Exam 2 Study Guide PDF

Chapter 5 metabolism Metabolism: sum of chemical processes in living system Catabolism: metabolism involved in energy generation anabolism: metabolism involved in biosynthesis What do cells need to grow ○ 1) Minor and major elements (ex carbon, nitrogen, sulfur etc) and water...

Chapter 5 metabolism Metabolism: sum of chemical processes in living system Catabolism: metabolism involved in energy generation anabolism: metabolism involved in biosynthesis What do cells need to grow ○ 1) Minor and major elements (ex carbon, nitrogen, sulfur etc) and water ○ 2) Energy (always from redox rxns) ○ 3) Reducing equivalents: [H+]= H+ + e- Carriers NAD+/NADH, NADP+/NADPH Approx half of E colis games are required for metabolism Both energy (high energy phosphate bonds) and reducing equivalents are required for biosynthesis Classification system for microbes based on energy and carbon source Energy Carbon troph Chemo (chemical) Organ (organic) Hetero (organic troph Litho (inorganic) C) photo Auto (inorganic ex troph CO2) Life is redox Delta G: free energy of rxn ○ Delta G 0 endergonic ○ ΔG ’ Where means standard conditions (conc=1 M, atm=1, temp=25 C) Where ‘ means pH=7 Redox rxn ○ Oxidation: removal of e- from substrate (e donor) Bred → Box + e- ○ Reduction: addition of e- to substrate (e acceptor) Aox + e- → Ared ○ Aox + Bred → Ared + Box Redox rxn rules (order of importance) ○ Balance C → CO2, balance other elements (respiration) ○ Use H2O to balance O ○ Use H+ to balance H  ○ Use e- to balance charges (charges need to cancel out in overall rxn) Oxidation states of H2 and O2 =0 Ex H2 +½ O2 → H2O ○ Oxidation state 0 0 → 2(+1)-2 ○ Oxidation rxn: H2 → 2H+ +2 e- ○ Reduction rxn: ½ O2 + 2e- + 2H+ → H2O ○ Overall rxn: H2 + ½ O2 → H2O Ex Iron (II) oxidation to Iron (III) with molecular oxygen. what are reductive and oxidative halves of rxn? what is electron donor? Acceptor? what is balanced equation? ○ Electron donor: Fe2+ ○ Electron acceptor: O2 ○ Oxidation rxn: (Fe2+ → Fe3+ + e-)*2 ○ 2Fe2+ → 2Fe3+ + 2e- ○ Reduction rxn: ½ O2 + 2H+ + 2e- → H2O ○ Overall rxn: 2Fe2+ + ½ O2 + 2H+ → H2O + 2Fe3+ Ex an organism grows on glucose (C6H12O6) and molecular oxygen. what is the e- acceptor and e donor? what are the half and full reaction? (*goes to CO2) ○ Electron acceptor: oxygen ○ Electron donor: glucose ○ Oxidation: C6H12O6 + 6H2O → 6CO2 + 24H+ + 24e- ○ Reduction rxn: (½ O2 + 2H+ + 2e- → H2O)*12 ○ 6O2 + 24H+ + 24e- → 12H2O ○ Overall rxn: C6H12O6 + 6O2 → 6H2O + 6CO2 Common electron acceptors for redox rxns ○ O2/H2O ○ NO3-/N2 ○ NO2-/N2 ○ SO4^2-/H2S ○ Fe3+/Fe2+ ○ Mn4+/Mn2+ Reduction potential: tendency of compound to become oxidized or reduced ○ Measured in volts (V) ○ E ’ is potential (‘ prime means pH=7, knot means standardized) ○ The more positive the E ’, the better the compound is at accepting electrons Proton motive force/electron transport  ○ ○ The greater the difference in redox potential between acceptor and donor, the greater the energy released ○ Not all e- are equal in redox potential ex glucose more negative than iron, smaller potentials have shorter chains ○ ΔE’= E ’ acceptor - E ’ donor ○ ΔG ’= -nFΔE ’ n= # of electrons transferred F= Faraday Constant = 96.48 KJ/V Ex H2 oxidation with oxygen electron acceptor ○ For 2H+/H2, n=2 ○ F= 96 kJ/V  ○ ΔE = E ’ accepting couple - E ’ donating couple ○ H2 + ½ O2 → H2O ○ For electron as electron acceptor ΔE= 0.82 - (-0.42)= 1.24 V ○ ΔG= -2(96)(1.24) = -239 kJ/mol H2 Energy is released, organism can live ○ For mole of O2, x2 on everything because rxn has ½ O2 mol ○ -478 kJ/mol O2 Chapter 6 Energy usable to drive endergonic rxns ○ 1) high energy phosphoryl bonds (ATP, GTP, etc) ○ 2) transmembrane ion gradients (H+, Na+) 2 ways for ATP generation ○ 1) substrate level phosphorylation (SLP) PO4^3- transfer from substrate to ADP → ATP ○ 2) transmembrane ion gradient E- move down electrochemical gradient to more positive acceptor and pump H+/Na+ gradient then used to make ATP Fermentation ○ ATP formation by SLP ○ Fermentation substrate is both oxidized and reduced and serves as carbon source (no net change in oxidation state) ○ Reducing agents (NADH) cant be oxidized by oxygen so it builds up and goes back to pyruvate to form back to NAD+ and makes lactate ○ Challenge of fermentation is getting rid of [H] ○ fermentation end products can vary (doesn't always start with glucose)  ○ Substrate level phosphorylation ○ 1) organic substrate becomes phosphorylated with PO4^3- in rxn that requires no energy Sred —(PO4^3-)→ SredPO4 ○ 2) phosphorylated substrate is oxidized then that energy used to make high energy bond ○ 3) ~PO4 transferred to ADP → ATP ○ All steps can occur in 1 rxn Ex What is the oxidation state of carbon in glucose (C6H12O6) and lactic acid (C3H6O3)? What are the half reactions (pyruvic acid = C3H4O3) ? ○ Glucose to pyruvic acid is oxidation state=0 ○ Pyruvic acid to lactic acid reduction state=0 ○ C6H12O6 → 2C3H4O3 + 4H+ + 4e- ○ (C3H4O3 + 2H+ + 2e- → C3H6O3)*2 ○ 2C3H4O3 + 4H+ + 4e- → 2C3H6O3 ○ Overall rxn: C6H12O6 → 2C3H6O3 ATP synthesis by ATPase ○ F0= hydrophobic membrane traversing subunit ○ F1= hydrophilic, on interior surface of membrane, uses ion flow to catalyze ADP + Pi → ATP ○ 3-4 H+ transferred per 1 ATP  ○ Reversible Transmembrane ion gradient and ATPase ○ In environment where H+ gradient not feasible (high pH) then microbes use Na+ gradient ○ Importance of ion gradient ATP generation Flagella rotation Ion coupled transport Maintain turgor pressure Maintain pH NAD+ → NADH via reverse e- flow ○ Generating ion gradients 1) Respiration on cytoplasmic membrane (takes place in cell membrane) Transfer e- from donor to acceptor via e- carriers down an electrochemical gradient 2) photosynthesis Generating ion gradient via respiration ○ 1) energy change of e- transfer → pumped H+ ○ 2) carriers of e- can be e- only carrier or [H] reducing eq carriers Alternating between e- only and [H] carriers ○ H+ outside forms gradient to be used by ATPase or other ○ O2 as e- acceptor Provides greatest amount of energy Cost is toxic byproducts of O2 Respiration byproducts are H2O2 and O2 ROS oxidation species To protect against ROS Superoxide dismutase, SOD ○ 2O2- + 2H+ → O2 + H2O2  Catalase ○ 2H2O2 → 2H2O + O2 Without SOD, high Mn (abiotic) Mn2+ + 2H+ + O2 → Mn3+ + H2O2 ○ Other e- acceptors can be used besides molecular oxygen ○ Reversible electron flow To produce NADH use by many chemolithoautotrophs (especially autotrophs need lots of NADH to fix CO2) Has to occur with microbes that don't have enough potential Microbial fuel cells ○ Electrode/anode can be electron acceptor and skip steps of chain Quiz ○ Hydrogen peroxide is electron acceptor ○ Electron donor redox potential more negative than electron acceptor redox potential ○ In anaerobic respiration, electron acceptor is nitrate ○ Oxidation state of C in cholesterol is -44/27 ○ Energy for SLP comes from oxidation ○ ATP reversible, hydrolyzes ATP → ADP Generating ion gradient via photosynthesis ○ In only 6 bacteria lineages Cyanobacteria ○ Light changes redox potential of e- on chlorophyll (to more negative) and e- then flows down electrochemical gradient ○ E- is in chlorophyll a or bacteriochlorophyll a ○ Chlorophyll part of reaction center (has proteins and pigments, e- changes in here) ○ Antenna pigments harvest light energy to funnel to reaction center ○ RC and antenna have 50-300 molecules in membrane Types of photosynthesis ○ 1) cyclic: e- flow from activated chlorophyll and