proline in ␤-turns, giving rise to rigidity in proteins. Change in amino acids from Lys to Arg, Ser to Ala, Gly to Ala, Ser to Thr, and Val to Ile have been observed among mesophilic to thermophilic organisms (Scandurra et al., 1998). Pertaining to secondary structure, thermostable proteins have high levels of ␣-helical and ␤-sheet content. They also have a slow unfolding rate, which helps to retain their near-native structures. For example, pyrrolidone carboxyl peptidase from Pyrococcus furiosus and ribonuclease HII from the archaeon Thermococcus kodakaraensis show slower unfolding rates than those of their mesostable homologs, which vary from one mesophile to another (Okada et al., 2010). An enhanced hydrophobic effect is one of the reasons for the phenomenon of slow unfolding of thermophilic proteins (Okada et al., 2010). Inte- gral membrane proteins of thermophiles avoid glutamine, lysine, and aspartate amino acid residues, unlike soluble proteins, as a mode of adaptation to increased temperature (Lobry and Chessel, 2003). Larger amounts of Ala, Gly, Ser, Asp, and Glu and smaller amounts of Cys have been reported in the transmembrane proteins of thermophiles (Lobry and Chessel, 2003). A high GC content in the genome leads to the coding of GC-rich codons for amino acids such as Ala, Pro, Trp, Met, Gly, Glu, Arg, and Val. Also synthesized are chaper- onins, which refold denatured protein: for example, the thermosome of hyperthermophiles capable of growth above 100◦C, such as Pyrolobus fumarii and Methanopyrus kandleri (Madigan, 2000).

1.3. MOLECULAR EVOLUTION OF PSYCHROPHILES

1.3.1. Habitat Cold environments constitute the largest biome on Earth. Almost 70% of Earth’s surface is made up of oceans that have a temperature of 4 to 5◦C, and 15% are polar regions. In such milieus the evolutionary pressures are high salt concentrations in oceans, ultraviolet (UV) radiation on the surface of glaciers and ice, and low water and nutrient concentrations in endolithic rocks of antarctic dry deserts (Feller and Charles, 2003).

1.3.2. Cellular Organization Psychrophiles have an evolved lipid bilayer to avoid gel-phase transition and drastic loss of membrane properties (Feller and Charles, 2003). This is achieved through reduction in the packing of acyl chains in the membrane and introduction of steric hindrance, which reduces membrane viscosity (Feller and Charles, 2003). Psychrophiles have a larger proportion of polyunsaturated and branched fatty acids, to increase membrane ﬂuidity. Cis- and trans- unsaturated double bonds are observed in the acyl chain, which induce a bend and a kink, respectively, resulting in lowered compactness in the lipid bilayer. Shorter fatty acyl chains, polar pigments, and membrane-bound carotenoids also increase membrane ﬂexibility. The presence of cryoprotectants enhances their nutrient uptake. Oxygen is more soluble at low temperatures; hence, cells are prone to oxidative damage. In response to this, metabolic reactions that produce reactive oxygen intermediates are eliminated. Elimination of molybdopterin-dependent metabolism in Pseudoalteromonas haplokantis is an example of this (Podar and Reysenbach, 2006). Psychrophiles also possess a large number of gas JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

MOLECULAR EVOLUTION OF PSYCHROPHILES 5

vesicles, which leads to reduction in the cytoplasmic volume, resulting in shorter diffusion times (Staley et al., 1989). Trehalose and exopolysaccharides (EPSs) in psychrophiles play a role in cryoprotection by preventing protein denaturation and aggregation (Phadtare, 2004). EPSs are highly hydrated molecules secreted by psychrophiles in the antarctic marine environment that assist their survival strategy by providing a protective envelope to the cells and the extracellular proteins (Nichols et al., 2005).

1.3.3. Genome Psychrophiles produce nucleic acidÐbinding proteins such as RNA helicase to relieve strong interactions between DNA strands and secondary structures in RNA which impair tran- scription, translation, and replication (Feller and Charles, 2003). For example, an antarctic archeon synthesizes an RNA helicase, which removes cold destabilized RNA secondary structures. To reduce oxidative damage the genome possesses a greater number of catalase and superoxide dismutatase genes (Podar and Reysenbach, 2006). In psychrophilic bacteria, incorporation of dihydrouridine in tRNA is observed, which adds to the conformational ﬂexibility of RNA (Feller and Charles, 2003).

1.3.4. Proteome Psychrophiles are adapted to survive below-freezing temperatures. Like thermophiles, they have evolved parallel strategies for their survival. A handsome strategy employed is that they translate cold-adapted enzymes. The adaptations can be achieved through numerous molecular mechanisms. Structural factors include reduction in electrostatic interaction and increase in hydrogen bonding (D’Amico et al., 2006). In 2003, Feller and Charles reported that nucleic acidÐbinding proteins play key roles in protecting psychrophilic genomes and identified genes that encode such proteins. Like molecular chaperones, cold-shock proteins assist in protein renaturation during the growth of psychrophiles (Berger et al., 1996). They also increase translation efficiency by destabilizing secondary structures in mRNA (Podar and Reysenbach, 2006). The presence of unique antifreeze proteins such as the proteins from Marinomonas primoryensis and Pseudomonas putida GR12-2 lowers the freezing point of cellular water (Feller and Charles, 2003). Moreover, cold-adapted enzymes possessing a flexible catalytic center show high specific activity at low temperatures (Podar and Reysenbach, 2006). Survival of psychrophiles in temperatures at the other end of the thermometer is brought about by cold acclimation proteins (Feller and Charles, 2003). An example is the RNA chaperone CspA. The presence of antifreeze peptides and glycopeptides in psychrophilic eukaryotes lowers the freezing point of cellular water by binding to ice crystals during their formation (Feller and Charles, 2003). An increase in the flexibility of proteins is the key to low-temperature adaptation. This is achieved by decreasing the number of Pro and Arg residues (which leads to the rigidity of proteins by restricting backbone rotations) and increasing the number of Gly residues in their sequences (Feller and Charles, 2003). The protein interiors are less hydrophobic, decreasing their compactness, and weak noncovalent interactions are minimized. The protein surface shows the exposure of nonpolar groups and acidic residues which interact strongly with the essential water layer required to maintain the integrity of the protein structures. JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

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1.4. MOLECULAR EVOLUTION OF HALOPHILES

1.4.1. Habitat Hypersaline environments are widely distributed on Earth. They are of various forms, including natural permanent saline lakes and salt marshes. They have also been created artiﬁcially, due primarily to anthropogenic activities and exist as solar salterns (Setati, 2010). Hypersaline environments can be classiﬁed as thalassohaline and athalassohaline. The former were created by the evaporation of seawater. Sodium and chloride ions dominate and the pH is nearly neutral. Athalassohaline environments have a higher concentration of divalent cations than the monovalent ions and a low pH (Oren, 2002).

1.4.2. Cellular Organization Halophiles need to survive in environments of high salt concentration; hence, they maintain cellular osmotic pressure by controlling the amount of salt inside a cell. First, they possess Na + /H + antiporters to maintain a low sodium ion concentration (Oren, 1999; Zou et al., 2008). The Halobacteriales, including fermentative or homoacetogenic anaerobes, accumulate K + and Na + ions to maintain osmotic balance. Second, enhanced levels of glycerol, amino acids, alcohols, and their derivatives (e.g., glycine, betaine, and ectoine) in their cells maintain their osmolarity (Galinski, 1995; Shivanand and Mugeraya, 2011). The lipid bilayer membranes of halophiles contain phosphatidyl glycerol and phosphatidyl glycerol sulfate (PGS). Glycolipids and PGS are the taxonomic markers of halophilic archaea (Kamekura, 1993; Upasani et al., 1994). An interesting feature of halobacterial cells is that they lack intracellular turgor pressure, leading to the formation of corners in their cells (Schleifer and Stackebrandt, 1982).

1.4.3. Genome The GC content of the halophilic genome is around 60 to 70% (Siddiqui and Thomas, 2008). High GC levels can avoid ultraviolet-induced thymidine dimer formation and mutations. Adaptations to a hypersaline environment are achieved through lateral gene transfer. At the DNA level, compared to nonhalophilic genomes the halophiles exhibit distinct dinucleotides (CG, GA/TC, and AC/GT) at the ﬁrst and second codon positions, reﬂecting an abundance of aspartate, glutamine, threonine, and valine residues in halophile proteins, which leads to their stability (Paul et al., 2008). The presence of high levels of CG dinucleotides leads to an increase in stacking energy and, thus, genome stability.

1.4.4. Proteome The halophilic proteins are magniﬁciently engineered naturally to possess less hydrophobicity, as at high salt concentrations, proteins are destabilized by high hydrophobic interactions, which leads to protein aggregation. One remarkable feature of halophilic proteins is that they are often also thermotolerant and alkaliphilic (Setati, 2010). Acidic amino acid residues dominate their surface because they can hold the essential hydration shell layer for stability and catalysis intact at the surface of the protein. This is reﬂected in the low pI values of halophilic proteins (Siddiqui and Thomas, 2008). Acidic residues also form salt bridges, which add to the rigidity and thus stability of protein structures. A decrease JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

MOLECULAR EVOLUTION OF ALKALIPHILES 7

in large hydrophobic residues and an increase in smaller residues also contribute to the stability of halophilic proteins. Moreover, secreted enzymes are attached by lipid anchors in the halophilic bacterium Salinibacter ruber (Podar and Reysenbach, 2006). In relation to the secondary structure of proteins it was reported that residues that have low propensities for forming ␣-helices are preferred more in halophiles than in nonhalophiles. This leads to an increase in protein ﬂexibility at high salt concentrations (Paul et al., 2008).

