Microbial metabolism and biochemical assays

By Dr. C. Rexach Microbiology Mt San Antonio College 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 • =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 Enzymes speed up reactions by reducing activation energy components

• Some enzymes require non-protein cofactors or coenzymes • Cofactors – Usually metal ions ++ ++ cofactor – Ca , Mg , etc. enzyme – Help form bridge between enzyme and substrate • Coenzymes – NAD, FAD, CoA, etc. Mechanism of enzyme action

substrate enzyme Enzyme-substrate complex

enzyme product Enzyme can be reused Factors influencing activity • Temperature • pH • Amount of substrate • Amount of enzyme • Competitive inhibition • Feedback inhibition Enzymes can be denatured by pH and temperature 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 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 = • Two most frequently used methods – Cellular respiration

• Complete breakdown of glucose into H2O, CO2 and energy • Four steps: glycolysis, intermediate step, Krebs cycle, ETS – • Partial breakdown into or 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 (3C) and energy transfer molecules • 4ATP (2 net) • 2 NADH Summary: 4ATP-2ATP = 2ATP net 2NADH Glycolysis glucose Glucose-6-phosphate Fructose-1,6 bisphosphate

ATP ATP

Dihydroxyacetone Glyceraldehyde phosphate phosphate

NADH

2 1,3 bisphosphoglycerate

2 ATP Phosphoenolpyruvate 2 2 (PEP) 3-phosphoglycerate 2 ATP 2 pyruvic acid 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 Entner-Doudoroff Pathway Glucose

• Independent of glycolysis ATP ADP • Produces NADPH & ATP Glucose-6-phosphate • Two key enzymes – 6-phosphogluconate dehydrogenase NADP+ NADPH – 2-keto-3- deoxyglucosephosphate 6-phosphogluconic acid aldolase • Absent in gram-positive bacteria 2-keto-3-deoxygluconic acid 6-phosphate • Found in some gram negative bacteria, such as Pseudomonas, Glyceraldehyde pyruvate Rhizobium,Agrobacterium, 3-phosphate Zymomonas, etc. glycolysis ATP

pyruvate ATP 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 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) Summary: 2 NADH

2 CO2 Intermediate step

GLYCOLYSIS 2 Pyruvic acid

2 NADH 2 CO2

2 acetyl CoA KREBS CYCLE Krebs Cycle

Summary: 6 NADH

2 FADH2 2 ATP

4 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

FMN NADH = 3ATP Fe-S FADH2 = 2ATP Q NADH Cyt b Fe-S Fe-S

Cyt c1 Cyt c Cyt a

FADH2 Cyt a3

½ O2 Chemiosmosis generates ATP Chemiosmosis 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: Alcohol fermentation: causes food spoilage, production of Many bacteria and yeasts yogurt, pickles, sauerkraut Examples: Saccharomyces Examples: Lactobacillus, Streptococcus 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 – 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 • 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

glycolysis Pyruvic acid Lactic acid

CO2

Succinic acid • CO2 is produced only from formic acid via Ethanol

formate hydrogen lyase Acetyl CoA Acetic acid

• HCOOH H2 + CO2 CO2

Formic acid • Therefore, equal H2 amounts of H2 & CO2 2,3 butanediol fermentation

2,3 butanediol + CO2

ethanol

glycolysis Pyruvic acid Lactic acid

Succinic acid

• Produce CO2 from formic acid and from formation of Acetic acid butanediol CO2 + H2 Fermentation in microbes 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.

Electron acceptor Products – – NO3 NO2 , N2 + H2O – SO4 H2S + H2O 2 – CO3 CH4 + H2O Lipid Catabolism Protein Catabolism

Extracellular proteases Protein Amino Acids

Deamination, decarboxylation, dehydrogenation

Organic acids

Krebs cycle Photosynthesis

Figure 4.15 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:

0 CO2 + 2 H2S + Light energy  [CH2O] + H2O + 2 S Cyclic Photophosphorylation Noncyclic Photophosphorylation

• 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) Metabolic Diversity Among Organisms

Energy Carbon Nutritional type source source Example

Photoautotroph Light CO2 Oxygenic: Cyanobacteria, plants Anoxygenic: Green, purple bacteria Photoheterotroph Light Organic Green, purple nonsulfur compounds bacteria

Chemoautotroph Chemical CO2 Iron-oxidizing bacteria

Chemoheterotroph Chemical Organic Fermentative bacteria, compounds Animals, protozoa, fungi, bacteria. Polysaccharide biosynthesis Lipid Biosynthesis Amino Acid & Protein Biosynthesis Transamination Biosynthesis of purines and pyrimidines Biochemical tests are used to ID bacteria 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 Carbohydrate fermentation MR-VP Medium • Medium = glucose broth + peptone & dipotassium phosphate • Used to differentiate gram neg enteric bacteria • Two tests in one – Mixed acid fermentation • Results = 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 Citrate test

+ - 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 Nitrate reduction test 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 Catalase test 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 Indole 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 Urea hydrolysis 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 Kligler’s Iron Agar Litmus Milk API 20E