Bacterial Metabolism &
Growth Characteristics
Stijn van der Veen
Differentiating bacterial species
Morphology (shape)
Composition (cell envelope and other structures)
Metabolism & growth characteristics
Genetics
Differentiating bacterial species
Morphology (shape)
Composition (cell envelope and other structures)
Metabolism & growth characteristics
Genetics
Bacterial metabolism
The bacterial metabolism is the combination of
all (bio)chemical reactions that occur within
bacterial cells that allows them to live,
replicate, and maintain cellular integrity.
Bacteria can be differentiated by the unique
combination of different (bio)chemical reactions
they are able o perform. These can be
identified by specific substrates they are able to
convert or metabolites they are able to
produce.
Metabolic pathways
The metabolism is structured into metabolic pathways
that consist of series of consecutive (bio)chemical
reactions that are connected through their start and
end products.
Biochemical reactions in the metabolic pathways are
either spontaneous reactions or reactions driven by
enzymes (proteins).
Metabolic pathways are regulated by enzymes that
determine the direction and speed of the biochemical
reactions.
Metabolic maps
Metabolic maps
reflect the
metabolic
pathways and
display how all
the metabolites
(dots) and
(bio)chemical
reactions (lines)
are connected.
Metabolic flux
Metabolic flux is the
turnover of metabolites
through metabolic
pathways.
Metabolic flux is regulated
by the enzymes that
perform the biochemical
reactions
Catabolism & Anabolism
The metabolism is generally divided into two
major groups:
Catabolism:
Biochemical reactions that
convert larger molecules
into smaller molecules,
thereby generating energy.
Anabolism:
Biochemical reactions that
consume energy to construct
larger cellular components
such as proteins, lipids, and DNA.
Catabolism & ATP
Catabolic degradation of larger molecules results in the generation of energy in the form of heat (which is lost) and adenosine triphosphate (ATP).
ATP is the most important storage molecule for chemical energy.
ATP provide the energy for most of the energy consuming metabolic processes.
Generation of ATP
Reduction-oxidation (redox) reactions
Aerobic respiration: Complete conversion of carbohydrates
into water, carbon dioxide and ATP, using oxygen as the
final electron acceptor in the electron transport chain (ETC).
Fermentation: Anaerobic conversion of carbohydrates into
acids, gases, and/or alcohols, and ATP.
Anaerobic respiration: Similar to aerobic respiration but
instead of oxygen, sulfate or nitrate are used as final
electron acceptors.
Sunlight
Photosynthesis: Use of light energy to energize electron
donors (photophosphorylation), which results in the
spontaneous movement of electrons through the ETC.
Oxygen tolerance
Bacteria can be classified according to their oxygen tolerance: Obligate aerobes
Require oxygen to stay alive
Aerobic respiration
Obligate anaerobes
Die in the presence of oxygen
Fermentation or anaerobic respiration
Facultative anaerobes
Survive with and without oxygen
Combination of aerobic respiration and fermentation
Microaerophiles
Require low levels of oxygen
Combination of growth modes
Aerotolerant anaerobes
Survive with and without oxygen
Fermentation
Aerobic respiration
Conversion of carbohydrates such as glucose into water, carbon dioxide and ATP is a 4-step process:
Glycolysis
Pyruvate decarboxylation
Krebs (TCA or citric acid) cycle
Oxidative phosphorylation
(in the ETC)
C6H12O6 + 6 O2 + 38 ADP + 38 Pi
6 CO2 + 6 H2O + 38 ATP
Oxidase test
Used to determine the presence of cytochrome c oxidase.
Cytochrome c oxidase is part of the ETC and uses oxygen as terminal electron acceptor.
The oxidase test uses reagents such as N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) that turn blue when oxidized and stay colorless when reduced.
Detoxification of oxygen radicals
During aerobic respiration reactive oxygen species (ROS) are generated as side products.
ROS such as superoxide anions(O2-), hydrogen
peroxide (H2O2), and hydroxyl radicals (OH•) are very reactive and damaging to cellular structures such as DNA, proteins, and lipids.
Aerobic respiring bacteria contain the detoxifying enzymes superoxide dismutase (SOD), catalase (Kat), and peroxidase.
Catalase test
The catalase test is used to identify bacteria that contain the catalase enzyme.
