Antecedentes internacionales

In order to withstand adverse conditions, microbes alter their physiology to adapt accordingly, and consequently thrive in extreme habitats.

In hotspots, such as geothermal reservoirs, physiological processes are generally less efficient than in mesophilic conditions. At high temperatures, microbes face an irreversible breakdown of their biomolecules and a disruptive high fluidity of their plasma membrane [62]. Consequently, thermophilic and hyperthermophilic organisms evolved thermostable proteins and enzymes and their cell membranes have a different

composition. The stability of their proteins is guaranteed through extra chemical bonds (S‐S bridges, H‐bonds, metal bindings) [63], by hydrophobic amino acids, by producing multiple small subunits for the formation of an enzyme, and by the presence of chaperone proteins. Stability of proteins by extra chemical bonds results in rigid enzymes that seem to be less productive catalysts than mesophilic enzymes. Thermophiles seem to use thermal energy to overcome this reduction. A second adaptation is the accumulation of chemicals, such as amines and polyamines, which increase the stability of NADH, ATP and amino acids [50]. A final adaptation concerns the integrity of the plasma membrane, as at high temperatures, the fluidity of the plasma membrane increases. The stability of the membrane is guaranteed through an increase in the length of carbon chains and an extension of the branching of the phospholipids, an accumulation of saturated phospholipids, and through changes in the heads of the phospholipids [50, 64]. At low temperatures, microorganisms face the low availability of liquid water, and the damages to cellular integrity caused by the formation of ice crystals. The cellular response to cold is a change of the membrane composition; as expected, the opposite modifications of those taking place in thermophiles are made: unsaturated and shorter fatty acids are incorporated into the cellular membrane, while branching between lipids is also limited [65]. These fatty acids make the membrane more fluid because they introduce gaps that push apart the components of the membrane. At the interior of the cell, they contain antifreeze agents (mainly sugars) and small acidic proteins, which prevent ice formation [62]. Moreover, their proteins tend to have more α‐helices than β‐ folded sheets, to allow flexibility [63]. Finally, psychrophiles are found to have a unique cold‐stable translational system [50].

In highly acidic or highly alkaline habitats, microbes enable processes for gaining energy and carrying out chemical reactions inside the cell in order to regulate the intracellular pH to neutral. This is mainly accomplished through pumping protons out or into the cell,

respectively. In an acidic environment, a K+/H+ antiporter is pumping K+ in and H+ out to

make the cytoplasm alkaline. In a basic environment, a Na+_/H+ _{antiporter is pumping H}+ _in

and Na+ out to produce the opposite effect [50].

In highly saline as well as dry habitats, microorganisms struggle with high osmotic pressure and low water availability. Moreover, the membrane integrity is threatened by disruption due to dryness or salinity. In both saline and dry habitats, microbes control the water loss from the cell by producing compatible solutes [66]. These molecules are polar, water‐soluble and are capable of stabilizing proteins [50]. Examples of organic compatible solutes in bacteria are glycine betaine, ectoine, and trehalose.

Osmoprotection can be accomplished by accumulation of potassium chloride. Cl‐ _{and K}+

are transported separately into the cytoplasm. KCl is formed to counterbalance the high concentration of NaCl that is found outside the membrane [67].

Osmotic pressure is only one challenge that prokaryotes face. There is also high atmospheric pressure, under which the tight packing of molecules and the loss of fluidity of the cellular membrane result in impaired cellular functions. Microorganisms have developed mechanisms of alternative gene expression in order to produce molecules

that enhance the uptake of nutrients and a change in the membrane structure, by incorporating unsaturated fatty acids to guarantee fluidity.

Microorganisms are in contact with light in most habitats, and for phototrophs light is their energy source. However, ultraviolet (UV) light and ionizing radiation are extremely harmful because the cells only perform a limited repair of damaged DNA. Bacteria can survive only low exposure to this radiation since they can only repair limited DNA damage. Some extremophilic bacteria can survive very high levels of radiation (thousand times higher than other cells) due to advanced DNA repair mechanisms, and due to their specialized cell membrane and cell wall. Moreover, these organisms often have multiple copies of their genomes at stationary phase. However, this resistance is highly costly: DNA damage repair and the replication of a 9‐fold genome both demand high amounts of energy [50].

