Microbiology Test 2

  1. DNA Replication
    • Duplicate of original molecule
    • Bidirectional
    • Semiconservative
    • Begins at origin of replication
  2. Transcription
    • From DNA to RNA
    • dsDNA to ssRNA
  3. Translation
    • mRNA to Protein
    • Genetic code
    • Reading frames
    • Ribosomes (30S and 50S)
  4. DNA Polymerase of Leading Strand
    • At origin of DNA replication
    • Adds nucleotides in 5’ to 3’ direction onto growing strand
  5. Primase of Leading and Lagging Strand
    • At origin of DNA replication
    • Helps polymerase begin new DNA strand
  6. Helicase of Leading Strand
    • Used in DNA replication
    • Keeps fork open
    • Breaks hydrogen bonds
  7. DNA Gyrase of Leading Strand
    • Used in DNA replication
    • Prevents breakage of strand
    • Will cut strands to help relieve tension for helicase
  8. Nucleotides
    • Are added to 3' of new DNA strands
    • Direction of synthesis from 3' to 5'
  9. DNA Polymerase of Lagging Strand
    • Used in DNA replication
    • Adds nucleotides 5’ to 3’
    • Moves in opposite direction
  10. Okazaki Fragment
    • Used in DNA replication
    • Short strands of DNA that form on lagging
    • side
  11. DNA Ligase of Lagging Strand
    • Used in DNA replication
    • Glues Okazaki fragments together
  12. mRNA in Transcription
    • Synthesized from minus strand
    • Almost identical to the plus strand
    • Monocistronic or polycistronic
    • Thymine is replaced with Urecil
  13. Monocistronic in mRNA
    Can transcribe one gene in mRNA
  14. Polycistronic in mRNA
    Can transcribe one or more genes making a giant mRNA
  15. Initiation in Transcription
    • Promoter (the address can go in any direction) recognized by RNA polymerase (the pizza) and
    • sigma factor (the driver)
  16. Elongation in Transcription
    • Synthesis in 5’ to 3’ direction
    • Same as replication
    • Will always pick up 3’ to 5
  17. Termination in Transcription
    Terminator and hairpin loop
  18. Steps to Make One Strand in DNA Replication Fork
    • DNA strand is going in opposite direction
    • RNA Primase
    • DNA polymerase III
    • DNA polymerase I
    • DNA ligase
    • DNA polymerase III coils down to bind the DNA strand back together
  19. It would not be able to “open the fork”, and
    then DNA polymerase couldn’t perform its job
    Can’t grow
    • What if there was a mutation in helicase that
    • prevented it from performing its function?
    • What would happen?
  20. Start Site of DNA (Replication)
  21. Start Site of RNA (Transcription)
  22. Direction of Synthesis of DNA (Replication) & RNA (Transcription)
    5' to 3'
  23. Enzymes Involved in DNA (Replication)
  24. Enzymes Involved in RNA (Transcription)
    • RNA polymerase
    • Sigma factor
  25. Resulting Strand of DNA (Replication)
  26. Resulting Strand of RNA (Transcription)
    ssRNA to mRNA
  27. Genetic Code of Translation
    • Three nucleotides = codon
    • Degenerate for encoding amino acids
  28. Reading Frames of Translation
    Which triplet of nucleotides to use
  29. Ribosomes (30S and 50S) of Translation
    • Protein and rRNA
    • tRNA with amino acid attached
  30. Initiation in Translation of mRNA
    • Binding of ribosome (30S) to mRNA
    • Start codon AUG
    • f-Met and 50S bind clamp onto mRNA to elongate
  31. Elongation in Translation of mRNA
    P-site; A-site; E-site
  32. Termination in Translation of mRNA
    • Stop codon UAA, UAG, UGA
    • Release factors
  33. Eukaryotic mRNA
    • Modified during transcription
    • 5’ end capped, 3’ end with poly A tail
    • After transcription precursor mRNA modified
    • -Splicing occurs
  34. Splicing
    Removing introns (non-encoding regions of mRNA)
  35. Types of Regulation in Transcription
    • Constitutive
    • Repressible
    • Inducible
  36. Constitutive Regulation in Transcription
    • Always on
    • Glycolysis
  37. Repressible Regulation in Transcription
    • Turn off
    • Amino acids
    • Aren’t always needed
  38. Inducible Regulation in Transcription
    • Turn on
    • Β-galactosidase for breakdown of lactose (alternate carbon/energy source)
    • Not always needed
  39. Inducer
    A component that helps to begin transcription
  40. Operon
    • Activator binding site, promoter and operator
    • Gene or genes to be transcribed
  41. Negative Control
    • Prevents transcription by RNA polymerase
    • Repressor
  42. Repressor of Negative Control
    • Encoded by a separate gene from operon
    • Binds to operator
    • Can require binding of a corepressor
    • Can be blocked by binding of an inducer
