Lecture Exam #2

metabolism

refers to the sum of all chemical reactions within a living organism 2 types: ① catabolism ② anabolism

protein synthesis inhibitors (MOA)

(antibacterials) *interrupts protein translation* all from genus streptomyces - disrupts 70s ribosomes ➤ *streptomycin* - binds to 30s subunit of ribosome -*changes shape of mRNA and is read mRNA incorrectly* -gram *negative bacteria* ➤ *tetracyclines* - binds to 30s subunit of ribosomes -*disrupts binding of tRNA* -broad spectrum: *gram negative and positive bacteria* ➤ *chloramphenicol* - binds to 50s subunit of ribosome; large subunit makes peptide bond between 2 amino acids -*disrupts peptide bond formation* -broad spectrum

cell wall synthesis inhibitors (MOA)

(antibacterials) *𝜷-lactam antibiotics* ➤ penicillins - mold penicillium ➤ cephalosporins - mold cephalosporium both have bactericidal effects - kill microbe -susceptible to osmotic lysis -cell wall ruptures *antibiotic resistance:* ⟶𝜷-lactamase cleaves 𝜷-lactam ring -whole molecule springs open and loses its shape -inhibits peptidoglycan *augmentin* ⟶ for humans amoxicillin (kills 80%; there's resistance) + clavulanic acid (kills 95%) (*clavulanic acid inhibits 𝜷-lactamases*) *clavamox* ⟶ for animals; amoxicillin with clavulanic acid

disruption of cell membrane targets (MOA)

(antibacterials) ➤ *polymyxins* *MOA*: -*membrane disruption* -effective *against Gram-negative* -polymyxin B -can only be used topically (it can disrupt the membrane of our cells) -antibiotic from genus bacillus

microaerophiles

grow at low concentrations of oxygen

properties of antimicrobial agents

➤ *selective toxicity* - drugs that kill microbes but don't harm us *CHEMOTHERAPEUTIC INDEX* = *max tolerable dose/kg body weight* ➗ *minimum effective dose/kg body weight* -better when number is bigger ex. *5 mg/kg* (max dose) ➗ *5 mg* (min effective does) = 1 *not good* ex. 50 mg/kg ➗ 5 mg/kg = 10 *much better* -still under the maximum tolerable dose -anything over max tolerable does = side effects ➤ *spectrum of activity* *narrow* -isoniazid: treats tuberculosis, inhibits fatty acid synthesis (acid fast cell wall) -polymyxins: effective against gram negative *broad* -tetraclyclines: effective against multiple taxonomic groups

how does resistance develop?

➤ antibiotic use selects for resistant microbes ➤ *superbugs* - resistant to several antibiotics (multidrug resistance) -natural selection MDR - multidrug resistance XDR - extensively drug resistance → tuberculosis is heading towards XDR

determining microbial sensitivities

➤ disk diffusion or *kirby-bauer method* ➤ *dilution method* -minimal inhibitory concentration (MIC) -minimal bactericidal concentration (MBC)

mechanisms of drug resistance

➤ enzyme development ➤ mutations in target molecules ➤ enzyme activity changes ➤ alterations in anabolic pathways ➤ alterations in membrane permeability

drug mechanisms of action (MOA)

➤ inhibition of cell wall synthesis ➤ disruption of cell membrane function ➤ inhibition of protein synthesis ➤ inhibition of nucleic acid synthesis ➤ antimetabolites

death phase

・exponential decrease ・no new cells are forming ・culture media is toxic ・nutrients are completely exhausted

log phase

・exponential growth ・conditions for growth are optimal ・bacteria are dividing with a rapid and constant rate doubling or generating time -period of growth -very short amount of time: ~8 hrs *chemostat* - continue exponential growth by constantly adding and removing equal amounts of culture media

stationary phase

・number of new cells formed is equal to the number of dying cells ・stabilization of the population ・not optimal conditions ・limiting factor -carbon source is being depleted -culture media is experiencing harmful changes in pH

nucleic acid synthesis inhibitors (MOA)

(antibacterials) ➤ *rifamycin* - *blocks transcription* very effective against *mycobacterium tuberculosis*: penetrates tubercles and kills mycobacteria -antibiotic from streptomyces -side effects: *rifampin* ⟶ by-product of metabolizing rifamycin (red man syndrome) ➤ *quinolones* - *blocks DNA replication* -synthetic ex. fluoroquinolones - treat UTIs and pneumonia side effect: can cause spontaneous tendon rupture

antimetabolites

(antibacterials) ➤ *sulfonamides* (sulfa drugs) - competes with PABA -*competitive inhibitor* ⟶ slows it down when it binds to enzyme -*inhibits metabolic pathway that makes folic acid* -kills bacteria ➤ *isoniazid* - *inhibits pathway to make fatty acid (mycolic acid)* -effective against *mycobacteria* -synthetic ⟶ narrow spectrum

oxygen (bacterial growth)

