Exam 3 new part of final - Lecture notes after exam 2 to end of course PDF

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lecture notes taken from slides and what prof said in class.
course with prof. Alenka...

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Exam reflection ○ Agar is used because bacteria (and most microorganisms) cannot break it down ■ This is helpful so we can see colony growth patterns and so we can distinguish colonies and count them ○ Gram negative lipopolysaccharides are in the outer membrane! ○ For 1:100 dilution, dilution factor is 100. To make 100 ml of this dilution, add 1 ml sample to 99 ml broth ○ Does Bacillus subtilis have to produce endospore specific tRNAs and ribosomes? ■ No

Regulation of gene expression of gene expression: Lac operon ● Lac operon helps to determine what genes are expressed ○ Expression is based on sugars ○ In case of lac operon, it is specific to lactose (monosaccharide) ○ Beta galactose and permease are inducible ■ In the absence of lactose, there are 1-2 molecules.cell ■ In the presence of lactose, there is 1,000,000 molecules/cell ■ These are carbs and these are good energy source for cell ● https://phet.colorado.edu/en/simulation/gene-machine-lac-operon ○ Phet simulation for lac operon!!! Remember alenka loved it last year ● Operon ○ Operator and structural genes ○ Produces polycistronic mRNA (more than one translated product can be made) ○ Lac operon consists of 3 structural gene ■ Means that it encodes 3 distinct polypeptides (must be 3 sections defined by start and stop codons) ■ On DNA template, there is regulatory gene, and then operon ● Operon goes promoter then operator then structural genes ● Repressor is usually on operator but in presence of enough lactose, repressor will be removed so therefore lactose being present enables the expression of lac operon function (gene present) ● When lactose is absent, the repressor is on operator so even though the regulatory gene and promoter is being coded, it cannot follow through due to the block from the repressor ● When lactose is removed from the system, synthesis will slow down and then stop because the repressor will go back on the operator when there is no lactose in the cell ■ Lac operon is induced but repressor is always on!!! ○ If an operon consists of 3 structural, protein encoding genes, how many sections that are defined by a start and stop codon will it contain? ■ 3 ● Genetic logic (evolutionary advantage) of cell’s negative and/or positive control of gene

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expression Lac operon consists of 3 structural, protein encoding genes. How many sections on mRNA are defined by the open reading frame that starts with a start and stops with a stop codon ○ 3 Lac operon includes an operator ○ The operator is a region of DNA ○ The operator is located between a promoter and the coding region of a structural gene Complete regulation of Lac operon depends on the presence of ○ Repressor protein ○ Promoter ○ Operator ○ Expression products of structural genes Lactose is absent from the medium E. coli is in. structural genes in lac operon ○ Are not expressed Lactose is absent from the medium e. Coli is in. you measure the products that were made. List the macromolecular products that were found here ○ Regulatory gene mRNA ○ Repressor protein Lactose is added to medium E. coli is in and after a few minutes you measure the products made in the E. coli cells. What macromolecular products were found ○ Lac operon mRNA ○ An enzyme that breaks down lactose ○ Transport protein for lactose ○ Regulatory gene mRNA ○ repressor protein The lac operon is inducible ○ The structural genes ■ Expressed in the presence of lactose ■ Not expressed in the absence of lactose Microbial biofilm ○ Believe that most of the time microbes act as members of large communities (don’t act as individuals) ○ Microbial biofilm is a community of biofilms (usually different organisms) where they work together and collaborate to thrive ○ It is the community of microbes in which microbes mostly collaborate assuming a functionality each microbe would not have had individually ■ Collaborate by exchanging DNA (horizontal gene transfer) ■ Communicate with each other ■ Quorum sensing ● Gene regulating through cell-to-cell communication ● Depends on cell density ● Environmentally controlled

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Concentration of autoinducers (AIs) depends on cell density ■ When cell density low they diffuse away from the cell ■ When cell density is high, autoinducers present inside and outside the cell and will interact with receptors that regulate transcription of certain genes ○ Autoinducer is similar to lac operon in that when lactose is present it will interact… lactose is analogous to autoinducer repressor protein How quorum sensing works in Vibrio fischeri; microorganism that is able to generate light ○ This is the microorganism that is housed by squid and when all cells reach threshold, they will glow and emit light ■ The squid will spit out some of the culture when light is not needed to decrease concentration which will turn off light and the cells will then regrow and divide and will emit light again when cell culture reaches high enough concentration

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Clicker: the gene for the receptor is expressed. Few autoinducer molecules are made and exported out of the cell. The cell ○ Cannot make luciferase ○ Cannot emit light

