Unformatted text preview: BioMI 2900 Study Questions #5 - Lectures 13-14 ANSWERS 4 Oct 2009 Part I: ANABOLISM (BIOSYNTHESIS) 1--Define these terms: Calvin cycle, A MAJOR ANABOLIC (BIOSYNTHETIC) PATHWAYS USED TO FIX (INCORPORATE, REDUCE) CO2 INTO ORGANIC CARBON IN BIOMASS. THIS PATHWAY IS USED BY PLANTS, CYANOBACTERIA , PURPLE BACTERIA AND MANY LITHOAUTOTROPHIC BACTERIA. primary production, THE PRODUCTION OF ORGANIC CARBON FROM CO2 (ON AN ECOSYSTEM BASIS). PRIMARY PRODUCERS ARE AUTOTROPHS. THEY INCLUDE PHOTOTROPHIC AND LITHOTROPHIC ORGANISMS carbon fixation, THE REDUCTION OF CO2 TO ORGANIC CARBON. THIS IS WHAT HAPPENS IN PRIMARY PRODUCTION RubisCO, A HALLMARK ENZYME THAT INITIATES THE CALVIN CYCLE BY INCORPORATING CO2 ONTO A 5-CARBON BACKBONE MOLECULE CREATING 2 3-CARBON INTERMEDIATES OF GLYCOLYSIS, WHICH ARE REDUCED BY NADPH AND EVENTUALLY FORM FRUCTOSE-6-PO4 . carboxysome, A SPECIALIZED INTERNAL GRANULE IN CELLS OF CYANOBACTERIA AND SOME LITHOAUTOTROPHIC BACTERIA CONTANING RubisCO AND OTHER ENZYMES OF THE CALVIN CYCLE. CARBOXYSOMES ARE RARE EXAMPLES OF PROKARYOTIC COMPARTMENTATION AS THEY REPRESENT SPECIALIZED COMPARTMENTS WHERE CO2 FIXATION TAKES PLACE IN THE CELL.. central metabolite, ANY OF 12 ORGANIC INTERMEDIATE COMPOUNDS FROM WHICH ALL CARBON BACKBONES OF PRECURSORS FOR MACROMOLECULES ARE SYNTHESIZED IN ANABOLISM (BIOSYNTHESIS). EXAMPLES INCLUDE ACETYL-CoA (2C), PYRUVATE AND OTHER GLYCOLYSIS INTERMEDIATES (3C), KETOGLUTARATE AND OTHER TCA CYCLE INTERMEDIATES (4C), RIBOSE-5-P (5C), GLUCOSE-6-P AND FRUCTOSE-6-P (6C). secondary metabolite, A PRODUCT EXCRETED BY A MICROORGANISM NEAR THEN END OF ITS GROWTH PHASE; INCLUDES ANTIBIOTICS AND OTHER INDUSTRIALLY IMPORTANT COMPOUNDS transaminase, AN ENZYME INVOLVED IN NITROGEN METABOLISM, WHICH TRANSFERS AN AMINO GROUP FROM AN AMINO ACID TO AN ORGANIC ACID FORMING THE CORRESPONDING AMINO ACID. amino acid family, A GROUP OF AMINO ACIDS THAT ARE SYNTHESIZED FROM AN INITIAL AMINO ACIC IN REALTED PATHWAYS (SEE BBOM 10e FIG. 5.26). E.G. PROLINE, GLUTAMINE ,AND ARGENINE ARE SYNTHESIZED BY PATHWAYS ORIGINATING WITH GLUTAMATE 1 BioMI 2900 Study Questions #5 - Lectures 13-14 ANSWERS 4 Oct 2009 nitrate reductase, A RESPIRATORY ENZYME THAT ACCEPTS ELECTRONS FROM AN ETS AND DONATES THEM TO THE TERMINAL ELECTRON ACCEPTOR, NITRATE, THUS FORMING NITRITE AND ,EVENTUALLY LEADING TO N2 PRODUCTION IN ANAEROBIC NITRATE RESPIRATION. ANABOLIC PATHWAYS INITIATED BY THIS ENZYME ALSO SUPPLY AMMONIA TO THE CELL WHEN NO3- IS USED AS A NITROGEN SOURCE DURING NITROGEN ASSIMILATION. nitrogenase, THE ENZYME COMPLEX THAT CARRIES OUT THE N2 REDUCTION IN N2 FIXATION nitrogen fixation REDUCTION OF N2 BY NITROGENASE TO FORM NH3 FOR BIOSYNTHESIS. THIS ENERGY INTENSIVE PROCESS REQUIRES A LOT OF REDUCING POWER AND CONSUMES 2-3X MORE ATP /MOLE OF N2 FIXED THAN CO2 FIXATION /MOLE C FIXED BY THE CALVIN CYCLE (SEE ABOVE). 2-- Consider the bacterium, Thiobacillus ferrooxidans. This Bacterium makes ATP via a respiratory electron transport/PMF/ATPase system by oxidizing Fe2+ as an electron donor with oxygen (O2) as the terminal electron acceptor (see reduction potentials). It gets carbon strictly from CO2 fixation. NAD+/NADH E’o= -0.32 V FAD/FADH E’o= -0.22 V Fe3+/Fe2+ E’o=+0.77 V O2/H2O E’o=+0.82 V a) Which of the following terms would apply? autotroph AND lithotroph. T .ferrooxidans is a lithoautotroph. b) You suspect this organism fixes carbon via the Calvin cycle. The presence of what two enzymes would indicate that this is true? RUBISCO AND PHOSPHORUBULOKINASE
