Lectures 3/18, 3/28, and 3/30
Lectures 3/18, 3/28, and 3/30 Bios 312
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This 7 page Class Notes was uploaded by Cara Cahalan on Wednesday April 6, 2016. The Class Notes belongs to Bios 312 at University of Nebraska Lincoln taught by Karrie Weber in Spring 2016. Since its upload, it has received 10 views. For similar materials see Microbiology in Biology at University of Nebraska Lincoln.
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Date Created: 04/06/16
3/18: Microbial Systematics and Phylogeny Readings: 12.812.10 IV: Microbial Systematics 12.8 Species Concept of Microbiology Systematics study of diversity of organisms and their relationships (taxonomy and phylogeny) Phylogenetic species concept microbial species is a group of strains that share certain diagnostic traits, genetically cohesive and share unique recent common ancestor 12.9 Taxonomic Methods in Systematics Multilocus sequence typing (MLST) several different genes from related organisms are sequenced, then used to collectively distinguish an organism o More genes concatenated, higher resolution. Limits ability to apply to other species 12.10 Classification and Nomenclature Binomial system nomenclature, Carl Linnaeus, genus name and species epithets Classification organization of organisms into more inclusive groups on basis of phenotypic similarity or evolutionary relationship Lecture: 16S rRNA gene sequence Carl Woese, showed 3 domains of life, unified phylogenetic framework of Bacteria o Small subunit rRNA (SSU rRNA) most widely used, functional homology, highly conserved regions, functionally constant, sufficient length Phylogeny evolutionary history of organisms, inferred indirectly from nucleotide sequences o Bacteria 80 major evolutionary groups (phyla), defined from environmental sequences alone, phenotypically diverse o Archaea 7 major phyla: o Eukaryaorganelles originated within Bacteria (endosymbiotic hypothesis) o 3 domains of life can be characterized by various phenotypic properties Tree generation: o Align sequence of interest with homologous sequences from other strains/species o Distance matrix calculated from number of sequence differences o Tree constructed by adding nodes to join linages Cladistic methods: relationship defined by examining nucleotide changes at individual positions in the sequence Common cladistic methods: o Algorithms: programmed series of steps o Optimality criteria: pick the best of many possible tree (parsimony maximum likelihood) Phylogenetic tree graph of the relationships among sequences o Branch length number of changes occurring along that branch o Assumptions: gene under analysis inherited from common ancestor or vertically mother daughter o Violations: Convergent evolution (trait not from common ancestor) Horizontal gene transfer (complicates evolutionary history of microorganisms) Taxonomy description of distinct life forms and their organization into different categories o Domain phylum class order family genus species Species concept: o Biological: breeding with conspecifics, reproductive isolation protects genotypes o Phonetic: operational classification based on numerical taxonomy Operational Taxonomic Unit (OTU) species do not exist as lineages under this concept o Evolutionary: groups that evolve separate from other lineages, possess their own roles and tendencies o Phylogenetic: terminal organism in a lineage, not dependent on rank above genus level Bacterial classified by polyphasic taxonomy (phenotypic and genotypic analyses) o Classifications require biochemical and physiological analysis o Problems: inability to grow most organisms in same manner and dynamic nature of tests o Fatty Acid Methyl Esters (FAME) profiles identify microbes when they are compared to database Find differences between genera Disadvantage: profiles vary as function of temp, growth phase, and growth medium PCR and primers: selection of primers determines which area of template is amplified o Primer sequence made from known DNA (limitation) specific for certain genus o Conserved primers similar sequences o Universal sequence in all known members of genus or 3 domains DNADNA hybridization rough index of similarity between organisms o Differentiate similar organisms >70% = suggest same species, ≥25%= same genus Prokaryotic species definition two organisms have <97% 16S rRNA gene identity and <70% DNADNA hybridization identity they are different Key concepts: 16S rRNA gene sequence revolutionized classification, cannot always distinguish