(can affect bacterial growth) during respiration, toxic by-products of O₂ are formed normally: O₂ + 4 H+ + 4 e- ⟶ 2 H₂O *superoxide free radicals and hydrogen peroxide occur during cellular respiration; toxic forms of oxygen* alternatively: *superoxide free radical* O₂ + e- ⟶ O₂⁻ *hydrogen peroxide* O₂⁻ + 2 H+ + e- ⟶ H₂O₂ (good disinfectant; bad antiseptic) *① obligate aerobes* *② facultative anaerobes* *③ obligate anaerobes* *④ microaerophiles*

dilution method

(determining microbial sensitivities) A method of testing antibiotic sensitivity in which organisms are incubated in a series of tubes containing known quantities of a chemotherapeutic agent *minimal inhibitory concentration (MIC)* looking for lowest concentration of antimicrobial agent and highest dilution that inhibits growth -dilution increases as concentration decreases (↑ dilution ↓ concentration) -*bacteriostatic* *minimal bactericidal concentration (MBC)* lowest concentration at which an antimicrobial agent will kill a microorganism; need to subculture into clean media without antibiotic -*bactericidal*

kirby-bauer method

(determining microbial sensitivities) *used for antibiotics* A disk diffusion test to determine the sensitivity or resistance of a microorganism to chemotherapeutic agents -standardized method for carrying out disk diffusion test -sensitivity and resistance determined using tables that relate zone diameter to degree of microbial resistance -table values plotted and used to determine if concentration of drug reached in body will be effective media used: *mueller-hinton agar* *weakness: doesn't determine whether a drug is bactericidal or bacteriostatic* variables: -size of zone -diffusion rate -large zone depends on concentration of antibiotic -chemotherapeutic index

oxidizers

(disinfectant) *MOA* oxidation; disrupt disulfide bonds ➤ hydrogen peroxidase is not an effective antiseptic - the catalase in our cells immediately breaks it down to water and oxygen gas ➤ effective disinfectant → table top -it can kill spores at the right concentrations

heavy metals

(disinfectant) *MOA* ➤ attach to SH (sulfhydryl groups) and *denature proteins* -strong covalent bond ➤ copper can be used to destroy green algae -can be algicide: prevent algae from growing

phenolics

(disinfectant) *disrupt plasma membrane and denature enzymes* can be used as an antiseptic and a disinfectant -derivatives of phenol phenol itself is rarely used b/c it has a bad odor and irritates skin *MOA* → *can injure plasma membrane (hydrophobic) and lipid-containing cell wall of mycobacterium spp* hydrophobic → like dissolves like bisphenols contain 2 rings

alcohols

(disinfectant) *effective against gram-positive and gram-negative* not the best antiseptic → denatures protein, traps bacteria in wounds *MOA* ➤ *dissolves lipids and denatures proteins*, kills bacteria, not endospores -needs to me mixed with water to achieve best results -water is required to denature proteins -low alcohol concentration is more effective

halogens

(disinfectant) *kills gram-positive and gram-negative; spores under right set of conditions* iodine and chlorine ➤ *iodine* - effective antiseptic *MOD* → *alters cell membrane and impairs protein synthesis* -ex. betadine: iodine is mixed with organic molecule, has longer lasting effects ➤ *chlorine* - disinfectant OCl⁻ (hypochlorite ion) *MOD* → very strong *oxidizing agent → MOA: protein denaturation* -bleach: sodium hypochlorite (NaOCl) hypochlorous acid: dissociates in water Cl₂ + H₂O → HOCl HOCl → H⁺ + OCl⁻

surfactants

(disinfectant) can decrease surface tension *MOA* → dissolve lipids → disrupt membranes → denature proteins → inactivate enzymes in high concentrations → act as wetting agents in low concentrations ➤ *soaps/detergents* - removes microbes (doesn't kill), emulsifies oil on skin, good de-germing agent → physical removal of microbes ➤ *anionic sanitizers* - negatively charged -used as surface active sanitizers -less effective, repelled by bacteria due to charge -used in restaurants ➤ *quaternary ammonium compounds* - more effective than anionic, they are cationic (positively charged) -"Quats" are the most widely used cationic detergent based on the ammonium ion (NH₄⁺) -*affects plasma membrane; changes the cell's permeability*

alkylating agents

(disinfectant) more effective with more time and higher concentration *MOA* protein denaturation and disrupts nucleic acid by adding a methyl or alkyl group ex. aldehydes ➤ glutaraldehyde can be used to disinfect hospital instruments -it is bactericidal, tuberculocidal, virucidal, and sporucidal ex. formaldehyde ➤ even though it kills everything, it's not a sterilant because it doesn't kill within 30 mins (takes longer)

feedback inhibition

(end-product inhibition) reversible non-competitive inhibition end-product accumulates and inhibits the first enzyme of a metabolic pathway, shutting down the pathway end-product can act as non-competitive inhibitor (allosteric inhibitor) saves the cell wasted energy if there are high concentrations of end-product