back Lots of ATP, no reducing eq [H] ○ 2) noncyclic Anoxygenic photosynthesis: e- are derived from H2S, Fe2+, or organic acids Oxygenic photosynthesis: e- derived from H2O (electron donor) Only cyanobacteria Requires 2 photosystems since its harder to raise e- potential) no external e- acceptor  Generating ion gradient via enzyme pumps ○ Not part of electron transport system, but pump H+, Na+ ○ Ex bacteriorhodopsin Generating ion gradient via scalar reactions ○ Reactions that create ion gradient without transporting ions ○ Ex oxalate decarboxylase Oxalate + H+ → formate + CO2 ○ Consumes protons inside Requires light Requires Requires Net redox rxn external e- external e- acceptor donor respiration no yes yes yes photosynthesi yes no Yes yes s (noncyclic) Enzyme yes no no no pump Scalar rxn no no no no Chapter 7, biosynthesis General observations for biosynthesis: ○ End products are more reduced than starting materials and reducing equivalents delivered by NADPH NAD+/NADH poised for energy rxn (more NAD+ than NADH) NADP+/NADPH poised for biosynthesis (more NADPH than NADP+) NADPH + NAD+ ←→ NADH + NADP+ (transhydrogenase: keeping reducing equivalents ready/poised) ○ Uses ATP ○ Key enzymes allosterically regulated (small regulator bound or not controls enzyme activity) Controlling ligand is often end product of biosynthesis rxn (feedback inhibition/negative feedback) ○ Made from 13 precursor metabolites to make everything for cell Transport for gram - cell (outer membrane as outermost layer) ○ Diffusion  porins: small 600-700, hydrophilic ○ Facilitated diffusion: selective for solute (channel for certain compound) ○ Active transport: TonB dependent transporter uses H+ gradient (ATPase on inner membrane) Fe siderophore transport by TonB dependent transporters (most cells require Fe) Fe III is highly insoluble which makes it scarce in environment, in host Fe is not available Heme, ferritin, lactoferrin all bound in host ○ How do microbes solve iron problem Siderophores: small molecules that tightly bind to Fe Secreted by bacteria under Fe limitation Uptake Fe-siderophores using TBDT (TonB dependent transporters) Microbes can take up own and other siderophores ○  ○ Cell wall transport has diffusion Cell membrane transport ○ 1) passive Diffusion Simple: can diffuse across membrane Facilitated: selective for solute ○ 2) active transport: requires energy, move against gradient Ion coupled: use H+/Na+ motive force Antiport: moves two different ions or solutes in opposite directions across a membrane Symbort: move two different ions or solutes in same direction across membrane ABC transporters: ATP hydrolysis drives transport (in gram + and -) Periplasmic binding protein (gram + can have it but not in the OM like gram -) Transmembrane domain (substrate goes through it in cell membrane) ATP binding hydrolysis (in cytoplasm) Phosphotransferase system/group translocation: transport of sugars only Once inside, phosphorylates substrate as part of transport (moving down gradient since more glucose outside) P-sugar moves down concentration gradient Phosphoenolpyruvate (PEP) supplies energy Transport and phosphorylation in one step with 1 ATP which saves more ATP  ○ Feeder pathways ○ Heterotrophs convert C substrate to central metabolite ○ Autotrophs fix CO2 to central metabolite ex calvin cycle ○ Central metabolism: glycolysis, TCA cycle, Pentose P pathway ○ Supplies 13 precursor metabolites ○ Core energy generating pathway in heterotrophs ○ Can work in forward and reverse directions depending on substrate and cell needs N assimilation ○ Sources: organic N, NH3, N2, NO^3- Ammonia NH3 ← → NH4+ in equilibrium (NH3 diffuses across membrane) ○ Sources converted to NH4+ ○ Glutamate and glutamine are carriers of