fundamentals ii: bacterial physiology and taxonomy janet yother, ph.d. department of microbiology...
TRANSCRIPT
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Fundamentals II:Bacterial Physiology and
Taxonomy
Janet Yother, Ph.D.
Department of Microbiology
4-9531
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Learning Objectives
• Requirements for bacterial growth• Culturing bacteria in the lab• Bacterial mechanisms for transporting
substrates• Methods for identifying, classifying
bacteria
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Bacterial Growth and Metabolism
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Growth Requirements
• Water - 70 to 80% of cell• Carbon and energy source (may be same)
– Most bacteria, all pathogens = chemoheterotrophs (use organic molecules for carbon and energy sources)
– monosaccharides - glucose, galactose, fructose, ribose– disaccharides - sucrose (E. coli can't use), lactose (S.
typhimurium can't use)– organic acids - succinate, lactate, acetate– amino acids - glutamate, arginine– alcohols - glycerol, ribitol– fatty acids
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Growth Requirements - Nitrogen
• Inorganic source– Ammonia (NH4
+) glutamate, glutamine– Nitrogen fixation N2 NH4
+ Glu, Gln– Nitrate (NO3
-) or nitrite (NO2-)
• Nitrate reduction NO3 NO2 NH4+
• Denitrification NO3 N2 (use NO3 as electron acceptor under anaerobic conditions, give off N2)
• Organic source – amino acids, e.g. (Glu, Gln, Pro)
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Growth Requirements - Oxygen
• Aerobe (strict) - requires O2– Cannot ferment (i.e., transfer electrons and protons
directly to organic acceptor); always transfers to oxygen (respires)
• Anaerobe (strict) - killed in O2
– lack enzymes necessary to degrade toxic O2 metabolites; always ferment
superoxide radical
O2 2H2O2 2H2O + O2
flavoproteins catalase
2O2 2O2- O2 + H2O2
Ferrous ion + 2H+
TOXIC
hydrogen peroxide
superoxide dismutase hydrogen peroxide
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Growth Requirements - Oxygen
• Aerobe (strict) - requires O2– Cannot ferment (i.e., transfer electrons and protons
directly to organic acceptor); always transfers to oxygen (respires)
• Anaerobe (strict) - killed by O2– lack superoxide dismutase, catalase; always ferment
• Facultative - grows + or - O2 (respire or ferment)• Aerotolerant anaerobe - grows + or - O2 (always
ferments)• Microaerophilic - grows best with low O2; can
grow without
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Growth Requirements
• Temperature– Thermophiles - >50oC– Psychrophiles - 4oC to 20oC– Mesophiles - 20oC to 40oC
• pH - mostly 6 to 8; can vary with environment• Other
– Sulfur, phosphorous, minerals (K, Mg, Ca, Fe), growth factors (aa, vitamins)
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Bacterial Growth in Culture
• Lag phase - actively metabolizing; gearing up for active growth
• Log phase - exponential growth
• Stationary phase - slowed metabolic activity and growth; limiting nutrients or toxic products
• Death phase - exponential loss of viability; natural or induced by detergents, antibiotics, heat, radiation, chemicals
Growth rate dependent on bacterium, conditionsMaximum attainable cell density ~1010/ml (species-dependent)
lag
exponential (log)
stationary
death
time, hr
log C
FU/m
l
log O
D
O
R
b-lactamseffective here
not here
Lysozyme – effective all
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Bacterial Culture Systems
• Closed system (batch culture) - typical growth curve
• Open system (continuous culture) - chemostat. Constant source of fresh nutrients - growth rate doesn’t change (linear).
• Synchronous growth - all cells divide at same time
lag
exponential (log)
stationary
death
time, hrlo
g C
FU/m
l
log O
D
O
R
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Bacterial Growth on Solid (Agar) Medium
Each colony arose from a single bacterial cell (or chain for streptococci, cluster for staphylococci)
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Nutrient Uptake
1. Hydrolysis of nonpenetrating nutrients by proteases, nucleases, lipases
2. Cytoplasmic membrane transport - protein mediated
a. facilitated diffusion
b. active transport - group translocation
c. active transport - substrate translocation
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Facilitated Diffusion
• Passive mediated transport• No energy required • Carrier protein equilibrates [substrate]
in/out of cell• Phosphorylation traps substrate in cell• Glycerol = example
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Active Transport - Group translocation
• Requires energy (PEP, ATP)• Carrier protein concentrates substrates in
cell• Substrate altered and trapped in cell• Glucose = example
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Active Transport - Substrate Translocation
• Requires energy (proton gradient or ATP)• Carrier protein concentrates substrate in cell• Substrate unchanged. Transport system has
higher affinity for substrate outside cell.
