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Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 凌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 [email protected] • References: 1. Chapters 4 in Medical Microbiology (Murray, P. R. et al; 5 th edition) 2. 凌凌凌凌凌凌 ( 凌凌凌 凌凌凌 , 4th edition)

Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 [email protected] References:

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Page 1: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

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)

Page 2: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

Outline

• Metabolic Requirements

• Metabolism & the Conversion of Energy- Glucose: Glycolysis (Embden-Meyerhof-

Parnas pathway) TCA cycles Pentose phosphate pathway

- Nucleic acid synthesis

• Bacterial Growth

Page 3: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

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

Page 4: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

Essential Elements

Page 5: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

# 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

Page 6: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

# 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

Page 7: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

# 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.

Page 8: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

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“)

Page 9: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

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

Page 10: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

Excluding oxygen

Reducing agents

Anaerobic jar

Anaerobic glove chamber

Anaerobic cultivation methods

Page 11: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

Outline

• Metabolic Requirements

• Metabolism & the Conversion of Energy- Glucose: Glycolysis (Embden-Meyerhof-

Parnas pathway) TCA cycles Pentose phosphate pathway

- Nucleic acid synthesis

• Bacterial Growth

Page 12: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

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

Page 13: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

Pyruvate: universal intermediate

Aerobic respiration

Fermentation

Glycolysis (EMP pathway)

Substrate-level phosphorylation

Catabolism

Page 14: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

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.

Page 15: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

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

Page 16: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

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

Page 17: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

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

Page 18: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

Saccharomycetes

E. coliClostridium

Propionebacterium Enterobacter

StreptococcusLactobacillus

Page 19: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

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.

Page 20: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

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

Page 21: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

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

Page 22: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

Aerobic Glucose Metabolism

x2

Page 23: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

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

Page 24: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:
Page 25: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

Nucleic acid synthesis

Page 26: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

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

Page 27: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:
Page 28: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

Bacterial Cell Division

1. Replication of chromosome

2. Cell wall extension

3. Septum formation

4. Membrane attachment of DNA pulls into a new cell.

Page 29: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

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

Page 30: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

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.

Page 31: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:
Page 32: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:
Page 33: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

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

Page 34: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

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.

Page 35: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

Biofilm: a community of microbes embedded in an organic polymeric matrix (glycocalyx, slime), adhering to an inert or living surface.

Page 36: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References:

The End

Page 37: Bacterial Physiology -Metabolism & Growth Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References: