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

• 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

Enzymes speed up reactions by reducing activation energy

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

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 = 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

Krebs Cycle

Summary:6 NADH2 FADH2

2 ATP4 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

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: 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

Fermentation in microbes

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.

Lipid Catabolism

Krebs cycle

Deamination,

decarboxylation,

dehydrogenation

Protein Catabolism

ProteinExtracellular proteases

Amino Acids

Organicacids

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: CO2 + 2 H2S + Light energy [CH2O] + H2O + 2 S0

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)

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

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 = 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

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

Catalasetest

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