pyruvate acetyl-CoA oxaloacetate c) Thiobacillus does not use organic carbon as an energy source; therefore, the TCA cycle is not used to generate reducing power(NADH/FADH) as is usual in aerobic succinate respiration-- yet you find it does have enzymes for the TCA cycle (at right). How can you explain the presence of these enzymes? THE ENZYMES ARE USED TO FORM INTERMEDIATE COMPOUNDS SUCH AS ORGANIC ACIDS THAT MAY BE USED FOR THE BIOSYNTHESIS OF AMINO ACIDS. ketoglutarate d) Considering the negative reduction potentials of the NAD+/NADH and FAD/FADH couples against the positive reduction potential of the Fe2+/Fe3+ couple (see above), explain how T. ferrooxidans gets the reducing power it needs for biosynthesis. LIKE OTHER STRICT LITHOAUTOTROPHS, T. FERROOXIDANS MUST REVERSE ELECTRON 2 BioMI 2900 Study Questions #5 - Lectures 13-14 4 Oct 2009 ANSWERS TRANSPORT IN ITS MEMBRANE-BOUND E.T.S TO REDUCE NAD+ TO NADH OR FAD TO FADH. THIS REQUIRES ENERGY IN PUT. THE ENERGY IS DERIVED PRIMARILY FROM PMF. BECAUSE T.FERROOXIDANS GROWS IN ACIDIC ENVIRONMENTS THAT PROVIDE PROTONS OUTSIDE THE CELL TO FORM THE PMF, IT DOES NOT NEED TO USE AS MUCH ATP TO REVERSE ITS ELECTRON TRANSPORT AS OTHER LITHOAUTOTROPHS DO. NEVERTHELESS, LIKE ALL LITHOAUTOTROPHS, T. FERROOXIDANS GROWS RELATIVELY SLOWLY BECAUSE OF THE SMALL AMOUNT OF FREE ENERGY AVAIABLE IN THE OXIATION OF Fe2+ AND THE NEED TO SPEND SIGNIFICANT AMOUNTS OF THAT ENERGY TO PRODUCE REDUCING POWER BY REVERSED ELECTRON TRANSPORT. 3-- Suppose you were a organotrophic bacterium, and you had NH4+, NO3-, organic N (R-NH2) and N2 available as nitrogen sources. a) Describe what you would have to go through to use each of these as a N source. • NH4+/NH3 WOULD BE TRANSPORTED INTO THE CELL AND THEN ASSIMILATED INTO GLUTAMINE (GLUTAMINE SYNTHETASE) OR GLUTAMATE (GLUTAMATE DEHYDROGENASE) CONSUMING ATP AND NADH AS PER FIG 5.27 • NO3-, WOULD BE TRANSPORTED, THEN REDUCED BY AN ASSIMILATORY NITRATE REDCUTASE ENZYME AND OTHER ENZYMES THAT WOULD PRODUCE NH3, WHICH WOULD BE ASSIMILATED AS ABOVE. • organic N (R-NH2) ; DEAMINASE ENZYMES WOULD STRIP OFF THE AMINE GROUP, THE RESUTING NH3 WOULD BE ASSIMILATED AS ABOVE. • N2: WOULD BE REDUCED BY NITROGENASE, IF PRESENT, USING ATP AND REDUCING POWER TO FORM NH3, WHICH WOULD BE ASSIMILATED AS ABOVE. b) Assuming you have the genes that code for the enzymes that are involved with the assimilation of each N source, which source would you prefer to use first? NH3 ASSIMILATION TAKES THE LEAST AMOUNT OF ENERGY AND REDUCING POWER; THEREFORE IT IS THE PREFERED N SOURCE c) Which last? N2 BECAUSE IT TAKES THE MOST ENERGY AND REDUCING POWER TO REDUCE N2 TO NH3 4) Review Sec. 6.1, 6.5-6.6 in the textbook. Define lag phase, exponential phase and stationary phase for microbial cell growth curves. Lag phase: early part of some growth curves where there is little or no increase in cell number as cells adjust to new growth conditions Exponential phase: period of the highest growth rates for those conditions Stationary phase: period of slowed growth due to limitation of nutrients or accumulations of toxic waste products. Would these curves look different if the culture was the yeast, Saccharomyces cerevisiae (a single-celled fungus that divides by a binary budding fission process) rather than Bacteria or Archaea? Why or Why not? 3 BioMI 2900 Study Questions #5 - Lectures 13-14 4 Oct 2009 ANSWERS No. All single cells that divide by binary fission would be expected to grow exponentially. 5) Use the semi-logarithmic graph at the right to answer the questions below. a) Identify the phases of growth for each growth curve. Lag-- the first 1/2 hour of graph 2; the first 1 1/2 hour of graph 1 Exponential -- the part of the curve with the steepest slope Stationary -- the last 1 1/2 hour of each curve b) Which experiment has the fastest growth rate? 2 has the steepest slope, therefore the fastest growth rate The highest total cell yield? 