Hyperthermophilic archaeal cellular structures

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An image of P. fumarii from a scanning electron microscope.4

Hyperthermophiles are organisms that can live at temperatures ranging between 70-125ºC. They have been the subject of intense study since their discovery in 1977 in the Galapagos Rift1. It was thought impossible for life to exist at temperatures a great as 100ºC until Pyrolobus fumarii was discovered in 19972. P. fumarii is an unicellular organism from the domain Archaea living in the hydrothermal vents in black smokers along the Mid-Atlantic Ridge2. These organisms can live at 106ºC at a pH of 5.52. In order to get energy from their environment these organisms are facultatively aerobic obligate chemolithoautotrophs, meaning these organisms build biomolecules by harvesting CO2 from their environment by using H2 as their primary electron donor and NO3- as its primary electron acceptor2. These organisms can even survive the autoclave, which is a machine designed to kill organisms through high heat and pressure2. Because hyperthermophiles live in such hot environments, they need to have DNA, membrane and enzyme modifications in order to withstand the intense thermal energy. Such modifications are currently being studied to better understand what allows an organism or protein to survive such harsh conditions. By learning what allows these organisms to survive such harsh conditions, researchers will be better able to synthesize molecules that are harder to denature that can be used in industry.


DNA structures of P. fumarii

Introduction to DNA

Two DNA strands are held together by base pairing that allows the nucleotide bases adenosine (A) to bind with thymine (T), and guanine (G) to bind with cytosine (C). It has been proposed that thermophilic archaea would be expected to have higher GC content within their DNA, because GC pairings have three hydrogen bonds, while AT pairings have only two. Increasing the number of hydrogen bonds would increase the stability of the DNA, thereby increasing the energy required to separate the two strands of DNA. This would help the DNA to remain double stranded while at temperatures high enough to denature DNA found in mesophiles.3

Stabilization of DNA

A phylogenetic tree comparing P. fumarii to other organisms within the Desulfurococcales order.4

P. fumarii was first sequenced in 2001 by the Diversa Corporation and the sequence was released to the public in 20114. The data from this analysis showed a GC content of 54.90%. This supports the hypothesis that thermophiles experience selective pressure to increase their GC content in order to stabilize their DNA5. However, research has not conclusively supported this hypothesis. A study done by Hurst and Merchant (2001) showed no correlation between higher GC content in prokaryotes and increased optimal growing temperatures. However, their analysis did show that there was higher GC content for the third amino acid within the codon. This demonstrates that within the wobble position there is likely a selective pressure for more hydrogen bonds to increase stability within the DNA, but less selective pressure for GC pairings within the DNA as a whole5. This supports what is seen in P. fumarii because there was only a slightly higher GC content than AT content. These results indicate that along with increasing GC pairing in the wobble position, thermophilic archaea have other mechanisms for stabilizing their DNA at such high temperatures5.


One possible mechanism for stabilizing DNA at such high temperatures are proteins such as a type I topoisomerase. This is a protein that supertwists the DNA, which makes spontaneously untwisting of the DNA more difficult. The presence of this protein in multiple evolutionarily distant organisms supports the hypothesis that this protein plays a role in DNA stabilization.6

Membrane Adaptations

Introduction to membranes

All microbes ranging from the smallest bacteria to the largest multicellular eukaryote contain membranes with phospholipids. A phospholipid molecule is composed of a long fatty acid, often called the tail of the molecule, and a phosphate group, which serves as the head of the molecule. Phospholipid membranes can range widely in the structure of the fatty acid tail, which is composed of mostly hydrocarbons. These phospholipid molecules form bilayers. . The bilayers use the hydrocarbon tails as the middle of the membrane, while the phosphate heads face out either into the extracellular environment or into the cell’s cytosol. The membrane is then combined with proteins. These proteins then control which molecules are allowed in or out of the cell. For this reason, the membrane plays a crucial role in the survival of the cell. A faulty membrane can allow too many solutes into the cell, resulting in cell death.3


