molecular evolution of extremophiles

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MOLECULAR EVOLUTIONOF 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. INTRODUCTION

Extremophiles have evolved to adapt to severe geological conditions. Their adaptationcannot be justified merely as stress responses, as they not only survive but thrive in suchmilieus. The term extreme is used anthropocentrically to designate optimal growth for ex-tremophiles under such conditions. This can be proved through the time-evolved complexitypertaining to their molecular mechanism of adaptation. Their evolution can be mapped onthe geological time scale of life, in which a thermophile is argued to be the last commonancestor from which life has arisen. The early existence of methanogens has been proved

Extremophiles: Sustainable Resources and Biotechnological Implications, First Edition. Edited by Om V. Singh.© 2013 Wiley-Blackwell. Published 2013 by John Wiley & Sons, Inc.

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2 MOLECULAR EVOLUTION OF EXTREMOPHILES

by isotopic records about 2.7 Gya (gigayears ago). Extremophiles span the three domains oflife 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 foundin all Earth habitats (Romano, 2003). Extremophiles such as archaea have evolved throughthe phenomenon of lateral gene transfer by orthologous replacement or incorporation ofparalogous genes (Allers and Mevarech, 2005). It has been suggested that the switch froman 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 genetransfer (Allers and Mevarech, 2005).

According to the 16S rRNA classification domain, archaea can be divided intofour kingdoms: Crenarchaeota, Euryarchaeota, Korarchaeota, and the recently discoveredNanoarchaeota (Grant and Larsen, 1989; Huber et al., 2002). Crenarchaeota includes ther-mophiles and psychrophiles, Euryarchaeota comprises hyperthermophiles and halophiles,hyperthermophilic archaea represent Korarchaeota, and to date, Nanoarcheota is repre-sented by only a single species, Nanoarchaeum equitans.

Recently, sequencing of extremophilic genomes and their global analysis has shed lighton their genetic evolution. An appropriate example of such an evolutionary process is that ofthe radioactive damage-resistant microorganism Deinococcus radiodurans. Deinococcuspossesses a robust DNA repair system that performs interchromosomal DNA recombinationto cope up with radiation damage of its genome. The natural selection pressure that led tothis novel evolution is known as desiccation (Cavicchioli, 2002). These organisms educateus on exactly what extremophilic evolution means.

The evolutionary history of extremophiles will aid us in understanding the adaptation ofmicrobes to extreme conditions and environments. Research on extremophiles speeded uponly recently after the first genome sequence of the methanogenic archaeon Methanococcusjannaschii was published (Bult et al., 273). Success in extremophilic research is limited, dueto the lack of proper in vitro conditions and genetic systems for extremophile cloning, librarycreation, and expression (Allers and Mevarech, 2005). Knowing the evolutionary route ofextremophiles can lead to novel discoveries, adding to industrial economy. In-depth under-standing of extremophilic evolution will help in engineering extremophiles and hence add tothe multibillion-dollar biotechnology industry. In this chapter we discuss the natural routesfollowed 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 hotsprings, volcanic areas, geothermal vents and mud holes, and solfataric fields (Huberet 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%) andslightly acidic-to-alkaline pH values (pH 5 to 8.5) (Bock, 1996). In volcanic regions largeamounts 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 pilesand hot outflows from geothermal power plants constitute artificial high-temperature mi-lieus (Huber et al., 2000). Domestic and industrial hot-water systems are the anthropogenichabitats for thermophiles.


MOLECULAR EVOLUTION OF THERMOPHILES 3

1.2.2. Cellular Organization

In the cell membranes of thermophiles, the lipid side-chain branching and ether linkages ofthe 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 Pyrobolusand Thermoplasma acidophilum use a modified lipid that forms a monolayer instead of abilayer, 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 metabolicpathways. Synthesis of heme, acetyl-CoA, acyl-CoA, and folic acid are either reduced orabsent in thermophiles (Kawashima et al., 2000).

1.2.3. Genome

Thermophilies have adapted to temperature maxima by the evolution of a wealth ofstructural 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 thermophilicenvironments (Podar and Reysenbach, 2006). It has been observed that the primary structureof DNA is prone to denaturation at increased temperature by hydrolysis of the N-glycosylbond and due to deamination of the cytosine bases (Kampmann, 2004). The genomes of suchthermophiles as T. kodakaraensis show a high guanine–cytosine (GC) content (Bao et al.,2002; Saunders et al., 2003). They also show a preference for purine-rich codons, favoringcharged amino acids (Paz et al., 2004). DNA shows positive supercoils in thermophiles,hence greater stability. The positive supercoils are catalyzed by the enzyme reverse gy-rase, which is present only in hyperthermophiles, in contrast to the negative supercoils inmesophiles (Madigan, 2000). Additionally, monovalent and divalent salts enhance the sta-bility of nucleic acids by screening the negative charges of the phosphate groups, protectingDNA from depurination and hydrolysis (Rothschild and Mancinelli, 2001). In some hyper-thermophiles, heat-resistant protein (histone-like protein) binds and stabilizes the DNA bylowering its melting point (Tm) (Madigan, 2000). Recently, work carried out by Zeldovichet al. (2007) concludes that an increase in purine (A + G) of thermophilic bacterial genomesdue to the preference for isoleucine, valine, tyrosine, tryptophan, arginine, glutamine, andleucine, which have purine-rich codon patterns, is responsible for the possible primaryadaptation mechanism for thermophilicity. These amino acid residues increase the contentof hydrophobic and charged amino acids, enhancing thermostability.