1 has more cells at the end of the experiment, therefore has the highest total cell yield. c) In one experiment, cells were taken from a complex yeast-extract containing medium and were put into a tube of identical medium. For the other experiment, cells were taken from a complex yeast-extract containing medium and put into a defined glucose medium. Which is which? How can you tell? (HINT: look at the lag phase times) Exp.1 was switched from complex yeast extract medium and put into a defined glucose medium whereas in Exp. 2 cell were taken from a complex yeast extract medium and were put into a tube of identical growth medium. If cells go from one medium to an identical medium there is little or o adjustment or LAG time (as in Exp. 2) 6--You are working with a purple bacterium to investigate nitrogen utilization. You grow the cells in 3 tubes containing the following medium with no other N source except:
Acetate K2HPO4 MgSO4 CaSO4 CaCO3 Water 0.2 g 0.5g 0.2 g 0.1 g 5.0 g I liter Tube 1: add N2; grow under oxic conditions Tube 2: add N2; and grow under anoxic conditions Tube 3: add NH4+ and grow under anoxic condiitons You monitor growth over time, and get the graph at the right. Unfortunately, your lab partner forgot to label which tube was which. Looking at the data, predict which curve (A, B or C) came from which tube above. Explain your reasoning. Assume that these purple bacteria, like many others, can fix N2 under anoxic (anaerobic) conditions, while growing on light energy.
log of cells/ml A B C time (hrs) 4 Study Questions #5 - Lectures 13-14 4 Oct 2009 ANSWERS Curve A = Tube 3. In this case the cells have NH4+/ NH3 supplied in the medium. They can put more of their energy toward carbon backbone biosynthesis from acetate instead of nitrogen reduction (assimilation) and, therefore, they will grow faster. Curve B = Tube 2. The cells grow more slowly than those in tube 3 because they must spend more energy to fix nitrogen, which means they will not have as much energy to spend on carbon backbone biosynthesis. Curve C = Tube 1. The cells cannot grow in the presence of oxygen because oxygen inhibits N2 fixation. Although some purple bacteria can grow as organotrophs by aerobic respiration of acetate, even these cells will not grow if they cannot fix N2 because there is no other source of nitrogen. PART III: ENVIRONMENTAL FACTORS AFFECTING GROWTH 7. Refer to the growth vs. temperature curves (see BBOM 12e, Fig. 6.19 ) for psychrophiles, mesophiles, thermophiles, and hyperthermophiles. a) Based on the information shown, what do you know about lag and stationary phases of growth in these organisms at various temperatures? NOTHING-- these graphs show growth rates determined in the exponential phase of growth versus temperature b) List 2 adaptations of key macromolecules of thermophiles at high temperatures. • DNA HAS HIGH G+C CONTENT; POSITIVE SUPER COILS, AND HISTONELIKE DNA-BINDING PROTEINS • PROTEINS ADAPTED TO HIGH TEMPS, CONTAIN CHAPERONINS AND/OR CATION BRIDGES • LIPIDS BRANCHED, SATURATED FATTY ACIDS, SOMETIMES CONSIST OF TETRA ETHER MONOLAYERS, WHICH ARE TWICE AS WIDE AS PHOSPHOLIPID BILAYERS, AND HAVE COVALENT LINKAGES IN THE CENTER c) List 2 adaptations of key macromolecules that psychrophiles at low temperatures. • TRANSPORT PROTEINS AND ENZYMES ADAPTED TO LOW TEMPS, • PHOSPHOLIPIDS WITH SHORT CHAIN UNSATURATED FATTY ACIDS d) What happens to certain important macromolecules in psychrophilic cells when their environment is 10° C? proteins denature, membranes get too fluid (melt & leak) What happens to certain important macromolecules in mesophilic cells at 10° C? Enzymes slow, membranes get too solid (gel), transport slows down. e) What happens to certain important macromolecules in mesophilic cells when their environment is 50° C? proteins denature, membranes get too fluid (melt) What happens to certain important macromolecules in thermophilic cells at 50° C? enzymes slow, membranes get too solid (gel) BioMI 2900 5 BioMI 2900 Study Questions #5 - Lectures 13-14 ANSWERS 8. Answer Review Questions 10-13 in BBOM 12e Ch.6, p173. 