1.5. MOLECULAR EVOLUTION OF ALKALIPHILES

1.5.1. Habitat Alkaliphiles live in soils rich in carbonate and in soda lakes and can be classified into alkaliphiles and haloalkaliphiles. Along with their extreme alkaline habitats, alkaliphiles also coexist where neutrophilic microorganisms dwell. They also thrive in deep-sea sediments and hydrothermal areas (Horikoshi, 1998). Naturally occurring alkaline environments are soda deserts and soda lakes. The alkaline lakes and desserts are geographically widely distributed. The Wadi Natrun lakes in Egypt are the best example of alkaline milieus. Alkaline environments have formed through the leaching of metal bicarbonates from rocks. Saline brines are also rich in divalent cations. Saturation of groundwater with respect to CaCO3 results in the deposition of calcite and leads to their alkalinity (Seckbach, 2000). Small natural environments such as the gut of termites and artificial environments such as fer- mented foods also harbor alkaliphiles (Horikoshi, 2010). Artificial alkaline environments are created by permanent alkaline effluents through anthropogenic activities.

1.5.2. Cellular Organization Alkaliphiles maintain nearly neutral pH by constantly pumping protons into their cytoplasm (Horikoshi, 1999). For example, Bacillus subtilis and Vibrio alginolyticus have evolved a mechanism for acidiﬁcation of cytoplasm relative to the external pH (Speelmans et al., 1993). The cell membrane is constructed of acidic polymers such as galacturonic acid, gluconic acid, glutamic acid, aspartic acid, teichuronic acid, and phosphoric acid, which reduce the pH at the cell surface (Aono and Horikoshi, 1983). The acidic polymers permit absorption of sodium and hydronium ions and repel hydroxide ions, permitting the cell to grow at alkaline pH. Passive regulation of cytoplasmic pools of polyamines, low membrane permeability (Bordenstein, 2008), and Na + /K + antiporters (Hamamoto et al., 1994) maintain pH homeostasis in alkaliphiles.

1.5.3. Genome Alkaliphiles have evolved genetically to cope with their environment. Recently, the complete genome of Bacillus subtilis and Bacillus halodurans C-125 has been sequenced. Genes responsible for the alkaliphily of B. halodurans C-125 and Bacillus ﬁrmus OF4 have been analyzed (Takami et al., 1999). In their genome, several open reading frames for Na + /H + antiporters responsible for pH homeostasis in alkaliphiles have been characterized (Horikoshi, 1999). The tupA gene was identiﬁed in the B. halodurans genome, which is responsible for the synthesis of teichuronopeptide, a major structural component in the cell wall important for maintaining pH homeostasis (Takami et al., 1999). Alkylphosphonate JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

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ABC transporter genes coding for two permeases, one phosphonate-binding protein and one ATP-binding protein, are the most frequent class of protein coding gene expressed in alkaliphiles. These transporters couple the hydrolysis of ATP to solute transport (Takami et al., 1999).

1.5.4. Proteome Most proteins from alkaliphiles are extracellular enzymes. Comparative studies along with experimental and theoretical analysis have led to three main conclusions (Siddiqui and Thomas, 2008). First, the pKa modulation of a catalytic residue toward higher pH is responsible for alkaline protein stability. This is achieved through modiﬁcation of the hydrogen bonds and reduction in solvent exposure of the catalytic residue. Second, an increase in the surface exposure of acidic residues with respect to basic residues changes the net charge of the molecule toward negative. GH10 alkaline xylanase BSX from Bacillus sp. NG- 27 and alkaline phosphosereine aminotransferase from Bacillus alkalophilus show such a trend (Siddiqui and Thomas, 2008). Third, the gain of glutamate plus arginine residues and the loss of aspartate plus lysine residues are key players in alkaline adaptation of proteins. Moreover, during the adaptation process it was observed that smaller hydrophobic residues were gained and larger ones lost when enzymes from alkalophiles and nonal- kalophiles were compared (Siddiqui and Thomas, 2008). Modiﬁcation of the proteome by increasing the fraction of acidic amino acids and reducting the protein hydrophobicity for alkaline stability has been observed in the haloalkaliphilic archaeon Natronomonas pharaonis (Horikoshi, 1998).

1.6. MOLECULAR EVOLUTION OF ACIDOPHILES

1.6.1. Habitat Acidic environments are generally formed by natural processes. In such environments, ferrous iron and reduced forms of sulfur are often very abundant; thus, acidic environments are rich in sulfur (Nancucheo and Johnson, 2010). To a certain extent, anthropogenic activities contribute in the creation of acidic and metal-polluted milieus. In such environments, microbial aerobic and anaerobic metabolism generate acidity by the formation of inorganic acids, which results in most of the acidic environments. Nitrification and sulfur oxidation by microorganisms are potent contributors in the generation of such environments (Johnson et al., 2009). Elemental sulfur and sulfide minerals are oxidized to sulfuric acid by acidophilic bacteria and archaea in geothermal areas and in mine environments. Geothermal sulfur-rich sites known as solfatars are home to a variety of acidophiles (Johnson et al., 2009): for example, the solfatara fields in Yellowstone National Park, located near the Norris Geyser Basin, and at Sylvan Springs.

1.6.2. Cellular Organization Acidophiles maintain a circumneutral intracellular pH (Baker-Austin and Dopson, 2007) by membrane impermeability to protons by the presence of tetraether lipids (Apel et al., 1980). Ether linkages characteristic of acidophilic membranes are less prone than ester linkages JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

MOLECULAR EVOLUTION OF ACIDOPHILES 9

+ (i) K+ (ii) H H+ –– –

++ + ADP H+ ATP K+ (iii) – + (vi) H+ + –

H+ (vii) CO2 + H2 (v) H3PO4 + – (iv) HCOO– + H+ H3PO4– +

– + HCOOH H

Figure 1.1. Processes associated with pH homeostasis in acidophiles. (i) Reversal of charge distribution occurs to stop the inward ﬂow of protons through potassium-transporting ATPases. (ii) Impermeability of cell membranes to stop proton inﬂux. (iii) Active proton export by transporters. (iv) Secondary transporters reduce energy demands of importing nutrients into the cell. (v) Cer- tain enzymes bind and sequester protons. (vi) DNA and protein repair systems. (vii) Uncoupling of organic acids. [From Baker-Austin and Dopson (2007), with permission from Elsevier. Copyright C 2007.]

to acid hydrolysis (Golyshina and Timmis, 2005). Membrane channel proteins show a reduction in size (Amaro et al., 1991). An intracellular chemiosmotic gradient is created by the Donnan potential by positively charged molecules (Baker-Austin and Dopson, 2007). The presence of proton efﬂux protein systems is another interesting evolutionary feature (Tyson et al., 2004). Cytoplasmic buffer molecules having basic amino acids are capable of sequestering protons, hence maintaining pH homeostasis. For example, the glutamate and arginine are decarboxylated in Escherichia coli, resulting in cell buffering by proton consumption (Baker-Austin and Dopson, 2007). The overall process of pH homeostasis can be explained stepwise and is illustrated in Figure 1.1.

1.6.3. Genome The genome size of acidophiles is smaller than that of neutrophiles. The smallest genome belongs to Thermoplasmatales (<2 Mb). Acidophiles possess genes for an organic acid degradation pathway (Angelov and Liebl, 2006). In extreme acidophilic genomes, functional characterization of a large number of DNA and protein repair genes (e.g., chaperones) gives a clue about their mechanism of acid homeostasis (Baker-Austin and Dopson, 2007). The genomes of acidophiles also contain a large number of pyramidine codons, which are less susceptible to acid hydrolysis for protection from acid stress (Baker-Austin and Dopson, 2007; Paul et al., 2008). Tyson et al. (2004) also reported a variety of genes JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

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involved in their unique cell membrane biosynthesis, indicative of a complex structure in microorganisms’ acid tolerance capacity. Comparative genome analysis suggests that the acidophilic genome sequences show the presence of a higher proportion of secondary transporters and a larger proportion of DNA and protein repair systems than in neutrolophiles, such as in the genome of the acidophile Picrophilus torridus (Baker-Austin and Dopson, 2007). Purines undergo acid hydrolysis. Thus, the genomes of thermoacidophiles such as P. torridus have evolved by lowering the purine-containing codons in long open reading frames (Baker-Austin and Dopson, 2007).

1.6.4. Proteome Proteins of acidophiles were observed to be rich in acidic residues, which also show low solvent exposure. Stability is also obtained by replacement of charged amino acids by neutral polar amino acids in proteins reduces the electrostatic repulsion that occurs between charged groups at low pH (Norris, 2001). The presence of a high proportion of iron proteins contributes to acidic pH stability, as iron maintains the secondary structure of proteins at acidic pH by functioning as an “iron rivet” (Baker-Austin and Dopson, 2007). Chaperones involved in protein refolding are expressed strongly in acidophiles (Baker-Austin and Dopson, 2007). An increase in the isoleucine content of the proteins in acidophiles such as P.torridus was another trend observed, which was assumed to contribute to acid stability (Baker-Austin and Dopson, 2007). Another report by Settembre et al. (2004) brings forth the fact that an increase in the intersubunit hydrogen-bonding number of arginine-containing salt bridges in Acetobacter aceti PurE enzyme accounts for its increased acid stability.

