Environmental Flashcards
Pure culture (culture-dependent) methods
- Nutrients available in lab culture are typically higher than in nature
- Difficult to replicate environmental conditions in lab
- Only minor components of ecosystem
16S rRNA gene
- Functions as part of small subunit (SSU)
- Good marker gene
- Extensive databases with sequences from a large number of microorganisms
- PCR
- Align rRNA gene sequences
- Find number of differences in gene
- Less differences –> closer in evolution
- Three distinct lineages of cells called domains
- Bacteria (prokaryotic)
- Archae a (prokaryotic)
- Eukarya (eukaryotic)
- Archaea more closely related to Eukarya than Bacteria
Domain Bacteria
- 60 phyla
- Majority defined from environmental sequences
- Many groups are phenotypically diverse
- Physiology and phylogeny not necessarily linked
Domain Archaea
Euryarchaeota
Crenarchaeota
Euryarchaeota
- Methanogens: degrade organic matter, produce methane - Extremophiles: require high salt concentrations for metabolism and reproduction - Grow in moderately high temperatures and low pH environments
Crenarchaeota
- Vast majority of cultured Crenarchaeota grow in high temperatures (extremophiles)
Marine, freshwater, soil, etc
Extremophiles
Temperature
Psychrophile: min <0oC, optimum 15oC, max <20oC
Psychrotolerant: can grow 0oC, optimal 20oC-40oC
Thermophile: optimal 45-80oC
Hyperthermophile: optimal >80oC
Extremophiles
pH
Acidophile: optimum pH <6
Alkaliphile: optimal pH >8
Extremophiles
Salinity
Halophile: optimal 1-15% NaCl
Extreme halophile: optimal 15-30% NaCl
Halotolerant: can tolerate some, but grow best in absence
Extremophiles
Pressure
Barophile: thrives at high pressure, typically light sensitive
Barotolerant: can survive high pressures, but can exist in less extreme
Obligate barophiles: cannot survive outside of high pressure
Chemical energy store
ATP
Phosphoenolpyruvate
Long term energy storage
Insoluble polymers that can be oxidised to generate ATP
- Glycogen
- Poly-B-hydroxybutyrate
- Elemental sulfur
Long term energy storage
Insoluble polymers that can be oxidised to generate ATP
- Glycogen
- Poly-B-hydroxybutyrate
- Elemental sulfur
Chemoorganotrophy
Fermentation
- Anaerobic
- Substrate level phosphorylation
- ATP directly synthesised from energy-rich intermediate
- Glycolysis
- Glucose –> 2 pyruvate + 2 ATP + fermentation products
Fermentation product: Lactic acid, ethanol, CO2
Chemoorganotrophy
Respiration
- Aerobic and anaerobic
- Oxidative phosphorylation
- ATP produced from proton motive force formed by transport of electrons
- Oxidation using O2 as terminal electron acceptor
Higher ATP yield than fermentation
Associated Electron Carriers
NADH dehydrogenases
Flavoproteins
Quinones
Cytochromes
-NADH dehydrogenases: active site binds NADH and accepts 2 electrons and 2 protons and passes to flavoproteins
Proteins bound to inside surface of cytoplasmic membrane
-Flavoproteins: accepts 2 electrons and 2 protons, donates only electrons to the next protein
-Quinones: Accept electrons and protons but only pass on electrons
Non-protein-containing molecules that participate in electron transport
-Cytochromes: Accept and donate a single electron via the iron atom in heme
Proteins that contain heme prosthetic groups
Proton Motive Force
- pH gradient
- Electrochemical potential across membrane
- ATP synthase (ATPase) converts proton motive force to ATP
- 38 ATP
The Citric Acid Cycle (CAC)- Krebs Cycle
-Pyruvate completely oxidised to CO2
- Glucose –> pyruvate (same as glycolysis)
- 1 Glucose –> 6 CO2 + NADH + FADH
- Key role in catabolism
Energetic advantage to aerobic respiration over fermentation
Anaerobic Respiration
-Use electron acceptors that aren’t oxygen
Nitrate (NO3-), ferric ion (Fe3+), sulphate (SO4-), carbonate (CO32-)
-Less energy released than aerobic respiration
-Dependent on electron transport, generation of proton motive force, ATPase activity
Nitrogen