4 Oct 2009 10. The optimum temperature for growth occurs when all the enzymes in the cell are functioning at their maximal possible rates. Above the Topt the enzymes proteins are denatured and membranes collapse, so the growth rate falls off rapidly. Below the Topt the enzymes function at less than their maximum, but they can continue to function at lower and lower temperatures, albeit at slower rates. Thus the low end of the Growth Rate vs. T curves fall off more gradually as temperatures decline. 11. Psychrophiles are found in constant low-temperature habitat such as Antarctic sea ice, high mountain snow and ice, the deep sea, and your refrigerator. Hyperthermophiles are found in constant high-temperature enviroments such as the hot springs at Yellowstone National Park and other volcanic areas and in deep sea thermal vents. 12. Acidophiles live in low pH environments. Alkaliphiles live in high pH environments. Although the known environmental pH range for these organisms varies widely from pH~ 0-10, the internal pH of both types of cells is usually around pH 7. However, it has been found that the internal pH in some extreme cases varies from ~pH 4. 5 in some extreme acidophiles to ~ pH 9.5 in some extreme alkaliphiles. These values are thought to be the pH limits of macromolecule stability. 13. Halophile cells accumulate a compatible solute, usually betaine or KCl, in side the cell to achieve a higher salt concentration inside than outside the membrane, thus water can move into the cell by osmosis. 9. Many anaerobic prokaryotes are killed by oxygen. These are called strict anaerobes. a. What makes O2 so toxic to strict anaerobes? Reactive oxygen species (e.g. superoxide-- O2-, hydrogen peroxide--H202, and hydroxyl radicals—OH.) are produced during respiration to O2. These are very reactive oxidants that can destroy enzyme proteins and nucleic acids How do aerobes overcome this? They produce enzymes like catalase, peroxidase, and superoxide dismutase that destroy the toxic oxygen species. b. Why can't strict anaerobes just keep O2 out? (How does it get into the cell?) O2 diffuses freely through the membrane 10. For each blank below, use the term best describes each organism’s oxygen preference (see list of terms: BBOM 12e in Fig. 6.27).. a) E. coli can be described as a(n) facultative anaerobe b. S. volutans is a(n) microaerophile c) Desulfotomaculum and Clostridium are both strict anaerobes d) P. denitrificans is a facultative anaerobe e) Lactobacillus does have enzymes to convert oxygen radicals to harmless endproducts, therefore it is an aeroltolerant anaerobe 11. Refer to the example bacteria given in the previous question to answer the following questions. 6 BioMI 2900 Study Questions #5 - Lectures 13-14 4 Oct 2009 ANSWERS a) Which two of the organisms above make virtually all their ATP by substrate-level phosphorylation? Clostridium and Lactobacillus. Both are primarily fermenters. Without respiratory electron transport systems, they can only make ATP from substrate level phosphorylation. b) Which THREE organisms absolutely require oxidative phosphorylation? Desulfotomaculum, Pseudomonas, Spirillum. All three organisms depend on respiratory metabolism, SO42- reduction, Aerobic respiration or denitrification (NO3 respiration), and aerobic respiration at low O2 concentrations, respectively. c) Which is the only organism in A-E that can survive on either oxidative- or substrate levelphosphorylation? E. coli. Yes, E. coli is a facultative aerobe. 7 ...
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