Courtesy of Dr' Deborah Kelley

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Courtesy of Dr. Deborah Kelley
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Microbiology of High Temperature Environments

• Matt
• Doug
• Kana
• Wes

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Thermophiles

• Discovered by Dr. Thomas Brock, 1967 in
  Yellowstone.
• First thermophile found was Thermus aquaticus
  Yellowstone type-1 (Taq YT-1)
• Thrive at temperatures above 45C

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Hyperthermophiles
Thermophiles
Rothschild Mancinelli, Nature 2001
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Taxonomy of Thermophiles

• Polyphyletic
• Archaea, Prokaryotes, Eukaryotes
• Thermophiles vs. Hyperthermophiles
• 50-70C vs. 70
• Not a phyletic distinction

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Thermophilic Eukaryotes

• Compost-dwelling fungus
• Cyanidium caldarium
• Rhodophyte of hot, acidic waters
• Alvinella pompejana
• Ephedra thermophila
• No known hyperthermophilic eukaryotes
• Possibility of such is much debated

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Thermophilic Prokaryotes

• Cyanobacteria
• Alkaline water of 70-73C
• Other photosynthetics with similar tolerances
• Heterotrophics
• Up to 90C
• Includes common genera such as Bacillis and
  Pseudomonas

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Thermophilic Archaea

• Thermophilic Heavyweights
• All four phyla contain thermophilic genera
• Euryarchaeota
• Crenarchaeota
• Korarchaeota
• Nanoarchaeota

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Euryarchaeota

• Largest phylum within the Archaea
• Methanopyrus
• Hyperthermophilic methanogen
• Thermococcus and Pyrococcus
• Optimum range of 70-100C
• Archaeoglobus
• True sulfate reducer
• Ferroglobus
• Shallow water hydrothermals around 80C
• Reduces nitrate and oxidizes iron
• May be responsible for Banded Iron Formations
  (1.9-2.6 Ga) previously attributed to
  cyanobacteria

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Crenarchaeota

• Small group, found in hot and cold environments
• Sulfabolus
• Aerobic chemolithotrophs and chemoorganotrophs
• Oxidizes iron, utilizes sulfate and organics
• 75C optimum
• Acidianus
• Aerobic and anaerobic chemolithotrophs and
  chemoorganotrophs
• 65-95C
• Pyrolobus fumerii
• Obligate chemolithotroph of hydrothermal vents
• Optimum temp. 106C, survives up to 121C
• Will survive an autoclave

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Korarchaeota

• Type locality- Obsidian Pool, Yellowstone
• First discovered through PCR analysis
• Currently being cultured, optimum temperature of
  85C determined
• Primitive
• 16S rRNA places it far from other Archaea
• Possibly ancestral to the division

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Nanoarchaeota

• Nanoarchaeum equitans (2002)
• External symbiote of Ignicoccus (Prokaryote)
• Undetectable by normal PCR tests
• 0.5 megabase genome
• Smallest known
• Humans have 3,000 megabases

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Effect of High Temperature

• Why cant all organisms survive?
• Microorganisms vary widely in temperature
  response
• Most microorganisms readily inactivate above 50C
• Bacterial spores often maintain viability at
  temps over 100C
• Some Archaea remain viable at temps well above
  100C
• Mechanisms of thermal inactivation
• Cytoplasmic membrane
• Typically consists of phospholipid bilayer with
  embedded (structural) proteins
• Fatty acids main constituents of lipids
• Temperatures increase - fatty acid chains begin
  to melt/separate
• Membrane becomes too fluid - cytoplasm begins to
  leak
• Cells lyse and death can occur
• Proteins break down as well
• No longer function as transport mechanism
• Interaction between proteins and lipids fall
  apart

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• Enzymes (Catalytic proteins)
• Not stable over wide range of temp
• Chain of amino acids covalently linked via
  peptide bonds (primary structure)
• Polypeptide chain folded back on itself - held
  together by hydrogen bonds (secondary structure)
• Hydrogen bonds in secondary structure breakdown -
  polypeptide chain unfolds
• Proteins denature become unstable, unravel,
  tangle with other proteins, form precipate
• Boiled egg - protein (egg white) precipitates,
  forms solid
• Once protein is unfolded chemical modifications
  take place, irreversibly inactivating proteins
• Peptide bond hydrolysis, deamidation, cysteine
  oxidation
• Unfolding irreversible in most mesophilic
  proteins
• Essential cellular processes stop - leads to cell
  death

