41st Saas‐Fee course from Planets to Life 3‐9 April 2011 Lecture 2: The “top down” approach to understanding the origin of life – cont.

• Understanding the characteriscs of the organisms close to the root of the tree – Most are extremophiles (grow at high temperatures, high and low pH, high salt, etc) – The origin of metabolism – The “RNA” world – The possible role of viruses in the origin of life – The possible importance of “biofilms” in the early evoluon of life Characterisc differences between the three domains of life: Archaea, Bacteria and Eukarya Characteristics* Archaea Bacteria Eukarya Cells with membrane-bound nucleus and other organelles No No Yes DNA circular1 Yes Yes No Ribosome size 70S 70S 80S Membrane lipids Ether linked Ester linked Ester linked Cell walls No PDG2 PDG No Histone proteins Yes No Yes Operons in DNA Yes Yes No Ribosome structure distinct distinct Archaeal-like Antibiotic sensitivity No Yes No Photosynthesis No Yes Yes Growth at temperatures >80°C Yes Yes No *There are many physiological characteriscs that are found only in bacteria and archaea. 1There are some excepons. 2PDG is pepdoglycan; Archaea do not have PDG but do have at least 7 different cell surface layers (protein, lipid, etc) The endoplasmic reculum is an interconnected network of tubules, vesicles and is involved in the synthesis of proteins, lipidss, sugar metabolism, etc

Longitudinal secon through the flagella area The kinetosome (basil body) that is the anchoring site for a flagellum

Limits of Life and Limits of Diversity

• Are their limits to evolutionary diversity of life as we know it? • What environmental conditions limit where life can exist?

What did Darwin have to say that is germane to these questions? Quotes from The Origin of Species

In reference to natural selecon: “I can see no limit to this power, in slowly and beautifully adapting each form to the most complex relations of life”

Darwin’s ending paragraph: “…from so Darwin's most famous book, was simple a beginning endless published in 1859. Within 20 years it forms most beautiful and convinced most of the internaonal most wonderful have been, scienfic community that evoluon and are being evolved” was a fact. “I can see no limit to this power etc”

The blobfish (Psychrolutes marcidus) is found at depths greater than 5000 m off the coast of Australia and Tasmania. To remain This crustacean invades a fishes buoyant, the flesh of the blobfish is a mouth, devours its tongue, and takes gelanous mass with a density slightly less the tongues place. It then acts like a than water. This allows the fish to float above tongue; the fish can use it to grip and the sea floor without expending energy on swallow prey ‐ the parasite gets first swimming. The relave lack of muscle is not dibs at the food. (From: Carl a disadvantage as it primarily swallows edible Zimmer, Parasite Rex, Simon & maer that floats by in front it (adult blobfish Schuster) ~30 cm long). “there are no limits” ‐ The water bear (Tardigrade) could easily survive on Mars and in the ice of Europa Targrades are between 0.05 and 1.2 mm in length, have feet with claws like bears and walk like bears. They are found everywhere including hot springs, in a 5m layer of solid ice, on the top of the Himalayas, stone walls etc but mostly live in moss. They could survive on Mars because: The water bear is capable of surviving for more than 12 years in a completely dry state called the “tun” state or in “cysts”. In the “tun”state they will survive in liquid helium, absolute alcohol or even ether and brine. Just add water and they come back to life ‐ just like instant coffee

“gummy bear”

Dry form “tun” Asphyic state ‐O2 coming back to life with addion of water 41st Saas‐Fee course from Planets to Life 3‐9 April 2011 The “top down” approach to understanding the origin of life

• Understanding the characteriscs of the organisms close to the root of the tree – Most are extremophiles (grow at high temperatures, high and low pH, high salt, etc) The Universal Phylogenec Tree: Origin of Life and Evoluon Implicaons What does this tree tell us about the Universal Phylogenetic Tree evoluon of organisms? 1. There are three domains of life 2. All extant life arose from a common ancestor 3. Bacteria and Archaea thought to be part of the same group of organisms (prokaryotes, Monera etc) are disnctly different 7. The Eukarya evolved from the archaea 8. The deepest rooted organisms are thermophiles (hyperthermophiles) 11. The prosts are polyphylec (see diplomonads and ciliates) 12. The cyanobacteria (the mother of all oxygen producing photosynthec oganisms) are not deeply rooted The microbial world, its limits and our search for life elsewhere EXTREMOPHILES – Organisms that live in the most extreme environmental condions (Temperature, salinity, pH, pressure, radiaon, heavy metals, low water acvity, and combinaon of extremes) Important note: There is sll much we don’t understand about Earth life and the limits of evoluon of carbon‐based life to live under extreme condions Evoluonary innovaons observed in Earth organisms THERE IS STILL MUCH TO BE DISCOVERED • During the past 10 years the Census of Marine Life has discovered thousands of new species of animals and plants • This is even more pronounced for marine microorganisms and it is estimated that more than 99% of the microbes in the ocean are uncharacterized new species The microbial world, its limits and our search for life elsewhere EXTREMOPHILES – Organisms that live in the most extreme environmental condions (Temperature, salinity, pH, pressure, radiaon, heavy metals, low water acvity, and combinaon of extremes) Why study extremophiles? • Limits of carbon‐based life • Some extremophiles deeply rooted in global phylogenec trees (parcularly thermophiles) • The range of habitat condions for extremophiles may be analogous to environmental condions on other planets and moons • Paleomicrobiology (metabolic history) and the changing environmental condions throughout Earth history • “Top down” approaches to studying the origin of life Limits of Life Parameter Extreme range on Extreme level for growth Earth of organisms Temperature ~-50 - >1200°C Lowest Temperature -15°C Highest Temperature - 122°C Eukaryotes to 62°C ;metazoans to ~50°C pH 0 - 14 Bacteria, Archaea and fungi at pH 0 - 13