Hydrogen peroxide is added to a small amount of bacteria and observed for bubble formation (oxygen).
Fermentation
Conversion of carbohydrates such as glucose into acids, ethanol,
and ATP.
To maintain the redox balance, after glycolysis pyruvate is
converted into waste products (acids, ethanol, etc.).
There are many different types of fermentation processes and end products, but the most common types are homolacticfermentation (lactate as end product), heterolactic fermentation (mix of lactate and other acids), and alcohol fermentation.
Carbohydrate conversion and acidification
Carbohydrate conversion Most bacteria are able to convert glucose into energy.
The ability to utilize specific more complex carbohydrates as energy source is variable between bacteria, and this ability is useful for identification.
Acidification Homolactic and heterolactic fermentation results in the
production of acids.
Acidification of media can be detected with pH indicators.
Carbohydrate fermentation tests
Carbohydrate fermentation tests with pH indicator can show production of acids (color shift).
Carbohydrate fermentation tests with durham tube can also show production of gases (collected in durham tube).
Citrate conversion
Simmon’s citrate medium is
used to test whether bacteria
can utilize citrate as the sole
carbohydrate.
Citrate conversion results in
alkalization of the medium
which is indicated with
bromothymol blue.
Catabolism of proteins and amino acids
Proteins are degraded by
proteases into peptides
and peptides are further
degraded into amino acids.
Amino acids are converted
in various pathways to feed
into the TCA (Krebs) cycle
and further converted into
ATP.
Tryptophan conversion (indole test)
Some bacteria are able to convert the
amino acid tryptophan using the
tryptophanase enzyme.
Cleavage of tryptophan results in the
production of indole.
Indole reacts with para-dimethylamino
benzaldehyde from Kovacs reagent
and produces a red-violet color.
Cysteine / methionine conversion
Some bacteria are able to convert
the sulfide containing amino acids
methionine and cysteine.
Cleavage of these amino acids
results in the production of hydrogen
sulfide.
The hydrogen sulfide combines with
ferrous sulfide (Fe2S) in the triple
sugar iron (TSI) agar to form a black
to dark insoluble precipitate.
Urea conversion
Urea agar slants are used to test
whether bacteria can convert urea
into ammonia and carbon dioxide.
Production of ammonia results in
alkalization of the medium which
is indicated with phenol red.
Urease activity is important for
bacteria that pass through the
gastro-intestinal tract and need to
survive the acid environment
Anabolism
The anabolism is the general term for the biochemical reactions leading to the synthesis of cell structures.
Anabolism can be divided into four steps:
Collection and transport of elements and growth factors
Synthesis of monomers
Synthesis of polymers
Structural assembly of polymers
Elements and growth factors
The most abundant elements that make up 95 % of the dry weight of a bacterial cell are Carbon, Oxygen, Hydrogen, and Nitrogen.
The other 5% consists of Phosphorus, Calcium, Sodium, Potassium, Iron, Copper, Magnesium, and Manganese.
Other required elements are trace elements.
Growth factors are molecules that are required for growth but bacteria are unable synthesize themselves. These are variable and depend on the specific abilities of each species, but may include some vitamins, amino acids, or nucleic acid precursors.
Bacterial structures
The bacterial anabolism combines the elements into
many different metabolites with the majority finally
forming large bacterial structures.
Proteins: Forming 50-80% of the dry weight.
Sugars: Mainly in the cell wall and capsule.
Lipids: Mainly in the cell membrane and outer membrane.
Nucleic acids: Mainly DNA and RNA.
Bacterial growth
Bacterial growth is the asexual replication or division of a bacterium into two daughter cells in a process called binary fission.
Generation time
Generation time (doubling time) is the average time required for a population of bacteria to double in number.
The doubling time for bacteria is variable ranging from 10 min to 30 h or more and also depends on the growth conditions.
Organism Generation Time
Clostridium perfringens 10-15 min
Escherichia coli 20-25
Bacillus cereus 25-30 min
Staphylococcus aureus 25-30 min
Mycobacterium tuberculosis 18 – 24 hrs
Treponema pallidum 30 hrs
Generation Cell
Number Count
0 1
1 2
2 4
3 8
4 16
5 32
10 1,024
20 1,048,576
30 1,073,741,824
So, in 15 hrs a single cell can
turn into a billion cells!!!