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• DNA structure
• Double stranded (primary) structure - held
  together by hydrogen bonds
• Adenine-Thymine base pair (two hydrogen bonds)
• Guanine-Cytosine base pair (three hydrogen bonds)
• As temperature rises hydrogen bonds begin to
  break
• DNA strands separate/melt
• Typical DNA separates 65
• Strands can reform if cooled
• Secondary structure lost with continued temp rise
• RNA structure
• Single strand folded back on itself (secondary
  structure) - held together by hydrogen bonds
• As temp rises hydrogen bonds break - RNA unfolds
• Small molecules
• Molecules like ATP, GTP, NAD, FAD not very heat
  resistant, unstable
• Substrates
• Denature more rapidly with increased temperature
• No food source

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Mechanisms/Adaptations of Thermophiles to High
Temps

• Adaptation may be misnomer
• Two types of fundamentally different Thermophiles
• Ancestral Thermophiles (e.g. Themotoga, Aquifex)
• Only thermophilc ancestors (foundation for origin
  of life?)
• Evolved in thermophilic environments, thermophily
  built in from beginning
• Recent Thermophiles (e.g. thermophilic Bacillius
  and Clostridium sp.)
• Evolved from mesophilic organisms
• Recent adaptations
• Cytoplasmic membrane
• Bacteria and Eukarya - modify lipid bilayer
• Increase saturated fatty acids
• Strengthens hydrophobic interactions, holds
  membrane together
• Ratio of saturated to unsaturated fatty acids
  modified to optimize fluidity, strength
• Hydrocarbon chains vary in length (12-24 carbons)
  - longer higher Tm
• Bacterial hyperthermophile T. maritina contain
  glycerol ether lipids, not ester lipids like
  other bacteria - more heat resistant

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• Archaea different, more stable membrane all
  together
• Ether linkages between glycerol and hydrophobic
  side chains (fatty acids)
• Ether bonds more stable than ester
• Lack fatty acids altogether
• C40 hydrocarbons composed of repeating chains of
  five carbon compound isoprene
• Structure comprised of lipid monolayer in lieu of
  lipid bilayer
• Spans entire membrane not just inner or outer
  leaf
• Branching side chains
• In addition to being built from different
  components, in Archaea theside chains themselves
  are branched
• Branches join together, create more strength
• Five carbon rings are formed when one side branch
  bonds with another
• Much more stable at high temps
• Proteins
• More stable

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• Enzymes (Catalytic proteins)
• Little difference between proteins of
  hyperthermophiles and mesophiles
• Critical amino acid substitutions in one or a few
  locations
• Allows protein to fold in more dense, stable way
• Increased number of Disulfide bridges - increase
  stability, unfolding resistance
• Less of the amino acid glycine - which increases
  flexibility decreases stability
• Chaperones are also synthesized
• Proteins that help other proteins fold more
  densely
• Can also refold denatured proteins
• Other intracellular factors like coenzymes,
  substrates, general stabilizers such as
  thermamine also help resist unfolding
• Enzymatic function tuned to the organisms growth
  temp
• Exoenzymes are exception - optimal temp typically
  much higher than parent organism

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• DNA structure
• Contains many small, basic proteins
• Proteins bind duplex DNA, substantially
  increasing temperature stability
• Also compact or bend double stranded DNA upon
  binding - stronger yet
• Some contain Histone or histone-like proteins
  allowing formation of nucleosome like structure -
  raises melting temp
• Most Bacteria and Archaea have negatively
  supercoiled DNA
• Supercoiling altered with increasing temp,
  providing increased strength
• Extreme thermophiles in both Archaeal and
  Bacterial taxa possess reverse DNA gyrase protein
• Enables positive supercoiling of DNA
• Over winds DNA helix - much tighter, more
  stable
• Increased intracellular salt - strengthens DNA
• Contain variety of polyamines - increased
  structural strength
• Hyperthermophiles also thought to have unusually
  effective DNA repair mechanisms

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• RNA structure
• Modest changes in sequences and structures of RNA
  provides more stability
• Thermophilic RNA - rich in G-C base pairs
• More importantly - very low in G-U base pairs,
  mismatches, other irregularities that, in
  mesophiles lead to flexibility in the RNA
• RNA unusually short, no extra sequences
• Shorter sequences - fewer nonfunctional folding
  possibilities
• Alterations in protein binding can also
  stabilize RNA
• Small molecule stability
• May be biggest problem for thermophiles
• Tm of guanosine triphosphate (GTP) only a few
  seconds at 100C
• GTP required for translation, RNA synthesis and
  other processes
• ATP, NAD, FAD and others not very heat resistant
  either
• Critical to cell processes
• Likely synthesized on as needed basis so
  degradation loss not to great

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Is There an Upper Limit for Life? What is it
and Why?