Water activity Distilled H2O to total dryness Highest salt - 35% NaCl (many microbes and animals can survive desiccation) (Aw) Radiation Generally less than 1 kGy Some microbes survive levels 10X higher than found naturally on Earth Heavy metals Depends on environments Bacteria and algae grow in 2-5mM Cd, and specific metals (>10mM) Zn, Ni etc Pressure <1 to ~1,100 atm High diversity of bacteria, invertebrates (subseafloor habitas possibly and fish in ocean trenches to >6 km in the crust) Limits: Some key environmental variables regulang life processes • Temperature and Pressure: Together they determine the boundary condions for liquid water • Salinity: relates to the availability of water and in combinaon with pressure or low temperature can result in added stress to cells • pH: in most cases organisms evolve mechanisms to maintain pH’s near neutrality inside the cell • Organic solvents: destroys lipid membranes • Other combinaons: dryness, radiaon, redox condions, heavy metals, etc in combinaon with T, P, S, and pH What are the limits for C‐based life?

Only temperature and availability of water limit Earth life

Note: toxic levels of metals, radiaon, etc can kill life Temperature range for microbial growth and survival:

1. Microbial growth at ‐15°C and up to at least 122°C 2. Enzyme acvity at low temperature depends on liquid solvent Viable microbes observed at 250°C 3. Salts and extracellular polysaccharides (EPS) can protect cells; some (122°C) hyperthermophiles have >4M K at high temperatures 4. Bacterial spores and vegetave Maximum growth T for eukaryotes (70°C) cells have been observed from Maximum growth T for metazoans (~50°C) million year ice cores 5. Anaerobes including methanogens (along with methane) in ice cores 6. Anaerobic methane oxidizing Enzyme acvity in water/organic solvent mixture archaea associated with (Bragger et al., 2000) methane hydrates (Modified from Deming and Eiken, 2007) Temperature Classes of Microorganisms Hyperthermophile (Temp. Opmum >80°C) – Early microbiology studies pioneered by Thomas Brock in the 1970’s

Octobus Springs, Yellowstone Naonal Park (The site where Thermus aquacus was Boulder Spring, isolated. T aquacus provides the Yellowstone Naonal polymerize enxyme used in the Polymerase Park Chain Reacon) Hydrothermal vents discovered 1977, black smokers, 1979 Juan deFuca Ridge – NE Pacific (2,500 m depth, 350°C hot fluid) Different Edifice Morphologies, Endeavour Highest Temperature Organism on Earth from Finn (Mothra) Highest Temperature Organism on Earth from Finn 3 days growth 2 m

121°C organism grown under anaerobic condions with acetate, 1.03 m FeIII forms magnete, doubles 24 hrs Kashefi et al., Science 2003 Pyrolobus fumarii* (Tmax = 113ºC, Topt= 106ºC,

Tmin= 90ºC)

FISH staining of vent chimney TEM, P. fumarii cell (Reinhard Rachel) Red, Archaea; Green, Bacteria (Chrisan Jeanthon)

A Pyrodicum species has been described (Science 301:934, 2001) that can grow up to 121ºC, and a strain of Methanopyrus kandleri has been shown to grow up to 122ºC (PNAS 105:10949, 2008) Even hyperthermophiles have parasites

Nanoarchaeum 0.4 µm

Red, Nanoarchaeum Green, Ignicoccus

Photos by Reinhard Rachel Nanoarchaeum genome (PNAS 100:12984, 2003; J. Bacteriol. 190:1743, 2008) a. Circular, 0.49 Mbp–smallest genome of any species of Archaea. b. Contains no recognizable genes encoding biosynthec enzymes for amino acids, nucleodes, or coenzymes. c. Lacks genes encoding proteins for major catabolic pathways (e.g., glycolysis). d. Missing genes for some ATPase subunits. e. Most gene dense genome of any cell (99% of genes encode proteins). Hyperthermophiles: Overcoming the negave effects of high temperature

