39
41st Saas‐Fee course from Planets to Life 3‐9 April 2011 Lecture 3: The “top down” approach to understanding the origin of life – cont.
• Understanding the characterisFcs of the organisms close to the root of the tree – Most are extremophiles (grow at high temperatures, high and low pH, high salt, etc) • Growth at low temperatures • Growth at high and low pH • Growth in high salt • Growth and survival in dry environments
• Growth in extremely low nutrient environments
Bacteria Archaea Eukarya
Bacteria Archaea Eukarya
Only one tree of life
Present tree of life is a branch of an exFnct tree
Was there only one tree of life on Earth? Does conFngency play a significant role in the origin of life as some think it did in the evoluFon of diversity of life forms?
Baross, 1998
(Deming, 2009)!
Psychrophiles
• Lowest temperature for growth ‐12°C, acFve metabolism <‐22°C.
• Evidence for survival at temperatures as low as ‐80°C (liquid nitrogen)
• Spores found in ice cores that are >1 million years old
Psychrophiles: Tempmax < 20ºC
←Photos by Jim Staley→ Polaromonas Topt = +4ºC
Losest temperature for growth: Psychromonas ingrahami Topt= +4°C, Tmin= –12°C, Tmax= +10°C
Marine sea ice
Cultured phage‐bacterial host systems acFve at –1°C
Middelboe et al., 2002 (seawater) Borriss et al., 2003 (sea ice) Wells and Deming, 2006 (both)
Colwellia psychrerythraea strain 34H
3 µm
3 µm
3 µm
(Borriss et al., 2003)
(Wells and Deming, 2006)
Problems and Solutions: Psychrophiles Problems!Prevent ice-crystal formation and cell
death!
Enable protein activity: enzymes must maintain significant catalytic activity at low temperature!
Maintain membrane function: the organism must maintain significant levels of nutrient transport at low temperature!
Solutions!Live in a briny habitat, produce
compatible solutes and/or exopolysaccharides (EPS) !
Make more flexible proteins (higher α-helix; lower β-sheet content)!
Make more polar and less hydrophobic proteins, with fewer weak bonds (ionic, hydrogen)!
Make lipids with greater content of short-chained, branched, and unsaturated fatty acids!
The Antarc>c ice‐fish (Channichthyidae) are the only known vertebrates without hemoglobin. Consequently, their blood is transparent. Their metabolism relies on the oxygen dissolved in the liquid blood and is absorbed directly through the skin from the water. This works because of the increased solubility of oxygen in cold water and is an adaptaFon to life at temperatures that are less than 0°C (icefish size 25 cm long) (Wikipedia)
“I can see no limit to this power etc” (Darwin referring to natural selecFon”
Summary – Temperature and life
• To date, the lowest temperature for growth is ‐12°C and the maximum temperature for growth is 122°C
• Low temperature microbes (psychrophiles) do not have ancient lineages – Spore‐forming psychrophilic bacteria are of concern regarding planetary protecFon issues to icy planetary bodies
• High temperature microbes (hyperthermophiles) have ancient lineages – Hyperthermophiles are of interest regarding the origin of life and the origin of metabolism and eukaryotes
– The highest temperature for growth of a eukaryote is >60°C lower than the maximum temperature for a microbe
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
RoFfers
Natronobacterium
Bacillus firmus OF4
Spirulina
ProFsts
Lost City methanogens
Plectonema
Cyanidium
Synechococcus
Archaea
Sphagnum
Heather sedges
pH range in hydrothermal vents
Fungi
Carp
Ephydrid flies
Soda lakes Acid mine drainage; geo‐ thermal sulfur sites
Natronobacterium
pH limits for life
Acidic mud pot in Yellowstone Park ‐ home to the acidophile Sulfolobus acidocaldarius
Red coloraFon on rocks near Naples, Italy produced by the hyperthermophile Sulfolobus solfataricus
Sulfolobus acidocaldarius is an hyperthermophile that grows in acid hot springs, mud pots etc at temperatures from 60‐100°C and at pH from <1‐5. EM is X85,000 and fluourescent photomicrograph shows S. acidocaldarius amached to sulfur. The organisms oxidizes S° to H2SO4
S0 + 1+1/2 O2 + H2O → H2SO4
CO2 → Cell material
Sulfolobus spp growat pH <1
The record holder for growth at low pH
Picrophilus oshimae pHopt = 0.7 (J. Bacteriol. 177: 7050, 1997)
Picophilis oshimae is an Archaea that can grow at ph of 0.06 and is incapable of growing at pH 3 or higher. P. oshimae growth at 45‐65°C and was isolated from solfutaric soils. This organisms cannot maintain membrane integrity at pH higher than 4
Growth curves of Picophilis osphimae
Growth curves of P. oshimae at various pH values (OD = op>cal density) (from Puehler et al., J. Bacteriology 1995)
Unusual Characteristics of Acidophiles The genome of Picrophilus torridus (PNAS 101:9091, 2004)
reveals: 1. A very small genome ‐ the 1.55 Mbp P. torridus genome is near the smallest for free‐living heterotrophic aerobes. 2. High raFo of genes encoding Proton‐Motor‐Force ‐driven versus ATP‐driven transporters. 3. High coding density; ~91% of genome encodes protein. 4. Extremely acid‐stable membrane proteins. 5. Extensive respiratory systems are necessary because of energeFc needs plus the need to consume protons. Many genes encoding respiratory funcFons have been obtained by lateral transfer.
