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Module V
BACTERIAL METABOLISM

The ability of microbial cells to live, function and replicate in an

appropriate chemical environment and the chemical changes that result during
this transformation constitute the scope of bacterial metabolism. Metabolism
refers to all the biochemical reactions that occur in a cell or organism.
The study of bacterial metabolism focuses on the chemical diversity of
substrate oxidations and dissimilation (catabolic) reactions in which molecules
are functionally broken down to generate energy in bacteria. The processes that
bring about uptake and utilization of the inorganic or organic compounds
required to generate energy in order to maintain a cellular steady state
(assimilation reactions) are included within the scope of bacterial metabolism.
The role of bacterial cell integrated enzyme systems in catalyzing relevant energy-
yielding (exergonic) and energy-requiring (endergonic) reactions are explored to
understand fundamental mechanisms that sustain bacterial metabolism.

Objectives:
On successful completion of the module, students will be able to:

1. Recognize groups of bacteria based on their energy sources.

2. Identify the 5 metabolic pathways in bacteria.

3. Define the principles of thermodynamics relative to the study of bacterial

metabolic processes.

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A source of energy is required by bacteria as this is necessary to fuel

bacterial metabolism. The energy is obtained by the transfer of an electron from
an electron donor to an electron acceptor. Four (4) basic sources of energy that
support metabolism have been identified in bacteria and these classify bacteria
as follows:

1. Phototrophs are microbes that use photosynthesis to generate cellular energy

in the form of adenosine triphosphate (ATP) from light energy.

2. Chemotrophs are microbes that obtain their energy from chemicals (organic
and inorganic compounds). Some chemotrophs are seen as either chemo-

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heterotrophs which obtain their energy sources from organic compounds

and chemo-lithotrophs which obtain their energy from inorganic salts.

3. Autotrophs are microbes that obtain energy from a carbon source.

4. Heterotrophs need organic substances as sources of energy.

5. Obligate are living forms that strictly require a living host.

The bacterial cell as a thermodynamic system

One of the fundamental means of organizing bacteria is based on energy

source to meet their nutritional needs. The bacterial cell is a highly specialized
energy transformer. Chemical energy generated by substrate oxidations is
conserved by formation of high-energy compounds such as adenosine
diphosphate (ADP) and adenosine triphosphate (ATP) or compounds containing
the thio-ester bonds (like acetyl ~ SCoA or succinyl ~ SCoA). ADP and ATP
represent adenosine monophosphate (AMP) plus one and two high-energy
phosphates (AMP ~ P and AMP ~ P~ P, respectively) and the energy is stored in
these compounds as high-energy phosphate bonds. In the presence of proper
enzyme systems, these compounds can be used as energy sources to synthesize
new complex organic compounds needed by the cell.

All living cells maintain a steady-state of biochemical reactions for the

formation and use of high-energy compounds. It is recognized that bacterial cells
are in many respects similar chemically to all other living cells. Bacteria like
mammalian and plant cells use ATP or the high-energy phosphate bond (~ P) as
the primary chemical energy source and that hydrogen transfer is a common and
fundamental feature of all metabolic processes.

The individual bacterial cell bounded by the outer surface of the cell wall
is a thermodynamic system whose properties and behavior are related with
metabolism. A living bacterium grows and some point during its growth, each
bacterium divides into 2 bacteria. The size of the single cell at the beginning of
division varies and also does the relative size of the two daughter cells but there
could be lower and upper bounds on cell volume. Bacterial life is then a cyclic
process beginning with the newly separated daughter cell and commence again
when the next generation proceeds to grow as a free entity. It seems that there
is no end to bacterial life, given the proper environment.

No distinction can be made between a single cell as a thermodynamic

system and the sum of all cells in a culture as a thermodynamic system. The
system, considered as a single cell or as the sum of all cells in a culture, is an
open one in the thermodynamic sense. If a matter can pass across a boundary,
then the system is said open, otherwise it is a closed system. Energy, entropy (a

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thermodynamic property that measures a system thermal energy per unit of

temperature which is unavailable for doing useful work) and matter flow into the
system from the environment.

Chemical and physical transformations occur within the system,

accompanied by the net production of entropy. The environment of the system
includes the suspending aqueous solution of cell nutrients and substances
discarded by the cells. The gaseous atmosphere above the suspending solution,
the vessel that contains it and the remainder of the world outside the bounding
surface of the system are parts of the environment or surroundings.

Three laws of thermodynamics

1. The first law of thermodynamics is known as the law of conservation of

energy which states that energy can neither be created nor destroyed.

2. The second law says that the entropy of any isolated system always
increases.

3. The third law states that the entropy of a system approaches a constant
value as the temperature approaches absolute zero.

