MicroBio Phages 4/5/6

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  1. microbiology 4
  2. capsid
    • protein coat of a virus that sometimes contains carbohydrate and/or lipid
    • it may contain appendages that can act as adherence factors
  3. bacteriophage (phage)
    a virus that affects bacteria
  4. why bacterial viruses are obligate parasites
    • the temporal regulation of the viral growth cycle
    • how prokaryotic gene transcription stops
  5. Coliphage T4
    Image Uploada complex well-characterized bacteriophage
  6. What are the 3 classes of bacteriophages?
    • 1. virulent or lytic
    • 2. temperate or lysogenic
    • 3. pseudotemperate
  7. virulent or lytic phage infection
    results in cell lysis (cell death) and production of many progeny phages
  8. temperate or lysogenic phage infection
    • can either cause the cell to lyse and produce more phage
    • or can lead to lysogeny: maintenance of the virus WITHIN the living cell in a dormant state
  9. pseudotemperate phages
    establish a permanent relationship with the host cell so that the viruses are continuously produced and secreted into the environment without causing lysis or death of the host cell
  10. Phage Growth Cycle - Stages of Infection
    • 1. adsorption
    • 2. penetration
    • 3. gene expression
    • 4. nucleic acid replication
    • 5. synthesis of structural proteins
    • 6. assembly of virions
    • 7. lysis --> release

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  11. Adsorption & Penetration (steps 1-2)
    • 1. phage tail fibers attach to a gram-negative bacterium's outer membrane OR to the cell wall of a gram-positive bacterium
    • 2. phage tail fibers contract to allow the base plate to attach to the cell surface
    • 3. the contractile sheath contracts
    • 4. the needle passes through cell wall
    • 5. phage DNA is injected into the bacteria's intramembrane space
    • 6. the phage DNA is transported across the cytoplasmic membrane

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  12. If the phage in an enveloped phage, how does it insert its genetic information?
    • membrane fusion
    • all it has to do is come physically close to a membrane bound bacterium --> find its receptor --> & the membranes fuse, allowing the phage entrance into the cell
  13. Why can't phages be phagocytosed into a bacteria cell?
    because bacteria are incapable of phagocytosing ANYTHING
  14. What is another method by which a bacteriophage can enter a host bacterial cell?
    • some bacterial viruses, containing either ss DNA or ss RNA, are known to attach to a special pilus and use the connection to enter the bacterium
    • RNA viruses (round) attach to the side of the pilus, roll down the side of the pilus until they reach the surface --> nucleic acid enters the cell
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    • DNA viruses (thin & rod-shaped) attach to the tip of the pilus & cause the pilus to retract --> at the cell surface phage DNA is transferred into the cell
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  15. What are some examples of cell surface molecules that phages can use as receptors to adhere to? (4)
    • O-Antigen/LPS/endotoxin (in gram-negative bacteria)
    • Porins (in gram-negative bacteria)
    • Pili
    • Capsule - polysaccharide coating
    • these receptors can be polysaccharides or proteins
  16. What is the E.coli receptor for the T4 phage?
    • E. coli Omp C porin protein
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  17. How does bacteriophage MS2 insert it's genomic information into a bacterium?
    • it has a SINGLE STRANDED RNA (ssRNA) genome
    • the spikes on it's surface will recognize a pilus on a bacterium
    • the pilus retracts bringing capsid close to membrane --> the genome can enter

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  18. plaque
    a visible hole that appears in a confluent layer (lawn) of bacterial host cells after bacteriophages have completed several rounds of infection and killing of host cells
  19. Which step of bacteriophage assembly happens spontaneously (i.e. don't require chaperone proteins)?
    • only the final step of phage assembly - the joining of a tail to a head - happens spontaneously
    • (there is no covalent modification of any of the components)

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  20. Holins (Lysins)
    • phage-encoded proteins (degradative enzymes) that destroy the bacterium cell wall and cytoplasmic membrane
    • are activated in conjunction with lysis of the bacterial host & release of the newly developed bacteriophages
  21. How are single-stranded DNA bacteriophages released from bacteria cell hosts?
    due to the fact that they're enveloped viruses they're 'extruded' through the membrane, usually without destruction of the cell
  22. Describe early and late phage genes and what the timing of their transcription looks like?
    • Early: code for proteins required for viral DNA replication (eg. a specific DNA pol or polymerase component)
    • Late: code for the structural components (capsid, tail fibers) of the virus + the lysis proteins
    • the timing of their transcription tends to follow a biphasic or multiphasic pattern