  43. Positive Control
    • Transcription by RNA polymerase is facilitated
    • Transcription enhanced by an activator
    • Activator-binding site upstream of promoter
  44. Activator of Positive Control
    • Sometimes requires an inducer to bind
    • •Extra symbol to find
  45. Control by Negative Transcription
    • Lac operon
    • 3 genes for utilization of lactose :lacZ, lacY and lacA- break down lactose
    • Activator: CAP and cAMP (inducer)
    • Repressor that is induced by allolactose
    • A lot of glucose= low cAMP= not enough inducers to bind to CAP (activator)
    • If lactose is present, repressor doesn’t bind
  46. Lac Operon
    • Transcription dependent upon presence/absence of glucose or lactose
    • Glucose is preferred
    • Must “turn on” genes to utilize lactose
  47. Diauxic Growth in Presence of Glucose and Lactose
    • Goes from lag to log
    • lag to log
  48. Mutation in Bacteria
    • Change in DNA sequence (genotype) passed to daughter cells
    • Vertical gene transfer
  49. Gene Transfer in Bacteria
    • Acquisition of genes from another organism
    • Horizontal gene transfer
  50. Cause of Bacterial Mutations
    • Spontaneous
    • Induced
  51. Spontaneous Bacterial Mutation
    • Base substitution
    • Remove or add nucleotides
    • Transposable elements
    • Mistake in DNA synthesis
  52. Induced Bacterial Mutation
    • Chemical mutagens
    • Transposons
    • Radiation
  53. Base Substitution of Spontaneous Bacterial Mutation
    • Point mutation (one nucleotide)
    • Silent mutation
    • Missense mutation
    • Nonsense mutation
    • - Knockout mutation
  54. Silent Mutation of Spontaneous Bacterial Mutation
  55. Missense Mutation of Spontaneous Bacterial Mutation
  56. Nonsense Mutation of Spontaneous Bacterial Mutation
  57. Knockout Mutation of Spontaneous Bacterial Mutation
    Any mutation that renders the protein product dysfunctional
  58. Removal or Addition of Nucleotides
    • Three nucleotides added/removed
    • One or two nucleotides added/removed
    • Frameshift mutation
    • Shift in reading frame of codons
    • Stop codon may be created
  59. Frameshift Mutation of Spontaneous Bacterial Mutation
  60. Transposable Element of Spontaneous Bacterial Mutation
    • Transposon
    • -Short segment of DNA
    • -Transposition: Moves itself into a new position
    • Barbara McClintock
  61. Induced Bacterial Mutation
    • Chemical mutagens
    • Base analogs
    • Intercalating agents
  62. Chemical Mutagens of Induced Bacterial Mutation
    Alkylating agents change purine and pyrimidine structure
  63. Base Analogs of Induced Bacterial Mutation
    • 5-bromouracil pairs with wrong base
    • Once incorporated into DNA, the analog can shift in structure and pair with a different base
  64. Intercalating Agents of Induced Bacterial Mutation
    • Ethidium bromide inserts into ds DNA
    • Frameshift mutation occurs
    • Once the agent inserts into dsDNA, DNA polymerase will recognize the extra space as a need to insert a new base pair
  65. UV Irradiation
    • In induced mutation
    • Thymine dimers
    • Mutation from SOS repair
  66. X-Rays
    • In induced mutation
    • ds and ss breaks in DNA
  67. Repair in Error of DNA Synthesis
    • Proofreading
    • Mismatch Repair
  68. Proofreading in Repair of DNA Synthesis
    Immediately correcting nucleotide
  69. Mismatch Repair in Repair of DNA Synthesis
    • After DNA synthesis
    • Endonucleases to cut and trim away mistake
    • DNA polymerase to make new strand
    • DNA ligase to seal
  70. Repair in Thymine Dimers
    • Light repair
    • -Enzyme requires light to work
    • Excision (dark) repair
  71. SOS Repair
    • Last ditch effort for too many dimers
    • New DNA polymerases made
    • Many mutations created to compensate dimers
  72. Recombinants
    • Having a combination of properties from two cells
    • -Use a defined medium
  73. Homologous Recombination
    Integration of DNA from an outside source only if there is a similar region in the recipient's genome
  74. DNA-Mediated Transformation
    • Uptake of a "naked" DNA
    • Natural competence
  75. "Naked" DNA
    Not associated with a chromosome or plasmids
  76. Natural Competence of DNA-Mediated Transformation
    • Entry of single strand DNA- degrade one side of DNA
    • Integration of donor DNA by homologous recombination
    • Multiplication by selection
  77. Repair
    Based on "Bacterial Transformation," which of the following terms accurately describes the removal of the mismatched strand after transformation?
