Bacteriology Module 2: Microbial Metabolism PDF

Metabolism - refers to the sum of all chemical reactions within a living organism, more specifically, within a cell. Metabolite - any molecule that is a nutrient, an intermediary product, or the end- product in a metabolic reaction. 2 Classes of Chemical Reaction...

Metabolism - refers to the sum of all chemical reactions within a living organism, more specifically, within a cell. Metabolite - any molecule that is a nutrient, an intermediary product, or the end- product in a metabolic reaction. 2 Classes of Chemical Reactions / Components of Metabolism 1. Anabolic Reaction -- synthetic; energy-consuming ( Endergonic) Includes processes that utilize energy stored in ATP to synthesize and assemble the subunits or building blocks of macromolecules that make up the cell. The chemical reaction that combines simple substances into more complex molecules. This requires energy in the form of ATP. These biosynthetic reactions generate the materials for growth. These biosynthetic reactions generate the materials for growth. Formation of Nucleic acids from Mononucleotides Formation of Polysaccharides from simple sugars  2. Catabolic Reactions -- degradative; Energy-yielding (Exergonic) the chemical reaction that breaks down complex organic compounds with the release of energy and is stored in ATP. The energy is then utilized to drive anabolic reactions this encompasses processes that harvest energy released from the breakdown of compounds (e.g., Glucose) and using that energy to synthesize ATP. Catabolic reactions are a cell’s major source of energy. ▪ Metabolism is a kind of energy-balancing act that occurs within a cell, with some metabolic reactions releasing energy and other metabolic reactions requiring energy. ▪ It is also a dynamic balance between those reactions that provide the cell with energy or building blocks / materials and those that utilize them. ▪ In addition to the energy required for metabolic pathways, energy is also required by the organism for growth, reproduction, sporulation, movement and the active transport of substances across membranes. ▪ In living systems, energy is passed from one source to another in the form of molecules. ▪ To produce energy, a cell must have an energy source that functions as an electron donor. Energy sources (electron donor): Glucose, Elemental Sulfur, Ammonia, Hydrogen gas or light. ▪ Electrons removed from these energy sources are next transferred to electron carriers, such as coenzymes NAD, then finally to its final Hydrogen acceptors. The initial energy source is oxidized as the electron carrier is reduced. (this is an oxidation-reduction reaction) Aerobic respiration - when oxygen serves as the final electron acceptor. Anaerobic respiration - inorganic substances such as NO3 or SO4 ions serve as the final electron acceptor. Fermentation - organic compounds serve as the final hydrogen acceptor. ▪ Regardless of their energy sources, all microorganisms use similar oxidation-reduction reactions to transfer electrons and similar mechanisms to use the energy released to produce ATP. ▪ In order for bacterial growth to occur, the organism must be provided with all the substances essential for the synthesis and maintenance of its protoplasm, a source of energy and suitable environmental condition. ▪ Most of the bacteria encountered in clinical specimens utilize carbohydrate to achieve their energy needs. ▪ The bacterial degradation of carbohydrates proceeds by several metabolic pathways involving a series of steps in which hydrogen ions (electrons) are successively transferred to compounds of higher redox potential, with the ultimate release of energy in the form of ATP. ▪ Glucose serves as the main carbohydrate source for bacteria and depending upon the enzymatic composition and the presence or lack of oxygen, degradation proceeds by the following pathways: 1. Embden-Meyerhoff Parnas Pathway also called Glycolytic or Anaerobic Pathway or Fermentative Pathway used primarily by anaerobic bacteria and to some degree by facultatively anaerobic bacteria as well. glucose is degraded without oxygen. Intermediate Steps: a.) Initial phosphorylation of Glucose b.) conversion of Fructose-6-phosphate by the enzyme Isomerase. c.) Cleavage to form 2 molecules of Glyceraldehyde PO4 by the enzyme Aldolase → ultimate formation of Pyruvic acid.  