phenol coefficient

(evaluation of chemical antimicrobials) phenol is used as a standard to which other disinfectants are compared (good/better than phenol?) *procedure*: ➤ prepare separate dilutions of chemical agent to be tested and phenol (4 sets of dilution) -tested against gram-negative; usually *salmonella typhi* → more resistant -another tested against gram-positive; *staphylococcus aureus* → more susceptible ➤ add standard amount of culture to each dilution tube ➤ after specified amount of time, transfer a loopful of dilution tube to clean tube of nutrient broth ➤ after 48 hrs, look for the highest dilution (lowest effective concentration) that killed all the microbes ➤ compare to dilution of phenol with same effect

filter paper method

(evaluation of chemical antimicrobials) *used for chemical agents* ➤ filter paper discs are soaked with disinfectants and placed on lawns of Gram-positive and Gram-negative bacteria ➤ clear area around filter paper = zone of inhibition ➤ wildest zone of inhibition DOES NOT necessarily mean the chemical agent is the most effective. Chemical may be smaller and diffuse more easily ➤ can compare effectiveness of agent on Gram-positive vs Gram-negative (ex. merthiolate is more effective on Gram-positive than negative) a lot of factors that affect diffusion -rate of diffusion -molecular weight -etc. a large zone of inhibition doesn't mean that it is more effective

turbidity

(indirect measurement of growth) *an increase of absorption is an increase in bacterial growth* uses spectrophotometer -optical density can read transmittance or absorbance -*as absorption increases, transmission decreases* *100% transmittance means no absorption* color in solution will absorb light ↑ absorption = ↑ bacterial growth serial dilution can be correlated in absorbance reading (direct measurement) turbidity = cloudy *disadvantage* more than a million cells/mL have to be present for first trace of turbidity -not useful for small numbers of bacteria

metabolic activity

(indirect measurement of growth) making the assumption that if a product of metabolism is used up, that correlates with the growth of culture products: -O₂ -lactic acid -CO₂ *more waste product/product correlates to more growth of culture* *disadvantage* assumption, only applicable to certain circumstances

doubling times

(log phase) *E. coli* ⟶ 20 min *Mycobacterium tuberculosis* ⟶ 360 min *Bacillus stearothermophilis* ⟶ 6 min -thermophile, bacillus, spore-former, food spoilage muramil dipeptide???

membrane damage

(mechanisms of action) membranes regulate the passage of nutrients damage can cause cell contents to leak out ex ionized surfactant molecule -one end has positively charge cation and a negatively charged anion -attached to hydrocarbon -soluble when placed in water -*hydrocarbon can dissolve insoluble substances like the membrane, it surrounds hydrophobic molecule* -substance now becomes soluble and is washed away

protein damage

(mechanisms of action) ➤ heat can denature (unfold) proteins ➤ chemicals: hydrogen bonds and disulfide bonds are broken (not covalent bonds); hydrophobic core is exposed; denatured protein bonds with hydrophobic core → permanently denatured protein -can be renatured if only some bonds are broken *denaturation*: ➤ oxidation - the breaking of bonds ➤ attachment of atoms or bulky chemical groups like: -heavy metals -alkylating agents bactericidal = permanently denatures

alterations in membrane permeability

(mechanisms of drug resistance) *genetic change in membrane protein* -can deter drug from getting into microbe due to alteration of membrane proteins (*carrier proteins*)

enzyme activity changes

(mechanisms of drug resistance) alteration of enzyme development ex. sulfonamide competes with PABA and binds to enzyme and inhibits the synthesis of folic acid if *enzyme is mutated*, it can develop a higher affinity to normal substrate (PABA) and lower affinity to sulfonamide (competitive inhibitor)

alterations in anabolic pathway

(mechanisms of drug resistance) microbes can *use an alternative source* of folic acid (for example) to build complex molecules

mutations in target molecules

(mechanisms of drug resistance) ➤ *MRSA* has a *modified Penicillin Binding Brotein (PBP)* → *PBP2a* - which has a low affinity for methicillin -doesn't bind to methicillin -no 𝜷-lactamase -cell wall is not compromised -methicillin normally binds to tranpeptidase as competitive inhibitor, prevents from cross-linking the peptidoglycan ➤ PBPs function as transpeptidases - enzymes that cross-link the cell wall

enzyme development

(mechanisms of drug resistance) ➤ *methicillin* - semi-synthetic derived from penicillin; *has 𝜷-lactam ring* -*developed to be resistant to 𝜷-lactamase* -*random mutation in 𝜷-lactamase that can lead to higher affinity for modified substrate than antibiotic* ➤ *penicillin* - *has 𝜷-lactam ring* directional selection - natural selection where a phenotype is favored over the other ex. giraffes with short necks vs long necks clonal population - variation due to mutation -susceptibility or resistance to antibiotics

strong visible light

(physical antimicrobial method) *oxidation of light-sensitive materials* can be used with dyes to destroy bacteria and viruses; may help sanitize clothing