- Inorganic nitrogen compounds are the most common electron acceptors in anaerobic respiration (nitrate reduction, denitrification)
- Denitrification is the main biological source of gaseous N2
Nitrate (NO3-) –> Nitrite (NO2-) –> Nitric oxide (NO) –> Nitrous oxide (N2O) –> Dinitrogen (N2)
Manganese Oxide
Insoluble MgO2 + 4H+ + 2e- –> soluble Mn2+ + 2H2O
Manganese Oxide
Insoluble MgO2 + 4H+ + 2e- –> soluble Mn2+ + 2H2O
Chemolithotrophy
-Uses inorganic molecules as electron donors (oxidation)
Hydrogen Sulphide (H2S), Hydrogen gas (H2), ferrous iron (Fe+), ammonia (NH3)
- Aerobic (oxygen as electron acceptor)
- Oxidation of inorganic electron donor
- Different inorganic compounds yield different energy
- Uses phosphorylation (electron transport chain and proton motive force)
- Autotrophic (uses CO2 as carbon source)
Sulfur Oxidisers
- Many reduced sulfur compounds are used as electron donors
- Sulfur-oxidising bacteria
- Oxidise sulfide (H2S/HS-), sulfur (S), thiosulphate (S2O32-) and sulfite (SO32-)
- Sulfate (SO42-) is the final product of sulfur oxidation
- Deposit internal granules of sulfur
- Oxidation –> respiratory electron transfer + generate proton motive force
Nitrifiers (ammonia and nitrite oxidisers)
- NH3 and NO2- are oxidised by nitrifying bacteria through nitrification
- Two groups of bacteria work in concert to fully oxidise ammonia –> nitrate
- Nitrosomonas: oxidise NH3 –> NO2- (ammonia oxidisers)
- Nitrobacter: oxidise NO2- –> NO3- (nitrite oxidisers)
- Nitrate is a key nutrient for plants
- Nitrification in aerobic zones –> nitrate –> denitrification in anaerobic zones
Iron Oxidisers
- Oxidise ferrous iron Fe2+ –> ferric iron Fe3+
- Fe2+ is a weak electron donor
- Acidophiles use ferrous iron (Fe2+ is unstable at neutral pH)
Phototrophy
- Metabolism that uses light as an energy source
- Phototrophs carry out photosynthesis
- Photoautotrophs use ATP for assimilation of CO2for biosynthesis
- Photoheterotrophs use ATP for assimilation of organic carbon for biosynthesis
- Photophosphorylation is light-mediated ATP synthesis
Oxygenic vs anoxygenic photosynthesis
- Oxygenic photosynthesis produces oxygen
- Anoxygenic does not produce oxygen
Photopigments
Oxygenic produces chlorophylls
Anoxygenic produces bacteriochlorophylls
Different chlorophylls have different absorption spectra to adapt to various environments
Reaction Centres
- Bacteria and archaea do not have chloroplasts
- Reaction centres (RC) participate directly in the conversion of light –> ATP
- Antenna pigments (LH) funnel light –> RC-Chlorosomes function as massive antenna complexes
- Found in green sulfur bacterial (Chlorobium)
- Green non sulfur bacteria (Chloroflexus)
- Capture low intensity light
Carotenoids
- Accessory pigments in addition to chlorophyll/bacteriochlorophyll
- Found in all phototrophic organisms
- Yellow, red, brown, green, pink colour in phototrophs
- Energy absorbed by carotenoids can be transferred to a RC
- Primary role as photoprotective agents
Prevent photo-oxidative damage to cells caused by toxic oxygen species
Anoxygenic Photosynthesis
-At least five phyla of bacteria
(Proteobacteria, Chlorobi, Chloroflexi, Firmicutes, Acidobacteria_
-One photosystem
-Poor electron donor –light–> strong electron donor
-Reducing power for CO2 comes from reductants present in environment
-Electrons transported in membrane through series of proteins and cytochromes
Oxygenic Photosynthesis
- Two photosystems (PSII –> PSI)
- Use light to generation ATP and NADPH
Cyclic or noncyclic photophosphorylation
- Use light to generation ATP and NADPH
Oxygenic Photosynthesis
- Two photosystems (PSII –> PSI)
- Use light to generation ATP and NADPH
Cyclic or noncyclic photophosphorylation
- Use light to generation ATP and NADPH
Biogeochemical Cycles
- Recycling by microorganisms
- C, N, P
- Flow of nutrients/elements through ecosystem
Anaerobic respiration
- Utilisation of electron acceptors other than O2
- Denitrification: NO3- –> NO2- –> NO –> N2O –> N2
Chemolithotrophy
