Physiology of Bacteria.Growth and reproduction of

Bacteria

Chair of Microbiology, Virology, and Immunology

Lecture schedule

1. Chemical composition of Bacteria2. Cell metabolism3. Contstructive metabolism метаболізм4. Types of microbial nutrition5. Bacterial transport systems6. Types of respiration7 Growth and reproduction of microbes8. Nutrient media

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 reactions (reactions by which substrate molecules are broken down), which normally function in bacteria to generate energy.

Chemical composition of bacteria

Protein 55 %

Total RNA 20.5 %

DNA 3.1 %

Phospholipid 9.1 %

  Lipopolysaccharide  3.4 %

Murein 2.5 %

Inorganic ions  1.0 %

Bacterial cell consists of:

Water – 70-90 % Dry weight – 10-30 % Proteins – 55 %, 2,35 million of molecules, 1850 different types of molecules

RNA – 20,5 %, 250000 molecules, 660 different types of molecules

DNA – 3,1 %, 2 molecules

Lipids – 9 %, 22 million of molecules

Lipopolysaccharides –3,4 %, 1,5 million of molecules

Peptidoglycan – 1 molecule

Microbial metabolism1. Catabolism (Dissimilation)

- Pathways that breakdown

organic substrates

(carbohydrates, lipids, &

proteins) to yield metabolic

energy

for growth and maintenance.

2. Anabolism (Assimilation)

- Assimilatory pathways for the

formation of key

intermediates and then to end

products (cellular

components).

4. Intermediary metabolism -

Integrate two processes

Pyruvate: universal intermediate

Aerobic respiration

Fermentation

Glycolysis (EMP pathway)

Substrate-level phosphorylation

Catabolism

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 thioester bond

O

║

(R –C ~ S – R), such as acetyl ~ S-coenzyme A

Another form of energy - transmembrane potential - ΔμН+

Chemiosmosis

• Production of ATP in Electron Transport

• Electrochemical Gradient Formed between membranes

• H+ (Protons) generated from NADH

• Electrical Force (+) & pH Force (Acid)

• Gradient formed• ATPase enzyme that channels H+

from High to Low concentration– 3 ATP/NADH– 2 ATP/NADH

Fermentation: metabolic process in which the final electron acceptor is an organic compound.

Sources of metabolic energyRespiration: chemical reduction of an electron acceptor through a specific series of electron carriers in the membrane. The electron acceptor is commonly O2, but CO2, SO4

2-, and NO3- are employed by some microorganisms.

Photosynthesis: similar to respiration except that the reductant and oxidant are created by light energy. Respiration can provide photosynthetic organisms with energy in the absence of light.

Substrate-level phosphorylation

The Krebs cycle intermediate compounds serve as precursor molecules (building blocks) for the energy-requiring biosynthesis of complex organic compounds in bacteria. Degradation reactions that simultaneously produce energy and generate precursor molecules for the biosynthesis of new cellular constituents are called amphibolic.

Energy RequirementsOxidation of organic compounds - Chemotrophs

Sunlight - Phototrophs

Carbon source- Autotrophs (lithotrophs): use CO2 as the C source

Photosynthetic autotrophs: use light energy

Chemolithotrophs: use inorganics

- Heterotrophs (organotrophs): use organic carbon (eg.

glucose) for growth.

- Clinical Labs classify bacteria by the carbon sources

(eg. Lactose) & the end products (eg. Ethanol,…).

Nitrogen sourceAmmonium (NH4

+) is used as the sole N source by most

microorganisms. Ammonium could be produced from N2 by

nitrogen fixation, or from reduction of nitrate (NO3-)and nitrite

(NO2).

Metabolic Requirements

Physiologic types of bacterial existence

Carbon Source

Energy Source

Oxidation of organic compounds - Chemotrophs

Sunlight - Phototrophs

Organic - Heterotrophs Inorganic - Autotrophs

Electrone donor

Inorganic - Lithotrophs Оrganic -Organotrophs

Chemoorganoheterotrophic bacteria

Sulfur source

A component of several coenzymes and amino acids.

