extremophiles km

YellowstonePinwheel Geyser : 

YellowstonePinwheel Geyser Bacteria Thrive Near Boiling: T ~ 80 C Water: Acidic as battery acid Each color a different species of bacterium

Yellowstone National Park : : 

Yellowstone National Park : Suitable for Life ? Boiling Geysers Freezing winter Sulfuric acid. One of the Least Hospitable Places on Earth:

Life at High Temperatureand High Acidity : 

Life at High Temperatureand High Acidity Temp = 65 C pH = 2 Yellowstone

YellowstoneChurning Cauldron : 

YellowstoneChurning Cauldron Boiling Temp

What are Alkaliphiles? : 

What are Alkaliphiles? These are organisms that require growth at pH 9 and above.

Biotechnological Impact? : 

Biotechnological Impact? Cellulase103 (Genecor NY based) Enzyme can function regularly at pH 10 Function in hot or cold water Breaks down cellulose fibers that trap dirt on cotton textiles Does this without as much harm as other additives

What are Halophiles? : 

What are Halophiles? Organisms that require and prosper in high salt concentrations

Biotechnological Impact? : 

Biotechnological Impact? Dunaliella Macroalgae Glycerol 80% of dry weight Food industry Beta-carotene 12% of dry weight Fight against cancer

What are Acidophiles? : 

What are Acidophiles? Organisms that grow at pH 3 or below

Where are Acidophiles? : 

Where are Acidophiles? Volcanic areas Acidic mud pots Yellowstone Acid springs and streams

Biotechnological Impact? : 

Biotechnological Impact? Leach metals from insoluble ores Mainly copper Thiobacilli Uranium Leaching

What are Thermophiles? : 

What are Thermophiles? Organisms that thrive between 60º-80°C

Where are Thermophiles? : 

Where are Thermophiles? Hot springs Mud pots Geysers Hot water beaches Heated lakes

Biotechnological Impact? : 

Biotechnological Impact? Waste treatment Fuel production Remove sulfur compounds from crude oil

Final thought? : 

Final thought? Extremophiles have opened the door to a new era of biotechnology “We have yet to fully realize the benefits of these organisms” -Mark Madden, biochemist

Extremophiles : 

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

Extremophile : 

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

Slide 19: 

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

Slide 20: 

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 106 km2 = 8.7 x 1012 m3) 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)

Slide 21: 

Thermophiles – Great Artesian Basin, Australia Bore well 17263 Temp: 88oC

Chemical Extremes : 

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

Slide 23: 

Acidophiles pH 0-1 of waters at Iron Mountain

Slide 24: 

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

Slide 25: 

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

Survival : 

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

Survival : 

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? : 

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 : 

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 : 

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 : 

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 : 

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

PCR - Polymerase Chain Reaction : 

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

Acidophiles : 

Acidophiles Enzymes used to increase efficiency of animal feeds enzymes help animals extract nutrients from feed more efficient and less expensive

Alkaliphiles : 

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

Osmoregulation : 

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 : 

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 : 

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 : 

“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 : 

“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 : 

“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 : 

“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: 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 : 

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 : 

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 : 

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 : 

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 : 

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.

Slide 49: 

Life in hot salts Thermohalophiles which adapt to high temperatures and salts Halothermothrix orenii Thermohalobacter berensis What is their habitat? Isolation sources? Salt lakes Oil fields What are the adaptation, protection and evolutionary strategies? Structural Cellular Molecular

The thermohalophilic extermophiles : 

The thermohalophilic extermophiles Only two truly halothermophiles known to date: Halothermothrix orenii: (optimum 60oC + 10% NaCl; <65oC + < 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 65oC + 5% NaCl; 70oC 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 : 

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)

Slide 52: 

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?? Combination

More on molecular adaptation strategies : 

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)

Slide 54: 

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 : 

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

Slide 56: 

Thermophiles – Great Artesian Basin, Australia Bore well 17263 Temp: 88oC

Slide 58: 

Thermostable dextranases Great Artesian Basin, Australia

Unique characteristics of Archaea : 