Fig. 1. The Laws of thermodynamics

The laws of thermodynamics are important in unifying principles that

govern the processes involved in the metabolism of all living organisms. All
biological organisms require energy to survive. This energy is not consumed but
is converted or transformed from one form to another (First law). Microorganisms
perform a number of important processes and these processes require energy.
For phototrophs, a radiant energy is supplied, absorbed by cells, is converted to

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chemical energy and stored in the form of glucose (or any form of metabolite) to
form complex compounds needed by the cell. In a biological process, the transfer
of energy is not always complete and not all of the light energy is absorbed by a
phototroph. Some energy is lost as heat and the loss of energy to the environment
results in an increase of disorder or entropy in the cell/organism surroundings
(Second law). (Entropy is referred to as the measure of disorder in a closed
system). The entropy of a system approaches a constant value (equilibration) as
the temperature approaches absolute zero (third law).

5 Major metabolic pathways of bacteria

The five major metabolic pathways in bacteria include glycolysis, pentose
phosphate pathway, Entner-Doudoroff pathway, tricarboxylic acid cycle and
glyoxylate cycle.

1. Glycolysis

Glycolysis (glyco-sugar of sweet, lysis-breakdown) is the initial phase of

metabolism during which the organic molecule glucose and other sugar are
partially oxidized to smaller molecules (e.g. pyruvate) usually with the
generation of some ATP and reduced co-enzymes. This is a mechanism by which
organisms obtain energy from organic compounds in the absence of molecular
oxygen. As the metabolic pathway takes place in the absence of oxygen it is also
called anaerobic fermentation. Some microbes that thrive in an environment that
lacks oxygen utilize anaerobic fermentation as the method to obtain energy from
glucose. Glycolysis serves as an emergency mechanism in anaerobic organisms
to produce energy in the absence of oxygen (anaerobic fermentation).

Glycolysis also occurs in aerobic organisms and this takes place as a

preparatory pathway for the complete aerobic degradation of glucose to pyruvic
acid. In case of aerobic bacteria such as E. coli and Bacillus spp., the breakdown
(catabolism) of carbohydrates like glucose (also called aerobic respiration)
involves the EMP pathway. It is through this Embden-Myerhof pathway (EMP)
that glycolysis is made possible for some aerobes, to differentiate it from the
process by which anaerobic organisms obtain energy from glucose.

EMP pathway in bacteria initiates by using the phospho-enol pyruvate

phosphotransferase system (PEP:PTS) that converts glucose to glucose 6-
phosphate during nutrient transport across the cell membrane. The glucose 6-
phosphate is isomerized to fructose 6-phosphate which is further converted to
fructose 1, 6-bi-phosphate. This conversion requires ATP as a source of energy
and an enzyme called phospho-fructokinase. It is particularly important during
growth on pyruvate-related C3 compounds and C2 units. The several classes of
carbon from pyruvate maintains a supply of hexoses which are required for the
synthesis of cell wall component.

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The complete pathway of glycolysis from glucose to pyruvate were

reportedly elucidated by Gustav Embden (who described the manner of cleavage
of fructose 1, 6-diphosphate and pattern of subsequent steps) and Otto Meyerhof
(who confirmed the work of Embden and studied the energetics of glycolysis) in
late 1953s. The sequence of reactions from glucose to pyruvate is also called
Embden-Meyerhof pathway (EMP) or glycolysis.

Glycolysis is carried out by the help of ten enzymes for ten reactions of
the glycolytic pathway. These enzymes are present in the soluble portion of the
cytoplasm. All the intermediates of the glycolytic pathway are phosphorylated
compounds. The most important use of phosphate groups is in the production
of ATP from ADP and phosphate. The involvement of phosphate groups has to be
mentioned in several reactions such as phosphorylation which refers to a
process by which a phosphate group is added to an organic compound while
the removal of phosphate group/s from an organic compound refers to
dephosphorylation.

The complete reactions of glycolytic pathway is visibly divided into two

stages; the energy investment phase and the energy yielding phase. In the first
stage, 2 molecules of ATP is utilized (not produced) and glucose is converted into
two molecules of three carbon compounds, glyceraldehyde 3-phosphate and
dihydroxy acetone phosphate. This stage does not produce NADH or ATP.

In the energy yielding phase is where ATP is produced. The glyceraldehyde

3-phosphate is converted into pyruvic acid resulting in the production of 4 ATP
molecules (a net synthesis of two molecules of ATP in the entire process since
two ATPs have been used in the energy investment phase). When this phase of
glycolysis proceeds, two molecules of NADH (reduced form of Nicotinamide
Adenine Dinucleotide) is produced. The three key regulatory enzymes,
hexokinase, phosphofructokinase and pyruvate kinase act irreversibly to
complete the process.

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Fig. 2. The glycolytic pathway that shows the synthesis of 2 ATP molecules and
pyruvic acid (with respective enzymes)

In a typical aerobic glycolysis, 2 molecules of ATP, 2 molecules of NADH,

and 2 molecules of pyruvic cid (3-Carbon molecule) are produced. Glycolysis
takes place in the cytosol immediately outside the mitochondria.

