Bacterial Physiology -Metabolism & Growth

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: 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)

2. Outline • Metabolic Requirements • Metabolism & the Conversion of Energy - Glucose: Glycolysis (Embden-Meyerhof- Parnas pathway) TCA cycles Pentose phosphate pathway - Nucleic acid synthesis • Bacterial Growth

3. 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

4. Essential Elements

5. 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-)andnitrite (NO2).

6. 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.

7. # 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.

8. 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“)

9. Toxicity of O2 for Anaerobes • 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.

10. Anaerobic cultivation methods Excluding oxygen Reducing agents Anaerobic jar Anaerobic glove chamber

11. Outline • Metabolic Requirements • Metabolism & the Conversion of Energy - Glucose: Glycolysis (Embden-Meyerhof- Parnas pathway) TCA cycles Pentose phosphate pathway - Nucleic acid synthesis • Bacterial Growth

12. 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

13. Catabolism Substrate-level phosphorylation Fermentation Glycolysis (EMP pathway) Aerobic respiration Pyruvate: universal intermediate

14. 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

15. 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

16. Sources of metabolic energy 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 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 Fermentation: metabolic process in which the final electron acceptor is an organic compound.

17. 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.

18. Saccharomycetes Clostridium E. coli Propionebacterium Enterobacter Streptococcus Lactobacillus

19. 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.

20. 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

21. 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

22. Aerobic Glucose Metabolism x2

23. Pentose phosphate pathway (hexose monophosphate shunt) Nucleotide synthesis • Functions: • Provides various sugars as precursors of biosynthesis, and NADPH for use in biosynthesis • The various sugars may be shunted back to the glycolytic pathway.

25. Nucleic acid synthesis

26. Nucleic acid synthesis • 1. Ribose-5-P (product of HMP) synthesis of purine ring from sugar moiety inosine monophosphate purine monophosphate • Pyrimidine orotate orotidine monophosphate (pyrimidine orotate attaches to ribose phosphate) cytidine or urine (pyrimidine) monophosphate • Reduction of ribonucleotides at the 2’ carbon of the sugar portion deoxynucleotides

28. Bacterial Cell Division • 1. Replication of chromosome • 2. Cell wall extension • 3. Septum formation • 4. Membrane attachment of • DNA pulls into a new cell.

29. 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. Alarmonesis induced. Some bacteria undergo sporulation. Decline phase (the death phase)

30. 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. Medium Basic media Rich media Enrichment media Selective media Differential media Agar:an acidic polysaccharide extracted from red algae

33. 10-1 10-2 10-3 10-4 10-5 10-6 10-7 0.1 ml > 1000 220 18 Bacterial concentration: 220 x 106 x 10 = 2.2 x 109/ml Growth, survival and death of microorganisms 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.

34. Bacterial growth in nature Interaction 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.

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