  78. Artificial Competence of DNA-Mediated Transformation
    • Escherichia coli
    • Single and double stranded DNA can be introduced
    • Use plasmid DNA
    • -Can force DNA into a cell
  79. Transduction
    • Transfer of DNA by a bacteriophage
    • -Phage chops up chromosome making the bacteria no longer functional
    • Generalized transduction
    • Specialized transduction
  80. Mistake during Transduction
    • Picks up bacteria DNA instead of phage DNA
    • -If attached to new bacteria, homologous recombination can occur
  81. Generalized Transduction
    • Phage randomly selects host’s genes
    • After lysis infects new cell, iransfers previous host’s genes
  82. Genetic Transfer System and Transformation
    • Homologous recombination
    • Competence
    • Artificial: plasmid, fragment
    • Natural: fragment
  83. Genetic Transfer System and Transduction
    • Homologous recombination
    • Bacteriophage
    • Generalized
    • Specialized
  84. Plasmids
    • Sitting outside of chromosome
    • Used to benefit cells
    • -Degrade a type of sugar
    • Double stranded DNA
    • -Origin of replication
    • Usually encode for properties that may increase cell viability
    • Low copy vs. high copy
    • Narrow vs. broad host range
  85. Low Copy of Plasmids
    10 cells
  86. High Copy of Plasmids
  87. Narrow Host Range of Plasmids
    Can only be used by one species/genus
  88. Broad Host Range of Plasmids
    Can be used in multiple species/genus
  89. Conjugation
    • Plasmid transfer by cell-to-cell contact
    • Resistant plasmids are among those transferred
    • -Contains one or more drug resistant genes
  90. F plasmid
    Sex pilus
    Conjugation Requires...
  91. Chromosomal Transfer in Conjugation
    • F plasmid integrates into chromosome
    • Hfr – high frequency recombination
    • Donor cell has F+
  92. 1. Hfr extends sex pilus
    2. Rolling replication begins at F plasmid
    3. Only a portion of plasmid and chromosome transferred
    4. Homologous recombination may occur in recipient
    Steps for Conjugation
  93. Transposons
    • A DNA segment that carries the gene(s) required for excision from and insertion into DNA
    • -Often can insert randomly
    • -Can move from one location to another within the chromosome
    • Insertional inactivation of genes
    • Knock-out mutation
  94. Antibiotic resistant genes
    Virulence factor genes
    Genes that allow for adaptability to new environment
    Transposons can Contain...
  95. Insertion Sequence in Transposons
    Encodes for transposase, enzyme which transfers the transposon
  96. No, they can only jump inside a host cell
    It would require an F plasmid through conjugation or through transduction
    • If transposons only jump from one part of a genome to another, could they jump from one cell to another?