GLUCOSE EMBDEN MEYERHOFF PARNAS PATHWAY (Glycolytic or Anaerobic Pathway or Glucose 6-Phosphate Fermentative Pathway) Fructose 6-Phosphate Fructose 1.6-Diphosphate 1.3 Phosphoglycerate Pyruvic Acid 2H 2H Lactic Acid “Mixed Acids” Fermentation - an oxidation-reduction metabolic process that takes place in an anaerobic environment, with an organic substrate serving as the final hydrogen (electron) acceptor in place of Oxygen. ▪ Thus, Pyruvic acid acts as intermediate hydrogen acceptor but is oxidized by giving up its hydrogen ions to Sodium lactate to form Lactic acid or to other organic salts to form “Mixed Acids”. ▪ These acids are the end-products of glucose metabolism by the EMP Pathway, accounting for the drop in pH in fermentation tests used for identifying bacteria. ▪ Bacteria that possess the necessary enzyme systems can further degrade these mixed acids into alcohols, CO2, gas or other organic compounds. 2. Entner- Duodoroff Pathway (EDP) also called the Aerobic Pathway Oxygen is required for glycolysis to occur. Steps: a.) Phosphorylation of glucose b.) Unlike in the EMP, glucose is not broken into 2 triose carbon molecules. c.) Glucose is oxidized to 6-phosphogluconate and 2-keto-3-deoxy-6- phosphogluconate before forming Pyruvic acid. d.) Lacking the dehydrogenase enzymes necessary to oxidize Pyruvic acid to lactic acid or other “mixed acids”, oxidative bacteria rather transfer the available hydrogen ions from pyruvic acid into the Kreb’s cycle in which they ultimately link with elemental oxygen to form water. ENTNER DOUDEROFF GLUCOSE PATHWAY (Aerobic Pathway) Glucose 6-Phosphate Glucone-τ-Lactone 6-Phosphate 6-Phosphogluconate 2-keto-3-deoxy-6-phosphogluconate Pyruvic Acid Krebs Cycle 2H H2O ▪ Thus, the oxidative metabolism of carbohydrates is defined as the energy-yielding reaction that requires molecular oxygen (or other nonorganic elements) to serve as terminal hydrogen acceptor. ▪ Because the end-product of oxidative metabolism is water, gas is not formed from carbohydrates by oxidative organisms. ▪ The acids that are formed in the ED Pathway (glucuronic acid and its derivatives) and those produced in the Kreb’s cycle (citric acid and its derivatives) are extremely weak compared to the mixed acids → absence of drop in the reaction and conversion in the pH indicator) ▪ Some bacteria use Shunt pathways through which glucose is oxidized directly into glucuronic acid without initial phosphorylation.  3. Warburg-Dickens Hexose Monophosphate Pathway (HMP) used by aerotolerants (nonoxidative) bacteria that are capable of growing in the presence of oxygen but grow better in its absence (anaerobic) a hybrid between the EMP and ED Pathways, Steps: Initial steps in the degradation of glucose in the HMP parallels those of ED. Later, however, the glyceraldehyde-3-P04 is formed as the precursor of Pyruvic acid similar to the EMP. Therefore: the HMP provides a means for nonoxidative bacteria to degrade glucose ultimately forming pyruvic acid. (Those incapable of using the Kreb’s Cycle and passing hydrogen on to molecular O2, And, those that lack the isomerase or aldolase enzymes necessary to carry out the initial steps in the EMP.) ▪ The precursor to the formation of glyceraldehyde-3-PO4 in the HMP is Ribulose- 5-phosphate. Ribulose is a pentose thus, this pathway is otherwise known as the Pentose Pathway. ▪ It provides the major avenue by which pentoses are metabolized by a number of bacterial species. WARBURG DICKINS HEXOSE GLUCOSE MONOPHOSPHATE PATHWAY (Metabolic Pathways for the bacterial Glucose 6-Phosphate degradation of glucose) Glucono-τ-Lactone 6-Phosphate 6-Phosphogluconate Ribulose 5-Phosphate Glyceraldehyde 3-Phosphate Pyruvic Acid 2H 2H Krebs Cycle “Mixed Acids” H2O  Bacterial Growth Growth - an orderly increase in mass or number of all components of the cell. - an orderly increase of all the chemical constituents of the cell. ▪ It entails the replication of all cellular structures, organelles and protoplasmic components from the nutrients present in the surrounding environment. ▪ The main indicator of microbial viability. In fact, we do not know if an organism is alive unless it multiplies. Binary Fission - the division of a single bacterium into two daughter bacteria in a suitable environment. ▪ Bacteria reproduce by binary fission. The bacterium, which is a single cell, divides into two identical daughter cells.... The bacterial cell then elongates and splits into two daughter cells each with identical DNA to the parent cell. Each daughter cell is a clone of the parent