Different organisms have devised different strategies in order to control what goes in and out of the cell. Bacteria and eukaryotic cells contain phospholipid bilayers containing ester linkages, while archaea contain ether linkages. While these mechanisms work very well for organisms that live in mesophilic environments, they do not work for extremophiles. Mesophiles are organisms that live within relatively moderate temperatures (20-45ºC). They are organisms that live around sea level and can survive around the same temperatures as humans.7


Bacteria and eukaryotic cells contain phospholipid bilayers containing ester linkages. While ester linkages work very well for organisms that live in mesophilic environments, they do not work for extremophiles. Mesophiles are organisms that live within relatively moderate temperatures (20-45ºC). These organisms live around sea level and can survive around the same temperatures as humans.7


Extremophiles are organisms that grow best extremely cold, acidic, basic or hot environments. P. fumarii is a hyperthermophile, indicating that this organism grows best at extremely high temperatures (70-125ºC)8. P. fumarii grows best at 106ºC2. Due to the extremely high temperatures this archaea is subjected to, this organism needs to have extremely stable biomolecules in order to survive. Without increased stability in the membrane the cell would fall apart, and too many molecules would flow in and out of the membrane destroying the chemical gradients the cell uses for energy, while also allowing all the proteins the cell had synthesized to diffuse away, stopping the cell's metabolic processes.3

Tetraether membranes

The top molecule represents the ether linkage found in archaea membranes, which is shown by the yellow on the molecule. The second molecule represents the phospholipid found in eukaryotic and bacterial bilayers. The molecule labeled 9 is a phospholipid bilayer found in most bacteria and eukaryotes. The molecule labeled 10 shows a tetraether forming a membrane with only one layer of molecules, which are found only in archaea. 17

To deal with the stability problem, archaea have changed their membrane lipid compositions. They still contain phosphate groups and long fatty acid tails, but they also contain ether linkages instead of ester linkages. The ether linkages make the bonds between phosphate groups and hydrocarbons more stable because the carbon connecting the phosphate group and glycerol molecule is more electron-rich than it would be in an ester, making that carbon less electrophilic and therefore less chemically reactive. This allows the ester-linked phospholipid to be more stable and less susceptible to breakdown from large amounts of thermal energy. This contributes to the archaea's ability to live in such extreme environments.7


Another membrane adaptation seen in some archaea is tetraether phospholipids. This specific adaptation has been found in P. fumarii along with other hyperthermophiles. A tetraether phospholipid is a molecule containing two phosphate groups attached by one hydrocarbon chain. These phospholipids form monolayers instead of the typical bilayers seen in most bacteria and all eukaryotes. Therefore, instead two different molecules interacting with each other, only one molecule spans the entire width of the membrane. The monolayer then allows for tighter packing of molecules within the membrane because fewer molecules must fit into membrane, however these large molecules are less able to move within the membrane. This then decreases membrane fluidity, allowing the cell to keep more molecules from crossing the membrane. . This is an extremely important adaptation because at such high temperatures molecules will be moving much more quickly than they would be mesophilic temperatures, therefore it is more likely that a molecule will cross the membrane going in or out of the cell. To combat this problem hyperthermophiles decrease their membrane fluidity. The decreased fluidity allows hyperthermophilic cells to decrease the movement of the phospholipid molecules, which stops the unwanted movement of molecules across the membrane.9

Cyclopentane rings to increase stabilization

Another extremely important membrane modification, which helps decrease influx and efflux of solutes is the addition of cyclopentane rings within the hydrocarbon tails of the ester-linked phospholipids. The addition of these rings into the membrane allows for even tighter packing of the membrane molecules. These cyclopentane rings can exist in tetraether lipids or diether lipids. Cyclopentane rings help to crowd the membrane’s inner structure making it more difficult for the solutes to get through the membrane to the other side of cell. This is important for the cell, because at hyperthermophilic conditions the solutes will travel very fast carrying a lot of thermal energy from their environment. If the cell did not have these rings, too many unwanted molecules would likely make it past the membrane either out or into the cell. This would result in the slowing or complete stop of metabolic processes, resulting in cell death.9


