2. Extremophiles Images from NASA, http://pds.jpl.nasa.gov/planets/ Extraterrestrial microbial life-does it exist? What will it be like?

3. Lecture Aims What are Extremophiles- an introduction Strategies for growth & survival Biotechnology

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

5. Introduction to Extremophiles What are Extremophiles Live where nothing else can How do they survive? Extremozymes (more details later) Why are they are interesting? Extremes fascinate us  Life on other planets  Life at boiling temperatures Practical applications are interesting Interdisciplinary lessons Genetic Prospecting

6. Extremophile Definition - Lover of extremities History First suspected in 1950’s Extensively studied since 1970’s Temperature extremes Boiling or freezing, 100 0 C to -1 0 C Chemical extremes Vinegar or ammonia ( 9 pH) Highly saline, up to x10 sea water How we sterilize & preserve foods today

7. Extreme Temperatures Thermophiles - High temperature Thermal vents and hot springs Subsurface (oil wells, aquifers eg GAB) May go hand in hand with chemical extremes Psychrophiles - Low temperature Arctic and Antarctic 1/2 of earth’s surface is oceans between 1-4 0 C Deep sea –1 0 C to 4 0 C Most rely on photosynthesis

8. Thermophiles - volcanic Hydrothermal Vents- Black smokers at 350 o C Obsidian Pool, Yellowstone National Park

9. Thermophiles – Great Artesian Basin, Australia  Brisbane The Great Artesian Basin (GAB) is a unique deep subsurface thermal non-volcanic aquifer Worlds largest underground water reserve (1.7 x 10 6 km 2 = 8.7 x 10 12 m 3 ) 5000 free-flowing bore wells have been drilled thro’ 4 sequential interbeded sedimentary bases of different geological ages– distinct water chemistry. Natural discharge via mound springs (basin edges)

10. Thermophiles – Great Artesian Basin, Australia Bore well 17263 Temp: 88 o C

11. Psychrophiles

12. Chemical Extremes Acidophiles - Acidic Again some thermal vents & hot springs Alkaliphiles - Alkaline Soda lakes in Africa and Western U.S. Halophiles - Highly saline Natural salt lakes and manmade pools Sometimes occurs with extreme alkalinity

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

14. Alkaliphiles Mono Lake- alkaline soda lake, pH 9 & salinity 8%

15. Halophiles Dead Sea Great Salt Lake coastal splash zones Solar salterns Owens Lake

16. Survival 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

17. Survival 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 concs of a solute inside to avoid “pickling” Some enzymes from these microbes are interesting

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

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

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

21. What are enzymes? Enzymes are just a protein They can be destroyed by Heat, acid, base They can be inhibited by Cold, salt Heat an egg white or add vinegar to milk Protein is a major component of both- denatures

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

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

24. Extremophiles got there first 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

25. Thermophiles Most interesting, with practical applications Many industrial processes involve high heat 45 0 C (113F) is a problem for most enzymes First Extremophile found in 1972 Life at High Temperatures, Thomas M. Brock

26. PCR - Polymerase Chain Reaction Allows amplification of small sample of DNA using high temperature process Technique is about 20 years old DNA fingerprints - samples from crime scene Genetic Screening - swab from the mouth Medical Diagnosis - a few virus particles from blood Thermus aquaticus or Taq Life at High Temperatures, Thomas M. Brock

27. Psychrophiles 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

28. Acidophiles 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

29. Alkaliphiles “Stonewashed” pants Alkaliphilic enzymes soften fabric and release some of the dyes, giving worn look & feel Detergents Enzymes dissolve proteins or fats Detergents do not inhibit alkaliphilic enzymes

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

31. What is a halophile? Halophile = “salt loving; can grow in higher salt concentrations Based on optimal saline environments halophilic organisms can be grouped into three categories: extreme halophiles, moderate halophiles, and slightly halophilic or halotolerant organisms Some extreme halophiles can live in solutions of 25 % salt; seawater = 2% salt

32. Diversity of Halophilic Organisms Halophiles are a broad group &t can be found in all three domains of life. Found in salt marshes, subterranean salt deposits, dry soils, salted meats, hypersaline seas, and salt evaporation ponds.

33. Unusual Habitats A Pseudomonas species lives on a desert plant in the Negev Desert- the plant leaves secretes salt through salt glands. A Bacillus species is found in the nasal cavities of desert iguanas- iguanas nasal cavities have salt glands which secrete KCl brine during osmotic stress.

34. Osmoregulation Halophiles maintain an internal osmotic potential that equals their external environment. Osmosis is the process in which water moves from an area of high concentration to an area of low concentration.

35. Osmoregulation In order for cells to maintain their water they must have an osmotic potential equal to their external environment. As salinity increases in the environment its osmotic potential decreases. 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.

36. Osmoregulation Halophiles have adapted to life at high salinity in many different ways. Structural modification of external cell walls- posses negatively charged proteins on the outside which bind to positively charged sodium ions in their external environments & stabilizes the cell wall break down.