Aerobic Glycolysis – 2 Pyruvic Acid, 2 ATP, 2 NADH (2-PAN)

Anaerobic fermentation can be divided into two possible tracks. One

happens in the absence of oxygen where pyruvate is further converted to lactate
(lactic acid fermentation), while the other one converts pyruvate into alcohol
(alcoholic fermentation). Like aerobic glycolysis, anaerobic fermentation also
produces 2 molecules of ATP at the end of the reaction. In lactic acid
fermentation, along with 2 ATP molecules, 2 molecules of lactic acid are formed.
In alcoholic fermentation, 2 molecules of Carbon dioxide and 2 molecules of
ethanol are formed.

Lactic Acid Fermentation – 2 ATP, 2 Lactic Acid (2-AL)

Alcoholic Fermentation – 2 ATP, 2 Carbon Dioxide, 2 Ethanol (2-ACE)

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Lactic acid fermentation usually takes place in animals (milk and cheese
production). It is also evident in humans, when too much muscular strength is
exerted leading to lack of oxygen, thereby forming lactic acid which causes
muscle cramps. While alcoholic fermentation usually happens in plants and
fungi (in the production of beer and wine).

At the completion of aerobic glycolysis, there is a transitional phase before

the Krebs Cycle (also known as Tricarboxylic acid cycle or Citric Acid Cycle)
starts. This is called the grooming phase and it occurs when pyruvate is
transported to the matrix of the aerobic bacterial mitochondrion. The pyruvate
molecules are converted to acetyl CoA (a 2-carbon molecule) at the end of this
step and this is a preparatory step or a doorway into the tricarboxylic acid cycle
for the complete oxidation of glucose.

Grooming Phase – 2 Acetyl CoA, 2 Carbon Dioxide, 2 NADH (2-ACN)

It is clear that two molecules of carbon dioxide are released from the 2
molecules of pyruvate (3-Carbon molecule) as end-products since acetyl CoA is
only a 2-Carbon molecule. One carbon molecule (converted to CO2 after binding
with oxygen) is removed from each pyruvate molecule.

2 molecules of pyruvate
C C C C C C produced from the
previous reaction
Pyruvate Pyruvate

One carbon
C C C C C C separates from the
pyruvate molecule
Acetyl CoA Free carbon Acetyl CoA Free carbon

C The two carbon molecules

C C C C C become Acetyl CoA, the free
carbon molecules is then
Acetyl CoA for Becomes CO2 released and combines with
Acetyl CoA for Becomes CO2
Krebs Cycle Krebs Cycle oxygen to become CO2

Other types of sugar (polysaccharides, disaccharides, monosaccharides),

aside from glucose can also enter the glycolytic pathway as intermediate/s of
glycolysis.

1. Polysaccharides in the glycolytic pathway (in the form of glycogen)

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2. Disaccharides in the glycolytic pathway (Sucrose)

3. Mono-saccharides in the glycolytic pathway (Fructose). Fructose can enter the

glycolysis by changing to glyceraldehyde 3-phosphate. Dihydroxy-acetone
phosphate can enter the glycolysis after enzymatically converting to dihydroxy-
acetone phosphate.

Monosaccharides include Glucose, Fructose, and Galactose (GFG) while

disaccharides include Maltose (Glucose + Glucose), Lactose (Glucose + Galactose),
and Sucrose (Glucose + Fructose) (MaLaS)

Methyl Glyoxal pathway (an alternate EMP Pathway)

The methyl glyoxal metabolic pathway operates in the presence of low

concentration of phosphate in some bacteria like E. coli, Clostridium spp. and
Pseudomonas spp.. In this pathway, the dihydroxy-acetone formed is converted
to methyl glyoxal which later on gives rise to pyruvate. There is complete absence

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of the phosphorylation step which involves the formation of glyceraldehyde 3-

phosphate forms 1, 3-bis-phospho-glycerate. The methyl glyoxal pathway
consumes O2 and ATP, and no ATP is generated.

Fig. 3. The Methyl Glyoxal Pathway as an alternate EMP Pathway for glycolysis
(Pseudomonas spp.)

2. Pentose phosphate pathway (PPP) or Hexose monophosphate shunt

(HMP)

Pentose phosphate pathway is seen as an alternate of glucose degradation.

This pathway is also called hexose monophosphate shunt (HMP) or phospho-
gluconate pathway which has 3 functions.

Primarily, it generates a reducing power in the extra-mitochondrial

cytoplasm in the form of NADH (where CO2 is a by-product). Its secondary
function is to convert hexoses (sugar molecules with 6 carbons) into pentoses
(sugar molecules with 5 carbons) required in the synthesis of nucleic acids; and
its tertiary function involves the complete oxidative degradation of pentose. The
action of the phospho-gluconate pathway take place in the soluble portion of
extra-mitochondrial cytoplasm of cells.