1.7. MOLECULAR EVOLUTION OF BAROPHILES

1.7.1. Habitat Barophiles are deﬁned as those organisms displaying optimal growth at pressures above 40 MPa, whereas barotolerant bacteria display optimal growth at pressures below 40 MPa and can grow well at atmospheric pressure (Horikoshi, 1998). Aquatic environments with high pressure accompanied by low temperature are home to barophiles. The bottom of the deep sea is a world exposed to extremely high pressure and low temperature (1 to 2◦C). But in the vicinity of hydrothermal vents, the temperature can raise to 400◦C (Horikoshi, 1998), which is another temperature extreme of barophiles. Hydrostatic pressure increases at a rate of 10.5 kPa per meter of depth in the sea and decreases with altitude, and the boiling point of water increases with pressure, keeping it in the liquid state at the bottom of the sea. Thus, increased pressure increases the optimal temperature for microbial growth. Such microorganisms need to adapt to pressure challenges which cause volume change and a decrease in membrane ﬂuidity (Rothschild and Mancinelli, 2001).

1.7.2. Cellular Organization An increase in pressure leads to tighter packing of the molecules in lipid membranes, decreasing membrane ﬂuidity (Rothschild and Mancinelli, 2001). Barophiles circumvent JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

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such a situation, as they have evolved tightly packed lipid membranes with high levels of unsaturated fatty acids (Lauro and Bartlett, 2007) (e.g., docosahexaenoic acid) with low-melting-point lipids (Yano et al., 1998). Fungal strains such as Graphium species isolated from deep-sea calcareous sediment were reported to show microconidiation or the absence of hyphal growth (Raghukumar and Damare, 2008). In cultures of Aspergillus ustus under an elevated pressure of 100 bar, the hyphae showed thick swellings and beaded structures. Moreover, Rhodosporidium sphaerocarpum was reported to show multiple germ tube formation in yeasts (Raghukumar and Damare, 2008).

1.7.3. Genome Survival at high pressure requires robust DNA repair systems (Rothschild and Mancinelli, 2001). Pressure-regulated operons have evolved in barophiles (Kato et al., 1995, 1996). Bartlett et al. (1989) discovered pressure-regulated promoter (ompH) and two open reading frames (ORF1 and ORF2) in Photobacterium sp. SS9. The promoter is activated at high pressure. Another pressure-regulated operon, designated ORF3, was identiﬁed in bacterial strain DSS12, which encodes for cytochrome d dehyrogenase (CydD) protein, which is required for the assembly of cytochrome bd complex and may be important for barophily (Kato et al., 1997; Horikoshi, 1998). Another such promoter was screened from the barophilic bacterium DB6705, which controlled chloremphenicol acetyl transferase gene expression at moderate pressure in Escherichia coli (Kato et al., 1997). It has also been reported that transcriptional efﬁciency of various ribosomal proteins is responsible for high-pressure adaptations in barophiles (Nakasone, 2005). Lauro and Bartlett (2007) reported that in barophiles, elongated helices occur in the 16S rRNA genes and thet their frequency increases with increased pressure. These helix changes are correlated with improved ribosome function under high-pressure conditions. Pressure-regulated ompH and ompL gene expression was studied through transposon and gene replacement mutagenesis experiments in Photobacteriun sp. SS9. ompH was found to be necessary for a greater range of nutrient uptake at high pressure than was ompL (Horikoshi, 1998).

1.7.4. Proteome Research work carried out in 2005 provided important insights into the role of amino acids in rendering proteins stable at high pressure and concluded that polar and small amino acids contribute more to barophilicity. These two amino acid properties are important for the origin of universal genetic code and support the hypothesis that genetic code structuring took place under high hydrostatic pressure (Giulio, 2005). The presence of proteins related to the heat-shock proteins in barophiles such as Thermus barophilus also aid in survival at elevated pressures (Marteinsson et al., 1999). Protease from Methanococcus jannaschii was the ﬁrst enzyme to be characterized from a barophile (Horikoshi, 1998). This enzyme was reported to have narrow substrate speciﬁcity and higher activity at elevated pressure than under atmospheric pressure conditions (Horikoshi, 1998). Certain membrane proteins, such as the ToxR and ToxS proteins in Photobacterium sp. strain SS9, were also studied to assist in pressure adaptation controlling gene expression by the oomph/ompL pressure- regulated operons (Fig. 1.2) (Horikoshi, 1998). The enzymes of extreme barophiles are often folded differently as an evolutionary strategy under high pressure. Mutational studies by Horikoshii and others suggested that tryptophan uptake and trehalose accumulation in JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

12 MOLECULAR EVOLUTION OF EXTREMOPHILES

RR high presure RR S S Periplasm Inner membrane Cytoplasm low presure

–+

ompH ompL

Figure 1.2. Model of Tox R/S function in the regulation of omp gene expression. [From Kato and Bartlett (1997), with permission from Springer Science + Business Media. Copyright C 1997.]

Sacharomyces cerevisiae are the key processes for survival under hydrostatic pressure by preventing the formation of protein aggregates (Abe et al., 2008).

1.8. ENGINEERING EXTREMOPHILES

Ground-breaking research on discovering extremophiles is based on their potential for industrial and biomedical applications (Hough and Danson, 1999; Cavicchioli and Thomas, 2000). However, extremophiles present a number of challenges for the development of bioprocesses because of their slow growth and low yield (Ludlow and Clark, 1991). These extreme conditions for culturing extremophiles are also incompatible with standard industrial fermentation and downstream processing equipment (Gomes and Steiner, 2004). “Microbes are available now but they are not effective for the most part,” says marine micro- biologist Jay Grimes of the University of Southern Mississippi (Biello, 2010). Therefore, a crucial goal in this ﬁeld is determining the features that are critical for their extremophily so that engineering extremophiles will be possible. To engineer extremophiles it is of prime importance to understand their physiology and biochemistry. Knowledge of experimental evolution is one way for naturally engineered extremophiles to modify their metabolic pathways as well as to optimize growth rates. This can be accomplished using genomic informatics, genetic engineering, and directed evolution strategies. The methods are described brieﬂy in the following sections.

1.8.1. Microbiology Manipulation of the milieus of extremophiles through selection pressure to make them robust is one of the oldest approaches. Routine microbial techniques have been employed to enhance extremophilicity. The organisms are grown in minimal or auxotrophic media, subjected to various combinations of selection pressure (e.g., heat, pressure, temperature), or to physical and chemical mutagens such as ultraviolet irradiation and hydroxylamine, respectively. Chemical mutagens such as hydroxylamine cause DNA damage and change in base pairing by tautomeric shift. Brieﬂy, the method of engineering extremophiles involves growing cells in a culture medium to the early exponential phase and subjecting them to centrifugation. The pellets are then resuspended in a fresh culture medium and JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

ENGINEERING EXTREMOPHILES 13

transferred to tubes containing a different concentration of the chemical mutagen. To stop the reaction the cells are again centrifuged and washed with a salty solution (Rodr«õguez- Valera et al., 1980). The washed pellets are then suspended in a liquid medium and grown overnight. The appropriate dilution is selected and the mixture is plated in a solidiﬁed medium. For example, hydroxylamine has been used to mutate halophiles of the family Halomonadaceae (Vargas and Nieto, 2004). Llamas et al. (1999) mutated Halomonas eurihalina using hydroxylamine and obtained nonmucoid mutants of its F2-7 strain which can be used as a genetic tool to discover and study the genetic determinants of bacterial exopolysaccharides. The surviving colonies were then screened for any changes, and experiments were performed regularly to study interesting developments in the population. Microbial techniques such as vertical and horizontal gene transfer through conjugation and protoplast fusion can be employed to engineer extremophiles. An interesting approach in this regard, which has been patented, involves plasmid transfer by mating of Pyrococcus ﬁtriosis with E. coli. The method involves interkingdom gene transfer by cocultivating an isolated recipient extremophile and a member of the family Enterobacteriaceae. Further steps involve identifying an exconjugant that includes at least a portion of the conjuga- tive gene that has been introduced, integrated into its genomic DNA (Michael et al., 2009). Horizontal gene transfer has also been used successfully to transfer vectors from E. coli to halophiles such as Chromohalobacter, Halomonas, and Salinivibrio (Vargas and Nieto, 2004).

