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This 9 page Class Notes was uploaded by Cara Cahalan on Thursday April 7, 2016. The Class Notes belongs to Bios 312 at University of Nebraska Lincoln taught by Karrie Weber in Spring 2016. Since its upload, it has received 10 views. For similar materials see Microbiology in Biology at University of Nebraska Lincoln.
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Date Created: 04/07/16
2/22: Metabolic Regulation Readings: Ch. 7.77.13 III: Sensing and Signaling Transduction 7.7 TwoComponent Regulatory System Signal transduction pathway components: o Sensor protein kinase in membrane, detect a signal from environment and autophosphorylate. Phosphate is transferred from sensor to response regulator o Regulator protein in cytoplasm, binds to DNA tro regulate transcription (positive or negative) Feedback loop complete regulatory circuit to terminate response, employs phosphatase (removes phosphate from response regulator) Example of 2 component system: o Low osmotic pressure OmpF larger pore o High osmotic pressure OmfC smaller pore o EnvZ sensor kinase, detects changed in osmotic pressure 7.8 Regulation of Chemotaxis Regulation of chemotaxis: o Two component: 1. Response to signal. 2. Controlling Flagellar Rotation o Modification: 3.Adaptation 1. Response to signal o Sensory proteins response to presence of attractants/repellents, interact with sensor kinases, monitor environment Methylaccepting chemotaxis proteins (MCPs) o MCP binds changes conformation of CheA (sensor kinase) CheAP o Attractant decreases autophosphorylation Repellents increase o CheAP passes P to CheY (response regulator) that controls rotation CheB (adaptation) 2. Controlling rotation: o CheY governs direction of rotation (CCWrun, CW tumble) o CheYP induce CW (tumble) CheY induce CCW (run) o CheZ dephosphorylates CheY, allowing for runs o Repellents increase CheYP tumbling. Attractants lower levels of CheYP run 3. Adaptation must stop responding to signal (feedback loop) o CheR methylates: Methylated sensitive to repellants. Unmethylated sensitive to attractants o Attractant level high: CheA autophosphorylation is low swim o CheB is phosphorylated, CheBP demethylates MCPs cell stops swimming if conc. remains high Only swims if encounters higher level of attractant 7.9 Quorum Sensing Quorum sensing sensing how many cells are present in environment o If not enough cells, waste of nutrients to produce toxins. Wait until enough are present to make a diff. o Triggers expression of subset of genes necessary for biofilm formation Autoinducer molecule synthesized when quorum sensing used, freely diffuses, binds to transcriptional activator protein transcription of genes 7.10 Other Global Control Networks Heat shock proteins counteract damage in cells that experience heat stress, HSP70, 60, and 10 o 70 Stabilizes unfolded proteins. 60 and 10 catalyze correct refolding Response o DnaK binds to unfolded proteins. More denatured proteins lower levels of DnaK and higher of RpoH heat shock gene expression IV: Regulation of Development in Model Bacteria 7.11 Sporulation in Bacillus Adverse conditions lead to production of sporulation factors Spo0A highly phosphorylated sporulation proceeds 4 sigma factors control development of endospore: o F and G developing endospore. E and K activate mother cell genes Endospore cannibalizes neighboring sister cells to gain nutrients o Cells making Spo0A lyse nearby cells that are not producing it. Also make antitoxic proteins to protect from own toxin release 7.12 Caulobacter Differentiation CtrA phosphorylated activated genes swarmer. CtrAP represses GcrA inhibits DNA replication. CtraAP degraded rise in DnaA origin of replication unblocked DNA replication DnaA degraded GcrA rises cell elongation forms immobile stock Decrease Increase Result CtrA DnaA Deblock origin of replication DnaA GcrA Stalk formation GcrA CtrA Swarmer formation Lecture: Quorum sensing bioluminescence (ensures enough members are present to make a difference) o Examples: Virulence factors, switching from freeliving to growing as biofilm o Exists in microbial eukaryotes and Archaea Biofilm formation: Pseudomonas aeruginosa o Produces polysaccharides increase pathogenicity and antibiotic resistance (2 quorumsensing systems) o Produces AHLs and cyclic diguanosine monophosphate (cdiGMP) o Leads to exopolysaccharide production and flagella