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Fundamentals II: Bacterial Physiology and Taxonomy

Fundamentals II: Bacterial Physiology and Taxonomy. Janet Yother, Ph.D. Department of Microbiology jyother@uab.edu 4-9531. Learning Objectives. Requirements for bacterial growth Culturing bacteria in the lab Bacterial mechanisms for transporting substrates

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Fundamentals II: Bacterial Physiology and Taxonomy

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  1. Fundamentals II:Bacterial Physiology and Taxonomy Janet Yother, Ph.D. Department of Microbiology jyother@uab.edu 4-9531

  2. Learning Objectives • Requirements for bacterial growth • Culturing bacteria in the lab • Bacterial mechanisms for transporting substrates • Methods for identifying, classifying bacteria

  3. Bacterial Growth and Metabolism

  4. 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

  5. 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)

  6. TOXIC catalase flavoproteins O2 2H2O2 2H2O + O2 hydrogen peroxide + 2H+ Ferrous ion 2O2 2O2- O2 + H2O2 superoxide dismutase superoxide radical 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 in O2 • lack enzymes necessary to degrade toxic O2 metabolites; always ferment

  7. 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

  8. 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)

  9. 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 b-lactams effective here not here Lysozyme – effective all Growth rate dependent on bacterium, conditions Maximum attainable cell density ~1010/ml (species-dependent)

  10. 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

  11. Bacterial Growth on Solid (Agar) Medium Each colony arose from a single bacterial cell (or chain for streptococci, cluster for staphylococci)

  12. Nutrient Uptake • Hydrolysis of nonpenetrating nutrients by proteases, nucleases, lipases • Cytoplasmic membrane transport - protein mediated a. facilitated diffusion b. active transport - group translocation c. active transport - substrate translocation

  13. Facilitated Diffusion • Passive mediated transport • No energy required • Carrier protein equilibrates [substrate] in/out of cell • Phosphorylation traps substrate in cell • Glycerol = example

  14. Active Transport - Group translocation • Requires energy (PEP, ATP) • Carrier protein concentrates substrates in cell • Substrate altered and trapped in cell • Glucose = example

  15. 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.

  16. Protein-Mediated Transport (Uptake) Mechanisms

  17. Bacterial Taxomony How bacteria are named, classified, and identified

  18. 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

  19. Bacterial Nomenclature • Kingdom Eubacteria • Division Gracilicutes • Class Scotobacteria • Subclass • Order Spirochaetales • Family Spirochaetaceae • Tribe • Genus Borrelia • Species Borrelia burgdorferi • Subspecies

  20. 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)

  21. 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)

  22. 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

  23. 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

  24. Multilocus Enzyme Electrophoresis 1 2 ref Starch gel; enzyme assays to detect proteins; shifts in mobility due to changes in protein (amino acid) sequence

  25. 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

  26. Restriction Fragment Length Polymorphism (RFLP) analysis 2 4 1 3 DNA Cut with restriction enzyme Agarose gel stained with ethidium bromide

  27. 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

  28. DNA Hybridization heat ds DNA ss DNA Total DNA or specific sequence + labeled DNA (ss; 3H, fl) of known http://members.cox.net/amgough/ Fanconi-genetics-PGD.htm

  29. DNA Hybridization - PCR http://www.246.ne.jp/~takeru/chalk-less/lifesci/images/pcr.gif

  30. 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

  31. 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 transcription translation Phylogenetic trees are based on rRNA sequences

  32. 3’ end of 16S rRNA 3’ 5’ A N U N UCCUCCA 5’-NNNNNNAGGAGGU-N5-10-AUG-NNNn-3’ mRNA Shine- Delgarno sequence Initiation Codon Translation Initiation Ribosome Ribosome Binding Site

  33. 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

  34. http://asiago.stanford.edu/RelmanLab/supplements/Nikkari_EID_8/nikkari2002.htmlhttp://asiago.stanford.edu/RelmanLab/supplements/Nikkari_EID_8/nikkari2002.html

  35. 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

  36. Domains (Kingdoms)Based on evolutionary relationships • Eukaryote (Plants, Animals, Protists, Fungi) • Eubacteria (Eubacteria) • Archaea (Archaea)

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