1.8.2. Molecular Biology Recombinant extremophiles can be evolved in vitro by incorporation of foreign genes into the native genome. The foreign gene required can be inserted in the host strain following molecular biology protocols. The ﬁrst step involves cloning the desired recombinant DNA in large copy numbers, integrating vector plasmids lacking an origin of replication for archaea or shuttle vector systems possessing the origin of replication indigenous to the host (Allers and Mevarech, 2005). The geneÐvector construct can also be transformed into an extremophile through biolistics, polyethylene glycol, and transposon-mediated integration of plasmid containing the gene(s) of interest, depending on the target extremophile. For example, Cline and others performed polyethylene glycolÐmediated transfection of Halobacterium halobium with naked DNA from phage FH; this method of transfection has been adapted for other archaea, such as Methanococcus maripaludis and Pyrococcus abyssi (Cline and Doolittle, 1992; Allers and Mevarech, 2005). However, one drawback of all the methods described above is that it is only effective in species for which spheroplast can be generated. Similarly, electroporation can be used for Methanococcus voltae but not for Methanosarcina acetivorans. To overcome this problem, autonomously replicating plasmid vectors for such extremophiles as Thermus thermophilus have been constructed by several groups using trpB, ␤-galactosidase, or kanamycin resistance genes as selectable markers (Tamakoshi et al., 1997). Moreover, shuttle integration vector systems have also been developed which can integrate in the extremophilic genome by homologous recombination and can also be recovered from recombinant hosts. One major problem in cloning genes in extremophiles such as Haloferax volcanii is that they have a restriction system that recognizes adenine-methylated GATC sites frequently found in vectors that are based on E. coli plasmids, resulting in DNA fragmentation followed by plasmid loss (Blaseio and Pfeifer, 1990; Allers and Mevarech, 2005). This can be overcome by cloning the DNA ﬁrst JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

14 MOLECULAR EVOLUTION OF EXTREMOPHILES

in an E. coli dam− strain, which is deficient in GATC methylation (Holmes et al., 1991; Allers and Mevarech, 2005). The next step is selection of recombinant clones. In the case of extremophiles, puromycin and novomycin are used routinely as selectable markers. One problem associated with such markers is that their genes are prone to instability, owing to homologous recombination. This problem can be overcome using marker genes in vectors from distantly related species. An indirect and much simpler way than genetic modification of engineering extremophiles is by expressing their targeted genes in recombinant hosts. The first step in this regard would be cloning genomic DNA from extremophiles. Several commercially available kits can also be used for this purpose. The second step is molecular library creation and screening. This is achieved through DNA fragmentation with restriction enzymes and further cloning in vectors such as pBR322 (Mellado et al., 1995). As genomes of a number of extremophiles have already been sequenced and characterized, the genome size of a particular strain can be assessed, and thus a complete genome can be successfully cloned into a library. One well-known vector system is the pET series from Novagen. Construction of genomic libraries with bacteriophages leads to high-quality libraries of extremophilic genomes. The third step involves the expression of desired products such as enzymes in a recombinant host such as E. coli strains or Halomonadaceae (Vargas and Nieto, 2004). An example of such an approach is the expression of ice-nucleation protein from Pseudomonas syringae and amylases from Pyrococcus woesei in halophiles using native or heterologous promoters (Vargas and Nieto, 2004). Another approach to genetically engineered extremophiles is through gene-knockout strategies. Extremophiles are transformed with the construct for gene knockout using circular DNA, which is more stable than linear DNA fragments, and selection of the recombinant host is achieved most efficiently through reusable uracil auxotrophic counter-selectable marker systems (Allers and Mevarech, 2005). Details of this strategy are presented in Fig- ure 1.3. One problem is that Gelrite, used in solid media for culturing hyperthermophiles, contains trace amounts of uracil. Another drawback is the failure of the gene construct to recombine with the host chromosome. Thus, the selection of strains proficient in homologous recombination can overcome such problems (Worthington et al., 2003; Allers and Mevarech, 2005). One example where gene-knockout strategy was employed successfully to generate genetically modified extremophiles was through complete replacement of the leuB gene with the pyrE gene and further deletion of the pyrE gene by using 5-fluoroorotic acid in the host strain, Thermus thermophilus (Tamakoshi et al., 1997). The results of such an interesting effort supports the assumption that this gene-knockout strategy is useful for the reconstruction of a reliable plasmid vector system and that it can be used in the selection of stabilized enzymes (Tamakoshi et al., 1997). Directed evolution is another molecular biology strategy used to evolve extremophilic native enzymes in the laboratory by applying a directional approach for emulating the enzyme. Based on the blueprints of well-characterized extremophilic proteins, unknown proteins lacking those stability properties can be engineered by directed evolution experiments which comprise random mutagenesis steps using physical (UV irradiation) or chemical mutagens or by error-prone polymerase chain reaction (PCR), recombination, and screening processes. Directed evolution was performed on a psychrophilic enzyme, subtilisin S41 (Davail et al., 1994), to increase its thermostability (Miyazaki et al., 2000; Wintrode et al., 2001). Raffaele et al. (2001) characterized a mutated version of the hy- gromycin B phosphotransferase gene from Escherichia coli, isolated by directed evolution JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in 5-FOA R R strain Ð ura ura Mev Mev ura 2005.] C Linear DNA transformation Linear DNA Requires Direct selection Marker cannot be re-used (d) pop-in pop-out Two-step gene deletion or mutation R R strain ura Ð ura ura Mev Mev ura R marker) and the pop-in pop-out method, suitable for Mev ura Marker cannot be re-used Circular DNA transformation Circular DNA Direct selection Requires (c) Pop-in pop-out replacement (c) Pop-in gene 5-FOA 5-FOA strain or ura ura ura Ð ura Wild type Requires Circular DNA transformation Circular DNA Marker cannot be re-used No direct selection gene deletion pop-out (b) Pop-in gene R R Mev Mev Deletion mutant Deletion mutant Deletion mutant Point mutant Simple, any strain can be used Direct selection transformation Linear DNA Marker cannot be re-used (a) Gene replacement (a) Gene Gene-knockout methods used in archaeal genetics. (a) Direct replacement of a gene with a selectable marker, by recombination Figure 1.3. between linear DNA, which comprises flankingand regions selection of for the transformation gene, to and uracilby a prototrophy. a chromosomal (c) target. marker Variant of (b) that the The allows pop-in pop-in direct pop-out pop-out method method selection. uses for (d) circular gene Combination DNA deletion, of in which gene the replacement gene (with is replaced generating point mutations. [From Allers and Mevarech (2005), with permission from Macmillan Publishers Ltd. Copyright Advantages Disadvantages

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16 MOLECULAR EVOLUTION OF EXTREMOPHILES

in transformants of Sulfolobus solfataricus with respect to its genetic stability in both the original mesophilic and the new thermophilic hosts. One drawback of the method is that it is time consuming, requiring several generations of mutagenesis recombination and screening. However, random mutagenesis employed for directed evolution strategy is time consuming, owing to generations of mutagenesis recombination and screening. In directed evolution a specific property is subjected to selection pressure. Thus, a site-directed mutagenesis technique of directed evolution is gaining popularity among researchers worldwide. The procedure involves the synthesis of a short DNA primer containing the desired base-change mutation subjected to PCR with the target DNA of interest in plasmid. After a sufficient number of PCR cycles, the mutated fragment will be amplified sufficiently with respect to the unmutated plasmid by gel electrophoresis.

1.8.3. Bioinformatics Functional genomics and genomic informatics can be used to manipulate an organism’s biology (Daly, 2001). A significant number of genomes from extremophiles have been sequenced and deposited in databases such as the NCBI (National Centre for Biotechnology Information). Complete genome sequencing of Methanococcus jannaschii, Colwellia psy- chrerythraea, Thermoplasma acidophilum, and Sulfolobus acidocaldarius are a few such examples. From this vast catalog of information about the open reading frame of interest, first, knowledge of their in vitro/in vivo manipulation can be gained. Such knowledge can lead to expression of novel gene products in recombinant host strains. Second, multiple alignments of extremophilic protein sequences through servers such as ClustalW, which can be accessed through http://www.ebi.ac.uk/Tools/msa/clustalw2/, can aid in the design of degenerate PCR primers to fish out genes from newly isolated organisms. Degenerate primers are mixtures of similar primers. They are used when homologous genes are to be amplified from uncharacterized genomes from newly isolated organisms or genomes isolated from the environment through metagenomic approaches. Rose et al. (2003) developed specific degenerate primers known as CODEHOPs (Consensus Degenerate Hybrid Oligonucleotide Primers). A CODEHOP is degenerate at the 3 core region and is non- degenerate at the 5 consensus clamp region. The workflow of designing CODEHOPs is represented schematically in Figure 1.4. CODEHOPs have been used to fish out new genes from extremophiles. For example, Han et al. (2010) used CODEHOP PCR to clone selected regions of the phaE and phaC genes’ haloarchae, encoding PHA synthases (type III), which were sequenced further. Moreover, structural analysis of proteins from extremophiles through homology modeling, molecular superimposition, and molecular docking can guide in the molecular design of their properties in homologous nonextremophilic counterparts by site-directed mutagenesis and directed evolution strategies and thus lead to their in vitro evolution. Moreover, extremophilic databases have been created which are freely accessible to the public. One database is CCCryo (Culture Collection of Cryophilic Algae), which can be accessed through http://extrem.igib.res.in/, created by the Institute of Genomics and Integrative Bi- ology in India. The halophile genome site, which is a database of annotated halophilic genomes, can be accessed through http://edwards.sdsu.edu/halophiles/. The annotation was done by the Rapid Annotation Using Subsystems Technology (RAST) server, and the database can be accessed through http://edwards.sdsu.edu/halophiles/. JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

CASE STUDIES 17

Input An aligned array of amino acid sequence with out gap

Computation of an amino acid position specific scoring matrix (PSSM)

Selection of most common coded amino acid codon from the PSSM matrix

DNA PSSM is calculated from the amino acid matrix

Calculation and identification of degenerate region

Identification of consensus from degenerate region

CODEHOP primers

Figure 1.4. Flowchart showing the process of designing CODEHOPs. [Adapted from Rose et al. (1998), by permission of Oxford University Press.]