1.2.4. Proteome

Thermophiles are under constant threat of high temperature on their proteins. Thus, ther-mophilic intracellular protein and enzymes, compatible solutes, molecular chaperones, andtranslational modifications lead synergistically to their dramatic stability at high tempera-ture (England et al., 2003). Hyperthermophilic proteins are more resistant to denaturationdue to restriction on the flexibility of these proteins (Scandurra et al., 1998). Factors suchas increased van der Waals interactions, higher core hydrophobicity, hydrogen bonds, ionicinteractions, 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



thermophiles has been a unanimous observation claimed by many researchers (Scandurraet al., 1998). In thermophiles we also observe proline in �-turns, giving rise to rigidity inproteins. Change in amino acids from Lys to Arg, Ser to Ala, Gly to Ala, Ser to Thr, andVal to Ile have been observed among mesophilic to thermophilic organisms (Scandurraet 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 retaintheir near-native structures. For example, pyrrolidone carboxyl peptidase from Pyrococcusfuriosus and ribonuclease HII from the archaeon Thermococcus kodakaraensis show slowerunfolding rates than those of their mesostable homologs, which vary from one mesophileto another (Okada et al., 2010). An enhanced hydrophobic effect is one of the reasons forthe phenomenon of slow unfolding of thermophilic proteins (Okada et al., 2010). Inte-gral membrane proteins of thermophiles avoid glutamine, lysine, and aspartate amino acidresidues, unlike soluble proteins, as a mode of adaptation to increased temperature (Lobryand Chessel, 2003). Larger amounts of Ala, Gly, Ser, Asp, and Glu and smaller amounts ofCys 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 aminoacids 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 hyperthermophilescapable 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 ismade up of oceans that have a temperature of 4 to 5◦C, and 15% are polar regions. In suchmilieus 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 inendolithic rocks of antarctic dry deserts (Feller and Charles, 2003).


Psychrophiles have an evolved lipid bilayer to avoid gel-phase transition and drastic loss ofmembrane properties (Feller and Charles, 2003). This is achieved through reduction in thepacking of acyl chains in the membrane and introduction of steric hindrance, which reducesmembrane viscosity (Feller and Charles, 2003). Psychrophiles have a larger proportion ofpolyunsaturated 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 akink, respectively, resulting in lowered compactness in the lipid bilayer. Shorter fattyacyl chains, polar pigments, and membrane-bound carotenoids also increase membraneflexibility. The presence of cryoprotectants enhances their nutrient uptake. Oxygen is moresoluble at low temperatures; hence, cells are prone to oxidative damage. In response to this,metabolic reactions that produce reactive oxygen intermediates are eliminated. Eliminationof molybdopterin-dependent metabolism in Pseudoalteromonas haplokantis is an exampleof this (Podar and Reysenbach, 2006). Psychrophiles also possess a large number of gas


MOLECULAR EVOLUTION OF PSYCHROPHILES 5

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

1.3.3. Genome

Psychrophiles produce nucleic acid–binding proteins such as RNA helicase to relieve stronginteractions between DNA strands and secondary structures in RNA which impair tran-scription, translation, and replication (Feller and Charles, 2003). For example, an antarcticarcheon synthesizes an RNA helicase, which removes cold destabilized RNA secondarystructures. To reduce oxidative damage the genome possesses a greater number of catalaseand superoxide dismutatase genes (Podar and Reysenbach, 2006). In psychrophilic bacte-ria, incorporation of dihydrouridine in tRNA is observed, which adds to the conformationalflexibility of RNA (Feller and Charles, 2003).

1.3.4. Proteome

Psychrophiles are adapted to survive below-freezing temperatures. Like thermophiles, theyhave evolved parallel strategies for their survival. A handsome strategy employed is thatthey translate cold-adapted enzymes. The adaptations can be achieved through numerousmolecular mechanisms. Structural factors include reduction in electrostatic interaction andincrease in hydrogen bonding (D’Amico et al., 2006). In 2003, Feller and Charles reportedthat nucleic acid–binding proteins play key roles in protecting psychrophilic genomesand identified genes that encode such proteins. Like molecular chaperones, cold-shockproteins assist in protein renaturation during the growth of psychrophiles (Berger et al.,1996). They also increase translation efficiency by destabilizing secondary structures inmRNA (Podar and Reysenbach, 2006). The presence of unique antifreeze proteins such asthe proteins from Marinomonas primoryensis and Pseudomonas putida GR12-2 lowers thefreezing point of cellular water (Feller and Charles, 2003). Moreover, cold-adapted enzymespossessing a flexible catalytic center show high specific activity at low temperatures (Podarand Reysenbach, 2006). Survival of psychrophiles in temperatures at the other end of thethermometer is brought about by cold acclimation proteins (Feller and Charles, 2003). Anexample is the RNA chaperone CspA. The presence of antifreeze peptides and glycopeptidesin psychrophilic eukaryotes lowers the freezing point of cellular water by binding to icecrystals during their formation (Feller and Charles, 2003). An increase in the flexibility ofproteins is the key to low-temperature adaptation. This is achieved by decreasing the numberof Pro and Arg residues (which leads to the rigidity of proteins by restricting backbonerotations) and increasing the number of Gly residues in their sequences (Feller and Charles,2003). The protein interiors are less hydrophobic, decreasing their compactness, and weaknoncovalent interactions are minimized. The protein surface shows the exposure of nonpolargroups and acidic residues which interact strongly with the essential water layer requiredto maintain the integrity of the protein structures.



