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

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

Lecture Aims What are Extremophiles- an introduction Strategies for growth & survival Biotechnology Info on me, in NZ on volcanic hot springs and than here on subsurface GAB ***Any Questions, go ahead and ask ** 1) You need to understand them before you can teach 2) A great deal of Info on web sites & new text books due to interest in astrobiology & who we are etc 3) These are fascinating creatures and I enjoyed putting presentation together

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 2) Review of enzymes, then extremozymes Why is this the second talk??? A natural lead in to BioTech/Computing 3) - Interesting to scientists, and are interesting to us too. - Life on other planets - DNA fingerprinting, stonewashed jeans, and oil extraction - Interdisciplinary lessons

Extremophile Definition - Lover of extremities History First suspected in 1950’s Extensively studied since 1970’s Temperature extremes Boiling or freezing, 1000C to -10C Chemical extremes Vinegar or ammonia (<5 pH or >9 pH) Highly saline, up to x10 sea water How we sterilize & preserve foods today

Extreme Temperatures Thermophiles - High temperature Thermal vents and hot springs May go hand in hand with chemical extremes Psychrophiles - Low temperature Arctic and Antarctic 1/2 of earth’s surface is oceans between 1-40C Deep sea –10C to 40C Most rely on photosynthesis Who lives in the temperature extremes? Photosynthesis???

Thermophiles Obsidian Pool, Yellowstone National Park Hydrothermal Vents- Black smokers at 350 oC Thermophiles Obsidian Pool, Yellowstone National Park

Psychrophiles

Chemical Extremes Acidophiles - Acidic Alkaliphiles - Alkaline 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 Can see that there is some overlap Thermophile/Acidophile and Halophile/Alkaliphile White Sands???

Acidophiles pH 0-1 of waters at Iron Mountain

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

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

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

Survival Chemical extremes Different from temperature 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 Different from temperature Halophiles - Osmosis 3) Because interior tends to be normal...

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

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

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

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 Wrapping up review of enzymes, they have limitations Remember definition, a protein that catalyses chemical reactions without being changed

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? Remember last slide on enzymes, each of these situations destroy or inhibit an enzyme

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 2) leads to genetic prospecting, but first

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

Thermophiles Most interesting, with practical applications Many industrial processes involve high heat 450C (113F) is a problem for most enzymes First Extremophile found in 1972 Life at High Temperatures, Thomas M. Brock

PCR - Polymerase Chain Reaction Life at High Temperatures, Thomas M. Brock 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 Thermus aquaticus or Taq

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

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

Alkaliphiles “Stonewashed” pants Detergents 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 Here is a wild application that caught my eye… Enzyme needs to work in soapy (alkaline) solution

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

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

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.

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.

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.

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.

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.

“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.

“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.

“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.

“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.

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.

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.

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.

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.

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)

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.

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

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