What, if anything, is an Extremophile?
Milton S. da Costa Universidade de Coimbra, Portugal
Microbiotec 15 11 Dezembro 2015 Universidade de Évora What, if anything, is an Extremophile?
Albert E. Wood (1957)What, If anything, is a Rabbit? Evolution, Vol. 11, 417-425.
Stephen J. Gould (1983) What, If anything, is a Zebra?, Hen's Teeth and Horse's Toes: .
Equus koagga
Equus caballus Sorraia
Equus koagga What, if anything, is an Extremophile?
The term Extremophile was first used by:
Macelroy, R. M., (1974) Some Comments of the Evolution of Extremophiles. Biosystems, 6: 74-75.
(The same issue of Biosystems contained other papers about “microorganisms living in extreme environments”)
The term extremophile is sometimes used to justify biotechnological research; ”Biotechnology of Extremophiles”, “Hotsolutes”. “Hypersolutes”, etc.
We now tend to define extremophiles as organisms that live in environments where diversity is low.
The problem then, becomes circumscribing and defining a microbial environment.
Should we consider Mycobacterium tuberculosis to be an extremophile? A Journal Called “EXTREMOPHILES”
Extremophiles
Chief Editor: Garo Antranikian Examples of Extremophiles
TEMPERATURE Psychrophiles grow at very low temperatures Thermophiles grow at high temperatures pH Acidophiles grow in environments of low pH Alkaliphiles grow in environments of high pH
WATER ACTIVITY Osmophiles grow environments with high concentrations of salts or sugars Halophiles grow in environments with high NaCl concentrations
HYDROSTATIC PRESSURE Piezophiles grow better at hydrostatic pressures higher than atmospheric pressure, some are not able to grow at normal atmospheric pressure
RADIATION and/or DESICCATION Extremely radiation-resistant organisms survive and grow under extreme UV and gamma radiation and survive extreme desiccation Extremophiles in the Tree of Life
EUKARYA Animals Fungi PROTEOBACTERIA (Debaryomyces) Algae Hydrogenophilus (Dunaliella) Colwelia ARCHAEA Halomonas Sulfolobus Thiobacillus Polaromonas Aeropyrum Hyperthermus BACTERIA GRAM-POSITIVE Alicyclobacillus Bacillus Hyperthermophiles CYTOPHAGALES Clostridium Pyrobaculum Thermophiles Rhodothermus Rubrobacter Halophiles Polaribacter Pyrodictium Thermus pH Extremes Deinococcus CYANOBACTERIA Psicro-/Piezophiles Synechococcus Thermotoga Radiation Resistant Pyrococcus Methanothermus Dyctioglomus Archaeoglobus
Methanopyrus Halococcus Aquifex Thermoplasma Picrophilus Natronococcus Why Study Extremophiles?
Extremophiles inhabit unusual environments,
Extremophiles have peculiar physiological, biochemical and molecular characteristics,
Extremophiles represent the rare products of 4 billion years of evolution,
Extremophiles have important enzymes and products for industry and health.
Extremophiles may be useful to understanding life on other planets.
When asked why he was attempting to climb Mount Everest in 1924, George Mallory answered; “Because it is there”. He died in this attempt. Thermophiles Optimum Growth Temperatures of Some Bacteria and Archaea
Strain 121 120
Reported to grow at 121 Pyrolobus fumarii ºC (autoclave temp.). Methanopyrus kandleri Crenarchaeote (Kashefi 100 and Lovley, 2003). Pyrococcus furiosus Thermococcus celer Thermotoga maritima Acidianus infernus 80 Thermus aquaticus
Rhodothermus marinus (ºC) Meiothermus ruber 60 Meiothermus chliarophilus Deinococcus murrayi 40 Escherichia coli
Deinococcus grandis
Vibrio marimus 20 Micrococcus cryophilus
Polaromonas vacuolata 0 Thomas D. Brock in Yellowstone National Park
Tom Brock went to Yellowstone National Park in 1965 and brought an ecological perspective to life at high temperatures. It was now possible to isolate thermophiles from specific environments. Microbiologists now knew where to look for thermophiles. Optimum Temperature for Activity of Mannosylphosphoglycerate Synthase
T. thermophilus O.G.T.- 70ºC 100 D. ethenogenes P. horikoshii 80 O.G.T. - 30ºC O.G.T.- 98ºC
60
40
20 Relative Relative activity (%)
0 0 20 40 60 80 100 120
Temperature (ºC)
O.G.T., Optimum Growth Temperature pH pH Range for Growth of Some Bacteria and Archaea
Picrophilus Esherichia Bacillus Clostridium torridus Thiobacillus Alicyclobacillus coli alkalophilus paradoxus
acidophilus acidocaldarius Growth rate Growth
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 pH Truepera radiovictrix
The strains were isolated from fresh water hot springs with neutral pH.
Truepera radiovictrix is extremely radiation resistant, like the species of the Deinococcus.
Thermus spp. are not.
