Microbiology Exam 3

Required macronutrients by ALL cells

Carbon (C), Nitrogen (N), Oxygen (O) and hydrogen (H): from water, Phosphorus (P), Sulfur (S), Potassium (K), Magnesium (Mg)

Endosymbiotic Origin of Eukaryotes

Divergence of Eukarya from Archaea resulted in membrane-enclosed nucleus, and organelles gave rise to eukaryotic cell structures

Evolution

a change in allele (alternative version) frequencies in a population over time

Glycolysis (Embden-Meyerhof-Parnas pathway)

a common pathway for catabolism of glucose that forms two ATP •Glucose can be fermented or respired •ATP produced by substrate-level phosphorylation: energy-rich phosphate bond from organic compound is transferred to ADP, making ATP

respiration

aerobic or anaerobic catabolism in which a donor is oxidized with O2 (aerobic) or another compound (anaerobic) as an electron acceptor

fermentation

anaerobic catabolism in which organic compounds donate and accept electrons •Fermentative diversity •Some fermentations allow additional ATP synthesis from substrate-level phosphorylation •involves coenzyme-A derivatives •Some fermentations are beneficial for humans •Fermentation-respiration switch is based on energetic benefit

pan genome

core genome plus genes not shared and often acquired through horizontal gene transfer

Species

fundamental units of diversity •What constitutes a microbial species remains controversial

taxonomy

how organisms are classified and named

classification

organization of organisms into progressively more inclusive groups on the basis of either phenotypic similarity or evolutionary relationship

endosymbiotic hypothesis

well-supported hypothesis for origin of eukaryotic cells •contends that mitochondria arose from stable incorporation of an aerobic respiring bacterium into the cytoplasm of early eukaryotic cells

ABC (ATP-binding cassette) systems

•200+ different systems identified in prokaryotes for organic and inorganic compounds •High substrate affinity •ATP drives uptake •Requires transmembrane and ATP-hydrolyzing proteins plus: -Gram-negatives employ periplasmic binding proteins -Gram-positives and Archaea employ substrate-binding proteins on external surface of cytoplasmic membrane.

example: Rhodobacter

•Anoxygenic phototrophic purple bacterium •In anaerobic culture, bacteriochlorophyll and carotenoids are synthesized •In light, pigments lead to ATP synthesis; in dark, no benefit •In constant darkness, mutants with reduced levels of photopigments have an advantage and rapidly take over due to fitness •In the light, pigment loss is not advantageous, and mutants are lost

Macronutrients

nutrients required in large amounts

Discuss the coupling of the carbon, nitrogen, and oxygen cycles and how environmental changes (such as increased CO2) impact the cycles.

-Carbon Cycle: =The rate of carbon fixation and plant growth is often limited by the availability of nitrogen. =The carbon cycle and the nitrogen cycle are closely coupled =Microbial decomposition releases CO2 in the atmosphere =Photosynthesis reduces inorganic carbon dioxide to organic carbohydrates =Respiration oxidizes organic carbohydrates to inorganic carbon dioxides two end products of decomposition are methane and carbon dioxide =Methane is converted to carbon dioxide by methanotrophs -Nitrogen Cycle: =Nitrogen is a key constituent of cells and exists in a number of oxidation states =Undergoes four major transformations: =Nitrification: oxidized ammonia converted to nitrite, then that nitrite gets oxidized to nitrate. Nitrogen cycle step occurs in soil. =Denitrification: reduces nitrate to gaseous nitrogen products (N2) and is the primary mechanism by which N2 is produced biologically. =Anammox: anaerobic oxidation of ammonia to N2 gas =Nitrogen Fixation: energy-intensive process that converts inorganic nitrogen (N2) into usable organic nitrogen. =N2 is the most stable form of nitrogen and is a major reservoir, as it is 70% of earth's air. =Denitrification and anammox result in losses of organic nitrogen from the biosphere. =Ammonia produced by nitrogen fixation or ammonification can be assimilated into organic matter or oxidized to nitrate -Nutrient Coupling =All nutrient cycle are linked to the carbon cycle, but nitrogen is very closely linked because nitrogen and carbon are macronutrients =Nutrient cycles are coupled as changes in either nitrogen or carbon cycles will affect other cycles. -Impacts of CO2: =Carbon dioxide is a greenhouse gas that has increased 40% since the start of the indistruial revolution. =Carbon dioxide functions to trap long wave heat from the sun that hit earth and try to bounce off =Radiative forcing is the difference in sunlight absorbed by earth and the energy radtiated back into space =Carbon dioxide is highly soulable in water and produces carbonic acid. =The carbonic acid makes the water more acidic and increases the surface temeperature. =The exapansion of the oxygen minimum zone also contributes to ocean warming. -Biological impacts carbon: =Corals will die due to seasurface temperatures increasing. =Foraminifera calcification will be impaired due to ocean acidification =Over a period of time the invasion of anthropogenic carbon dioxide intot he deep ocean will reduce the levels of calcium carbonate sequestered there and this will likey affect the carbon cycles in major ways that are not yet known. -Biological impacts Nitrogen: =Most of the industrial produced nitrogen is added to farm lands but a significant fraction runs off to the ocean and contributes to eutrophiication. =Large portions of nitrogen are also lost as nitrogenous gas through nitrification and denitrification due to the close coupling of nutrient cycles that the significant changes in the carbon and nitrogen cycle will impact the other nutrient cycles

Describe the timeline for the origin of life on Earth. Include in your answer the current hypotheses relative to the origin of Bacteria, Archaea, and Eukarya.

-Earth is approximately 4.5 bya -Water appeared approximately 4.3 bya -Evidence for life was approximately 4.1 bya -Fossilized remains of cells can be found in the rocks ~3.86 bya -Life may have originated at hydrothermal systems on the ocean floor =more nutrients, energy, most stable than surface geochemistry could support abiotic production of molecules =The structure of Hydrothermal vents facilitated a energetically favorable environment that resulted in production of molecules =Lipid Bilayers replaced the mineral compartments, allowing dispersion of cells into new habitats. -Cells may have begun in an RNA world =An essential cofactor and molecules (ATP) =Has catalytic activity =Early Viruses may have evolved from RNA =Proteins replaced RNA as catalysts =DNA became a genome and template due to stability. -LUCA!!! then bacteria and archaea diverged ~3.8-3.7 bya -Origin of Eukarya and Archaea =Great oxidation event ==Rise of oxygen gas led to the evolution of life to exploit energy from oxygen gas respiration ~2.5-3.3 bya ; because of evolved cyanobacteria ==Ozone shield allowed organisms to range over the surface exploiting new habitat and evolving diversity. (after oxidation event) ==Endosymbiosis (Origin of Eukarya; split form Archaea) divergence of Eukarya from Archaea resulted in membrane-enclosed nucleus,and organelles gave rise to eukaryotic cell structures ===well-supported hypothesis for origin of eukaryotic cells ===mitochondria arose from stable incorporation of aerobic respiring bacterium into the cytoplasm ===chloroplasts arose from stable incoordination of a cyanobacterium cell into cytoplasm of eukaryotic cell physiology, metabolism, and genome ===structures/sequences of mitochondria and chloroplasts support endosymbiotic hypothesis ===2 hypotheses for the formation of eukaryotic cells: ====Serial endosymbiosis hypothesis: Eukaryotes began as nucleus-bearing line that split from Archaea and later acquired mitochondria and chloroplasts by endosymbiosis ====Symbiogenesis hypothesis: Eukaryotic cell arose from symbiotic relationship between Bacteria and Archaea, bacteria engulfed to form mitochondria Hydrogen hypothesis:eukaryotic cells arose from a hydrogen gas producing bacterium and a hydrogen gas consuming archaea -Timing of origin of nucleus is unclear

