By Dr. C. Rexach Microbiology Mt San Antonio College
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1 Microbial metabolism and biochemical assays By Dr. C. Rexach Microbiology Mt San Antonio College
2 Metabolism Sum total of all chemical reactions in living organisms Two general types Anabolism: building bonds,capturing energy Catabolism: breaking bonds, releasing energy Coupled reactions Enzymes=biological catalysts
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4 Characteristics of enzymes Almost all enzymes are proteins Exception: ribozymes Enzymes can only speed up reactions that would occur anyway Enzymes are able to work at biological temperatures Enzymes are sensitive to certain conditions Remember: functional proteins work on the basis of their 3-D shape Enzymes can be regulated
5 Enzymes speed up reactions by reducing activation energy
6 Enzyme components Some enzymes require non-protein cofactors or coenzymes Cofactors Usually metal ions Ca ++, Mg ++, etc. Help form bridge between enzyme and substrate Coenzymes NAD, FAD, CoA, etc. cofactor enzyme
7 Mechanism of enzyme action enzyme substrate Enzyme-substrate complex enzyme product Enzyme can be reused
8 Factors influencing activity Temperature ph Amount of substrate Amount of enzyme Competitive inhibition Feedback inhibition
9 Enzymes can be denatured by ph and temperature
10 Competitive inhibition
11 Feedback inhibition
12 Energy production Biochemical pathway Sequence of enzyme catalyzed chemical reactions in cell Oxi-redux reactions Electrons pulled off and passed along in series of reactions Oxidation = removal of one or more electrons from substance (often along with a H+) Reduction = substance gains one or more electrons
13 Oxidation-Reduction Rxns
14 Oxidation-Reduction Rxns In biological systems, the electrons are often associated with hydrogen atoms. Biological oxidations are often dehydrogenations.
15 Carbohydrate catabolism Oxidation of carbohydrates = one of primary energy sources in cell Most common = glucose Two most frequently used methods Cellular respiration Complete breakdown of glucose into H 2 O, CO 2 and energy Four steps: glycolysis, intermediate step, Krebs cycle, ETS Fermentation Partial breakdown into lactic acid or ethanol and CO 2 Note: Bacteria have many different pathways for carbohydrate metabolism based on the enzymes they are able to produce.
16 Glycolysis = Embden-Meyerhof pathway Overview Begin with 1 mole of glucose = C 6 H 12 O 6 Series of enzyme mediated reactions result in formation of 2 moles of pyruvic acid (3C) and energy transfer molecules 4ATP (2 net) 2 NADH
17 Glycolysis Summary: 4ATP-2ATP = 2ATP net 2NADH glucose Glucose-6-phosphate Fructose-1,6 bisphosphate ATP ATP Dihydroxyacetone phosphate Glyceraldehyde phosphate NADH 2 1,3 bisphosphoglycerate Phosphoenolpyruvate (PEP) ATP 2 ATP 3-phosphoglycerate 2 pyruvic acid
18 Entner-Doudoroff Pathway Each step in glycolysis is enzyme mediated Phosphofructokinase is an enzyme which phosphorylates fructose-6-phosphate, producing fructose 1,6 bisphosphate Glucose-6-phosphate Fructose-1,6 bisphosphate phosphofructokinase If organisms lack this enzyme, they can t progress down Embden-Meyerhof pathway Entner-Doudoroff pathway provides alternative way to go from glucose-6-phosphate to pyruvic acid
19 Entner-Doudoroff Pathway Independent of glycolysis Produces NADPH & ATP Two key enzymes 6-phosphogluconate dehydrogenase 2-keto-3- deoxyglucosephosphate aldolase Absent in gram-positive bacteria Found in some gram negative bacteria, such as Pseudomonas, Rhizobium,Agrobacterium, Zymomonas, etc. Glucose Glucose-6-phosphate 6-phosphogluconic acid 2-keto-3-deoxygluconic acid 6-phosphate pyruvate ATP NADP+ ADP NADPH glycolysis Glyceraldehyde 3-phosphate ATP pyruvate ATP
20 Pentose phosphate pathway Major uses 1. generate pentoses from hexoses 2. generate hexoses from pentoses (gluconeogenesis) 3. break down pentoses as a source of cellular energy Produces acetate and pyruvate 4. generate NADPH Important coenzyme used by cells for reductive biosynthesis 5. generates sugar diversity Produces a variety of sugar derivatives in ancillary reactions Key intermediate = ribulose-5-phosphate Source of ribose and deoxyribose for nucleic acid production
21 Aerobic respiration More ATP produced by oxidative phosphorylation Final electron acceptor is inorganic = O 2 Results in complete catabolism of glucose Three steps Intermediate step Krebs cycle Electron Transport System (ETS)
22 Summary: 2 NADH 2 CO 2 Intermediate step GLYCOLYSIS 2 Pyruvic acid 2 CO 2 2 NADH 2 acetyl CoA KREBS CYCLE
23 Krebs Cycle Summary: 6 NADH 2 FADH 2 2 ATP 4 CO 2
24 Electron transport system Electrons from NADH and FADH 2 passed along series of carrier molecules embedded in cristae (eukaryotes) or plasma membrane (prokaryotes) 3 types of carrier molecules Flavoproteins Cytochromes ubiquinones Energy released drives generation of ATP via chemiosmosis
25 Electron Transport System NADH FMN Fe-S Fe-S Q Cyt b Fe-S NADH = 3ATP FADH 2 = 2ATP Cyt c 1 Cyt c Cyt a FADH 2 Cyt a 3 ½ O 2
26 Chemiosmosis generates ATP
27 Chemiosmosis
28 Grand total for aerobic cellular respiration step #ATP #NADH/FADH2 #CO2 prod end products Glycolysis 2ATP net 2 NADH 0 CO2 2 pyruvic acid Intermediate Step 0 ATP 2 NADH 2 CO2 2 acetyl CoA Krebs Cycle 2 ATP 6NADH/2FADH2 4 CO2 H2O & CO2 ETS 34 ATP Grand total = 38 ATP (prokaryotes) or 36 ATP (eukaryotes)
29 Without oxygen: fermentation Final electron acceptor is organic = pyruvic acid Anaerobic respiration: less ATP produced Results Lactic acid Ethanol + CO 2 Lactic Acid Fermentation: causes food spoilage, production of yogurt, pickles, sauerkraut Examples: Lactobacillus, Streptococcus Alcohol fermentation: Many bacteria and yeasts Examples: Saccharomyces
30 Summary for fermentation No new electron transfer molecules (either NADH,FADH 2, or ATP) produced in intermediate step The electrons from the 2NADH made during glycolysis are removed and transferred to pyruvic acid, the final electron acceptor. Therefore, they are unavailable for making more ATP in the ETS. If lactic acid is end product, no CO 2 is produced during fermentation If ethanol is the end product, 2 CO 2 are produced during fermentation The total ATP produced net in fermentation = 2
31 Homolactic vs. heterolactic fermentation Two types of lactic acid fermentation Homolactic fermentation Produces only lactic acid using pyruvic acid Usually begins with Embden-Meyerhof pathway Characteristic of Streptococci and some Lactobacilli Heterolactic fermentation Produces lactic acid, ethanol and CO2 using pyruvic acid and acetate Begins with the pentose phosphate pathway Characteristic of some Lactobacilli and Leuconostoc
32 Fermentation in enteric bacteria Type and proportion of products of anaerobic fermentation used to separate enteric bacteria into various genera Two major patterns Mixed-Acid Fermentation Produces acetic, lactic, and succinic acid Also produces ethanol and CO2 and H2 CO2 and H2 are produced in equal amounts 2,3 butanediol fermentation Major products are butanediol, ethanol, CO2, and H2 Much more CO2 is produced than H2 Also produces small amounts of succinic, lactic, and acetic acids
33 Mixed-Acid Fermentation glycolysis Pyruvic acid Lactic acid CO2 CO 2 is produced only from formic acid via formate hydrogen lyase Acetyl CoA Succinic acid Ethanol Acetic acid HCOOH H 2 + CO 2 Therefore, equal amounts of H 2 & CO 2 Formic acid CO2 H2
34 2,3 butanediol fermentation 2,3 butanediol + CO2 ethanol glycolysis Pyruvic acid Lactic acid Produce CO 2 from formic acid and from formation of butanediol Succinic acid Acetic acid CO2 + H2
35 Fermentation in microbes
36 Anaerobic Respiration The final electron acceptor in the electron transport chain is not O 2. Yields less energy than aerobic respiration because only part of the Krebs cycles operations under anaerobic conditions. Electron acceptor NO 3 SO 4 CO 2 3 Products NO 2, N 2 + H 2 O H 2 S + H 2 O CH 4 + H 2 O
37 Lipid Catabolism
38 Protein Catabolism Protein Extracellular proteases Amino Acids Deamination, decarboxylation, dehydrogenation Organic acids Krebs cycle
39 Photosynthesis Figure 4.15
40 Photosynthesis Photo: Conversion of light energy into chemical energy (ATP) Light-dependent (light) reactions Synthesis: Fixing carbon into organic molecules Light-independent (dark) reaction, Calvin-Benson cycle
41 Oxygenic: Photosynthesis 6 CO H 2 O + Light energy C 6 H 12 O H 2 O + 6 O 2 Anoxygenic: CO H 2 S + Light energy [CH 2 O] + H 2 O + 2 S 0