• YES!
• Until the 60s it was thought to be around 60C
• 1967 Brock discovered Thermus Aquaticus in
  Yellowstone hot springs
• First organism to be identified capable of growth
  over 70C
• More recent studies
• Reported growth of Strain 121 at 121C (Autoclave
  temp)
• Survival up to 130C

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• Lab studies may underestimate upper limit
• Thermus aquaticus readily isolated from
  hydrothermal systems at 90-100C
• Wont grow reliably in lab above 80C
• Temp gradient around black smokers very step
• Can vary from 450C to 5C over tens of
  centimeters
• Horizontal transects have shown biological
  signals (16S rRNA) across much of transect
• Location makes correlating biological signatures
  to temp gradient difficult
• Consequently, upper limit likely higher than
  121C

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• Current understanding places upper limit around
  150C
• Energetic burden imposed by molecular repair and
  resynthesis unsustainable
• Could not prevent dissolution of chemical bonds
  maintaining integrity of DNA and other molecules
• Amino acids relatively stable up to 150C,
  degrade in seconds at 250C
• High temp amino acids become racemized (flip from
  L to D)
• Flipping would lead to irreversible denaturation
• Small molecules (GTP, ATP , NAD, etc.) denature
  very rapidly above 150C
• May actually dictate upper temp of life

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Thermozymes not just for the exam

• Stabilization of mesophilic enzymes
• Industrial applications
• Novel proteins
• Fame and fortune

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Modified Mesozymes

• Reaction stability
• High temperature reactions
• Chemical resistance
• Long-term stability
• Shelf life and activity length

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Novel Proteins

• 42 of the 25 top selling drugs in the US are
  from natural and derived natural products. (Bull
  et al. 2000)
• Possibility that deep sea diversity could exceed
  10 million species

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Fame and Fortune

• 1993 Nobel prize in chemistry awarded to Dr. Kary
  Mullis for inventing PCR.
• In 1991 Cetus sold Hoffman-La Roche the PCR
  patent for 300 million.

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Methods of Enzyme Manufacture

• Mesophile enzyme mutants
• Site Directed Mutagenesis
• Directed Evolution
• Hyperthermophile enzymes
• Direct extraction
• Mesophilic host

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Site Directed Mutagenesis
http//www.food.rdg.ac.uk/online/fs916/lect11/t5a2
.gif
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Directed Evolution

• Random point mutations introduced into the target
  gene followed by a screen for desired phenotype.
  Repeat for multiple mutants.
• More common approach as less prior knowledge of
  the protein is required.

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Bacillus subtilis substillin E engineering
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• Hyperthermophile extraction
• Cultivation of organism and isolation of enzyme

• Mesophilic Host
• Transfection of a hyperthermophilic gene into a
  host for expression.

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Commercial Uses of Enzymes

• Biotech
• DNA polymerases
• DNA ligases
• Restriction Enzymes
• Industry (potential uses)
• Starch degradation - amylases
• Cellulose degradation cellulases, xylanases

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Polymerase Chain Reaction
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Polymerase Fidelity

• Pfu 1.3 x 10-6
• exo- Pfu 5 x 10-5
• Vent 2.8 x 10-6
• Deep Vent 2.7 x 10-6
• Taq 8.0 x 10-6
• UlTma 5 x 10-6
• (mutation frequency/bp)

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Uses of PCR

• DNA cloning
• DNA fingerprinting
• Producing point mutations
• Quantifying mRNA
• Identification of diseases
• and much more!

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Courtesy of Dr. Deborah Kelley
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Where The Heat Comes From
Magma chamber acts as heat source and sets up
convection cells Entrained water is heated near
chamber and exits heated and full of dissolved
minerals Most hot springs are volcanically
created, but at least one is not.
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The Lost City Hydrothermal Field
Is Unlike Any Known Submarine Vent System
Hosted on 1-2 my old mantle rocks
60 m tall carbonate towers
Venting lt40-90C diffuse fluids, low metals,
low silica, pHs 9-11
Fluids are enriched in methane, hydrogen,
and other hydrocarbons
Macrofaunal communities sparse, unique
microbial populations
Fueled by rock-altering reactions does not
require volcanic heat
May be our closest analogue to early Earth?
Courtesy of Dr. Deborah Kelley
Courtesy of Dr. Deborah Kelley
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Early Atmosphere

• No free oxygen.
• Contained CH4, H2O, N2, NH3, H2S, CO2
• H2 and He were escaping to space because of the
  Earths weak gravity.
• Atmospheric pressure 10x what it is today.
• Temperature around 90C because of greenhouse
  effects.
• Venus and Mars have similar atmospheres, but are
  too close or too far from the sun to have liquid
  water.