    • What would they require if they could jump from one cell to another
  97. Antimicrobial Medication
    A metabolic product of an organism or a chemical compound that is inhibitory to microorganisms
  98. Paul Ehrlich
    • German physician began systematic search for treatment for syphilis
    • Noted that some dyes stain bacteria but not human tissue
    • Identified Salvarsan- arsenic derivative
  99. Gerhard Dogmagk
    • Screened large numbers of chemicals against infectious disease for Bayer in Germany
    • Discovered Prontosil kills streptococci in mice but not in the test tube
    • Prontosil broken down in body to yield active ingredient sulfanilamide
  100. Alexander Fleming
    • Scottish physician who had treated wounds during World War I
    • Worked with Staphylococcus aureus
    • Noted that there was no growth of the bacteria in a zone surrounding the fungal contamination
  101. Ernst Chain and Howard Florey
    Picked up Fleming’s work on penicillin and develop mass production of drug
  102. Chemical Alteration of Antibiotics
    Changing the side chain “R” of penicillin, other types of antibiotics were created
  103. Antibiotic Characteristics
    • Majority from Streptomyces and Bacillus (bacteria), Penicillium and Cephalosporium (fungal)
    • Bacteriocidal
    • Bacteriostatic
    • Selective Toxicity
    • Broad and narrow spectrum
    • Synergistic, antagonistic, additive
    • Uptake
    • Half life
  104. Bacteriocidal
    Antimicrobials kill their targets
  105. Bacteriostatic
    • Allows the body to kill infection, but can kill healthy cells/bacteria
    • -Secondary infections can be worse, so eat yogurt
    • Antimicrobials inhibit the growth but do not kill their targets
    • Allows immune system to catch up
    • May be a problem if immunodeficient
  106. Selective Toxicity
    • Causes greater harm to microbes than human
    • Therapeutic index
  107. Therapeutic Index
    • Lowest toxic dose/dose required for treatment
    • Example: 10/2= 5 (low)
    • Higher numbers= better, body will take care of it
  108. Broad Spectrum of Antibiotics
    • Can treat almost everything, except possibly microbacteria
    • Kills all bacteria, but kills good body bacteria
    • Useful in acute life-threatening disease
  109. Narrow Spectrum of Antibiotics
    • Specific to just gram positive or gram negative
    • Targets just pathogens leaving body alone, but does take a lot of time
    • Requires identification of pathogen
  110. Synergistic, antagonistic, additive
    When Antibiotics are Combined…
  111. Synergisitic
    Greater affect than the two added together, use less
  112. Additive
    Adding antibiotics together to double effect
  113. Antagonistic
  114. Half Life
    How many times a day you have to take an antibiotic for it to be affective
  115. Adverse Affects of Antibiotics
    • Allergic reactions
    • Toxic effects (e.g. aplastic anemia with chloramphenicol)
    • Suppression of normal flora
    • -Antibiotic associated colitis
    • •Clostridium difficile
    • Resistance
  116. Resistance
    • Innate
    • Acquired
  117. Innate Resistance
  118. Acquired Resistance
  119. Mechanisms of Action for Antibiotics
    • Step 1- Inhibit cell wall synthesis
    • Step 2-Disrupt cell membrane
    • Step 3- Inhibit nucleic acid synthesis
    • Step 4- Inhibit protein synthesis
    • Step 5- Anti-metabolite (metabolic analog)
  120. Inhibit Cell Wall Synthesis
    • Inhibit transpeptidation & activation of cell wall lytic enzymes
    • -Penicillin
    • -Cephalosporin
    • Inhibit transpeptidation
    • -Vancomycin
    • Inhibit transport by carrier
    • -Bacitracin stops NAG and NAM from entering cell wall
    • Vary in ability to inhibit Gram + or Gram -
  121. Disrupt Cell Membrane
    • Binds membrane & disrupts integrity
    • Effective against gram negative
    • -Polymyxin B targets and pops a hole in the cell wall/membrane
  122. Inhibit Nucleic Acid Synthesis
    • Broad spectrum
    • Interfere with DNA gyrase
    • -Ciprofloxacin
    • Interfere with transcription
    • -Rifampin
  123. Replication
    RNA polymerase, not making mRNA
    No protein
    • DNA gyrase
    • Transcription
    • Not making RNA
  124. Inhibit Protein Synthesis
    • Aminoglycosides and tetracyclines – 30S
    • -Streptomycin
    • -Tetracycline
    • Macrolides, chloramphenicol, lincosamides, oxazolidinones, streptogramins - 50S subunit
    • Varies in spectrum and action
    • Attacking ribosome
  125. DNA gyrase relieves tension of fork, when not allowed, DNA starts to break apart
    If DNA gyrase is inhibited, how does DNA begin to unwind?
