Extremophiles

Life on edge

Life at High Temperatures, Thomas M. Brock

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

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

Thermophiles

Hydrothermal Vents- Black smokers at 350 oC

Obsidian Pool,Yellowstone National Park

Psychrophiles

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

Acidophiles

pH 0-1 of watersat Iron Mountain

Alkaliphiles

Mono Lake- alkalinesoda lake, pH 9 &salinity 8%

Halophiles

Dead Sea

Great Salt Lake coastalsplash zones

Solar salterns Owens Lake

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– 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

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

Substrate A

Product B

Product C

What are enzymes?

• Activation energy– Enzymes allow reactions with lower

energyEn

erg

y

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

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?

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

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

• 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

Life at High Temperatures, Thomas M. Brock

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– 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

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• Extremophiles–Where they live & how they survive

• They are interesting because– They have enzymes that work in

unusual conditions– The practical applications are

interesting