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 createdartificially, 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 dominateand the pH is nearly neutral. Athalassohaline environments have a higher concentration ofdivalent cations than the monovalent ions and a low pH (Oren, 2002).


Halophiles need to survive in environments of high salt concentration; hence, they main-tain cellular osmotic pressure by controlling the amount of salt inside a cell. First, theypossess Na+ /H+ antiporters to maintain a low sodium ion concentration (Oren, 1999; Zouet al., 2008). The Halobacteriales, including fermentative or homoacetogenic anaerobes,accumulate K+ and Na+ ions to maintain osmotic balance. Second, enhanced levels ofglycerol, amino acids, alcohols, and their derivatives (e.g., glycine, betaine, and ectoine) intheir cells maintain their osmolarity (Galinski, 1995; Shivanand and Mugeraya, 2011).

The lipid bilayer membranes of halophiles contain phosphatidyl glycerol and phos-phatidyl glycerol sulfate (PGS). Glycolipids and PGS are the taxonomic markers ofhalophilic archaea (Kamekura, 1993; Upasani et al., 1994). An interesting feature ofhalobacterial cells is that they lack intracellular turgor pressure, leading to the formation ofcorners 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 theDNA 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 abundanceof aspartate, glutamine, threonine, and valine residues in halophile proteins, which leads totheir stability (Paul et al., 2008). The presence of high levels of CG dinucleotides leads toan increase in stacking energy and, thus, genome stability.

1.4.4. Proteome

The halophilic proteins are magnificiently engineered naturally to possess less hydropho-bicity, as at high salt concentrations, proteins are destabilized by high hydrophobic inter-actions, which leads to protein aggregation. One remarkable feature of halophilic proteinsis that they are often also thermotolerant and alkaliphilic (Setati, 2010). Acidic amino acidresidues dominate their surface because they can hold the essential hydration shell layerfor stability and catalysis intact at the surface of the protein. This is reflected in the lowpI values of halophilic proteins (Siddiqui and Thomas, 2008). Acidic residues also formsalt bridges, which add to the rigidity and thus stability of protein structures. A decrease


MOLECULAR EVOLUTION OF ALKALIPHILES 7

in large hydrophobic residues and an increase in smaller residues also contribute to thestability of halophilic proteins. Moreover, secreted enzymes are attached by lipid anchorsin the halophilic bacterium Salinibacter ruber (Podar and Reysenbach, 2006). In relation tothe secondary structure of proteins it was reported that residues that have low propensitiesfor forming �-helices are preferred more in halophiles than in nonhalophiles. This leads toan 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 alka-liphiles and haloalkaliphiles. Along with their extreme alkaline habitats, alkaliphiles alsocoexist where neutrophilic microorganisms dwell. They also thrive in deep-sea sedimentsand hydrothermal areas (Horikoshi, 1998). Naturally occurring alkaline environments aresoda deserts and soda lakes. The alkaline lakes and desserts are geographically widely dis-tributed. The Wadi Natrun lakes in Egypt are the best example of alkaline milieus. Alkalineenvironments have formed through the leaching of metal bicarbonates from rocks. Salinebrines 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). Smallnatural environments such as the gut of termites and artificial environments such as fer-mented foods also harbor alkaliphiles (Horikoshi, 2010). Artificial alkaline environmentsare created by permanent alkaline effluents through anthropogenic activities.


Alkaliphiles maintain nearly neutral pH by constantly pumping protons into their cytoplasm(Horikoshi, 1999). For example, Bacillus subtilis and Vibrio alginolyticus have evolved amechanism 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, whichreduce the pH at the cell surface (Aono and Horikoshi, 1983). The acidic polymers permitabsorption of sodium and hydronium ions and repel hydroxide ions, permitting the cellto grow at alkaline pH. Passive regulation of cytoplasmic pools of polyamines, low mem-brane 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 com-plete 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 OF4have been analyzed (Takami et al., 1999). In their genome, several open reading frames forNa+ /H+ antiporters responsible for pH homeostasis in alkaliphiles have been character-ized (Horikoshi, 1999). The tupA gene was identified in the B. halodurans genome, which isresponsible for the synthesis of teichuronopeptide, a major structural component in the cellwall important for maintaining pH homeostasis (Takami et al., 1999). Alkylphosphonate



