Recent advances in understanding extremophiles

pH: acidophiles

With few exceptions, these organisms maintain a cellular pH near neutral, which requires maintaining a pH gradient that is several orders of magnitude different on either side of the plasma membrane¹¹. One benefit of this is that there is always a ready supply of protons to power the proton motive force and form adenosine triphosphate (ATP) via the electron transport chain. However, the flip side is that the unusually high differences in concentration of protons (compared with neutrophiles) mean that, if left unchecked, the incoming protons have a greater capacity to dramatically change the internal cellular pH, which would lead to cell death.

To counteract these conditions, acidophiles have evolved several mechanisms. One of these is a cell membrane that is fairly impermeable to protons¹². In the Archaea branch of Life, this impermeability has been shown via tetraether lipids, differences in lipid head-group structures, a bulky isoprenoid core, and the fact that its ether linkages are less sensitive to acid hydrolysis compared with the ester linkages in the Bacteria and Eukarya Domains¹³. Another mechanism is the reduced pore size of membrane channels, which has been shown for Acidithiobacillus ferrooxidans¹⁴. Acidophiles also have a net positive potential charge inside the cell, which can counteract the high concentration of H⁺ ions in their surroundings. They also employ active proton pumping as has been observed in Bacillus and Termoplasma species¹⁵. It has not been shown that there is an identifiable acid response signature in the genomes of acidophiles. However, it is interesting to note that the genomes of acidophiles are predominately smaller than neutrophiles¹³. It is currently unclear why this should be and whether it confers any evolutionary advantage to these organisms.

pH: alkaliphiles

When adaptations are discussed, alkaliphiles are often grouped with halophiles, as they are typically found in saline environments¹⁶. However, the response to high pH is specific to these organisms and is worth discussing. The cytoplasm of alkaliphiles, like that of acidophiles, is typically near neutral pH¹⁷; therefore, alkaliphiles also have to overcome an imbalance of H⁺ ions¹⁸. Where acidophiles are “swimming” in H⁺ ions, alkaliphiles are in a relative desert by comparison.

In response to this challenge, alkaliphiles have developed a negatively charged cell wall, which lowers the pH of the environment just outside the cell. They also produce an acidic secondary cell wall composed of teichurono-peptide and teichuronic acid or polyglutamic acid. These acids attract H⁺ and repel OH⁻, possibly helping to generate the proton motive force needed to drive ATP synthesis. In several alkaliphilic Bacillus species, the proton motive force for ATP synthesis is driven by Na⁺ or K⁺ antiporters that catalyse an electrogenic exchange of outwardly moving ions (Na⁺ or K⁺) and an increased number of entering H⁺¹⁹. More generally, alkaliphiles are able to use these antiporters (Na⁺/H⁺ and K⁺/H⁺)²⁰ and also produce acids to reduce the internal pH when it is too high for metabolism to occur⁷.

Salinity: halophiles

Organisms that require a saline environment to grow (also known as halophiles) can be found along a continuum of salinity. These organisms have adopted either a “salt in” or “salt out” approach as the main adaptation for their ability to thrive in these conditions²¹.

As suggested by the name, the salt-in approach means that these organisms have salinity/ion concentrations (up to 4 or 5 M) that are similar both inside and outside the cell membrane. As such, the cytosol of these organisms presents a significant challenge for the regular biochemistry of life. High-salinity conditions typically strip water molecules from proteins, resulting in aggregation and precipitation, and often this is due to exposed hydrophobic patches binding to one another. To counteract this, these organisms have evolved a proteome that is composed of primarily acidic proteins²², and the acidic residues (aspartic and glutamic acid) are typically found on the surface of most of their proteins. These acidic residues have been shown to coordinate water molecules (that is, H⁺ of water interacts with the COO⁻ of the acidic side chain) around the proteins forming a “water cage” that protects the proteins from being dehydrated and precipitating out of solution^23,24. As a result of the large-scale evolutionary changes needed for this survival (that is, changes to the proteome), these organisms tend to live mainly in environments where salinity does not dramatically fluctuate frequently. Thus far, only prokaryotes (bacteria and archaea) have been shown to adopt this strategy²⁵.

In contrast to salt-in organisms, the salt-out organisms have differing concentrations of salt/ions inside and outside the cell membrane (similar to H⁺ with acido- and alkaliphiles). This strategy is more energy-intensive than the salt-in strategy¹⁶, as these organisms actively accumulate ions and organic osmolytes (for example, glycine betaine, ectoine/5-hydroxyectoine, glucosylglycerol, and dimethylsulfoniopropionate²⁶) to maintain turgor pressure. The accumulated compatible solutes eventually can be released into the environment via mechanosensitive channels or used as an energy source during times of lower external salinity²⁵. As the salt-out approach requires fewer large-scale evolutionary changes, organisms that have adopted it are able to grow over a wider range of salinity.