While these cyclopentane rings are extremely useful for keeping unwanted solutes from entering or leaving the cell, not all archaea use them. However, they have been seen in psychrophiles, which are archaea that require very cold conditions to survive (-15ºC or below). This is counterintuitive because cyclopentane molecules help to make the membrane more rigid, which is something that happens naturally at very low temperatures. It is unclear why these rings are seen at both ends of the temperature spectrum. However, their presence at in both hyperthermophiles and psychrophiles makes it clear that they serve other functions than simply slowing molecules from entering or leaving a cell.9

Metabolism

Introduction to hyperthermophile metabolism

Because organisms like P. fumarii live in such harsh environments, these archaea have needed to devise unusual ways to gather energy from the environment and protect themselves against heat stress. P. fumarii, like plants, are able to harvest CO2 from the environment to build their biomolecules, but unlike plants, they take electrons from H2 instead of H2O and transfer those electrons to NO3-, SO42- or O22. This type of metabolic process is classified as chemolithoautrophism, meaning their carbon comes from an inorganic source, their final electron acceptor is not O2 and they produce and consume their own food.7

Stabilization of proteins through heat shock proteins

Another way in which hyperthermophiles ensure their proteins’ proper function is through the use of heat shock proteins (HSPs). These are proteins that help to keep other proteins in their proper shapes. They will often refold denatured proteins and them functional again. In an environment where proteins are regularly subjected intense heat, HSPs help to keep proteins functional for longer. This keeps the cell from wasting energy by constantly making new proteins. While these HSPs are not unique to extremophiles, they are extremely important to study because HSPs found in hyperthermophiles are the most stable of their kind. HSPs are also able to prolong the life of a hyperthermophile even beyond its optimal growing temperature. By studying these proteins it may be possible to learn the mechanisms proteins use to stabilize other proteins, which may help in biosynthesis of new molecules. HSPs act as chaperone proteins that help enzymatic proteins maintain their proper conformation for longer than they would by themselves at such high temperatures. This is part of what allows P. fumarii to perform metabolic processes at temperatures that were long thought to be much too hot for life to exist.10


Different mechanisms for carbon fixation

The TCA cycle can be seen on the left, while the 3-HP/4-HP cycle is seen on the right. These metabolic pathways can be found in anaerobic bacteria. The rTCA cycle is simply the TCA cycle run backwards. The rTCA cycle is used to create new sugars and biosynthesis, while the TCA cycle breaks down sugar derivatives to obtain energy. The 3-HP/4-HP cycle is also used to create new sugar molecules for biosynthesis. 15

The most common organisms that harvest CO2 to build biomolecules are plants and photosynthetic bacteria. Those particular organisms use the Calvin cycle for their carbon fixation. However, P. fumarii and other similar organisms contain particular enzymes that allow them to harvest CO2 at temperatures well above those tolerated by plants and photosynthetic bacteria with slightly different mechanisms. The alternate pathways used by these extremophiles are either the rTCA cycle11, 3-HP cycle, 3-HP/4-HP cycle, or DC/4-HP cycle. These are likely some of the first pathways to evolve because the bacteria and archaea who use them live in environments that mirror the early Earth environments12. Therefore it is likely that these are some of the first carbon fixation pathways to evolve. The rTCA cycle is usually seen in organisms living at temperatures between 20-90ºC, while organisms living at temperatures above 90ºC most often use the DC/4-HP cycle. The 3-HP/4-HP cycle is most often used by thermoacidophiles in the Sulfolobales genus.12


The rTCA cycle is simply the Calvin Cycle run backwards. Therefore instead of producing CO2 and NADH, they cycle uses CO2 and NAD+. The CO2 is then used to make new sugars or proteins that the cell can use for energy of synthesis of new enzymes. This cycle allows organisms to produce their own food from the environment and not need to eat, making these organisms autotrophs.12