synthesis Caulobacter differentiation to form: o Swarmed cell dispersal, cannot replicate Stalked cell reproductive role o Controlled by three major regulatory proteins CtrA activated by phosphorylation in response to stimuli, concentrations rise, GcrA declines DnaA degraded and GcrA increases allowing for cell elongation, levels rise when CtrA degraded Access to origin of replication when CtrA is absent o Externa stimuli and internal factors play a role in affecting life cycle During a growth curve of Aliivibrio fischeri, when would you expect to see the strongest bioluminescence? o late log to early stationary phase o early to middle log phase o lag phase o middle to late stationary phase Key Concepts: o Twocomponent regulatory systems respond to environmental factors with a kinase that senses and environmental signal and phosphorylates a response regulator which results in expression or activity o Quorum Sensing allows for communication between microorganisms and expression of genes o Five sensor kinases detect conditions which control endospore formation in a system that is similar to a twocomponent regulatory system but is much more complicated involving sigma factors. o Three major global regulators control differentiation of cells of Caulobacter to yield a swarmer cell that can freely disperse in the environment but cannot divide and a stalked cell for reproduction 2/24: Viruses Readings: Ch. 8.18.8 I: The Nature of Viruses 8.1 What is a Virus? Viruses possess nucleic acid genome, and the virion (infectious virus particle; nucleic acid genome surrounded by protein coat, extracellular) allows movement between host cells, cannot replicate without host cell (infection) Virus structure: o Capsid protein shell, contains viral genome, arranged in repetitive pattern o Most are naked (no further layers), some are enveloped (protein plus lipid layer) Virulent (lytic) infection replicate and destroy host cell, host metabolism used to support viral replication Lysogenic infection host cell isn’t destroyed, but genetically altered because of incorporation of viral genome Viral genomes can be: o Either DNA or RNA o Single stranded or double stranded o Linear or circular o Plus sense (exact same base sequence as viral mRNA) or minus (complementary base sequence to mRNA) Viruses classified on what organisms they infect (i.e. bacteriophage bacterial viruses) 8.2 Structure of the Virion Capsomeressubunit of capsid Small size of viral genomes restricts the number of distinct viral proteins that can be encoded Folding and assembly of viral proteins: o Often embedded in amino acid sequence (selfassembly) o Some require assistance (i.e. lambda require assistance from E. coli chaperone GroE) Viral symmetry: o Helical symmetry (rod shaped) o Icosahedral symmetry (helical) most efficient arrangement because it requires the smallest amount of capsomeres. (smallest needs 60, can be 180, 240, or 360) Enveloped viruses infect animal cells, assist in fusing with membrane and allow easy exit from animal cell Enzymes in viruses o Resemble lysozyme to allow a hole to form in infection and also in lysis o Nucleic acid polymerases cells cannot make RNA from RNA template o Reverse transcriptase cannot make RNA from DNA template 8.3 Overview of Virus Life Cycle Steps of replication cycle: duration from 206 minutes in bacteria, 840 hours in animals o Attachment of virion to cell o Penetration of virion nucleic acid into host cell o Synthesis of virus nucleic acid and protein by host cell machinery redirected by virus o Assembly and packaging of viral genomes into new virions o Release of new virions Latent period: o Eclipsevirus is attaches, cannot infect another cell. If infected cell breaks no, virion no longer infectious o Maturation phase new nucleic acid packaged inside capsids, number of virions increase End of maturation mature virions are released (burst size number of virions released per cell) 8.4 Enumeration of Viruses Titer virions per volume of fluid Plaque assay virus infects host cell growing on flat surface, zone of lysis (plaque) appears as a clear area Counting number of plaques (one virus), can calculate titer (plaqueforming units) Plating efficiency higher than 50% for bacterial viruses, 0.11% for animal viruses II: Bacteriophage Life Cycles 8.5 Attachment and Entry of Bacteriophage T4 Attachment: lock and key, external surface interact with specific