Nitrogen fixation
Methanogenesis
Methanotrophy
-Utilisation of inorganic energy sources
Hydrogen, iron and sulfur oxidizers
-Nitrification: NH3 –> NO2- –> NO3-
Nitrogen fixation: N2 –> 2NH3
Methanogenesis: H2 + CO2 –> CH4
Methanotrophy: CH4 + O2 –> CO2
The Carbon Cycle
Atmosphere- most rapid
Photosynthesis
Respiration -> atmosphere
Microbial decomposition -> largest source
Methanogenesis
- Carbon cycling
- Archaea
- CO2 –> CH4
Autotrophic: CO2 + H2 → CH4 + H2O + CH2O (biomass)
Heterotrophic: CH3COOH → CH4 + CO2 + CH2O (biomass)
The Nitrogen Cycle
Anammox
-Anaerobic ammonium oxidation
NH4+ + NO2- –> N2 + 2H2O
- Treatment of wastewater
- 50% of ammonia removal from marine environments
Anammoxosome
-Compartment where anammox reactions occur
- Protects cell from reactions occurring during anammox
Hydrazine is an intermediate of anammox
Importance of Nitrogen Fixers
- Atmosphere = N sink
- 80% N
- N fixing only in bacteria nad archaea
- Supply N to planet
Nitrogen Fixers
N2 –reduction/nitrogenase–> NH3
-Two distinct proteins: dinitrogenase and dinitrogenase reductase
-Dinitrogenase has iron-molybdenum cofactor (FeMoco)
-Sensitive to the presence of oxygen
Rapidly and irreversibly inactivated by oxygen
-Fermentation, respiration, photosynthesis –> reducing power, ATP –> energy for nitrogen fixation
-Nitrogen fixation carried out by free-living and symbiotic bacteria
Control and Regulation of N2 fixation
- Complex regulon (the Nif regulon)
- 24 kb DNA
- 20 genes
- Several transcription units
- Genes for nitrogenase, FeMo-co, electron transport are present
- Nif genes are highly conserved across the phylogenetic lineages
- N-fixation is blocked by O2, NH3, NO3-, certain amino acids
- Highly regulated process (because energy demanding)
- Nitrogenase has strict regulatory controls at transcriptional level
Aerobic cells: nitrogenase is protected from O2
- High respiration rates
- Slime layers (e.g. in Azotobacter)
- Compartmentalization (e.g. in heterocysts in Cyanobacteria)
Oxygen scavenging by leghemoglobin in Rhizobium nodules
Symbiotic N2 Fixation
- Mutalistic relationship between leguminous plants and nitrogen-fixing bacteria
- Proteobacteria that live in soil and can infect leguminous plants
- Rhizobia enters plant through root hairs
- Forms root nodules
- Root nodules contains rhizobia in bacteroid form
The Nitrogen Cycle
- N2 is most stable form of nitrogen (major reservoir)
- Few prokaryotes have ability to use N2 as a cellular nitrogen source (nitrogen fixation)
- Ammonia produced by nitrogen fixation or ammonification
- Can be assimilated into organic matter or oxidized to nitrate
- Denitrification is reduction of nitrate to gaseous nitrogen products and is the primary mechanism by which N2 is produced biologically
- Anammox is the anaerobic oxidation of ammonia to N2 gas
- Denitrification and anammox result in losses of nitrogen from the biosphere
Early History of Earth
- 4.5-4.6 billion years old
- First cells ~3.8-3.9 billion years ago
- Atmosphere anoxic until ~2 billion years ago
- Metabolisms were exclusively anaerobic until evolution of oxygen-producing phototrophs
- Life exclusively microbial until ~1 billion years ago
Ancient and Modern Stromatolites
-Fossilized microbial mats consisting of layers of filamentous prokaryotes and trapped sediment
-Found in rocks 3.5 billion years old
-Comparison gives evidence that:
- Anoxygenic phototrophic filamentous bacteria formed ancient stromatolites
Oxygenic phototrophic cyanobacteria dominate modern stromatolites
Conditions on Early Earth
- Anoxic and much hotter
- Molten surface under intense bombardment from space by masses of accreted materials
- Water originated from icy comets and volcanic outgassing
- First biochemical compounds made by abiotic systems
- Earth cooled –> solid crust
- Water condensing into oceans
4. 4-4.3 billion years ago
Surface Origin Hypothesis
- First membrane-enclosed, self-replicating cells arose out of primordial soup rich in organic and inorganic compounds in ponds on Earth’s surface