Most microorganisms can use sulfate (SO42-) as the S

source.

Phosphorus source

- A component of ATP, nucleic acids, coenzymes,

phospholipids, teichoic acid, capsular polysaccharides;

also is required for signal transduction.

- Phosphate (PO43-) is usually used as the P source.

Metabolic Requirements

Mineral source- Required for enzyme function.- For most microorganisms, it is necessary to provide sources

of K+, Mg2+, Ca2+, Fe2+, Na+ and Cl-.

- Many other minerals (eg., Mn2+, Mo2+, Co2+, Cu2+ and Zn2+)

can be provided in tap water or as contaminants of other

medium ingredients.- Uptake of Fe is facilitated by production of siderophores

(Iron-chelating compound, eg. Enterobactin).

Growth factors: organic compounds (e.g., amino acids, sugars, nucleotides, vitamines) a cell must contain in order to grow but which it is unable to synthesize. Purines and pyrimidines: required for synthesis of nucleic acids (DNA and RNA);Amino acids: required for the synthesis of proteins; Vitamins: needed as coenzymes and functional groups of certain enzymes.

Transport systemsThe proteins that mediate the passage of solutes through

membranes are referred to as transport systems, carrier proteins, porters, and permeases. Transport systems operate by one of three transport processes.

In a uniport process, a solute passes through the membrane unidirectionally. In symport processes (cotransport) two solutes must be transported in the same direction at the same time; in antiport processes (exchange diffusion), one solute is transported in one direction simultaneously as a second solute is transported in the opposite direction.

Transport systems

Diffusion systems

• passive diffusion

• facilitated diffusion

• ion-driven transport

• binding protein dependent transport

• group translocation

• Membrane is selectively permeable– Few molecules pass through freely– Movement involves both active and passive

processes

Passive processes – no energy (ATP) required – Along gradient – simple diffusion, facilitated diffusion, osmosis

• Simple diffusion

• Facilitated diffusion

Can reduce Can reduce concentration gradient but concentration gradient but can’t create onecan’t create one

Osmosis• Osmotic pressure

Active processes• energy (ATP)

required– Active transport

– Group translocation

Facilitated diffusion

Active transport

Transport systems

TEMPERATURE

• One of the most important factors

• optimal growth temperature – temperature range at which the

highest rate of reproduction occurs

• optimal growth temperature for human pathogens ????

• Microorganisms can be categorized based on their optimal temperature requirements– Psychrophiles

• 0 - 20 ºC– Mesophiles

• 20 - 40 ºC– Thermophiles

• 40 - 90 ºC• Most bacteria are mesophiles

especially pathogens that require 37 ºC

TEMPERATURE

BACTERIAL TEMPERATURE REQUIREMENTS

Variable

100

50

0

0 0C

% Max Growth

37 0C 90 0C

Psychrophile

Mesophile

Thermophile

Effects of Temperature on Growth

Mesophiles10o-50o

Thermophiles70o-110o

BC YangFor lecture only

• PsychrophilesPsychrophiles– some will exist below 0 oC if liquid water is

available• oceans• refrigerators• freezers

TEMPERATURE

Pigmented bacteria in Antarctic ice

• MesophilesMesophiles– most human flora and

pathogens

TEMPERATURE

• ThermophilesThermophiles– hot springs– effluents from

laundromat– deep ocean thermal

vents

TEMPERATURE

Respiration in Bacteria

Obligate Aerobe

Microaerophile

Obligate Anaerobe

Facultative Anaerobe (Facultative Aerobe)

Aerotolerant Anaerobe

Capneic bacteria

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Categories of Oxygen Requirement

Aerobe – utilizes oxygen and can detoxify it obligate aerobe - cannot grow without oxygen

(Mycobacterium tuberculosis, Micrococcus spp., Bacillus spp., Pseudomonas spp.

facultative anaerobe – utilizes oxygen but can also grow in its absence (Echericihia spp., Salmonella spp., Sta[phylococcus spp.)

microaerophylic – requires only a small amount of oxygen (Helycobacter spp., Lactobacillus spp.)