Unique characteristics of Archaea Cell membrane Single layer Pseudopeptidoglycan or protein L-glycerol (stereoisomer) Ether linkage (C-20 diether lipids) Some tetraether molecules (C-40 tetraether lipids) Branching hydrophobic side chain Carbon ring formation Resistant to lysozyme and beta-lactam antibiotics Flagella have unique composition and development

Cell Membrane : 

Cell Membrane

Unique Characteristics : 

Unique Characteristics Metabolic differences ADP dependent kinase (not ATP) Pyrophosphate-linked kinases (not pyrophosphate dependent phosphofructokinases) Organotrophs, autotrophs, and an unusual form of photosynthesis No Archaea uses the full respiration or photosynthetic cycles, but instead employs many of the steps individually Methanogens and some extreme thermophiles use glycogen instead of glucose

Unique Characteristics : 

Unique Characteristics Intracellular bodies rRNA (16S) sequence tRNA Plasmids Lack of organelles (similar to bacteria)

Unique Characteristics : 

Unique Characteristics Genetic Material Resistance to denaturation by heat seen in thermophiles Similar structure to bacteria Some sequencing has revealed sections of DNA that are shared with bacteria (gene sharing between bacteria and archaea?) Primary protein sequence is similar to Eukarya Genes with similar functions organized together (similar to operons) Introns are found in rRNA and tRNA genes

Unique Characteristics : 

Unique Characteristics Replication DNA Polymerase similar to that of eukaryotes, eukaryal virues and E. coli 3’-5’ exonuclease (proofreading) Restriction endonuclease Topoisomerase Gyrase Halobacterium halobium has reverse transcriptase

Unique Characteristics : 

Unique Characteristics Transcription RNA polymerase has up to 14 subunits (E. coli has only 4) and is similar to eukaryotes Requires general transcription factors to initiate (like eukarya) Promoters have an A-T rich sequence similar to eukarya TATA box Translation Signals similar to bacteria

Archaea : 

Archaea Extremophiles Evolutionarily Primitive Formerly known as Archaeabacteria

History : 

History Originally grouped with Bacteria Recognized in 1977 Carl Woese and George Fox 16S rRNA sequencing Greek archaea “ancient” Common ancestor thought to be a simplistic prokarya with poorly organized genetic material Thought to be involved in evolution of Eukarya-not accepted

Morphology : 

Morphology Spherical, rod-shaped, spiral, lobed, filamentous, or rectangular

Morphology : 

Morphology 0.1-15 microns Single circular chromosome Single cell membrane Flagella No organelles

Ecology : 

Ecology Extremophiles (coined 1974) Thermophiles (up to 113C) Black smokers Geysers Psycrophiles Acidophiles and Alkaliphiles Halophiles Some combine extremes, ie Picrophilus (~60C and 0.5pH) Methanogens Often found in the guts of ruminants, termites and even humans Found in all known environments

Adaptations to Extremes : 

Adaptations to Extremes In extreme pH must avoid hydrolysis of proteins-achieved by changing internal pH Anaerobes do not maintain stasis, while aerobes do Specific enzymes are active at optimal pH Structure of cell membrane stabilized in high temperature environments by: Allows for formation of carbon rings which increases stability Ether linkage is less reactive than ester linkage Tetraether molecules Can form monolayers (Sulfolobus and Thermoplasma)

Adaptations to Extremes : 

Adaptations to Extremes Protection of genetic material High salt concentrations in cytoplasm DNA binding proteins similar to eukaryotic histones Share amino acid homology MC1-Methanosarcinaceae HMf-Methanobacteriales Organizes DNA in sturctures similar to chromatin Allows for positive supercoiling Eukarya have negative supercoiling (nucleosome) HTa-Thermoplasma HTa (like)-Sulfolobus

Evolution : 

Evolution Primitive form Related to Eukarya tRNA Ribosomes TATA binding proteins and TFIIB (transcription) Similar initiation and elongation factors for translation Similarities to bacterial genetic material

Archaea : 

Archaea Hot springs Deep sea vents