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Bacterial Physiology 王淑鶯 微生物免疫學所 國立成功大學醫學院 分機: 5634 Email: [email protected] Microbial metabolism Microbial growth •Reference: Chapter 3 in Medical Microbiology (Murray, P. R. et al; 6th edition) What is Metabolism? The Greek metabole, meaning change It includes all the biochemical reactions that occur in the cell. - Catabolism - Anabolism Why do we must know the metabolism of bacteria ? Because we want to control their metabolism and know how to inhibit or stop bacteria growth Why Study Metabolism? Classification of bacteria Oxygen Tolerance Biochemical reactions Fermentation Products Food Products Commercial Products Yogurt, Sour Cream, Bread, Alcohol Citric Acid Environmental Cleanup e.g. common soil bacteria could clean up nuclear contamination Common Soil Bacteria Could Clean Up Nuclear Contamination COLUMBUS, Ohio, March 17, 2009 (ENS) - An international team of scientists has found a common soil bacterium that might one day be used to clean up radioactive toxics left from nuclear weapons production decades ago. The bacteria's cleaning power comes from their ability to "inhale" toxic metals and "exhale" them in a non-toxic form, explains team member Brian Lower, assistant professor in the School of Environment and Natural Resources at Ohio State University. Using a unique combination of microscopes, researchers at Ohio State University and scientists from Austria, Sweden, Switzerland and the United States were able to see how the bacterium Shewanella oneidensis breaks down metal to chemically extract oxygen. The study, published online this week in the journal "Applied and Environmental Microbiology," provides the first evidence that the Shewanella bacterium maneuvers proteins within the bacterial cell into its outer membrane to contact metal directly. The proteins then bond with metal oxides, which the bacteria utilize the same way we use oxygen - to breathe. "We use the oxygen we breathe to release energy from our food. But in nature, bacteria don't always have access to oxygen," said Lower. "Whether the bacteria are buried in the soil or underwater, they can rely on metals to get the energy they need. It's an ancient form of respiration." Catabolism Anabolism Metabolites degrade, break bonds, convert large molecules into smaller component often produce energy synthesis of cell molecules and structures usually requires the input of energy compounds given off by the complex networks of metabolism Overview of cell metabolism Microbes need three things to grow Energy source Nutrients (C) Suitable environmental conditions Nutrient Requirements Energy Source Phototroph Uses light as an energy source Chemotroph Uses energy from the oxidation of reduced chemical compounds Nutrient Requirements Carbon source All bacteria require carbon for growth Bacteria can been classified on the basis of their carbon source Autotroph Can use CO2 as a sole carbon source Heterotroph use more complex organic compounds such as carbohydrates and amino acids as source of carbon Energy/Carbon classification Where microbes get their energy? How do they obtain carbon? Sunlight vs. Chemical Photo- vs. Chemo- trophs Carbon Dioxide (or inorganic cmpds.) vs. Organic Compounds (sugars, amino acids) Auto- vs. Hetero- trophs Examples Photoautotrophs vs. Photoheterotrophs Chemoautotrophs vs. Chemoheterotrophs Nutrient Requirements Nitrogen source Organic nitrogen Primarily Oxidized forms of inorganic nitrogen Nitrate from the catabolism of amino acids (NO32-) and nitrite (NO2-) Reduced inorganic nitrogen Ammonium (NH4+) Dissolved nitrogen gas (N2) (Nitrogen fixation) Nutrient Requirements Inorganic nutrients (ions) contain no carbon and hydrogen atoms : phosphates, potassium, magnesium, nitrogen, sulfur, iron and numerous trace metals Organic nutrients contain carbon and hydrogen atoms. Include carbohydrates, lipids, amino acids, Nucleic acids etc. Nutrient