The polyphasic approach to taxonomy uses three methods:

1. phenotypic (morphological, metabolic, physiological, chemical characteristics) analysis 2. genotypic (genome) analysis 3. phylogenetic (evolutionary) analysis

Taxonomy

Characterizes, names, classifies organisms •Bacterial taxonomy incorporates multiple methods for identifying and describing new species

Symbiogenesis hypothesis

Eukaryotic cell arose from symbiotic relationship between Bacteria and Archaea; bacterial partner was engulfed to form mitochondria

Phylogeny

Evolutionary history of related DNA sequences

anoxygenic photosynthesis

Generation of reducing power •For a purple bacterium to grow autotrophically, the formation of ATP is not enough •Reducing power (NADH) is also necessary to reduce CO2 to cell material •Reducing power for purple bacteria comes from many sources, especially sulfur compounds like H2S •Requires reverse electron transport (against electrochemical gradient) for NADH production in purple phototrophs -also mechanism chemolithotrophs use to get reducing power for CO2 fixation

Compare fermentation and respiration (what are their end goals? How much ATP is produced? Who are the electron receptors and donors?

Glycolysis- A common catabolic pathway using glucose which produces 2 ATP in the process. -Glucose can be both fermented or respired, making it important to know for both electron pathways. -The ATP is produced by substrate level phosphorylation, in which ADP is converted into ATP. Fermentation- Stage 3 of glycolysis: An anaerobic catabolism in which organic compounds donate and accept electrons. This process creates two ATP produced via substrate level phosphorylation and its end product is glucose. Fermentation electron donors and receptors are different for different types of fermentation. Some fermentations allow additional ATP synthesis from substrate- level phosphorylation (involving coenzyme A derivatives). Respiration- Aerobic or anaerobic catabolism where a donor is oxidized with O2 (aerobic) or another compound (anaerobic) as an electron donor. Pyruvate is fully oxidized to CO2 through citric acid and glyoxylate cycles. ATP produced via oxidative phosphorylation. The flavoproteins and iron are electron carriers Electron donor: substance oxidized Electron Acceptor: substance released

How would you generate electricity?

How can you produce protons using iron or sulfur? Iron oxidizing bacteria break down ferious iron into ferric iron. The process requires cytochrome, rusticyanin, and the iron oxidizing membrane protein to function in its entierity. Initially Ferreous Iron is oxidized by the outer membrane cytochromes c which oxidizies the Fe2+ and transfers the electrons to the periplasm. Once that is complete Rusticyanin reduces the periplasmic cytochrome c to aa3 which is then reduced to oxygen and water. Using the oxygen ATP is generated when the oxygen is reduced during respiration How does the proton motive force work? electron transport systems reside in the cytoplasmic membrane. Protons originating from NADH and the dissociation of water are released to the outside of the membrane via different complexes. A pH gradient and an electrochemical potential across the membrane creates the proton motive force as each successive carrier complex operates in increasingly positive E0'. the generated PMF will be used by ATP synthases to convert ADP + Pi to ATP. This can cause the cell to act as a sort of battery to store energy that can be used in processes requiring ATP, while the electrochemical gradient can be harvested into a microbial battery.

How would you make drinkable water?

How can you treat the water to remove ammonia, nitrates and nitrites? By denitrification, ion exchange which is the process of nitrogen fixation and distillation to remove nitrogen. Also the process of Nitrogenase which allows for the the reduction of nitrogen to ammonia How can you treat the water to decompose organic material through aerobic and anaerobic digestion? Anaerobic Digestion: This is the process by which certain microorganisms are kept in closed top anoxygenic tanks. The microorganisms from here digest the pollutants. Aerobic Digestion: In this process microorganisms are kept in open top tanks with free access to oxygen from the atmosphere. In doing this the microorganisms are feeding on the sludge it self.

How would you produce biofuels?

How would you produce methane using organic compounds? using CO2 as a terminal electron acceptor and H2 as an electron donor, methanogens can produce CH4. Rhodoferax ferrireducens

Phosphorus (P)

nucleic acids and phospholipids

How would you make your food? How can oxygenic and anoxygenic photoautotrophs make sugars?

Oxygenic Phtotrophs follow the standard recipe of photosynthesis. This uses CO2 + H2O = O2 Sugar Anoxygenic Phototrophs instead of using H2O as their reducing agent use H2S. Using this molecule sugars are produced. How can you get enough nitrogen to make new cells and proteins? Atmospheric nitrogen is the most abundant form of nitrogen on Earth, so a majority of the time, N2 must be reduced and fixated for it to be used by cells and proteins. No eukaryotic organism exists which can fixate nitrogen so a prokaryotic cell must be used.

Two hypotheses explaining formation of eukaryotes

Serial endosymbiosis hypothesis & Symbiogenesis hypothesis

You are stuck in a place with no food, a polluted pond, and a car without fuel. How would you use an ampule of microalgae Spirulina platensis, and Rhodoferax ferrireducens to get food, drinking water, electricity and fuel?

Spirulina platensis can perform: Oxygenic photosynthesis Calvin cycle (dark cycle of photosynthesis) Denitrification and nitrification Production of triacylglycerides (storage lipids) Oxygenic photosynthesis Calvin cycle (dark cycle of photosynthesis) Denitrification and nitrification Production of triacylglycerides (storage lipids) Rhodoferax ferrireducens Iron oxidation Aerobic and anaerobic digestion Organic decomposition

Systematics

Study of diversity of organisms and relationships, links phylogeny with taxonomy

Metabolism

The sum of all chemical reactions that occur in a cell

fermentation and respiration

Two reaction series are linked to energy conservation in chemoorganotrophs

Photosynthesis

is the conversion of light energy to chemical energy •Phototrophs carry out photosynthesis •Most phototrophs are also autotrophs that use CO2 as sole carbon source •Photoautotrophs use energy from light to reduce CO2 to organic compounds •Photoheterotrophs are phototrophs that use organic carbon as a carbon source •Photosynthesis originated in Bacteria •Photosynthesis also evolved in Eukarya •Photosynthesis requires light-sensitive pigments called chlorophylls and bacteriochlorophylls that absorb light energy

Calcium (Ca) and sodium (Na)

required by some microbes (e.g., marine microbes)

Photoautotrophy

requires two sets of parallel reactions •Light reactions produce ATP •Dark reactions reduce CO2 to cell material for growth -requires ATP and electrons (NADH or NADPH) -NADH/NADPH requires an electron donor from the environment (e.g., water, H2S, H2) -Oxygenic photosynthesis: Oxidation of H2O produces O2 (cyanobacteria) -Anoxygenic photosynthesis: all other phototrophic bacteria -NADH/NADPH requires an electron donor from the environment (e.g., Water, H2S, H2).

