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 toleranceBacteria 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 homolactic fermentation (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 11 22 43 84 165 3210 1,02420 1,048,57630 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 neutrophiles and 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 environmental conditions (pH, temperature, nutrients, etc.).

Enzymes and intermediates are formed and accumulate until they 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 agar composed 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 colour and 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 mannitol fermentation. Staphylococcus aureus

produce 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:ClostridiaAerobic:BacillusFacultative anaerobes:CorynebacteriaLactobacillus

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:ClostridiaAerobic:BacillusFacultative anaerobes:CorynebacteriaLactobacillus

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