microbial physiology and genetics 微生物及免疫學研究所 何漣漪

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

Back

MacConkey agar plate

Blood agar plate

Back

Back

Caused by tautomeric shift

Back

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Transposition by transposable elements (Insertion sequences and transposons)

Back

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