37. “Compatible Solute” Strategy Cells maintain low concentrations of salt in their cytoplasm by balancing osmotic potential with organic, compatible solutes. They do this by the synthesis or uptake of compatible solutes- glycerol, sugars and their derivatives, amino acids and their derivatives & quaternary amines such as glycine betaine. Energetically synthesizing solutes is an expensive process. Autotrophs use between 30 to 90 molecules of ATP to synthesize one molecule of compatible solute. Heterotrophs use between 23 to 79 ATP.

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

39. “ Salt-in” Strategy Potassium ions enter the cell passively via a uniporter. Sodium ions are pumped out. Chloride enters the cell against the membrane potential via cotransport with sodium ions. 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 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 extreme halophile Halobacterium are members of domain archaea. Widely researched for their extreme halophilism and unique structure. Require salt concentrations between 15% to saturation to live. Use the “salt-in” strategy. Produce ATP by respiration or by bacteriorhodopsin.

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

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

44. Applications 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 Other possible applications being explored: Increasing crude oil extraction (MEOR) Genetically engineering halophilic enzymes encoding DNA into crops to allow for salt tolerance Treatment of waste water (petroleum)

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

47. Life in hot salts o Thermohalophiles which adapt to high temperatures and salts Halothermothrix orenii Thermohalobacter berensis o What is their habitat? Isolation sources? o Salt lakes o Oil fields oWhat are the adaptation, protection and evolutionary strategies? o Structural o Cellular o Molecular

48. The thermohalophilic extermophiles  Only two truly halothermophiles known to date: Halothermothrix orenii: (optimum 60 o C + 10% NaCl; <65 o C + < 13% NaCl  )  Cayol, J-L, Ollivier, B., Patel, B.K.C., Prensier, G., Guezennec, J. and Garcia, J-L (1994). Isolation and characterization of Halothermothrix orenii gen. nov. sp. nov., a halophilic, thermophilic, fermentative strictly anaerobic bacterium. Int. J. of Bacteriol. 44:534-540 Thermohalobacter berrensis: (optimum 65 o C + 5% NaCl; 70 o C with 15% NaCl  )  Cayol, J.-L.,, Ducerf, S., Patel, B.K.C., Garcia, J.-L., Thomas, P. and Ollivier,B. (2000). Thermohalobacter berrensis gen. nov., sp. nov., a thermophilic, strictly halophilic bacterium from a solar saltern. Int. J. Syst. Evol. Microbiol. 50:559–564.

49. A Reminder on the molecular strategies adopted by halophiles Salt out- biomolecular structures maintained by cytoplasmic solutes (eg betaine) Salt In- structures maintained by surface charges of acidic aa (asp & glu)

50. THERMOPHILES  Structural Adaptations  Lipid Bilayer Structure  Cellular Adaptations  Molecular Chaperones  Histone-like DNA Binding Proteins  Molecular Adaptations  Excess glutamate, valine, tyrosine, & proline residues  Salt-bridges, packing density etc. HALOPHILES  Structural Adaptations  Lipid Bilayer Structure  Cellular Adaptations  Salt-in Cytoplasm Strategy  Compatible Solute Strategy  Molecular Adaptations  Excess acidic amino acids on protein surface Halothermophiles Do they exist, than their limits to life?? What Adaptation Strategies?? What Adaptation mechanisms??

51. More on molecular adaptation strategies The surface charges of proteins of halophiles, mesophiles & thermophiles show individual specific AA composition but the AA composition of the core (interior) is similar  Thermophiles – surface posses equal concentrations of acidic & basic AA (Arg, His, Lys)  Halophiles – surface posses excess acidic amino acid residues (Asp & Glu)

52. Hyperthermophiles Mesophiles & psychrophiles Thermophiles Psychrohalophiles, mesohalophiles & thermohalophiles have so far been reported but no hyperthermohalophiles (growth temp > 70oC with >10% NaCl) have been reported.

53. Genetic prospecting What is it? Think of a hunt for the genetic gold

54. Thermophiles – Great Artesian Basin, Australia Bore well 17263 Temp: 88 o C

55. Sequencing HMW DNA Monitoring Real Time PCR T-RFLP D. Population dynamism: trend & quantitation Mrl.thaut3 O5L10 O5L20 O5L2 O5L4 Dfm.thbenz O5L12 Spi. stenos O5L33 Mlb. organ2 C. Community structure, organism ID & relative frequency Develop Probes / primers PCR Gene library (Structural / Functional) Data analysis PCR Bharat Patel © A systems biology approach: Integrative bioprospecting assists fundamental research (evolution, adaptation) & biotechnology Preservation Novel microbes A.Ecosystems B. Microbiology Electron microscopy Micromanipulation Anaerobes Direct isolation (plating) Directed enrichment CORE Unix Linux PC Antimicrobials Novel Products F. Structural / Functional Genomics Rapid screening of native & recombinant products Genomic library (BAC,pBSK, Expression vectors) G. Biotechnology E. Community Functions Enzymes Others Screening - Mass spectrometry Purification Crystal growth Data collection

56. Thermostable dextranases Great Artesian Basin, Australia

57. Thermostable dextranases Great Artesian Basin, Australia 0 40 45 50 55 60 65 70 75 80 85 90 95 20 40 60 80 100 Relative Activity (%) Temperature ( o C)              Chaetomium gracile Anaerobe GAB11A Anaerobe Rt364

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