Three (3) enzymes are involved in PPP or HMP and these include trans-
ketolase, trans-aldolase and ribulose-phosphate 3-epimerase. Trans-ketolase
catalyzes the transfer of glycol-aldehyde group (CH, OH-CO-) from xylulose 5-
phosphate to ribose 5-phosphate to yield sedo-heptulose 7-phosphate and
glyceraldehyde-3-phosphate. Trans-aldolase acts on the products of
transketolase and transfer dihydroxy-acetone group to form fructose 6-
phosphate and erythrose 4-phosphate. Ribulose phosphate 3-epimerase
catalyzes the conversion of ribulose 5-phosphate into the epimer xylulose 5-
phosphate.

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Fig. 4. The Pentose Phosphate Pathway

Pentose phosphate pathway may possibly function according to the needs

of the cell. If the requirement of reducing power is more, then it proceeds towards
the formation of NADPH (CO2 is a by-product) but if pentoses are required it
functions in the direction of pentose formation. But if the cell requires instant
energy the PPP stops, then glycolysis and TCA pathway may proceed. The
bacteria which show PPP are Bacillus subtilis, E. coli and Streptococcus faecalis.
Aside from microorganisms, physiological processes that take place in the
tissues of the liver, mammary gland and adrenal cortex utilize the pentose
phosphate as a metabolic pathway.

3. Entner-Doudoroff pathway (ED)

The Entner-Doudoroff pathway is another track for the oxidation of

glucose to pyruvic acid. In this pathway, each molecule of glucose forms two
molecules of NADPH and one molecule of ATP. Glucose 6-phosphate is oxidized
to 6-phosphogluconate, then converted to 2- keto-3-deoxy-6-phosphoglucose
(kDPG) cleaved using enzyme to give rise to glyceraldehyde 3- phosphate and
pyruvate directly without generation of ATP. The catabolism (breakdown) of
glucose results in production of only one ATP molecule (in comparison to the
EMP pathway where 2 ATP molecules are produced).

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Fig. 5. The Entner-Doudoroff pathway

In the ED pathway, a reduced NADPH is generated from NADP (where H2O

is a by-product to differentiate from the PP pathway). The ED pathway provides
an important mechanism for producing NADPH and the 3-carbon building
blocks used in biosynthetic reactions.

A non-phosphorylated ED pathway is seen in some bacteria such as

Clostridium spp. and Achromobacter spp.. In this case, intermediate product
formed prior to KDPG is non-phosphorylated and phospho-gluconate is
dehydrated to give rise to KDPG which gives to pyruvic acid (3-Carbon molecule).

A phosphorylated ED pathway is also possible and this involves conversion

of glyceraldehyde 3-phosphate form to pyruvic acid resulting in a net synthesis
of two molecules of ATP (similar to EMP/glycolysis). The phosphorylated ED
pathway is followed by Alcaligenes spp..

4. Tricarboxylic acid cycle (TCA)

The tricarboxylic acid cycle (also known as Krebs Cycle or Citric acid
cycle) occurs in all the aerobic organisms and leads to complete oxidation of
glucose to CO2 and H2O in comparison to the incomplete oxidation of glucose to
pyruvate in the glycolysis pathway and of pyruvate to Acetyl CoA in the grooming
phase. All the reactions of tricarboxylic acid cycle take place in the inner
compartment of mitochondrion. Some of these enzymes occur in the matrix of
inner compartment while other enzymes occur on the inner mitochondrial
membrane. For the start of the cycle, the pyruvate formed in the glycolysis is
first converted to acetyl Co-A by a preparatory reaction.

Pyruvate + NAD+ + CoA → acetyl CoA + NADH + H+ + CO2

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Tri-carboxylic acid cycle completely oxidizes glucose to release large

amount of energy in the form of NADH + H+ mainly and GTP. NADH + H+ enter
the respiratory chain where each NADH + H+ produces 3 ATP molecules. GTP
is then converted to ATP by substrate level oxidation.

Another form of energy is in the form of substrate of FADH2 (Reduced flavin

adenine dinucleotide) which enters the respiratory chain to form 2 molecules of
ATP. The reaction is irreversible and is not a part of the tricarboxylic acid cycle.
It is carried out with the help of the enzyme pyruvate dehydrogenase. Acetyl CoA
(2-Carbon molecule) then enters the cycle after combining with oxalo-acetate (4-
Carbon molecule) to form citrate (6-Carbon molecule) after which a cycle of
reactions occurs leading to the formation of six molecules CO2, eight molecules
NADH + H+, one FADH2 and one molecule of glucose.

Krebs Cycle – 1 Glucose, 1 FADH2, 6 CO2, 8 NADH + H+ (1168-GFCN)

There are few key steps in the tricarboxylic acid cycle which control the
cycle depending upon the need of the cell. The first of these controls is the
preparatory reaction. The activity of pyruvate dehydrogenase is reduced in the
presence of excess ATP and again increases in the absence of ATP.

The cycle is primarily controlled by the reaction carried out by citrate

synthase. There are other steps which control the cycle and these include the
isocitrate dehydrogenase reaction (which requires ADP as positive regulation)
and succinate dehydrogenase reaction (promoted by succinate, phosphate and
ATP).

