For the beam and loading shown, determine (a) the slope at point A, 615(b) the deflection at point D.
2/8: Replication Online Lecture See notes from 2/5 Replication allows the organism to grow, must be accurate to create genetically identical daughter cells Replication begins at the origin of replication (AT rich) o New strand proceeds from 5’ 3’ o Template strand replicated from 3’ 5’ Bidirectional DNA replication in prokaryotes o 2 original strands are templates for the new daughter strands (semiconservative) o Replication forks will proceed in each direction in bidirectional 2 forks are moving in opposite direction speeds up replication rate (about 40 min. to replicate) Multiple replication forks allow for cell to decrease doubling time o DNA Pol III stops when it reaches synthesized DNA o DNA Pol I replaced Pol III, removes RNA primer and adds nucleotides o DNA ligase makes last phosphodiester bond sealing nicks (also helps in DNA repair) DNA pol catalyze the addition of nucleotides (dNTPs deoxyribonucleotide triphosphate) o DNA Pol III primary enzyme replicating chromosomal DNA o Can only add dNTP to existing 3’ –OH (only synthesize DNA from existing template) o Requires an RNA primer to start replication Errors in replication introduced mutations into the system o Low rates of mutations: 10 – 10 errors per base pair due to proofreading by DNA pol III Replication fork: o Single stranded binding proteins stabilize unwound parental DNA o RNA primer 1112 nucleotides complement to DNA One on leading, multiple on lagging Primosome helicase and primase subcomplex Chromosome is replicated bidirectionally in prokaryotes 2/10: Microbial Growth Readings: 5.15.1, 5.55.7 I: Bacterial Cell Division 5.1 Binary Fission Binary fission cell elongates to twice its size and constricts to form two daughter cells o Septum inward growth of cytoplasmic membrane and cell wall from opposing directions Generation time time it takes for one cell to divide into two daughter cells Balanced growth each cell receives the same organelles and genetic information 5.2 Fts Proteins and Cell Division Divisome division apparatus in cell, orchestrates synthesis of new cytoplasmic membrane and cell wall (division septum). Proteins involved (Fts filamentous temperaturesensitive) o FtsZ tubulin, attachment of FtsZ ring around the center of cell, guided by Min proteins to cell midpoint o FtsA actin, connects FtsZ ring and recruits other divisome proteins o FtsI penicillin binding proteins o ZipA anchors FtsZ ring to cytoplasmic membrane o MinD localize MinC, oscillates, preventing FtsZ ring from forming o MinE sweep MinC/D aside, keeping lowest concentration of these in the middle septum forms Cell constricts FtsZ ring depolymerized inward growth of cell wall Short generation times aided by multiple replication forms each cell has at least 1 (maybe more) copy of genes II: Population Growth 5.5 Quantitative Aspects of Microbial Growth Exponential growth number of cells double in time interval o Log graph straight line, cells number is growing, but the rate is constant N=N 0 n n=3.3(log N – log N0) o N= final number of cells. 0 = initial number of cells. n= number of generations in time t G generation time (g= n ¿ log2 k= ¿ K specific growth rate ( g 1 V division rate (= ¿g , h1 o Number of generations per unit time 5.6 Growth Cycle Lag phase between inoculation and beginning of growth, transfer from rich culture medium to poorer one Exponential phase healthiest state, most desirable for study Stationary growth is limited due to depletion of essential nutrient or accumulation of waste products, growth rate=0, some growing some dying Death phase slower rate of exponential death than growth, some cells will remain for long periods of time Events are characteristic of populations of cells, not individual cells 5.7 Continuous Culture Chemostat continuous culture (open system microbial culture of fixed volume) o Growth rate (how fast) and cell density (how many cells per mL obtained) controlled independently. o Dilution rate rate at which fresh medium pumped in and spent medium pumped out (affects growth rate) Too high organisms washed out because cannot grow fast enough Too low cells die from starvation because not enough limiting nutrient o Concentration of limiting nutrient entering the chemostat, C (affects cell density) Increasing concentration of a limiting nutrient results in