BMS1-3. Bacterial Metabolism - Assist. Prof. Dr. Güner Ekiz(1).pdf

BACTERIAL METABOLISM Assist. Prof. Dr. Güner Ekiz Dinçman [email protected] Faculty of Pharmacy Department of Pharmaceutical Microbiology 19.09.2023 1  Bacterial Metabolism § Before cell replication, a range of chemical r...

BACTERIAL METABOLISM Assist. Prof. Dr. Güner Ekiz Dinçman [email protected] Faculty of Pharmacy Department of Pharmaceutical Microbiology 19.09.2023 1  Bacterial Metabolism § Before cell replication, a range of chemical reactions must occur in the cell: these reactions are, collectively, referred to as metabolism Metabolism Overview - Metabolism is divided into two types of classes: - catabolism - anabolism 2  Bacterial metabolism: study of the uptake and utilization of the inorganic or organic compounds required for growth and maintenance of a cellular steady state (assimilation reactions). These respective exergonic (energy-yielding) and endergonic (energy-requiring) reactions are catalyzed within the living bacterial cell by integrated enzyme systems, the end result being self-replication of the cell. The capability of microbial cells to live, function, and replicate in an appropriate chemical milieu (such as a bacterial culture medium) and the chemical changes that result during this transformation constitute the scope of bacterial metabolism. 3  Metabolism Overview Catabolism is the chemical reactions that break down large compounds and release energy. Anabolism is the chemical reactions that require energy to build large compound Catabolic reactions furnish the energy needed to drive anabolic reactions. Energy harvested from catabolic reactions are stored in ATP molecules. ATP molecules are used to drive many anabolic reactions. 4 Cellular respiration: is a metabolic pathway that breaks down glucose and produces ATP. Bacteria have two ways of making energy aerobic respiration anaerobic respiration (involves oxygen) (does not involve oxygen) 2 ATP 38 ATP 5  Oxidation Reduction Energy is often transferred from one molecule to another by oxidation/reduction reactions. Energy is transferred when electrons from a molecule being oxidized are shifted to a molecule being reduced. a. Oxidation is the removal of electrons b. Reduction is the gaining of electrons c. Oxidation and reduction always occur together d. Most microorganisms oxidize carbohydrates as their primary source of energy. 6 Cellular Respiration 1. Cellular respiration oxidizes glucose to reduce NAD+ to NADH (NADH is an electron carrier) 2. Cellular respiration has three stages: Glycolysis, Krebs cycle and electron transport chain 7  Glucose (aerobic) Catabolism ATP as the cellular energy storage unit, can be formed during respiration or fermentation. Both contain the Glycolysis pathway; which produces ATP, the electron carrier molecule NADH, and pyruvate from glucose. Aerobic Respiration will proceed via Krebs Cycle and an ETC if there is oxygen to react as a (anaerobic) terminal electron acceptor. Oxygen is not the only possible Fermentation proceeds when there is no terminal electron acceptor in terminal electron some bacteria e.g. NO3 (nitrate) acceptor for respiration. or SO4 (sulphate) can be used; called Anaerobic Respiration. 8 (ETC)  Products of Fermentation Without any form of respiration, glycolysis products, pyruvate and NADH, will accumulate. To keep making more ATP by glycolysis, fermenting cells must convert NADH (red.) back to NAD+ (ox.) by passing its electrons to pyruvate. Reaction pathways that do this convert pyruvate to many other compounds, depending on the organism. 9  Glycolysis Glycolysis is an oxidation reduction reaction. 1. Glucose is oxidized to 2 Pyruvic acids. 2. 2NAD+ are reduced to 2 NADH. 3. Produces 2 ATP by substrate level phosphorylation 4. Occurs in the cytoplasm of both prokaryotes and eukaryotes 10  Glycolysis: 6C glucose goes to 2x 3C pyruvate plus 2 ATP net, and 2 NADH. ATP must first be invested to then yield energy from oxidation and substrate level phosphorylation of ATP. 11  Preparatory step (Pyruvate Decarboxylation) Preparatory step for Krebs cycle: Both pyruvic acid molecules from glycolysis are oxidized into two acetyl Co-A 2 NAD+ are reduced yielding two NADH Preparatory step in bacteria occurs in the cytoplasm 12 Pyruvate Decarboxylation: (Preparatory Step Before Kreb Cycle) Pyruvic acid loses a carbon in the form of CO2 ; an electron is removed to convert NAD+ to NADH, and coenzyme-A (CoA) binds to the 2C acetyl group. Acetyl CoA enters the Krebs Cycle by binding with 4C oxaloacetate to form 6C citric acid. Krebs Cycle: The cycle converts a citric acid back to oxaloacetate; losing 2 CO2 ; releasing electrons to yield 3 NADH, 1 FADH, and one ATP by substrate level phosphorylation. For one glucose the cycle runs twice. 