1. CHAPTER 5 Nutrition, Laboratory Culture, and Metabolism of Microorganisms

2. Nutrition and Culture of Microorganisms

3. The hundreds of chemical compounds present inside a living cell are formed from nutrients. Elements required in fairly large amounts are called macronutrients, whereas metals and organic compounds needed in very small amounts are called micronutrients (Table 5.2) and growth factors (Table 5.3), respectively.

8. Some prokaryotes are autotrophs, able to build all of their cellular structures from carbon dioxide. Nitrogen is important in proteins, nucleic acids, and several other cell constituents. Iron plays a major role in cellular respiration, being a key component of cytochromes and iron-sulfur proteins involved in electron transport. To obtain iron from various insoluble minerals, cells produce agents called siderophores that bind iron and transport it into the cell (Figure 5.1).

9. Siderophores - Iron-chelating agents produced by microorganisms Anoxic conditions - iron is Fe 2+ (soluble) Oxic conditions – iron is Fe 3+ (insoluble minerals)

10. Ferric entrobactin produced by E. coli and S. typhimurium

11. Siderophore aquachelin produced by marine microorganisms (iron is low in picogram conc. (10 -12 g) Tail assists in transferring iron through the membrane into the cell

12. How to grow cells in the Lab? Culture media supply the nutritional needs of microorganisms and can be either chemically defined (defined medium) or undefined (complex medium). Selective, differential, and enriched are terms that describe media used for the isolation of particular species or for comparative studies of microorganisms.

13. Successful cultivation and maintenance of pure cultures of microorganisms can be done only if aseptic technique (Figure 5.3) is practiced to prevent contamination by other microorganisms.

14. Aseptic Technique

15. Medium containing growth factors

16. Such solid culture media immobilize cells, allowing them to grow and form visible, isolated masses called colonies (Figure 5.2). Culture media (Table 5.4) are sometimes prepared in a semisolid form by the addition of a gelling agent (e.g. agar) to liquid media.

17. Confluent growth of Serratia marcescens on MacConkey agar Isolated colonies

18. Pseudomonas aeruginosa on Trypticase-Soy agar

19. Shigella flexneri on MacConkey agar

20. Energetics and Enzymes Free energy (G) is the energy in a chemical reaction that is available to do useful work. The change in free energy during a reaction is  G0'. Table 5.5 shows the free energy of formation for a few compounds of biological interest.

21. Can not be formed spontaneously. It can decompose to Nitrogen and Oxygen

22. The chemical reactions of the cell are accompanied by changes in energy, expressed in kilojoules. A chemical reaction can occur with the release of free energy (exergonic reactions, or catabolism) or with the consumption of free energy (endergonic reactions, or anabolism).

23. Catalysis and Enzymes Activation energy is the energy required to bring all molecules in a chemical reaction into the reactive state (Figure 5.5).

24. The reactants in a chemical reaction must first be activated before the reaction can take place, and this requires a catalyst. Enzymes are catalytic proteins that speed up the rate of biochemical reactions by raising the activation energy. Enzymes are highly specific in the reactions they catalyze, and this specificity is found in the three-dimensional structure of the polypeptide(s) in the protein. Catalytic cycle of enzyme

25. Oxidation-Reduction and Energy-Rich Compounds Oxidation - removal of electron(s) from a substance Reduction – addition of electron(s) to a substance Energy (ATP) is released or consumed during oxidation or reduction reactions

26. Oxidation-reduction (redox) reactions (Figure 5.8) involve the transfer of electrons from electron donor to electron acceptor.

27. The tendency of a compound to accept or release electrons is expressed quantitatively by its reduction potential, E 0 '. In a redox reaction The substance oxidized is the electron donor. The substance reduced is the electron acceptor.

28. One way to view electron transfer reactions in biological systems is to imagine a vertical tower. The tower represents the range of reduction potentials possible for redox couples in nature, from those with the most negative E 0 's on the top to those with the most positive at E 0 's on the bottom (Figure 5.9). Anaerobic (anoxic) H 2 + fumarate (e’-acceptor) succinate Aerobic (oxic) Succinate + ½ O 2 fumarate + H 2 O (e’- donor) Others Succinate + NO 3 fumarate + NO 2 + H 2 O

30. NAD as a Redox Electron Carrier In a cell, the transfer of electrons from donor to acceptor typically involves one or more electron carriers. Some electron carriers are membrane-bound, whereas others—such as NAD + /NADH–are freely diffusible (Figure 5.10), transferring electrons from one place to another in the cell.

31. Nicotinamide adenine dinucleotide

32. Coenzymes (NAD and NADP) increase the diversity of redox reactions possible in a cell by allowing chemically dissimilar molecules to interact as primary electron donor and terminal electron acceptor, with the coenzyme acting as an intermediary (Figure 5.11).

