microbial physiology and genetics 微生物及免疫學研究所 何漣漪
TRANSCRIPT
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
Required elements
C, H, O sources (amino acids, lipids, nucleic acids, sugars)
N source (amino acids and nucleic acids)
S source (amino acids)
P source (nucleic acids, membrane lipids, ATP)
K, Mg, Ca, Fe (enzyme cofactors, etc.)
Growth factors
Compounds that bacteria require but cannot synthesize
Nutritional factors
Energy sources
Sunlight for phototrophs
Oxidation of chemical compounds for chemotrophs
Nutritional diversity (concerning the energy source and carbon source)
Photoautotrophs
(primary producers)
Photoheterotrophs
Chemoautotrophs
Chemoheterotrophs
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.
Temperature
Psychrophile (15 oC - 20 oC)
Mesophile (30 oC - 37 oC)
Thermophile (50 oC - 60 oC)
pH
Neutrophile (pH 6 - 8)
Acidophile (pH 1-5)
Alkaliphile (pH 9-11)
Environmental factors for microbial growth
Oxygen availability
Obligate aerobe
Obligate anaerobe
Facultative anaerobe
Microaerophile (5-10% O2)
Water availability
Osmophile
Halophile
Obtaining a pure culture
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.
Cultivating bacteria on a solid medium (bacterial isolation)
Colony: population of bacterial cells arising from a single cell.
Streak-plate method
Pour plate method
Culture of microorganisms
Complex (rich) media
nutrient agar or broth;
blood agar or chocolate agar for more fastidious bacteria.
Chemically defined (minimal media)
Selective media
Inhibitors for organisms other than the one being sought are added.
Culture mediaDifferential media
Substances that certain bacteria change in a recognizable way are added.
Nutrient broth Glucose-salt
Peptone GlucoseDipotassium
Meat extract phosphateMonopotassium
Water phosphateMagnesium sulfateAmmonium sulfateCalcium chlorideIron sulfateWater
Principles of bacterial growth
Bacteria multiply by binary
fission.
Microbial growth is defined
as an increase in the
number of cells in a
population. Bacterial growth curve
Bacterial growth in laboratory conditions
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.
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.
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.
Assimulation (anabolism): energy-requiring
Dissimulation (catabolism): energy-acquiring
Bacterial Metabolism
Focal metabolites: metabolic intermediates that link
anabolic and catabolic pathways.
Glycolysis
Pentose phosphate pathway
TCA cycle
Respiration (aerobic and anaerobic)
Fermentation
Glycolysis (the Embden-Meye
rhoff-Parnas path
way)
Substrate-level phosphorylation
water
Oxidative phosphorylation
The electron transport chain
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
E. coliClostridium
Propionebacterium Enterobacter
StreptococcusLactobacillus
Microbial GeneticsProkaryotic microbes: bacteria
Prokaryotic genome
Chromosomal DNA: double-stranded; circular; haploid.
Extrachromosomal genetic elements
Plasmids (autonomously self- replicating)
Phages (bacterial viruses)
Transposons (DNA sequences that move within the same or between two DNA molecules)
Eukaryotic microbes:
fungi, yeasts
Eukaryotic genome
Chromosomal DNA
Mitochondrial DNA
Plasmids in yeast
Bacterial GeneticsGene mutation
Spontaneous mutationBase substitution; addition and deletion of
nucleotides; transposition
Induced mutationChemical mutagens; transposition; radiation
Gene transferTransformation
Natural and artificial competence
TransductionConjugation
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)
TransformationNatural competence
Electroporation
TransformationArtificial competence
plasmid
Plasmid
or
Transduction
F+ cell F+ cell
F+ cell (donor) F- cell (recipient)
Conjugation Transfer of plasmid
F plasmid
R plasmid
R plasmid
R: drug resistance
RTF: transfer of R plasmid
Obligate aerobe
Facultative anaerobe
Obligate anaerobe
Microaerophile
Increased CO2 (for capnophils)
Candle jar; CO2 incubator
Microaerophilic
Culture methods
AnaerobicAnaerobic jar; anaerobic chamber; reducing agents
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MacConkey agar plate
Blood agar plate
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Caused by tautomeric shift
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Transposition by transposable elements (Insertion sequences and transposons)
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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
Enrichment culturesIsolating an organism from natural sources
Maintaining stock cultures
Agar slant
Store agar slant cultures in a re
frigerator.
Stock at –70 oC
Store a pure culture in the pres
ence of 17% glycerol.
Lyophilization (freeze drying)
Dry a pure culture with a lyophi
lizer. This can be stored at roo
m temperature for years.
Direct cell count
Count under a microscope;
cell-counting instrument
Measuring biomass
Turbidity;
total weight;
chemical constituents
Viable cell count
Plate counts;
membrane filtration;
Detecting cell products
Methods to detect and measure bacterial growth