host cell surface components (receptors including proteins, glycoproteins, lipids, lipoproteins, or complexes) o Attachment changes host cell and virus to facilitate penetration o Mutation of cell may lead to resistance to virus o Carbs on LPS outer membrane of gram negative bacteria recognize T4, which binds to E. coli Penetration capsid remains outside cell and viral genome enters cytoplasm (bacteriophage T4) o Viral proteins must also enter host cell to allow replication o Tail fibers interact with LPS layer and retract o T4 lysozyme forms a small pore in peptidoglycan and tail sheath contracts, T4 DNA enters cytoplasm 8.6 T4 Genome T4 genome encodes DNA polymerase, helicase, primases, and DNA replisome complex Circular permutation genomes with same set of genes arranged in different order, open identical circular genomes at different places o Terminally redundant DNA sequences are duplicated on both ends of DNA Concatemer long DNA unit composed of genomic units recombined end to end Headful packaging linear segments of DNA fill phage heads, heads can hold more so terminal repeats are added Restriction restriction endonucleases cleave foreign DNA, prevent invasion, specific to double stranded DNA o Host protects its own DNA through modification (methylation of nucleotides) T4 protects through substituting 5hydroxymethylcytosine in place of C in viral DNA, glucose added and DNA is resistant to attack 8.7 Replication of Bacteriophage T4 3 sets of proteins: o Early proteins enzymes for synthesis of 5hydrozyl.., to produce copies of phagespecific genome, and modify host RNA pol o Middle and late RNA pol modifying proteins, and virion structural and release proteins Antisigma factor binds to host RNA sigma factor preventing recognition of promoters on host genes, SWITCH o T4 late genes direct host RNA pol to promoters for these genes only, assembly now begins Pumping process of T4 genome: o Proheads (precursor to bacteriophage head) assembled but empty o Packaging motor assembled at opening to prohead, ATP is driving force o Motor discarded and head is sealed After, T4 tail and tai fibers selfassemble and cell osmotically lyses and virion are released (100burst size) 8.8 Temperate Bacteriophages and Lysogeny Bacteriophage lambda Temperate viruses infect host and establish longterm stable relationship Lysogeny genes are not transcribed, genome is replicated with host genome and passed to daughter cells Prophage replicated along with host cell o Lambda genome integrated into host genome o P1 plasmid in the cytoplasm Induction to virulent phase DNA excised virions produced cell lysis Bacteriophage lambda double stranded DNA with head and tail, enters host cell and form the cos site (base pair) to circularize genome o Lytic pathway rolling circle replication (one strand in circular genome nicked and rolled out as template) cut into lengths at cos sites packaged into heads o DNA damage can initiate lytic cycle Transduction package chromosomes from lysed host in newly synthesized virions and transfer, gene transfer Lysis vs. Lysogeny: genetic switch o C1 protein represses Cro lysogenic pathway o Cro represses CII and CIII (required for lysogeny) lytic pathway Lecture: Five sensor kinases detect conditions which control endospore formation in a system that is similar to a two com31nent regulatory system but is much more complicated involving sigma factors. 10 viruses on Earth Virus particle: extracellular form of a virus; allows virus to exist outside host and facilitates transmission from one host cell to another Nucleocapsid: complete complex of nucleic acid and protein packaged in the virion Question: A sample of ground water impacted by direct runoff from a cattle feedlot into an open well is collected and diluted. The diluted sample is equal to a 0.001ml aliquot of the dilution was added to molten soft agar with Escherichia coli and poured on to nutrient agar plates. After incubating the plates at 37°C for 24 hours, the plate contained 100 plaque forming units. How many bacteriophage infecting E. coli are present in the ground water? 