core genome

shared by all strains of a species

Nutrients

supply of monomers (or precursors of) required by cells for growth

Other pathways of CO2 fixation

•At least four other pathways known •Chloroflexus uses the 3-hydroxypropionate bi-cycle to fix CO2 -May have been one of earliest mechanisms for autotrophy -Also found in several hyperthermophilic Archaea -Cyclic photophosphorylation: electrons move within closed loop; no net input or consumption •Also 3-hydroxypropionate/4-hydroxybutyrate cycle and dicarboxylate/4-hydroxybutyrate cycle (in Archaea) •Reductive acetyl-coenzyme A pathway found in obligate anaerobes including methanogenic Archaea, acetogens, Planctomyces -Most efficient: requires only six to eight ATP per six CO2/1 glucose -Can be coupled directly to energy conservation

Metabolic diversification: Consequences for Earth's biosphere

•Because early Earth was anoxic, energy-generating metabolism of primitive cells was exclusively anaerobic •Obtained carbon from CO2 (autotrophy) •Evolved ability to use N2 (nitrogen fixation) •Obtained energy from H2 •S may have been an early electron acceptor •Early forms of chemolithotrophic metabolism would have supported production of large amounts of organic compounds •Accumulated organic material provided conditions needed for evolution of chemoorganotrophic metabolisms •Eventually Earth became highly oxic

Biosynthesis: Amino Acids and Nucleotides

•Biosynthesis often involves long, multistep pathways •Amino acid biosynthesis -Carbon skeletons come from intermediates of glycolysis or citric acid cycle -Ammonia is incorporated by glutamine dehydrogenase or glutamine synthetase -Amino group transferred by transaminase and aminotransferase/synthase •Purine and pyrimidine biosynthesis are complex •Purines (A, G) -inosinic acid precursor to adenine and guanine •Pyrimidines (C, T) -orotic acid precursor to thymine, cytosine, and uracil

The Carbon Cycle

•Carbon is cycled through all of Earth's major carbon reservoirs -includes atmosphere, land, oceans, sediments, rocks, and biomass -all nutrient cycles are linked to the carbon cycle, but nitrogen is very closely linked because nitrogen and carbon are macronutrients •Reservoir size and turnover time are important parameters in understanding the cycling of elements •While the sediments and rocks in the Earth's crust are the largest carbon reservoir, CO2 in the atmosphere is the most rapidly transferred carbon reservoir •CO2 is removed from the atmosphere by photosynthetic land plants and marine microbes, so a large amount of carbon is found there •More carbon is found in humus, or dead organic material, than is found in the living organisms •CO2 is returned to the atmosphere by respiration and decomposition as well as by human-related (anthropogenic) activities •Microbial decomposition is the largest source of CO2 released to the atmosphere •Since the Industrial Revolution, human (anthropogenic) activities have increased atmospheric carbon by 40 percent •This rise in carbon dioxide has led to steadily increasing temperatures worldwide (global warming) because carbon dioxide is a greenhouse gas •Phototrophic organisms produce organic or fixed carbon and reduce the level of carbon dioxide in the atmosphere -Oxygenic phototrophic organisms can be divided into two groups: plants and microorganisms =Plants dominate terrestrial environments =Microorganisms dominate aquatic environments •Photosynthesis and respiration are part of redox cycle •Photosynthesis -reduces inorganic carbon dioxide to organic carbohydrates -CO2 + H2O (->CH2O) + O2 •Respiration -oxidizes organic carbohydrates to inorganic carbon dioxide -(CH2O) + O2 ->CO2 + H2O •The two major end products of decomposition are methane (CH4) and carbon dioxide (CO2 ) •CH4 is a potent greenhouse gas and is produced in anoxic (or oxygen-free) environments •Most methane is converted to carbon dioxide by methanotrophs; however, some enters the atmosphere •Methane hydrates -form when high levels of methane are under high pressure and low temperature -Huge amounts of methane are trapped underground as methane hydrates -Methane hydrates can absorb and release methane -Methane hydrates fuel deep-sea ecosystems called cold seeps •Coupled cycles -In nature, nutrient cycles are interconnected and feed back upon one another -Major changes in one cycle affect the functioning of other cycles -For example, the rate of carbon fixation and plant growth is often limited by the available nitrogen. This is why adding nitrogen to farm fields will increase yield. - The carbon cycle and the nitrogen cycle are very closely coupled

Enzyme catalysis

•Catalysis depends on -substrate binding -position of substrate relative to catalytically active amino acids in active site •Endergonic and exergonic reactions coupled -example: ATP hydrolysis or proton motive force

Autotrophic Pathways

•Cells require carbon and nitrogen to form biomass •Atmospheric sources (CO2 and N2) must be chemically reduced for assimilation (CO2 fixation and N2 fixation). •Requires ATP and reducing power •Autotrophy: process by which CO2 is reduced and assimilated into cells •In phototrophs, autotrophy is often called the "dark reactions" •In oxygenic photosynthesis, Calvin cycle reduces CO2 to glyceraldehyde-3-phosphate •Many alternative pathways exist, reducing CO2 to the central metabolite acetyl-CoA

Energy-Rich Compounds

•Chemical energy released in redox reactions is primarily stored in certain phosphorylated compounds -ATP; the prime energy currency -phosphoenolpyruvate •Chemical energy also stored in coenzyme A derivatives •Long-term energy storage involves biosynthesis of insoluble polymers that can be oxidized to generate ATP -examples in prokaryotes =glycogen (polyglucose) =poly-β-hydroxybutyrate and other =polyhydroxyalkanoates =elemental sulfur (S) -examples in eukaryotes =starch (also polyglucose) =lipids (simple fats)

Energy Classes of Microorganisms

•Chemoorganotrophs conserve energy from organic chemicals •Chemolithotrophs oxidize inorganic compounds (H2, H2S, NH4+) •Phototrophs convert light energy into ATP •Heterotrophs obtain carbon from organics •Autotrophs obtain carbon from CO2

Photosynthetic membranes, chloroplasts, and chlorosomes

•Chlorophyll pigments and light-gathering apparatus are located within special membranes -In eukaryotes, photosynthesis occurs in chloroplasts (intracellular organelles containing thylakoids: sheet-like membrane systems) -In prokaryotes, pigments are integrated into cytoplasmic membrane: chromatophores or lamellae in purple bacteria, thylakoids in cyanobacteria •Chlorosomes capture low light intensities -found in anoxygenic green sulfur bacteria, filamentous green nonsulfur bacteria, and photosynthetic Acidobacteria -function as giant antenna systems -contain bacteriochlorophyll in dense arrays -transfers light energy through FMO protein •Green bacteria grow at lowest light intensities -often found in deepest waters where light cannot support other phototrophs

Reaction centers and antenna pigments

•Chlorophyll/bacteriochlorophyll is not free in the cell and is found in photocomplexes containing proteins housed within membranes •Reaction centers contain some pigments and participate directly in energy conservation •Antenna pigments surround and funnel light energy to reaction centers.