Fig. 6. The Krebs Cycle

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The tricarboxylic acid cycle was first given by HA Krebs in 1937 then
eventually gave the name citric acid cycle. Citric acid is the first product of the
Krebs cycle and the reason it is also referred to as TCA cycle, due to the presence
of 3 carboxylic groups in a molecule of citric acid.

5. Glyoxylate cycle or anaplerotic pathway

This is a reaction in which oxalo-acetate is taken from the TCA cycle to

meet the demand of bacteria for carbon needed in amino acid biosynthesis. These
intermediates have to be replenished via an alternate route called anaplerotic or
glyoxylate pathway which reportedly operates for gluconeogenesis.

In this cycle, the CO2 evolving steps of tricarboxylic acid cycle are by-
passed and instead a second molecule of acetyl CoA is utilized (which condenses
with glyoxylate to form malate). Succinate is a by-product used for biosynthesis
particularly in gluconeogenesis.

2 Acetyl Co-A + NAD+ + 2H2O → Succinate + 2CoA + NADH + H+

The two key enzymes, isocitrate lyase and malate synthase of glyoxylate
cycle are localized in cytoplasmic organelles called glyoxysomes. Glyoxylate cycle
goes on simultaneously with the tricarboxylic acid cycle while tricarboxylic acid
provides energy, glyoxylate cycle provides succinate for the formation of new
carbohydrate from fats.

Fig. 7. The glyoxylate cycle

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This cycle is a modified form of tricarboxylic acid cycle found in plants and
those microorganisms which utilize fatty acids as the source of energy in the
form of acetyl CoA. Glyoxylate cycle was first defined by Krebs and HR Kornberg.

Metabolic classification of bacteria

1. Autotrophic metabolism

All autotrophs use CO2 as a carbon source for growth while their nitrogen
comes from inorganic compounds such as NH3, NO3 or N2. The energy source for
these organisms is the oxidation of specific inorganic compounds. Among the
autotrophic microorganisms are the sulfur-oxidizing or sulfur-compound-
oxidizing bacteria and the obligate nitrifying bacteria. The representative sulfur
compounds oxidized by such bacteria are H2S, S2 and S2O3. All autotrophic
bacteria assimilate CO2 which is reduced to glucose from which organic cellular
matter is synthesized. The energy for this biosynthetic process is derived from
the oxidation of inorganic compounds.

The most common pathways for synthesizing organic compounds from

carbon dioxide are the reductive pentose phosphate cycle,
the reductive tricarboxylic acid cycle and the acetyl-CoA pathway which takes
place in most aerobic autotrophic bacteria and photosynthetic bacteria. This
cycle involves the synthesis of one molecule of glyceraldehyde-3-phosphate
which requires consumption of nine molecules of ATP and the oxidation of six
molecules of the electron donor and the reduced form of nicotinamide adenine
dinucleotide phosphate (NADPH). Metabolic energy is made available from the
oxidation of these electron donors which transfer electrons from an organic
molecule to oxygen. As electrons are passed along the electron transport chain
to oxygen, a proton gradient is generated across the cell membrane and this
gradients is used to generate molecules of ATP.

2. Phototrophic metabolism

This mechanism requires the action of two separate light-absorbing

systems to raise the energy of the electrons from water to a level high enough for
their transfer to NADP. Two distinct photo-reaction centers are present in these
organisms, one for the oxygen-generating reaction and the other for the cyclic
process for energy generation. The general process of phototrophic metabolism
makes use of pigments (like chlorophyll) that absorbs light energy and releases
an electron with a higher energy level. This electron is passed through an
electron transport chain, with the generation of energy from a proton gradient
and concomitant ATP synthesis. For the cell to grow, the reductive pentose
phosphate cycle of carbon dioxide fixation is activated and electrons are
transferred to the co-factor NADP to form NADPH which is needed in large

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amounts for the cycle to keep going. Phototrophic cell growth requires a source
of electrons that replaces the electrons that are consumed during biosynthetic
reactions.

3. Heterotrophic metabolism

Heterotrophic bacteria obtain energy from oxidation of organic

compounds. Carbohydrates (particularly glucose), lipids and protein are the
most commonly oxidized compounds. Biologic oxidation of these organic
compounds by bacteria results in synthesis of ATP as the chemical energy
source. This process also permits generation of simpler organic compounds
(precursor molecules) needed by the bacteria cell for biosynthetic or assimilatory
reactions.

The Krebs cycle intermediate compounds serve as precursor molecules

(building blocks) for the energy-requiring biosynthesis of complex organic
compounds in bacteria. These carbon- and nitrogen-containing compounds are
growth substrates which are used aerobically or anaerobically to generate
reducing equivalents (reduced nicotinamide adenine dinucleotide/NADH + H+).

Glucose is the most common substrate used for studying heterotrophic

metabolism. Most aerobic organisms oxidize glucose completely by the following
reaction equation:

This equation expresses the cellular oxidation process called respiration.