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

1.5.4. Proteome

Most proteins from alkaliphiles are extracellular enzymes. Comparative studies along withexperimental and theoretical analysis have led to three main conclusions (Siddiqui andThomas, 2008). First, the pKa modulation of a catalytic residue toward higher pH is respon-sible for alkaline protein stability. This is achieved through modification of the hydrogenbonds and reduction in solvent exposure of the catalytic residue. Second, an increase in thesurface exposure of acidic residues with respect to basic residues changes the net chargeof the molecule toward negative. GH10 alkaline xylanase BSX from Bacillus sp. NG-27 and alkaline phosphosereine aminotransferase from Bacillus alkalophilus show such atrend (Siddiqui and Thomas, 2008). Third, the gain of glutamate plus arginine residuesand the loss of aspartate plus lysine residues are key players in alkaline adaptation of pro-teins. Moreover, during the adaptation process it was observed that smaller hydrophobicresidues were gained and larger ones lost when enzymes from alkalophiles and nonal-kalophiles were compared (Siddiqui and Thomas, 2008). Modification of the proteomeby increasing the fraction of acidic amino acids and reducting the protein hydrophobicityfor alkaline stability has been observed in the haloalkaliphilic archaeon Natronomonaspharaonis (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 environmentsare rich in sulfur (Nancucheo and Johnson, 2010). To a certain extent, anthropogenic activ-ities contribute in the creation of acidic and metal-polluted milieus. In such environments,microbial aerobic and anaerobic metabolism generate acidity by the formation of inorganicacids, which results in most of the acidic environments. Nitrification and sulfur oxidationby microorganisms are potent contributors in the generation of such environments (Johnsonet al., 2009). Elemental sulfur and sulfide minerals are oxidized to sulfuric acid by aci-dophilic bacteria and archaea in geothermal areas and in mine environments. Geothermalsulfur-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 theNorris Geyser Basin, and at Sylvan Springs.


Acidophiles maintain a circumneutral intracellular pH (Baker-Austin and Dopson, 2007) bymembrane 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


MOLECULAR EVOLUTION OF ACIDOPHILES 9

(i)– –

–

–

–

–

–

+

+

+

+

+ +

+

K+

K+ATP

CO2 + H2 H3PO4

H3PO4–HCOO– + H+

HCOOH

ADP

(ii)

(iii)

(vii)

(vi)

(iv)

(v)

H+

H+

H+

H+

H+

H+

Figure 1.1. Processes associated with pH homeostasis in acidophiles. (i) Reversal of charge dis-

tribution 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 areduction in size (Amaro et al., 1991). An intracellular chemiosmotic gradient is created bythe 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 capableof sequestering protons, hence maintaining pH homeostasis. For example, the glutamateand arginine are decarboxylated in Escherichia coli, resulting in cell buffering by protonconsumption (Baker-Austin and Dopson, 2007). The overall process of pH homeostasiscan 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 genomebelongs to Thermoplasmatales (<2 Mb). Acidophiles possess genes for an organic aciddegradation pathway (Angelov and Liebl, 2006). In extreme acidophilic genomes, func-tional 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, whichare less susceptible to acid hydrolysis for protection from acid stress (Baker-Austin andDopson, 2007; Paul et al., 2008). Tyson et al. (2004) also reported a variety of genes



involved in their unique cell membrane biosynthesis, indicative of a complex structure inmicroorganisms’ acid tolerance capacity. Comparative genome analysis suggests that theacidophilic genome sequences show the presence of a higher proportion of secondary trans-porters 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 asP. torridus have evolved by lowering the purine-containing codons in long open readingframes (Baker-Austin and Dopson, 2007).

1.6.4. Proteome

Proteins of acidophiles were observed to be rich in acidic residues, which also showlow solvent exposure. Stability is also obtained by replacement of charged amino acidsby neutral polar amino acids in proteins reduces the electrostatic repulsion that occursbetween charged groups at low pH (Norris, 2001). The presence of a high proportion ofiron proteins contributes to acidic pH stability, as iron maintains the secondary structureof 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 inacidophiles such as P. torridus was another trend observed, which was assumed to contributeto 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 ofarginine-containing salt bridges in Acetobacter aceti PurE enzyme accounts for its increasedacid stability.

1.7. MOLECULAR EVOLUTION OF BAROPHILES

1.7.1. Habitat

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


An increase in pressure leads to tighter packing of the molecules in lipid membranes,decreasing membrane fluidity (Rothschild and Mancinelli, 2001). Barophiles circumvent