Phylogenetic trees

•Diagrams depicting evolutionary history •Difference in nucleotide sequence between two organisms is a function of number of mutations accumulated since they shared a common ancestor

Simple transport

•Driven by proton motive force •Either symport: solute and H+ cotransported in one direction -E. coli lac permease, phosphate, sulfate, other organics •or antiport: solute and H+ transported in opposite directions

Origin of Earth

•Earth is ~4.5 billion years old •Liquid water required for life; first appeared ~4.3 billion years ago •Evidence for life ~4.1 billion years ago •Fossilized remains of cells can be found in rocks ~3.86 billion years old

Electron Transport and the Proton Motive Force

•Electron transport system oriented in cytoplasmic membrane so that electrons are separated from protons •Two electrons + two protons enter when NADH oxidized to NAD+ by NADH dehydrogenase •The final carrier in the chain donates the electrons and protons to the terminal electron acceptor (e.g., O2) •During electron transfer, protons are released on outside of the membrane -Protons originate from 1) NADH and 2) dissociation of water •Results in generation of pH gradient and an electrochemical potential across the membrane (the proton motive force) -The inside becomes electrically negative and alkaline (OH-) -The outside becomes electrically positive and acidic (H+) •Common characteristics: -Membrane carrier complexes are arranged in an order of increasingly positive E0′ -Complexes alternate between those that carry electrons only and those that carry electrons plus H+ -Each system generates a PMF, which is characterized by a pH gradient and an electrochemical potential across the membrane, during electron transport. The PMF will be used to synthesize ATP via oxidative phosphorylation

Electron Donors and Acceptors

•Energy from oxidation-reduction (redox) reactions is used in synthesis of energy-rich compounds (e.g., ATP) •Redox reactions occur in pairs (two half reactions) •Electron donor: the substance oxidized Electron acceptor:the substance reduced •Substances can be either electron donors or electron acceptors under different circumstances (redox couple) •Reduction potential (E0′): tendency to donate electrons -expressed as volts (V) compared with reference (H2) •Reduced substance of a redox couple with a more negative E0′ donates electrons to the oxidized substance of a redox couple with a more positive E0′ •The redox tower represents the range of possible reduction potentials •Substances toward the top (reduced) prefer to donate electrons •Substances toward the bottom (oxidized) prefer to accept electrons •The farther the electrons "drop," the greater the amount of energy released (ΔE0′) •Oxygen (O2): strongest significant natural electron acceptor •NAD+ and NADH facilitate redox reactions without being consumed; they are recycled •Allows many different donors and acceptors to interact •Coenzyme acts as intermediary •Another example: NADP+/NADPH facilitate anabolic (biosynthetic) redox reactions

Principles of Bioenergetics

•Energy is measured in units of kilojoules (kJ) of heat energy •In any chemical reaction, energy is either required or released •Free energy (G): energy released that is available to do work •The change in free energy during a reaction is referred to as ΔG0′ (standard conditions) •Exergonic: Reactions with -ΔG0′ release free energy •Endergonic: Reactions with +ΔG0′ require energy •To calculate free-energy yield of a reaction, we need to know the free energy of formation -Gf0; the energy released or required during formation of a given molecule from the elements •For the reaction A + B C + D, -ΔG0′ = Gf0 [C + D] - Gf0[A + B] •ΔG0′ is not always a good estimate of actual free energy changes •ΔG: free energy that occurs under actual conditions -ΔG = ΔG0′ + RT ln Keq -where R and T are physical constants and Keq is the equilibrium constant for the reaction •Only exergonic reactions yield energy that can be conserved by the cell

New traits can evolve quickly in microorganisms

•Environmental change or introduction of new cells can cause rapid evolutionary changes •Microbes form large populations and reproduce quickly

Nitrogenase

•Enzyme complex consisting of dinitrogenase and dinitrogenase reductase •Iron-molybdenum cofactor (FeMo-co) is where N2 reduction occurs •Alternative nitrogenases lack molybdenum and contain either vanadium (V) and iron or iron-only •Inhibited by oxygen •In obligate aerobes, nitrogenase is protected, for example, by removal by respiration, oxygen-retarding slime layers, anoxic heterocyst formation

Serial endosymbiosis hypothesis

•Eukaryotes began as nucleus-bearing line that split from Archaea and later acquired mitochondria and chloroplasts by endosymbiosis •Endosymbiosis occurred when line engulfed a bacterial cell that survived and replicated •Eukaryotic genes that resemble bacterial genes were acquired through gene transfers from endosymbiont to nucleus •does not account for similarities in bacterial and eukaryotic membrane lipids

Hydrogen hypothesis

•Eukaryotic cell arose from an H2-producing bacterium and an H2-consuming Archaea •Genes for lipid biosynthesis were transferred from bacterial symbiont to archaeal host

Formation of the eukaryotic cell

•Eukaryotic cell is chimeric and made up of genes from both Bacteria and Archaea -Eukaryotes have transcription and translational machinery similar to those of Archaea -Eukaryotes have metabolisms similar to those of Bacteria •timing of origin of nucleus unclear •formation may be associated with evolution of RNA processing (i.e., Nuclear membrane may have evolved to separate spliceosomes from ribosomes)

Biosynthesis: Fatty Acids and Lipids

•Fatty acids are biosynthesized two carbons at a time -Acyl carrier protein (ACP) holds the growing fatty acid as it is being synthesized -each C2 actually originates from C3 malonate; CO2 released -varies between species and at different temperatures =lower temps: shorter, more unsaturated =higher temps: longer, more saturated -can be unsaturated, branched, or contain odd numbers of carbon atoms •In Bacteria and Eukarya, assembly of lipids involves addition of fatty acids to glycerol •In Archaea, lipids contain phytanyl side chains instead of fatty acids •In all three kingdoms, polar groups necessary for canonical membrane architecture (hydrophobic interior, hydrophilic surfaces)

Respiration: Citric Acid and Glyoxylate Cycles

•First catabolize glucose with glycolysis •Pyruvate is fully oxidized to CO2 through citric acid and glyoxylate cycles

The ozone shield

•Formation of ozone (O3) shield that protects Earth's surface from UV radiation •Before, Earth's surface was inhospitable •Ozone shield allowed organisms to range over surface, exploiting new habitats and evolving diversity

Electron flow and ATP synthesis in oxygenic photosynthesis

•PSII splits water into oxygen and electrons at water-oxidizing complex •Proton motive force generated by electron transport through quinones and cytochromes (like aerobic respiration) •PSII transfers energy to PSI, terminating with reduction of NADP+ to NADPH •12 H+ translocated per O2 produced •Noncyclic photophosphorylation: Electrons do not cycle back and reduce NADP+ to NADPH •Cyclic photophosphorylation can occur if cell requires less NADPH to produce more ATP

The Calvin cycle

•Found in purple bacteria, cyanobacteria, algae, green plants, most chemolithotrophic Bacteria, few Archaea •Requires CO2, a CO2 acceptor, NADPH, ATP, ribulose bisphophate carboxylase (RubisCO), and phosphoribulokinase •First step catalyzed by RubisCO, forming two molecules 3-phosphoglyceric acid (PGA) from ribulose bisphophate and CO2 •PGA then phosphorylated and reduced to glyceraldehyde-3-phosphate •Glucose formed by reversal of glycolysis •Easiest to consider cycle as six molecules of CO2 required to make one molecule of glucose •12 NADPH and 18 ATP required •Carboxysomes: inclusions containing and improving efficiency of RubisCO in many autotrophs •Inorganic carbon first incorporated as bicarbonate (HCO3-), which is converted to CO2 by carbonic anhydrase •CO2 cannot escape carboxysome •Carboxysome also protects RubisCO from O2, which competes with CO2