Requirements Carbohydrates are used as the initial carbon source for many biosynthetic pathways and as electron donors (energy source) by many bacteria Amino acids are important source of carbon and nitrogen. The nitrogen is converted to ammonia. Nutrient Requirements Phosphorus Minerals is present as phosphates salts They function in energy metabolism and as constituents of nucleic acids, phospholipids, teichoic acids, ATP, etc K, Mg, Ca, Fe are required in relatively high levels Function as cofactors in enzyme reactions and as cations they act as buffers within the cells Vitamins function as coenzymes General Pathways of Metabolism -- Catabolism -1. Breakdown of macromolecules to building blocks protein amino acids polysaccharide glucose, other sugars lipid glycerol, fatty acids nucleic acids ribose, bases, phosphate no useable energy yield here - only building blocks obtained 2. Breakdown of monomers to common intermediates amino acids glucose, other sugars glycerol, fatty acids pyruvate NH4+ acetyl CoA citric acid cycle ETS/Ox Phos ATP CO2 3. Breakdown of intermediates to CO2 and electrons is accomplished through a central oxidative pathway: The Citric Acid Cycle or TCA or the Krebs Cycle. This cycle leads to the production of ATP by processes called electron transport and oxidative phosphorylation. --Anabolism-proteins polysaccharides lipids amino acids glucose, other sugars glycerol, fatty acids NH4+ pyruvate acetyl CoA citric acid cycle Anabolism, cont’d 1. utilization of critical Common Intermediates including components of TCA cycle to make building blocks 2. making building block requires energy = ATP 3. synthesis of macromolecules requires energy = ATP -- Some General Principles -•Processes of metabolism are highly controlled •Anabolism and catabolism are not necessarily balanced- one or the other may predominate in certain cells or at different times depending on cell needs •The pathway to synthesize a complex substance is not simply the reverse of the degradative pathway. Carbohydrate Catabolism Microorganisms oxidize carbohydrates as their primary source of energy Glucose - most common energy source Energy obtained from Glucose by: Respiration Fermentation Aerobic Respiration Glucose to Carbon dioxide + Water +Energy C6H12O6 + O2 6CO2 + 6H2O + 38 ATP Electrons released by oxidation are passed down an Electron Transport System with oxygen being the Final Electron Acceptor Requires Oxygen 4 subpathways 1. Glycolysis 2. Transition Reaction 3. Kreb’s Cycle 4. Electron Transport System 1. Glycolysis (splitting of sugar) Oxidation of Glucose into 2 molecules of Pyruvic acid Embden-Meyerhof Pathway End Products of Glycolysis: 2 Pyruvic acid 2 NADH2 2 ATP 2. Transition Reaction Connects Glycolysis to Krebs Cycle Pyruvic Acid Acetyl - Co A + CO2 + NADH End Products: 2 Acetyl CoEnzyme A 2 CO2 2 NADH 3. Krebs Cycle (Citric Acid Cycle) Metabolic Wheel Series of chemical reactions that begin and end with citric acid Fats, amino acids, etc. enter or leave Energy Produced: 2 6 2 ATP NADH FADH2 4. Electron Transport System Occurs within the cell membrane of Bacteria How 34 ATP from E.T.S. ? 3 ATP for each NADH 2 ATP for each FADH2 NADH Glycolysis FADH2 Glycolysis R. Krebs Cycle 2 2 6 Total 10 Total T. 10 x 3 = 30 ATP T.R. Krebs 2 Cycle x 2 = 4 ATP 0 0 2 2 Total ATP production for the complete oxidation of 1 molecule of glucose in Aerobic Respiration Glycolysis Transition Reaction Krebs Cycle E.T.S. Total ATP 2 0 2 34 38 ATP Anaerobic Respiration Electrons released by oxidation are passed down an E.T.S., but oxygen is not the final electron acceptor Nitrate (NO3-) ----> Nitrite (NO2-) Sulfate (SO24-) ----> Hydrogen Sulfide (H2S) Carbonate (CO24-) ----> Methane (CH4) Anaerobic Respiration Examples of anaerobic respiration glucose + 3NO3- + 3H2O glucose + 3SO42- + 3H+ glucose + 12S + 12H2O 6HCO3- + 3NH4+ 6HCO3- + 3SH6HCO3- + 12HS- + 18H+ All of these terminal electron acceptors have smaller