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Microbiology Notes 7 - 10.docx

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BIOL 240
Heidi Engelhardt

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Microbiology Topic 5 Cultivation of Bacteriophage 1. Mix Bacterial host cells and phage samples together 2. Then add molten liquid agar (provides limit on the extent that the virus can clear cells 3. Incubate the plate 4. Where you will find infectious particles is shown as plaque • You can grow them if you know the bacteria host and the virus well enough Cultivation of Animal Viruses 1. Grow the viruses on host cells (in tissue culture)  HeLa cells Or 2. Grow them in embryo (fertilized) chicken or duck eggs. Viral Purification 1. Step 1 (not concentrated): Filter (take micron filters)  push through the solution  bacterial/eukaryotic will stay on filter, virus will pass through 2. Method 1 : differential centrifugation a. Take the debris of bacteria/eukaryotic cells b. Begin with low speed centrifugation c. Largest particles will pellet d. Medium speed centrifugation e. High speed centrifugation f. Pellet = virus 3. Method 2: gradient centrifugation Topic 6 Metabolism Macronutrients are nutrients that all the cells need • Topic 7 Proteobacteria • Metabolically diverse • Morphologically diverse Anoxygenic Photosynthesis (Gammaproteo) • Use bacteriochlorophyll for blue + carotenoid for red • Photoautotrophs  Chromatium – high sulfur (found lower on winogradsky)  Rhodomicrobium – lower sulfur (found higher)  Sulfur = electron  Light = energy  CO 2 carbon source Methylotrophs • Oxidizes methyl groups/derivatives for energy • Methanotrophs are subset  Oxidize only methane for energy  Both groups are obligate aerobes  Enzyme – methane monooxygenase Nitrifiers • (Chemolitho)autotroph – can be photo • Oxidize Ammonia (Nitroso-)  Enzyme – Ammonia Monooxygenase • Oxidize NO (3itro-)  Enzyme – Nitrite Oxidase Pseudomonads • Polar flagella • Very versatile  Big genes • Produce siderophore (pyoverdin) – grabs iron  Fluorescent Nitrogen Fixers (Alphaproteo) • Reduces nitrogen • Free-living  aerobes  enzyme – nitrogenase  slime layer to protect nitrogenase from O 2 • Symbiotic  Agrobacterium  Causes crown-gall tumour o Once inside, send DNA into plant cells o Gets into plant cell and their DNA is expressed o Forces plant to make amino acid that only agrobacterium can eat  Rhizobium  Mutual relationship o Plant sends out flavonoids o When bacteria there – sends nod factor o Plant allows bacteria to enter o Plants give protection + carbon for energy o Leghemoglobin brings oxygen to rhizobium o Ammonia is given to plant Enteric (Gammaproteo) • E.coli, Salmonella • Peritrichous flagella • Facultative anaerobes • Coliforms • Many are pathogens Bdellovibrio • Prey on other bacteria • Virus-like • Really fast Myxobacteria • Hunting packs  Needs quorum – for fruiting body  Large genome • Feed  replicate  spore  cycle repeats Gram-positive (Non-sporulating) • Both:  Often make pigments  Aerobic + love high salt • Staphylococcus  Firmicutes – high GC • Micrococcus  Actinobacteria – low GC Lactic acid bacteria • Aerotolerant anaerobes • Fastidious • 2 type fermenters  Homofermenter  Lactic acid  Heterofermenter  Ethanol, CO ,2lactic acid • Streptococcus pyogenes  Flesh-eating disease Gram-positive (Sporulating) Bacillus Group • Facultative/obligate aerobes • Produce antibiotics • Bacillus thuringiensis forms BT toxin  Crystalline protein (Cry protein)  Toxic in insect bellies Clostridium Group • Obligate anaerobes • Some need to fix N 2 • Clostridium botulinum – toxic to humans  At right [] – botox! Endospore • Terminal • Subterminal • Central Actinobacteria (Gram positive) • Mycobacterium  Acid-fast  Lipid-rich cell wall  Grow slowly  Many human pathogens  Leprosy o Primary infection – External lesions o Secondary infections – limbs falling off o Cannot be cultured in lab – cannot be proven by Koch’s postulate  only grows in armadillos/humans • Filamentous actinobacteria  Dessication-resistant spores  Smell of soil – geosmins (means water)  Produce antibiotics  Streptomyces  Aerial hypha (sporophores)  Wiggles – forms conidia (spores) Cyanobacteria • Oxygenic phototroph  Possess pigments  chlorophyll a  phycocyanins (blue)  phycoerythrin (red) • Found everywhere • Intracellular membranes – thylakoid • Heterocysts  Thick cell wall that keeps oxygen out and because nitrogen fixing Topic 8 Recall: • Transcription – DNA to RNA • Translation – DNA to protein • Sigma factors are accessory to RNA polymerase  Directs to a promoter • Termination loop of transcription  Rho-dependent  RNA pol sees termination  slows  rho (following pol) will kick off RNA pol  Rho-independent  RNA hairpin loop o Due to high GC content o Pulls back RNA pol  kicks off • Shine-Dalgarno  Ribosome recognizes where to start making polypeptides  Multiple Shine-Dalgarno = polycistronic  Multiple polypeptides being made at once Regulation • Constitutive genes  always on  TCA cycle  ATP synthases • Inducible genes  turned on when needed • Level of gene expression: transcription, translation, post-translation Post-translational • Proteins  Covalently modification  Adding sugar/methyl/phosphor groups  Allosteric modification  Effector binds to allosteric site causes: o Activation o Inhibition Transcription • Lac Operon  Structural genes  Operator before structural genes  Allosteric protein  Negative control o Repression – putting there  Anabolic enzymes  E.g. trytophan o Induction – removing it  Catabolic enzymes  E.g. lac operon  Promoter before the operator  Activator binding site before promoter  Allosteric protein  Positive control o E.g. maltose catabolism o promotes binding of polymerase to its promoter  physically putting it onto its promoter  altering its DNA to help it bind more strongly  E.coli has genes under 1/both/neither  Effector molecules  Co-inducers or co-activators  Co-repressors  Diauxic growth  Switching to new substrate  E.g. running out of glucose – using lactose instead o LacY (forms permease) brings lactose in and needs to break down o LacZ (forms Beta-galactosidase) – cleaves lactose  glucose, galactose, (little bit) allolactose  Allolactose is signal to E.coli there is lactose to be metabolized  LacI – repressor  binds to operator  Effector molecule (allolactose)  transcription  Cyclic AMP receptor protein (activator protein)  cAMP levels go up – no glucose  protein binds to promoter better when [] of lactose is high, glucose is low  transcription  Attenuation  RNA pol beings to transcribe – RNA never finishes its job  Leader region/sequence  Scenario 1: lots of tryptophan o Leader peptide passes region 1 pauses occupying 2  Leaves region 3 and 4 free – make nice pair GC linked in hairpin (terminator loop)  Stop polymerase  Scenario 2: low levels of tryptophan o Leader peptide doesn’t get into region 2  Region 2 and region 3 form huge hairpin but not terminator loop  RNA polymerase doesn’t get knocked off and can keep transcribing  Doesn’t happen in eukaryotes – only in prokaryotes o Eukaryotes have spatial separation of transcription/translation Quorum Sensing • Chemical signaling • Positive feedback loop  Rapid • E.g. aliivibrio fischeri  Squid spends day under sand  Population of aliivibrio grows during rest  Night comes out to hunt  Big population inside light organs o High density produces lots of N-acyl-homoserine lactone (AHL)  stimulates luminescence  AHL = co-inducer o LuxI protein catalyzes AHL synthesis  Hunt without fear of predation from below  When sun comes out – squid goes to rest o Aliivibrio is flushed out (little remain)  Scenario 1: Low concentration of AHL  Low population
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