1.9. CASE STUDIES

1.9.1. Biofuel Production A microbial strain with high ethanol tolerance and yield is a good candidate for biofuel production and an efﬁcient extremophile. Thermoanaerobacterium, a heterofermentative thermophile, was genetically engineered for a single knockout mutant for lactate dehydrogenase in Thermoanaerobacterium, resulting in reduced levels of lactate production and a fourfold increase in ethanol production (Desai et al., 2004). Clostridium thermocellum is a good candidate for bioethanol production; however, the strain is sensitive to high ethanol concentration. Thus, a strain tolerant of high ethanol concentration was genetically engineered (Lynd et al., 2005). In other research, a C. thermocellum lactate dehydrogenase mutant, with higher ethanol production and enhanced product tolerance, was constructed through electrotransformation of a foreign gene for ethanol production (Tailliez et al., 1989). The UK-based company Green Biologics genetically modiﬁed extremophiles to produce butanol, which is commercially sold as Butafuel (Barnard et al., 2010). Xylose utilizing the thermophile Thermoanaerobacterium saccharolyticum was subjected to end-product JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

18 MOLECULAR EVOLUTION OF EXTREMOPHILES

metabolic engineering for ethanol production, as thermophilic microbes isolated to date cannot produce ethanol in consistently high yields. Genes involved in lactate (ldh) and acetate (ack/pta) production were knocked out, resulting in a strain able to produce ethanol as the only detectable organic product (Shaw et al., 2008). A lactate dehydrogenase mutant, Thermoanaerobacter BG1L1, was engineered with resistance to 8.3% ethanol. This strain is also robust in utilizing xylose from lignocellulosic hyrolysates, stover pretreated with dilute acid, and wheat straw hydrolysate (Georgieva et al., 2007).

1.9.2. Bioremediation Bioremediation is the use of biological agents to reduce toxic substances in the environment to a nontoxic or less toxic state. The most radioactivity-resistant organism, Deinococcus radiodurans, has been genetically engineered for use in bioremediation in oil, heavy metal, and mixed radioactive metal contaminations. It is robust because of its unique capability to repair damaged DNA (Clark et al., 2009). Mercuric reductase gene (mer A) has been cloned from E. coli strain BL308 into Deinococcus to detoxify the ionic mercury residue found in radioactive waste generated from nuclear weapons manufacture (Brim et al., 2000). More- over, a recombinant strain of Deinococcus radiodurans which degrades organopollutants under radioactive conditions has been engineered by cloning toluene dioxygenase from Pseudomonas putida F1 into its genome, enabling it to oxidize toluene, chlorobenzene, and indole in a highly irradiating environment. An added advantage of the strain is that it remains tolerant of trichloroethylene at appreciable levels (Lange et al., 1998). The only disadvantage is that it cannot operate in the thermophilic range; thus, Deinococcus geother- malis was transformed genetically with plasmids designed for Deinococcus radiodurans. This made it possible to reduce Hg(II) at elevated temperatures in the presence of 50 Gy/h (Brim et al., 2000).

1.9.3. Pesticide Biodegradation Pesticides persist in the environment for a long time without undergoing biological transformation. Extremophilic microorganisms are a potent source for degrading such xenobiotics, as they are adapted to grow and thrive in such environments. Thermophiles such as Pseu- domonas spp. are good candidates for pesticide bioremediation (Margesin and Schinner, 2001). However, the limitation is that a single microbial species is speciﬁc for a particular xenobiotic compound. Pesticide bioremediation increased dramatically following characterization and cloning of pesticide-degrading genes from such microorganisms. A pioneering work in this ﬁeld was the creation of a superbug from genetically engineered Pseudomonas putida by Ananda Mohan Chakrabarty at General Electric in 1975. The genetically engineered P. putida was potent in degrading camphor, naphthalene, xylene, toluene, octanes, and hexanes (Chakrabarty, 1976). The genes responsible for the degradation of octane, xylene, camphor, and naphthalene were cloned from different plasmids of various microbial strains and transformed into a single cell of P. putida. The plasmids of recombinant P. putida degrading various chemical compounds are TOL (for toluene and xylene), RA500 (for 3,5-xylene), pAC 25 (for 3-cne chlorobenxoate), and pKF439 (for salicylate toluene). Researchers have also transformed Pseudomonas with two genes responsible for Y-HCH or lindane degradation present in halogenated pesticides: linA and JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

CASE STUDIES 19

linB (Nagata et al., 1994). These genes are a member of the haloalkanedehalogenase family with a broad substrate speciﬁcity range. Another pioneering work in this ﬁeld relates to a halophile, Halobacterium sp., subjected to a patented selection process of environmental adaptation to high concentrations of various xenobiotics, such as phenolics and aromatic compounds (Oesterhelt et al., 1998).

1.9.4. Escherichia coli: A Candidate Extremophile E. coli has been the most favored workhorse for genetic engineering with the introduction of genes from extremophiles for stable expression of enzymes for industrial and biomedical applications. For example, E. coli is unable to produce butanol naturally, thus has been engineered with the introduction of genes from extremophiles for the production of higher-chain alcohols. The plasmid WWO of P. putida, which is useful for hydrocarbon degradation, has been propagated in E. coli (Franklin et al., 1981). Another example is the citramalate synthase enzyme from Methanococcus jannaschii, which was overexpressed in E. coli for the production of both 1-propanol and 1-butanol (Atsumi and Liao, 2008). Researchers also attempted to induce radioactive resistance in E. coli by directed evolution (Clark et al., 2009). In 2011, directed evolution strategy was employed to educate the model bacterium E. coli to develop the ability to survive exposure to high temperature or pressure (Vanlinta et al., 2011). Recent research in Japan subjected E. coli to an extreme of gravity. The bacteria were cultured after being rotated in an ultracentrifuge at a high speed: 403,627 × g. Analysis showed that the small size of prokaryotic cells is essential for successful growth under hypergravity. This research implicates the feasibility of panspermia (Deguchi et al., 2011).

1.9.5. Oil-Spill-Cleaning Bacteria Many halophiles have the unique ability to degrade specific hydrocarbons in oil to access energy, but oil spills contain a mixed variety of hydrocarbons. Thus, it is necessary to engineer extremophiles to make them robust for degrading more than one type of hydrocarbon. Alcanivorax borkumensis, which can decompose hydrocarbons aerobically, is a blueprint organism for genetic modification for the same purpose. Evolugate, a Florida company, developed microbes for bioremediation of oil spills. Microbes are grown in continuous cell culture vessels under selection pressure to increase cell proliferation (Dimond, 2010). Such extremophiles can therefore be engineered for degrading high-molecular-weight paraffins, asphaltenes, and sulfur heterocycles (Seckbach, 2000).

1.9.6. Potential Applications and Beneﬁts The potential use of genetically engineered extremophilic microorganisms is an important and exciting prospect. They are used in bioremediation, medicine, and commercial products such as detergents. Deinococcus radiodurans is being engineered for the remediation of radioactive waste (Brim et al., 2000). Engineered extremophiles build up revenues for companies. For example, Genencor, a proﬁtable biotechnology company, has the genetic material of 15,000 strains of microbes stored in deep-freeze in Palo Alto, California, and the Netherlands. It is using alkaline resistance genes from alkaliphiles in East Africa and Kenya JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

20 MOLECULAR EVOLUTION OF EXTREMOPHILES

to create enzymes for laundry detergent. For example, enzymes from alkaliphiles have been used in Tide detergent and to give jeans a faded look. In the field of medicine a eukaryotic homolog of the myc oncogene product from halophilic archaeae has been used to screen the sera of cancer patients. The archaeal homolog produced a higher number of positive reactions than the recombinant protein expressed in Escherichia coli (Satyanarayana et al., 2005). Interestingly, the Defense Advanced Research Projects Agency in the United States is sponsoring experiments on genetically engineering extremophiles to extend the shelf life of blood-clotting platelets in extreme conditions to treat battlefield wounds. The ultimate effort in this field was made by the Craig Venter Institute, which produced a synthetic organism, Mycoplasma laboratorium (Reich, 2000). An article in the Scientific American by David Biello in 2010 stated that “the first microbe to live entirely by genetic code synthesized by humans has started proliferating at a lab in the J. Craig Venter Institute.” Moreover, the government of Kuwait is using engineered extremophiles to clean up gallons of spilled oil.

1.10. IMPLICATIONS OF ENGINEERED EXTREMOPHILES ON ECOLOGY, ENVIRONMENT, AND HEALTH

Whether engineered extremophiles are a boon or a bane to ecology and health is a hot topic of discussion among scientiﬁc communities worldwide. On the negative side, once genetically modiﬁed extremophiles (GMEs) are introduced into the environment it may be impossible to eliminate them. Their persistence in the milieu is almost equivalent to that of wild-type organisms. GMEs can also lead to elimination of a wild-type microbial species due to competition. Horizontal gene transfer to wild-type and pathogenic organisms can cause adverse effects. They can also be opportunistic pathogens in immunosuppressed hosts. On the other hand, they may be targeted for development as biowarfare agents (Daly, 2001). Advantages of GMEs include their use as sources of enzymes and other molecules for diagnostic and pharmaceutical purposes (Irwin and Baird, 2004). A good example is that extremophile extracts such as the iron-binding antifungal compound pyochelin from Pseudomonas spp. were found to have positive activity against pathogenic Candida and Aspergillus spp. (Phoebe et al., 2001). In the food industry, dried Dunaliella (halophlie) has shown potential as a food supplement with antioxidant properties (Irwin and Baird, 2004).