Catalysis and Enzymes

•Free energy calculations do not provide information on reaction rates •Activation energy: minimum energy required for molecules to become reactive -A catalyst is usually required to overcome activation energy barrier •Many enzymes contain small nonprotein, nonsubstrate molecules that participate in catalysis •Prosthetic groups -tightly bound -usually bind covalently and permanently (e.g., heme in cytochromes) •Coenzymes -Loosely bound -Most are derivatives of vitamins

The Evolution of Microbial Genomes

•Genome sequencing of three strains of Escherichia coli showed only 39 percent of genes were shared

Photosynthetic electron flow in other anoxygenic phototrophs

•Green sulfur bacteria, Acidobacteria, and Heliobacteria use FeS-type reaction centers •Reverse electron flow is unnecessary in green sulfur bacteria and Heliobacteria •Ferredoxin is critical for electron transfer •Unclear whether electron transfer in green sulfur bacteria and Heliobacteria is cyclic or noncyclic

Human Impacts on the Carbon and Nitrogen Cycles

•Human activity is believed to have a major impact on the carbon cycle -CO2 levels have increased more than 40 percent since the start of the Industrial Revolution and are now higher than they have been in the last 800,000 years -CO2 is a greenhouse gas that traps long-wave heat waves from the Earth's surface, in effect making the entire planet a large greenhouse. This phenomenon is called radiative forcing. -Dissolved carbon dioxide decreases the pH of the ocean. This acidification endangers coral reefs, which will release calcium carbonate as they die. -Air and ocean water temperatures are increasing, which increases the oxygen minimum zones (OMZ) in the ocean •Human activity believed to have a major impact on nitrogen cycle -Humans produce large amounts of nitrogenous fertilizers -Ecological effects of fertilizers are unknown, but the alteration of nitrogen cycles will also change iron availability and the carbon cycle •Nutrient cycles are coupled -Change in either nitrogen or carbon cycles will affect other cycles

Anoxygenic photosynthesis in oxygenic phototrophs

•If PSII is blocked, some oxygenic phototrophs can do photosynthesis with just PSI •Cyclic photophosphorylation occurs exclusively •Reducing power for CO2 reduction comes from sources other than water (H2S in cyanobacteria, H2 in green algae) •Anoxygenic photosynthesis must have been first on Earth •Cyanobacteria evolved to connect PSI and PSII and use H2O as an electron donor

micronutrients required

•Iron (Fe): cellular respiration •trace metals: enzyme cofactors •Growth factors: Organic compounds required in small amounts by certain organisms -examples: vitamins, amino acids, purines, pyrimidines •Vitamins: Most frequently required growth factors & Most function as coenzymes

Iron cylce

•Iron is one of the most abundant elements in Earth's crust but often a limiting nutrient for microbial growth •On Earth's surface, iron exists naturally in two oxidation states -ferrous (Fe2+) and ferric (Fe3+) -The redox reactions in the iron cycle include both oxidations and reductions •Fe3+ can be used by some microorganisms as an electron acceptor in anaerobic respiration •In nature, humic substances can serve as an electron shuttle to reduce Fe3+ •The oxidation of ferrous iron to ferric iron yields very little energy, so bacteria with this type of metabolism need to oxidize large amounts of ferrous iron to fuel their growth

Catabolism

•Large molecules are broken down into small ones. •Energy-releasing metabolic reactions

Origin of cellular life

•Life may have originated at hydrothermal systems on ocean floor -Conditions would have been more stable than surface -Steady and abundant supply of energy (e.g., H2 and H2S) may have been available at these sites -Geochemistry can support abiotic production of molecules required for life (e.g., amino acids, lipids, sugars, and nucleotides) -Mineral structures may have produced compartments for conserving energy •Life may have begun in an RNA world -RNA is part of essential cofactors and molecules (e.g., ATP, NADH, coenzyme A) -RNA can bind small molecules (e.g., ATP, other nucleotides, amino acids) -RNA has catalytic activity; may have catalyzed its own synthesis -Earliest viruses may have evolved from RNA genome cell-like structures •Proteins eventually replaced RNAs as catalysts •DNA (more stable) became genome and template •Earliest cells probably had DNA, RNA, protein, and membrane system for energy conservation •Last universal common ancestor (LUCA) existed 3.8-3.7 billion years ago, then Bacteria and Archaea diverged

Example: Escherichia coli

•Long-term evolution experiment (LTEE) started in 1988 and has tracked 12 parallel lines over 50,000+ generations •Minimal glucose medium represents an adaptive environment in which E. coli can evolve over time •A marker that colors cells red or white enables measurement of evolved strains relative to ancestor by competition •dramatic increase in fitness over first 500 generations, then slowed down •New ability to use citrate found in only one of 12 lines

Manganese Cycle

•Manganese (Mn) is also present on Earth's surface -Manganese exists mainly in two oxidation states -Manganese cycles between oxidized and reduced states with iron in aquatic ecosystems

Mercury Transformations

•Mercury has a tendency to concentrate in living tissues and is highly toxic •The major form of mercury in the atmosphere is elemental mercury (Hg0), which is volatile and oxidized to mercuric ion (Hg2+) photochemically •Most mercury enters aquatic environments as Hg2+ •Hg2+ readily adsorbs to particulate matter where it can be metabolized by microorganisms •Microorganisms form methylmercury (CH3Hg+), an extremely soluble and toxic compound •Several bacteria can also transform toxic mercury to nontoxic forms •Bacterial resistance to mercury is often linked to specific plasmids that encode enzymes capable of detoxifying or pumping out the metals

Methanogenesis

•Methanogenesis is central to carbon cycling in anoxic environments •Most methanogens use carbon dioxide as a terminal electron acceptor, reducing CO2 to CH4 with H2 as an electron donor; some can reduce other substrates (e.g., acetate) to form CH4 •Methanogens team up with partners (syntrophs) that supply them with necessary substrates •Methanogenic symbionts can be found in some protists. There are protists that live within termite guts that have methanogenic symbionts. •Possible that endosymbiotic methanogens benefit protists by consuming H2 generated from glucose fermentation •The combination of protists and their endosymbionts enables termites to digest cellulose •Acetogenesis is another H2-consuming process competing with methanogenesis in some environments -occurs in termite hindgut with the methanogens and protists •Methanogenesis is energetically more favorable than acetogenesis. •Acetogens can ferment glucose and methoxylated aromatic compounds (like lignin, which is found in wood), whereas methanogens cannot. This expands the diet of termites.

