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
PROTEOBACTERIA
Algae
Hydrogenophilus (Dunaliella)
Colwelia
Halomonas
Thiobacillus
Polaromonas
BACTERIA
CYTOPHAGALES
Rhodothermus
Polaribacter
CYANOBACTERIA
Synechococcus
Fungi
(Debaryomyces)
ARCHAEA
Sulfolobus
Aeropyrum
GRAM-POSITIVE
Alicyclobacillus
Bacillus
Clostridium
Rubrobacter
Thermus
Deinococcus
Hyperthermus
Pyrobaculum
Hyperthermophiles
Thermophiles
Halophiles
Pyrodictium
pH Extremes
Psicro-/Piezophiles
Thermotoga
Radiation Resistant
Pyrococcus
Methanothermus
Dyctioglomus
Archaeoglobus
Methanopyrus
Aquifex
Thermoplasma
Picrophilus
Halococcus
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
Reported to grow at 121
ºC
(autoclave
temp.).
Crenarchaeote
(Kashefi
and Lovley, 2003).
120
Pyrolobus fumarii
Methanopyrus kandleri
Pyrococcus furiosus
100
Thermococcus celer
Thermotoga maritima
Acidianus infernus
80
Thermus aquaticus
Rhodothermus marinus
Meiothermus ruber
(ºC)
60
Meiothermus chliarophilus
Deinococcus murrayi
Escherichia coli
40
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
Relative activity (%)
100
D. ethenogenes
O.G.T. - 30ºC
80
P. horikoshii
O.G.T.- 98ºC
60
40
20
0
0
20
40
60
80
Temperature (ºC)
O.G.T., Optimum Growth Temperature
100
120
pH
pH Range for Growth of Some Bacteria and Archaea
Growth rate
Picrophilus
Esherichia Bacillus Clostridium
torridus Thiobacillus Alicyclobacillus
alkalophilus paradoxus
coli
acidophilus acidocaldarius
0.0
2.0
4.0
6.0
8.0
pH
10.0
12.0
14.0
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
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
Truepera radiovictrix
Extreme UV- and gamma-radiation resistance
Nuclear explosions, nuclear reaction cores, nuclear waste and
nuclear disasters, as at Chernobyl, produce extreme gammaradiation.
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 extremeradiation 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
Deinococcus navajoensis
Deinococcus hopiensis
Deinococcus apachensis
Deinococcus maricopensis
Deinococcus pimensis
Deinococcus yavapaiensis
Deinococcus papagoensis
Deinococcus sonorensis
100
NA
RM
PCA
10
% survival
1
0.1
0.01
0.001
0.0001
0
5
10
15
20
Radiation exposure (kGy)
25
30
35
Fred Rainey et al. 2005, AEM,
Gamma-Radiation Resistance of Deinococcus and Rubrobacter spp.
100
Surviving fraction
10-1
Rubrobacter radiotolerans
10-2
10-3
Deinococcus radiodurans
10-4
Esherichia coli
10-5
Rubrobacter xylanophilus
10-6
0
4
8
12
16
Dose (kGy)
20
24
28
What makes a organism radiation resistant?
There is a close relationship between
bacterial ionizing-radiation resistance and
desiccation tolerance.
10 1
10 0
Dried bacterial cells exhibit a substantial
number of DNA DSBs, single-strand
breaks, and DNA crosslinks as happens
during ionizing-radiation exposure.
10 -1
10 -2
10 -3
Surviving Fraction
10 -4
10 1
10 -5
0
2
4
6
8
10
12
14
16
10 0
Gamma (kGy)
10 -1
10 -2
Callegan et al. 2008, IJSEM 58:1252-8
10 -3
Surviving Fraction
Effect of Gamma-radiation and desiccation on the
survival of strains () D. radiodurans, () D. claudioa,
() D. radiomolis, (), and () D. alpinitundrae .
10 -4
10 -5
0
10
20
Desiccation (Days)
30
40
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
0,16
0,6
0,5
0,12
0,4
0,08
0,3
0,2
0,04
0,1
0
Specific growth rate
Solutes (umol/mg)
0,7
Mannosylglycerate
Trehalose
di-myo-Inositol-P
di-N-Acetylglucosamine-P
Glycine betaine
Growth Rate
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
Surviving Fraction
10-1
10-2
R. xylanophilus
10-3
R. radiotolerans
10-4
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
Growth rate
Non-halotolerant
Slight halophile Moderate halophile
Extreme halophile
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
CH2OH
Glycerol, arabitol, mannitol
O
1
1
OH
ECTOINES
HO
O
OH
CH2OH OH
O
Ectoine, hydroxyectoine
OH
Trehalose
Compatible solutes of (hyper)thermophiles
MANNOSYLGLYCERATE
DI-MYO-INOSITOL-PHOSPHATE
DIGLYCEROL PHOSPHATE
MANNOSYL-DI-MYO-INOSITOL-PHOSPHATE
GALACTOSYL-5-HYDROXYLYSINE
MANNOSYLGLYCERAMIDE
GLUCOSYLGLYCERATE
CH2 OH
HO
O
OH
HO
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 classlevel 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
Salinibacter ruber
Archaeon
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
Nuno Empadinhas
Joana Costa
Susana Alarico
Ana Luisa Nobre
Zélia Silva
André Antunes
Fernanda Nobre
Igor Tiago
Catarina Ferreira
Célia Manaia
Olga Nunes
Prof. Helena Santos
Pedro Lamosa
Lígia Martins
Nuno Borges
Louisiana State University
Prof. Fred A. Rainey
DSMZ
Peter Schumann