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Protein-Mediated Transport (Uptake) Mechanisms
Energy Substrate Example
Facilitated Diffusion no Trapped by P; equilibrated
Gly Gly-P
Active Transport(Group Translocation)
PEP, ATP Altered (P);Concentrated
Glc Glc-6-P(phosphotransferase system, PTS)
Active Transport(Substrate Translocation)
ATP, PMF
Unchanged;Concentrated
Mal, aa, peptides (ABC transporters)
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Bacterial Taxomony
How bacteria are named, classified, and identified
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Bacterial Taxonomy
• Nomenclature - assignment of names by international rules. Latinized, italicized (Escherichia coli, E. coli)
• Classification - arrangement into taxonomic groups based on similarities.
• Identification - determining group to which new isolate belongs
• Bergey’s Manual of Systematic Bacteriology - standard reference
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Bacterial Nomenclature
• Kingdom Eubacteria• Division Gracilicutes• Class Scotobacteria• Subclass• Order Spirochaetales• Family Spirochaetaceae• Tribe• Genus Borrelia • Species Borrelia burgdorferi
– Subspecies
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Numerical Classification - enumerates similarities and differences
• Morphology – Microscopic - size, shape, motility, spores,
stains (gram, acid fast, capsule, flagella)– Colony - shape, size, pigmentation
• Biochemical, physiological traits - growth under different conditions (sugars, C, pH, temp, aeration)
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Serological Classifications
• Reactivity of specific antibodies with homologous antigens of different bacteria
• Usually surface antigens - capsules, flagella, LPS (O-Ag), proteins, polysaccharide, pili
• Important in epidemiology (E. coli O157:H7)
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Genetic relatedness
• DNA base composition - %GC– Very different - unrelated – Very similar - may be related
• Multilocus enzyme electrophoresis• Ability to exchange and recombine DNA• DNA restriction profile
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Genetic relatedness
• DNA base composition - %GC– Very different - unrelated – Very similar - may be related
• Multilocus enzyme electrophoresis• Ability to exchange and recombine DNA• DNA restriction profile
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Multilocus Enzyme Electrophoresis
1 2 ref
Starch gel; enzyme assays to detect proteins; shifts in mobility due to changes in protein (amino acid) sequence
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Genetic relatedness
• DNA base composition - %GC– Very different - unrelated – Very similar - may be related
• Multilocus enzyme electrophoresis• Ability to exchange and recombine DNA• DNA restriction profile
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Restriction Fragment Length Polymorphism (RFLP) analysis
DNACut with restriction
enzyme
1 2 3 4
Agarose gel stained with ethidium bromide
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Genetic relatedness
• DNA sequence - genes, whole genomes; true % identity
• DNA hybridization - total or specific sequences• DNA-RNA homology - hybridization between
DNA and rRNA (highly conserved, small part of genetic material)
• rRNA sequence - most useful – Determine sequence of DNA encoding rRNA
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DNA Hybridizationds DNA ss DNATotal DNA or specific sequence
+ labeled DNA (ss; 3H, fl) of known
heat
http://members.cox.net/amgough/Fanconi-genetics-PGD.htm
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DNA Hybridization - PCR
http://www.246.ne.jp/~takeru/chalk-less/lifesci/images/pcr.gif
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Genetic relatedness
• DNA sequence - genes, whole genomes; true % identity
• DNA hybridization - total or specific sequences• DNA-RNA homology - hybridization between
DNA and rRNA (highly conserved, small part of genetic material)
• rRNA sequence - most useful – Determine sequence of DNA encoding rRNA
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Sensitivity of rRNA
rRNA - associated with ribosome; critical for protein synthesis
(DNA ------------> mRNA -------------> protein)• binds initiation site (Ribosome binding site, Shine-
Delgarno sequence) in mRNA• must have 2o structure (base pairs with self)• Changes in critical areas likely detrimental• DNA that encodes rRNA is highly conserved among
bacteria of common ancestry
Phylogenetic trees are based on rRNA sequences
transcription translation
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Translation Initiation
3’ 5’ A N U N
UCCUCCA5’-NNNNNNAGGAGGU-N5-10-AUG-NNNn-3’
3’ end of16S rRNA
mRNA
Shine-Delgarnosequence
InitiationCodon
Ribosome
Ribosome Binding Site
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Sensitivity of rRNA
rRNA critical for protein synthesis• binds initiation site (Ribosome binding site,
Shine-Delgarno sequence) in mRNA• must have 2o structure (base pairs with self)• Changes in critical areas likely detrimental• DNA that encodes rRNA is highly
conserved among bacteria of common ancestry
Phylogentic trees are based on rRNA sequences
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http://asiago.stanford.edu/RelmanLab/supplements/Nikkari_EID_8/nikkari2002.html
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Sensitivity of rRNA
rRNA critical for protein synthesis• binds initiation site (Ribosome binding site,
Shine-Delgarno sequence) in mRNA• must have 2o structure (base pairs with self)• Changes in critical areas likely detrimental• DNA that encodes rRNA is highly
conserved among bacteria of common ancestry
Phylogenetic trees are based on rRNA sequences
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Domains (Kingdoms)Based on evolutionary relationships
• Eukaryote (Plants, Animals, Protists, Fungi)• Eubacteria (Eubacteria)• Archaea (Archaea)
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