bacterial physiology 王淑鶯 微生物免疫學所 國立成功大學醫學院 分機 : 5634...
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Microbial metabolism
Microbial growth
Reference:
Chapter 3 in Medical Microbiology
(Murray, P. R. et al; 6th edition)
What is Metabolism?
The Greek metabole, meaning change
It includes all the biochemical reactions that occur in the cell.
- Catabolism
- Anabolism
Why do we must know the metabolism of bacteria ?
Because we want to control their metabolism
and know how to inhibit or stop bacteria growth
Why Study Metabolism? Classification of bacteria
Oxygen Tolerance Biochemical reactions
Fermentation Products Food Products
Yogurt, Sour Cream, Bread, Alcohol
Commercial Products Citric Acid
Environmental Cleanup e.g. common soil bacteria could clean up nuclear contamination
Common Soil Bacteria Could Clean Up Nuclear ContaminationCOLUMBUS, Ohio, March 17, 2009 (ENS) - An international
team of scientists has found a common soil bacterium that might one day be used to clean up radioactive toxics left from nuclear weapons production decades ago.
The bacteria's cleaning power comes from their ability to "inhale" toxic metals and "exhale" them in a non-toxic form, explains team member Brian Lower, assistant professor in the School of Environment and Natural Resources at Ohio State University.
Using a unique combination of microscopes, researchers at Ohio State University and scientists from Austria, Sweden, Switzerland and the United States were able to see how the bacterium Shewanella oneidensis breaks down metal to chemically extract oxygen.
The study, published online this week in the journal "Applied and Environmental Microbiology," provides the first evidence that the Shewanella bacterium uses proteins within the bacterial cell into its outer membrane to contact metal directly.
The proteins then bond with metal oxides, which the bacteria utilize the same way we use oxygen - to breathe.
"We use the oxygen we breathe to release energy from our food. But in nature, bacteria don't always have access to oxygen," said Lower. "Whether the bacteria are buried in the soil or underwater, they can rely on metals to get the energy they need. It's an ancient form of respiration."
Catabolism degrade, break bonds, convert large
molecules into smaller component
often produce energy
Anabolism synthesis of cell molecules and structures
usually requires the input of energy
Metabolites compounds given off by the complex
networks of metabolism
Overview of cell metabolism
Microbes need three things to grow
Energy source Nutrients (C) Suitable environmental conditions
Nutrient Requirements
Energy Source Phototroph
Uses light as an energy source Chemotroph
Uses energy from the oxidation of reduced chemical compounds
Nutrient Requirements Carbon source
All bacteria require carbon for growth Bacteria can been classified on the basis of their
carbon source Autotroph
Can use CO2 as a sole carbon source
Heterotroph use more complex organic compounds such as carbohydrates and
amino acids as source of carbon
Energy/Carbon classification Where microbes get their energy?
Sunlight vs. Chemical Photo- vs. Chemo- trophs
How do they obtain carbon? Carbon Dioxide (or inorganic cmpds.) vs. Organic
Compounds (sugars, amino acids) Auto- vs. Hetero- trophs
Examples Photoautotrophs vs. Photoheterotrophs Chemoautotrophs vs. Chemoheterotrophs
Nutrient Requirements Nitrogen source
Organic nitrogen Primarily from the catabolism of amino acids
Oxidized forms of inorganic nitrogen Nitrate (NO
32-) and nitrite (NO
2-)
Reduced inorganic nitrogen Ammonium (NH
4+)
Dissolved nitrogen gas (N2) (Nitrogen fixation)
Nutrient Requirements
Inorganic nutrients (ions) contain no carbon and hydrogen atoms : phosphates,
potassium, magnesium, nitrogen, sulfur, iron and numerous trace metals
Acquisition of Fe is facilitated by production of siderophores
Organic nutrients contain carbon and hydrogen atoms. Include
carbohydrates, lipids, amino acids, Nucleic acids etc.
