Part 2 Chris Microbiology Exam 1 Study Guide.pdf

- o Descriptive names consist of genius and specific epithet (binomial nomenclature) ▪ Genus always a noun, written first, and capitalized ▪ Epithet lowercase, usually adjectives Domains o Based on ribosomal RNA Phage Typing/Serological Testing o Compare phages that form plaques for bacterial diseas...

- o Descriptive names consist of genius and specific epithet (binomial nomenclature) ▪ Genus always a noun, written first, and capitalized ▪ Epithet lowercase, usually adjectives Domains o Based on ribosomal RNA Phage Typing/Serological Testing o Compare phages that form plaques for bacterial diseases Prokaryotic taxonomy based on GC content Microbial Metabolism Metabolism – anabolic, catabolic - The collection of controlled biochemical rxns within a microbe o 8 elementary statements that guide metabolism ▪ Every cell acquires nutrients as building blocks and energy for metabolism ▪ Metabolism requires energy from light or from catabolism/breakdown of acquired nutrients ▪ Energy often stored in ATP bonds ▪ Using enzymes, cells catabolize nutrients to form precursor metabolites ▪ Cells use precursors, enzymes, E from ATP to construct larger building blocks in anabolism ▪ Cells use enzymes + ATP to anabolically link blocks together to form macros in polymerization rxns ▪ Cells typically divide in 2 when doubled in size - Two major classes of metabolic rxns o Catabolism ▪ Break large molecules into smaller products and make ATP ▪ Major source of cellular energy ▪ Energy made here drive anabolic pathways ▪ Involve hydrolysis of bonds, emergencies (release E/energetically favorable) ▪ Examples: Cellular respiration o Anabolism ▪ Synthesize large molecules from smaller ones, utilizing ATP in the process ▪ Dehydrate bonds to build polymers ▪ Endergonic (E trapped in bonds, requires more E than E released in formation) ▪ Examples Photosynthesis - Basic Chem Reactions underlying Metabolism o Redox ▪ Simultaneous with eachother ▪ Use e- carriers NAD+, NADP+, FAD o ATP Production and E storage ▪ Phosphorylation - Cells phosph ADP to ATP in 3 ways o Substrate-level o Oxidative o Photo- Enzymes o Catalysts of rxns (not consumed) ▪ Incr rxn rate Functions of enzymes in cells, enzyme regulation, inhibition, Enzyme-Substrate Interactions, Cofactors, Coenzymes, Enzyme activity is affected by temperature, pH and salt concentration, allosteric control, feedback inhibition, In competitive, inhibition, non-competitive inhibition - Function of enzymes in cells o Serve to catalyze rxns by lowering activation energy needed for rxn to proceed forward o Enzyme function types ▪ Hydrolases – For catabolic hydrolysis of bonds (i.e. Lipase) ▪ Isomerases – Rearrangement of atoms within molecule (neither anabolism nor catabolism) ▪ Ligase/Polymerase – Joins two+ chemicals together (anabolic), (i.e. synthetases) ▪ Lyase – spits a chemical into smaller parts WITHOUT using water (catabolic), (i.e. aldolase) ▪ Oxidoreductase – transfer of e- or H atoms from one molecule to another ▪ Transferase – moves a functional group from one molecule to another (may be anabolic), (i.e. hexokinase) - Enzyme regulation o Activation by binding to molecules (apoenzymes) ▪ Nonprotein molecules are cofactors, i.e. Mg2+ ▪ Organic molecules/proteins are coenzymes (NAD+, NADP, FAD, Tetraflouride, CoA, Pyridoxal Phosphate, Thiamine Pyrophosphate) o Once activated they become holoenzymes o RNA molecules called ribozymes (ribosomes themselves are ribozymes) - Enzyme-Substrate Interactions o Active site holds substrate in favorable position for catalysis ▪ Hydrolysis of substrate bonds Bond stretched and weakened to break ▪ Dehydration into new bonds Molecules held by enzyme such that collision occurs and new bonds are formed - External factors affecting enzymatic activity o Temperature ▪ Optimal T; lower T slows down activity, higher T denatures enzymes o pH ▪ optimal pH; lower pH or higher pH slows activity ▪ extremes denature o Chemical agents ▪ Alcohols and phenols inactive enzymes and precipitate proteins ▪ - Several anti microbial interfere with enzymatic action, kill cells by blocking metabolism Modulation o Feedback inhibition ▪ End product inhibits enzyme earlier in path, prevents too much product o Competitive inhibition ▪ Inhibitor molecule blocks