reduction potentials than O2, so it is less energetically efficient than aerobic respiration Commercial applications of anaerobic respiration Anaerobic digestion is a series of processes in which microorganisms break down biodegradable material in the absence of oxygen. Anaerobic digestion is a renewable energy source because the process produces a methane suitable for energy production helping replace fossil fuels. Also, the nutrient-rich solids left after digestion can be used as fertilizer. http://www.wikipedia.com Fermentation Anaerobic process that does not use the E.T.S. Usually involves the incomplete oxidation of a carbohydrate which then becomes the final electron acceptor. Glycolysis - plus an additional step Fermentation may result in numerous end products 1. Type of organism 2. Original substrate 3. Enzymes that are present and active 1. Lactic Acid Fermenation Only 2 ATP End Product - Lactic Acid Food Production Yogurt - Milk Pickles - Cucumbers Sauerkraut - Cabbage 2 Genera: Streptococcus Lactobacillus 2. Alcohol Fermentation Only 2 ATP End products: alcohol CO2 Alcoholic Beverages Bread dough to rise Saccharomyces cerevisiae (Yeast) 3. Mixed - Acid Fermentation Only 2 ATP End products- “FALSE” formic acid acetic acid lactic acid succinic acid ethanol Escherichia coli and other enterics Propionic Acid Fermentation Only 2 ATP End Products: Propionic acid CO2 Propionibacterium spp. Saccharomycetes Clostridium Propionebacterium E. coli Enterobacter Streptococcus Lactobacillus Summary of Respiration Aerobic Respiration Glycolysis Transition Rx. Kreb’s Cycle Electron Transport Chain Anaerobic Respiration Pyruvate Lactic Acid Mixed Acids Alcohol + CO2 Recycle NADH 2 ATP / Glucose Pentose phosphate pathway Function NADPH production Reducing power carrier Synthetic pathways Role as cellular antioxidants Ribose synthesis Nucleic acids and nucleotides Microbes need three things to grow Energy source Nutrients (C) Suitable environmental conditions Environmental factors Oxygen Requirement Obligate aerobes The growth of bacteria is inhibited by absence of oxygen Pseudomonas aeruginosa, Mycobacterium tuberculosis Obligate anaerobes Growth is inhibited by the presence of oxygen Clostridium spp and Bacteriodes spp. Environmental factors Oxygen Requirement Facultative anaerobes are able to grow in the presence or absence of molecular oxygen Staphylococci, Streptococci, E. coli, etc Microaerophilic bacteria grow best under increased carbon dioxide tension Neisseria gonorrhoeae, Haemophilus influenzae Environmental factors Oxygen Requirement Aerotolerant bacteria can survive (but not grow) for a short period of time in the presence of atmospheric oxygen Tolerance to oxygen is related to the ability of the bacterium to detoxify superoxide and hydrogen peroxide produced as bye products of aerobic respiration. Enzymes that detoxify the toxic byproducts of aerobic metabolism Superoxide dismutase which converts superoxide ( a toxic metabolite) into hydrogen peroxide is present in aerobic and aerotolerant bacteria but not in obligate anaerobes. 2O2− + 2H+ H2O2 + O2 SOD Catalase which converts hydrogen peroxide into water and oxygen is also present in all aerobic bacteria but is lacking in aerotolerant organisms. Strict anaerobes lack both enzymes 2 H2O2 catalase 2H2O + O2 Environmental factors Temperature There are three critical temperature ranges for growth: (a) Minimum temperature (b) Maximum temperature (c) Optimum temperature Environmental factors Temperature Psychrophiles: Mesophiles: Has optimum temperature below 15°C but capable of growth at 0°C grow at a range of 20° – 40°C. Include most bacterial pathogens with optimum temp. at 37°C Thermophiles: microbes that has optimum temperature above 45°C with a general range of 45-80°C Most thermophiles form spores e.g. Bacillus steareothermophilus Environmental factors pH Optimum pH for most bacteria is near pH 7.0 (pH 6.5- pH 7.5) Bacteria