ammonia  ○ Organic N Uptake with transporters and use deaminase inside Excrete deaminase ○ Nitrate, NO^3- NO^3- → NO^2- by nitrate reductase NO^2- → NH3 by nitrite reductase ○ N2 fixation N2 → NH3 nitrogenase with FeMoCo cofactor Difficult to break triple bond Uses 16 ATP and 8 reducing equivalents Nitrogenase highly O2 sensitive and results in breakage Fixes sensitivity problem by separating cells that do oxygenic photorespiration and N2 fixation Cyanobacteria that fix N2 Heterocysts: spatial separation Temporal separation (day time produce oxygen and night time fix N2) ○ Glutamate and glutamine synthesis ○ 1) glutamate dehydrogenase: high NH3 required More energy savable option 2oxoglutarate + NH3 + H+ + NADPH → glutamate + H2O + NADP+ ○ 2) glutamine synthetase: uses ATP Glutamate + NH3 + ATP → glutamine + Pi + ADP  Glutamine + 2oxoglutarate + H+ + NADPH → (GOGAT) → 2 glutamate + NADP+ Make glutamine with ATP then make glutamate Glutamate-oxoglutarate amido transferase (GOGAT) makes glutamate from glutamine ○ Ex based on the following data, what growth condition has low levels of ammonia Growth Glutamate Glutamine GOGAT condition dehydrogenas synthetase activity e activity activity A 34 162 114 B 126 150 12 A has low levels because glutamate more active with more ammonia S assimilation ○ H2S ← → HS- ← → S^2- in equilibrium ○ Sources: H2S (only in anaerobic, can diffuse in), organic S, SO^4- (sulfate) ○ All forms go to H2S ○ H2S + O-acetely-L-serine → cysteine which is carrier of sulfur for cell ○ Sulfate expensive since needs ATP and reducing equivalents ○ P assimilation ○ Sources: PO4^3- (cant diffuse and needs transporter), organic P ○ PO4^3- converted to ATP (ATP is carrier and used for energy metabolism ○ ATP is carrier ○ Organic P transformed to PO4^3- by phosphatase ○ No redox change  ○ Assimilation sources Sources first Carrier in cell Does of converted to assimilation require redox N N2 NH3 Glutamate no and glutamine NH3 no Organic N yes NO^3- yes S H2S H2S cysteine no Organic S Sulfate then yes H2S SO4^2- yes P PO4^3- PO4^3- ATP no Organic P no Quiz ○ Noncyclic photosynthesis requires e- donor ○ Can O2 be used as e- donor? No, in noncyclic photosynthesis iron can be e- donor ○ What is required for biosynthesis? NADPH/NADP+ > 1 ○ B→D→C→A regulated by A in B→D ○ Feeder pathway ex is calvin cycle ○ TonB dependent transporter powered by proton gradient across CM ○ There is hydrolysis of phosphate bond in ABC transporters  What is the state where DNA replication begins? Origin of replication From the origin, what direction does replication proceed? Bidirectional Identify the leading and lagging strands? 5 → 3 direction is leading What bacterial enzyme relieves supercoiling? Gyrate What enzyme catalyzes strand elongation? Polymerase What are the DNA fragments formed on the lagging strand called? Okazaki fragments What enzyme joins Okazaki fragments? Ligase How does the cell prevent errors in replication? Proofreading and mismatch repair What is the direction of DNA synthesis? 5 → 3 Can DNA polymerase start polymerization de novo? No What is transcription? DNA to RNA What enzyme catalyzes transcription? RNA polymerase Where does RNA polymerase bind to DNA? Promoter What gives RNA polymerase specificity for binding? Sigma factor Are all RNA transcripts used to make proteins? No What does polycistronic mean? One mRNA codes for multiple peptides What is operon? Cluster of genes on DNA strand that are transcribed together (promoter, operator, structural genes) What catalyzes translation? Ribosomes Chapter 8-9, Proteins Protein processing ○ 1) covalent modification Cleavage: F-met signal peptide ( transport to cell envelope) S-S bond formation Formed by cysteines Cytoplasm is too reducing for