13  Krebs cycle Oxidation of 1 acetyl co-A to carbon dioxide produces – 3 NADH – 1 FADH – 1 molecule of ATP Occurs in the cytoplasm of bacteria Substrate-level phosphorylation is the production of ATP from ADP by a direct transfer of a high-energy phosphate group from a phosphorylated intermediate metabolic compound FAD: Flavin adenine dinucleotide 14 NAD: Nicotinamide adenine dinucleotide  Energy Perspective on the Electron Transport Chain (ETC) Function * The ETC is a series of membrane bound electron carriers that transports electrons from high to low energy state, ending with oxygen accepting electrons to water. Energy release is first used to pump protons (H+) across the membrane; a proton motive force (PMF) then drives ATP synthesis. Each NADH will make 3 ATP. Each FADH will make 2 ATP. Energy State 15  Electron Transport Chain The bacterial ETC is a series of protein complexes, electron carriers, and ion pumps that is used to pump H+ out of the bacterial cytoplasm into the extracellular space. H+ flows back down the electrochemical gradient into the bacterial cytoplasm through ATP synthase, providing the energy for ATP production by oxidative phosphorylation. 16  Electron Transport Chain - Occurs in the plasma membrane of prokaryotes 17  Production of ATP H+ ions pass through ATP synthase stimulating it to produce ATP from ADP 18 19 I F erm entation. O rg an ism s p rodu ce A T P in th e absence o f o xyg en. A. Fermentation produces ATP through glycolysis. 1. Fermentation does not use the krebs cycle or the electron transport. 2. NADH is used to reduce pyruvic acid to either lactic acid or alcohol. a. NADH is converted back to NAD+ 20 Fermentation In fermentations, simple organic end products are formed from the anaerobic dissimilation of glucose (or someother compound). Energy (ATP) is generated through the dehydrogenation reactions that occur as glucose is broken down enzymatically. The simple organic end products formed from this incomplete biologic oxidation process also serve as final electron and hydrogen acceptors. 21 22 The Theoretical Maximum yield per glucose = 38 ATP According to some newer sources, the ATP yield during aerobic respiration is not 36–38, but only about 30–32 ATP molecules / 1 molecule of glucose. 23 Why? BACTERIAL NUTRITION & GROWTH Assist. Prof. Dr. Güner Ekiz Dinçman [email protected] Faculty of Pharmacy Department of Pharmaceutical Microbiology 24 AUTOTROPHS 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 Ø Culture media 43  What Is Culture Media? Ø Culture media are mediums that provide essential nutrients and minerals to support the growth of microorganisms in the laboratory. Ø Microorganisms have varying nature, characteristics, habitat, and even nutritional requirements, thus it is impossible to culture them with one type of culture media. Ø However, there are also microorganisms that can’t grow on a culture media at all in any condition – these are called obligate parasites. Ø Culturing microorganisms is essential for diagnosing infectious diseases, obtaining antigens, developing serological assays for vaccines, genetic studies, and identification of microbial species. 44  A. Classification of culture media based on consistency 1. Solid media 2. Semi-solid media 3. Liquid media Solid culture media contain agar at a concentration of 1.5-2.0% or some other primarily inert solidifying agent. Solid medium has a physical structure and allows bacteria to grow in physically informative or useful ways (e.g., as colonies or in streaks). MacConkey agar, MHA agar, nutrient agar, blood agar, etc., are some 45 examples of solid culture media.  A. Classification of culture media based on consistency 1. Solid media 2. Semi-solid media 3. Liquid media Liquid media do not contain any traces of solidifying agents, such as agar or gelatin, and large growth of bacterial colonies can be observed in the media. Liquid media are also called broths, they allow for uniform and turbid growth of bacterial strains when incubated at 37ºC for 24h. Broth medium serves various purposes such as propagation of many organisms, fermentation studies, and various other tests. Tryptic soy broth, phenol red carbohydrate broth, MR-VP broth, and 46 nutrient broth, etc.  