bacterial physiology -metabolism & growth pin lin ( 凌 斌 ), ph.d. departg ment of...
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Bacterial Physiology -Metabolism &
Growth
• Pin Lin ( 凌 斌 ), Ph.D.
Departg ment of Microbiology & Immunology, NCKU
ext 5632
• References:
1. Chapters 4 in Medical Microbiology (Murray, P. R. et al; 5th edition)
2. 醫用微生物學 ( 王聖予 等編譯 , 4th edition)
Outline
• Metabolic Requirements
• Metabolism & the Conversion of Energy- Glucose: Glycolysis (Embden-Meyerhof-
Parnas pathway) TCA cycles Pentose phosphate pathway
- Nucleic acid synthesis
• Bacterial Growth
Metabolic Requirements1. Bacteria must obtain or synthesize Amino acids,
Carbohydrates, & Lipids => build up the cell.
2. Minimum requirements for bacterial growth – C, N, H2O, Ion & energy
3. Growth requirements & metabolic by-products => Classify different bacteria
4. O2 is essential for animal cells but not for all bacteria. - Obligate aerobes: Mycobacterium tuberculosis - Obligate anaerobes: Clostridium perfringens - Facultative anaerobes: Most bacteria
Essential Elements
# 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 (e.g. Lactose) & the end products (e.g. 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-I
# Sulfur sourceA component of several coenzymes and amino acids.Most microorganisms can use sulfate (SO4
2-) 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-II
# 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, e.g. Enterobactin).
# Growth factors: organic compounds (e.g., amino acids, sugars, nucleotides) a cell must contain in order to grow but which it is unable to synthesize.
pH value Neutrophiles ( pH 6-8) Acidophiles ( pH 1-5) Alkalophiles ( pH 9-11) Internal pH is regulated by variou
s proton transport systems in the cytoplasmic membrane.
Temperature Psychrophiles (<15 or 15-20 oC) Mesophiles ( 30-37 oC) Thermophiles ( at 50-60 oC)
Heat-shock response is induced to stabilize the heat-sensitive proteins of the cell.
Environmental factorsAeration
Obligate aerobes
Facultative anaerobes
Microaerophilics
Obligate anaerobes
(Capnophilics: bacteria that do not produce enough CO2
and, therefore, require additional CO2 for growth.)
Ionic strength and osmoti
c pressure
Halophilic (Greek for
"salt-loving“)
1. O2 reduced to H2O2 by enzymes.
2. O2 reduced to O2- by ferrous ion.
3. In aerobes and aerotolerant anaerobes, O2- is remov
ed by “Superoxide dismutase”, while H2O2 is removed by “Catalase”.
4. Strict anaerobes lack both Catalase and Superoxide dismutase.
Toxicity of O2 for
Anaerobes
Excluding oxygen
Reducing agents
Anaerobic jar
Anaerobic glove chamber
Anaerobic cultivation methods
Outline
• Metabolic Requirements
• Metabolism & the Conversion of Energy- Glucose: Glycolysis (Embden-Meyerhof-
Parnas pathway) TCA cycles Pentose phosphate pathway
- Nucleic acid synthesis
• Bacterial Growth
Microbial metabolism
1. All cells require the energy supply to survive. The common energy form => ATP (Adenosine Tri-Phosphate)
2. Catabolism (Dissimilation)- Pathways that breakdown organic substrates (carbohydrates, lipids, & proteins) to yield metabolic energy for growth and maintenance.
3. 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
Metabolism of Glucose
1. Here we focus on discussing the metabolism of glucose. For the metabolism of other organic compounds (e.g. 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: (1) Glycolysis (EMP pathway), (2) TCA cycle, & (3) Pentose phosphate pathway.
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
Fermentation: metabolic process in which the final electron acceptor is an organic compound.
Sources of metabolic energyRespiration: chemical reduct
ion 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
1. In fermentation, Pyruvate produced from glycolysis is converted to various end products via bacterial species.
2. The NADH produced during glycolysis is recycled to NAD.
3. Many bacteria are identified on the basis of their fermentative end products.
4. Fermentation of bacteria produces yogurt, sauerkraut, flavors to various cheeses and wines.
5. Alcoholic fermentation is uncommon in bacteria.
Fermentation
Saccharomycetes
E. coliClostridium
Propionebacterium Enterobacter
StreptococcusLactobacillus
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 a
s precursors of biosynthesi
s, and NADPH for use in bi
osynthesis
2. The various sugars may b
e shunted back to the glyc
olytic pathway.
Pentose phosphate pathway (hexose monophosphate shun
t) Nucleotide synthesis
Nucleic acid synthesis
1. Ribose-5-P (product of HMP) synthesis of purine r
ing from sugar moiety inosine monophosphate
purine monophosphate
2. Pyrimidine orotate orotidine monophosphate (pyri
midine orotate attaches to ribose phosphate)
cytidine or urine (pyrimidine) monophosphate
3. Reduction of ribonucleotides at the 2’ carbon of the sug
ar portion deoxynucleotides
Nucleic acid synthesis
Bacterial Cell Division
1. Replication of chromosome
2. Cell wall extension
3. Septum formation
4. Membrane attachment of DNA pulls into a new cell.
Lag phase (adaptation)
Exponential phase (Log phase)
Determination of the generation time (doubling time)
The ending of this phase is due to exhaustion of nutrients in the medium and accumulation of toxic metabolic products.
Stationary phase
A balance between slow loss of cells through death and formation of new cells through growth.
Alarmones is induced.
Some bacteria undergo sporulation.
Decline phase (the death phase)
Bacterial growth curve
Cultivation methodsMedium
Basic media
Rich media
Enrichment media
Selective media
Differential media
Agar: an acidic polysaccharide extracted from red algae
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 cultureDifferential mediumSelective medium
Isolation of microorganisms in pure culture
Pour plate methodStreak method
For growing bacterial cells
Provide nutrients and conditions reproducing the organism's natural environment.
Most bacteria reproduce by binary fission.
Measurement of microbial concentrations:
Cell concentration (no. of cells/unit vol. of culture)
Viable cell count
Turbidimetric measurements
Biomass concentration (dry wt. of cells/unit vol. of culture): can be estimated by measuring the amount of protein or the volume occupied by cells.
Growth, survival and death of microorganisms
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
Bacterial growth in natureInteraction of mixed
communities
A natural environment may be similar to a continuous culture.
Bacteria grow in close association with other kinds of organisms.
The conditions in bacterial close association are very difficult to reproduce in the laboratory. This is part of the reason why so few environmental bacteria have been isolated in pure culture.
Biofilms
Polysaccharide encased community of bacteria attached to a surface.
Attachment of bacteria to a surface or to each other is mediated by glycocalyx.
About 65% of human bacterial infection involve biofilms.
Biofilms also causes problems in industry.
Bioremediation is enhanced by biofilms.
Biofilm: a community of microbes embedded in an organic polymeric matrix (glycocalyx, slime), adhering to an inert or living surface.
The End