MOLECULAR EVOLUTION OF BAROPHILES 11

such a situation, as they have evolved tightly packed lipid membranes with high levelsof unsaturated fatty acids (Lauro and Bartlett, 2007) (e.g., docosahexaenoic acid) withlow-melting-point lipids (Yano et al., 1998). Fungal strains such as Graphium speciesisolated from deep-sea calcareous sediment were reported to show microconidiation or theabsence of hyphal growth (Raghukumar and Damare, 2008). In cultures of Aspergillusustus under an elevated pressure of 100 bar, the hyphae showed thick swellings and beadedstructures. Moreover, Rhodosporidium sphaerocarpum was reported to show multiple germtube 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 read-ing frames (ORF1 and ORF2) in Photobacterium sp. SS9. The promoter is activated athigh pressure. Another pressure-regulated operon, designated ORF3, was identified inbacterial strain DSS12, which encodes for cytochrome d dehyrogenase (CydD) protein,which is required for the assembly of cytochrome bd complex and may be important forbarophily (Kato et al., 1997; Horikoshi, 1998). Another such promoter was screened fromthe barophilic bacterium DB6705, which controlled chloremphenicol acetyl transferasegene expression at moderate pressure in Escherichia coli (Kato et al., 1997). It has alsobeen reported that transcriptional efficiency of various ribosomal proteins is responsiblefor 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 theirfrequency increases with increased pressure. These helix changes are correlated with im-proved ribosome function under high-pressure conditions. Pressure-regulated ompH andompL gene expression was studied through transposon and gene replacement mutagenesisexperiments in Photobacteriun sp. SS9. ompH was found to be necessary for a greaterrange 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 acidsin rendering proteins stable at high pressure and concluded that polar and small aminoacids contribute more to barophilicity. These two amino acid properties are important forthe origin of universal genetic code and support the hypothesis that genetic code structuringtook place under high hydrostatic pressure (Giulio, 2005). The presence of proteins relatedto the heat-shock proteins in barophiles such as Thermus barophilus also aid in survivalat elevated pressures (Marteinsson et al., 1999). Protease from Methanococcus jannaschiiwas the first enzyme to be characterized from a barophile (Horikoshi, 1998). This enzymewas reported to have narrow substrate specificity and higher activity at elevated pressurethan under atmospheric pressure conditions (Horikoshi, 1998). Certain membrane proteins,such as the ToxR and ToxS proteins in Photobacterium sp. strain SS9, were also studiedto assist in pressure adaptation controlling gene expression by the oomph/ompL pressure-regulated operons (Fig. 1.2) (Horikoshi, 1998). The enzymes of extreme barophiles areoften folded differently as an evolutionary strategy under high pressure. Mutational studiesby Horikoshii and others suggested that tryptophan uptake and trehalose accumulation in



R R high presure Periplasm

Inner membrane

Cytoplasm

ompH

low presure

– +

SR R

S

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 bypreventing 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 in-dustrial and biomedical applications (Hough and Danson, 1999; Cavicchioli and Thomas,2000). However, extremophiles present a number of challenges for the development ofbioprocesses because of their slow growth and low yield (Ludlow and Clark, 1991). Theseextreme conditions for culturing extremophiles are also incompatible with standard in-dustrial 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, acrucial goal in this field is determining the features that are critical for their extremophilyso that engineering extremophiles will be possible. To engineer extremophiles it is of primeimportance to understand their physiology and biochemistry. Knowledge of experimentalevolution is one way for naturally engineered extremophiles to modify their metabolicpathways as well as to optimize growth rates. This can be accomplished using genomic in-formatics, genetic engineering, and directed evolution strategies. The methods are describedbriefly in the following sections.

1.8.1. Microbiology

Manipulation of the milieus of extremophiles through selection pressure to make themrobust is one of the oldest approaches. Routine microbial techniques have been employedto 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 changein base pairing by tautomeric shift. Briefly, the method of engineering extremophiles in-volves growing cells in a culture medium to the early exponential phase and subjectingthem to centrifugation. The pellets are then resuspended in a fresh culture medium and


ENGINEERING EXTREMOPHILES 13

transferred to tubes containing a different concentration of the chemical mutagen. To stopthe reaction the cells are again centrifuged and washed with a salty solution (Rodrıguez-Valera et al., 1980). The washed pellets are then suspended in a liquid medium and grownovernight. The appropriate dilution is selected and the mixture is plated in a solidifiedmedium. For example, hydroxylamine has been used to mutate halophiles of the familyHalomonadaceae (Vargas and Nieto, 2004). Llamas et al. (1999) mutated Halomonas eu-rihalina using hydroxylamine and obtained nonmucoid mutants of its F2-7 strain whichcan be used as a genetic tool to discover and study the genetic determinants of bacterialexopolysaccharides. The surviving colonies were then screened for any changes, and ex-periments were performed regularly to study interesting developments in the population.Microbial techniques such as vertical and horizontal gene transfer through conjugation andprotoplast fusion can be employed to engineer extremophiles. An interesting approach inthis regard, which has been patented, involves plasmid transfer by mating of Pyrococcusfitriosis with E. coli. The method involves interkingdom gene transfer by cocultivating anisolated recipient extremophile and a member of the family Enterobacteriaceae. Furthersteps 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 fromE. coli to halophiles such as Chromohalobacter, Halomonas, and Salinivibrio (Vargas andNieto, 2004).