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  23. Phage T7 Infection of E. coli
    • during phage T7 infection, early & late genes are expressed in the order they are arranged on the viral genome
    • early genes: transcribed by host cell RNA pol
    • late genes: transcribed by phage RNA pol (encoded by early genes)

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  24. dyad symmetry
    • a symmetrical region in the bacterial DNA that when encoded by RNA polymerase, the RNA forms a “stem loop” or hairpin structure
    • RNA pol senses a stem-loop in the newly synthesized RNA & transcription stops --> nascent RNA is released
    • example of a factor-Independent termination site
  25. Rho factor
    • binds to RNA pol & forces it to release bacterial DNA transcript --> terminates transcription
    • example of factor dependent termination
    • innate in bacteria
  26. Does T7 bacteriophage undergo factor-dependent or independent transcription termination?
    • BOTH Rho factor & RNA stem loop is found in the T7 bacteriophage genome to separate early and late genes
    • "stacking the deck"
  27. For T7 phage gene transcription, why weren't the late RNAs made at the very beginning? Why did the early RNAs stop after the first 12 minutes?
    • E. coli "host" RNA pols transcribe early RNA but phage encoded RNA pol is the only protein that can recognize late gene promoters --> transcribe late genes
    • early promoter is recognized by E. coli host RNA pol
    • late gene promoters are different and can't be recognized by E. coli host RNA pol
  28. GP1 (gene 1 product)
    • phage encoded RNA pol otherwise known as T7 pol
    • protein produced by 'gene I', found in T7 early genes
  29. What genes do phage T7 RNA pol (GP1) transcribe?
    late genes for capsid, tail fibers
  30. Gp2
    • late T7 gene product that binds to host E. coli RNA pol (the holoenzyme) & completely inhibits host RNA pol transcription initiation
    • prevents transcription of all its own bacterial transcription as well as early T7 genes
  31. What are some strategies a host bacterium can use to defend itself against viral attack?
    • 1. host can alter its surface proteins used by virus to adsorb; altered proteins wouldn't be recognized as receptors --> the virus couldn't hijack the cell
    • 2. restriction-modification systems (restriction enzymes)
  32. restriction enzymes
    • arose originally so that bacteria could recognize and cut foreign DNA, eg. phage DNA
    • bacterial enzymes that recognize & cut certain sequences in order to destroy potential phage or foreign DNA
  33. microbiology 5
  34. temperate bacteriophage
    can undergo a lytic or a lysogenic life cycle (a lytic phage cannot lysogenize)
  35. lysogenic
    • the state of existing in a repressed viral state
    • the viral DNA genome is inserted into the host chromosome, ensuring stable inheritance & maintenance
  36. prophage (provirus)
    integrated phage DNA of a lysogenic life cycle virus
  37. lysogen
    • a bacterial cell containing a prophage
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  38. bacteriophage λ
    • quintessential temperate phage
    • classical model for understanding how a temperate virus decide whether to replicate itself killing its host OR to shut off the lytic cycle and form a stable relationship with the cell

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  39. Lysis or Lysogeny?
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  40. early λ gene expression
    • initiates at PL and PR and terminates at the left end of the N gene and at the right end of the cro gene, respectively
    • leads to production of only two proteins, N and Cro

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  41. N gene
    • produces anti-termination N protein during early λ gene transcription that acts as a transcriptional “anti-terminator” to bypass the early terminators (TL & TR)
    • allows leftward transcription to proceed through the int gene & rightward transcription to proceed through the Q gene
    • promoter = PL
  42. cro gene
    • produces cro protein which inhibits cI gene expression
    • occurs during early λ gene transcription
    • promoter: PR
  43. middle λ gene expression
    • initiates from PL and PR after N protein synthesis, allows transcription to proceed through TL & TR
    • leads to synthesis of:
    • left: Int, Xis
    • right: Q protein, cII
    • misc: O & P (phage DNA replication proteins)

