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 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, FractionSurviving () 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 growthSpecific rate 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 A good theory destroyed by a nasty little fact 100 D. radiodurans 10-1 -2 10 Fraction 10-3 R. xylanophilus Surviving 10-4 R. radiotolerans RG-1T 10-5 RG-3T 10-6 0 200 400 600 800 1000 1200 UV flux (Joules/m2) R. xylanophilus survived 2 weeks but not 4 weeks R. radiotolerans survived 16 weeks but not 20 weeks Strains RG-1T and RG-3T survived beyond 44 weeks all at <5% relative humidity. 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 Growth rate 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