1.11. CONCLUSIONS AND RECOMMENDATIONS

The evolutionary mileage of the various classes of extremophiles are speciﬁc for their sub- types and not accounted for in neutrophiles, indicating the role of evolution in determining precise and highly speciﬁc molecular mechanisms for extreme adaptations. The evolution of extremophiles can be summarized, emphasizing adaptions in cell membranes, metabolic pathways, genome, and proteome. Important differentiation landmarks are observed in the phospholipid bilayer of thermophiles and acidophiles, which is composed of ether linkages, compared to ester linkages in neutrophiles. The compactness of the cell membrane in psychrophiles and barophiles, with its high levels of unsaturated fatty acids, and the presence of phosphatidyl glycerol sulfate in halophiles can be cited as their evolutionary trademarks. JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

REFERENCES 21

Natural metabolic pathway engineering is observed in thermophiles and psychrophiles, wherein the synthesis pathway of heme, acetyl-CoA, acyl-CoA, and folic acid and the pathway for formation of reactive oxygen intermediates are eliminated. Pertaining to the genome of extremophiles, an important evolutionary marker is the high GC content of thermophilic, psychrophilic, and halophilic genomes. The presence of reverse gyrase in thermophiles, nucleic acidÐbinding proteins such as RNA helicases in psychrophiles, purine-rich codons in thermophilic and psychrophilic genomes, pyrimidine-rich tracts in acidophiles, robust DNA repair systems, and the presence of pressure-regulated operons in barophiles are some noteworthy exceptions and discoveries. On the proteome level, thermophiles and psychrophiles differ markedly from neutrophiles by synthesizing chaperones and antifreeze proteins, respectively, which leads to refolding of denatured proteins at such extremes. Bulky hydrophobic and charged residues show higher frequency in thermophilic proteins, whereas smaller ones are preferred in psychrophiles. Acidophiles and alkalophiles show a preference for acidic and small hydrophobic residues in their proteins. Protein compactness and increased hydrogen bonding in thermophiles and psychrophiles are other noteworthy trends. In the secondary structure of extremophilic proteins, high ␣-helix and ␤-sheet content are observed in thermophiles, whereas halophilic proteins favor loops over helices as a mode of extreme adaptation. Despite the foregoing, extensive capital funding and concerted efforts are required to understand the diversity of extremophiles and hence their exploitation. The bottlenecks are that proper genetic systems for high-level expression are still rare for extremophiles, and although the complete genome sequences of thermophiles such as Thermoplasma acidophilum and Sulfolobus acidocaldarius have recently been published, they still lack global annotation. Culturing extremophiles in vitro is challanging, and researchers thinking of switching to archaea should ponder the fact that only a single model organism is available for this entire domain. We hope that our work will be helpful to researchers in engineering potent extremophiles.

REFERENCES

Abe, F., Minegishi, H. 2008. Global screening of genes essential for growth in high-pressure and cold environments: searching for basic adaptive strategies using a yeast deletion library. Genetics 178(2):851Ð872. Allers, T., Mevarech, M. 2005. Archaeal genetics: the third way. Nature Reviews Genetics 6:58Ð73. Amaro, A.M. Chamorro, D., Seeger, M., Arredondo, R., Peirano, I., Jerez, C.A. 1991. Effect of external pH perturbations on in vivo protein synthesis by the acidophilic bacterium Thiobacillus ferrooxidans. Journal of Bacteriology 173:910Ð915. Angelov, A., Liebl, W. 2006. Insights into extreme thermoacidophily based on genome analysis of Picrophilus torridus and other thermoacidophilic archaea. Journal of Biotechnology 126:3Ð10. Aono, R., Horikoshi, K. 1983. Chemical composition of cell walls of alkalophilic Bacillus strains. Journal of General Microbiology 129:1083Ð1087. Apel, W.A., Dugan, P.R., Tuttle, J.H. 1980. 5-Triphosphate formation in Thiobacillus ferrooxidans vesicles by H + ion gradients comparable to those of environmental conditions. Journal of Bacte- riology 142:295Ð301. Atsumi, S., Liao, J.C. 2008. Directed evolution of Methanococcus Jannaschii citramalate synthase for biosynthesis of 1-propanol and 1-butanol by Escherichia coli. Applied and Environmental Microbiology 74(24):7802Ð7808. JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

22 MOLECULAR EVOLUTION OF EXTREMOPHILES

Baker-Austin, C., Dopson, M. 2007. Life in acid: pH homeostasis in acidophiles. Trends in Microbi- ology 15:165Ð170. Bao, Q.Y., Tian, W., Li, Z., Xu, Z., et al. 2002. A complete sequence of the T. tengcongensis genome. Genome Research 12:689Ð700. Barnard, D., Casanueva, A., Tuffin, M., Cowan, D. 2010. Extremophiles in biofuel synthesis. Envi- ronmental Technology 31:871Ð888. Bartlett, D.H., Wright, A., Yayanos, A., Silverman, M. 1989. Isolation of a gone regulated by hydrostatic pressure in a deep-sea bacterium. Nature 342:572Ð574. Berezovsky, I.N., Zeldovich, K.B., Shakhnovich, B.E. 2007. Positive and negative design in stability and thermal adaptation of natural proteins. PLoS Computational Biology 3:1Ð10. Berger, F., Morellet, N., Menu, F., Potier, P. 1996. Cold shock and cold acclimation proteins in the psychrotrophic bacterium Arthrobacter globiformis SI55. Journal of Bacteriology 178:2999Ð 3007. Biello, D. 2010. Slick solution: How microbes will clean up the Deepwater Horizon oil spill: Bacteria and other microbes are the only thing that will ultimately clean up the ongoing oil spill in the Gulf of Mexico. Scientific American, May 25. Blaseio, U., Pfeifer, F. 1990. Transformation of Halobacterium halobium: development of vectors and investigation of gas vesicle synthesis. PNAS USA 87(17):6772Ð6776. Bock, G. 1996. Evolution of Hydrothermal Ecosystems on Earth (and Mars?), Vol. 202. Chichester, UK: Wiley. Bordenstein, S. 2008. Microbialife in alkaline environments. http://serc.carleton.edu/microbelife/ extreme/alkaline/index.html. Brim, H., McFarlan, S.C., Fredrickson, J.K., Minton, K.W., Zhai, M., Wackett, L.P., Daly, M.J. 2000. Engineering Deinococcus radiodurans for metal remediation in radioactive mixed waste environments. Nature Biotechnology 18:85Ð90. Bult, C.J., White, O., et al. 1996. Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science 273:1058Ð1073. Cavicchioli, R. 2002. Extremophiles and the search for extraterrestrial life. Astrobiology 2(3):281Ð 292. Cavicchioli, R., Thomas, T. 2000. Extremophiles. In Encyclopaedia of Microbiology, Edited by Lederberg, J., et al., pp. 317Ð337. San Diego, CA: Academic Press. Chakrabarty, A.M. 1976. Plasmids in Pseudomonas. Annual Review of Genetics 10:7Ð30. Clark, D.P., Dunlap, P.V., Madigan, M.T., Martinko, J.M. 2009. Brock Biology of Microorganisms. Upper Saddle River, NJ: Pearson. Cline, S.W., Doolittle, W.F. 1992. Transformation of members of the genus Haloarcula with shuttle vectors based on Halobacterium halobium and Haloferax volcanii plasmid replicons. Journal of Bacteriology 174:1076Ð1080. Daly, M.J. 2001. The emerging impact of genomics on the development of biological weapons: threats and benefits posed by engineered extremophiles. Clinics in Laboratory Medicine 21(3):619Ð629. D’Amico, S., Collins, T., Marx, J.C., Feller, G., Gerday, C. 2006. Psychrophilic microorganisms: challenges for life. EMBO Reports 7(4):385Ð389. Davail, S., Feller, G., Narinx, E., Gerday, C. 1994. Cold-adaptation of proteins: purification, characterization, and sequence of the heat-labile subtilisin from the antarctic psychrophile Bacillus TA41. Journal of Biological Chemistry 269:17448Ð17453. Deguchi, S., Shimoshige, H., Tsudome, M., Mukai, S.-A., Corkery, R.W., Ito, S., Horikoshi, K. 2011. Microbial growth at hyperaccelerations up to 403,367 × g. PNAS USA 108(19):7997Ð 8002. JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