osmotic control

(physical antimicrobial method) *plasmolysis* high concentrations of sugar and salt create a *hypertonic* environment → water moves out of the cell (draws out moisture) ex. pickling, jam bacterium cannot perform any metabolic processes without water

refrigeration/freezing

(physical antimicrobial method) *refrigeration*: → bacteriostatic effects → slows growth → metabolic rate is reduced and pathogens do not produce toxins *freezing*: → completely stops the rate of growth → disadvantage: thawing can produce more bacterial growth psychotrophs can grow

freeze-drying

(physical antimicrobial method) = lyophilization *inhibits microbial growth* drying from frozen state does not kill microbes

dessication

(physical antimicrobial method) the *removal of water* bacteria *cannot grow or perform metabolism without water* *slows metabolism* they can remain viable in frozen state for years and once rehydrated, they return to vegetative state

radiation

(physical antimicrobial method) ➤ *UV* - UVB is absorbed by the nitrogenous bases of DNA and adjacent thymine residues can create dimers *damages DNA* -*bonds break and reform* A═T ☞ T═T -they *inhibit the correct replication of DNA* -can be used to disinfect operating rooms ➤ *ionizing* - can dislodge electrons and form ions; *ionizes water to release hydroxyl group -OH* *damages DNA* -can kill cells -*can be used for food preservation* ex. x-rays, gama rays, and electron beams

heat application

(physical antimicrobial method) ➤ *dry heat* - *MOA*: oxidizes molecules ex. direct flaming - incinerates use: sterilize glassware and metal objects ➤ *moist heat* - autoclaving -moist heat under pressure -temp 121℃ -15 lbs/in² pressure for 20 mins → pressure increases temp -*sterilization* kills everything including spores ➤ *pasteurization* - kills pathogens in milk ex. mycobacterium and salmonella *inhibits microbial growth* -not sterilization, prolongs shelf life -denatures protein -*phosphatase*: cleaves phosphate; if there is phosphatase activity in milk = not pasteurized

prep step (krebs cycle)

*Pyruvate is oxidized into Acetyl CoA* with an accompanying reduction of NAD⁺ to NADH ① decarboxylation of pyruvate -removing a carboxyl group (-COOH) and releasing CO₂ *2 CO₂* ② *2 NAD⁺ is reduced to 2 NADH* -acetyl is being oxidized ③ product: 2 pyruvates = *2 acetyl CoA*

standard plate count

*advantages* counting viable cells (living bacteria) *disadvantages* -takes about 24 hrs for colonies to form and count -can be several colonies serial dilution

enzyme

*biological catalyst* substances that can speed up a chemical reaction at a compatible temperature without being permanently altered themselves *substrate* specific substance *enzyme-substrate complex* temporary binding of enzyme and reactants that enables the collisions to more effective and lowers the activation energy of reaction -usually proteins -have specificity -inhibited -temp and pH preferences

controlling oxygen content

*candle jar* ・candles in the jar use up all of the oxygen and create some CO₂ ・doesn't create a complete anaerobic environment ・can grow *microaerophiles, anaerobes and facultative anaerobes* *gas peak* ・reduces all of the oxygen and water ・produces an anaerobic environment ・can grow obligate *anaerobes*

nutritional factors (bacterial growth)

*carbon source* ・*heterotrophs* - use organic carbon; use ready-made organic molecules for food ・*autotrophs* - use "inorganic" carbon (CO₂); making of own food by reducing CO₂ *energy* ・*phototrophs* - use light energy ・*chemotrophs* - use energy obtained from oxidation of organic/inorganic chemicals

protein catabolism

*exoenzyme ⟶ protease* hydrolyzes into individual amino acids *endoenzymes* ⟶ further hydrolyzes peptide *tripson* = protease, recognizes sequence of AA and cleaves it -digest larger proteins into smaller peptides *deamination* the removal of an amino group (NH₃) and converted into ammonium ion (NH₄⁺) so it can be excreted from the cell; needs to be removed before cellular respiration ⟶ krebs cycle ex. pyruvate, acetyl CoA *protease and peptidase* breakdown proteins into their component amino acids -2 ATP (substrate-level phosphorylation) -8 NADH x 3 ATP = 24 -2 FADH₂ -4 ATP 2 + 24 + 4 = 30 ATP 1 amino acid = 30 ATP

growth and cell division

*growth* increase in population, growth of a culture *binary fission* = divide in 2 ・analogous to mitosis in eukaryotes ・simpler process ・asexual reproduction - producing a clonal population -mechanisms: transfer DNA, high mutations, highly adaptable, no variation

fermentation

*homolactic acid fermentation* only produce lactic acid ① molecule of glucose is oxidized to pyruvate (2) ② pyruvate is reduced by NADH forming 2 molecules of lactic acid ⟶ end product ex. lactobacillus and streptococcus *can result in food spoilage* -pyruvate is final electron acceptor -NAD+ needs to be regenerated to continue glycolysis *alcohol fermentation* *heterolactic* produce lactic acid as well as other acids ① glucose is oxidized to 2 pyruvate molecules ② pyruvate is converted into *2 molecules of acetaldehyde and 2 molecules of CO₂* ③ acetaldehyde is *reduced to 2 molecules of NADH to form 2 molecules of ethanol* ⟶ end product ex. saccharomyces