A. Classification of culture media based on consistency 1. Solid media 2. Semi-solid media 3. Liquid media Semi-solid media has 0.2-0.5% agar concentration, and due to the reduced agar concentration, it appears as a soft, jelly-like substance. It’s mainly used to study the motility of microorganisms, distinguish between motile and non-motile bacterial strains, and cultivate microaerophilic bacteria. Hugh and Leifson’s oxidation fermentation medium, Stuart’s and Amies media, and Mannitol motility media. 47 48 B. Classification of culture media based on application/chemical composition 1. General-purpose media 2. Enriched media 3. Selective and Enrichment media 3.1. Selective media 3.2. Enrichment media 4. Differential/Indicator media 5. Transport media 6. Anaerobic media 7. Assay media 49 B. Classification of culture media based on application/chemical composition 1. General-purpose media Basal media, also called general-purpose media, are simple media that support the growth of most non-fastidious bacteria. Peptone Water, nutrient broth, and nutrient agar (NA) are basal media. These media are generally used for the primary Nutrient Agar isolation of microorganisms. 50 B. Classification of culture media based on application/chemical composition 2. Enriched media Adding extra nutrients, such as blood, serum, egg yolk, etc., to the basal medium makes an enriched medium. Enriched media are used to grow nutritionally exacting (fastidious) bacteria. Blood agar, chocolate agar, Loeffler’s serum slope, etc., are a few examples of enriched Blood Agar media. Blood agar is prepared by adding 5-10% (by volume) blood to a blood agar base. 51 B. Classification of culture media based on application/chemical composition 3. Selective and Enrichment media 3.1. Selective media This media allows the growth of certain microbes while inhibiting the growth of others. The selective growth of microbes is decided by adding substances like antibiotics, dyes, bile salts, or by pH adjustments. 52 B. Classification of culture media based on application/chemical composition 3. Selective and Enrichment media 3.1. Selective media A list of common selective media and the bacteria they’re used to culture: 53 B. Classification of culture media based on application/chemical composition 3. Selective and Enrichment media 3.2. Enrichment media This medium increases the relative concentration of specific microorganisms in the culture before plating on a solid selective medium. Unlike selective media, enrichment culture is typically used as a broth medium. Enrichment media are liquid media that also serves to inhibit commensals in the clinical specimen. Selenite F broth, tetrathionate broth, and alkaline peptone water (APW) recover pathogens from fecal samples. 54 B. Classification of culture media based on application/chemical composition 4. Differential/Indicator media It contains certain indicators like dyes or metabolic substrates in the medium composition which gives different colors to colonies of different microbial species when they utilize or react with these components. The bacterial colonies are differentiated based on their color when a chemical change occurs in the indicator, such as neutral red, phenol red, methylene blue. 55 B. Classification of culture media based on application/chemical composition 5. Transport media Transport media are useful for clinical specimens which are required to be transferred immediately to labs to maintain the viability of potential pathogens and to prevent overgrowth of commensals or contaminating microorganisms. Cary Blair transport medium and Venkatraman Ramakrishnan (VR) medium transport feces from suspected cholera patients. Sach’s buffered glycerol saline is used to transport feces from patients suspected of suffering from bacillary dysentery. Pike’s medium is used to transport streptococci from throat specimens 56 B. Classification of culture media based on application/chemical composition 6. Anaerobic media This media is for anaerobic bacteria which require low oxygen levels, extra nutrients, and reduced oxidation-reduction potential. It is supplemented with hemin and vitamin K nutrients and oxygen is removed by boiling it in a water bath and sealing it with paraffin film. Thioglycollate broth and Robertson Cooked Meat (RCM) medium which is commonly used to grow Clostridium spp. 57 B. Classification of culture media based on application/chemical composition 7. Assay media It’s used for amino acids, vitamins, and antibiotics assays. For example, antibiotic assay media is used to determine the antibiotic potency of microorganisms. 58 59 60 61 62 63 Basic Culture Technique: Streak Plate 64 65 66 67