REFERENCES 23

Desai, S.G., Guerinot, M.L., Lynd, L.R. 2004. Cloning of l-lactate dehydrogenase and elimination of lactic acid production via gene knockout in Thermoanaerobacterium saccharolyticum JW/SL- YS485. Applied Microbiology and Biotechnology 65:600Ð605. Dimond, P.F. 2010. Can microbes help stem the BP oil-spill disaster? Genetic Engineering and Biotechnology News Analysis & Insight, June 17. England, J.L., Shakhnovich, B.E., Shakhnovich, E.I. 2003. Natural selection of more designable folds: a mechanism for thermophilic adaptation. PNAS USA 100:8727Ð8731. Feller, G., Charles, G. 2003. Psychrophilic enzymes: hot topics in cold adaptation. Nature Reviews Microbiology 1(3):200Ð208. Franklin, F.C., Bagdasarian, M., Bagdasarian, M.M., Timmis, K.N. 1981. Molecular and functional analysis of the TOL plasmid pWWO from Pseudomonas putida and cloning of genes for the entire regulated aromatic ring meta cleavage pathway. PNAS USA 78(12):7458Ð7462. Futterer, O., Angelov, A., Liesegang, H., Gottschalk, G., et al. 2004. Genome sequence of Picrophilus torridus and its implications for life around pH 0. PNAS USA 101:9091Ð9096. Galinski, E.A. 1995. Osmoadaptation in bacteria. Advances in Microbial Physiology 37:272Ð328. Georgieva, T.I., Mikkelsen, M.J., Ahring, B.K. 2007. Ethanol production from wet-exploded wheat straw hydrolysate by thermophilic anaerobic bacterium Thermoanaerobacter BG1L1inacontin- uous immobilized reactor. Applied Biochemistry and Biotechnology 145(1Ð3):99Ð110. Giulio, M.D. 2005. Structuring of the genetic code took place at acidic pH. Journal of Theoretical Biology 237:219Ð226. Golyshina, O.V.,Timmis, K.N. 2005. Ferroplasma and relatives,recently discovered cell wallÐlacking archaea making a living in extremely acid, heavy metalÐrich environments. Environmental Mi- crobioogy 7:1277Ð1288. Gomes, J., Steiner, W. 2004. The biocatalytic potential of extremophiles and extremozymes. Food Technology and Biotechnology 42:223Ð235. Grant, W.D.,Larsen, H. 1989. Extremely halophilic archaeobacteria. In Bergey’s Manual of Systematic Bacteriology, edited by Staley, J.T., Bryant, M.P., Pfennig, N., Holt, J.G., Vol. 3, pp. 2216Ð2219. Baltimore: Williams & Wilkins. Hamamoto, T., Hashimoto, M., Hino, M., Kitada, M., Seto, Y., Kudo, T., Horikoshi, K. 1994. Characterization of the gene responsible for Na + /H + antiport system of alkaliphilic Bacillus species strain. Molecular Microbiology 14:939Ð946. Han, J., Hou, J., Liu, H., Shuangfeng, C., Feng, B., Zhou, J., Xiang, H. 2010. Wide distribution among halophilic Archaea of a novel polyhydroxyalkanoate synthase subtype with homology to bacterial type III synthases. Applied Environmental Microbiology 76(23):7811Ð7819. Holmes, M.L., Nuttall, S.D., Dyall-Smith, M.L. 1991. Construction and use of halobacterial shuttle vectors and further studies on Haloferax DNA gyrase. Journal of Bacteriology. 173:3807Ð 3813. Horikoshi, K. 1998. Barophiles: deep-sea microorganisms adapted to an extreme environment. Ecol- ogy and Industrial Microbiology 1(3):291Ð295. Horikoshi, K. 1999. Alkaliphiles: some applications of their products for biotechnology. Microbiology and Molecular Biology Reviews 63:735Ð750. Horikoshi, K. 2010. Extremophiles Handbook. Tokyo: Springer-Verlag. Hough, D.W., Danson, M.J. 1999. Extremozymes. Current Opinion in Chemical Biology 3:39Ð46. Huber, R., Huber, H., Stetter, K.O. 2000. Towards the ecology of hyperthermophiles: biotopes, new isolation strategies and novel metabolic properties. FEMS Microbiology Reviews 24:615Ð623. Huber, H., Hohn, M.J., Rachel, R., Fuchs, T., Wimmer, V.C., Stetter, K.O. 2002. A new phylum of archaea represented by a nanosized hyperthermophilic symbiont. Nature 417:27Ð28. JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

24 MOLECULAR EVOLUTION OF EXTREMOPHILES

Irwin, J.A., Baird, A.W. 2004. Extremophiles and their application to veterinary medicine. Irish Veterinary Journal 57(6):348Ð354. Johnson, D.B., Bacelar-Nicolau, P., Okibe, N., Thomas, A., Hallberg, K.B. 2009. Characteristics of Ferrimicrobium acidiphilum gen. nov., sp. nov., and Ferrithrix thermotolerans gen. nov., sp. nov.: heterotrophic iron-oxidizing, extremely acidophilic actinobacteria. International Journal of Systematic Evolution and Microbiology 59:1082Ð1089. Kampmann, M., Stock, D. 2004. Reverse gyrase has heat-protective DNA chaperone activity inde- pendent of supercoiling. Nucleic Acids Research 6;32(12):3537Ð3545. Kamekura, M. 1993. Lipids of extreme halophiles. In: Vreeland, R. H., Hochstein, L. L. (Eds.), The Biology of Halophilic Bacteria. CRC Press. Inc. Boca Raton: Florida. Kato, C., Bartlett, D.H. 1997. The molecular biology of barophilic bacteria. Extremophiles 1:111Ð116. Kato, C., Sato, T., Horikoshi, K. 1995. Isolation and properties of barophilic and barotolerant bacteria from deep-sea mud samples. Biodiversity and Conservation 4:1Ð9. Kato, C., Masui, N., Horikoshi, K. 1996. Properties of obligately barophilic bacteria isolated from a sample of deep-sea sediment from the IzuÐBonin trench. Journal of Marine Biotechnology 4:96Ð99. Kato, C., Li, L., Nakamura, Y., Nogi, Y., Tamaoka, J., Masui, N., Horikoshi, K. 1997. Biodiversity in the world’s deepest sediment of the Mariana Trench, at a depth of 11,000 m and isolation of hyperbarophilic bacteria from the same sediment. JAMSTEC Journal of Deep Sea Research 13:119Ð130. Kawashima, T., Amano, A., Koike, A., Makino, S., et al. 2000. Archaeal adaptation to higher temperatures revealed by genomic sequence of Thermoplasma canium. PNAS USA 97:14257Ð 14262. Kennedy, S.P., Ng, W.V., Salzberg, S.L., Hood, L., DasSarma, S. 2001. Understanding the adaptation of Halobacterium species NRC-1 to its extreme environment through computational analysis of its genome sequence. Genome Research 11:1641Ð1650. Lange, C.C., Wackett, L.P., Minton, K.W., Daly, M.J. 1998. Engineering a recombinant Deinococcus radiodurans for organopollutant degradation in radioactive mixed waste environments. Nature Biotechnology 16:929Ð933. Lauro, F.M., Bartlett, D.H. 2007. Prokaryotic lifestyles in deep sea habitats. Extremophiles 12:15Ð25. Llamas, I., Bejar,« V., Argandona,˜ M., Quesada, E., del Moral, A. 1999. Chemical mutagenesis of Halomonas eurihalina and selection of exopolysaccharide-deﬁcient variants. Biotechnology Letters 21(5):367Ð370. Lobry, J.R., Chessel, D. 2003. Internal correspondence analysis of codon and amino acid usage in thermophilic bacteria. Journal of Applied Genetics 44:235Ð261. Ludlow, J.M., Clark, D.S. 1991. Engineering considerations for the application of extremophiles in biotechnology. Critical Reviews in Biotechnology 10:321Ð345. Lynd, L.R., van Zyl, W.H., et al. 2005. Consolidated bioprocessing of cellulosic biomass: an update. Current Opinion in Biotechnology 16(5):577Ð583. Madigan, M.T., Martinko, J.M., Parker J. 2000. Brock Biology of Microorganisms, 9th ed. Prentice Hall: USA. Madigan, M., Martinko, J., Parker, J. 2003. Brock biology of microorganisms, 10th ed. Upper Saddle Rive: Pearson Education. Margesin, R., Schinner, F. 2001. Biodegradation and bioremediation of hydrocarbons in extreme environments. Applied Microbiology and Biotechnology 56:650Ð663. Marteinsson, V.T., Birrien, J.L., Reysenbach, A.L., Vernet, M., Marie, D., et al. 1999. Thermococ- cus barophilus sp. nov., a new barophilic and hyperthermophilic archaeon isolated under high JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

REFERENCES 25

hydrostatic pressure from a deep-sea hydrothermal vent. International Journal of Systematic Bacteriology 49:351Ð359. Mathai, J.C., Sprott, G.D., Zeidel, M.L. 2001. Molecular mechanisms of water and solute transport across archaebacterial lipid membranes. Journal of Biological Chemistry 276:27266Ð 27271. Mellado, E., Moore, E.R.B., Nieto, J.J., Ventosa, A. 1995. Phylogenetic inferences and taxonomic consequences of 16S ribosomal DNA sequence comparison of Chromohalobacter marismortui, Volcaniella eurihalina,andDeleya salina and reclassiﬁcation of V. eurihalina as Halomonas eurihalina comb. nov. International Journal of Systematic Bacteriology 45(4):712Ð716. Michael, A., Fei, W.W.Y., Janet, W. 2009. Methods for genetic manipulation of extremophiles. University Of Georgia Research. World Patent WO/2009/055699. Miyazaki, K., Wintrode, P.L., Grayling, R.A., Rubingh, D.N., Arnold, F.H. 2000. Directed evolution study of temperature adaptation in a psychrophilic enzyme. Journal of Molecular Biology 297:1015Ð1026. Nagata, Y. Ohtomo, R., Miyauchi, K., Fukuda, M., Yano, K., Takagi, M. 1994. Cloning and sequencing of a 2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase gene involved in the degradation of ␥-hexachlorocyclohexane in Pseudomonas paucimobilis. Journal of Bacteriol- ogy 176(11)L:3117Ð3125. Nakasone, K. 2005. Gene expression and genome analysis of high pressure-adapted marine bacteria Shewanella violacea. Research Reports of the School of Engineering 39:23Ð26. Nancucheo, I., Johnson, D.B. 2010. Production of glycolic acid by chemolithotrophic iron- and sulfur- oxidizing bacteria and its role in delineating and sustaining acidophilic sulﬁde mineral-oxidizing consortia. Applied and Environmental Microbiology 76(2):461Ð467. Nichols, C.M., Lardiere, S.G., Bowman, J.P., Nichols, P.D., Gibson, J.A.E., Guezennec,« J. 2005. Chemical characterization of exopolysaccharides from antarctic marine bacteria. Microbial Ecol- ogy 49:578Ð589. Norris, P. R. 2001. Acidophiles. In Nature Encyclopedia of Life Sciences. London: Nature Publishing Group. Oesterhelt, D., Patzelt, H., Kesler, B.1998. Decomposition of halogenated hydrocarbons by halophilic bacteria. German Patent DE19639894, Apr. 9. Okada, J., Okamoto, T., Mukaiyama, A., Tadokoro, T., You, D.-J., Chon, H., Koga, Y., Takano, K., Kanaya, S. 2010. Evolution and thermodynamics of the slow unfolding of hyperstable monomeric proteins. BMC Evolutionary Biology 10:207. Oren, A. 1999. Bioenergetic aspects of halophilism. Microbiology and Molecular Biology Reviews 63:334Ð348. Oren, A. 2002. Diversity of halophilic microorganisms: environments, phylogeny, physiology, and applications. Industrial Microbiology and Biotechnology 28:56Ð63. Paul, S., Bag, S.K., Das, S., Harvill, E.T., Dutta, C. 2008. Molecular signature of hypersaline adaptation: insights from genome and proteome composition of halophilic prokaryotes. Genome Biology 9:R70. Paz, A., Mester, D., Baca, I., Nevo, E., et al. 2004. Adaptive role of increased frequency of polypurine tracts in mRNA sequences of thermophilic prokaryotes. PNAS USA 101:2951Ð2956. Phadtare, S. 2004. Recent developments in bacterial cold-shock response. Current Issues in Molocular Biology 6:125Ð136. Phoebe, C.H., Jr., Combie, J., Albert, F.G., Van Tran, K., Cabrera, J., Correira, H.J., Guo, Y., Lindermuth, J., Rauert, N., Galbraith, W., Selitrennikoff, C.P. 2001. Extremophilic organisms as an unexplored source for antifungal compounds. Journal of Antibiotics 54:56Ð65. JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