  126. Anti-Metabolite (Metabolic Analog)
    • Broad spectrum
    • Inhibits folic acid synthesis (needed for nucleotide synthesis)
    • -Sulfonamides- competitive inhibitor
    • Make folic acid themselves
    • Blocks tetrahydrofolate synthesis
    • -Trimethoprim
    • Protein, RNA, mRNA, enzyme
  127. Susceptibility to Antimicrobial Drugs
    • Bacterial culture spread over petri plate
    • Discs saturated with specific concentration of antibiotic added
    • Measure areas of clearing where culture did not grow
    • -Zone of Inhibition
  128. Selection for Drug Resistance
    • A simple case scenario is forgetting to finish your prescribed antibiotics
    • Conjugation and mutation
  129. Decreased Uptake
    • 1. Decreased Uptake
    • 2. Inactivation
    • 3. Alteration of target
    • 4. Elimination
  130. Inactivation
    • Enzyme that cuts penicillin to make “resistant”
    • Beta lactamase
  131. Alteration of Target
    Mutation of enzyme
  132. Elimination
    Efflux pump finds antibiotic and pushes out of body before any affects
  133. Acquiring Resistance of Antimicrobial Drugs
    • Vertical
    • Horizontal
  134. Acquiring Resistance-Vertical
    Spontaneous mutation
  135. Acquiring Resistance-Horizontal
    • Gene transfer
    • -Conjugative transfer of R plasmids
    • -Plasmids contain more than one resistance
    • -Resistance genes can be carried on transposons
  136. Metabolic Capabilities of Pseudomonas
    • Most are strict aerobes
    • -Some can grow if nitrate is available for e- acceptor
    • Can metabolize 80 different substrates
    • -Ability to use this variety of substrates are encoded on plasmids
    • Can be found growing in water of respirators
  137. Where to Find Pseudomonas
    Found in a biofilm, attached to some surface or substrate, or in a planktonic form, as a unicellular organism, actively swimming by means of its flagellum
  138. Problems with Pseudomonas
    • 1. The species' inherent resistance to many drug classes
    • 2. Its ability to acquire resistance, via mutations, to all relevant treatments
    • 3. Its high and increasing rates of resistance locally
    • 4. Its frequent role in serious infections
    • A few isolates are resistant to all reliable antibiotics, and this problem seems likely to grow
  139. Opportunistic pathogen
    -The bacterium almost never infects uncompromised tissues, yet there is hardly any tissue that it cannot infect if the tissue defenses are compromised in some manner
    Pseudomonas are...
  140. Types of Infections by Pseudomonas
    Urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia, bone and joint infections, gastrointestinal infections and a variety of systemic infections, particularly in patients with severe burns and in cancer and AIDS patients who are immunosuppressed
  141. Pseudomonas in Hospitals
    A serious problem in patients hospitalized with cancer, cystic fibrosis, and burns. The case fatality rate in these patients is near 50 percent
  142. Tuberculosis Statistics
    1/3 infected worldwide; 2 million die annually
  143. Tuberculosis Symptoms
    Slight fever, weight loss, chronic cough with bloodied sputum
  144. Epidemiology and Pathogenesis of Tuberculosis
    • Inhalation Mycobacterium tuberculosis
    • Multiply within macrophages
    • Granulomas form to wall off bacteria
    • -Called tubercles
    • Latent infection
    • Active tuberculosis
    • Reactivation tuberculosis
  145. Latent Infection
    Asymptomatic, not contagious
  146. Active Tuberculosis
    Symptomatic, can transmit bacteria
  147. Reactivation Tuberculosis
    • Renewed growth after latent period
    • Takes years to reactiviate in the body
  148. Tuberculosis
    • Acid-fast
    • Gram positive bacillus
    • Slow growth (generation time = 12 hours)
    • Cell wall contains layer of complex glycolipids
    • Resistant to drying, disinfectants and low/high pH
    • Takes 12 hours to go through 1 binary fission
  149. Tuberculosis Pathogenesis
    • Inhaled droplets, bacteria are attacked in lungs by macrophages
