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Transcript of Microbial metabolism and biochemical assaysinstruction2.mtsac.edu/crexach/microbiology 1/pdf...
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
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
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.
enzymecofactor
Mechanism of enzyme action
enzymesubstrate
Enzyme-substrate complex
enzyme product Enzyme can be reused
Factors influencing activity
• Temperature
• pH
• Amount of substrate
• Amount of enzyme
• Competitive inhibition
• Feedback inhibition
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
Oxidation-Reduction Rxns
• In biological systems, the electrons are often associated with hydrogen atoms.
• Biological oxidations are often dehydrogenations.
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 H2O, CO2
and energy• Four steps: glycolysis, intermediate step,
Krebs cycle, ETS– Fermentation
• Partial breakdown into lactic acid or ethanol and CO2
Note: Bacteria have many different pathways for carbohydrate metabolism based on the enzymes they are able to produce.
Glycolysis = Embden-Meyerhof pathway
• Overview– Begin with 1 mole of glucose
= C6H12O6
– Series of enzyme mediated reactions result in formation of 2 moles of pyruvic acid (3C) and energy transfer molecules
• 4ATP (2 net)
• 2 NADH
Glycolysisglucose Glucose-6-phosphate Fructose-1,6 bisphosphate
Phosphoenolpyruvate(PEP)
ATP ATP
2 pyruvic acid
Glyceraldehydephosphate
Dihydroxyacetonephosphate
1,3 bisphosphoglycerate
3-phosphoglycerate
NADH
2 ATP
2 ATP
Summary:4ATP-2ATP =2ATP net2NADH
2
2 2
Entner-Doudoroff Pathway
• Each step in glycolysis is enzyme mediated
• Phosphofructokinase is an enzyme which phosphorylates fructose-6-phosphate, producing fructose 1,6 bisphosphate
• 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
Glucose-6-phosphate Fructose-1,6 bisphosphate
phosphofructokinase
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
pyruvateGlyceraldehyde
3-phosphate
pyruvate
ATP ADP
ATP
ATP
glycolysis
NADP+ NADPH
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
Aerobic respiration• More ATP produced by oxidative
phosphorylation• Final electron acceptor is inorganic = O2
• Results in complete catabolism of glucose• Three steps
– Intermediate step– Krebs cycle– Electron Transport System (ETS)
Intermediate step
2 Pyruvic acid
2 acetyl CoA
2 NADH
GLYCOLYSIS
KREBSCYCLE
Summary:2 NADH2 CO2
2 CO2
Electron transport system
• Electrons from NADH and FADH2 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
Electron Transport System
FMNFe-S
Q
Cyt b
Fe-S
Cyt c1
Cyt cCyt a
Cyt a3
Fe-SNADH
FADH2
½ O2
NADH = 3ATPFADH2 = 2ATP
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 0 0 0
Grand total = 38 ATP (prokaryotes) or 36 ATP (eukaryotes)
Without oxygen: fermentation
• Final electron acceptor is organic = pyruvic acid
• Anaerobic respiration: less ATP produced
• Results
Lactic acid Ethanol + CO2
Lactic Acid Fermentation: causes food spoilage, production of yogurt, pickles, sauerkrautExamples: Lactobacillus, Streptococcus
Alcohol fermentation: Many bacteria and yeastsExamples: Saccharomyces
Summary for fermentation
• No new electron transfer molecules (either NADH,FADH2, 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 CO2 is produced during fermentation
• If ethanol is the end product, 2 CO2 are produced during fermentation
• The total ATP produced net in fermentation = 2
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
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
Mixed-Acid Fermentation
• CO2 is produced only from formic acid via formate hydrogen lyase
• HCOOH H2 + CO2
• Therefore, equal amounts of H2 & CO2
glycolysis Pyruvic acid Lactic acid
Succinic acid
Formic acid
Acetyl CoA
Ethanol
Acetic acid
CO2
H2
CO2
2,3 butanediol fermentation
• Produce CO2 from formic acid and from formation of butanediol
glycolysis Pyruvic acid
2,3 butanediol + CO2
ethanol
Lactic acid
Succinic acid
Acetic acid
CO2 + H2
Electron acceptor ProductsNO3
– NO2–, N2 + H2O
SO4– H2S + H2O
CO32 – CH4 + H2O
Anaerobic Respiration
• The final electron acceptor in the electron transport chain is not O2.
• Yields less energy than aerobic respiration because only part of the Krebs cycles operations under anaerobic conditions.
Krebs cycle
Deamination,
decarboxylation,
dehydrogenation
Protein Catabolism
ProteinExtracellular proteases
Amino Acids
Organicacids
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
Photosynthesis• Oxygenic:
6 CO2 + 12 H2O + Light energy C6H12O6 + 6 H2O + 6 O2
• Anoxygenic: CO2 + 2 H2S + Light energy [CH2O] + H2O + 2 S0
• Halobacterium uses bacteriorhodopsin, not chlorophyll, to generate electrons for a chemiosmotic proton pump.
Nutritional classification
Photoautotrophs
Source of energy = light
Carbon source = CO2
Photoheterotrophs
Source of energy = light
Carbon source = organic
Chemoautotrophs
Source of energy = reduced inorganic compounds
Carbon source = CO2
Chemoheterotrophs
Source of energy and carbon = glucose
saprophytes (decaying matter), parasites (living matter)
Nutritional type Energy source
Carbon source Example
Photoautotroph Light CO2 Oxygenic: Cyanobacteria, plants
Anoxygenic: Green, purple bacteria
Photoheterotroph Light Organic compounds
Green, purple nonsulfur bacteria
Chemoautotroph Chemical CO2 Iron-oxidizing bacteria
Chemoheterotroph Chemical Organic compounds
Fermentative bacteria, Animals, protozoa, fungi, bacteria.
Metabolic Diversity Among Organisms
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
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
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
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
Catalase tests• H2O2 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 H2O2 to cells on a glass
slide and watching for bubbles
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
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
Phenylalanine deamination
• Differentiates some gram negative organisms
• Oxidative deamination of phenylalanine catalyzed by phenylalanine deaminase
• Detects presence of enzyme by adding 10% ferric chloride
Kligler’s Iron Agar• Differentiates gram negative
enterics• Multiple test medium
– Fermentation of glucose and lactose– Production of H2S from cysteine
catabolism
• Phenol red