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: 1. Chapters 4 in Medical Microbiology (Murray, P. R. et al; 5 th edition) 2. 醫用微生物學 ( 王聖予 等編譯, 4th edition)

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

4. Metabolic Requirements 1. Bacteria must obtain or synthesize Amino acids, Carbohydrates, & Lipids => build up the cell. 2. Minimum requirements for bacterial growth – C, N, H 2 O, Ion & energy 3. Growth requirements & metabolic by-products => Classify different bacteria 4. O 2 is essential for animal cells but not for all bacteria. - Obligate aerobes: Mycobacterium tuberculosis - Obligate anaerobes: Clostridium perfringens - Facultative anaerobes: Most bacteria

5. Essential Elements

6. # Carbon source - Autotrophs (lithotrophs): use CO 2 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 source Ammonium (NH 4 + ) is used as the sole N source by most microorganisms. Ammonium could be produced from N 2 by nitrogen fixation, or from reduction of nitrate (NO 3 - )and nitrite (NO 2 ). Metabolic Requirements-I

7. # Sulfur source A component of several coenzymes and amino acids. Most microorganisms can use sulfate (SO 4 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 (PO 4 3- ) is usually used as the P source. Metabolic Requirements-II

8. # Mineral source - Required for enzyme function. - For most microorganisms, it is necessary to provide sources of K +, Mg 2+, Ca 2+, Fe 2+, Na + and Cl -. - Many other minerals (eg., Mn 2+, Mo 2+, Co 2+, Cu 2+ and Zn 2+ ) 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.

9. 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 o C) Mesophiles ( 30-37 o C) Thermophiles ( at 50-60 o C) Heat-shock response is induced to stabilize the heat-sensitive proteins of the cell. Environmental factors Aeration Obligate aerobes Facultative anaerobes Microaerophilics Obligate anaerobes (Capnophilics: bacteria that do not produce enough CO 2 and, therefore, require additional CO 2 for growth.) Ionic strength and osmotic pressure Halophilic (Greek for "salt-loving“)

10. 1.O 2 reduced to H 2 O 2 by enzymes. 2. O 2 reduced to O 2 - by ferrous ion. 3. In aerobes and aerotolerant anaerobes, O 2 - is removed by “Superoxide dismutase”, while H 2 O 2 is removed by “Catalase”. 4. Strict anaerobes lack both Catalase and Superoxide dismutase. Toxicity of O 2 for Anaerobes

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

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

13. 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

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

15. 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.

16. 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

17. Fermentation: metabolic process in which the final electron acceptor is an organic compound. 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 O 2, but CO 2, SO 4 2-, and NO 3- 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

18. 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

19. Saccharomycetes E. coliClostridium PropionebacteriumEnterobacter Streptococcus Lactobacillus

20. 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.

21. 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

22. 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, NO 3 - ) as terminal acceptor  Anaerobic respiration  Produce less ATP

23. Aerobic Glucose Metabolism x2

24. 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

26. Nucleic acid synthesis

27. 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 Nucleic acid synthesis

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

30. 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

31. Cultivation methods Medium 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 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.

34. 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 10 6 x 10 = 2.2 x 10 9 /ml

35. 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.

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

37. The End