cell. Generation Time -- - the time required to accomplish cell division, or doubling. - estimation of this can be done by: a.) actual bacterial counts at specific time intervals b.) determination of the rate of increase of a specific enzymatic activity of the bacterium Growth Rate Constant - the factor that expresses the average number of times the cells will divide in a given time. Microbial growth can be measured in terms of: 1.) Cell Concentration - the number of cells per unit volume of culture. - viable cell count 2.) Cell Density - dry weight of cells per unit volume of culture. - replaced by: a.) photoelectric measurements b.) Nitrogen determination c.) Centrifugation in special vessels A. Lag Phase Otherwise known as “Phase of Rejuvenescence” or “Physiological Youth”. this represents the period during which the cells, depleted of metabolites and enzymes as a result of unfavorable conditions that was obtained at the end of their previous culture history, adopt to their new environment. Activities: 1.) increased metabolic activity in preparation for bacterial division. 2.) increase in bacterial RNA and protein synthesis 3.) increase in bacterial cell size 4.) increase in macromolecular components 5.) increase susceptibility to physical and chemical agents B. Exponential Phase this represents the peak of growth activity in the culture medium. cells are dividing at a constant rate; multiplying by geometric progression. multiplication is steady until one of this happens: a.) either one or more nutrients in the medium become exhausted. b.) toxic metabolites/ metabolic products accumulate and inhibit growth. ❑ during this stage, bacteria are especially susceptible to agents, such as antibiotics, probably because the physiologic efficiency provides more frequent opportunities for interaction with the antibiotic and the bacterial envelope barriers are more easily traversed. C. Maximum Stationary Growth Phase decreased growth rate occurs as a result of the deleterious effect on the culture of the following: a.) accumulation of waste products b.) exhaustion of nutrients- especially Carbon and Nitrogen sources. c.) change in pH ▪ There is a slow loss of cells through death which is just balanced by the formation of new cells through growth and division. ▪ Some cannibalistic variants in the population may use the dead remnants of their own kind for nutrients. D. Death Phase / Phase of Decline this occurs when the majority of the cells have died and the death rate increases drastically. a small number of survivors may persists for months or even years – a few cells growing at the expense of nutrients released from cells that die or lyse. Death - the irreversible loss of the ability to reproduce. (grow & divide) Growth Requirements (Review of Past Lessons) A.) Physical Requirements - Oxygen, Temperature, Radiation, Osmotic Pressure B.) Chemical Requirements 1. Water: - source of moisture for bacteria (in culture medium) - Loss of water in Medium - a.) less water for metabolic pathways; and b.) relative increase of solute concentration of the medium Note: Increase solute concentration can osmotically shock the bacterial cell and can cause lysis. 2. Carbon -- all microorganisms demand small quantities of Carbon in the form of inorganic CO2 for growth. the majority of microorganisms utilize carbon in its organic form expressly for the biosynthesis of cell material. 3. Nitrogen -- Inorganic Nitrogen sources: Nitrate, Ammonia & gaseous Nitrogen Two important roles of Nitrates: a.) source of Nitrogen b.) it is associated with ATP production by serving as terminal electron acceptor during the process of anaerobic respiration. 4. Sulfur -- - an important constituent of the amino acids, Cysteine & Methionine - particularly important because of its ability to stabilize protein molecules. - it is utilized by bacteria in the form of Sulfates. 5. Phosphorus -- plays an important part in Nucleic acid metabolism Inorganic phosphates is the most widely available source of the cell. 6. Minerals – Magnesium, Potassium, Iron, Calcium, Zinc, Cobalt, Molybdenum 7. Vitamins (Organic growth factor) -- Thiamine, Riboflavin, Niacin, Biotin, Folic acid, Vit B12, Pantothenic acid Nutritional Classification (Review of Past Lessons) Nutritional patterns among microorganisms can be distinguished on the basis of 2 criteria: energy source and principal source of Carbon a.) Photoautotroph b.) Photoheterotroph c.) Chemoautotroph d.) Chemoheterotroph