Nutrient Requirements Carbohydrates
are used as the initial carbon source for many biosynthetic pathways and as electron donors (energy source) by many bacteria
Amino acids are important source of carbon and nitrogen.
The nitrogen is converted to ammonia.
Nutrient Requirements Phosphorus
is present as phosphates salts They function in energy metabolism and as
constituents of nucleic acids, phospholipids, teichoic acids, ATP, etc
Minerals K, Mg, Ca, Fe are required in relatively high levels Function as cofactors in enzyme reactions and as
cations they act as buffers within the cells Vitamins
function as coenzymes
General Pathways of MetabolismGeneral Pathways of Metabolism-- Catabolism --
Breakdown of macromolecules to building blocks
protein polysaccharide lipid nucleic acids
amino acids
glucose, other sugars
glycerol,fatty acids
ribose,bases,
phosphate
no useable energy yield here - only building blocks obtained
Breakdown of monomers to common intermediates
amino acids
glucose, other sugars
glycerol,fatty acids
pyruvate
acetyl CoANH4
+
citric acid cycle ETS ATP
CO2
--Anabolism--
amino acids
glucose, other sugars
glycerol,fatty acids
pyruvate
acetyl CoA
NH4+
proteins
polysaccharide
s lipids
citric acid cycle
1. utilization of critical Common Intermediates including components of TCA cycle to make building blocks
2. making building block requires energy = ATP
3. synthesis of macromolecules requires energy = ATP
Anabolism, cont’d
Carbohydrate Catabolism Microorganisms oxidize carbohydrates as
their primary source of energy Glucose - most common energy source Energy obtained from Glucose by:
Respiration Fermentation
Pyruvate: universal intermediate
Aerobic respiration
Fermentation
Glycolysis (EMP pathway)
Substrate-level phosphorylation
Catabolism
Substrate-level phosphorylation
High energy phosphate group of one of the intermediates is used under the direction of the enzyme (kinase) to generate ATP from ADP
Glycolysis
(Embden-Meyerhof-
Parnas pathway)
1. The most common pathway for bacteria in the catabolism of glucose.
2. Reactions occur under both aerobic and anaerobic conditions
3. One Glucose => 2 ATP (2X2-2=2) 2 NADH 2 Pyruvate
Metabolism of Glucose
1. Here we focus on discussing the metabolism of glucose. For
the metabolism of other organic compounds (eg. Proteins or
lipids), please refer to a textbook of Biochemistry.
2. Bacteria can produce energy from glucose by fermentation
(w/o O2), anaerobic reaction (w/o O2), or aerobic
respiration.
3. Three major metabolic pathways are used by bacteria to
catabolize glucose: Glycolysis (EMP pathway), TCA cycle, &
Pentose phosphate pathway
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
Anaerobic Respiration Electrons released by oxidation are passed
down an E.T.S., but oxygen is not the final electron acceptor
Nitrate (NO3-) ----> Nitrite (NO2-)
Sulfate (SO24-) ----> Hydrogen Sulfide (H2S)
Carbonate (CO24-) ----> Methane (CH4)
Anaerobic Respiration Examples of anaerobic respiration
glucose + 3NO3- + 3H2O 6HCO3
- + 3NH4+
glucose + 3SO42- + 3H+ 6HCO3
- + 3SH-
glucose + 12S + 12H2O 6HCO3- + 12HS- + 18H+
All of these terminal electron acceptors have smaller reduction potentials than O2, so it is less energetically efficient than aerobic respiration
Fermentation Anaerobic process that does not use the
E.T.S. Usually involves the incomplete oxidation of a carbohydrate which then becomes the final electron acceptor.
