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Bacterial Physiology
-Metabolism & Growth
• Pin Lin (凌 斌), Ph.D.
Departg ment of Microbiology & Immunology, NCKU
ext 5632
[email protected]
• 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-MeyerhofParnas pathway)
TCA cycles
Pentose phosphate pathway
- Nucleic acid synthesis
•
Bacterial Growth
Metabolic Requirements
1. 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
Metabolic Requirements-I
# 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 source
Ammonium (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-II
# 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.
# 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) a cell must contain in order to grow but
which it is unable to synthesize.
Environmental factors
pH value
Neutrophiles ( pH 6-8)
Acidophiles ( pH 1-5)
Alkalophiles ( pH 9-11)
Internal pH is regulated by
various 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.
Aeration
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
osmotic pressure
Halophilic (Greek for
"salt-loving“)
Toxicity of O2 for Anaerobes
1. O2 reduced to H2O2 by enzymes.
2. O2 reduced to O2- by ferrous ion.
3. In aerobes and aerotolerant anaerobes, O2- is
removed by “superoxide dismutase”, while H2O2 is
removed by “catalase”.
4. Strict anaerobes lack both catalase and superoxide
dismutase.
Anaerobic cultivation methods
Excluding oxygen
Reducing agents
Anaerobic jar
Anaerobic glove chamber
Outline
•
Metabolic Requirements
•
Metabolism & the Conversion of Energy
- Glucose: Glycolysis (Embden-MeyerhofParnas 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 Triphosphate)
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
Catabolism
Substrate-level
phosphorylation
Fermentation
Glycolysis
(EMP pathway)
Aerobic
respiration
Pyruvate: universal intermediate
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), TCR cycle, &
Pentose phosphate pathway
Glycolysis
(Embden-MeyerhofParnas 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
Sources of metabolic energy
Substrate-level phosphorylation
Fermentation: metabolic process in
which the final electron acceptor is
an organic compound.
Respiration: chemical
reduction of an electron
acceptor through a specific
series of electron carriers in
the membrane. The electron
acceptor is commonly O2,
but CO2, SO42-, and NO3are 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.
Fermentation
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.
Saccharomycetes
Clostridium
Propionebacterium
E. coli
Enterobacter
Streptococcus
Lactobacillus
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-CoA
1x 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
Pentose phosphate pathway
(hexose monophosphate shunt)
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.
Nucleotide
synthesis
Nucleic acid synthesis
Nucleic acid synthesis
1. Ribose-5-P (product of HMP)
synthesis of purine
ring from sugar moiety
inosine monophosphate
purine monophosphate
2. Pyrimidine orotate
orotidine monophosphate
(pyrimidine orotate attaches to ribose phosphate)
cytidine or urine (pyrimidine) monophosphate
3. Reduction of ribonucleotides at the 2’ carbon of the
sugar portion
deoxynucleotides
Bacterial Cell Division
1. Replication of chromosome
2. Cell wall extension
3. Septum formation
4. Membrane attachment of
DNA pulls into a new cell.
Bacterial growth curve
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)
Cultivation methods
Medium
For microbiologic examination
Rich media
Use as many different media and conditions
of incubation as is practicable. Solid media
are preferred; avoid crowding of colonies.
Enrichment media
For isolation of a particular organism
Basic media
Selective media
Differential media
Agar: an acidic
polysaccharide
extracted from red
algae
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.
Growth, survival and death of microorganisms
Most bacteria reproduce by
binary fission.
10-1
10-2
10-3
10-4
10-5
10-6
Measurement of microbial
concentrations:
Cell concentration (no. of
cells/unit vol. of culture)
0.1 ml
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.
> 1000
220
18
Bacterial concentration:
220 x 106 x 10 = 2.2 x 109/ml
10-7
Bacterial growth in nature
Interaction of mixed
communities
Biofilms
A natural environment may be
similar to a continuous culture.
Polysaccharide encased
community of bacteria attached
to a surface.
Bacteria grow in close
association with other kinds of
organisms.
Attachment of bacteria to a
surface or to each other is
mediated by glycocalyx.
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.
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.