disulfide bonds so its SH (except cyanos b/O2 production) Additions: can be permanent or reversible PO4, CH3, sugars, nucleotides, lipids ○ 2) protein folding: chaperones Trigger factor: rotates proline bonds DnaK: repairs hydrophobic patches GroE: provides refolding environment ○ 3) transformation: Sec system Sec system  ○ 1) Sec A recognize and binds signal sequence and leads protein to channel (SecYEG) ○ 2) SecB (chaperone) coats protein and keeps it from folding ○ 3) at SecYEG channel, protein is translocated across cell membrane using ATP 4) as protein crosses membrane, Lep cuts signal sequence ○ 5) protein folds ○ 6) protein in periplasm (of outer membrane) of gram - and in gram + protein is excreted ○ Following refers to protein translocation of protein, ProA by Sec pathway ○ Mutation in Phenotype No mutation, WT ProA is correct size, correctly folded, and present outside cell SecB Slightly longer ProA in cytoplasm bound to SecA, correctly folded, not present outside cell (not correct size since not going to channel, correctly folded unless there's disulfide bond in cytoplasm) Lep Slightly longer ProA found outside cell, correctly folded, present outside cell Classes of protein secretion  ○ Type I: ABC transporter ○ Type II: two step secretion ○ Type III contact dependent secretion Common in many pathogens ○ Type IV: conjugal transfer system ○ Type V: autotransport ○ Type VI: contact dependent secretion Type III protein secretion ○ In pathways ○ Contact dependent ○ All bacterial proteins ○ Used to evade and deter host defenses ○ Coevolved from flagella ancestor Chapter 17, viruses virus/virion (infectious virus) ○ Shape evolution ○ Present in large numbers ○ Not organisms (dont have metabolism) ○ Nucleic acid (DNA or RNA) + protein shell called capsid Some surrounded by envelope aka membrane called envelope virus ○ Common shapes Icosahedron (20 sided triangles) Helical filament ○ Classification Host Size and shape Genome (DNA or RNA and single or double stranded) Enveloped or not Mode of replication Viral replication: attachment then penetration then uncoating then replication then assembly then release ○ Requires host and is fast ○ 1) attachment of virus to host cell: ligands (capsid proteins or envelope proteins) bind to host receptors ○ 2) penetration: virus’s nucleic acids enters cell, virus can be intact or disassemble on entry but always ends up disassembled Membrane fusion: Enveloped viruses can fuse with host membrane  Endocytosis Injection (done by bacteriophage) ○ 3) make mRNA and complying genome for viral proteins DNA viruses gDNA –(host) → mRNA using RNA polymerase gDNA –(host) → gDNA using DNA polymerase, can proofread gssDNA –(host) → complementary/template cDNA using DNA polymerase –(host) → gssDNA using DNA polymerase RNA viruses (generally single stranded) Positive polarity: gRNA=mRNA (used to make proteins) Negative polarity: gRNA= complement of mRNA ○ First make mRNA then proteins ○ RNA –(viral RNA replicase which cant proofread) → mRNA, genome ○ gRNA(-) –(viral RNA replicase) → mRNA (+), template –(viral RNA replicase) → gRNA (-) Retroviruses: RNA based genome but not RNA virus gRNA –(viral reverse transcriptase) → ssDNA –(host DNA polymerase) → dsDNA –(viral integrase) → host genome becoming provirus –(host RNA polymerase) →mRNA, gRNA ○ 4) viral protein synthesis mRNA –(host ribosome) → protein Subvert host metabolism so only viral proteins made Ex sigma factor to control mRNA produced Host genome broken down so no host proteins made Viral translational regulators made which stops host translation so only viral proteins made ○ 5) virus assembly Self assembled Capsids filled with nucleic acids ○ 6) virus release Budding for enveloped viruses Cell lysis Ex how does an RNA virus with positive polarity like hepatitis A replicate its genome and make viral proteins? What host and viral proteins are involved?  ○ Quiz ○ Sec transport system has covalent modification, ATP hydrolysis, and chaperones ○ Trigger factors rotates proline bonds ○ Main carrier of nitrogen assimilation when ammonia is high is glutamate ○ RNA polymerase catalyzes transcription ○ Gyrate fixes bacterial supercoiling ○ Phosphatase which removes phosphate from organic P is not ABC transporters and not redox reaction ○ Aerobic marine organism uses sulfate for s assimilation Quantifying viruses ○ 1) plaque count ○ 2) # of lesions ○ 3) electron microscopy ○ 4)fluorescence microscopy ○ 5) qPCR= quantitative PCR for DNA viruses qRT-PCR for RNA viruses because you have to do reverse transcriptase Types of viral lifestyles ○ Virulent: actively replicating, kill host by inducing lysis ○ temperate/latent: can live in harmony with host Reversibly integrates into host DNA and is replicated with the host chromosome Becomes episome (plasmid like virus) (bacteriophage is temperate) Temperate phage lifecycle ○ Lysogeny → lysis Process of induction: when conditions threaten host life ex DNA damage  ○ Lambda phage ○ Infects E.coli ○ Ds linear DNA ○ Becomes circularized when injected into E. coli using complementary ends ○ Integrates at specific site using viral integrase ○ Lambda repressor keeps phage genes from being expressed ○ Induction when things go poorly for E.coli (SOS response) ○ SOS response → cleavage of lambda repressor → expression of lambda genes including excisionase → removal of prophage  ○ Lysis or lysogeny? (depends on lambda repressor) ○ Environment ex lysis in rich media with fast growing cells (diluting repressor) ○ # of infecting particles/cell More phage/cell→ more repressor → lysogeny ○ Physiological state of cell Ex stationary phase is lysogeny since repressor builds up Viroids ○ Circular RNA particles, no coat proteins ○ Infects plants only because they use plant RNA dependent RNA polymerase (specific RNA replicase) ○ Do not encode protein ○ Enter through wound Prions: protein molecules that are misfolded and can catalyze misfolding of other proteins with same sequence ○ Can cause degeneration of central nervous system ex mad cow, scrapie, Kuru, cancer, alzherimers, Parkinsons ○ PrP^c → PrP^sc (irreversible, happens spontaneously) Normal (high in alpha helices) → diseased prion (high in B sheets) → amyloid fibers Resistant to heat and harsh chemicals  ○ Influenza ○ Negative polarity RNA virus ○ Enveloped ○ RNA has 8 segments ○ Influenza strains very specific for host but occasionally strains infect multiple species ○ Enters human epithelial cells in nasal passages ○ Evolution of influenza No proofreading Rarely see same strain twice Influenza types ○ Type A: birds and mammals (pandemic causing) Types based on HA and NA antigens which can bind different types of sugar residues on cell surfaces Characterized by 2 surface proteins (uses RNA replicase) Hemagglutinin: 6 variances Neuraminidase: 9 variances ○ Type B,C: predominantly found in humans ○ Type D: found in cattle Antigenic drift: gradual changes in proteins so our immune systems no longer recognize them ○ Caused by high error in replication and always happening Antigenic shift: genome shuffling with 2 strains that infect same host ○ If viruses encode different types of HA or NA antigens sometimes replicated genome pieces get mixed up and create new virus Flu vaccine ○ Protects you and vulnerable people (50% effective) ○ Public monitoring of patients with flu like symptoms ○ Worldwide public health officials meet and discuss data ○ Shot is inactivated influenza (cant replicate) or weakened virus called live attenuated vaccine