active site ▪ Can overcome by increasing [S] o Noncompetitive Inhibition/Allosteric inhibition ▪ Inhibitor binds away from AS, changes enzyme shape so no S can bind Diverse sources of energy and the cellular management of energy - Energy sources o Carbohydrates primary energy sources for anabolic reactions ▪ Glucose most common CHO used Catabolized in two processes: o Cellular respiration o Fermentation o Glycolysis ▪ Breakdown of glc to extract energy ▪ A cellular respiration, uses ETC to power ATP synthesis Aerobic = O2 gas utilized in final step Anaerobic = inorganic molecules utilized in final step o Fermentation follows glycolysis in microbes without ETCs roles of ATP in cells, energy strategies in microorganisms - ATP hydrolysis provides energy needed for endergonic reactions o Flagellar motion o Active transport o Spore formation o Biomolecule synthesis - ATP cannot be stored o Must reserve energy via different methods o Store in high E bonds o Long-term storage is in glycogen or lipids o Catabolism of such molecules to reform ATP ~3mn per second main catabolic pathways and their locations in aerobic respiration – glycolysis, Krebs cycle, oxidative phosphorylation/ETC, its input and output, and how it is linked to oxidative phosphorylation. - Glycolysis o Within cytosol/cytoplasm o Split 6C glc into two 3C molecules o A substrate-level phosphorylation – the direct transfer of phosphate between two substrates o NET GAIN OF 2 ATPs, 2 NADHs, 2 Pyruvate ▪ Input 2 ATP in energy-required/preparatory reactions (endergonic) ▪ Gain/output of 4 ATP in energy harvesting reactions (exergonic) - - - - - Pre-Krebs/TCA o Resultant 2 pyruvates decarboxylated and sent to mitochondrial matrix (of eu-) or in cytosol (pro-) o 2 pyruvate decarboxylated, 2 NAD+ reduced to NADH ▪ Net molecules of synthesis of acetyl-CoA 2 Acetyl-CoA 2 NADH 2 CO2 o TCA starts with Acetyl-CoA Krebs/TCA (substrate level phosphorylations) o Breaking of high E bonds through e- transfer to NAD+ and FAD to form NADH and FADH2 o Net products ▪ 2 ATP ▪ 2 FADH2 ▪ 6 NADH ▪ 4 CO2 All substrate-level phosphorylation reactions (glycolysis + TCA) o 10 NADH o 2 FADH2 o 4 ATP Oxidative Phosphorylation/ETC o Refers to oxidation – the loss of e- pairs from NADH and FADH2 to drive ATP synthesis o Occurs in cell membrane of prokaryotes o Within mito inner membrane (cristae) of eukaryotes o Occurs in 3 events: ▪ Electron transfer ▪ Electron transport ▪ Chemiosmosis o Electron Transport (similar to light-dependent photosynthesis in that both have ETC and a proton gradient) ▪ Where most significant production of ATP occurs in a series of redox rxns ▪ Carrier molecules pass e- from each other to final e- acceptor Flavoproteins Ubiquinones (lipophilic, ring structure, nonprotein) Metal-containing proteins Cytochromes (heme + protein) ▪ Establishes proton gradient that pump ATP synthase ▪ O2 the final e- acceptor (hence why oxidative phosphorylation) ▪ In aerobic respiration: O2 the final e- acceptor ▪ Anaerobic: not O2 o Chemiosmosis uses EC gradient to generate ATP ▪ Creates 34 ATP molecules Beta oxidation can breakdown fatty acids to generate acetyl-CoA and start TCA without encountering glycolysis results of aerobic respiration - Net product Table Pathway ATP Produced Glycolysis (SLP) Acetyl-CoA Synth + TCA (SLP) ETC (OP) Total Net Total 4 2 34 40 38 ATP Used 2 0 0 2 NADH Produced (make 3 ATP) 2 8 -10 0 FADH2 Produced (make 2 ATP) 0 2 -2 0 reactions of anaerobic respiration - Same reactions aside from final e- acceptor o Can use SO4, 2-to reduce to H2S (obligate anaerobes) o Bacillus and Pseudomonas use NO3 to produce NO2-, N2O, N2 (facultative) o Archaea and Methanogens reduce CO3 2- to CH4 - Leads to lower ATP yield and slower growth of cultures Fermentation and how fermentation is used by natural biological systems, process of fermentation and the products that result - When glc cannot be completely oxidized in respiration - Cells req constant source of NAD+ o Cannot be obtained only using glycolysis + krebs - Provides cells with alt source NAD+ o A partial oxidation of sugar to release E using organic molecule from within cell as final eacceptor, to recycle NAD+ back into glycolysis for ATP production - ATP synthesis by SLP o 2 produced - Types of fermentation detected o Methyl Red Test ▪ Detects