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  44. Int, Xis gene
    PL controlled genes that produce integration and excision proteins during middle λ gene expression
  45. Int protein
    • combines circular phage DNA w/ host cell chromosome
    • recognizes attP (phage DNA sequence) & attB (bacterial DNA sequence) & brings them together, catalyzing a breakage-rejoining reaction between them
    • result is a larger circle that includes all of the phage and bacterial DNA

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  46. Q gene
    • produces Q protein
    • during middle λ gene expression
  47. cII gene
    • produces cII protein
    • during middle λ gene expression
  48. late λ gene expression
    • initiates from Plate and requires the Q protein (encoded in middle λ gene expression) to act as an anti-terminator of transcription from Plate
    • late genes encode phage structural components & lysis enzymes

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  49. cI
    • protein that represses lytic growth --> only gene of the λ genome not transcribed during the lytic cycle
    • controlled by PRE (promoter for repressor establishment) and PRM (promoter for repressor maintenance)
  50. PRM
    • second mechanism for synthesizing repressor cI; avoids the switch of a phage from the lysogenic (repressed) to lytic state after cII is no longer being synthesized
    • cI λ repressor acts as a positive regulator (repressor must already be present in the cell for more to be synthesized)
  51. What are two things that must be done by a phage (QUICKLY) in order to choose a lysogenic response over a lytic one?
    • 1. synthesize a high concentration of cI repressor
    • 2. synthesize Int: a DNA recombination protein that integrates the phage genome DNA into the host cell chromosome
  52. repressor
    a protein that binds to DNA at a site within or downstream of a promoter site blocking transcription by competing with RNA pol for interaction with the DNA
  53. What is a repressor binding site called?
    an “operator” site
  54. the response due to the nutritional status of the host cell is mediated by:
    • cII (phage regulatory protein)
    • proteases (host) that uses cII as a substrate
  55. On initial infection, the stability of ___ determines the lifestyle of the phage
    • cII
    • stable cII leads to production of cI --> repression of PR & PL --> lysogenic pathway (low temperature, cell starvation, high multiplicity of infection)
    • if cII is degraded by host proteases --> lytic pathway
  56. cIII gene
    • cIII protein acts to protect the cII protein from proteolysis by FtsH (a membrane-bound essential E. coli protease)
    • acts as a competitive inhibitor
  57. What is the only way a lysogen can return to the lytic cycle?
    • if the repressor protein cI is inactivated or destroyed
    • eg. by DNA damage & subsequently RecA*
  58. RecA
    • initiates the termed the “SOS” response: protein binds to certain kinds of repressors & causes them to degrade themselves
    • activated by DNA damage (eg. UV light, toxic agents)
  59. LexA
    • repressor protein that has autoproteolytic activity activated upon RecA* interaction
    • once degraded, ~40 genes LexA had repressed are immediately induced --> tend to be DNA repair genes coding for DNA repair enzymes
  60. aberrant excision
    • when phage enzymes Int + Xis make a mistake during excision
    • mistake: Int and Xis proteins recombine phage genes with nearby bacterial DNA, resulting in circular "phage" DNA missing some phage genes & incorrectly including bacterial DNA
  61. Specialized Transduction
    • a result of aberrant exision; when packaged hybrid phage/bacterial DNA is injected into new cells, the bacterial genes from the previous host can recombine with chromosomal genes of the newly infected cell, generating NEW genotypes
    • this has significant implications for the acquisition of VIRULENCE GENES by bacteriophages
  62. microbiology 6
  63. What are the only type of phages that can undergo specialized transduction?
    temperate phages - they are the only phages that can be incorrectly excised from a host chromosome, carrying with them host DNA
  64. What is the only usual active phage gene when the temperate phage is in a lysogenic state?
    most genes of a temperate phage are silent EXCEPT for the phage REPRESSOR (λ = cI) gene, which is continually expressed to prevent lytic growth
  65. Lysogenic Conversion
    • the acquisition of a new property (phenotype) by a host bacterium as a result of temperate phage lysogeny
    • (how temperate viruses can convert NONPATHOGENIC bacteria to PATHOGENIC bacteria)
  66. epsilon-15
    • a salmonella-specific bacteriophage that possesses genes which alter the structure of the polysaccharide O-antigen present on salmonella's surface (gram -)
    • the first epsilon-15 to infect uses the O-antigen as a receptor & following lysogenization, the phage alters O-antigen's structure so that it's no longer a receptor for other epsilon-15 phage's
    • the change in O-antigen structure confers a survival advantage to the salmonella
  67. Scarlet Fever
    • β-hemolytic Streptococcus (gram +) lysogenized by a phage that carries an exotoxin gene produces an exotoxin that can cause a rash, aka scarlet fever
    • Streptococcus is normally the strep throat causing bacteria
  68. food poisoning
    caused by Staphylococcus aureus (gram +) is often due to an exotoxin encoded by a prophage
  69. Botulism, Tetanus & Gas Gangrene
    • all are due to production of exotoxins encoded by prophages in Clostridium (gram +)
    • Clostridium botulinum & Clostridium tetani differ principally with respect to which prophage they carry --> which phage-encoded toxin they produce --> which disease results
  70. Vibrio cholerae bacteria
    • in the small intestine V. cholerae bacterium creates a single polar flagellum, becomes highly motile, & chemotaxes (swims) through layers of mucus to the intestinal epithelial cells
    • at the epithelium surface the bacteria express TCP (toxin-co-regulated pili), adhere to the epith. surface, & promote colonization
    • *cholera toxin is secreted, resulting in severe diarrhea releasing some bacteria into the environment to potentially infect other hosts