26 MOLECULAR EVOLUTION OF EXTREMOPHILES

Podar, M., Reysenbach, A.-L. 2006. New opportunities revealed by biotechnological explorations of extremophiles. Current Opinion in Biotechnology 17(3):250Ð255. Raffaele, C., Contursi, P., Rossi, M., Bartolucci, S. 2001. Thermoadaptation of a mesophilic hy- gromycin B phosphotransferase by directed evolution in hyperthermophilic Archaea: selection of a stable genetic marker for DNA transfer into Sulfolobus solfataricus. Extremophiles : Life Under Extreme Conditions 5(3):153Ð159. Raghukumar, C., Damare, S. 2008. Deep sea fungi. In High-Pressure Microbiology,editedby Michiels, C., Bartlett, D.H., Aertsen, A. New York: ASM Press. Reich, K.A. 2000. The search for essential genes. Research in Microbiology 151:319Ð324. Rodr«õguez-Valera, F., Ruiz-Berraquero, F., Ramos-Cormenzana, A. 1980. Isolation of extremely halophilic bacteria able to grow in defined inorganic media with single carbon sources. Journal of General Microbiology 119:535Ð538. Romano, F.A. 2003. On water bears. Florida Entomologist 86(2):134Ð137. Rose, T.M., Schultz, E.R., Henikoff, J.G., Pietrokovski, S., McCallum, C.M., Henikoff, S. 1998. Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly-related sequences. Nucleic Acids Research 26(7):1628Ð1635. Rose, T.M., Henikoff, J.G., Henikoff, S. 2003. CODEHOP (COnsensus-DEgenerate Hybrid Oligonu- cleotide Primer) PCR primer design. Nucleic Acids Research 31(13):3763Ð3766. Rothschild, L.J., Mancinelli, R.L. 2001. Life in extreme environments. Nature 409:1092Ð1101. Satyanarayana, I., Raghukumar, C., Shivaji, S. 2005. Extremophilic microbes: diversity and perspec- tives. Current Science 89(1):78Ð87. Saunders, N.F., Thomas, T., Curmi, P.M., Mattick, J.S., et al. 2003. Mechanisms of thermal adaptation revealed from the genomes of the antarctic archaea Methanogenium frigidum and Methanococ- coides burtonii. Genome Research 13:1580Ð1588. Scandurra, R., Consalvi, V., Chiaraluce, R., Politi, L., Engel, P.C. 1998. Protein thermostability in extremophiles. Biochimie 80:933Ð941. Schleifer, K.-H., Stackebrandt, E. 1982. The Prokaryotes. In A Handbook on the Biology of Bac- teria: Ecophysiology, Isolation, Identification, Applications, 3rd ed. New York: Springer-Verlag (electronic publication). Seckbach, J. 2000. Journey to Diverse Microbial Worlds: Adaptation to Exotic Environments,Dor- drecht, The Netherlands: Kluwer Academic. Setati, M.E. 2010. Diversity and industrial potential of hydrolase-producing halophilic/halotolerant eubacteria. African Journal of Biotechnology 9(11):1555Ð1560. Settembre, E.C., Chittuluru, J.R., Mill, C.P., Kappock, T.J., Ealick, S.E. 2004. Acidophilic adaptations in the structure of Acetobacter aceti N5-carboxyaminoimidazole ribonucleotide mutase (PurE). Acta Crystallographica D 60(Pt. 10):1753Ð1760. Shaw, A.J., Podkaminer, K.K., Desai, S.J., Bardsley, J.S., Rogers, S.R., Thorne, P.G., Hogsett, D.A., Lynd, L.R. 2008. Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield. PNAS 105(37):13769Ð13774. Shivanand, P., Mugeraya, G. 2011. Halophilic bacteria and their compatible solutes: osmoregulation and potential applications. Current Science 100(10):1516Ð1521. Siddiqui, K.S., Thomas, T. 2008. Protein Adaptation in Extremophiles: New York: Nova Biomedical Books. Speelmans, G., Poolman, B., Konnings, W.N. 1993. Amino acid transport in the thermophilic anaerobe Clostridium fervidus is driven by an electrochemical sodium gradient. Journal of Bacteriology 175:2060Ð2066. JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in

REFERENCES 27

Staley, J., Irgens, R., Herwig, R. 1989. Gas vacuolated bacteria from the sea ice of Antarctica. Applied Environmental Microbiology 55:1033Ð1036. Tailliez, P., Girard, H., Millet, J., Beguin, P. 1989. Enhanced cellulose fermentation by an asporoge- nous and ethanol-tolerant mutant of Clostridium thermocellum. Applied and Environmental Mi- crobiology 55(1):207Ð211. Takami, H., Nakasone, K., Hirama C., Takai, Y., Masui, N., Fuji, F., Nakamura Y., Inoue A. 1999. An improved physical and genetic map of the genome of alkaliphilic Bacillus sp. C-125. Extremophiles 3:21Ð28. Tamakoshi, M., Uchida, M., Tanabe, K., Fukuyama, S., Yamagishi, A., Oshima, T. 1997. A new ThermusÐEscherichia coli shuttle integration vector system. Journal of Bacteriology 179:4811Ð 4814. Tyson, G.W., Chapman, J., Hugenholtz, P., Allen, E.E., Ram, R.J., Richardson, P.M., Solovyev, V.V., Rubin, E.M., Rokhsar, D.S., Banﬁeld, J.F. 2004. Community structure and metabolism through reconstruction of microbial genomes from the environment. Nature 428:37Ð43. Upasani, V.N., Desai, S.G., Moldoveanul, N., Kates, M. 1994. Lipids of extremely halophilic archaeobacteria from saline environments in India: a novel glycolipid in Natronobacteriurn strains. Microbiology 140:1959Ð1966. van de Vossenberg, J.L., Ubbink-Kok, T., Elferink, M.G., Driessen, A.J., et al. 1995. Ion permeability of the cytoplasmic membrane limits the maximum growth temperature of Bacteria and Archaea. Molecular Microbiology 18:925Ð932. Vanlinta, D., Mitchell, R., Bailey, E., Meersman, F., McMillan, P.F., Michiels, C.W., Aertsen, A. 2011. Rapid acquisition of gigapascal-high-pressure resistance by Escherichia coli. mBio 2:e00130Ð10. Vargas, C., Nieto, J.J. 2004. Genetic tools for the manipulation of moderately halophilic bacteria of the family halomonadaceae. Methods in Molecular Biology 267:183Ð208. Wintrode, P.L., Miyazaki, K., Arnold, F.H. 2001. Patterns of adaptation in a laboratory evolved thermophilic enzyme. Biochim Biophys Acta 1549(1):1Ð8. Worthington, P., Hoang, V., Perez-Pomares, F., Blum, P. 2003. Targeted disruption of the ␣-amylase gene in the hyperthermophilic archaeon Sulfolobus solfataricus. Journal of Bacteriology 185:482Ð 488. Yano, Y., Nakayama, A., Ishihara, K., Saito, H. 1998. Adaptive changes in membrane lipids of barophilic bacteria in response to changes in growth pressure. Applied Environmental Microbiol- ogy 64(2):479Ð485. Zeldovich, K.B., Berezovsky, I.N., Shakhnovich, E.I. 2007. Protein and DNA sequence determinants of thermophilic adaptation. PLoS Computational Biology 3(1):e5. Zou, Y.J., Yang, L.F., Wang, L., Yang, S.S. 2008. Cloning and characterization of a Na + /H + an- tiporter gene of the moderately halophilic bacterium Halobacillus aidingensis AD-6. Journal of Microbiology 46(4):415Ð421. JWBS091-c01 JWBS091-Singh Printer: Yet to Come September 24, 2012 17:57 Trim: 7in × 10in