The 3-HP/4-HP cycle uses acetyl-CoA as its starting and ending substrate just like the rTCA cycle. However, instead of creating citrate, it creates malonyl-CoA and other unique substrates to create the molecules needed for making sugars and amino acids. This pathway uses more ATP and NADH than the rTCA cycle, making it less efficient. This makes sense because the 3-HP/4-HP cycle is thought to have evolved earlier; making it likely that the enzymes this pathways uses less efficient. The 3-HP/4-HP cycle is found almost exclusively in Sulfolobales, where the rTCA cycle is not found. Sulfolobales are usually acidothermophiles. This means these cells likely use the chemical gradients outside the cells (in the form of many hydrogen ions in solution) and thermal energy to help drive their biosynthesis. This environment helps to explain why their biosynthesis pathway has not experienced evolutionary pressure to consume less energy and become more like the rTCA cycle. By using the environment to power some of their cellular processes, Sulfolobales can afford to use more energy on their biosynthesis pathways without running out of cellular energy.12


The DC/4-HP cycle

The DC/4-HP cycle is show in this image. The DC/4-HP cycle uses part of the TCA cycle running forwards, but splits once it gets to succinyl-CoA. This intermediate then becomes succinic semialdehyde. That intermediate follows through different intermediates to reform acetyl-CoA, which then goes on to create sugars for biosynthesis.16

The DC/4-HP cycle is a combination of the rTCA cycle and the 4-HP half of the 3-HP/4-HP cycle. The cycle begins with acetyl-CoA, which is the starting substrate for all three carbon fixation pathways. The acetyl-CoA is then converted to pyruvate, then PEP, then oxaloacetate. It then follows the rTCA cycle until it forms the succinyl-CoA. Once this substrate is formed it follows the 4-HP half of the 3-HP/4-HP cycle. This cycle is found in Desulfurococcales and Crenarchaeota. Because it uses enzymes present in the rTCA and 3-HP/4-HP cycles making it hard for researchers to discover it. Due to the lack unique enzymes, it wasn’t found in P. fumarii until 201013. This pathway is also uses ATP and NADH, however it uses less than the 3-HP/4-HP cycle but more than the rTCA cycle. This makes sense because all the thermal energy in the thermophiles’ environment can also help drive cellular processes similar to the acidothermophiles.12


Signal sugars found in hyperthermophiles

One molecule that has been identified as being related to hyperthermophilic organisms is di-myo-inositol phosphate (DIP). Inositol and other phosphate derivatives of this molecule are sugars often used as secondary messenger in eukaryotic cells. However, DIP has only ever been found in thermophilic cells and their use within the cells is largely unknown. A derivative of this sugar called UDP-α-GlcNAc3NAc-(4←1)-β-GlcpNAc3NAc has been found only in P. fumarii. The function of this sugar is unknown. However, phosphorylated forms of this sugar have been found in conjunction with UDP-α-Glc- NAc3NAc, which is a sugar known to participate in the formation of the S-layer. These UDP sugars are only found when the cells are in extremely favorable growing conditions. This suggests that these sugars are building blocks within the cell that allow for the creation of the S-layer protecting Gram positive bacteria14. This connection to the S-layer is extremely important, because it is hypothesized that the S-layer is used to help protect the cell from the heat stress associated with hyperthermophilic environments. The S-layer is also thought to help slow molecules from exiting or entering the cell9. While these results are not conclusive, they do help to elucidate how the S-layer is created, which has largely remained a mystery for years.14

Further Reading

A rapidly evolving topic within the study of hyperthermophiles is the evolutionary history of these organisms. The environment that hyperthermophiles occupy closely resembles the early environment on Earth. By studying how these organisms evolved, it may be possible to shed light on how early life evolved on Earth.7


The use of unique hyperthermophilic structures has been explored many times. One current area of exploration is the use of tetraether membranes in biotechnology. These membranes may provide new ways of synthesizing biofilms and other nanotechnologies. These films would have more stability, better insulating properties and lower permeability than current films. These advances could then be applied to many new technologies to improve human life.18


The hyperthermophilic community of researchers is also trying to determine how hyperthermophile proteins become so stable. Research in this area is focused on how to make proteins more stable to increase knowledge about protein structure and organization. So little is known about how proteins are formed and what allows them to function. By understanding more about such stable proteins, researchers will be able to understand how biological organisms are able to do all the amazing things they do.19

References

1. Lonsdale, P. Clustering of suspension-feeding macrobenthos near abyssal hydrothermal vents at oceanic spreading centers. Deep Sea Res. 24, 857–863 (1977).