42 Cyclic Photophosphorylation
43 Noncyclic Photophosphorylation
44
45
46 Halobacterium uses bacteriorhodopsin, not chlorophyll, to generate electrons for a chemiosmotic proton pump.
47 Nutritional classification Photoautotrophs Source of energy = light Carbon source = CO 2 Photoheterotrophs Source of energy = light Carbon source = organic Chemoautotrophs Source of energy = reduced inorganic compounds Carbon source = CO 2 Chemoheterotrophs Source of energy and carbon = glucose saprophytes (decaying matter), parasites (living matter)
48 Metabolic Diversity Among Organisms Nutritional type Energy source Carbon source Example Photoautotroph Light CO 2 Oxygenic: Cyanobacteria, plants Photoheterotroph Light Organic compounds Anoxygenic: Green, purple bacteria Green, purple nonsulfur bacteria Chemoautotroph Chemical CO 2 Iron-oxidizing bacteria Chemoheterotroph Chemical Organic compounds Fermentative bacteria, Animals, protozoa, fungi, bacteria.
49 Polysaccharide biosynthesis
50 Lipid Biosynthesis
51 Amino Acid & Protein Biosynthesis
52 Transamination
53 Biosynthesis of purines and pyrimidines
54 Biochemical tests are used to ID bacteria
55 Carbohydrate fermentation Investigates ability of particular bacterium to metabolize specific sugars and determines method they use Phenol red used as ph indicator Durham tube captures gas Results A, AG, AGR, negative
56 Carbohydrate fermentation
57 MR-VP Medium Medium = glucose broth + peptone & dipotassium phosphate Used to differentiate gram neg enteric bacteria Two tests in one Mixed acid fermentation Results = methyl red added to determine ph change Durham tube used to visualize gas production 2,3 butanediol fermentation Voges-Proskauer test Gram negative enterics which do not use mixed-acid fermentation sometimes produce 2,3 butanediol Add Barritt s reagent to convert butanediol to acetoin Pink to red color change after 30 minute incubation is positive
58
59 Citrate Test Citrate in media is only source of oxidizable carbohydrate Citrate split to produce oxaloacetate + pyruvate Products fermented Also contains ammonium salts as nitrogen source ph indicator called Brom thymol blue Color change when citrate is used due to production of ammonia, which makes ph alkaline
60 Citrate test + -
61 Nitrate reduction tests Used to detect gram negative rods Nitrate is final electron acceptor in anaerobic respiration, reducing nitrate to nitrite Durham tube for gas, reagents used to determine presence of nitrite Negative tests are double-checked with Zinc dust
62 Nitrate reduction test
63 Catalase tests H 2 O 2 produced as by-product of aerobic respiration using oxygen Protect themselves against oxidation by producing catalase Produced by aerobes + facultative anaerobes, but not by obligate anaerobes Test by adding H 2 O 2 to cells on a glass slide and watching for bubbles
64 Catalase test
65 Indole production Some bacteria can cleave amino acid tryptophan to prod indole + pyruvic acid Presence of indole detected by Kovac s reagent Forms pinkish red layer on surface
66 Indole
67 Urea hydrolysis Produced when protein and nucleic acids broken down Organisms able to make urease convert urea to ammonia and CO2 Ammonia becomes ammonium hydroxide in water ph increases Phenol red indicator used to detect change
68 Urea hydrolysis
69 Phenylalanine deamination Differentiates some gram negative organisms Oxidative deamination of phenylalanine catalyzed by phenylalanine deaminase Detects presence of enzyme by adding 10% ferric chloride
70 Kligler s Iron Agar Differentiates gram negative enterics Multiple test medium Fermentation of glucose and lactose Production of H 2 S from cysteine catabolism Phenol red
71 Kligler s Iron Agar
72 Litmus Milk
73 API 20E
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