Radiation: radiophiles

Organisms’ responses to primarily two main types of radiation—ionizing (gamma) radiation and UV radiation—have been studied. Although on the surface it may seem that the same mechanisms of adaptation should be involved in both, there are, in fact, quite a few differences, which are probably due to the types of damage caused by each.

Ionizing radiation is responsible mainly for double-stranded breaks in the genome of organisms. However, it has also been shown to damage both proteins and lipids and induce persistent oxidative stress²⁷. Therefore, ionizing radioresistant organisms have developed all, or a combination of, the following strategies: novel and adaptive DNA repair mechanisms, antioxidant and enzymatic defense systems, and a condensed nucleoid. Fast and accurate repair of genomes is essential in surviving doses of ionizing radiation. This has been shown to be accomplished through the use of the nucleotide excision repair pathway (uvrA1B), base excision repair pathway (ung and mutY), and homologous recombination pathway (recA, ruvA, ddrA, and pprA) in D. radiodurans²⁸ and single-stranded binding proteins in Halobacterium sp. NRC-1 (Rfa-like genes)⁴ and D. radiodurans (DdrB and SSB)^29,30. In some especially sensitive species, protein damage causes death before double-stranded breaks start to form. It has been suggested that, especially for bacterial species, the role of protein damage in cell death due to ionizing radiation is underestimated. For example, D. radiodurans cells contain several oxidative stress prevention and tolerance mechanisms: cell cleaning through elimination of oxidized macromolecules, selective protection of proteins against oxidative damage, and suppression of reactive oxygen species production. A condensed nucleoid has also been shown to promote the efficiency/accuracy of DNA repair³¹ and to limit the diffusion³² of radiation-generated DNA fragments.

Unlike ionizing radiation where DNA damage is primarily double-stranded breaks, UV radiation damages DNA in more subtle ways through the formation of cyclobutene pyrimidine dimers (that is, thymine dimers) and pyrimidine (6-4) pyrimidone photoproducts (that is, 6-4 photo products). These two types of damage account for roughly 80% of photolesions induced by UV radiation³³. However, cyclobutene pyrimidine dimers are far more numerous and typically outnumber pyrimidine (6-4) pyrimidone photoproducts 3 to 1. To repair these DNA lesions, organisms use a combination of photoreactivation (phr) genes, nucleotide excision repair (uvrABCD, xpf, and rad), base excision repair (mutY and nth), and homologous recombination (recA and radA/51)³³. Additionally, organisms have evolved a suite of photoprotection devices to protect themselves from continual exposure to UV radiation. These include carotenoids, superoxide dismutases and hydroperoxidases, gene duplication via polyploidy, and genome composition (that is, reduction in the number of bipyrimidine sequences)³³. However, as in ionizing radiation, reactive oxygen species interference with normal metabolic processes is a more typical cause of cell death³⁴.

Pressure: piezophiles

These organisms are typically found in deep lakes such as Baikal (1.6 km) and Tanganyika (1.5 km), the ocean, and subsurface communities. To have an idea of how much pressure is involved, one must remember that hydrostatic pressure increases roughly 10.5 kPa per meter depth while lithostatic (overburden) pressure increases about 22.6 kPa per meter. This means that microbes growing at the bottom of the Mariana Trench (10.9 km below the ocean surface) are subjected to 114.4 kPa of pressure but that those in the South African gold mines (3.5 km below the Earth’s surface) face 79.1 kPa.

With increasing pressures, membranes lose fluidity and permeability as lipids pack more tightly and enter a gel phase similar to what happens at cold temperatures. To counteract this, bacterial piezophiles have been shown to incorporate polyunsaturated and monounsaturated fatty acids¹¹ or phosphatidylglycerol and phosphatidylcholine instead of phosphatidylethanolamine³⁵. Little is known about the adaptations in their archaeal counterparts, but alterations in the amount of certain archaeols seem to be important³⁶. Other mechanisms of adaptation are generally not well known, but a few reports suggest that changes are not due to a specific group of genes/enzymes but rather are the result of an overall change in metabolism^37–39 — similar to how the salt-in halophiles have evolved an acidic proteome to keep proteins soluble and active at high salinities rather than altering the expression of a few genes.