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Europa

• Discovered January 7, 1610 by Galileo

• Second of the four Galilean moons

• Surface composition is Ice

• Tidal heat creates fissures and keeps
• interior water melted

• Tectonically active, ice rather than rock

• Believed to have hydrothermal vents
• under ice

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Hydrobot
Plans to test Hydrobot on Lake Vostok in
Antarctica Cryobot melts through the ice and
deploys AUV Hydrobot
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Courtesy of Dr. Deborah Kelley
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References

• Andrade et al, 1999. Extremely thermophilic
  microorganisms and their polymer-hidrolytic
• enzymes. Revista de Microbiologica
  30287-298
• Arnet, Bill. "Bilder av Jupiter og dens
  satellitter", 1999.
• http//www.astro.uio.no/ita/DNP/nineplanets/p
  xjup.htmlBull et al, 2000. Search and Discovery
  strategies for biotechnology the paradigm shift.
• Microbiology and Molecular Biology Reviews
  64 no. 3575-606.Cline et al, 1996. PCR fidelity
  of Pfu DNA polymerase and other thermostable DNA
  polymerases.
• Nucleic Acids Research 24, no. 18
  3546-3551.
• Daniel, R. 1996. The Upper Limits of Enzyme
  Thermal Stability. Enzyme and Microbial
• Technology. 1974-79.
• Eichler, J., 2001. Biotechnological uses of
  archaeal extremozymes. Biotechnology Advances
• 19261-278.
• Grogan, D. 1998. Hyperthermophiles and the
  Problem of DNA Instability. Molecular
• Microbiology. 28(6)1043-1049.
• Haki, G., and S. Rakshit. 2003. Developments in
  Industrially Important Thermostable Enzymes
• A Review. Bioresource Technology.
  8917-34.
• Hively, W. 1993. Life Beyond Boiling.
  Discover. May87-91.
• Huber, R., H. Huber, and K. Stetter. 2000.
  Towards the Ecology of Hyperthermophiles
• Biotopes, New Isolation Strategies and Novel
  Metabolic Properties. FEMS Microbiology
• Reviews. 24615-623.
• Itoh, Y., A. Sugai, I. Uda, and T. Itoh. 2001.
  The Evolution of Lipids. Adv. Space Res.

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Kelley, Deborah. Personal Communications April
27, 2005. University of Washington School of
Oceanography. Lurquin, Paul F. The Origins of
Life and the Universe, New York Columbia
University Press, 2003. Madigan, M., and B.
Mars. 1997. Extremophiles. Scientific
American, April82-87. Madigan, M. T., J. M.
Martinko, and J. Parker (2003) Brock Biology of
Microorganisms, Tenth Edition, Prentice
Hall, Pearson Education, Inc., 1019 pp. NASA.
"Life on Europa", http//www.resa.net/nasa/europa_
life.htm NASA. "Solar System Exploration
Jupiter Moons", http//solarsystem.nasa.gov/
planets/profile.cfm?ObjectJupiterDisplayMoons R
obb, F., and D. Maeder. 1998. Novel
Evolutionary Histories and Adaptive Features of
Proteins from Hyperthermophiles. Current
Opinion in Biotechnology. 9288-291. Rothschild,
L. and Mancinelli, R., 2001. Life in extreme
environments. Nature 4091092-1101 Russell, A.
2003. Lethal Effects of Heat on Bacterial
Physiology and Structure. Science Progress.
86115-137. Stetter, K. 1999. Minireview
Extremophiles and Their Adaptation to Hot
Environments. FEBS Letters.
45222-25. Takai, K., T. Komatsu, F. Inagaki, and
K. Horikoshi. 2001. Distribution of Archaea in
a Black Smoker Chimney Structure. Applied
and Environmental Microbiology.
67(8)3618-3629. Tritt, Charles S. "Possibility
of Life on Europa", http//people.msoe.edu/tritt/
sf/europa.life.html Vieille, C., and G. Zeikus.
2001. Hyperthermophilic Enzymes Sources, Uses,
and Molecular Mechanisms for
Thermostability. Microbiology and Molecular
Biology Reviews. 65(1)1-43.