Glycolysis - plus an additional step
Fermentation may result in numerous end products
1. Type of organism
2. Original substrate
3. Enzymes that are present and active
1. Lactic Acid Fermenation Only 2 ATP End Product - Lactic Acid Food Production
Yogurt - Milk Pickles - Cucumbers Sauerkraut - Cabbage
2 Genera: Streptococcus Lactobacillus
2. Alcohol Fermentation Only 2 ATP End products:
alcohol CO2
Alcoholic Beverages Bread dough to rise Saccharomyces cerevisiae (Yeast)
3. Mixed - Acid Fermentation Only 2 ATP End products- “FALSE”
formic acid acetic acid lactic acid succinic acid ethanol
Escherichia coli and other enterics
Propionic Acid Fermentation Only 2 ATP End Products:
Propionic acid CO2
Propionibacterium spp.
Saccharomycetes
E. coliClostridium
Propionebacterium Enterobacter
StreptococcusLactobacillus
Pyruvate: universal intermediate
Aerobic respiration
Fermentation
Glycolysis (EMP pathway)
Substrate-level phosphorylation
Catabolism
Function of TCA cycle
1. Via the TCA cycle, Pyruvate from glycolysis or other
catabolic pathways can be completely oxidized (w/ O2)
to H2O & CO2
2. Generation of ATP
3. Supplies key intermediates for amino acids, lipids,
purines, and pyrimidines
4. The final pathway for the complete oxidation of amino
acids, fatty acids, and carbohydrates.
Tricarboxylic Acid (TCA) cycle
1. Pyruvate => Acetyl-CoA1x NADH => 3ATP
2. TCA cycle: 3x NADH => 3x 3 ATP 1x FADH2 => 1x 2 ATP 1x GTP => 1x ATP
3. NADH & FADH2 go to the Electron transport chain
Electron transport chain
1. Electrons carried by NADH (FADH2) A series of donor-acceptor pairs Oxygen: terminal electron acceptor Aerobic respiration
2. Some bacteria use other compounds (CO2, NO3
-) as terminal acceptor Anaerobic respiration Produce less ATP
Aerobic Glucose Metabolism
x2
Functions:
1.Provides various sugars
as precursors of
biosynthesis, and
NADPH for use in
biosynthesis
2.The various sugars may
be shunted back to the
glycolytic pathway.
Pentose phosphate pathway (hexose monophosphate shunt)
nucleotide synthesis
Microbes need three things to grow
Energy source Nutrients (C) Suitable environmental conditions
Environmental factors Oxygen Requirement
Obligate aerobes The growth of bacteria is inhibited by absence of
oxygen Pseudomonas aeruginosa, Mycobacterium
tuberculosis
Obligate anaerobes Growth is inhibited by the presence of oxygen Clostridium spp and Bacteriodes spp.
Environmental factors Oxygen Requirement
Facultative anaerobes are able to grow in the presence or absence of
molecular oxygen Staphylococci, Streptococci, E. coli, etc
Microaerophilic bacteria grow best under increased carbon dioxide tension Neisseria gonorrhoeae, Haemophilus influenzae
Environmental factors
Oxygen Requirement Aerotolerant bacteria
can survive (but not grow) for a short period of time in the presence of atmospheric oxygen
Tolerance to oxygen is related to the ability of the bacterium to detoxify superoxide and hydrogen peroxide produced as bye products of aerobic respiration.
Superoxide dismutase which converts superoxide ( a toxic metabolite) into
hydrogen peroxide is present in aerobic and aerotolerant bacteria but not in obligate anaerobes.