Respiration occurs within the cells of plants and animals, normally generating
38 ATP molecules (as energy) from the oxidation of 1 molecule of glucose.
Respiration takes place when any organic compound (usually carbohydrate) is
oxidized completely to CO2 and H2O. In aerobic respiration, molecular O2 serves
as the final acceptor of electrons.

For anaerobic respiration (fermentation), NO3–, SO4–, CO2 or fumarate can

serve as final electron (or hydrogen) acceptors (rather than O2). The end result of
the respiratory process is the complete oxidation of the organic substrate
molecule and the end products formed are primarily CO2 and H2O. Ammonia is
formed if protein (or amino acid) is the substrate oxidized.

A chemical reaction that involves the

coupled oxidation and reduction of amino acids to organic acids is called
Stickland fermentation or Stickland reaction. This is the manner by which
Clostridium sporogenes and Clostridium botulinum derive energy from amino
acids. The electron donor amino acid is oxidized to a volatile carboxylic

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acid which is one-carbon atom shorter than the original amino acid. For
example, alanine (amino acid) with a 3-carbon chain is converted
to acetate (organic acid) with two carbons. The electron acceptor amino acid is
reduced to a volatile carboxylic acid with the same length as the original amino
acid. For example, glycine with two carbons is converted to acetate. In this way,
amino acid fermenting microbes can avoid using hydrogen ions as electron
acceptors to produce hydrogen gas. Amino acids can be Stickland acceptors,
Stickland donors or act as both donor and acceptor.

In bacteria, glycolysis represents one of several pathways by which

bacteria can catabolically attack glucose. The glycolytic pathway is commonly
associated with anaerobic or fermentative metabolism in bacteria and yeasts.
Bacteria differ from cyanobacteria (blue-green algae) and eukaryotes where
glucose oxidation may occur by more than one pathway. Other minor hetero-
fermentative pathways such as the phospho-ketolase pathway also exist in
bacteria. In addition, two other glucose-catabolizing pathways are found in
bacteria and these are the oxidative pentose phosphate pathway (hexose
monophosphate shunt) and the Entner-Doudoroff pathway which is almost
exclusively found in aerobic bacteria.

Glucose oxidation is the most commonly studied dissimilatory reaction

leading to energy production or ATP synthesis. The complete oxidation of glucose
may involve three fundamental biochemical pathways. The first is the glycolytic
or Embden- Meyerhof-Parnas pathway, the second is the Krebs cycle (also called
the citric acid cycle or tricarboxylic acid cycle) and third is the series of
membrane-bound electron transport oxidations coupled to oxidative
phosphorylation.

Glucose catabolism also occurs by the hexose monophosphate shunt. This

oxidative pathway was discovered in tissues that actively metabolize glucose in
the presence of two glycolytic pathway inhibitors (iodoacetate and fluoride).
Neither inhibitor had an effect on glucose dissimilation and NADPH +
H+ generation occurred directly from the oxidation of glucose-6-phosphate (to 6-
phosphoglucono-δ-lactone) by glucose-6phosphate dehydrogenase. The pentose
phosphate pathway subsequently permits the direct oxidative decarboxylation of
glucose to pentoses. The capability of this oxidative metabolic system to bypass
glycolysis explains the term shunt.

Electron transport

A series of oxidation-reduction electron transfer reactions provides the

energy to initiate oxidative phosphorylation that produces ATP at the final stage
of respiration. The enzymes involved in electron transport and oxidative
phosphorylation reside on the bacterial inner (cytoplasmic) membrane which is
equipped with invaginations that form structures called respiratory vesicles,

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lamellar vesicles or mesosomes which function as the bacterial equivalent of the

eukaryotic mitochondrial membrane.

Respiratory electron transport chains vary greatly among bacteria while

these are absent in some organisms. The bacterial mitochondrion electron
transport chain oxidizes the compounds NADH + H+, NADPH + H+ and succinate
(as well as the co-acylated fatty acids such as acetyl~CoA) while it can directly
oxidize a larger variety of reduced substrates such as lactate, malate, formate,
α-glycerophosphate, H2 and glutamate via non-pyridine nucleotide-dependent
pathways.

The electron transfer reaction is the primary mode of generating energy

where electrons (2e-) from a low-redox-potential compound such as NADH +
H+ are sequentially transferred to a specific flavoprotein dehydrogenase or oxido-
reductase (flavin mononucleotide [FMN] type for NADH or flavin adenine
dinucleotide [FAD] type for succinate). This electron pair is transferred to a non-
heme iron center (FeS) and finally to a specific ubiquinone or a naphtho-quinone
derivative. The transfer of electrons causes a differential chemical redox potential
change so that within the membrane enough chemical energy is conserved to be
transferred by a coupling mechanism to a high-energy compound (e.g. ADP + Pi
→ ATP). ATP molecules represent the final stable high-energy intermediate
compound formed.