33. Schematic of an oxidation- reduction reaction Glyceraldehyde 3-phosphate dehydrogenase Glyceraldehyde 3-phosphate (glycolysis) or Isocitrate or malate (citric acid cycle) NADH dehydrogenase Quinone Quinol 1,3-bisphosphoglycerate (glycolysis) Alpha ketoglutarate or oxaloacetate

34. Energy-Rich Compounds and Energy Storage The energy released in redox reactions is conserved in the formation of certain compounds that contain energy-rich bonds. 1. phosphate bonds 2. sulfur bond

35. The most common of these compounds is adenosine triphosphate (ATP), the prime energy carrier in the cell. Long-term storage of energy is linked to the formation of polymers (e.g. glycogen, PHB), which can be consumed to yield ATP.

36. Major Catabolic Pathways, Electron Transport, and the Proton Motive Force Glycolysis as an Example of Fermentation

37. Glycolysis is a major pathway of fermentation and is a widespread method of anaerobic metabolism. The end result of glycolysis is the release of a small amount of energy that is conserved as ATP and the production of fermentation products. For each glucose consumed in glycolysis, two ATPs are produced. Glycolysis is an anoxic process and can be divided into three major stages, each involving a series of individually catalyzed enzymatic reactions (Figure 5.14).

38. Glycolysis (Embden-Meyerhof pathway) Substrate level phosphorylation = ATP is synthesized during catabolism of an organic compound Oxidative phosphorylation = ATP is produced at the expense of proton motive force Photophosphorylation = ATP is produced during photosynthesis using a mechanism similar to oxidative phosphorylation

40. Respiration and Membrane- Associated Electron Carriers

41. Electron transport systems consist of a series of membrane-associated electron carriers that function in an integrated way to carry electrons from the primary electron donor to oxygen as the terminal electron acceptor.

42. Energy Conservation from the Proton Motive Force When electrons are transported through an electron transport chain (Figure 5.19), protons are extruded to the outside of the membrane, forming the proton motive force (Figure 5.20).

43. Electron Transport Chain

44. Generation of proton motive force during aerobic respiration Acidic environment

45. Key electron carriers include flavins, quinones, the cytochrome complex, and other cytochromes, depending on the organism. The cell uses the proton motive force to make ATP through the activity of ATP synthase (ATPase) (Figure 5.21), a process called chemiosmosis. FMN Quionone

46. ATP synthase (ATPase) Complex V Reversible reaction Two part enzyme F1 – multisubunit F0 – proton conducting channel Mechanism Proton movement Through F0 derives Rotation of c Proteins, resulting torque is transmitted to εγ. This causes a conformational change in β to allow binding of ADP and Pi. ATP is formed when β returns to original conformation

47. Carbon Flow in Respiration and Catabolic Alternatives The Citric Acid Cycle

48. Respiration involves the complete oxidation of an organic compound with much greater energy release than occurs during fermentation. The citric acid cycle (Figure 5.22) plays a major role in the respiration of organic compounds.

49. Citric acid cycle

51. Catabolic Alternatives In anaerobic respiration, electron acceptors other than O 2 can function as terminal electron acceptors for energy generation.

52. Chemolithotrophs use inorganic compounds as electron donors, whereas phototrophs use light to form a proton motive force. The proton motive force is involved in all forms of respiration and photosynthesis (Figure 5.23).

53. Energetics and carbon flow

56. Biosynthesis of Sugars and Polysaccharides

57. Polysaccharides are important structural components of cells and are biosynthesized from activated forms of their monomers.

58. For organisms growing in culture media or in nature that are not provided with these building blocks, they must be biosynthesized from simpler components, a process called anabolism (Figure 5.24).

59. Summary

60. Gluconeogenesis is the production of glucose from nonsugar precursors (Figure 5.25).

61. Biosynthesis of polysaccharides

62. Glycogen biosynthesis ADPG = adenosine diphosphoglucose

63. Gluconeogenesis Glucose formation

64. Pentoses for nucleic acid synthesis

65. Biosynthesis of Amino Acids and Nucleotides Amino acids are formed from carbon skeletons generated during catabolism (Figure 5.26).

66. Biosynthesis of amino acids

67. Nucleotides (purines and pyrimidines) are biosynthesized using carbon from several sources (Figure 5.28).

68. Biosynthesis of purines and pyrimidines, monomers of nucleotides (b) Inosinic acid, precursor of purines (c) Orotic acid and (d) uridylate are precursors of pyrimidines (a) Precursors of purines

69. Biosynthesis of Fatty Acids and Lipids Fatty acids are synthesized two carbons at a time and then attached to glycerol to form lipids (Figure 5.29).

70. Fatty acids are synthesized 2 carbon at a time Biosynthesis of fatty acids (b) Inosinic acid, precursor of purines ACP = acyl carrier protein hold growing fatty acids