Viruses of hyperthermophilic/acidophilic Archaea are extremely
unusual (Res. Microbiol. 154:474, 2003).
Problems and Solutions: Acidophiles
Problems Solutions 1. Internal pH Remains above pH 4.5 due to impermeability of
the cytoplasmic membrane; DNA hydrolysis would be a problem at lower cytoplasmic pH values
2. Protein Stability/Function Proteins in wall and membrane contacting high [H+] are extremely acid-stable; cytoplasmic proteins must be somewhat acid-stable
3. Bioenergetics Proton motive force drives nutrient transport and ATP synthesis; ATP synthesis by ATPase is not a “free lunch”, since respiration requires electrons: 1/2 O2 + 2 e– + 2H+→ H2
ATPase H+ Pump
H+ H+
H+ H+
Redox reacFons alternaFng H and e‐ carriers (REDOX H+ PUMP)
Chemical energy Light energy
(oxidaFve phosphorylaFon) (photophos‐ phorylaFon)
Coupling membrane
The “bare bones” of chemiosmoFc coupling (Raven and Smith, 1978)
An ion gradient has a potenFal energy and can be used to power chemical reacFons when the ions pass through a channel
Chemiosmosis
ATP
Growth at high pH - Alkaliphiles: pHopt > 9
SODA LAKE CHARACTERISTICS
NaCl → low to high
SO42– → low to high
Mg2+, Ca2+ → extremely low
HCO3– /CO3
2– → extremely high
pH → 9–12 Lake Hamara, Libyan Desert, Egypt (Madigan)
Record Holder: Natronobacterium magadii pHopt= 10; pHmin= 8; pHmax= 11.5
Properties of Alkaliphiles 1. Diversity:
a. Many uncultured (presumably alkaliphilic) Bacteria and Archaea exist in halo‐alkaline habitats (Extremophiles
`````8:63, 2004).
b. Many alkaliphiles are species from well‐known phyla of Bacteria and Archaea.
2. Alkalithermophiles: a. Many rapidly growing alkalithermophiles are known and
some have generaFon Fmes as short as 10 minutes.
b. Some alkalithermophiles are also halophiles–the first “triple extremophiles” known (temperature, salt, pH).
Overview of Alkaliphiles: Prokaryotes 2: 283, 2006
Problems and Solutions: Alkaliphiles
Problems Solutions 1. Internal pH Remains below pH 9.5 due to
impermeability of the cytoplasmic membrane; RNA hydrolysis would be a problem at higher cytoplasmic pH values
2. Protein Stability/Function Proteins contacting the environment are stable to alkali and alkaliphilic; have applications as laundry proteases, lipases
3. High Salt Many alkaliphiles are also halophilic
4. Bioenergetics Na+ gradient (instead of H+ gradient) drives motility and transport, but a proton-motive force drives ATP synthesis
Mono Lake in California is very alkaline at pH 10 (2.5g NaOH/liter). The towers consist of calcium carbonate (FW mixes with alkaline springs). Towers can be 10 m high. The salinity of Mono Lake is about 8.5% salts. Brine shrimp and a diverse groups microorganisms live in the lake.
Science 2008
Arsenic driven photosynthetic bacteria discovered at Mono Lake
New Electron Donor for CO2 FixaFon Cyanobacteria and green plants get their electrons from H2O
Anoxygenic photosyntheac bacteria use : H2, H2S, Fe2+, NO2
–, ‐ now arsenite (AsO3‐2)
(Calvin Cycle: 6 CO2 + 24 H → C6H12O6 + 12 H2O)
The pelagic ocean Hydrothermal vents*
*Besides the use of hydrogen sulfide as an energy source, some microorganisms use hydrogen and methane gas or some metals like iron and manganese as sources of energy
PosiFon of chemosynthesis within autotrophic metabolism. Instead of light, the energy required for the reducFon of CO2 to organic‐carbon (CH2O) by photosyntheFc organisms, vent microbes use chemical energy including hydrogen sulfide (H2S)
Other electron donors include H2, FeII, Arsenic
AsO32– → AsO4
2– + 2 e‐
Arsinite Arsinate
These electrons are available for photosynthesis by microbes in Mono Lake (Science 2008)
Can arsenic subsFtute for phosphate in nucleic acids?
2010
Some issues with the Arsenic paper
Statement in IntroducFon: “.. However, there are no prior reports of subs>tu>ons for any of
the six major elements essen>al for life.”