chemoautotroph

*inorganic compounds = energy source* us the electrons reduced inorganic compounds as a source of energy, and they use CO₂ as their principal source of carbon use inorganic compounds like hydrogen sulfide (H₂S), sulfur (S), ammonia (NH₃), nitrite ions (NO₂), hydrogen gas (H₂), ferrous iron (Fe²⁺), and carbon monoxide (CO)

photoautotrophs

*light = energy source* photosynthetic bacteria: green sulfur purple sulfur cyanobacteria algae, plants *most common*

photoheterotroph

*light = energy source* use light as a source of energy but cannot convert CO₂ to sugar use carbon organic compounds like alcohols, fatty acids, etc. ex. green nonsulfur bacteria, purple nonsulfur bacteria

chemoheterotroph

*organic compounds = energy source* can use glucose as its carbon and energy source use electrons from hydrogen atoms in organic compounds as their energy source heterotrophs - further classified according to their source of organic molecules ・saprophytes - live on dead organic matter ・parasites - derive nutrients from living host most bacteria, all fungi, protozoa, and animals *most common*

energy capture

*oxidation* the removal of electrons (e-) or hydrogen from an atom or molecule *reaction often produces energy* → exothermic, exergonic (gives off heat) -dehydrogenases -coenzymes *free electrons are damaging, they have to go somewhere* *reduction* it has gained one or more electrons (e-), gain of hydrogen *gain of energy* → endothermic, endergonic (requires energy, like heat) *energy comes from covalent bonds* *redox* the pairing of these reactions; each time one substance is being oxidized, another is being reduced OIL = oxidation is losing electrons RIG = reduction is gaining electrons

mechanisms of ATP production

*oxidative phosphorylation* *substrate-level phosphorylation* *photophosphorylation*

osmotic pressure (bacterial growth)

*plasmolysis* - shrinkage of cell's cytoplasm ・when a microbe is in a solution whose concentration of solutes is higher than in the cell (hypertonic solution), water from cell leaves to higher solute concentration *obligate/extreme halophiles* an organism that requires high salt ・extreme halophile ・ higher than 15% salt *facultative halophile* an organism that does not require high salt, but can tolerate it ・2-15% salt pseudomonas spp tolerate 3-4% adding salt to food increases osmotic pressure, can be used to preserve foods

culture media purposes

*selective media* selects for gram positive/negative -bile salts *differential media* allows you to differentiate between species ・pH indicator, fermenters *enriched media* ・blood agar - TSA with 4% sheep's blood, complex media; contains a lot of proteins ・fastidious - require growth factors ⟶ AA, vitamins, ions

factors that influence enzymatic activity

*temperature* the rate of most chemical reactions increase as temp increases optimal temp: 37℃ *pH* extreme changes in pH can cause denaturation optimal pH: 7 pH *substrate concentration* the maximum rate at which a certain amount of enzyme can catalyze a specific reaction -*saturation*: an enzyme's active site is always occupied by substrate or product -under normal conditions, enzymes are not saturated with substrate -the activity of the enzyme is dependent on the substrate -*Vmax* = maximal velocity of enzyme *inhibitors*

sulfur

*uses*: sulfur containing amino acids ・cysteine, methionine ・required for synthesis of 2 vitamins: biotin and thiamine *sources*: ・*organic*: sulfur containing AA ⟶ peptone, beef extract ・*inorganic*: sulfate ion

nitrogen

*uses*: ・building nitrogenous bases of nucleotides ・building AA ・peptidoglycan *sources*: ・*organic*: AA ⟶ peptone ・*inorganic*: NH₄+, N₂, nitrate ion (NO₃⁻)

phosphorous

*uses*: ・makes ATP, phospholipids *source*: ・*inorganic*: phosphate ion

trace elements

*uses*: ・electron transport chain -cytochromes, iron sulfur proteins (FeS) ⟶ exist in oxidized/reduced state -enzyme cofactors: required for enzyme activity; part of *holoenzyme* *source*: ・*inorganic*: zinc, copper, iron, Mg²+, Ca²+

two key players in the oxidation of organic compounds

1) *dehydrogenase* remove hydrogen from substrate (each hydrogen represents an electron) ➞ enzymes *oxidoreductase* perform redox reactions ➞dehydrogenase: remove ➞oxidase: add oxygen 2) *coenzymes* vitamin-derived organic molecules -Nicotinamide Adenine Dinucleotide (NAD) • reduced to NADH -Flavin Adenine Dinucleotide (FAD) • reduced to FADH₂ H atom: 1 proton and 1 electron

temperature (bacterial growth)

