What, if anything, is an Extremophile?

Milton S. da Costa

Universidade de Coimbra, Portugal

Microbiotec 15

11 Dezembro 2015

Universidade de Évora

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 koagga

Equus caballus

Sorraia

What, if anything, is an Extremophile?

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

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

Examples of Extremophiles

CYANOBACTERIA

Synechococcus

PROTEOBACTERIA

HydrogenophilusColwelia Halomonas

Thiobacillus

Polaromonas

GRAM-POSITIVE

Alicyclobacillus

Bacillus

Clostridium

Rubrobacter

ThermusDeinococcus

Thermotoga

Aquifex

Sulfolobus

Aeropyrum Hyperthermus

Pyrobaculum

Pyrodictium

Pyrococcus

Methanopyrus

Methanothermus

HalococcusThermoplasma

Animals

Dyctioglomus

Archaeoglobus

CYTOPHAGALES

Rhodothermus

Polaribacter

Picrophilus Natronococcus

BACTERIA

EUKARYA

ARCHAEA

Hyperthermophiles

Thermophiles

Halophiles

pH Extremes

Psicro-/Piezophiles

Radiation Resistant

Fungi (Debaryomyces)

Algae(Dunaliella)

Extremophiles in the Tree of Life

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.

Why Study Extremophiles?

Thermophiles

Meiothermus chliarophilus

Polaromonas vacuolata

Micrococcus cryophilus

Vibrio marimus

Deinococcus grandis

Escherichia coli

Deinococcus murrayi

Meiothermus ruber

Rhodothermus marinus

Thermus aquaticus

Acidianus infernus

Thermotoga maritima

Thermococcus celer

Pyrococcus furiosus

Methanopyrus kandleri

Pyrolobus fumarii

Optimum Growth Temperatures of Some Bacteria and Archaea

(ºC)

0

20

40

60

80

100

120Strain 121

Reported to grow at 121

ºC (autoclave temp.).

Crenarchaeote (Kashefi

and Lovley, 2003).

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.

Thomas D. Brock in Yellowstone National Park

Optimum Temperature for Activity of Mannosylphosphoglycerate Synthase

T. thermophilus

O.G.T.- 70ºC

0

20

40

60

80

100

0 20 40 60 80 100 120

Temperature (ºC)

Re

lati

ve a

cti

vit

y (

%) D. ethenogenes

O.G.T. - 30ºCP. horikoshii

O.G.T.- 98ºC

O.G.T., Optimum Growth Temperature

pH

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0

pH

Picrophilus

torridusEsherichia

coli

Bacillus

alkalophilusClostridium

paradoxusAlicyclobacillus

acidocaldarius

Thiobacillus

acidophilus

Gro

wth

ra

te

pH Range for Growth of Some Bacteria and Archaea

Truepera radiovictrix

Truepera radiovictrix

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)

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.

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

Radiation exposure (kGy)

0 5 10 15 20 25 30 35

% s

urv

iva

l

0.0001

0.001

0.01

0.1

1

10

100

NA

RM

PCA

Deinococcus hohokamensis

Deinococcus navajoensis

Deinococcus hopiensis

Deinococcus apachensis

Deinococcus maricopensis

Deinococcus pimensis

Deinococcus yavapaiensis

Deinococcus papagoensis

Deinococcus sonorensis

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.

Fred Rainey et al. 2005, AEM,

Gamma-Radiation Resistance of Deinococcus and Rubrobacter spp.

0 164 128 20

Dose (kGy)

Su

rviv

ing

fra

cti

on

24 28

10-6

10-5

10-4

10-3

10-1

10-2

100

Deinococcus radiodurans

Rubrobacter xylanophilus

Rubrobacter radiotolerans

Esherichia coli

Gamma (kGy)

0 2 4 6 8 10 12 14 16

Surv

ivin

g F

ractio

n 10 -5

10 -4

10 -3

10 -2

10 -1

100

101

Desiccation (Days)

0 10 20 30 40

Su

rviving

Fra

ction 10 -5

10 -4

10 -3

10 -2

10 -1

100

101

Effect of Gamma-radiation and desiccation on the

survival of strains () D. radiodurans, () D. claudioa,

() D. radiomolis, (), and () D. alpinitundrae .