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○ Will not synthesize more autoinducer molecules Clicker: Vibrio fischeri is not emitting light. Select condition that would activate expression of luciferase genes ○ The increase in the concentration of receptor proteins (LuxR) ○ The increase in the concentration of autoinducer (AHL) molecules outside the cell Clicker: Vibrio fischeri is not emitting light. Because of the successful research project you have 1 mL of .00004 M solution of AHL. if you add 5 ul to the culture V. fischeri culture ○ Will emit light Biofilm gene expression ○ When there is high cell density, there will be high AHL density ○ Overall, there will be increasing reactions (ReDOx) which will result in light emission and production Biofilms, quorum sensing in Vibrio cholera & pathogenicity ○ Very often, the interactions with the environment is tied with the ability to produce biofilms ○ It is a common organism in water in several forms ■ It has flagella so it swims ○ It is also found in sedentary areas What is biofilm ○ Free swimming microorganisms will give up freedom of mobility under certain conditions and they will settle on the surface of the environment ■ Can be caused by concentration of microorganisms or environment itself ■ Requires a gene change to allow for settlement and become sedentary organism ■ Once it is sedentary, it produces components that allow it to become stuck to surface (get moved easily by water and more adhesion, the more likely it will stay put) ● Cell membrane adherence is helped by capsule. This component has polymers and loose structure which help it become stuck ■ Once sedentary, the cells are communicating and are creating a large and growing community ● Cells within biofilm continue division ● Can be change in metabolism due to the genetic change based on environmental change due to being in the biofilm (some stuff is limited such as oxygen) ○ There is horizontal gene transfer while in biofilm and while bacteria are swapping and gathering more genes, they are becoming more adverse and gaining more traits and components from others in the community and this increase in diversity helps the biofilm survive ○ There can be cheaters who take genes but don’t provide or supply and if there are too many cheaters, the environment will not survive ○ After, cells can separate and go on their own way (dispersion)

○ This process is a cycle and occurs over and over again Cholera ● Where does V. cholera live in nature ○ Cholera mode of transmission of disease ■ Lives in resivouir and is found on vector (such as kelp or algae) and anything that consumes the vector will take on the organism (serve as host) and anything that consumes this organism will take it on and so on… if a human eats shellfish that consumed vector the person will get cholera ○ Sources of cholera are humans and animals (fecal matter) ○ Vibrio cholerae are metabolically quiescent and they are metabolically active cells ● In its natural environment, water, Vibrio cholerae is found ○ In a planktonic, free swimming form ○ As a sedentary microorganism ○ Within biofilms ● How does V. cholera harm human host ○ V. cholerae virulence factor ■ Enterotoxin causes an increase in intracellular cAMP which causes stimulation of pumping Cl- ions into the intestine ● This is what causes the diarrhea and vomiting to pull water into cell and push the Cl- ions out ■ Influence on intracellular water is that it pulls water into the cell ■ Influence on intracellular electrolytes is ● V. cholerae ○ Quorum sensing regulates ■ Production of virulence factors ■ Motility ■ Biofilm formation ○ When vibrio gets into host, it will create enterotoxin or not based on the concentration of the cell in the host ■ For disease to occur, there needs to be a sufficient number of cells and microbes to make the pathogenic reaction Staphylococcus aureus ● Lab determined traits ○ This was organism that turned agar from red to yellow ○ Gram+ cocci ○ In liquid medium they formed clusters (grape like) ● It is common organism. ~30% of humans have staph aureus colonized and it is fairly commonly found on the skin ● Staph aureus interaction ○ Causes bacteremia- when bacteria is in the bloodstream and is extremely dangerous ○ Bacteremia causes:

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■ Infective endocarditis ■ Osteoarticular infections ■ Skin and soft tissue infection ■ Pleuropulmonary infections ■ Device-related infection ○ Major cause of nosocomial infections ■ Host-commensal ● Host is the living organism that harbors and is affected by another living organism (microbe) ■ Host-pathogen-parasite ● Parasite is thing that invades the organism and damages the host and lives off the host ● Pathogen and parasite must have set of viral infectors for transmission (illness) to occur ■ Disease ● Communicable ○ Agfent can be passed on from one host to another ● Non-communicable ○ Agent causes illness and it cannot be passed on ● Infectious ○ Means that infection is caused by biotic agent (virus, microbe, etc.) ● Non-infectious ○ Infection caused by abiotic agent ● Acute ○ Does not last too long ● Chronic ○ Long lasting How S. aureus switches from a commensal to a pathogen ○ What makes S. aureus virulent ■ Gene regulation and getting stuck to the surface is influenced by microbe concentration ■ Cytoxic factors (compounds that kill cells and can even kill host cells which is dangerous and harmful to the host organism) ● Leukocidin ● Protease ● Enterotoxin ● Exfoliative toxin ● TSST-1 ■ Antibiotic resistance (genes are expressed based on conditions) ● Beta-lactams ● Quinolone ● Aminoglycoside Clicker: nutrient agar plate was streaked with an overnight E. coli culture. Antibiotic