Options for Energy Conservation

•Microorganisms demonstrate a wide range of mechanisms for generating energy: •anaerobic respiration •chemolithotrophy •phototrophy

The rise of oxygen: Banded iron formations

•Most iron would have been reduced (Fe0 and Fe+2) and dissolved in anoxic oceans •O2 produced reacted spontaneously with reduced iron forming iron oxides instead of accumulating •By 2.4 billion years ago, O2 rose to one part per million (Great Oxidation Event) •Iron oxides precipitated and formed banded iron formations: laminated sedimentary rocks •Atmosphere gradually became oxic •New metabolisms evolved (e.g., sulfide oxidation, nitrification, other aerobic chemolithotrophy) •Respiring O2 energetically advantageous because of high reduction potential, allowing aerobes to reproduce much faster than anaerobes

Origins of genetic diversity

•Mutations: random changes in DNA sequence occurring over time •Most mutations are neutral or deleterious; some are beneficial •Several forms including substitutions, deletions, insertions, duplications •Recombination breaks and rejoins DNA segments to make new combinations of genetic material -can reassort genetic material already present -required for integration of acquired DNA -can be classified as homologous (requiring short flanking segments of similar sequence) or nonhomologous (does not require high similarity) -the ability to produce progeny and contribute to genetic makeup of future generations

Respiration: Electron Carriers

•NADH dehydrogenases: active sites bind NADH, accept two electrons and two protons that are transferred to flavoproteins, generate NAD+ •Flavoproteins: contain flavin prosthetic group (e.g., FMN, FAD) that accepts two electrons and two protons but donates only electrons •Cytochromes -proteins that contain heme prosthetic groups -accept and donate a single electron via the iron atom in heme (Fe2+ or Fe3+) -sometimes form complexes (e.g., cytochrome bc1) •Other iron proteins -nonheme iron -contain clusters of iron and sulfur, (e.g., ferredoxin) -Reduction potentials vary -only carry electrons •Quinones -small hydrophobic nonprotein redox molecules -can move within membrane -accept electrons and protons but transfer electrons only -typically link iron-sulfur proteins and cytochromes -ubiquinone (coenzyme Q) and menaquinone most common

Culture collections

•National microbial culture collections are an important foundation •catalog and store microorganisms and provide cultures on request for a fee •protect diversity •store viable cultures (frozen or free-dried) •act as repositories for type strains (serve as nomenclatural type for future comparison with other strains of the species)

Nitrogen Fixation

•Nitrogen needed for proteins, nucleic acids, other organics •Only certain prokaryotes can form ammonia (NH3) from gaseous dinitrogen (N2): nitrogen fixation •Some nitrogen fixers are free-living, and others are symbiotic (fixing only in association with certain plants) •No eukaryotes fix N2

Assaying nitrogenase: Acetylene reduction

•Nitrogenases reduce other triply bonded compounds, including acetylene, to form ethylene •Definitive proof requires 15N2 as tracer to form 15NH3

Bergey's Manual and The Prokaryotes

•No official classification, but most widely accepted is Bergey's Manual of Systematic Bacteriology •The Prokaryotes available online

Phenotypic analysis

•Observable characteristics (phenotype) provide differentiable traits •example: fatty acid analysis -types and proportions of fatty acids in cytoplasmic membrane lipids and gram-negative outer membrane lipids often used in taxonomic analyses -technique used is FAME (fatty acid methyl ester) -widespread use in clinical, public health, and food- and water-inspection laboratories to identify pathogens -relies on variation in composition of fatty acids in membrane lipids for specific prokaryotic groups -analysis by gas chromatography compared with database -requires rigid standardization because FAME profiles can vary as a function of temperature, growth phase, and growth medium

Endosymbiosis

•Oldest eukaryotic microfossils ~two billion years old •Fossils of multicellular and more complex eukaryotes are found in rocks 1.9 to 1.4 billion years old •By 0.6 billion years ago, O2 was near present levels, and large multicellular organisms (Ediacaran fauna) were present in sea, then diversified into ancestors of algae, plants, fungi, animals •Chloroplasts arose from stable incorporation of a cyanobacterium-like cell into cytoplasm of a eukaryotic cell, leading to eukaryotic photosynthesis •Oxygen spurred evolution of organelle-containing eukaryotic microorganisms -consumed by mitochondria, produced by chloroplast •Physiology, metabolism, and genome structures/sequences of mitochondria and chloroplasts support endosymbiotic hypothesis -70S ribosomes including 16S rRNA -mitochondria ancestor likely Alphaproteobacteria, chloroplast ancestor likely Cyanobacteria

Redox (Reduction-oxidation)

•Oxidation involves the removal of an electron (or electrons) from a substance •Reduction involves the addition of an electron (or electrons) to a substance

Oxygenic Photosynthesis

•Oxygenic phototrophs use both FeS-type (photosystem I, PS I, or P700) and Q-type reaction centers (photosystem II, PSII, or P680) •"Z scheme" of photosynthesis -Photosystem II transfers energy to photosystem I •In eukaryotes, occurs in chloroplast •In cyanobacteria, occurs in stacked membranes in cytoplasm

How many species of Bacteria and Archaea exist?

•no firm estimate on the number of prokaryotic species •10,000 species of Bacteria and Archaea currently known •Nearly all plant and animal species have microbiomes with countless unique microbes

Respiration: Citric acid cycle

•Pathway through which pyruvate is completely oxidized to CO2; much greater ATP yield than fermentation (38 vs. 2) -Decarboxylation of pyruvate to CO2, NADH, and acetyl-CoA -Acetyl-CoA + oxaloacetate forms citric acid =2 CO2, 3 NADH, 1 FADH2 =oxaloacetate regenerated •Per pyruvate, total = 3 CO2, 4 NADH, 1 FADH2 •Per glucose molecule, 6 CO2 molecules released and NADH and FADH2 generated -NADH and FADH oxidized in electron transport chain: consumes electrons and produces ATP •Another role of the citric acid cycle: Biosynthesis! -α-Ketoglutarate and oxaloacetate (OAA): precursors of several amino acids; OAA also converted to phosphoenolpyruvate, a precursor of glucose -succinyl-CoA: required for synthesis of cytochromes, chlorophyll, and other tetrapyrrole compounds -acetyl-CoA: necessary for fatty acid biosynthesis

Photosynthesis and the Oxidation of Earth

•Phototrophs use energy from sun to oxidize H2S, S, and H2O to synthesize complex organic molecules from CO2 or simple organics •First phototrophs were anoxygenic •Cyanobacteria (O2 producers; oxygenic phototrophs) evolved •Stromatolites (fossilized microbial formations) found in rocks 3.5 billion years old -phototrophic bacteria (cyanobacteria and Chloroflexus) from modern stromatolites -Ancient stromatolites contain fossils similar to modern phototrophic bacteria •Between 2.5 and 3.3 billion years ago, cyanobacteria evolved a photosystem that could use H2O instead of H2S, generating O2 •Rise of O2 allowed evolution of life to exploit energy from O2 respiration

Phycobiliproteins and phycobilisomes

•Phycobiliproteins are main light-harvesting systems of cyanobacteria and red algae chloroplasts -Consist of red or blue-green tetrapyrroles called bilins bound to proteins -Give characteristic colors -Phycoerythrin absorbs ~550 nm, phycocyanin absorbs ~620 nm, and allophycocyanin absorbs ~650 nm. -Pigments are integrated into cytoplasmic membrane in prokaryotes, into chromatophores or lamellae in purple bacteria, and into thylakoids in cyanobacteria •Phycobiliproteins assemble aggregates called phycobilisomes that attach to thylakoids •Allow cell to grow at lower light intensities

Sequence alignment

•Phylogeny requires homology (inheritance from common ancestor) -Orthologs have same function and originate from a single ancestral gene in a common ancestor -Paralogs have evolved to have different functions resulting from gene duplication -Phylogenetic analyses typically focus on orthologs •Estimate evolutionary changes from number of sequence differences across a set of homologous nucleotides •Sequence alignment adds gaps to establish positional homology

Biosynthesis: Sugars and Polysaccharides

•Prokaryotic polysaccharides are synthesized from activated glucose -uridine diphosphoglucose (UDPG) =precursor of some glucose derivatives needed for biosynthesis of important polysaccharides (e.g., N-acetylglucosamine and N-acetylmuramic acid) -adenosine diphosphoglucose (ADPG) =precursor for glycogen biosynthesis •Gluconeogenesis: synthesis of glucose from phosphoenolpyruvate(from oxaloacetate •Pentoses (C5 sugars) -formed by the removal of one carbon atom from a hexose -required for the synthesis of nucleic acids •Major pathway for pentose production is the pentose phosphate pathway -major means for direct synthesis of NADPH for deoxyribonucleotide and fatty acid biosynthesis

Calcium cycle

•Reservoirs are rocks and oceans •Marine phototrophic microorganisms, such as foraminifera, use Ca2+ to form exoskeleton. This is what formed a large part of the White Cliffs of Dover.