3 primary groups: ① *psychrophile* cold loving microbes -live in ocean, arctic, involved in food spoilage (in fridge) ・4℃ ex. flavobacterium ② *mesophiles* moderate-temperature loving microbes ・room temp to 42℃ ex. escherechia ③ *thermophiles* heat-loving microbes ・60℃ ex. thermus ・*extreme thermophile*, 90℃ ・*thermus aquaticus* (extremophile) retain DNA polymerase, useful in PCR, withstand heat and does not denature ex. thermococcus

biofilm

A surface-coating colony of one or more species of prokaryotes that engage in metabolic cooperation.

substrate-level phosphorylation

ATP is generated when a phosphate is transferred from an organic compound directly to ADP directly phosphorylating ADP with a phosphate and energy provided from a coupled reaction -will only occur if there is a reaction that releases sufficient energy to allow the direct phosphorylation of ADP occurs during *glycolysis* and *krebs cycle*

cellular respiration

C₆H₁₂O₆ + 6O₂ ➝ 6CO₂ + 6H₂O + ATP occurs in 3 stages: ① glycolysis ② kreb's cycle ③ chemiosmosis/ electron transport chain

activation energy

Energy needed to get a reaction started enzymes lower activation energy (Eᴬ)

electron transport chain

NADH and FADH₂ donate their electrons to ETC embedded in plasma membrane of cell *electron carriers* - must exist in an oxidized and reduced state ・*FMN* - flavin mononucleotide, a coenzyme ⟶ accept and release protons and electrons ・*coenzyme Q* - lipid-soluble, non-protein carrier ⟶ accept and release protons and electrons ・*cytrochromes* - proteins containing an iron group ⟶ transfer electrons only *function of ETC* transfer of electrons to lower energy containing compounds in a stepwise manner, so that the energy that's released is manageable -otherwise spontaneous combustion transport of electrons is associated with transport pumping⟶creating a gradient -comes from release of energy (1 NADH = 3 ATP) (1 FADH₂ = 2 ATP) *input* 10 NADH 2 FADH₂ *output* 30 + 4 ATP *net* 34 ATP TOTAL YIELD = 38 ATP per glucose

disinfection

a *reduction in the number of pathogenic microbes* on an object or in material so that they pose no threat of disease -sanitation is a form of disinfection can kill endospores but don't meet the definition of sterilant bc it doesn't kill endospores within 30 mins -disinfectant needs to be applied for about 4 hours *disinfectant* - usually chemical, used for the disinfection of inanimate objects *antiseptic* - disinfectant applied to living tissue

amphibolic pathway

a pathway that is both anabolic and catabolic all reactions are reversible to make a carbon source -glucose -proteins -lipids

active site

a region on an enzyme that binds to a protein or other substance during a reaction *induced-fit model*: hand shaking *enzyme-substrate complex*

krebs cycle

aerobic metabolism *substrate level phosphorylation* occurs in cytoplasm highly oxidative pathway⟶glucose oxidized *decarboxylation*⟶made CO₂ (4x) (FAD picks up 2 H⟶less energy = 2 ATP) (NADH = 3 ATP) (FADH₂ = 2 ATP) *input* 0 *2 Acetyl CoA*: 8 NAD⁺ reduced to *8 NADH* (2 NADH from prep step) 2 FAD⁺ reduced to *2 FAD₂* *4 CO₂* *2 ATP*

glycolysis

anaerobic metabolism; occurs in cytoplasm substrate-level phosphorylation you add 2 phosphates on each end (from ATP) of glucose product is 2 molecules of pyruvate *invest 2 ATP* 2 NAD⁺ reduced to *2 NADH* 4 ATP = gained *net of 2 ATP *final product*: glucose is *oxidized to 2 pyruvate*

direct microscopic count

can be counted directly by placing solutions on special microscope slide (opposite from standard plate count) *advantages* no incubation period *disadvantages* counting both dead and viable cells trypan blue exclusion - a viability stain used to differentiate dead cells (blue) from living cells (clear)

facultative anaerobes

can use *oxygen* when it is present but are able to continue growth by using fermentation or anaerobic respiration when oxygen is not present *can perform respiration and fermentation* *produce SOD and catalase* ex. E. coli

biosynthesis of polysaccharides, lipids, and proteins

carbon structures to build large molecules may come from glycolysis and the krebs cycle may need modifications

lag phase

cell walls are getting ready to build; not completely acclimated to particular culture media ・cells are not dormant ・time of active metabolism ・cellular adaptation (carbon and energy source; turn on/off certain enzymes) ・analogous to interphase

substrate analog

chemical compounds with a chemical structure that resemble the substrate molecule in an enzyme-catalyzed chemical reaction substrate analogs can act as competitive inhibitors of an enzymatic reaction ex. penicillin, sulfa drugs → looks like substrate that binds to enzyme

aerobic respiration

complete oxidation of glucose CO₂ best outcome in terms of ATP, compared to fermentation at this point, energy is held by reduced coenzymes: NADH and FADH₂ energy is "cashed" in using electron transport chain/chemiosmosis

