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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 • 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 O2 2O2 flavoproteins TOXIC 2H2O2 catalase hydrogen peroxide + 2H+ Ferrous ion 2O2- superoxide radical superoxide dismutase 2H2O + O2 O2 + H2O2 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 stationary b-lactams effective here growth • Stationary phase - slowed metabolic activity and growth; limiting nutrients or toxic products • Death phase - exponential l og OD OR log CFU/ m l • Log phase - exponential death exponential (log) not here lag loss of viability; natural or induced by detergents, antibiotics, heat, radiation, chemicals Growth rate dependent on bacterium, conditions Maximum attainable cell density ~1010/ml (species-dependent) Lysozyme – effective all time, hr Bacterial Culture Systems stationary death l og OD OR log CFU/ m l • 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 exponential (log) lag time, hr 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 mediated a. facilitated diffusion b. active transport - group translocation c. 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 Facilitated Diffusion no Active Transport (Group Translocation) Active Transport (Substrate Translocation) Substrate Trapped by P; Gly Gly-P equilibrated PEP, ATP Altered (P); Concentrated ATP, PMF Example Unchanged; Concentrated Glc Glc-6-P (phosphotransferase system, PTS) 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 Division Class Subclass Order Family Tribe Genus Species – Subspecies Eubacteria Gracilicutes Scotobacteria Spirochaetales Spirochaetaceae Borrelia Borrelia burgdorferi 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 Electrophoresis 1 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 1 2 3 4 DNA Cut with restriction enzyme 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 Hybridization ds DNA heat ss DNA Total DNA or specific sequence + labeled http://members.cox.net/amgough/ Fanconi-genetics-PGD.htm DNA (ss; 3H, fl) of known 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 rRNA rRNA - associated with ribosome; critical for protein synthesis transcription translation (DNA ------------> mRNA -------------> protein) • binds initiation site (Ribosome binding site, ShineDelgarno 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 Translation Initiation Ribosome 3’ end of 16S rRNA 3’ A 5’ N U N UCCUCCA mRNA 5’-NNNNNNAGGAGGU-N5-10-AUG-NNNn-3’ ShineInitiation Delgarno Codon sequence 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)