2. Extremophiles Life on the edge Life at High Temperatures, Thomas M. Brock

3. Extremophiles Images from NASA, http://pds.jpl.nasa.gov/planets/

4. Goals u Overview of Extremophiles –Review some biology –Give some applications –Motivate you to study Microbiology!!

5. Introduction to Extremophiles u What are they? –Microbes living where nothing else can u How do they survive? –Extremozymes u Why are they are interesting? »Extremes fascinate us u Life on other planets »Practical applications are interesting –Interdisciplinary lessons »Genetic Prospecting

6. Extremo - phile u Definition - Lover of extremes u History –Suspected about 30 years ago –Known and studied for about 20 years u Temperature extremes –boiling or freezing, 100 0 C to -1 0 C (212F to 30F) u Chemical extremes –vinegar or ammonia ( 9 pH) –highly salty, up to ten times sea water u How we sterilize & preserve foods today

7. Extreme Temperatures u Thermophiles - High temperature –Thermal vents and hot springs –May go hand in hand with chemical extremes u Psychrophiles - Low temperature –Arctic and Antarctic »1/2 of Earth’s surface is oceans between 1 0 C & 4 0 C »Deep sea –1 0 C to 4 0 C »Most rely on photosynthesis

8. Thermophiles Obsidian Pool, Yellowstone National Park Hydrothermal Vents

9. Psychrophiles

10. Chemical Extremes u Acidophiles - Acidic –Again thermal vents and some hot springs u Alkaliphiles - Alkaline –Soda lakes in Africa and western U.S. u Halophiles - Highly Salty –Natural salt lakes and manmade pools –Sometimes occurs with extreme alkalinity

11. Acidophiles pH 0-1 of waters at Iron Mountain

12. Alkaliphile e.g. Mono Lake alkaline soda lake, pH 9 salinity 8%

13. Halophiles solar salterns Owens Lake, Great Salt Lake coastal splash zones Dead Sea

14. Survival u Temperature extremes –Every part of microbe must function at extreme »“Tough” enzymes for Thermophiles »“Efficient” enzymes for Psychrophiles –Many enzymes from these microbes are interesting Life at High Temperatures, Thomas M. Brock

15. Survival u Chemical extremes –Interior of cell is “normal” –Exterior protects the cell »Acidophiles and Alkaliphiles sometimes excrete protective substances and enzymes »Acidophiles often lack cell wall »Some moderate halophiles have high concentrations of a solute inside to avoid “pickling” –Some enzymes from these microbes are interesting

16. What are enzymes? u Definition - a protein that catalyses (speeds up) chemical reactions without being changed

17. What are enzymes? u Enzymes are specific –Lock and key analogy Enzyme Substrate A Product B Product C

18. What are enzymes? u Activation energy –Enzymes allow reactions with lower energy Energy Time Without Enzyme With Enzyme

19. What are enzymes? u Enzymes are just a protein –They can be destroyed by »Heat, acid, base –They can be inhibited by »Cold, salt u Try doing this with an egg white or milk –Protein is a major component of both

20. Practical Applications u Extremozymes »Enzyme from Extremophile u Industry & Medicine u What if you want an enzyme to work –In a hot factory? –Tank of cold solution? –Acidic pond? –Sewage (ammonia)? –Highly salty solution?

21. One solution u Pay a genetic engineer to design a “super” enzymes... –Heat resistant enzymes –Survive low temperatures –Able to resist acid, alkali and/or salt u This could take years and lots of money

22. Extremophiles got there first u Nature has already given us the solutions to these problems –Extremophiles have the enzymes that work in extreme conditions Endolithic algae from Antarctica; Hot springs in Yellowstone National Park, © 1998 Reston Communications, www.reston.com/astro/extreme.html

23. Thermophiles u Most interesting practical applications so far –Many industrial processes involve high heat –45 0 C (113F) is a problem for most enzymes –First Extremophile found 30 years ago Life at High Temperatures, Thomas M. Brock

24. PCR - Polymerase Chain Reaction u Allows amplification of small sample of DNA using high temperature process –Technique is about 10 years old –DNA fingerprints - samples from crime scene –Genetic Screening - swab from the mouth –Medical Diagnosis - a few virus particles from blood u Thermus aquaticus or Taq Life at High Temperatures, Thomas M. Brock

25. Psychrophiles u Efficient enzymes to work in the cold –Enzymes to work on foods that need to be refrigerated –Perfumes - most don’t tolerate high temperatures –Cold-wash detergents Algal mats on an Antarctic lake bottom, © 1998 Reston Communications, www.reston.com/astro/extreme.html

26. Acidophiles u Enzymes used to increase efficiency of animal feeds –enzymes help animals extract nutrients from feed »more efficient and less expensive Life at High Temperatures, Thomas M. Brock

27. Alkaliphiles u “Stonewashed” pants –Alkaliphilic enzymes soften fabric and release some of the dyes, giving worn look and feel u Detergents –Enzymes to dissolve proteins or fats –Alkaliphilic enzymes can work with detergents

28. Halophiles u What is a halophile? u Diversity of Halophilic Organisms u Osmoregulation u “Compatible Solute” Strategy u “Salt-in” Strategy u Interesting Facts and Applications

29. What is a halophile? u The word halophile means “salt loving”. u A halophile is an organism that can grow in higher salt concentrations than the norm. u Based on optimal saline environments halophilic organisms can be grouped into three categories: extreme halophiles, moderate halophiles, and slightly halophilic or halotolerant organisms. u Some extreme halophiles can live in solutions of 35 % salt. This is extreme compared to seawater which is only 3% salt.