Dramatic temperature fluctuations, mixing from meteor impacts, dust clouds, storms argue against hypothesis
Miller-Urey Experiment (1952)
Water was boiled to vapour –> high temperatures
Gases (including H2, CH4, NH3) –> reducing atmosphere (no oxygen)
Electrical discharge –> lightning as an energy source for reactions
The mixture was then allowed to cool (concentrating components) and left for a period of ~1 week
The condensed mixture was analysed and found to contain traces of simple organic molecules
Subsurface Origin Hypothesis
- Life originated at hydrothermal springs in ocean floor
- Less hostile, less stable
- Steady and abundant supply of energy (e.g. H2, H2S)
Submarine Mound Formed at Ocean Hydrothermal Spring
- Slightly acidic, iron-rich waters
- Precipitates of collodial pyrite (FeS), silicates, carbonates, mangenium-containing montmorillonite clays formed
- Structured mounds on ocean floor
- Gel-like absorptive surfaces, semipermeable enclosures and pores
Model for Origin of Cellular Life
- Three part systems (DNA, RNA, protein) evolved and became universal mong cells
- DNA (more stable) became genetic repository
- RNA world theory: first self-replicating systems may have been RNA based
- Phosphate in seawater and nucleotides polymerised in RNA
- Prebiotic chemistry –> self-replicating systems
- Synthesis of phospholipid membrane vesicles to enclose biochemical/replication machinery is also an important step
- Bacteria and archaea diverged ~3.7-3.8 billion years ago
- Strong selective pressure –> divergence in lipids, cell walls, enzymatic machinery
From LUCA (~4.3 billion years ago) cellular life began to evolve in two distinct directions
Panspermia
Hypothesis that life exists throughout the Universe, distributed by meteoroids, asteroids and planetoids
Early Earth Metabolism
- Anoxic –> exclusively anaerobic, likely chemolithotrophic (autotrophic)
- These forms of metabolism –> production of large amounts of organic matter –> evolution of chemoorganotrophs
Major Landmarks in Biological Evolution
- 2.4 bya: O2 concentrations raised to 1 ppm (Great oxidation event)
- 2.7-3.0 bya: cyanobacterial lineages developed photosystem to use H2O (not H2S)
- 3.2-3.3 bya: first phototrophs (anoxygenic)
- Methanogenesis
- 1.9-1.4 bya: multicellular eukaryotes
- 2 bya: oldest eukaryotic fossil
- Bacteria and archaea develops respiration
Banded Iron Formations
- Laminated sedimentary rocks
- Prominent feature in geological record
- O2 accumulates when it reacts with abundant reduced materials in oceans (FeS, FeS2)
- Oxidation Fe2+ –> Fe3+ (iron oxide formation)
Microbial diversification
-Development of oxic atmosphere –> evolution of metabolic pathways that yield more energy than anaerobic mechanisms
-Formation of ozone layer (barrier against UV radiation)
Life under ocean surface without ozone layer
-Oxygen –> evolution of organelle-containing eukaryotic microorganisms
- Oldest eukaryotic fossils: 2 byo
- Multicellular/complex eukaryotic fossils: 1.9-1.4 byo
Endosymbiotic Origin of Eukaryotes
-Mitochondria and chloroplasts arose from symbiotic association of prokaryotes within another type of cell
Supported by:
- Physiology and metabolism of mitochondria nad chloroplast
- Sequence and structure of genomes (16 S RNA, circular genome)
Endosymbiotic Hypothesis 1
Eukaryote began as nucleus bearing lineage that later acquired mitochondria and chloroplasts by endosymbiosis
Endosymbiotic Hypothesis 2
- Hydrogen hypothesis
- Eukaryotic cell arose from intracellular association between H2-producing bacterium (symbiont), which gave rise to mitochondria, and an H2 consuming archaeal host
Endosymbiotic Evaluation
- Both suggest eukaryotic cell is chimeric
- Hydrogen hypothesis is supported by:
- Eukaryotes have similar lipids to bacteria (ester linked lipids)
- Eukaryotes have transcriptional and translational machinery most similar to archaea