1.8.2. Molecular Biology

Recombinant extremophiles can be evolved in vitro by incorporation of foreign genes intothe native genome. The foreign gene required can be inserted in the host strain followingmolecular biology protocols. The first step involves cloning the desired recombinant DNAin large copy numbers, integrating vector plasmids lacking an origin of replication forarchaea or shuttle vector systems possessing the origin of replication indigenous to thehost (Allers and Mevarech, 2005). The gene–vector construct can also be transformed intoan extremophile through biolistics, polyethylene glycol, and transposon-mediated integra-tion of plasmid containing the gene(s) of interest, depending on the target extremophile.For example, Cline and others performed polyethylene glycol–mediated transfection ofHalobacterium halobium with naked DNA from phage FH; this method of transfectionhas been adapted for other archaea, such as Methanococcus maripaludis and Pyrococcusabyssi (Cline and Doolittle, 1992; Allers and Mevarech, 2005). However, one drawback ofall the methods described above is that it is only effective in species for which spheroplastcan be generated. Similarly, electroporation can be used for Methanococcus voltae butnot for Methanosarcina acetivorans. To overcome this problem, autonomously replicatingplasmid vectors for such extremophiles as Thermus thermophilus have been constructedby several groups using trpB, �-galactosidase, or kanamycin resistance genes as selectablemarkers (Tamakoshi et al., 1997). Moreover, shuttle integration vector systems have alsobeen developed which can integrate in the extremophilic genome by homologous recom-bination and can also be recovered from recombinant hosts. One major problem in cloninggenes in extremophiles such as Haloferax volcanii is that they have a restriction systemthat recognizes adenine-methylated GATC sites frequently found in vectors that are basedon E. coli plasmids, resulting in DNA fragmentation followed by plasmid loss (Blaseio andPfeifer, 1990; Allers and Mevarech, 2005). This can be overcome by cloning the DNA first



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 caseof extremophiles, puromycin and novomycin are used routinely as selectable markers. Oneproblem associated with such markers is that their genes are prone to instability, owing tohomologous recombination. This problem can be overcome using marker genes in vectorsfrom distantly related species.

An indirect and much simpler way than genetic modification of engineering ex-tremophiles is by expressing their targeted genes in recombinant hosts. The first stepin this regard would be cloning genomic DNA from extremophiles. Several commerciallyavailable kits can also be used for this purpose. The second step is molecular library creationand screening. This is achieved through DNA fragmentation with restriction enzymes andfurther cloning in vectors such as pBR322 (Mellado et al., 1995). As genomes of a numberof extremophiles have already been sequenced and characterized, the genome size of aparticular strain can be assessed, and thus a complete genome can be successfully clonedinto a library. One well-known vector system is the pET series from Novagen. Constructionof genomic libraries with bacteriophages leads to high-quality libraries of extremophilicgenomes. The third step involves the expression of desired products such as enzymes in arecombinant host such as E. coli strains or Halomonadaceae (Vargas and Nieto, 2004). Anexample of such an approach is the expression of ice-nucleation protein from Pseudomonassyringae and amylases from Pyrococcus woesei in halophiles using native or heterologouspromoters (Vargas and Nieto, 2004).

Another approach to genetically engineered extremophiles is through gene-knockoutstrategies. Extremophiles are transformed with the construct for gene knockout using circu-lar DNA, which is more stable than linear DNA fragments, and selection of the recombinanthost is achieved most efficiently through reusable uracil auxotrophic counter-selectablemarker 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 torecombine with the host chromosome. Thus, the selection of strains proficient in homol-ogous recombination can overcome such problems (Worthington et al., 2003; Allers andMevarech, 2005). One example where gene-knockout strategy was employed successfullyto generate genetically modified extremophiles was through complete replacement of theleuB gene with the pyrE gene and further deletion of the pyrE gene by using 5-fluorooroticacid in the host strain, Thermus thermophilus (Tamakoshi et al., 1997). The results of suchan interesting effort supports the assumption that this gene-knockout strategy is useful forthe reconstruction of a reliable plasmid vector system and that it can be used in the selectionof stabilized enzymes (Tamakoshi et al., 1997).

Directed evolution is another molecular biology strategy used to evolve extremophilicnative enzymes in the laboratory by applying a directional approach for emulating theenzyme. Based on the blueprints of well-characterized extremophilic proteins, unknownproteins lacking those stability properties can be engineered by directed evolution ex-periments which comprise random mutagenesis steps using physical (UV irradiation) orchemical 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


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in transformants of Sulfolobus solfataricus with respect to its genetic stability in both theoriginal mesophilic and the new thermophilic hosts. One drawback of the method is that it istime 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 evolutiona specific property is subjected to selection pressure. Thus, a site-directed mutagenesistechnique of directed evolution is gaining popularity among researchers worldwide. Theprocedure involves the synthesis of a short DNA primer containing the desired base-changemutation subjected to PCR with the target DNA of interest in plasmid. After a sufficientnumber of PCR cycles, the mutated fragment will be amplified sufficiently with respect tothe unmutated plasmid by gel electrophoresis.

