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Fundamentals II: Bacterial Physiology and Taxonomy. Janet Yother, Ph.D. Department of Microbiology [email protected] 4-9531. Learning Objectives. Requirements for bacterial growth Culturing bacteria in the lab Bacterial mechanisms for transporting substrates - PowerPoint PPT Presentation
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Fundamentals II:Bacterial Physiology and
Taxonomy
Janet Yother, Ph.D.Department of Microbiology
Learning Objectives
• Requirements for bacterial growth• Culturing bacteria in the lab• Bacterial mechanisms for transporting
substrates• Methods for identifying, classifying
bacteria
Bacterial Growth and Metabolism
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
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)
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
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
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)
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
OD
OR
b-lactamseffective here
not here
Lysozyme – effective all
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, hrlog
CFU
/ml
log
OD
OR
Bacterial Growth on Solid (Agar) Medium
Each colony arose from a single bacterial cell (or chain for streptococci, cluster for staphylococci)
Nutrient Uptake
1. Hydrolysis of nonpenetrating nutrients by proteases, nucleases, lipases
2. Cytoplasmic membrane transport - protein mediateda. facilitated diffusion b. active transport - group translocationc. active transport - substrate translocation
Facilitated Diffusion
• Passive mediated transport• No energy required • Carrier protein equilibrates [substrate]
in/out of cell• Phosphorylation traps substrate in cell• Glycerol = example
Active Transport - Group translocation
• Requires energy (PEP, ATP)• Carrier protein concentrates substrates in
cell• Substrate altered and trapped in cell• Glucose = example
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.
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)
Bacterial Taxomony
How bacteria are named, classified, and identified
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
Bacterial Nomenclature• Kingdom Eubacteria• Division Gracilicutes• Class Scotobacteria• Subclass• Order Spirochaetales• Family Spirochaetaceae• Tribe• Genus Borrelia • Species Borrelia burgdorferi
– Subspecies
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)
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)
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
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
Multilocus Enzyme Electrophoresis1 2 ref
Starch gel; enzyme assays to detect proteins; shifts in mobility due to changes in protein (amino acid) sequence
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
Restriction Fragment Length Polymorphism (RFLP) analysis
DNACut with restriction
enzyme
1 2 3 4
Agarose gel stained with ethidium bromide
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
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
DNA Hybridization - PCR
http://www.246.ne.jp/~takeru/chalk-less/lifesci/images/pcr.gif
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
Sensitivity of rRNArRNA - 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
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
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
http://asiago.stanford.edu/RelmanLab/supplements/Nikkari_EID_8/nikkari2002.html
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
Domains (Kingdoms)Based on evolutionary relationships
• Eukaryote (Plants, Animals, Protists, Fungi)• Eubacteria (Eubacteria)• Archaea (Archaea)