This is wrong – two prominent examples: Arsenolipids (As for P) have been known since 2004, and
Selenocysteine (Se for S) enzymes are well known
“Alien organism” – no, the organism is a common, and late branching bacterium (Universal Tree) and known to tolerate high concentraFons of metals
It is extremely difficult to achieve P limitaFon in microbes – they have very creaFve ways of storing P, and besides there was some P in the medium used for growth
Bioessays 2011
These authors discuss the literature of what is known about arsenic and biology and parFcularly where arsenic can subsFtute for phosphate in living organisms. Arsenic can subsFtute for phosphate in lipids (membranes), for example.
However, the authors point our that “there is an extremely rapid rate of spontaneous hydrolysis of arsenate esters that would make small molecules such as sugar/arsenates unstable
and arsenic DNA and RNA rapidly fall apart”. Authors conclude “considering the later consideraFon indicates that arsenic life seems unlikely”.
Dry environments – related to high salt stress and the availability of water
Most soils during summer months – microbes forms spores or resistant cycts AntarcFc dry valleys and high salt lakes Deserts (Death Valley, etc) 1. desert varnish (ferromanganese deposits) 2. Rock hosted microbes
Chile’s Atacama Desert (viable microbes below the surface) Death Valley 1. Evaporites and brine areas
Desert Varnish
The sun‐baked boulders of the Alabama Hills in Owens Valley, California look like they were blackened by ancient campfires. They are actually coated with a black layer of clay and manganese oxide precipitated by colonies of bacteria living on the rock surface for countless centuries.
The thin layer of reddish iron oxide varnish on this rock surface has been etched to reveal the lighter granodiorite beneath. Indian tribes uFlized desert varnish to create petroglyphs.
Images and text from Eayne Armstrong, Palomar College, California
Microbes in desert soils
Sampling soil at Chile’s Alacama Desert – life found generally in suspended animaFon between desert rains.
“Extreme microbes drink dew on spiderwebs to live”
A spiderweb in a cave in the Atacama Desert where novel extremophile microbes were found living on the web’s silken threads. Credit: Armando Azua‐Bustos
Deinococcus species are the most radiaFon resistant life form
Deinicoccus radiodurans can survive extremely high doses of ionizing raFaFon (10,000 Gy). For comparison, 5 Gy is lethal for humans, and 2000 Gy will sterilize a culture of Escherichia coli. The resistance to radiaFon is due to the ability of D. radiodurans to repair 2S DNA breaks
It is generally believed that the radiaFon resistance of D. radiodurans is from an adaptaFon to desiccaFon – a common occurrence for this organisms in soils
Extreme Halophiles: NaClopt > 1.5 M
Record Holder: Halobacterium salinarum NaClopt = 4.5 M (~26%)
Great Salt Lake, Utah (Brock)
Sea Salt Plant, San Francisco Bay (NASA) SEM of a Spanish
Saltern (F. Rodriquez‐Valera)
Overview of halophiles: Halophilic microorganisms and their environments (A. Oren ed.) Springer, 2002
Brines: Science 342:1523, 2009
Salt flats at Lake Magadi, Kenya; the red color Is from haloarchaea produced bacteriorhodopsin
Salt flat in Australia
Halobacterium salinariumis grows in 4‐5 M salt and can’t grow below 3M salt. Freeze‐etch micrograph shows the surface structure of the cell membrane and reveals smooth patches of “purple membrane” (bacterio‐rhodopsin) embedded in the plasma membrane
The only square microorganism 1. Diversity: “Walsby’s square
bacterium” (FEMS Le\s 238:469, 2004; Environ. Microbiol. 6:1287, 2004): Haloquadratum walsbyi
2. Genomes: Halobacterium (PNAS 97:12176, 2000); Haloarcula (Genome Res. 14:2221, 2004).
a. MulFple chromosomes (2–4) and mulFple large plasmids (2–5).
b. High surface negaFve charge on proteins (average pI of proteome, 4.5–5).
c. Considerable variability in genome size (the Haloarcula genome at 4.27 Mbp is twice the size of the Halobacterium genome).
1 µm
Photos from FEMS LeFs 238: 469, 2004
Haloquadratum walsbyi
Problems and Solutions: Extreme Halophiles Problems Solutions 1. Dehydration Compatible solutes (glycine
betaine, alcohols, sugars, or K+) Halobacterium cytoplasm, 5.3M K+
2. Protein Stability
Cytoplasmic proteins are highly polar and acidic; require high [K+]. Proteins typically have low nonpolar amino acid contents
3. Solubility of O2 is low
Bacteriorhodopsin: a light-driven proton pump synthesized under anoxic conditions. This boosts ATP levels when respiration is impossible
The range of habitat condiFons for extremophiles may be analogous to environmental condiFons on other planetary
bodies • Cold, low nutrient oceans – icy moons
• Sea ice – icy moons
• Dry environments ‐ Mars
• High and low pH environments ‐ ?
• Atmospheres – Hot, sulfurous like Venus, etc
• Geothermal and hydrothermal systems including peridoFte‐hosted systems
• Early Earth analogues?
How complex can metazoan animals get ayer long evoluFon in the absence of oxygen?