between species. Bacteria and Archaea do not fit into the described species concepts and are classified using polyphasic taxonomy 3/28: Microbial Diversity: Intro and Phototrophy Readings: 13.113.4 I: Phototrophy 13.1 Photosynthesis and Chlorophylls Photoautotrophs require 2 reactions in parallel: ATP production and CO reduction to cell material 2 Different chlorophyll units have different absorption spectra allow coexistence Chloroplasts contain chlorophyll attached to sheet like membranes (thylakoids) tacked in grana. Two regions: o Stroma matrix that surrounds thylakoid and inner space, allows light driven proton motive force ATP Chlorosome present in anoxygenic green S bacteria, giant antenna systems, not attached to proteins o Allow green bacteria to grow phototrophically with only minimal light 13.2 Carotenoids and Phycobilins (accessory pigments) Carotenoids always in phototrophs, typically yellow, red, brown, or green, energy absorbed can be transferred to reaction center, prevent photooxidative damage to cells Phycobiliproteins main antenna pigment of cyanobacteria and red algae o Phycobilisomes aggregates in cell, more light energy captured than chlorophyll alone, facilitate energy transfer to cyanobacteria reaction centers allowing growth at low light intensities 13.3 Anoxygenic Photosynthesis (occurred before oxygenic) Reverse electron transport quinone is not electro enough to reduce NAD+ to NADH so reverse is required 13.4 Oxygenic Photosynthesis Electron flow: begins with splitting of water into O and electrons, light energy into strong reductant o Electron from water donated to oxidized P680 to return to ground state If PSII is blocked, photosynthesis can be performed only with PSI (cyclic) Lecture: Metabolic description of microorganisms: o Energy source: Chemical (Chemotroph) Organic/ inorganic Light (Phototroph) o Carbon Source Organic (Heterotroph) InorganicCO 2(Autotroph) inorganic to organic C Redox reaction: all written as reduction reactions o Electron donor: more negative, on right o Electron acceptor: more positive, on left Electron transport and energy storage during transport protons are continually generated Proton motive force energized membrane from charge separation and elements of water across membrane Phototrophs: o Photophosphorylation: ATP made from a proton motive force formed from light driven electron transport, cyclic (no net input or consumption of electrons) o All use light as energy source Use inorganic C photoautrophs, others use organic carbon photoheterotrophs o Anoxygenic oxygen not produced, purple and green bacteria o Oxygenic oxidation of water produces O 2, nobacteria o Photoautotrophy required ATP production and CO 2eduction Question: To describe a type of photosynthesis as oxygenic implies (that) _________. o O is produced 2 Banded iron formations result of the oxidation of Fe(II) 2 O generated by cyanobacteria, but possible formed without oxygen anaerobic metabolisms Phototrophs: o 1. Capture light must produce some form of chlorophyll (related to porphyrins) to be photosynthetic Number of different types of chlorophyll exist Cyanobacteria chlorophyll a Prochlorophytes produce chlorophyll a and b Anoxygenic phototrophs produce bacteriochlorophylls Antenna pigments funnel light to reaction center (conversion of light energy to ATP) Reaction center of purple phototrophic bacterium: Anoxygenic photosynthesis: photosynthesis apparatus embedded in membrane, electron transport occurs in reaction center o 2. Convert light energy to chemical energy and 3. Generate reducing power for CO 2ixation (autotrophy) Cyclic generated proton motive force, something must be r2+enerate2– Reducing power for CO f2xation from reductants (H S2 Fe , or NO ) Weak electron donor (bacteriochlorophyll) strong electron donor by light Requires reverse transport for NADH production in only purple phototrophs o ATP production not enough for autotrophic growth, reducing power needed (NADH) o Reducing substances are oxidized and electrons end up in “quinone pool” of photosynthetic membrane Electrons transported in membrane through series of proteins/cytochromes Electron flow in oxygenic photosynthesis light generates ATP and NADPH with two light reactions (photosystem I and II) Z scheme II transfers E to I, ATP produced by cyclic photophosphorylation Key concepts: Unlike oxidative phosphorylation, there is no net input of electrons required for photophosphorylation. In addition to oxygenic photosynthesis, some bacteria are also capable of anoxygenic photosynthesis. During anoxygenic photosynthesis, reduced chemical species are oxidized when electrons flow to the bacteriochlorophyll in an oxidized (more positive reduction potential). 