ACID end products o Votes-Proskaeuer Test ▪ Detects NEUTRAL end products o Helps to ID pathogens o Improper fermentation of food —> illness and tissue damage - Comparing Aerobic Respiration, Anaerobic Respiration, Fermentation Aerobic Resp Anaerobic Resp Fermentation Oxygen Required Yes No No Type of Phosphorylation SLP + OP SLP and OP SLP Final E- (Hydrogen) Acceptor O2 NO3, SO4, CO3, externally Cellular organic mol acq molecules Potential Molecules ATP Prod 38 (pro-) 4-36 2 per Molecule Glc 36 (eu-) Amphibolic Sources of Cellular Building Blocks - Other anabolic pathways have amphibolic sources o Amphibolic rxns can proceed in either direction (catabolism or anabolism) o Incl: ▪ Glucose-6-Phos (generated in glycolysis, can synthesize into lipopolysaccharides) ▪ Fructose-6-Phos (generated in glycolysis, can synth into PG) ▪ G3P (generated in glycolysis, can synth into glycerol portion of lipids) ▪ Phosphoglyceric Acid (glycolysis, can synth into AAs: C,G,S) ▪ PEP (glycolysis, can synth into AAs: F,W,Y) ▪ Pyruvic Acid (glycolysis, synth into AAs: A,L,V) Formation of Macromolecules - Anabolic pathways to make proteins, carbohydrates, lipids, nucleotides, amino acids - Reversal of catabolic pathways (rxns in either direction are amphibolic – seen in both forward and backward pathways of catabolism/anabolism) - Inclusions: o Gluconeogenesis (carbohydrate formation) o Lipid synthesis/Steroid Synthesis (as seen in Mycobacterium, makes lipids in form of waxy mycolic acid) o Amino Acid synthesis (from Pentose Phosphate Pathway) o Fatty acid synthesis (reverse beta-oxidation) o Nucleotide synthesis (Pentose phosphate pathway) Carbohydrate Biosynthesis - Reversal of Glycolysis, called gluconeogenesis - Acetyl-CoA —> Pyruvate —> OAA —> G3P and DHAP —> F16BP —> F6P (leave here to make PG) —> G6P (leave here to make Glycogen)—> Glc —> Starch Pentose phosphate pathway, Entner-Doudoroff pathway, Calvin Benson cycle - Penrose Phosphate o Alternative to glycolysis o Less E-efficient o Makes precursor metabolites and NADPH ▪ To make DNA nuc, steroids, fatty acids (when glycolysis/E production not as important) - ED Pathway o Substitute to normal glycolysis ▪ Only in prokaryotes ▪ 1 ATP, 1 NADH, 1 NADPH produced - Calvin-Benson o In photosynthetic pathways ▪ Capture light E to use to synth CHOs from CO2 and H2O o Requires chlorophylls, photosystems (within thylakoids) o Can be light-independent or light-dependent ▪ Light dependent E- move down ETC and pump protons across membrane Phosphorylation uses proton-motive force to gen ATP Cyclic or noncyclic ▪ Light Independent Do not req light directly Use ATP and NADPH generated by light-dependent rxns Key rxn is carbon fixation by Calvin-Benson Cycle o 3 steps ▪ Fixation of CO2 ▪ Reduction of NADP+ to NADPH ▪ Regeneration of RuBP o How all O2 produced on Earth Microbial Nutrition and Growth media, and culture, and describe basic techniques for isolation, including isolated colony, pure culture, subculture, mixed culture, and contaminated culture - Media o What makes up the substance that a microbe population grows on, contains nutrients for growth - Culture o Act of cultivating microbes or the microbes that are cultivated - Inoculum of microbes introduced into nutrient-rich medium o Various sources to obtain – environmental specimens (from external env), clinical specimens (i.e. from patients), stored specimens (lab grown) - Isolation techniques (to obtain pure cultures – cultures of one single microbe species) o Pure cultures arise from a single progenitor (termed the Colony forming unit – CFU) o Aseptic techniques required o Two common techniques: ▪ Streak plates Heavy streaks that desaturate colony throughout plate to eventually only get small areas of colony formation that can be counted ▪ Pour plates Serial dilution of sample until colony can be counted - Culture Media o Nutrient broth (liquid) o Agar (make media solid) - Mixed culture has multiple cultures - Contaminated cultures can be mixed cultures if they are unwanted (contaminants) - After inoculating media, then culture it via incubation o Different incubation based on microbe type identifying microbes from samples (see below) chemically defined and complex media, Enriched media - Chemically defined o All ingredients to media is known - Enriched media (i.e. chocolate agar) o Broth/solid with rich supply of special nutrients that promote growth of desired organism - Selective media (i.e. PEA agar) o Added inhibitors to medium that discourage growth of certain microbes while promoting growth of others - Differential Media (i.e. MacConkey Agar) o Permits differentiation of organisms that grow - Complex media o Exact chemical comp unknown, useful when nutrient needs of an organism are unknown functional media; list several different categories, and explain what characterizes each type of functional media - Selective and Differential Media, Miscellaneous Media, Lactose fermentation and MacConkey Agar, hemolysis and blood agar - Differential Media o Different bacteria growing on media distinguished by: ▪ The presence of visible changes in the medium ▪ Difference in appearance of colonies o Examples ▪ MacConkey Agar differentiates bacterial colonies that produce lactose fermentation (pink color) vs non-lactose fermenters (white color) [i.e. E. Coli vs Salmonella Enterica or E.Coli vs S. Aureus] In E.Coli vs Salmonella enterica (differential) o Differentiation between Gram Neg bacteria based on ability to ferment lactose In E.Coli vs Staph Aureus (selective) o Selects for gram-negative bacteria (E.Coli) and inhibits Gram positive growth (Staph Aureus) ▪ Mannitol Salt Agar differentiates S. Aureus from other bacteria Changes mannitol plate from red normal color to yellow ▪ Durham Tubes A carbohydrate utilization tube that differentiates between acid fermenters that require O2 from obligate anaerobes Alcaligenes faecalis does not ferment/turn tube yellow while E.Coli ferments and produces acid to turn tube yellow ▪ Blood Agar (enriched and differential – enriched with blood) To culture fastidious orgs and differentiates between hemolytic microbes o Can differentiate between partial, complete, and no digestion of blood (different species of Streptococcus) ▪ Thioglycollate (THIO) broth Supports growth of all categories of bacteria from obligate aerobes to obligate anaerobes Have different concentration gradient of dissolved O2 in tube from high to low, bc loose fitting cap on tube o Obligate aerobes grow at surface o Obligate anaerobes killed at top, grow at bottom with no O2 o Facultative grow better near top but can grow near bottom but more sparsely o Aerotolerant can grow in O2 but not necessary, unaffected by O2 levels in tube and grow uniformly Obligate Parasites and Unculturable Microorganisms, fastidious organisms - Obligate parasite and unculturable microbes require special culturing techniques o Obligate parasites (i.e. viruses) require animal and cell culture ▪ Used when artificial media is inadequate o Low-O2 cultures ▪ CO2 incubators and Candle jars (low O2, high CO2) - Vast majority of bacteria and archaea are unculturable - Fastidious microorganisms require complex media to grow because nutrient needs are unknown/require too many for single plates main categories of nutritional types among organisms – obligate Aerobes, anaerobes, facultative aerobes & facultative anaerobes, microaerophilic, aerotolerant anaerobes, autotrophs, chemotrophs, phototrophs (see below) Growth requirements - Chemical and Energy Requirements, organic and inorganic nutrients, Chemical and Energy Requirements - Availability of nutrients o Nutrients are the E sources, obtain E by breaking chem bonds o Carbon Sources ▪ Autotrophs – Carbon source is CO2 ▪ Heterotrophs – Carbon source is organic mols o Source of Energy ▪ Chemotrophs – energy from molecular compounds ▪ Photographs – Energy source from light o Electron Source ▪ Organotrophs – heterotrophs acquire e- and H atoms from same organic molecules that provide them C source ▪ Lithotrophs – autotrophic organisms acquire e- or H atoms from inorganic molecules - Moisture o Water necessary for growth and to carry out metablism - Temperature o Thermophiles prefer high T o Hyperthermophiles prefer >80*C o Mesophiles grow best at moderate T (e.g. 37*C, the T of human body) o Psychrophiles prefer cold T (deep ocean water T) o Psychrotrophes (subset of psychrophiles) prefer moderately cold T (4*C, as in fridges and AC) - pH o Neutrophils, most microbes prefer moderate pH between 7-7.4 o Acidophiles prefer 214.7PSI) Gaseous Atmosphere o Obligate aerobes req O2 o Microaerophiles