The respiratory electron carriers in bacterial electron transport systems

are more varied (compared to eukaryotes) and the chain is usually branched at
the site(s) reacting with molecular O2. Some electron carriers such as non-heme
iron centers and ubiquinone (co-enzyme Q) are common to both the bacterial
and mammalian respiratory electron transport chains. In some bacteria, the
naphthoquinones or vitamin K may be found with ubiquinone. In other bacteria,
vitamin K serves in the absence of ubiquinone.

In mitochondrial respiration, only one oxygen-reactive cytochrome

(cytochrome oxidase) component is found (cytochrome a + a3 oxidase). In
bacteria there are multiple cytochrome oxidases, including cytochromes a, d, o,
and occasionally a + a3. In bacteria cytochrome oxidases usually occur as
combinations of a1: d: o and a + a3:o. Bacteria also possess mixed-function
oxidases such as cytochromes P-450 and P-420 and cytochromes c' and c'c'
which also react with carbon monoxide. There are diverse types of oxygen-
reactive cytochromes and when O2 became available as a metabolite, bacteria
advanced to use it in different ways which accounts for the diversity in bacterial
oxygen-reactive hemoproteins. Cytochrome oxidases in many pathogenic
bacteria are studied by the bacterial oxidase reaction (subdivides Gram-negative
organisms into two major groups, oxidase-positive and oxidase-negative. The
oxidase reaction is assayed by using N,N,N', N'-tetramethyl-p-phenylenediamine
oxidation (Wurster's blue) or by using indophenol blue synthesis (with dimethyl-
p-phenylenediamine and α-naphthol). Oxidase-positive bacteria contain

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integrated (cytochrome c type:oxidase) complexes where the oxidase component

frequently encountered is cytochrome o and occasionally a + a3.

Bacterial electron transfer systems carry out electron transfer (oxidation)

reactions with NADH + H+, NADPH + H+ and succinate. Energy generated from
such membrane oxidations is conserved within the membrane and then
transferred in a coupled manner to drive the formation of ATP. The electron
transfer sequence is accomplished entirely by membrane-bound enzyme
systems. As the electrons are transferred by a specific sequence of electron
carriers, ATP is synthesized from ADP + inorganic phosphate (Pi) or ortho-
phosphoric acid (H3PO4).

Redox reactions in bacteria

Many biological processes in bacteria involve redox reactions. Cellular

respiration involves the oxidation of glucose (C6H12O6) to CO2 and the reduction
of oxygen to water and is summarized as:

C6H12O6 + 6 O2 → 6 CO2 + 6 H2O

The process of cell respiration depends heavily on the reduction of NAD+ to

NADH and the reverse reaction (the oxidation of NADH to
NAD+). Photosynthesis and cellular respiration are complementary, but
photosynthesis is not the reverse of the redox reaction in cell respiration:

6 CO2 + 6 H2O + light energy → C6H12O6 + 6 O2

Biological energy is frequently stored and released by means of redox

reactions. Photosynthesis involves the reduction of carbon
dioxide into sugars and the oxidation of water into molecular oxygen. In the
reverse reaction, respiration oxidizes sugars to produce carbon dioxide and
water. As intermediate steps, the reduced carbon compounds are used to
reduce nicotinamide adenine dinucleotide (NAD+) to NADH which contributes to
the creation of a proton gradient that drives the synthesis of adenosine
triphosphate (ATP) and is maintained by the reduction of oxygen.

Free radical reactions are redox reactions that occur as a part

of homeostasis and killing microorganisms, where an electron detaches from a
molecule and then reattaches almost instantaneously. Free radicals are part of
redox molecules and can become harmful to the human body if these do not
reattach to the redox molecule or an antioxidant.

The term redox state is often used to describe the balance of Glucose/
Sugars/Hydrogen/GSSG, NAD+/NADH and NADP+/NADPH in a biological
system such as a cell or organ. The redox state is reflected in the balance of
several sets of metabolites (e.g. lactate and pyruvate, beta-hydroxybutyrate

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and aceto-acetate) wherein interconversion is dependent on these ratios. An

abnormal redox state can develop in a variety of deleterious situations such
as hypoxia, shock and sepsis. Redox mechanisms also control some cellular
processes. Redox proteins and their genes are co-located for redox regulation
(based on CoRedox regulation hypothesis) in mitochondria and chloroplasts.

Bacterial adaptation to the nutritional and physical environment

Bacteria can react to changes in their environment through changes in the

patterns of structural proteins, transport proteins, toxins and enzymes which
adapt them to a particular ecological situation. An example is E. coli which does
not produce fimbriae for attachment when living in a planktonic (free-floating)
environment. Vibrio cholerae does not produce its cholera toxin that associates
with diarrhea unless it is confined in the human intestinal tract. Bacillus
subtilis does not make the enzymes for tryptophan biosynthesis if it cannot find
an existing tryptophan in its environment. If E. coli is fed with glucose and
lactose together, glucose is used first because it takes two less enzymes to use
glucose than what it does to use lactose. The bacterium Neisseria
gonorrhoeae develops a sophisticated process for iron gathering and a transport
system as it senses that iron is in short supply in its environment.