discs were placed on the inoculated plate and then incubated. As you move out from the disc, what happens to the concentration of the antibiotic ○ It decreases (this is what forms the discs from the point) ○ Even past this ring, there is still antibiotic but the zone of inhibition boundary is the lowest concentration of antibiotic that would kill the microbe ○ When discussing, remember that bacteria ARE NOT IMMUNE TO ANTIBIOTICS because they dont have immune system ○ They are resistant or susceptable (this is terms to use) ● What makes S. aureus virulent ○ Exponential phase ■ Cell surface virulence factors ● Protein A ● Fibronectin-binding protein ● Clumping factor ● Other MSCRAMMs ■ Stationary phase ● Secreted virulence factors ○ Superantigens (SEs, SEIs, TSST-1) ○ Cytolysins Staphylococcus aureus ● When autoinducer (AIP) binds to receptor (green near ArgC) it turns on signal transduction component (P on ArgA) ● Arrow going from middle to ArgB blue thing is export mechanism for auto inducer ● Arrow is used to represent stimulation or expression of genes that do metabolism or gene transcription or gene expression ● Microorganism that has the genetic ability to go from harmless to pathogenic could be caused by environmental conditions (S. aureus is one of these) ○ The pathogenic genes are expressed under some conditions such as when they divide under enclosed areas such as in a wound that is healing ○ When this happens they become pathogenic and this can be dangerous Antibiotic activity and antibiotic resistance and antibiotics in general ● Effect on bacteria of antibiotics

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Can kill cells Can inhibit growth ■ This means that the moment you remove it, all cells that were there and continued to live will continue to grow Bacteria-cell structure; type of metabolism is necessary to know about antibiotic ○ Gram + ○ Gram Current antibiotics ○ They can act on different parts of the cells ■ Beta-lactams: target cell wall biosynthesis ■ Glycopeptides: Inbiy cell wall assembly ■ Quinolones: DNA gyrase targeted ● This is wide spectrum ■ Aminoglycosides: target ribosomal subunits and inhibit protein synthesis ■ Tetracyclines: inhibit protein synthesis ■ Rifampicins: influence transcription of genes ■ Sulfonamides:inhibits growth and replication Antimicrobial drugs methods of measuring ○ Effect on bacteria ■ Killing cells or inhibiting growth ○ Spectrum of activity ■ Narrow vs. broad range ○ Mode of action ■ How the bacteria is effected and what the antibiotic does to the microbe ■ Antibiotics are known to work on ● Cell wall construction ○ Can deteriorate it or prevent synthesis and without cell wall, bacteria loses osmotic regulation which can be detrimental and will succumb to pressure causing it to burst ○ Antibiotics include bacitracin, beta-lactams, fosfomycin, glycopeptides ● Cell membrane ○ Can change structure or functionality of membrane ○ Antibiotics include colistan and and polymyxin B ● Protein synthesis ○ Antibiotics include aminoglycosides, lincosamide, macrolides, and tetracyclines ● Transcription ○ Antibiotic includes rifampin ● Changes structure and function of DNA ○ Antibiotics include quinolones, nitrofurantoin ○ nitroimidazole Determining level of antimicrobial activity is done by

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○ Kirby Bauer - disk diffusion test ○ The E-test MIC and diffusion ○ Dilution susceptibility tests for MIC Clicker: Antibiotic resistance is a natural phenomenon Clicker: antibiotic resistant bacteria can spread to humans through ○ Contact with a person who has an antibiotic resistant infection ○ Contact with something that has touched a person who has an antibiotic resistant infection (such as healthcare workers hands or healthcare instruments with inadequate hygiene and safety protocols) ○ Contact with a live animal, food or water carrying antibiotic-resistant bacteria Clicker: i can prevent antibiotic-resistant infections when i ○ Don't take an antibiotic for a viral infection ○ Don’t save an antibiotic for the next time i am sick ○ Don’t take an antibiotic prescribed for someone else ○ Take my antibiotic exactly as my healthcare provider tells me Clicker: what can happen if i get an antibiotic resistant infection ○ I may have a longer lasting illness ○ I may require to be hospitalized ○ I may need more costly medicine that may cause side effects If there is no zone of inhibition then a microbe is resistant Clicker: if bacterial sample came from an island that has never been populated by humans ○ Few bacteria could be resistant to antibiotic even though it was never exposed to it Antibiotic targets ○ Cell wall ■ Beta-lactams ■ Vancomycin ○ DNA/RNA synthesis ■ Fluoroquinolones ■ Rifamycins ○ Folate synthesis ■ Trimethoprim ■ Sulfonamides ○ Cell membrane ■ Daptomycin ○ Protein synthesis ■ Linezolid ■ Tetracyclines ■ Macrolides ■ aminoglycosides Antibiotic resistance mechanisms ○ Efflux ○ Immunity and bypass (genetic)