greater biomass but same growth rate Concentration of incoming nutrient increased at a constant dilution rate, cell density will increase, but growth rate is constant Varying dilution rate and nutrient level can establish dilute, moderate, or dense cell populations at any growth rate Lecture: Production of new cell wall material is a major feature of cell division o In cocci, cell walls grow in opposite directions outward from the FtsZ ring o In rodshaped cells, growth occurs at several points along length of the cell Preexisting peptidoglycan needs to be severed to allow newly synthesized peptidoglycan to form o Beginning at the FtsZ ring, small openings in the wall are created by autolysins where new cell wall material is added o Wall band: junction between new and old peptidoglycan Bactoprenol: carrier molecule, insertion of peptidoglycan precursors o Bonds to Nacetylglucosamine/Nacetylmuramic acid/pentapeptide peptidoglycan precursor Glycolases: enzymes that interact with bactoprenol o Insert cell wall precursors into growing points of cell wall, catalyze glycosidic bond formation Generation time shorter for prokaryotes than eukaryotes In a batch culture, growth conditions are constantly changing; it is impossible to independently control both growth parameters Growth measured by: o PetroffHausser counting chamber cells counted in small square and multiple by plate size o Serial dilutionplate count x dilution factor = cells per milliliter of original sample 2/12: Factors Affecting Microbial Growth Readings: 27.11.18 IV: Antimicrobial Drugs 27.11 Synthetic Antimicrobial Drugs Antimicrobial drugs kill or control growth of microorganisms in the host Selective toxicity inhibit or kill pathogens without adversely affecting the host 2 categories of antimicrobial agents: o Synthetic antimicrobial drugs Growth factor analogs interfere with microbial metabolism, prevent the analog from functioning in the cell (sulfa drugs) Quinolones interfere with bacterial DNA gyrase, preventing supercoiling interfere with DNA packaging (fluoroquinolones ciprofloxacin UTI treatment) o Antibiotics (27.12) produced by microorganisms, inhibit or kill other microorganisms, 1% are clinically used Broad spectrum effective against gram +/ Narrow spectrum used when broad does not work 27.13 βlactam antibiotics: Penicillin’s and Cephalosporin’s βlactam antibiotics cell wall synthesis inhibitors o Penicillin first antibiotic o Cephalosporins bind to PBP and prevent cross linking of peptidoglycan, more resistant to βlactamase which destroys the βlactam ring 27.14 Antibiotics from Bacteria Aminoglycosides target 30S subunit, inhibits protein synthesis, used when other antibiotics fail Macrolide 20% of total antibiotics Tetracyclines broad spectrum, along with βlactam drugs, are two most important classes of antibiotics Novel antibiotics: o Daptomycin creates port and induces rapid depolarization of membrane o Platensimycin fatty acid biosynthesis, no host toxicity or potential of resistance 27.15 Antiviral Drugs Azidothymidine blocks reverse transcription Nucleoside reverse transcription inhibitors (NRTIs) inhibit elongation of viral nucleic acids, lost potency Nonnucleoside reverse transcriptase inhibitors (NNRTI) inhibits reverse transcription Protease inhibitors prevent viral maturation Fusion inhibitor stops conformational changes necessary for fusion of HIV and T lymphocyte Virus interference infection with one virus interferes with subsequent infection by another virus Interferons prevent viral replication by stimulating production of antiviral proteins in uninfected cells o Highly virulent viruses inhibit host protein synthesis before interferons can be produced o Hostspecific 27.16 Antifungal Drugs Toxic to hos so usually topical application used V: Antimicrobial Drug Resistance 27.17 Resistance Mechanisms and Spread Antimicrobial drug resistance acquired ability of microorganism to resist effects of antimicrobial agent to which it is normally susceptible Rplasmids contain drug resistant genes, allow horizontal transmission, modify or inactivate drugs and encode enzymes that prevent uptake of drug or actively pump it out Antibiotic use does not produce resistance, but rather selects for microorganisms with preexisting resistance mechanisms Overuse of antibiotics accelerates resistance 27.18 New Antimicrobial Drugs Drug combinations can decrease rate of resistance