1.8.3. Bioinformatics

Functional genomics and genomic informatics can be used to manipulate an organism’sbiology (Daly, 2001). A significant number of genomes from extremophiles have been se-quenced and deposited in databases such as the NCBI (National Centre for BiotechnologyInformation). Complete genome sequencing of Methanococcus jannaschii, Colwellia psy-chrerythraea, Thermoplasma acidophilum, and Sulfolobus acidocaldarius are a few suchexamples. 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 canlead to expression of novel gene products in recombinant host strains. Second, multiplealignments of extremophilic protein sequences through servers such as ClustalW, whichcan be accessed through http://www.ebi.ac.uk/Tools/msa/clustalw2/, can aid in the designof degenerate PCR primers to fish out genes from newly isolated organisms. Degenerateprimers are mixtures of similar primers. They are used when homologous genes are tobe amplified from uncharacterized genomes from newly isolated organisms or genomesisolated from the environment through metagenomic approaches. Rose et al. (2003) devel-oped specific degenerate primers known as CODEHOPs (Consensus Degenerate HybridOligonucleotide 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 isrepresented 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 phaCgenes’ 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 oftheir properties in homologous nonextremophilic counterparts by site-directed mutagen-esis 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. Onedatabase is CCCryo (Culture Collection of Cryophilic Algae), which can be accessedthrough 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 halophilicgenomes, can be accessed through http://edwards.sdsu.edu/halophiles/. The annotationwas done by the Rapid Annotation Using Subsystems Technology (RAST) server, and thedatabase can be accessed through http://edwards.sdsu.edu/halophiles/.


CASE STUDIES 17

InputAn aligned array of amino acid

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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 biofuelproduction and an efficient extremophile. Thermoanaerobacterium, a heterofermentativethermophile, was genetically engineered for a single knockout mutant for lactate dehydro-genase in Thermoanaerobacterium, resulting in reduced levels of lactate production and afourfold increase in ethanol production (Desai et al., 2004). Clostridium thermocellum is agood candidate for bioethanol production; however, the strain is sensitive to high ethanolconcentration. Thus, a strain tolerant of high ethanol concentration was genetically engi-neered (Lynd et al., 2005). In other research, a C. thermocellum lactate dehydrogenasemutant, with higher ethanol production and enhanced product tolerance, was constructedthrough electrotransformation of a foreign gene for ethanol production (Tailliez et al., 1989).The UK-based company Green Biologics genetically modified extremophiles to producebutanol, which is commercially sold as Butafuel (Barnard et al., 2010). Xylose utilizingthe thermophile Thermoanaerobacterium saccharolyticum was subjected to end-product



metabolic engineering for ethanol production, as thermophilic microbes isolated to datecannot produce ethanol in consistently high yields. Genes involved in lactate (ldh) andacetate (ack/pta) production were knocked out, resulting in a strain able to produce ethanolas the only detectable organic product (Shaw et al., 2008). A lactate dehydrogenase mutant,Thermoanaerobacter BG1L1, was engineered with resistance to 8.3% ethanol. This strainis also robust in utilizing xylose from lignocellulosic hyrolysates, stover pretreated withdilute 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 environmentto a nontoxic or less toxic state. The most radioactivity-resistant organism, Deinococcusradiodurans, 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 torepair damaged DNA (Clark et al., 2009). Mercuric reductase gene (mer A) has been clonedfrom E. coli strain BL308 into Deinococcus to detoxify the ionic mercury residue found inradioactive waste generated from nuclear weapons manufacture (Brim et al., 2000). More-over, a recombinant strain of Deinococcus radiodurans which degrades organopollutantsunder radioactive conditions has been engineered by cloning toluene dioxygenase fromPseudomonas 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 itremains tolerant of trichloroethylene at appreciable levels (Lange et al., 1998). The onlydisadvantage 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 transfor-mation. 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 par-ticular xenobiotic compound. Pesticide bioremediation increased dramatically followingcharacterization and cloning of pesticide-degrading genes from such microorganisms. Apioneering work in this field was the creation of a superbug from genetically engineeredPseudomonas putida by Ananda Mohan Chakrabarty at General Electric in 1975. Thegenetically engineered P. putida was potent in degrading camphor, naphthalene, xylene,toluene, octanes, and hexanes (Chakrabarty, 1976). The genes responsible for the degra-dation of octane, xylene, camphor, and naphthalene were cloned from different plasmidsof various microbial strains and transformed into a single cell of P. putida. The plasmidsof recombinant P. putida degrading various chemical compounds are TOL (for tolueneand xylene), RA500 (for 3,5-xylene), pAC 25 (for 3-cne chlorobenxoate), and pKF439(for salicylate toluene). Researchers have also transformed Pseudomonas with two genesresponsible for Y-HCH or lindane degradation present in halogenated pesticides: linA and