Gene sequence analyses

•SSU rRNA not always useful for distinguishing closely related species •other highly conserved genes (e.g., recA and gyrB) useful for distinguishing at species level

Selection

•Selection is defined by fitness (ability of an organism to produce progeny and contribute to genetic makeup of future generations) •Most mutations are neutral and accumulate over time •Deleterious mutations decrease fitness and are removed by natural selection over time •Beneficial mutations increase fitness and are favored by natural selection •Mutations occur by chance; environment selects for advantageous mutations

Anabolism

•Small molecules are assembled into large ones •Energy is required.

Speciation of microorganisms can take a long time

•Species can possess a variety of individuals with different traits •Sequence changes can be used as a molecular clock to estimate time since two lineages diverged •Major assumptions are that nucleotide changes accumulate in proportion to time, are generally neutral and do not interfere with function, and are random •Most reliable if calibrated with evidence from geological record •examples: E. coli harmless K-12 and O157:H7 pathogen diverged ~4.5 million years ago, E. coli and Salmonella enterica diverged ~100-140 million years ago

Taxonomy and describing new species

•Species is one to several strains •Genus (genera) groups several species •families, orders, classes, domains •Prokaryotes are given descriptive genus names and species epithets following the binomial system of nomenclature used throughout biology •Assignment of names for new species and higher groups of prokaryotes is regulated by the International Code of Nomenclature of Bacteria (the Bacteriological Code) •New isolate needs to be compared to see if it is sufficiently different to be described as a new taxon •Formal validation of a new prokaryotic species requires -detailed description of characteristics/traits and proposed name -deposition of viable cultures of the organism in at least two international culture collections -official publication for taxonomy and classification is International Journal of Systematic and Evolutionary Microbiology (IJSEM) -websites: List of Prokaryotic Names with Standing in Nomenclature (http://www.bacterion.net) and Prokaryotic Nomenclature Up-to-Date (http://www.dsmz.de) •Molecular and genomic techniques can characterize phenotypic and genotypic characteristics without cultivation •If an organism is well-characterized but not yet cultured, a provisional taxonomic name with Candidatus can be used (e.g., Candidatus Pelagibacter ubique) •The International Committee on Systematics of Prokaryotes (ICSP) oversees nomenclature and taxonomy of Bacteria and Archaea

three stages of glycolysis

•Stage I: "preparatory," form key intermediates •Stage II: redox •Stage III (fermentation): redox •Two ATPs are produced

Group translocation

•Substance transported is chemically modified •Energy-rich organic compound (not proton-motive force) drives transport •Best-studied system: •Phosphotransferase system in E. coli -best-studied group translocation system -glucose, fructose, and mannose -five proteins required -energy derived from phosphoenolpyruvate

The Sulfur Cycle

•Sulfur transformations by microorganisms are complex •The bulk of sulfur on Earth occurs in sediments and rocks as sulfate and sulfide minerals (e.g., gypsum, pyrite) •The oceans represent the most significant reservoir of sulfur (as sulfate) in the biosphere •Hydrogen sulfide is a major volatile sulfur gas that is produced by bacteria via sulfate reduction or emitted from geochemical sources •Sulfide is toxic to many plants and animals and reacts with numerous metals •Sulfur-oxidizing chemolithotrophs can oxidize sulfide and elemental sulfur at oxic/anoxic interfaces •Organic sulfur compounds can also be metabolized by microorganisms •The most abundant organic sulfur compound in nature is dimethyl sulfide (DMS). -produced primarily in marine environments as a degradation product of dimethylsulfoniopropionate (an algal osmolyte) •DMS can be transformed via a number of microbial processes

Chlorophyll and Bacteriochlorophyl

•Tetrapyrroles with magnesium •Several different types of chlorophyll exist, each with a distinct absorption spectrum •Bacteriochlorophylls found in anoxygenic phototrophs •Use of different pigments allows different phototrophs to absorb different wavelengths and coexist in same habitat

Silica cycle

•The marine silica cycle is controlled by unicellular eukaryotes that build cell skeletons called frustules •Examples: diatoms, silicoflagellates, and radiolarians

Electron flow in nitrogen fixation

•Triple bond stability makes activation and reduction very energy demanding •Six electrons needed; eight actually consumed because H2 must be produced •Electron donor → dinitrogenase reductase → dinitrogenase →N2 •ATP required to lower reduction potential (total 16)

The reverse citric acid cycle (TCA cycle)

•Used by green sulfur bacteria (e.g., Chlorobium) •CO2 reduced by reversal of steps in citric acid cycle •More efficient, requiring 4 NADH, 2 reduced ferredoxins, 10 ATP •Requires some unique enzymes not found in citric acid cycle (e.g., alpha-ketoglutarate synthase, pyruvate synthase, citrate lyase, fumarate reductase) •Occurs in some chemoautotrophs (e.g., Thermoproteus, Sulfolobus, Aquifex, Sulfurimonas)

Enzymes

•biological catalysts •typically proteins (some RNAs) •highly specific •active site: region of enzyme that binds substrate

Limitations of phylogenetic trees

•can be difficult to choose true tree if several fit data well -Bootstrapping can deal with uncertainty; indicates percentage of time a node is supported by data •Homoplasy (convergent evolution) complicates tree construction when sequence divergence is high •horizontal gene transfer

ATP synthase (ATPase)

•complex that converts proton motive force into ATP; two components •F1: multiprotein extramembrane complex extending into cytoplasm •Fo: membrane-integrated proton-translocating multiprotein complex •reversible catalysis of ADP + Pi to ATP •consumes three to four H+ per ATP; three ATP produced per two e- •ATPases in strict fermenters generate proton motive force for flagellar rotation and transport

Respiration: Electron Carriers: Electron transport systems

•cytoplasmic membrane-associated •mediate transfer of electrons •conserve some of the energy released during transfer and use it to synthesize ATP •many oxidation-reduction enzymes involved in electron transport (e.g., NADH dehydrogenases, flavoproteins, iron-sulfur proteins, cytochromes) •also quinones: nonprotein electron carriers increasingly more positive reduction potential

Phylogenetic trees: Composition and construction

•depicts evolutionary history and resembles family tree •composed of nodes and branches •branch tips are species that exist today •can have either rooted (show position of ancestor) or unrooted (do not show most ancestral node) trees •nodes show where an ancestor diverged into two lineages •branch length represents the number of changes that have occurred along that branch •only one correct tree possible, but challenging to build •structure inferred by applying an algorithm (programmed series of steps) or optimality criteria