3/31: Autotrophy and Chemolithotrophy Readings: 13.5, 13.613.11 13.5 Autotrophic Pathways Autotrophy energy poor and highly oxidized CO re2uced and assimilated into cell material Calvin cycle: requires CO , NADH, ATP and 2 enzymes. Incorporate 6 molecules of CO 2 2 o PGA phosphorylated and reduce to key intermediate (glyceraldehyde3P) o Glucose formed by reversal of early steps of glycolysis Carboxysomes concentrate CO in c2 l and make it available for RubisCO and blocks O from e2 yme o Allows for an increase in rate of CO2 fixation Green S bacteria (Chlorobium) reverse citric acid cycle, ferrodoxin enzymes catalyze fixation of CO 2 Green nonS (Chloroflexus) grows autotrophically with H or2H S a2 electron donor o Hydroxypropionate pathway 2 CO red2ced to glyoxylate One of the earliest mechanisms for autotrophy in anoxygenic phototrophs II: Chemolithotrophy 13.6 Inorganic Compounds as Electron Donors Chemolithotrophs conserve energy from oxidation of inorganic compounds. o Many inorganic compounds available, energy yield for oxidation of donors varies greatly o Reducing power gained: Directly from inorganic compound, if sufficiently negative reduction potential Reverse electron transport reactions if inorganic donor is more electro+ than NADH H bacteria oxidize strong electron donor (zero cost), whereas Fe oxidizes weak donor (more energy needed) 13.7 H 2 idation H + O →H O 0' 2 2 2 2 ∆ G =−237kJ Electrons from H 2 quinone acceptor cytochrome series proton motive force reduce O to w2ter H 2bacteria will use glucose as a C source when available facultative chemolithotroph o H may be low in oxic environments, needs backup plan for when levels are low 2 13.8 Oxidation of Reduced S Compounds Electron donors: H S2 elemental sulfur (S ). Final oxidation product is sulfate (SO4) S oxidation elemental sulfur, deposits in cell as potential energy reserve. o S taken up by cell but is insoluble so must be reduced to HS to transport into cell for metabolism S oxidation product H+ (environment becomes more acidic), S bacteria have become acidtolerant Oxidation of S: o Starting substrate oxidized SO , releases 6 electrons 2 3 o SO 3oxidized sulfate and 2 electrons Sox system: oxidizes reduced S directly to sulfate without sulfite intermediate 13.9 Iron Oxidation Fe to Fe , couple large oxidation of Fe to yield little ATP, acidophiles 2+ Iron oxidation: begins in outer membrane where contact with Fe or insoluble Fe which is oxidized o Cyt c transfers e to periplasm where Rusticyanin is electron acceptor 13.10 Nitrification and Anammox Nitrification microbial oxidation of ammonia (NH ) t3 nitrate (NO )3 Anammox anoxic ammonia oxidation, beneficial in treatment of wastewaters o Anammoxosome organellelike, aggregate in membrane structure (dense) preventing diffusion of substances into cytoplasm Membrane protects cell from toxic intermediates produced during anammox reactions Chemolithotroph reactions: inorganic substrate electron transport chain establish proton motive force drives ATP synthesis Nitrifiers convert ammonia to nitrate (more usable N source) III: Fermentations 13.11 Energetic and Redox Considerations Reaction: energy rich bonds, organic form fermentation ATP can be made via substrate level phosphorylation Fermentation products byproducts of fermentation reaction (alcohols/acids) secreted from cell Lecture: Autotrophs in prokaryotes: CO fixation pathways: Calvin cycle, reductive citric acid cycle (reverse TCA cycle), reductive acetylCoA 2 pathway, 3Hydroxypropinate cycle Calvin cycle fixes CO 2into cellular material for autotrophic growth, consuming cycle o Requires NADPH, ATP, ribulose RubisCO (enzyme), and phosphoribulokinase glyceraldehyde 3P o 6 molecules of CO 2re required to make 1 molecule of glucose o Each turn of the cycle condenses one CO 2 o Organisms that use: Oxygenic phototrophic bacteria (mainly cyanobacteria), chloroplasts, facultatively anaerobic purple bacteria, lithotrophic bacteria 3 main phases of Calvin cycle: o Carboxylation of splitting 6C 2 (3C) Catalyzed by RubisCO which catalyzes 2 PGA from RubisCO and CO 2 o Reduction of PGA to G3P o Regeneration of ribulose 1,5 biphosphate Reverse Citric Acid Cycle ATP citrate lyase reverses the TCA cycle o Found in Chlorobium and nonphototrophic prokaryotes o Key enzymes: pyruvate oxidoreductase, 2‐ketoglutarate oxidoreductase, and ATP citrate lyase. o Unlike