Exponential or logarithmic growth
Bacterial growth factors / conditions
Nutrient availability
Elements and growth factors.
Oxygen pressure
Growth mode (respiration, fermentation, etc.)
Temperature
Important for speed of enzymatic reactions and stability of
bacterial structures.
Acidity / alkalinity (pH)
Impact on proton motive force, stability of bacterial
structures, etc.
Water activity
Determines osmotic pressure
Temperature
Bacteria are divided into four classes for their ability to grow at specific temperature ranges.
This ability is particularly determined by their protein (enzyme) and cell membrane stability at these temperatures.
All bacterial pathogens are mesophiles.
Thermophiles
Acidity / alkalinity
Bacteria are divided in three groups for their ability to grow at different pH’s.
Most bacteria and bacterial pathogens are neutrophilesand have optimum growth around pH 6.5-7.5, which is the pH of most human organs and tissues.
Most important acidophile is Helicobacter pylori, which thrives in the human stomach.
Water activity
Water is important component of bacterial cells and is involved in many metabolic reactions.
Most bacteria die in the absence of water (desiccation).
Water activity is determined by the presence of salts and solutes.
Water activity determines osmotic pressure.
Halophiles (not bacterial pathogens) require high salt concentrations.
Measuring bacterial numbers
Turbidity of liquid cultures
Quantify total bacteria (live and
dead) by absorption at 600 nm
using a spectrophotometer.
Colony counting on agar plates
Count colony numbers after plating
a known volume of liquid (or serial
dilutions). Each colony is derived
from a single live bacterial cell.
Growth in liquid cultures
Growth in liquid media can be measured by turbidity or colony counting on agar plates.
Plotting the logarithmic values of turbidity or bacterial cell numbers against time results in a plot called a growth curve.
Growth curves are generally characterized by four phases: lag phase, log or exponential growth phase, stationary phase, and death phase.
Lag Phase
Bacteria are becoming "acclimatized" to the new environmentalconditions (pH, temperature, nutrients, etc.).
Enzymes and intermediates are formed and accumulate untilthey are present in concentrations that are permit growth.
Log or exponential growth phase
Bacteria have adapted to the environmental conditions and start the replicate.
The bacterial population is growing rapidly at an exponential rate.
This is the most homogeneous state of the bacteria and generally bacteria from this phase are used for most of the biochemical tests, including antibiotic sensitivity tests.
Stationary phase
When nutrients are becoming limited and metabolic waste products accumulate, growth rates decline until the point that growth rate equals death rate.
In this phase there is no increase in the population of live bacteria.
Generally, in this phase bacteria produce endospores,toxins, and antibiotics.
Death phase
The population of live bacteria decreases due to the lack of
nutrients and accumulation of toxic metabolic waste products.
Some bacteria autolyse in this phase, which might also result in
decreased turbidity.
Static liquid growth
Generally, liquid cultures are grown under shaking conditions, allowing uniform turbidity.
Static growth of liquid cultures can result in different patterns:
Uniform turbidity
Facultative or aerotolerant anaerobes
Ring or pellicle at the air-liquid interface
Aerobes
Sediment at the bottom
Anaerobes
Growth on solid media
Used to obtain a large number of bacteria, isolate
identical clones of bacteria (colony), and to perform
drug sensitivity test.
A colony is a cluster of bacterial cells that propagated
(multiplied) from a single cell.
Colony can be used to determine the original bacterial
numbers by counting colonies and to evaluate viability
of bacteria (colony forming units, CFU).
Differences in colony morphology
Procedure:
1. Flame the loop and streak a loop containing
bacteria as at A in the diagram.
2. Reflame the loop and cool it.
3. Streak as at B to spread the original inoculum
over more of the agar.
4. Reflame the loop and cool it.
5. Streak as at C.
6. Reflame the loop and cool it.
7. Streak as at D.
8. Incubate the plate inverted.
Streaking bacteria
By spreading bacteria over the surface of a plate, the amount of bacteria is diluted and individual cells are able to form a single pure colony.
Growth on semisolid media
Used to test the motility of bacteria (flagellum or pili).
+-
Bacterial cultivation media
Basic nutrient media
Supplies all the nutritional requirements for growth of most
of the common bacteria.
Minimal media
Supplies the minimal nutritional requirements for growth of
specific bacteria.