39

Categories of Oxygen Requirement

Anaerobe – does not utilize oxygen

• obligate anaerobe - lacks the enzymes to detoxify oxygen so cannot survive in an oxygen environment (Clostridium spp., Bacteroides spp.)

• aerotolerance anaerobes – do no utilize oxygen but can survive and grow in its presence (Streptococcus pyogenes)

40

Carbon Dioxide Requirement

All microbes require some carbon dioxide in their metabolism.

• capneic – grows best at higher CO2 tensions than normally present in the atmosphere (Brucella abortus)

OXYGENObligat

e Aerobe

Facultative

Anaerobe

Obligate Anaerob

e

Four Toxic Forms of Oxygen

Toxic Oxygen Forms Are formed:

Singlet oxygenduring photosynthesis as molecular oxygen with electrons are boosted to higher energy state

Superoxide radicalsduring incomplete reduction of oxygen in aerobic and anaerobic respiration

Peroxide anionduring reactions that neutralizes superoxide radicals

Hydroxyl radicalfrom ionizing radiation and from incomplete reduction of hydrogen peroxide

Four Toxic Forms of Oxygen

Toxic Oxygen Forms Are neutralized by:

Singlet oxygencarotenoids that remove the excess energy of singlet oxygen

Superoxide radicalssuperoxide dismutases, enzymes that detoxify them

Peroxide anioncatalase or peroxidase, enzymes that detoxify peroxide anion

Hydroxyl radicalcatalase, peroxidase, and antioxidants such as vitamins C and E that protect against toxic oxygen products

Enzymes and Their Role in Metabolism

Enzymes, organic catalysts of a highly molecular structure, are produced by the living cell. They are of a protein nature, are strictly specific in action, and play an important part in the metabolism of micro-organisms. Their specificity is associated with active centres formed by a group of amino acids.

Some enzymes are excreted by the cell into the environment (exoenzymes) for breaking down complex colloid nutrient materials while other enzymes are contained inside the cell (endoenzymes).

Bacterial enzymes are subdivided into some groups:

1. Hydrolases which catalyse the breakdown of the link between the carbon and nitrogen atoms, between the oxygen and sulphur atoms, binding one molecule of water (esterases. glucosidases, proteases. amilases, nucleases, etc.).

2. Transferases perform catalysis by transferring certain radicals from one molecule to another (transglucosidases, transacylases. transaminases).

3. Oxidative enzymes (oxyreductases) which catalyse the oxidation-reduction processes (oxidases, dehydrogenases, peroxidases, catalases).

4. Isomerases and racemases play an important part in carbohydrate metabolism. Rearrangement atoms of a molecule.

5. Lyases (remove chemical groups from molecules without adding water).

6. Lygases (join two molecules together and usually require energy from ATP).

Enzymes

Significance of the enzymes

With the help of amylase produced by mould fungi starch is saccharified and this is employed in beer making, industrial alcohol production and bread making. Proteinases produced by microbes are used for removing the hair from hides, tanning hides, liquefying the gelatinous layer from films during regeneration, and for dry cleaning.

Fibrinolysin produced by streptococci dissolves the thrombi in human blood vessels. Enzymes which hydrolyse cellulose aid in an easier assimilation of rough fodder.

Due to the application of microbial enzymes, the medical industry has been able to obtain alkaloids, polysaccharides, and steroids (hydrocortisone, prednisone, prednisolone. etc.).

Bacteria play an important role in the treatment of caouichouc, coffee, cocoa, and tobacco.

Enzymes permit some species of microorganisms to assimilate methane. butane, and other hydrocarbons, and to synthesize complex organic compounds from them.

With the help of the enzymatic ability of yeasts in special-type industrial installations protein-vitamin concentrates (PVC) can be obtained from waste products of petroleum (paraffin’s).