100PFU −1 o 0.001mL =100,000 PFU mL streamwater Permissive cell: host cell that allows the complete replication cycle of a virus to occur Bacteriophage Lambda linear, dsDNA genome o Complementary, singlestranded regions 12 nucleotides long at the 5′terminus of each strand o Upon penetration, DNA ends basepair forms cos site, DNA ligates and forms doublestranded circle o When lysogenic, integrates into E. coli chromosome at the lambda attachment site (att Lambda genome o At the 5’terminus of each strand is a single stranded region 12 nucleotides long o When lambda enters the cell they base pair, DNA is ligated and forms a double stranded circle o Lambda integrates into the E. coli chromosome o Lambda integrase recognizes phage and bacterial attachment sites Outcomes of lysogeny: o Via binary fission, host cell DNA & the provirus DNA are both replicated and passed on to the progeny. o Induction (i.e. stress) provirus DNA excises out of host genome initiates the lytic cycle. o Genetic information in the provirus can provide new characteristics for the host cell. 2/26: Viral Diversity Readings: 8.98.11, 9.19.2 III: Viral Diversity and Ecology 8.9 Overview of Bacterial Viruses Bacteriophage examples: o ϕX174 circular DNA, possess only a few genes, plus sense, converted to replicative form o MS2 RNA genome encodes four proteins, RNA replicase (replicate viral RNA genome), needed because RNA cannot be made from an RNA template Overlapping genes open reading frames overlap, allowing virus to produce more than 1 protein from a 1 gene 8.10 Overview of Animal Viruses Major tenets of virology: o Presence of capsid to carry viral RNA/DNA genome o Infection and takeover of host metabolic processes o Assembly and release from cell 2 differences in animal viruses: o Entire virion enters host cell o Eukaryotic cells have a nucleus, animal viruses replicate here Possible events in animal virus infection: o Virulent infection cell lysis, most common o Latent infection viral DNA does not replicate and host is unharmed o Persistent infection release of virions is slow, host may not be lysed o Transformation normal cell converted into cancer cell Cells in different tissues/organs have different surface proteins, so viruses infect only certain animal tissues Must lose outer coat to expose viral genome o Entire virion of animal virus enter cell (endocytosis) virion is uncoated in cytoplasm and genome passes into nucleus picks up part of cytoplasmic membrane on its way out Retrovirus inside host cell via DNA intermediate, transfer information from RNA to DNA (reverse transcriptase assists this flow of information) HIV is an example o Enveloped and carry reverse transcriptase, integrase, protease o Replication: virion enters host cell envelope removed reverse transcription begins reverse transcriptase uses complementary strand 2x stranded DNA nucleus with integrase incorporated into host genome nucleocapsid assembled bud out of membrane 8.11 Virosphere and Viral Ecology Lytic phages facilitate transfer of bacterial genes from one cell to another by transduction (horizontal gene transfer) bacterial evolution Lots of host cells lytic Few host cells lysogenic, survive as prophage Chapter 9: I: Viral Genomes and Evolution 9.1 Size and Structure of Viral Genomes Seven classes of viruses based on relationship of viral genome to its mRNA: o Class I: double stranded DNA, mRNA production and replication same as host cell o Class II: singlestranded plusstrand DNA, transcription yields minus sense replicative form needed (complementary DNA must be made) and plus becomes genome and minus discarded o Class III: start with double stranded (+/) RNA o Class IV: + strand RNA, genome is mRNA o Class V: strand RNA, RNA replicase synthesize + sense RNA off of – strand plus strand used as mRNA template make more – strand o Class VI: singlestranded RNA + but replicate through 2x stranded DNA intermediate (needs reverse transcription o Class VII: 2x stranded DNA, replicate through RNA intermediate 2 groups of viral proteins: o Early synthesized soon after infection, enzymes (small amounts), shut down host mechanisms o Late synthesized later in infection, structural components of virion 9.2 Viral Evolution Why viruses appeared: cells quickly move genes about enriching genetic diversity facilitate gene transfers Hypothesis: transition from RNA to DNA world o Viruses may have been the first DNA containing entities (DNA evolved as modification to protect against ribonucleases), DNA viruses infected all three domains o Gradually, each group was able to obtain necessary machinery to replicate DNA Evolution is so scrambled that viral phylogeny is impossible currently
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