Enrichment media
Supplies additional nutrients for the growth of fastidious
bacteria that do not grow on the basic nutrient media.
Bacterial cultivation media
Selective media
Supports the growth of desired bacteria while inhibiting the
growth of many or most of the unwanted ones. These
media contain selective agents that inhibit growth of
unwanted bacteria, while allowing growth of desired
bacteria (e.g. antibiotics, bile salts, etc.). Or alternatively,
specific nutrients are included or omitted to allow selection.
Differential medium
Supports the growth of two or more bacterial species, but
differentiates between them due to the addition of specific
components that react differently with these species (e.g.
pH indicators, blood, etc.).
Blood agar
These are the red plates that most of your cultures will be grown on.
The media is made of a basic nutrient agarcomposed mostly of a mixture of amino acids and peptides, combined with defibrinated blood.
When the bacteria produce a membrane toxin, this can lyse the red blood cells (haemolysis) and the media can change colourand become clearer around bacteria producing such toxins.
This is the most commonly used media because it is so nutrient rich, many bacteria make recognizable colony shapes on it, and you can see haemolysis.
Chocolate agar
These are the brown plates (which do not contain chocolate) and are very similar to blood agar.
After the blood has been added the media has been re-heated to above 56 degrees to damages the cells to releases more heme (also called growth factor X) and NAD (also called growth factor V) into the media where it is accessible to bacteria that cannot lyse the blood cells.
This medium is useful for growth of fastidious bacteria such as Neisseria sp. and Haemophilis sp.
MacConkey’s Agar
Combination of selective and differential medium.
It is selective because it contains bile salts that inhibit growth of most bacteria, except for the bacteria that colonize the gut and have adapted to bile salts (such as Enteric bacteria that contain long LPS).
It is a differential medium because it contains lactose as sole carbohydrate and the pH indicator neutral red. Acid production during lactose fermentation results in pink-red colonies.
Mannitol salt agar
Combination of selective and differential medium.
It contains high salt concentrations (<7.5% NaCl), which inhibits most bacteria except for Staphylococci (and few others).
It also contains the carbohydrate mannitol and pH indicator phenol red to detect acid production from mannitolfermentation. Staphylococcus aureusproduce yellow colonies, while other Staphylococci produce pink-red colonies.
Bacterial identification flowchart
Gram stain
-+Cell morphology Cell morphology
Cocci CocciRods Rods
Oxidase
+ -Neisseria Not a pathogen
+ -Grows with bile salts
HaemophilusFerments lactose
+ -
Oxidase
+ -Pseudomonas
Urease
+ -Proteus
H2S
+ -Salmonella Shigella
+ -Indole test
E. coli Klebsiella(check for capsule)
Atmospheres:
Anaerobic:
Clostridia
Aerobic:
Bacillus
Facultative
anaerobes:
Corynebacteria
Lactobacillus
Catalase
+ -Micrococcus# (or)
Staphylococcus
Streptococcus
# Micrococcus has larger cells and looks more yellow.
Coagulase
+ -Sta. aureus Sta. epidermidis
Haemolysis
Alpha (green)
Optichin sensitive
+ -Str. pneumonia Str. viridans
Gamma (not)Not a pathogen
Beta (clear)
Lancefield typing(can confirm D with growth on bile salts)
Gram stain
--++Cell morphology Cell morphology
Cocci CocciRods Rods
Oxidase
++ --Neisseria Not a pathogen
++ --Grows with bile salts
HaemophilusFerments lactose
++ --
Oxidase
++ --Pseudomonas
Urease
++ --Proteus
H2S
++ --Salmonella Shigella
++ --Indole test
E. coli Klebsiella(check for capsule)
Atmospheres:
Anaerobic:
Clostridia
Aerobic:
Bacillus
Facultative
anaerobes:
Corynebacteria
Lactobacillus
Catalase
++ --Micrococcus# (or)
Staphylococcus
Streptococcus
# Micrococcus has larger cells and looks more yellow.
Coagulase
++ --Sta. aureus Sta. epidermidis
Haemolysis
Alpha (green)
Optichin sensitive
++ --Str. pneumonia Str. viridans
Gamma (not)Not a pathogen
Beta (clear)
Lancefield typing(can confirm D with growth on bile salts)
This is a simplified version!!!
Next lecture
Bacterial Genetics