Calvin, intermediates can assimilate CO 2 o Key steps: SuccinylCoA assimilates CO 22 oxoglutarate (alphaketoglutarate) 2oxoglutarate assimilates CO 2 isoctrate AcetylCoA assimilates CO 2pyruvate Reductive AcetylCoA pathway: 2CO 2ATP + 4H + 2SCoA → CH COSCoA3+ 3H O +ADP + P2 i o CO 2educes formate then transferred on tetrahydrofolate (reduced form of folic acid) o Doesn’t need a lot of energy 1 ATP 3Hydroxypropinate cycle: o Two molecules of CO 2re reduced to glyoxylate by the hydroxypropionate pathway (may be oldest mechanism of autotrophy) o MalonylCoA reductase: key and distinguishing enzyme Autotrophy in Prokaryotes: CO fixation pathways 2 o CalvinBasshamBenson Cycle 6 CO 2 12 NADPH 18 ATP → C H O (P6 H12 6 123NA2P 18 ADP 17 P i o Reductive Citric Acid Cycle (reverse TCA cycle) 3 CO 2 12 H 5 ATP → glyceraldehyde 3P o Reductive AcetylCoA Pathway (Wood Ljungdahl Pathway) 2CO 2ATP + 4H + 2SCoA → CH COSCoA3+ 3H O +ADP + P 2 i o 3Hydroxypropionate Cycle 2 CO 2 6 H 3 ATP → glyoxylate NOT ALL phototrophs are capable of CO fixat2 n Photoheterotrophs: use light energy, but cannot use CO 2s only C source, use organic compounds instead The growth rate of an anoxygenic phototrophic purple bacterium Rhodobacter is about twice as fast when the organism is grown photoheterotrophically in medium containing malate (organic carbon) as the carbon source than when it is grown with CO a2 the carbon source and H a2 the electron donor. Why? o Under photoheterotrophic conditions all of the ATP formed from cyclic photophosphorylation is used for processes required for growth rather than carbon assimilation. o Under photoheterotrophic conditions all of the ATP formed from cyclic photophosphorylation is used for fixation of inorganic carbon into malate. o Under photoautotrophic conditions all of the ATP formed from cyclic photophosphorylation is used for processes required for growth rather than carbon assimilation. o Under photoautotrophic conditions all of the ATP formed from cyclic photophosphorylation is used for fixation of inorganic carbon into malate. Key Concepts: Numerous pathways/cycles are utilized in the prokaryotes to fix CO in2 cell material. Eukarya use fixed CO to2 uild cell material. The Calvin Cycle most expensive CO fix2tion (18 ATP and 12 NADPH) Hypothesis: rTCA and 3Hydroxypropiante pathway the first CO fixation pathway to have evolved. 2 Chemolithotrophs: inorganic chemical energy source (aerobic or anaerobic) Chemolithotrophic respiration: Oxidation of a reduced inorganic electron donor. Electrons are transported across and electron transport chain to O 2or an alternative electron acceptor. o ATP is generated via oxidative phosphorylation. o Can use any oxidized inorganic species that is energetically favored Respiration: compound oxidized with O or 2 bstitute as terminal electron acceptor ATP production through oxidative phosphorylation o NOTE: electrons move from components that is negative positive! o Functions: accept electron from electron donor and transfer to electron acceptor Conserve energy released during electron transport chain o Complex I: NADH donates e to FAD, FADH donates e to quinone + o Complex II : Bypasses Complex I, feeds e and H from FADH directly to quinone pool o Complex III: Transfers e from quinones to cyt c, cytochrome c shuttles e to cytochromes a and a 3 o Complex IV (cytochromes a and a ): 3erminal reductase; reduces O to 2 O 2 Sulfur oxidation: reduced S used as electron donors (H S2 S , S 2 )3 o One product: H+, lowers pH of surroundings o Sox system: oxidizes reduced sulfur compounds directly to sulfate Usually aerobic, but some organisms can use nitrate as an electron acceptor o Electrons from reduced sulfur compounds reach the electron transport system Transported through the chain to O 2 Generates a proton motive force that leads to ATP synthesis by ATPase Reverse electron transport consume ATP in order to generate required reducing power for processes Iron oxidation: utilized by Thiobacillus denitrificans, Gallionella ferruginea, Acidithiobacillus ferrooxidans 2+ o Fe(OH) 3Fe (0.014) 1 e o Acidophilic: iron is soluble at low pH Rusticyanin enzyme involved in reaction Some components of e transport on inner membrane of cell and another on outer (cyt c) Electron from exterior of cell (Fe doesn’t move into cell) converted to tri valent state Fe precipitate and cell doesn’t want accumulations of it inside (could cause damage) Oxygen final electron acceptor (aerobic) o At neutral pH: iron oxide is solid
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