holoenzyme

components of an active enzyme formed by apoenzyme and cofactor; can't function without each other

anabolism

endergonic *consume energy*: builds complex organic molecules *requires energy* *dehydration synthesis reactions* release water makes small molecules into large ones

direct measurements of growth

counting colonies ① standard plate count ② direct microscopic count ③ filtration

filtration

direct measurement; for quantities of bacteria that are small ・millipores (membrane filter) → good assay for small number of bacteria ・membrane filter placed in plate (with medium) and incubated ・pore size of filter: 0.2 μm ・8 bacteria/100mL *disadvantage* ・MPN limit: takes 24 hrs to get colonies (MPN = most probable number)

non-competitive inhibition

do not compete with the substrate for the enzyme's active site, instead they interact with another part of the enzyme -denatures protein *allosteric inhibition* the inhibitor binds to a site on the enzyme other than the substrate's binding site, called *allosteric site* *this binding causes the active site to change its shape*

proton motive force

electron flow is accompanied by H+ pumping -some electron carriers are organized into complexes that pump protons electrochemical gradient high concentration of protons on the outside of the membrane creates potential energy *proton gradient has potential energy that is used in chemiosmosis to make ATP*

oxidative phosphorylation

electrons are passed from coenzymes to electron carriers in the *Electron Transport Chain* - uses inorganic phosphate (Pᵢ) transfer of electrons from carrier to carrier releases energy used to make ATP ATP is generated from the oxidation of NADH and FADH2 and the subsequent transfer of electrons and pumping of protons. That process generates an electrochemical gradient, which is required to power the ATP synthase. occurs in the plasma membrane of prokaryotes

competition

enzymes are susceptible to inhibition 2 types: 1) competitive 2) non-competitive effective way to control bacteria is to control their enzymes

enzymatic mechanisms for elimination of toxic oxygen forms

enzymes needed in aerobic environment; mechanisms to eliminate toxins made by oxygen *superoxide dismutase (SOD)* converts superoxide free radical into hydrogen peroxide 2 O₂⁻ + 2 H+ ⟶ O₂ + H₂O₂ *catalase* breaks down hydrogen peroxide to water and oxygen gas (bubbling) 2 H₂O₂ ⟶ 2 H₂O + O₂ (gas) *peroxidase* inactivate hydrogen peroxide; no gas H₂O₂ + 2 H+ ⟶ 2 H₂O (no gas) bacteria that are aerobic or facultative anaerobic will have catalase or peroxidase

defined media

exact chemical composition is known broth media

complex media

exact chemical composition is not known media components that vary from batch to batch ・extracts from meat, plants, yeasts, or digests of proteins ・*agar* - all solid media is complex

catabolism

exergonic *produce energy*: a breakdown of complex organic compounds (larger molecules) into simpler ones (smaller) *release energy* *hydrolytic reactions* use water to break chemical bonds large → small

competitive inhibition

fill the active site of an enzyme and compete with the normal substrate for the active site *sulfa drugs compete with PABA* (para-aminobenzoic acid) at the active site -enzyme converts PABA to folic acid -PABA is an essential nutrient used by many bacteria in the synthesis of folic acid penicillin is a competitive inhibitor

heterotroph

get their carbon from ready-made *organic molecules* -carbohydrates, lipids, proteins other-feeders

photophosphorylation

occurs only in photosynthetic cells, which contain light-trapping pigments such as chlorophylls

non-reversible inhibition

inhibitor binds to enzyme and makes a covalent bond with enzyme and never becomes unbound called *suicide inhibition* ex. cyanide

reversible inhibition

inhibitor bonds noncovalently to the active site and prevents substrate from binding the binding and unbinding of inhibitor

autotroph

make their own carbohydrates from *inorganic carbon* (CO₂) self-feeders

filtration (physical antimicrobial method)

membrane filtration *can be used to sterilize* *prevents passage of anything bigger that the pore size* ➤ bacteria - 0.22 µm and 0.45 µm pore size ☞ self-free extract: filter out all cells but not viruses ➤ viruses - 0.01 µm (10 nm) pore size method: *can be used for antibiotics* as a cold filter method (autoclaving will kill it) -mix water with antibiotics -hook filter (.22 µm) with syringe -push antibiotic through filter into media -sterilizes -end product: media with antibiotics

other mechanisms to acquire carbon and energy

microbes can get their energy from lipids and proteins role of *exoenzymes* and *endoenzymes* exoenzymes = function outside of cell endoenzymes = function within the cell

chemical disinfectant types

not sterilizing agents - do not kill spores within 30 mins ① surfactants ② heavy metals ③ halogens ④ alcohols ⑤ phenolics ⑥ oxidizers ⑦ alkylating agents

obligate aerobes

organisms that require *oxygen* *produce SOD and catalase* are at a disadvantage b/c oxygen is poorly soluble in the water of their environment, many are facultative anaerobes

