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