Catalyst is a substance that

•facilitates a reaction without being consumed •lowers activation energy •does not affect energetics or equilibrium of a reaction •increases reaction rate

The Nitrogen Cycle

•four major nitrogen transformations -nitrification -denitrification: the reduction of nitrate to gaseous nitrogen products (N2) and is the primary mechanism by which N2 is produced biologically -anammox is the anaerobic oxidation of ammonia to N2 gas -nitrogen fixation: Only a few prokaryotes have the ability to use N2 as a cellular nitrogen source. They convert inorganic N2 to organic nitrogen through an energy-intensive process, Ammonia produced by nitrogen fixation or ammonification can be assimilated into organic matter or oxidized to nitrate •N2 is the most stable form of nitrogen and is a major reservoir, as it is ~70 percent of the Earth's air •Denitrification and anammox result in losses of organic nitrogen from the biosphere

Active transport

•how cells accumulate solutes against concentration gradient

Obtaining DNA sequences

•isolate genomic DNA and sequence directly or use polymerase chain reaction (PCR) •SSU (small subunit) ribosomal RNA (rRNA) genes highly conserved and easily sequenced and analyzed •can amplify SSU rRNA from environmental samples or to sequence environmental using metagenomics

Carbon (C)

•major element in ALL classes of macromolecules •Typical bacterial cell is ~50% carbon (by dry weight) •Most microbes (heterotrophs) use organic carbon •Autotrophs use carbon dioxide (CO2)

Multilocus sequence typing (MLST)

•method in which several different "housekeeping genes" (essential functions) from an organism are sequenced •has sufficient resolving power to distinguish between very closely related strains •useful in clinical microbiology (pathogenic strains), epidemiological studies (tracking), and environmental studies (geographic distribution)

Carotenoids

•most widespread accessory pigments •hydrophobic, embedded in photosynthetic membrane •example: β-carotene •typically yellow, red, brown, or green and absorb blue light •Some energy absorbed by carotenoids can be transferred to a reaction center •function primarily as photoprotective agents, quenching toxic oxygen species and preventing dangerous photooxidation

Micronutrients

•nutrients required in minute amounts •trace metals and growth factors

The dynamic nature of the Escherichia coli genome

•on average 4721 genes (4068-5379) •core genome: 1976 genes •only ~60 genes predicted to be universally present in all Bacteria and Archaea •number of unique genes and size of pan genome keeps increasing as each new strain is sequenced •genomes are highly dynamic and can shrink or enlarge quickly •prokaryotes regularly sample genes from other microbes through horizontal gene transfer

The Phosphorous cycle

•organic and inorganic phosphates (PO42-) •Phosphorus is a typical limiting nutrient that limits the growth of aquatic photosynthetic autotrophs •Alternate forms, such as phosphite and hypophosphite, rapidly cycle through aquatic ecosystems

Gene deletions in microbial genomes

•play important role in genome dynamics •deletions far more frequent than insertions •nonessential and nonfunctional materials are commonly deleted over time •genetic drift can promote deletion when population sizes are small or bottleneck •deletions streamlined genomes of obligate intracellular symbionts and pathogens because host provides many key metabolites

Nitrogen (N)

•proteins, nucleic acids, and many more cell constituents •Bulk of nitrogen in nature is ammonia (NH3), nitrate (NO3-), or nitrogen gas (N2) •Nearly all microbes can use NH3

genetic drift

•random process that can cause gene frequencies to change over time, resulting in evolution in the absence of natural selection •most powerful in small populations and those experiencing frequent "bottleneck" events (severe reduction in size followed by regrowth from remaining cells, such as pathogens)

Genome fingerprinting

•rapid approach for evaluating polymorphisms between strains •ribotyping: method of identifying microbes from analyzing DNA fragments generated from restriction enzyme digestion of genes encoding SSU rRNA, generating a pattern called a ribotype •highly specific and rapid •used in bacterial identification in clinical diagnostics and microbial analyses of food, water, and beverages •Other fingerprinting methods include repetitive extragenic palindromic PCR (rep-PCR) and amplified fragment length polymorphism (AFLP)

Potassium (K)

•required by enzymes for activity

A Phylogenetic Species Concept for Bacteria and Archaea

•should be genetically and phenotypically cohesive •should be distinct from other species •should be monophyletic (should share a recent common ancestor excluded by other species) •Phylogenetic species concept incorporates these principles •Bacterial and archaeal species are groups of strains sharing a high degree of similarity and a recent common ancestor for their SSU rRNA genes. •70 percent or less DNA-DNA hybridization and difference in SSU rRNA of 30 percent or more indicates two distinct species •Distinct genera have greater than five percent dissimilarity in SSU rRNA

Magnesium (Mg)

•stabilizes ribosomes, membranes, and nucleic acids •also required by many enzymes

Sulfur (S)

•sulfur-containing amino acids (cysteine and methionine) •vitamins (e.g., thiamine, biotin, lipoic acid)

Transporters

•three classes -simple transport -group translocation -ABC system •energy-driven (proton motive force, ATP, or another energy-rich compound)

Carl Woese and the tree of life

•universal tree of life based on nucleotide sequence similarity in ribosomal RNA (rRNA) •genealogy of all life on Earth •established the presence of three domains of life: Bacteria, Archaea, Eukarya •root represents when all life shared the last universal common ancestor (LUCA) •shows first life forms were microorganisms and that microbes have dominated most of history of life •Genomics supports three-domain concept through analysis of central cellular function genes -example: 60+ (including rRNA) genes shared by nearly all cells and must have been present in universal ancestor -Of these, eukaryotic and archaeal genes share more similarity -Bacteria and Archaea likely diverged before Eukarya existed -LUCA was likely prokaryotic with DNA genome and ability to transcribe and translate proteins •other influences affecting phylogeny -Many genes shared by two of three domains -One hypothesis: Horizontal gene transfer was extensive before primary domains had diverged -Barriers likely evolved to maintain genomic stability -Cells slowly sorted into primary lines of descent -Bacteria and Archaea likely diverged ~3.7 billion years ago -Eukarya diverged from Archaea ~1.2-2.7 billion years

Anaerobic respiration

•use of electron acceptors other than oxygen -Examples include nitrate (NO3-), ferric iron (Fe3+), sulfate (SO42-), carbon dioxide (CO2), and certain organic compounds (e.g., fumarate) •less energy conserved compared to aerobic respiration •requires electron transport, generates a proton motive force, uses ATPase

Multigene and whole genome analyses

•use of multiple genes and entire genomes increasingly common as sequencing improves and cost declines •Orthologs can be aligned and examined to determine average nucleotide identity (typically less than 95 percent between species) •Analyses of content (presence/absence of genes), order of genes (synteny), and GC content provide insight into relationships

Chemolithotrophy

•uses inorganic chemicals as electron donors -examples: hydrogen sulfide (H2S), hydrogen gas (H2), ferrous iron (Fe2+), ammonium (NH4+) -waste products of chemotrophs •typically aerobic •begins with oxidation of inorganic electron donor •electron transport generates proton motive force •autotrophic; uses CO2 as carbon source

Phototrophy

•uses light as energy source •photophosphorylation: light-mediated ATP synthesis •photoautotrophs: use ATP + CO2 for biosynthesis •photoheterotrophs: use ATP + organic carbon for biosynthesis