Optimum growth temperature: 50ºC Truepera radiovictrix Optimum pH for growth: 8.5 to 9.5 Optimum salinity for growth: 1.0 % NaCl Heterotrophic, Aerobic and fermentative (homolactic)
Albuquerque et al., 05 Extreme UV- and gamma-radiation resistance
Nuclear explosions, nuclear reaction cores, nuclear waste and nuclear disasters, as at Chernobyl, produce extreme gamma- radiation.
Natural environments with high gamma-radiation do not exist on the Earth’s surface and UV-radiation has low power of penetration through water and soil.
Which natural selective pressure drove the acquisition of extreme- radiation resistance? Deinococcus and Rubrobacter spp. survive extreme desiccation
From one gram of soil in the Sonora Desert in Arizona we recovered strains of nine new species of Deinococcus, plus several environclones of Rubrobacter.
Deinococcus hohokamensis 100 Deinococcus navajoensis
NA 10 RM Deinococcus hopiensis PCA Deinococcus apachensis 1 Deinococcus maricopensis
0.1 Deinococcus pimensis
% survival % Deinococcus yavapaiensis 0.01 Deinococcus papagoensis 0.001 Deinococcus sonorensis
0.0001 0 5 10 15 20 25 30 35 Fred Rainey et al. 2005, AEM, Radiation exposure (kGy) Gamma-Radiation Resistance of Deinococcus and Rubrobacter spp.
100
10-1 Rubrobacter radiotolerans
10-2
10-3
10-4 Deinococcus radiodurans Surviving fraction Surviving
Esherichia coli 10-5 Rubrobacter xylanophilus
10-6 0 4 8 12 16 20 24 28 Dose (kGy) What makes a organism radiation resistant?
There is a close relationship between
101 bacterial ionizing-radiation resistance and desiccation tolerance. 100 Dried bacterial cells exhibit a substantial 10-1 number of DNA DSBs, single-strand breaks, and DNA crosslinks as happens -2 10 during ionizing-radiation exposure.
10-3
Surviving Fraction Surviving
10-4
101 10-5 0 2 4 6 8 10 12 14 16 100 Gamma (kGy)
10-1
10-2
Effect of Gamma-radiation and desiccation on the 10-3 survival of strains () D. radiodurans, () D. claudioa, Surviving Fraction () D. radiomolis, (), and () D. alpinitundrae . 10-4
10-5 Callegan et al. 2008, IJSEM 58:1252-8 0 10 20 30 40 Desiccation (Days) Rubrobacter spp. Accumulate Compatible Solutes
The species of the genus Rubrobacter represent the most ancient lineage of the Actinobacteria; are extremely radiation resistant; have optimum growth temperatutes between 45 and 60ºC; and are halotolerant.
Minimal Medium, 60ºC 0,7 0,16 0,6
0,5 Mannosylglycerate 0,12 Trehalose 0,4 di-myo-Inositol-P 0,3 0,08 di-N-Acetylglucosamine-P Glycine betaine 0,2 0,04 Growth Rate
0,1
Specific growthSpecificrate Solutes(umol/mg)
0 0 0.0 2,5 4.0 5.0 NaCl (%)
These organisms accumulate trehalose and mannosylglycerate under all conditions examined. What is their role?
Empadinhas et al., 2007, Extremophiles
Surviving Fraction 10 10 10 10 10 10 10 ------6 5 4 3 2 1 0
0
A good theory destroyed by a nasty little fact all at <5% relative humidity.relative <5% all at Strains R. R. 200 RG RG R. radiotolerans xylanophilus
- - radiotolerans 3 1 T T RG 400 - 1 T
and RG UV survived 2 weeks but not 4 weeks 4 not but weeks 2 survived survived 16 weeks but not 20 weeksnot survived 16 but
flux R.
xylanophilus (Joules/m 600 - 3
T survived 44 beyondsurvived 2 )
800
D. radiodurans 1000
weeks weeks 1200
High Salt Environments
Artificial extremely saline evaporation ponds Salt composition similar to seawater
Natural terrestrial salt environment Salt composition different from seawater pH Range for Growth of Some Bacteria and Archaea
Haloterant Non-halotolerant
Slight halophile Moderate halophile
Extreme halophile Growthrate
NaCl Saturated Compatible Solutes of Mesophiles
AMINO ACIDS AND DERIVATIVES Glutamate, proline, alanine, glutamine Ne-acetyl-b-lysine, betaine
SUGARS AND HETEROSIDES Trehalose, glucosylglycerol, galactosylglycerol, glucosylglicerate
POLYOLS OH Glycerol, arabitol, mannitol CH2OH O 1 1 OH ECTOINES
HO OH O CH2OH OH Ectoine, hydroxyectoine O OH Trehalose Compatible solutes of (hyper)thermophiles
MANNOSYLGLYCERATE
DI-MYO-INOSITOL-PHOSPHATE
DIGLYCEROL PHOSPHATE
MANNOSYL-DI-MYO-INOSITOL-PHOSPHATE
GALACTOSYL-5-HYDROXYLYSINE CH2OH O HO MANNOSYLGLYCERAMIDE OH HO GLUCOSYLGLYCERATE O CH 2OH Mannosylglycerate COOH
CYCLIC 2,3-BISPHOSPHOGLYCERATE (methanogens)
ASPARTATE
GLUTAMATE
TREHALOSE Salt brines of the Red Sea Haloplasma contractile
Shaban Deep is an abyssal salt brine in the Red Sea.