2O2− + 2H+ H2O2 + O2
Catalase which converts hydrogen peroxide into water and
oxygen is also present in all aerobic bacteria but is lacking in aerotolerant organisms. Strict anaerobes lack both enzymes
2 H2O2 2H2O + O2
SOD
catalase
Enzymes that detoxify the toxic byproducts of aerobic metabolism
Environmental factors Temperature
There are three critical temperature ranges for growth: (a) Minimum temperature (b) Maximum temperature (c) Optimum temperature
Environmental factors Temperature
Psychrophiles: Has optimum temperature below 15°C but
capable of growth at 0°C Mesophiles:
grow at a range of 20° – 40°C. Include most bacterial pathogens with optimum temp. at 37°C
Thermophiles: microbes that has optimum temperature
above 45°C with a general range of 45-80°C Most thermophiles form spores e.g. Bacillus
steareothermophilus
Environmental factors pH
Optimum pH for most bacteria is near pH 7.0 (pH 6.5- pH 7.5)
Bacteria can be classified as alkalinophiles, neutrophiles or acidophiles according to their degree of tolerance to pH changes
Environmental factors Ionic strength and osmotic pressure
When a microbial cell is in a hypertonic solution cellular water moves out of the cell through the cell membrane to the hypertonic solution
This osmotic loss of water causes shrinkage of the cell (PLASMOLYSIS)
In a hypotonic solution such as in distilled water, water will enter the cell and the cell may be lysed by such treatment (PLASMOPTYSIS)
Halophiles require high salt concentrations for
growth. Some bacteria can tolerate 15% salt. E.g. S. aureus
Bacterial growth
Replication of chromosome
Cell wall extension Septum formation Membrane attachment
of DNA pulls into a new cell.
.
Cell division Generation or doubling time:
The average generation time for bacteria is 30-60 minutes under optimum conditions.
Most pathogens such as Staphylococcus aureus and Escherichia coli double in 20 – 30 minutes.
The longest generation time requires days. E.g. Mycobacterium leprae that causes leprosy doubles in 20 to 30 days.
The growth curve
The growth curve The lag phase
Cells adjust to new environment. There is no change in the number of cells but
metabolic activity is high leading to increase in cellular components
The log or exponential phase Bacteria multiply at the fastest rate possible under
the conditions provided. Are susceptible to cell wall active antibiotics Form metabolic end products
The growth curve The stationary phase
there is an equilibrium between cell division and cell death caused by : decrease in nutrient increase in cell population and accumulation of metabolic
waste /end products
Death or Decline phase The number of death cells exceeds the number of
new cells formed due to lack of nutrients and accumulation of toxic waste
Types of Culture media Basic media Rich media
contain additional nutrients to support the growth of fastidious organisms. E.g. blood agar and chocolate agar
Enrichment media is used to encourage the growth of a particular organism in a mixed
culture Selective media
contains salts, dyes or other chemicals that inhibit the growth unwanted microorganisms.
Differential media contain chemicals that allow the distinction between different types
of organisms e.g. Lactose in MacConkey agar
MacConkey agar with lactose(left) and non-lactose(right) fermenters
Cultivation methods For microbiologic examination
Use as many different media and conditions of incubation as is practicable. Solid media are preferred; avoid crowding of colonies.
For isolation of a particular organism Enrichment culture Differential medium Selective medium
Isolation of microorganisms in pure culture Pour plate method Streak method
For growing bacterial cells Provide nutrients and conditions reproducing the
organism's natural environment.
Excluding oxygen
Reducing agents
Anaerobic jar
Anaerobic glove chamber
Anaerobic cultivation methods
Measurement of Microbial Growth Viable cell counts
plating diluted samples (using a pour plate or spread plate) onto suitable growth media and monitoring colony formation
Serial Dilution 1. Carry out dilution series 2. plate known volumes on plates 3. count only plates with 30-300 colonies (best statistical
accuracy); 4. extrapolate to undiluted cell conc.
0.1 ml
10-1 10-2 10-3 10-4 10-5 10-6 10-7
> 1000 220 18
Bacterial concentration:
220 x 106 x 10 = 2.2 x 109/ml
Measurement of Microbial Growth
Turbidimetric measurements can estimate cell numbers accurately by
measuring visible turbidity Use a spectrophotometer to accurately
measure absorbance, usually at wavelengths around 400-600 nm
Light scattered is proportional to number of cells.
Summary Metabolic Requirements
Energy source Nutrients
Metabolism & the Conversion of Energy
- Glucose: Glycolysis (Embden-Meyerhof-Parnas pathway)
TCA cycles
Pentose phosphate pathway Bacterial Growth
Environment factors Growth curve Measurement of growth