can be classified as alkalinophiles, neutrophiles or acidophiles according to their degree of tolerance to pH changes Environmental factors Ionic strength and osmotic pressure When a microbial cell is in a hypertonic solution cellular water moves out of the cell through the cell membrane to the hypertonic solution This osmotic loss of water causes shrinkage of the cell (PLASMOLYSIS) In a hypotonic solution such as in distilled water, water will enter the cell and the cell may be lysed by such treatment (PLASMOPTYSIS) Halophiles require high salt concentrations for growth. Some bacteria can tolerate 15% salt. E.g. S. aureus Bacterial growth . Replication of chromosome Cell wall extension Septum formation Membrane attachment of DNA pulls into a new cell. Cell division Generation or doubling time: The average generation time for bacteria is 30-60 minutes under optimum conditions. Most pathogens such as Staphylococcus aureus and Escherichia coli double in 20 – 30 minutes. The longest generation time requires days. E.g. Mycobacterium leprae that causes leprosy doubles in 20 to 30 days. The growth curve The growth curve The lag phase Cells adjust to new environment. There is no change in the number of cells but metabolic activity is high leading to increase in cellular components The log or exponential phase Bacteria multiply at the fastest rate possible under the conditions provided. Are susceptible to cell wall active antibiotics Form metabolic end products The growth curve The stationary phase there is an equilibrium between cell division and cell death caused by : decrease in nutrient increase in cell population and accumulation of metabolic waste /end products Death or Decline phase The number of death cells exceeds the number of new cells formed due to lack of nutrients and accumulation of toxic waste Types of Culture media Basic media Rich media Enrichment media is used to encourage the growth of a particular organism in a mixed culture Selective media contain additional nutrients to support the growth of fastidious organisms. E.g. blood agar and chocolate agar contains salts, dyes or other chemicals that inhibit the growth unwanted microorganisms. Differential media contain chemicals that allow the distinction between different types of organisms e.g. Lactose in MacConkey agar MacConkey agar with lactose(left) and non-lactose(right) fermenters Cultivation methods For microbiologic examination For isolation of a particular organism Enrichment culture Differential medium Selective medium Isolation of microorganisms in pure culture Use as many different media and conditions of incubation as is practicable. Solid media are preferred; avoid crowding of colonies. Pour plate method Streak method For growing bacterial cells Provide nutrients and conditions reproducing the organism's natural environment. Anaerobic cultivation methods Excluding oxygen Reducing agents Anaerobic jar Anaerobic glove chamber Measurement of Microbial Growth Viable cell counts plating diluted samples (using a pour plate or spread plate) onto suitable growth media and monitoring colony formation Serial Dilution 1. Carry out dilution series 2. plate known volumes on plates 3. count only plates with 30-300 colonies (best statistical accuracy); 4. extrapolate to undiluted cell conc. 10-1 10-2 10-3 10-4 10-5 10-6 0.1 ml > 1000 220 18 Bacterial concentration: 220 x 106 x 10 = 2.2 x 109/ml 10-7 Measurement of Microbial Growth Turbidimetric measurements can estimate cell numbers accurately by measuring visible turbidity Use a spectrophotometer to accurately measure absorbance, usually at wavelengths around 400-600 nm Light scattered is proportional to number of cells. Summary Metabolic Requirements Energy source Nutrients Metabolism & the Conversion of Energy - Glucose: Glycolysis (Embden-Meyerhof-Parnas pathway) TCA cycles Pentose phosphate pathway Bacterial Growth Environment factors Growth curve Measurement of growth