PowerPoint Presentation - Extremophiles

beefzoologistΒιοτεχνολογία

21 Φεβ 2013 (πριν από 4 χρόνια και 5 μήνες)

171 εμφανίσεις

Extremophiles


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

Extraterrestrial microbial
life
-
does it exist?

What will it be like?

Lecture Aims



What are Extremophiles
-

an introduction


Strategies for growth & survival


Biotechnology

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, 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


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

Thermophiles
-

volcanic

Hydrothermal Vents
-

Black
smokers at 350
o
C

Obsidian Pool,

Yellowstone National Park


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)


Thermophiles



Great Artesian Basin, Australia

Bore well 17263

Temp: 88
o
C

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

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

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 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

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.

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


o
What are the adaptation, protection and evolutionary strategies?

o

Structural

o

Cellular

o

Molecular

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.



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)

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??

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)

NaCl (%)
0
20
40
60
80
100
120
140
0
5
10
15
20
25
Temperature (oC)
Hyperthermophiles

Mesophiles & psychrophiles

Thermophiles

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

Genetic prospecting


What is it?


Think of a hunt for the genetic gold


Thermophiles



Great Artesian Basin, Australia

Bore well 17263

Temp: 88
o
C

Sequencing

HMW DNA

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

PCR Gene library

Data analysis

PCR

Bharat Patel
©

A systems biology approach: Integrative bioprospecting assists fundamental research (evolution,
adaptation) & biotechnology

Preservation


Novel microbes

B. Microbiology

Electron microscopy

Micromanipulation

Anaerobes

Direct isolation (plating)

Directed enrichment

CORE

Unix

Linux
PC

Antimicrobials

Novel Products

Rapid screening of native

& recombinant products

Genomic library

(BAC,

pBSK,

Expression vectors)


Enzymes

Others

Screening
-

Mass
spectrometry

Purification

Crystal growth

Data collection

Thermostable dextranases

Great Artesian Basin, Australia

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

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