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
cofactor
– Ca++, Mg++, etc.
– Help form bridge between enzyme and
substrate
• Coenzymes
– NAD, FAD, CoA, etc.
enzyme
Mechanism of enzyme
action
enzyme
enzyme
substrate
product
Enzyme-substrate complex
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
Summary:
4ATP-2ATP =
2ATP net
2NADH
Glycolysis
glucose
Glucose-6-phosphate
ATP
Fructose-1,6 bisphosphate
ATP
Dihydroxyacetone
phosphate
Glyceraldehyde
phosphate
NADH
2
1,3 bisphosphoglycerate
2 ATP
2
Phosphoenolpyruvate
(PEP)
2
2 ATP
2 pyruvic acid
3-phosphoglycerate
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
• Independent of glycolysis
• Produces NADPH & ATP
• Two key enzymes
– 6-phosphogluconate
dehydrogenase
– 2-keto-3deoxyglucosephosphate
aldolase
• Absent in gram-positive
bacteria
• Found in some gram
negative bacteria, such as
Pseudomonas,
Rhizobium,Agrobacterium,
Zymomonas, etc.
Glucose
ATP
ADP
Glucose-6-phosphate
NADP+
NADPH
6-phosphogluconic acid
2-keto-3-deoxygluconic acid 6-phosphate
pyruvate
Glyceraldehyde
3-phosphate
glycolysis
pyruvate
ATP
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 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
GLYCOLYSIS
Summary:
2 NADH
2 CO2
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
NADH = 3ATP
FADH2 = 2ATP
Fe-S
Q
Cyt b
Fe-S
Fe-S
Cyt c1
FADH2
Cyt c
Cyt a
Cyt a3
½ O2
Chemiosmosis generates ATP
Chemiosmosis
Grand total for aerobic cellular
respiration
step
#ATP
Glycolysis
#NADH/FADH2
#CO2 prod
end products
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
ETS
34 ATP
0
H2O & CO2
0
Grand total = 38 ATP (prokaryotes) or 36 ATP (eukaryotes)
0
Without oxygen: fermentation
• Final electron acceptor is organic = pyruvic
acid
• Anaerobic respiration: less ATP produced
• Results
Lactic acid
Lactic Acid Fermentation:
causes food spoilage, production of
yogurt, pickles, sauerkraut
Examples: Lactobacillus, Streptococcus
Ethanol + CO2
Alcohol fermentation:
Many bacteria and yeasts
Examples: 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
glycolysis
Pyruvic acid
Lactic acid
CO2
• CO2 is produced only
from formic acid via
formate hydrogen lyase
• HCOOH
Succinic acid
Ethanol
Acetyl CoA
Acetic acid
H2 + CO2
CO2
• Therefore, equal
amounts of H2 & CO2
Formic acid
H2
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
butanediol
Acetic acid
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
CO32 –
CH4 + H2O
Lipid
Catabolism
Protein Catabolism
Protein
Extracellular proteases
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:
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)
Metabolic Diversity Among Organisms
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.
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
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