2. Blöchl, E. et al. Pyrolobus fumarii, gen. and sp. nov., represents a novel group of archaea, extending the upper temperature limit for life to 113°C. Extremophiles 1, 14–21 (1997).

3. Brooker, R. J. Biology. (McGraw-Hill Higher Education ; McGraw-Hill [distributor], 2010).

4. Göker, M. et al. Complete genome sequence of Pyrolobus fumarii type strain (1AT). Stand. Genomic Sci. 4, (2011).

5. Hurst, L. D. & Merchant, A. R. High guanine-cytosine content is not an adaptation to high temperature: a comparative analysis amongst prokaryotes. Proc. Biol. Sci. 268, 493–497 (2001).

6. Nakasu, S. & Kikuchi, A. Reverse gyrase; ATP-dependent type I topoisomerase from Sulfolobus. EMBO J. 4, 2705–2710 (1985).

7. Slonczewski, J., Foster, J. W. & Gillen, K. M. Microbiology: an evolving science. (2014).

8. Vieille, C. & Zeikus, G. J. Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability. Microbiol. Mol. Biol. Rev. 65, 1–43 (2001).

9. Oger, P. M. & Cario, A. Adaptation of the membrane in Archaea. Biophys. Chem. 183, 42–56 (2013).

10. Nath, I. V. A. & Bharathi, P. A. L. Diversity in transcripts and translational pattern of stress proteins in marine. Extremophiles 15, 129–153 (2011).

11. Campbell, B. J. & Cary, S. C. Abundance of Reverse Tricarboxylic Acid Cycle Genes in Free-Living Microorganisms at Deep-Sea Hydrothermal Vents. Appl. Environ. Microbiol. 70, 6282–6289 (2004).

12. Hügler, M. & Sievert, S. M. Beyond the Calvin Cycle: Autotrophic Carbon Fixation in the Ocean. Annu. Rev. Mar. Sci. 3, 261–289 (2011).

13. Berg, I. A., Ramos-Vera, W. H., Petri, A., Huber, H. & Fuchs, G.Study of the distribution of autotrophic CO2 fixation cycles in Crenarchaeota. Microbiology 156, 256–269 (2010).

14 Gonçalves, L. G., Lamosa, P., Huber, R. & Santos, H. Di-myo-inositol phosphate and novel UDP-sugars accumulate in the extreme hyperthermophile Pyrolobus fumarii. Extrem. Life Extreme Cond. 12, 383–389 (2008).

15. Ulas, T., Riemer, S. A., Zaparty, M., Siebers, B. & Schomburg, D. Genome-Scale Reconstruction and Analysis of the Metabolic Network in the Hyperthermophilic Archaeon Sulfolobus Solfataricus. PLoS ONE 7, (2012).

16. Siebers, B. et al. The Complete Genome Sequence of Thermoproteus tenax: A Physiologically Versatile Member of the Crenarchaeota. PLoS ONE 6, (2011).

17. File:Archaea membrane.svg. Wikipedia, the free encyclopedia at Archea Membrane

18. Jacquemet, A., Barbeau, J., Lemiègre, L. & Benvegnu, T. Archaeal tetraether bipolar lipids: Structures, functions and applications. Biochimie 91, 711–717 (2009).

19. Sawle, L. & Ghosh, K. How Do Thermophilic Proteins and Proteomes Withstand High Temperature? Biophys. J. 101, 217–227 (2011).

Edited by Libby Mannucci, a student of Nora Sullivan in BIOL168L (Microbiology) in The Keck Science Department of the Claremont Colleges Spring 2014.