Finally, the chemical composition of the environment plays an important role in microbial survival. Low nutrient availability or presence of harmful compounds result in a fatal reduction of biomolecule synthesis and enzyme productivity. Slow metabolic activity and production of extracellular ‐often polysaccharides‐ mucus or an impermeable cell wall, in the case of harmful chemicals, are the main adaptations for microorganisms under such conditions [62].

Needless to say that impaired cellular functions under the above‐mentioned extreme conditions are confronted by entering dormancy, as well. At the edges of extremity, this could be the last resort solution.

1.4.1 Adaptation under extreme conditions at a molecular level

From a genetic point of view, the above‐mentioned adaptations to extreme environments are imprinted as molecular modifications; they are either genomic imprints, or post‐transcriptional and post‐translational modifications. The metabolic adaptations that are imprinted in the genomes of microbes and the modifications at an intracellular level are summarized herein.

In thermophilic and hyperthermophilic bacteria and archaea a series of modifications at the genomic level are necessary for the cells to withstand high temperatures. Firstly, changes at the amino acid level are observed; proteins of thermophiles contain mostly alanine, threonine, arginine and glutamic acid residues, while amino acids that enable flexibility of the protein are rare [68]. Moreover, the overall codon usage and nucleotide content, especially concerning rRNA and tRNA, vary significantly between mesophiles and thermophiles [69]. Secondly, multiple chaperone genes are found in the genomes of thermophiles [50, 70]. Thirdly, genes that can be used as thermophily‐specific biomarkers have been defined and are related to the supercoiling of the circular DNA [71], DNA repair and transcription regulation [70]. Genes that encode for proteins related to metal detoxification have also been identified [50, 70]. Finally, differences at the level of gene expression are also observed between mesophiles and thermophiles, especially concerning genes for amino acid synthetases and the regulation of these genes [72].

To date, two major genomic imprints are known for psychrophiles, related to low temperature adaptation. On one hand, there are the general genomic characteristics, such as high GC content in specific genes that encode for RNAs, elongation factors, and RNA polymerases [73] and the specific amino acid composition: hydrophobic and charged residues are characteristics of psychrophilic proteins [74]. On the other hand, genes that encode for small acidic proteins (cold acclimation proteins) are psychrophily‐specific markers [75].

In acidophilic, acid‐tolerant, alkalophilic and alkalo‐tolerant prokaryotes, a series of universal genes are differentially expressed compared to mesophilic (neutrophilic) bacteria [50]. These genes encode for proteins that are mainly transporters and antiporters, but they also encode for enzymes, such as the glutamine decarboxylase (in acidophiles) and the cyclodextrin glycosyltransferase (in the case of alkalophiles) [50]. Halophiles may have two types of genetic halophilic markers in their genomes. On one hand, characteristic acidic proteins are the typical markers for halophilic bacteria, although this is not the case for all halophilic species [76]. On the other hand, there are genes that are related to the uptake of osmoprotectants. An example of such genes is the kdp operon that encodes and regulates a kinase, as part of a two‐component system of signal transduction. Another example is the transporters or symporters responsible for the uptake of compatible solutes. The transcription of all these genes is regulated by specific proteins that, themselves, are osmoregulated [50].

In prokaryotes that tolerate high ultraviolet or ionizing radiation exposure, typical repair systems are in place. Genes that encode for specific enzymes, which are related to the removal of thymine dimers, the SOS repair, or photoactivation, are some of the genomic markers of UV resistance. Genes that are involved in these processes are uvrA, uvrB and

uvrC that encode for proteins that have excision activities. Additionally, there is the gene

that encodes for the photolyase enzyme. Finally, a high level of RecA, which binds to the thymine dimer and influences the polymerase to proceed without stopping, is also an example for the T‐T dimer excision. LexA is a typical marker for the SOS repair mechanism. Finally, superoxide reductases, responsible for the removal of oxygen radicals, are highly active in UV/ionizing radiation‐resistant cells [50].

Metal tolerance is often related to EPS production or to reduction or oxidation of metals. Moreover, there are transport‐related mechanisms of resistance [62]. All these processes are controlled by reductases, oxidases or transporters.