    • Macrophages release signals to recruit other white blood cells
    • Tubercle forms with bacteria, macrophages and other immune cells
    • Turns latent in healthy individuals with walled off tubercles
  150. Tuberculosis Activation
    • Caseous lesion- cheese like tubercle
    • Tuberculous cavities
    • -Liquefied tubercle
    • -Spread of bacteria into rest of lung
    • -Cavity slowly enlarge
    • -Transmission occurs by coughing, spitting
  151. Mantoux Test
    • Injection of bacterial protein
    • Slow forming reaction shows either existing bacteria or previous exposure
    • Both latent and active TB are treated
  152. Treatment for Mantoux Test
    Rifampin and isoniazid for 6-9 months
  153. Tuberculosis Treatment
    • Limited antimicrobials used
    • -Chronic nature of disease
    • -Slow growth
    • -Waxy cell wall (mycolic acids)
    • First line drugs – five combined
    • -Target cell wall
    • -Isoniazid
    • -Ethambutol
    • -Pyrazinamide
  154. Isoniazid
    Inhibit mycolic acid synthesis
  155. Ethambutol
    Inhibits cell wall components synthesis
  156. Pyrazinamide
  157. Multi-Drug Resistance of Tuberculosis (MDR-TB)
    • Can be drug resistant way before anti-biotics
    • Increase of 13.3% in US
    • Occurs due to preexisting mutants
    • M. tuberculosis exhibits spontaneous chromosomal mutations
    • Results from
    • -Administering only one drug
    • -Improperly administered drug
  158. Extensively drug resistant tuberculosis (XDR-TB)
    4 cases reported to CDC in 2008
  159. Change form of antibiotic
    Make patient come back every week
    Up the dosage to take less days
    Scare the patients
    What are some ways that TB could be treated that would ensure that the patient finished their regimen of antibiotics?
  160. Directly Observed Therapy
    Doctor went to house or made patient come in to make sure they’re taking the anti-biotics
  161. DOT-SA
    Patient comes in every week for more pills
  162. Strictly Administered
    Send patient home with all anti-biotics
  163. CDC funding started to drop
    HIV/AIDS were on the rise, money went to that
    Politics- Raegan-Bush
    1992- Clinton put more money back into CDC
    What happened between 1980-1992?
  164. U.S. citizens start to decrease, while foreign born stay the same
    What is one trend on the “U.S. vs foreign born” chart?
  165. Tuberculosis Worldwide
    • China, India, the Russian Federation and South Africa account for 60% of the global number of MDR-TB cases and have increased their financing for TB control
    • Only 3% of the half million MDR-TB cases estimated to emerge each year worldwide are known to be receiving treatment according to WHO guidelines
  166. Staphylococcus
    • Gram positive
    • Releases coagulase
    • Clumping factor
    • Alpha toxin
    • Can be found in nose or skin
    • Opportunistic
  167. Staphylococcus Wound Infections
    • Pyrogenic with inflammation; if spread to blood, fever ensues
    • -Toxin producing strains can cause toxic shock
  168. Treatment of Staphylococcus
    • Penicillin or modified beta lactam drug (methicillin)
    • 90% now resistant to first penicillin due to plasmid-encoded resistance
  169. Types of Resistances of Staphylococcus
    • Methicillin resistant S. aureus (MRSA)
    • Vancomycin resistant S. aureus (VRSA)
  170. Methicillin resistant S. aureus (MRSA)
    • Produces a penicillinase (also found on plasmid)
    • Cell targets for beta-lactam drugs are not affected
    • Vancomycin used for treatment
  171. Vancomycin resistant S. aureus (VRSA)
    Synercid is made of two substances that block protein synthesis
  172. Epidemiology of Staphylococcus
    • Most resistant strains are traced to hospitals and clinics
    • 30-100% of surgical infections due to patient’s own staphylococcus strain in their nose
    • Over time, 20% of the population will almost always be colonized with S. aureus
    • -Termed carriers
    • 60% of the population will be colonized with S. aureus off and on
    • 20% are almost never colonized with S. aureus
Card Set
Microbiology Test 2