chemiosmosis

process that synthesizes ATP using ETC and the proton gradient generated protons diffuse through a channel protein that contains the enzyme ATP synthase ADP + Pᵢ ⟶ ATP (oxidative phosphorylation) *protein gradient* a gradient formed by the difference in proton concentrations across a membrane concentration gradient (chemical) and electrical gradient

apoenzyme

protein portion of an enzyme larger portion

nutritional factors that affect bacterial growth

sulfur nitrogen phosphorous trace elements

reaction rate

the frequency of collisions containing sufficient energy to bring about a reaction heat increases both the frequency of collisions and the number of molecules that attain activation energy number of collisions increase: -when pressure increases -when reactants are more concentrated

sterilization

the killing or removal of all microbes in a material or an object, including endospores physical method is necessary to kill endospores

cofactor

the nonprotein component of an enzyme *inorganic* ex. iron, zinc, magnesium, manganese, calcium, metal ions thought to make an ionic interaction between the substrate and apoenzyme → creates a bridge that increases affinity

coenzyme

the nonprotein component of an enzyme *organic* ex. vitamin derived coenzymes that act as electron carriers: NAD+ NADP+ FMN FAD coenzyme A (CoA)

fat catabolism

the process of lipids or phospholipids being broken down by lipases *exoenzyme* ⟶ *lipase* hydrolyzes (cleaves) into glycerol and fatty acids glycerol enters glycolysis where the 6-carbon glucose is split into 3-carbon molecules *beta oxidation* converts fatty acids (2-carbons) to *Acetyl CoA* to enter the krebs cycle 14 (carbon chain) x 3 = 42 42 ➗ 2 = 21 Acetyl CoA 1 ATP 3 NADH x 3 = 9 ATP 1 FADH₂ x 3 = 3 ATP = 12 ATP/Acetyl CoA 21 Acetyl CoA x 12 ATP = 252 ATP (not including glycerol) more energy in fat

obligate anaerobes

unable to use molecular oxygen for energy-yielding reactions, most are harmed by it -require anaerobic environment cannot grow in the presence of oxygen *do not produce either enzyme* (superoxide dismutase, catalse, or peroxidase)

pH (bacterial growth)

① *acidophiles* ・optimal pH between pH 1 - 5.5 ex. helicobacter pylori - produces urease (enzyme); can survive stomach acid ・causes ulcers ・hydrolyzes urea to ammonia and CO₂ ・ammonia neutralizes stomach acid urea ⟶ CO₂ + NH₃ ② *neutrophiles* ・optimal pH between pH 6.5 - 7.5 ・most microbes ③ *alkaliphiles* ・optimal pH between pH 7.0 - 11.5

chemotherapeutic agents

① *antibiotics* - naturally occurring ② *synthetic drugs* - not natural; man-made ex. sulfonamides, quinolones ③ *semi-synthetic drugs* - produced by microbes, given modified precursor (has xtra functional groups) -microbes makes it different ex. ampicillin, tetracylines - broader spectrum

physical antimicrobial methods

① heat application ② refrigeration/freezing ③ dessication ④ freeze-drying ⑤ radiation ⑥ filtration ⑦ osmotic control ⑧ strong visible light

bacterial growth curve

① lag phase ② log phase ③ stationary phase ④ death phase

evaluation of chemical antimicrobials

① phenol coefficient ② filter paper method

mechanism of action

① protein damage ② membrane damage ③ dyes can disrupt the formation of the cell wall -inhibit the formation of gram-positive cell wall ④ nucleic acids can be modified to disrupt the flow of genetic information -can be alkylated: interrupt replication ⑤ energy production may be disrupted

physical factors affecting bacterial growth

① temperature ② pH ③ osmotic pressure ④ oxygen

indirect measurements of growth

① turbidity (spectrophotometer) -optical density ② metabolic activity

side effects

➤ *disruption of microflora* *superinfection* susceptible to an overgrowth of yeast (for example) *when taking antibiotics* *candida * bacteria usually metabolizes and produces *lactic acid* which prevents the overgrowth of yeast -when bacteria is eliminated, eukaryotic yeast overgrows bc it is not affected by antibiotic *clostridium difficile* part of normal flora, spores of C. diff can germinate and can cause superinfection after taking antibiotics -more common in elderly people

resistance to drugs

➤ *non-genetic form of resistance* - infection is initiated by inhalation, engulfed by macrophage, presented to T-cells and create memory response -*mycobacterium resistant to phagocytosis*, can kill macrophage → sends signal (cytokine) that attracts other macrophages causing a formation of tubercle (necrosis - scar tissue) ➤ *genetic* - mutation in DNA, genetic form of mutation ex. random mutation in ribosome → it can no longer bind to antibiotic -antibiotic can kill all bacteria except for the one that has random mutation -natural selection -selecting for resistant bacteria ➤ *plasmid borne* - r-plasmids: multiple genes that are resistant to different classes of antibiotics -bacteria can exchange (transfer plasmid) chromosomal DNA, don't need to be related