Metabolism Results in Reproduction

• Microbial growth – an increase in a population of microbes rather than an increase in size of an individual

• Result of microbial growth is discrete colony – an aggregation of cells arising from single parent cell

• Reproduction results in growth

BINARY FISSION

• division exactly in half

• most common means of bacterial reproduction

– forming two equal size progeny

– genetically identical offspring

– cells divide in a geometric progression doubling cell number

BINARY FISSION

Doubling time is the unit of measurement of microbial growth

CULTURE GROWTH

• Growth of culture goes through four phases with time

• 1) Lag phase

• 2) Log or Logarithmic phase

• 3) Stationary phase

• 4) Death or Decline phase

BACTERIAL GROWTH CURVE

LAG PHASE• Organisms are adjusting to the environment

– little or no division

• synthesizing DNA, ribosomes and enzymes – in order to

breakdown nutrients, and to be used for growth

Mouse click for lag phase

adjustment

LOGARITHMIC PHASE• Division is at a constant rate (generation generation

timetime)

• Cells are most susceptible to inhibitors

STATIONARY PHASE

• Dying and dividing organisms are at an equilibrium

• Death is due to reduced nutrients, pH changes, toxic waste and reduced oxygen

• Cells are smaller and have fewer ribosomes

• In some cases cells do not die but they are not multiplying

STATIONARY PHASE

DEATH PHASE

in 37oC, pH 5.1 ; in 45oC, pH 6.2In bioreactors

BC YangFor lecture only

ENUMERATION OF BACTERIA

• 1) viable plate count

• 2) direct count

• 3) most probable number (MPN)1

2 3

45

6

7

8

910

VIABLE PLATE COUNT

• Most common procedure for assessing bacterial numbers– 1) serial dilutions of a suspension of bacteria

are plated and incubated

– 2) the number of colonies developing are then counted

• it is assumed that each colony arises from an individual bacterial cell

VIABLE PLATE COUNT

3) by counting the colonies and taking into account the dilution factors the concentration of bacteria in original sample can be determined

4) only plates having between 30 and 300 colonies are used in the calculations

VIABLE PLATE COUNT

See next slide for bigger diagram

VIABLE PLATE COUNT

– 5) multiply the number of colonies times the dilution factor to find the number of bacteria in the sample

– Example • Plate count = 54

• Dilution factor = 1:10,000 ml

• CalculationCalculation

– 54 X 10,000 = 540,000 bacteria/ml

VIABLE PLATE COUNT

• “TNTC”– if the number of colonies is too great (over 300) the

sample is labeled “TNTC”– Too Numerous To Count

• limitation of viable plate count – selective as to the bacterial types that will grow

given the incubation temperature and nutrient type

VIABLE PLATE COUNT

VIABLE PLATE COUNT

“TNTC”417 colonies

Dilution factor of 1/1,000 (10 -

3)

Click to incubat

e

VIABLE PLATE COUNT

22 colonies Too few the count is

less than 30

Click to incubat

e

Dilution factor of 1/1,000,000

(10 -6)

VIABLE PLATE COUNT

42 colonies

Dilution factor of 1/100,000

(10 -5)

Calculate the number of

bacteria per ml

Click to incubat

e

• Calculate:

– 42 colonies

– dilution factor of 100,000

• 42 X 100,000 = ???

• 4,200,000 bacteria/ml

VIABLE PLATE COUNT

Nutrient media• Ordinary (simple) media • Special media (serum agar, serum broth, coagulated

serum, pota toes, blood agar, blood broth, etc.).• Elective media • Enriched media • Differential diagnostic media: (1) proteolytic action; • (2) fermentation of carbohydrates (Hiss media); • (3) haemolytic activity (blood agar); • (4) reductive activity of micro-organisms; • (5) media containing substances assimilated only by

certain microbes.

Biochemical properties

Colonies

Colonies

Colonies

Pure Cultures Isolation

Isolated colonies obtaining