Haloplasma contractile was isolated from the brine/sediment interface at a depth of 1447 meters.
The organism represents a new class- level lineage within the Bacteria.
Antunes et al., 2008, J. Bacteriol. Way down below the ocean, where I want to be…
Discovery Urania Kryos Thetis
Medee
Palleronia abyssalis, Albuquerque et al., 2015. 5.000 meters in the Med
Natrinema salaciae, Albuquerque et al., 2014 Abyssal Salt brine, Thetis. “Extremophiles inhabit environments of low species diversity”
Obsidian Pool, YNP. Temp. 75 to 95ºC. Rich in sulfide, Fe+2,
CO2.
Rich bacterial diversity, but most clones would be expected to be thermophilic. Few archaea. Novel higher level taxa encountered.
This environment contains are large diversity Comprising many different bacterial groups.
Hugenholtz et al., JB, 1998 High salt environments
Lake Texcoco saline and alkaline soils, Mexico.
Using archaeal specific 16S rRNA primers, both extremely alkalihalophilic archaea and non-alkaliphilic extreme halophilic archaea were detected.
No other archaeal clones encountered.
Valenzuela-Encinas et al., Extremophiles, 2008 A String of Pearls in a bog
A two member biofilm called a String of Pearls was found in a bog near Regensburg, Germany by Rober Huber and collaborators. This biofilm is composed of one species of Thiothrix (red) and an unnamed Euryarchaeote (Yellow).
Henneberger et al. 2006. AEM 72:192
Is this an extreme environment? The salt pans of Maiorca
Two predominant species of saturated salt pans; the other other organisms are minor components of the ecosystem;
Haloquadratum walsby Archaeon Salinibacter ruber Bacteria “Sometimes I feel lonely”
Candidatus Desulfurodis audaxviator was found in a South African mine at a depth of 2.8 km. The organism lives along fractures with water at temperatures of about 60ºC and pH 9.3.
Candidatus Desulfurodis audaxviator is a member of the low G+C Gram + bacteria. And is closely related to the genus Clostridum. Forms spores and possesses genes for catabolism of sugars.
Fixes nitrogen and reduces sulfate. Fixes CO2. The organism may be chemoautotrophic obtaining energy from the radioactive
decay of uranium through the formation of H2. A big IF.
It is practically the only organism found in this environment No Bacteria, no Plants, only the Occasional Human
This area in the Central Atacama Desert, Chile, where there is no recorded rainfall, has no detectable Life. Courtesy of Fred Rainey What is an Extreme Environment?
Caldeira Grande, S. Miguel Sulfur on Vulcano Italy
Crater on Vulcano Island Italy
We now generally define an extreme environment as one with low species diversity. How do we circumscribe a microbial environment?
We now generally define an extreme environment as one with low species diversity. But how do we circumscribe an extreme environment?
Boiling water
About 250ºC No Life
About 2ºC
About 70ºC
In most hot spring there is considerable mixing between temperature gradients and we are insure where the sample came from.
We have isolated Legionella spp. from hot springs with temperatures of about 60ºC, where they survive for no more than half an hour. Should biofilms with only one species (strain) be considered extreme environments?
Biofilms can be communities of microorganisms where species consortia work depending on each other’s metabolic abilities.
Many biofilms have a high species diversity, but others do not.
Some biofilms are composed of only one strain.
Are lungs with cystic fibrosis an extreme environment?
Is septicemic blood an extreme environment?
Do macrophages infected with Legionella, Mycobacterium or Salmonella represent extreme environments? So what is an Extremophile?
1. Extremophiles live in low diversity environments.
In this case a huge number of different organisms are extremophiles. Perhaps, all organisms.
2. Extremophiles live where we cannot live.
In this case we are back to the old definition by which we are basing other forms of life on our own capabilities. We are being antrophomorphic. This definition is considered arcane and non-scientific.
The term extremophile serves as an umbrella definition for the organisms like the ones described here and many others. An it should remain as such. We should not try to define an extremophile, because it may not exist.
Esherichia coli Pseudomnas aeruginosa Epulopiscium sp. The People
Coimbra ITQB, Lisbon
Luciana Albuquerque Prof. Helena Santos Nuno Empadinhas Pedro Lamosa Joana Costa Lígia Martins Susana Alarico Nuno Borges Ana Luisa Nobre Zélia Silva André Antunes Fernanda Nobre Louisiana State University Igor Tiago Catarina Ferreira Prof. Fred A. Rainey Célia Manaia Olga Nunes DSMZ Peter Schumann