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Microbial Physiology and Genetics 微生物及免疫學研究所 何漣漪 Microbial Physiology Nutritional factors for microbial growth Environmental factors for microbial growth Pure culture Culture of microorganisms Culture media Dependence on oxygen Bacterial growth in laboratory conditions Growth curve; growth rate Microbial metabolism Growth of microorganisms Nutritional factors Required elements Energy sources C, H, O sources (amino acids, lipids, nucleic acids, sugars) Sunlight for phototrophs N source (amino acids and nucleic acids) Oxidation of chemical compounds for chemotrophs S source (amino acids) Nutritional diversity (concerning the energy source and C source) Photoautotrophs (primary producers) Photoheterotrophs Chemoautotrophs Chemoheterotrophs P source (nucleic acids, membrane lipids, ATP) K, Mg, Ca, Fe (enzyme cofactors, etc.) Growth factors Compounds that bacteria require but cannot synthesize Nitrogen source Ammonium (NH4+) is used as the sole N source by most microorganisms. Ammonium could be produced from N2 by nitrogen fixation, or from reduction of nitrate and nitrite. Sulfur source Most microorganisms can use sulfate (SO42-) as the S source. Phosphorus source Phosphate (PO43-) is usually used as the P source. Mineral source For most microorganisms, it is necessary to provide sources of K+, Mg2+, Ca2+, Fe2+, Na+ and Cl-. Many other minerals (e.g., Mn2+ , Mo2+, Co2+, Cu2+ and Zn2+) can be provided in tap water or as contaminants of other medium ingredients. Environmental factors for microbial growth Temperature Oxygen availability Psychrophile (15 oC - 20 oC) Obligate aerobe Mesophile (30 oC - 37 oC) Obligate anaerobe Thermophile (50 oC - 60 oC) Facultative anaerobe pH Neutrophile (pH 6 - 8) Microaerophile (5-10% O2) Water availability Acidophile (pH 1-5) Osmophile Alkaliphile (pH 9-11) Halophile Obtaining a pure culture Cultivating bacteria on a solid medium (bacterial isolation) A solid medium is required for obtaining a pure culture of microorganism. Agar: an algae extract, polysaccharide in nature, which very few bacteria can degrade. The agar plate contains 1.5% of agar. Colony: population of bacterial cells arising from a single cell. Streak-plate method Pour plate method Culture of microorganisms Culture media Complex (rich) media nutrient agar or broth; blood agar or chocolate agar for more fastidious bacteria. Differential media Substances that certain bacteria change in a recognizable way are added. Nutrient broth Peptone Chemically defined (minimal media) Meat extract Selective media Water Inhibitors for organisms other than the one being sought are added. Glucose-salt Glucose Dipotassium phosphate Monopotassium phosphate Magnesium sulfate Ammonium sulfate Calcium chloride Iron sulfate Water Bacterial growth in laboratory conditions Principles of bacterial growth Bacteria multiply by binary fission. (or decline) Microbial growth is defined as an increase in the number of cells in a population. Bacterial growth curve Growth rate is expressed as the doubling (generation) time E. coli: 20 min; M. tuberculosis: 12-24 h A balance between slow loss of cells through death and the formation of new cells through growth and division. The doubling time is measured during this period. The bacteria are most susceptible to antibiotics during this time. Bacteria stop growing due to decrease of nutrients and O2 supply, and accumulation of toxic metabolites. Bacteria synthesize macromolecules required for multiplication. The length of lag phase depends on the conditions in the original culture and the medium into which they are transferred. Bacterial Metabolism Assimulation (anabolism): energy-requiring Dissimulation (catabolism): energy-acquiring Glycolysis Pentose phosphate pathway TCA cycle Respiration (aerobic and anaerobic) Fermentation Focal metabolites: metabolic intermediates that link anabolic and catabolic pathways. Glycolysis (the EmbdenMeyerhoff-Parnas pathway) Substrate-level phosphorylation Oxidative phosphorylation The electron transport chain water ATP synthesis by proton motive force Fermentation: a metabolic process in which the final electron acceptor is an organic compound. The only ATP-yielding reactions of fermentation are those of glycolysis, and involve substrate-level phosphorylation. Saccharomycetes Clostridium Propionebacterium E. coli Enterobacter Streptococcus Lactobacillus Microbial Genetics Eukaryotic microbes: fungi, yeasts Eukaryotic genome Chromosomal DNA Mitochondrial DNA Plasmids in yeast Prokaryotic microbes: bacteria Prokaryotic genome Chromosomal DNA: doublestranded; circular; haploid. Extrachromosomal genetic elements Plasmids (autonomously selfreplicating) Phages (bacterial viruses) Transposons (DNA sequences that move within the same or between two DNA molecules) Bacterial Genetics Gene mutation Spontaneous mutation Base substitution; addition and deletion of nucleotides; transposition Induced mutation Chemical mutagens; transposition; radiation Gene transfer Transformation Natural and artificial competence Transduction Conjugation F and R plasmids Gene mutation Base substitution Removal or addition of nucleotides (insertion and deletion; frame shift and chain termination) Transposable elements Sources of diversity in microorganisms: gene mutation and gene transfer. Mechanisms of gene transfer Transformation: uptake of naked exogenous DNA by living cells. Conjugation: mediated by self-transmissible plasmids. Transduction: phage-mediated genetic recombination. Demonstration of transformation Avery, MacLeod, and McCarty (1944) Transformation Natural competence Transformation Artificial competence or plasmid Plasmid Electroporation Transduction Conjugation Transfer of plasmid F+ cell (donor) F- cell (recipient) F+ cell F plasmid R plasmid F+ cell R plasmid R: drug resistance RTF: transfer of R plasmid Microaerophile Obligate aerobe Facultative anaerobe Obligate anaerobe Culture methods Anaerobic Anaerobic jar; anaerobic chamber; reducing agents Back Increased CO2 (for capnophils) Candle jar; CO2 incubator Microaerophilic MacConkey agar plate Blood agar plate Back Back Back Caused by tautomeric shift Back Transposition by transposable elements (Insertion sequences and transposons) Transposable element: gene that moves from one DNA molecule to another within the same cell or from one site on a DNA molecule to another site on the same molecule Back Enrichment cultures Isolating an organism from natural sources Maintaining stock cultures Agar slant Store agar slant cultures in a refrigerator. Stock at –70 oC Store a pure culture in the presence of 17% glycerol. Lyophilization (freeze drying) Dry a pure culture with a lyophilizer. This can be stored at room temperature for years. Methods to detect and measure bacterial growth Direct cell count Viable cell count Count under a microscope; Plate counts; membrane filtration; cell-counting instrument Measuring biomass Turbidity; total weight; chemical constituents Detecting cell products