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 1950s
Extensively studied since 1970s
Temperature extremes
Boiling or freezing, 100
0
C to -1
0
C
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 earths surface is oceans between 1-
4
0
C
Deep sea 1
0
C to 4
0
C
Most rely on photosynthesis
Thermophiles
Hydrothermal Vents- Black
smokers at 350
o
C
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 waters
at Iron Mountain
Alkaliphiles
Mono Lake- alkaline
soda lake, pH 9 &
salinity 8%
Halophiles
Dead Sea
Great Salt Lake coastal
splash 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
energy
E
n
e
r
g
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
45
0
C (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 dont 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 cells 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