req reduced [O2] o Obligate Anaerobes killed by O2 presence o Capnophiles req incr [CO2] o Facultative Anaerobes can grow in no O2 presence but grow better in O2 presence o Facultative Aerobes can grow in O2 presence but grow better with no O2 presence Nitrogen Requirements o Anabolism ceases with lack of N, acquired from organic and inorganic mols o All cells recycle N for AA and nucleotides o N Fixation essential for life on Earth Trace elements o Req in small amounts Growth Factors o Cannot be synthesized by certain organisms o Incl: ▪ AAs ▪ Cholesterol ▪ Heme ▪ NADH ▪ Niacin/Vit B3 ▪ PABA mutual, commensal, and parasitic associations - As seen in bacterial associations and biofilms (resultant from quorum sensing) o Live in association with diff species o Can be: ▪ Antagonistic/parasitic – one organism benefits while other is harmed Bacterial antagonism occurs in the gut micro biome where indigenous microbes obtain the majority of nutrients available o Invading dx-causing microorganisms do not have enough nutrients for their survival and growth, an innate source of immunity provided by indigenous microbes of the gut and body ▪ Synergistic/commensal – individual members benefit from assc but may not be in direct contact with each other ▪ Symbiotic/mutual – members of assc are interdependent, maintain close nutritional/physical contact; both benefit binary fission, bacterial growth curve and its practical importance, importance of log phase, stationary phase and lag phase and implications in effect of antibiotic - Binary fission o Bacteria divide in two when reach optimum size, grow in log form with 2^n growth, n = generation number - Practical importance o Faster/lower generation time means more bacteria grow in same amount of time as a species with slower/higher generation time o Lower the generation time means culture division can rapidly spread in the body o Need to give Antibiotics more rapidly to a pt with a bacterial disease that has lower generation time; has higher severity and greater risk of death than higher gen time populations - Population growth curve determined by growing a pure culture of organisms in a liquid medium at a constant temperature o Collect samples at fixed intervals and plot o 4 phases ▪ Lag phase where growth is stationary To get accustomed to environment and feed on nutrients ▪ Log exponential phase Nutrients>number of microbes Allows for exponential growth of culture while nutrients outweigh the number of cells ▪ Stationary phase Cells dying = Cells growing ▪ Death phase Nutrient needs of cells outweigh the amount of available nutrients in media tools used to detect and count cells in culture - Tools used to count cells in a culture o Spectrophotometer ▪ Determine growth by measuring turbidity of medium – more turbid/less light extending through media means the more generations within the culture o Viable plate count ▪ Determine number of viable bacteria in liquid sample through serial dilutions of liquid, inoculating said liquid onto nutrient agar and count number of colonies o Chemostats ▪ Maintain microbial pops in a particular phase of growth ▪ An open system, req fresh medium and removal of old medium Microbial growth is defined as the increase of cell number in a population. The time it takes for a generation to double is the generation time; this varies among species. If the generation time of an organism and the starting number of a population of that organism are known, it is possible to calculate how many cells will be present after a given time, something that has medical significance. - 2^n - Can measure growth o Useful bc: ▪ Can determine severity of infections ▪ Determine effectiveness of food preservation techniques ▪ Measure degree of contamination of water supplies ▪ Evaluate antibiotic effectiveness o Direct methodology ▪ Microscopic counts Direct methodology not req incubation: electronic counters o Coulter counter ▪ Count cells as they interrupt electrical current o Flow cytometry ▪ Detect changes of light transmission as cells pass detector Direct methods req incubation: o Serial dilution o Membrane filtration o Most probable number o Indirect methodology ▪ Turbidity ▪ Metabolic activity ▪ Dry weight ▪ Molecular methods (isolate DNA seq of unculturable prokaryotes [archaea and bacteria])