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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