Temperature: psychrophiles

As mentioned above, the Earth and the Universe are both predominately cold environments. At first glance, psychrophile should be easy to define (that is, as something that grows in the cold) and it has often been defined as an organism with an optimum temperature of less than 15 °C⁴⁰. However, this definition has multiple problems: it is arbitrary, does not account for eukaryotes, and treats cells as mere thermal units⁴¹. Others have adopted the terms eurypsychrophile and stenopsychrophile to refer to a “broad” or “narrow” range of growth at low temperatures⁴². Still, some believe that the use of these terms does not “push” researchers enough to search for “true” psychrophiles – those organisms able to grow well below 0 °C. As such, there have been attempts to classify psychrophiles as those organisms that grow below 5 °C and to introduce the term cryophile, defined as organisms that can grow below 0 °C⁴³. So, whichever term you prefer, it is important to remember that in our efforts to communicate, scientists often impose strict restrictions that Nature does not create or recognize.

Psychrophiles have been isolated from a variety of natural (for example, polar regions, frozen lakes, and winter soils) and man-made (for example, refrigerators and cooling vents) environments. Microbes are generally subject to the temperature of their environment and as a consequence must find ways to adapt to the limitation placed on them by temperature.

To compensate for the negative effects of cold temperatures, organisms have developed several physiological adaptations, including regulating the fluidity of their membranes, synthesizing temperature-related chaperones, and producing antifreeze molecules⁴⁴. Psychrophiles regulate membrane fluidity through an increase in the number of branched-chain or unsaturated fatty acids or a shortening of the length of the fatty-acyl chains or both. Molecular chaperones are used to aid in the refolding of proteins and affect the levels of protein synthesis⁴⁵. Compatible solutes are used as cryoprotectors to lower the freezing point of the cytoplasm⁴⁶ and possibly prevent aggregation/denaturation of proteins, stabilize membranes, and scavenge free radicles in cold conditions⁴⁷. To reduce the damage caused by forming ice crystals, they may also use antifreeze or ice nucleation proteins^48,49. Antifreeze proteins act by binding seed ice crystals, thereby inhibiting their growth while ice nucleation proteins prevent the supercooling of water by ice crystal formation.

Structural proteins are also affected by temperature. Enzymes must overcome at least two obstacles in order to maintain activity at low temperatures: cold denaturation and slower reaction rates. Cold denaturation occurs at low temperatures because decreasing temperature results in more ordered water molecules surrounding a protein’s surface, which results in their being less associated with the protein and pushing the system equilibrium toward the unfolded state⁴⁴. The second problem for enzymes at low temperatures is slower reaction rates. According to the Boltzmann equation, reaction rates increase with increasing temperature and decrease two- to three-fold for every 10 °C decrease. Therefore, if cold-active enzymes are to have activities on par with their mesophilic counterparts, they must have developed structural changes to overcome these thermodynamic barriers⁵⁰.

Temperature: thermophiles

All portions of thermophiles, as with psychrophiles, are constantly exposed to temperature; therefore, they have adapted all macromolecules (DNA, lipids, and proteins) and complexes (cell surface, ribosomes, RNA polymerase, and so on) to remain functional. Among the most studied aspects of adaptation for thermophiles are those found in proteins. Additional networks of hydrogen bonds, decreased length of surface loops, enhanced secondary structure propensity, higher core hydrophobicity, increased van der Waals interactions, ionic interactions, and increased packing density have all been shown to contribute to protein thermostability²². It was more recently shown that, in addition to using the above mechanisms, archaeal cells use a structure-stabilization approach (that is, proteins are more compact than their mesophilic homologs) but that bacterial cells use a sequence-stabilization approach (that is, a small number of strong interactions)⁵¹. Another well-studied aspect of adaptation is the lipid composition of thermophilic membranes. Certain organisms, like Thermatoga maritima, have novel/specific lipids (15, 16-dimethyl-30-glycerylox-triacontanedioic acid)¹¹. The ether-based lipids of archaea have also been shown to be resistant to hydrolysis at high temperatures. These are also found in meso- and psychrophiles and so are not a specific thermal adaptation. However, some thermophilic archaeal cells do contain a monolayer composed of a “fused lipid bi-layer” that has also been shown to resist hydrolysis at higher temperatures²⁴.

The DNA of thermophiles also has a thermal resistance in that it has positive supertwists added by reverse gyrase⁵². Additionally, an increase in GC base pairs in specific regions (stem-loops) has been shown to stabilize DNA. Archaeal thermophiles also have histones that are closely related to the H2A/B, H3, and H4 core histone of eukaryotes. The binding of these histones has been shown to increase the melting temperature of DNA⁵³.