Complex mechanisms that regulate the control of catabolic and anabolic

pathways develop in bacteria. Bacteria do not synthesize catabolic enzymes
unless the substrates for these enzymes are present in their environment. For
instance, synthesis of enzymes that degrade lactose (lactase) is wasteful unless
the substrate for these enzymes (lactose) is available in the environment.
Bacterial cells shut down biosynthetic pathways when the end product of the
pathway is not needed or is readily obtained by uptake from the environment.
For instance, a bacterium finds a preformed amino acid like tryptophan in its
environment, it shuts down its own pathway of tryptophan biosynthesis and
conserve energy. The control mechanisms for all these metabolic pathways are
reversible as the environment can change quickly and drastically.

Conditions affecting enzyme formation in bacteria

Bacterial cells can change patterns of enzymes in order for them to adapt
to their specific environment. The concentration of an enzyme in a bacterial cell
often depends on the presence of the substrate for the enzyme. Enzymes for
metabolic functions come in 2 different forms and these are the 1) constitutive
and the 2) inducible or adaptive.

Constitutive enzymes are produced by cells independent of the

composition of the medium in which the cells are grown. The enzymes that
operate during glycolysis and the TCA cycle are recognized as constitutive in

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nature and these are present at more or less the same concentration in cells at
all times.

Inducible enzymes are produced (turned on) by bacterial cells in response

to a particular substrate which are produced only when needed. In the process
of induction, the substrate or a compound structurally similar to the substrate
evokes formation of the enzyme (called an inducer).

A repressible enzyme is one in which its synthesis is downregulated or

turned off by the presence of the end-product of a pathway that the enzyme
normally participates in.

Regulation of enzyme in metabolic processes

Bacteria exert control over metabolism at every possible stage at the level
of the gene that encodes for a protein and ending with alteration or modifications
in the protein after it is produced. For example, variation in gene structure can
change the activity or production of a protein, in the same way that a protein
can be modified after it is produced to alter or change its activity.

In bacterial cells, enzymatic reactions may be regulated by two unrelated

modes:

(1) control of enzyme activity (feedback inhibition or end-product

inhibition) that regulates the activity of pre-existing enzymes in the
cells

(2) control of enzyme synthesis which involves repression of end-product

in biosynthetic pathways, enzyme induction and catabolite repression
which normalize degradative pathways.

The processes which regulate the synthesis of enzymes may either be a

form of positive control or negative control. End-product repression and enzyme
induction are mechanisms of negative control because they lead to a decrease
the rate of protein transcription. Catabolite repression is considered a form
of positive control because it affects an increase in rates of transcription of
proteins.

Respiratory enzymes

Dehydrogenases are enzymes which can oxidize specific metabolites and

can be re-oxidized by other enzymes. Respiratory enzymes consist of an
apoenzyme and a co-enzyme.

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Types of dehydrogenases

1. Pyridinoproteins are enzymes which contain TPN (tri-phospho-pyridine) and

DPN (di-phospho-pyridine) as co-enzymes.
Ex: Malic acid, Lactic acid, Tri-phospho-glyceraldehyde

2. Cytochrome-linked dehydrogenases mediate transfer of hydrogen and their

substances to the cytochrome system.

3. Flavoproteins are respiratory enzymes that contain riboflavin phosphate or

flavin adenine dinucleotide as co-enzymes. Oxidation and reduction
processes are confined mainly to the flavin portion of the molecule.

4. Iron-porphyrin protein enzymes take up iron-porphyrin compounds which

resemble the heme portion of hemoglobin as co-enzyme.

Examples of iron-porphyrin protein enzymes

a. Cytochromes a, b and c - present only in aerobes and take part in

electron transport by accepting electrons from cytochrome reductase
and pass them on the cytochrome oxidase

b. Cytochrome oxidase (Warburg’s respiratory enzyme) - reduces

molecular oxygen to hydrogen peroxide

c. Catalase - decomposes hydrogen peroxide to water and oxygen.

(Catalase is present in all aerobes and some facultative anaerobes. It is
absent in species of Clostridium, Fusobacterium and Streptococcus).

d. Peroxidase catalyzes oxidation of substrates by hydrogen peroxide.

*****

Evaluation

1. Differentiate groups of bacteria based on their energy sources.

2. Discuss and differentiate the 5 metabolic pathways in bacteria.

3. Describe redox reactions in relation to metabolic processes in bacteria.

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References

http://textbookofbacteriology.net/regulation.html#.

Jurtshuk P. Bacterial metabolism. Medical Microbiology. 4th edition.

https://www.ncbi.nlm.nih.gov/books/NBK7919/.

https://www.biologydiscussion.com/metabolism/microbial-metabolism/5-
major-metabolic-pathways-in-organisms-microbiology/65509.

https://en.wikipedia.org/wiki/Stickland_fermentation#.

https://www.britannica.com/science/bacteria/Autotrophic-metabolism

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