CASE STUDIES 19

linB (Nagata et al., 1994). These genes are a member of the haloalkanedehalogenase familywith a broad substrate specificity range. Another pioneering work in this field relates to ahalophile, Halobacterium sp., subjected to a patented selection process of environmentaladaptation to high concentrations of various xenobiotics, such as phenolics and aromaticcompounds (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 introductionof genes from extremophiles for stable expression of enzymes for industrial and biomedicalapplications. For example, E. coli is unable to produce butanol naturally, thus has been engi-neered with the introduction of genes from extremophiles for the production of higher-chainalcohols. 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 citramalatesynthase enzyme from Methanococcus jannaschii, which was overexpressed in E. coli forthe production of both 1-propanol and 1-butanol (Atsumi and Liao, 2008). Researchersalso 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 bacteriumE. coli to develop the ability to survive exposure to high temperature or pressure (Vanlintaet al., 2011). Recent research in Japan subjected E. coli to an extreme of gravity. The bac-teria 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 growthunder 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 accessenergy, but oil spills contain a mixed variety of hydrocarbons. Thus, it is necessary to engi-neer extremophiles to make them robust for degrading more than one type of hydrocarbon.Alcanivorax borkumensis, which can decompose hydrocarbons aerobically, is a blueprintorganism for genetic modification for the same purpose. Evolugate, a Florida company,developed microbes for bioremediation of oil spills. Microbes are grown in continuous cellculture vessels under selection pressure to increase cell proliferation (Dimond, 2010). Suchextremophiles 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 importantand exciting prospect. They are used in bioremediation, medicine, and commercial productssuch as detergents. Deinococcus radiodurans is being engineered for the remediation ofradioactive waste (Brim et al., 2000). Engineered extremophiles build up revenues forcompanies. For example, Genencor, a profitable biotechnology company, has the geneticmaterial of 15,000 strains of microbes stored in deep-freeze in Palo Alto, California, and theNetherlands. It is using alkaline resistance genes from alkaliphiles in East Africa and Kenya



to create enzymes for laundry detergent. For example, enzymes from alkaliphiles have beenused in Tide detergent and to give jeans a faded look. In the field of medicine a eukaryotichomolog of the myc oncogene product from halophilic archaeae has been used to screenthe sera of cancer patients. The archaeal homolog produced a higher number of positivereactions than the recombinant protein expressed in Escherichia coli (Satyanarayana et al.,2005). Interestingly, the Defense Advanced Research Projects Agency in the United Statesis sponsoring experiments on genetically engineering extremophiles to extend the shelf lifeof blood-clotting platelets in extreme conditions to treat battlefield wounds. The ultimateeffort in this field was made by the Craig Venter Institute, which produced a syntheticorganism, Mycoplasma laboratorium (Reich, 2000). An article in the Scientific Americanby David Biello in 2010 stated that “the first microbe to live entirely by genetic codesynthesized 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 gallonsof spilled oil.

1.10. IMPLICATIONS OF ENGINEERED EXTREMOPHILESON ECOLOGY, ENVIRONMENT, AND HEALTH

Whether engineered extremophiles are a boon or a bane to ecology and health is a hottopic of discussion among scientific communities worldwide. On the negative side, oncegenetically modified extremophiles (GMEs) are introduced into the environment it maybe impossible to eliminate them. Their persistence in the milieu is almost equivalent tothat of wild-type organisms. GMEs can also lead to elimination of a wild-type microbialspecies due to competition. Horizontal gene transfer to wild-type and pathogenic organismscan cause adverse effects. They can also be opportunistic pathogens in immunosuppressedhosts. 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 moleculesfor diagnostic and pharmaceutical purposes (Irwin and Baird, 2004). A good example isthat extremophile extracts such as the iron-binding antifungal compound pyochelin fromPseudomonas spp. were found to have positive activity against pathogenic Candida andAspergillus spp. (Phoebe et al., 2001). In the food industry, dried Dunaliella (halophlie) hasshown 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 determiningprecise and highly specific molecular mechanisms for extreme adaptations. The evolutionof extremophiles can be summarized, emphasizing adaptions in cell membranes, metabolicpathways, genome, and proteome. Important differentiation landmarks are observed in thephospholipid bilayer of thermophiles and acidophiles, which is composed of ether linkages,compared to ester linkages in neutrophiles. The compactness of the cell membrane in psy-chrophiles and barophiles, with its high levels of unsaturated fatty acids, and the presenceof phosphatidyl glycerol sulfate in halophiles can be cited as their evolutionary trademarks.


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 path-way for formation of reactive oxygen intermediates are eliminated. Pertaining to the genomeof extremophiles, an important evolutionary marker is the high GC content of thermophilic,psychrophilic, and halophilic genomes. The presence of reverse gyrase in thermophiles,nucleic acid–binding proteins such as RNA helicases in psychrophiles, purine-rich codonsin thermophilic and psychrophilic genomes, pyrimidine-rich tracts in acidophiles, robustDNA repair systems, and the presence of pressure-regulated operons in barophiles aresome noteworthy exceptions and discoveries. On the proteome level, thermophiles and psy-chrophiles differ markedly from neutrophiles by synthesizing chaperones and antifreezeproteins, 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 apreference for acidic and small hydrophobic residues in their proteins.

Protein compactness and increased hydrogen bonding in thermophiles and psy-chrophiles are other noteworthy trends. In the secondary structure of extremophilic pro-teins, high �-helix and �-sheet content are observed in thermophiles, whereas halophilicproteins 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 ofextremophiles and hence their exploitation. The bottlenecks are that proper genetic sys-tems for high-level expression are still rare for extremophiles, and although the completegenome sequences of thermophiles such as Thermoplasma acidophilum and Sulfolobusacidocaldarius have recently been published, they still lack global annotation. Culturingextremophiles in vitro is challanging, and researchers thinking of switching to archaeashould 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.

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