There is a close relationship between

bacterial ionizing-radiation resistance and

desiccation tolerance.

Dried bacterial cells exhibit a substantial

number of DNA DSBs, single-strand

breaks, and DNA crosslinks as happens

during ionizing-radiation exposure.

What makes a organism radiation resistant?

Callegan et al. 2008, IJSEM 58:1252-8

Rubrobacter spp. Accumulate Compatible Solutes

MannosylglycerateTrehalose

di-myo-Inositol-P

di-N-Acetylglucosamine-P

Glycine betaine

Growth Rate

Minimal Medium, 60ºC

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0.0 2,5 4.0 5.0

So

lute

s (

um

ol/m

g)

0

0,04

0,08

0,12

0,16

Sp

ec

ific

gro

wth

ra

te

NaCl (%)

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.

These organisms accumulate trehalose and mannosylglycerate under all

conditions examined. What is their role?

Empadinhas et al., 2007, Extremophiles

Su

rviv

ing

Fra

cti

on

100

10-1

10-2

10-3

10-4

10-5

10-6

0 200 400 600 800 1000 1200

UV flux (Joules/m2)

RG-1T

RG-3T

R. radiotolerans

R. xylanophilus

D. radiodurans

A good theory destroyed by a nasty little fact

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

Haloterant

Non-halotolerant

NaCl

Slight halophile Moderate halophile

Extreme halophile

Gro

wth

rate

pH Range for Growth of Some Bacteria and Archaea

Saturated

Compatible Solutes of Mesophiles

POLYOLS

Glycerol, arabitol, mannitol

AMINO ACIDS AND DERIVATIVES

Glutamate, proline, alanine, glutamine

Ne-acetyl-b-lysine, betaine

SUGARS AND HETEROSIDES

Trehalose, glucosylglycerol, galactosylglycerol, glucosylglicerate

ECTOINES

Ectoine, hydroxyectoineHO O

CH2OH

O

OH CH2OH

O

1

OH

OH

OH1

OH

Trehalose

Compatible solutes of (hyper)thermophiles

MANNOSYLGLYCERATE

DI-MYO-INOSITOL-PHOSPHATE

DIGLYCEROL PHOSPHATE

MANNOSYL-DI-MYO-INOSITOL-PHOSPHATE

GALACTOSYL-5-HYDROXYLYSINE

MANNOSYLGLYCERAMIDE

GLUCOSYLGLYCERATE

CYCLIC 2,3-BISPHOSPHOGLYCERATE (methanogens)

ASPARTATE

GLUTAMATE

TREHALOSE

O

HO

HO

OH

CH2OH

CH OH

O

COOH

2

Mannosylglycerate

Salt brines of the Red Sea

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.

Haloplasma contractile

Discovery

Thetis

Medee

Thetis

KryosUrania

KryosUrania

Thetis

Discovery

Thetis

Medee

Thetis

KryosUrania

KryosUrania

Thetis

Discovery

Thetis

Medee

Thetis

KryosUrania

KryosUrania

KryosUrania

KryosUrania

Thetis

Way down below the ocean, where I want to be…

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

Valenzuela-Encinas et al., Extremophiles, 2008

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.

Henneberger et al. 2006. AEM 72:192

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).

Is this an extreme environment?

Two predominant species of saturated salt pans; the

other other organisms are minor components of the

ecosystem;

Haloquadratum walsby Archaeon

Salinibacter ruber Bacteria

The salt pans of Maiorca

“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

This area in the Central Atacama Desert, Chile, where there

is no recorded rainfall, has no detectable Life.Courtesy of Fred Rainey

No Bacteria, no Plants, only the Occasional Human

What is an Extreme Environment?

Sulfur on Vulcano

Italy

Crater on Vulcano Island

Italy

Caldeira Grande, S. Miguel

We now generally define an extreme environment as one with low species diversity.

How do we circumscribe a microbial environment?

Boiling water

About 250ºC

No Life

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.

About 2ºC

About 70ºC

We now generally define an extreme environment as

one with low species diversity. But how do we

circumscribe an extreme environment?

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.

Coimbra

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

ITQB, Lisbon

Prof. Helena Santos

Pedro Lamosa

Lígia Martins

Nuno Borges

Louisiana State University

Prof. Fred A. Rainey

DSMZ

Peter Schumann

The People