Problems Solutions 1. Protein Denaturation Heat-stable proteins; heat- shock proteins (chaperones) 2. DNA Denaturation Reverse DNA gyrase; introduces positive supercoiling into the chromosome, which raises the melting point; stabilizing proteins 3. Membrane Melting Tetra-ether lipid monolayer membranes; covalent bonds adjoining membrane halves resist membrane peeling

4. Low Solubility of O2 at High Temperatures Diverse anaerobic energy 0 metabolisms; S - and H2- based metabolisms Eukaryotes that live at high temperatures • Upper temperature for growth of a single‐cell eukaryotes is 62°C (fungi) • Upper temperature for growth of a metazoan is 50°C (polychaete worm from deep‐sea hydrothermal vents) Alvinella pompejana “Pompei worm” (A heat‐loving metazoan) Size: up to 150 mm Distribuon: East Pacific Rise from 21°N to 23°S Biology: Dwells inside organic tubes in acve chimney walls. Temperature growth range 20‐50°C but can tolerate exposure to temperatures >100°C; Feeds on bacteria; outer surface colonized by filamentous bacteria Inferno Palm worms – Axial Volcano Alvinella pompejana Worm

Photograph of a video taken from DSRV Alvin in hydrothermal vents at 21°N, East Pacific Rise, showing an Alvinella pompejana worm standing on a substrate measured at 105°C. Sketch to clarify the posion of the worm and the temperature probe Nature 1992 Alvinella pompejana (East Pacific Rise)

Scanning electron micrograph ‐ head size is 3 cm Cary et al., Nature 391:545‐546 (1998) There is an ongoing “discussion” as to the upper temperature for growth of the Alvinella worms that live on acve sulfide structures. This report by Cary and colleagues demonstrated that the rear part of the tube where Alvinella pompejana resides reaches temperatures close to 80°C. Some of the biochemistry data on these worms indicate that 40‐50°C may be the hoest temperature for these animals.It is clear, however, that Alvinella can survive exposure to temperatures approaching 100°C. Science 312:231 (2006) Schemac of the thermal gradient aquarium. Chamber consisted of : (A) aluminum reinforcement plate; (B) clear polycarbonate window; (C) PEEK oulow tubing; (D) holes drilled into the block to within 2.5 mm of the slot containing animals for inseron of temperature probe; (E) )‐ring face seal; (F) slot to contain animals; (G)PEEK inflow tubing; and (H) an anodized aluminum block with a slot to contain animals. Distribuon of P. sulfincola and P. palmiformis worms in temperature‐gradient experiments. Worms were uniformly dispersed within the aquaria before establishing the temperature gradient. (A to C) Plots of P. sulfincola distribuons over me within a 20° to 61°C gradient; N = 5, 9 and 4 individuals, respecvely. (D) Plot of P. palmformis distribuons over me within a 20° to 55°C gradient; N = 8 individuals.

Scale Worm (Polychaete) living on the edge Juan deFuca Ridge NE Pacific (320°C fluid) (Deming, 2009)! Psychrophiles

• Lowest temperature for growth ‐12°C, acve 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 Marine sea ice

Polaromonas ←Photos by Jim Staley→ Topt = +4ºC

Losest temperature for growth: Psychromonas ingrahami

Topt= +4°C, Tmin= –12°C, Tmax= +10°C Cultured phage‐bacterial host systems acve at –1°C

Middelboe et al., 2002 (seawater) Borriss et al., 2003 (sea ice) Wells and Deming, 2006 (both)

3 µm Colwellia psychrerythraea strain 34H

3 µm

3 µm (Borriss et al., 2003)

(Wells and Deming, 2006) Problems and Solutions: Psychrophiles Problems! Solutions! Prevent ice-crystal formation and cell Live in a briny habitat, produce death! compatible solutes and/or exopolysaccharides (EPS) ! Enable protein activity: enzymes must Make more flexible proteins maintain significant catalytic (higher α-helix; lower β- activity at low temperature! sheet content)! Make more polar and less hydrophobic proteins, with fewer weak bonds (ionic, hydrogen)!

Maintain membrane function: the Make lipids with greater content organism must maintain of short-chained, branched, significant levels of nutrient and unsaturated fatty acids! transport at low temperature! “I can see no limit to this power etc” (Darwin referring to natural selecon”

The Antarcc 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 adaptaon to life at temperatures that are less than 0°C (icefish size 25 cm long) (Wikipedia) 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 protecon 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