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○ Target modification (on DNA/RNA) ○ Inactivating enzymes in cell Molecular mechanisms of resistance ○ Intrinsic resistance ■ Innate ability because of inherent structural or functional properties ■ Ex. staph aureus has cell wall which is a form of resistance for some antibiotics ■ Enterococci ● Anaerobes -> insufficient oxidative metabolism -> does not support uptake of aminoglycosides ■ Gram negative -> outer membrane cannot be penetrated by vancomycin ● Too thick outer membrane and antibiotics cannot fit in porins! ○ This is protection from antibiotics ■ This does not work well with some antibiotics because they cannot get into the cell! ○ Acquired resistance ■ By preventing the antimicrobial from reaching its target by reproducing its ability to penetrate into the cell ● Ex. imipenem is antibiotic used on Pseudomonas aeruginosa, Enterobacter aerogenes, and Klebsiella epp. ○ Changes porin channel frequency, size and selectivity ● Antibiotic vancomycin ○ Vancomycin intermediate-resistant Staphylococcus aureus (VISA) has thicker cell wall which traps vancomycin so it doesn’t work ■ By expulsion of the antimicrobial agents from the cell via general or specific efflux pumps ● Antibiotic tetracycline ○ Does Not work on Enterobacteriaceae, E. coli, ● Antibiotic fluoroquinolones ○ Does Not Work on Staph aureus ■ By inactivation of antimicrobial agents via modification or degradation ● Antibiotic penicillin and cephalosporin ○ Does Not work on Staph aureus ○ Causes inactivation of antibiotic through hydrolytic deactivation caused by beta lactamase ■ By modification of the antimicrobial target within the bacteria ● Antibiotic streptomycin ○ Doesnt work on Mycobacterium spp. ○ Does modification of ribosomal proteins or of 16S RNA ● Antibiotic rifampicin ○ Doesnt work on Mycobacterium spp. ○ Does modification of RNA polymerae ■ mutation and horizontal gene transfer are other methods

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Antibiotic resistance vs. persisters ○ Molecular mechanisms of resistance ■ Intrinsic resistance ■ Acquired resistance ● Mutations ○ Mutations just happen and if it happens in a certain area that provides an advantage, the bacterial cell will survive and will divide with advantage causing a somewhat random resistance ● Horizontal gene transfer ○ Transformation ○ Transduction ○ Conjugation ● Causes of antibiotic resistance ○ Mutations ○ Horizontal gene transfer ○ Natural survival of fittest ○ Over prescription, over use, and misuse of antibiotics in humans and organisms consumed ○ Infection of people and surfaces coming in contact and spreading ○ Poor hygiene or sanitation ○ Lack of new antibiotic development Microbial metabolism; catabolism anabolism ● Metabolism ○ Energy ○ Matter ● Metabolism = interactions between energy and matter ● The laws of thermodynamics ○ 1st law ■ Energy cannot be created nor destroyed ○ 2nd law ■ Physical and chemical processes proceed in such a way that the disorder of the universe increases to the maximum possible ○ Cell has an order conditions that are separate from environment and they can resist tendency to become disordered. ■ Living cell is not disobeying these laws because they have processes that work to constantly put cell in order. Order isn’t naturally kept due to the processes that work ■ Living cell maintains homeostasis!!!! ■ Energy must be imput to maintain order and homeostasis\ ■ External energy taken in from environment allows cell to maintain its order. ● This energy comes from metabolism of molecules such as glucose, organic molecules, or sunlight

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Microbial cells must do work ○ Chemical work ■ Synthesis of complex molecules ■ Chemical reactions ● Exergonic vs. endergonic ○ Spontaneous vs nonspontaneous ○ Exergonic has energy release because end product is more stable ○ Endergonic requires more energy than is released because product is less stable that starting materials ● Endothermic vs. exothermic ○ Endothermic takes in heat ○ Exothermic releases heat ○ Transport work ■ Uptake of nutrients, elimination of wastes and maintenance of ion balances ○ Mechanical work ● Enzymes ○ Function is to allow process to work ○ Enzyme is a CATALYST ○ Chemical makeup is proteins (but can be RNA molecules...