30. Diversity of Halophilic Organisms u Halophiles are a broad group that can be found in all three domains of life. u They are found in salt marshes, subterranean salt deposits, dry soils, salted meats, hypersaline seas, and salt evaporation pools.

31. Unusual Habitats u The bacterium pseudomonas was found living on a desert plant in the Negev Desert. The plant secretes salt through salt glands on its leaves. u Bacillus was found in the nasal cavities of desert iguanas. These iguanas have salt glands in their nasal cavities that secrete KCl brine during osmotic stress.

32. Osmoregulation u Living in high salinity poses a serious stress that halophiles have overcome through special processes or adaptations. u The stress lies in the microbes ability to maintain an internal osmotic potential that equals their external environment. u Osmosis is the process in which water moves from an area of high concentration to an area of low concentration.

33. Osmoregulation u In order for cells to maintain their water they must have an osmotic potential equal to their external environment. u As salinity increases in the environment its osmotic potential decreases. u If you placed a non halophilic microbe in a solution with a high amount of dissolved salts the cell’s water will move into the solution causing the cell to plasmolyze.

34. Osmoregulation u Halophiles have adapted to life at high salinity in many different ways. u One way is through the modification of their external cell walls. They tend to have negatively charged proteins on the outside of their cell walls that stabilize it by binding to positively charged sodium ions in their external environments. If salt concentrations decline their cell walls may become unstable and break down.

35. “Compatible Solute” Strategy u There are two strategies that halophiles have evolved to deal with high salt environments. u In the “compatible solute” strategy cells maintain low concentrations of salt in their cytoplasm by balancing osmotic potential with organic, compatible solutes. u They do this by the synthesis or uptake of compatible solutes.

36. “Compatible Solute” Strategy u Compatible solutes include polyols such as glycerol, sugars and their derivatives, amino acids and their derivatives, and quaternary amines such as glycine betaine. u Energetically this is an expensive process. u Autotrophs use between 30 to 90 molecules of ATP to synthesize one molecule of the compatible solutes. Heterotrophs use between 23 to 79 ATP.

37. “ Compatible Solute” Strategy u Energy is also expended in pumping out salts that dissolve into the cell. u The uptake of available compatible solutes in the environment is an adaptation they have evolved to reduce the energy cost of living in high salt concentrations.

38. “Salt-in” Strategy u Cells can have internal concentrations that are osmotically equivalent to their external environment. u This “salt-in” strategy is primarily used by aerobic, extremely halophilic archaea and anaerobic bacteria. u They maintain osmotically equivalent internal concentrations by accumulating high concentrations of potassium chloride.

39. “Salt-in” Strategy u Potassium ions enter the cell passively via a uniport system. Sodium ions are pumped out. Chloride enters the cell against the membrane potential via cotransport with sodium ions. u For every three molecules of potassium chloride accumulated, two ATP are hydrolyzed making this strategy more energy efficient than the “compatible solute” strategy.

40. “Salt-in” Strategy u To use this strategy all enzymes and structural cell components must be adapted to high salt concentrations to ensure proper cell function.

41. Halobacterium: an example of an extreme halophile u Halobacterium are members of the halophile group in the domain archaea. They are widely researched for their extreme halophilism and unique structure. u They require salt concentrations between 15% to 35% sodium chloride to live. u They use the “salt-in” strategy. u They produce ATP by respiration or by bacteriorhodopsin.

42. Halobacterium u They may also have halorhodopsin that pumps chloride into the cell instead of pumping protons out. u The Red Sea was named after halobacterium that turns the water red during massive blooms.

43. Facts u The term “red herring” comes from the foul smell of salted meats that were spoiled by halobacterium. u There have been considerable problems with halophiles colonizing leather during the salt curing process.

44. Applications u Current applications using halophiles include: –the extraction of carotene from carotene rich halobacteria and halophilic algae that can then be used as food additives or as food-coloring agents. –the use of halophilic organisms in the fermentation of soy sauce and Thai fish sauce.

45. Applications u Many possible applications using halophiles are being explored such as: –increasing crude oil extraction –genetically engineering halophilic enzyme encoding DNA into crops to allow for salt tolerance –treatment of waste water

46. Conclusions u Halophiles are salt tolerant organisms. u They are widespread and found in all three domains. u The “salt-in” strategy uses less energy but requires intracellular adaptations. Only a few prokaryotes use it. u All other halophiles use the “compatible solute” strategy that is energy expensive but does not require special adaptations.

47. Genetic prospecting u What is it? –Think of a hunt for the genetic gold Pr. Patrick Forterre, Extremophiles Laboratory of IGM at Orsay, France http://www-archbac.u-psud.fr/

48. Summary u Now you know something about Extremophiles –Where they live & how they survive u They are interesting because –They have enzymes that work in unusual conditions –The practical applications are interesting