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  71. operon
    a group of contiguous genes transcribed from the same promoter
  72. ctxAB operon
    • contains ctxA and ctxB, two adjacent genes which encode the A & B subunits that make up the powerful toxin responsible for the diarrhea characteristic of cholera (toxin in total = 6-subunit protein complex)
    • A subunit: (one molecule per toxin complex) has all of the toxin activity
    • B subunit (5 molecules per toxin complex): attaches to host cell surface & brings the toxin to the host cell where it is taken up by endocytosis

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  73. CTXΦ
    • phage encoded in the bacterial chromosome which contains the CTX genes
    • the causative agent of cholera (gram -) is a LYSOGEN
    • the toxin genes are PHAGE genes
  74. What type of molecule activates transcription of the ctxAB operon?
    • even though the ctxAB operon is found within a lysogenic phage genome, it has its own promoter that ISN'T repressed by the CTXΦ repressor
    • instead, expression of CTX genes depends on HOST regulatory factors toxR & toxT
    • V. cholerae makes toxin when the phage is in EITHER the lysogenic or lytic cycles
  75. functions of TCP (toxin-co-regulated pili)
    • 1. to adhere to the small intestine epithelial surface & promote colonization of the V. cholerae bacteria
    • 2. act as receptors for the CTXΦ phage
  76. How did the CTXΦ phage acquire toxin genes?
    it's postulated that because the ctx genes in CTXΦ phage are located at the border between phage & bacterial DNA, a misexcision event in an ancestoral phage in a previous host occurred, capturing adjacent bacterial ctx toxin genes
  77. E. coli O157:H7
    • gram negative bacteria that produce dysentery Shiga toxin which damage the intestines
    • Shiga toxin genes come from an incorporated lysogenic λ-like temperate phage
    • the toxins cause severe food poisoning, hemorrhagic colitis, & in severe cases may progress to hemolytic-uremic syndrome (HUS, a condition that can lead to kidney failure --> death)
  78. What do the O157 & H7 refer to in E. coli O157:H7?
    • O157 refers to type of O-antigen
    • H7 refers to the type of the E. coli flagellum coded for by the H protein
  79. What is another bacteria that produces shiga toxin, similar to enterotoxigenic E. coli?
    Shigella, another bacterial gastrointestinal pathogen
  80. stxA and stxB
    • λ-like temperate phage genes that code for a shiga toxin
    • located in the late gene operon of the phage
    • activated during lytic growth & depend on Q anti-terminator protein for expression

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  81. antibiotics __________ disease caused by E. coli O157:H7
    antibiotics exacerbated disease caused by E. coli O157:H7
  82. Fluoroquinolone
    • antibiotics that kill bacteria by causing nicked (partially single-stranded) DNA to accumulate
    • these particular antibiotics exacerbate the symptoms of those infected with E. coli O157:H7 because single stranded phage DNA is in it's transcriptionally active form (as opposed to when it's in the bacterial chromosome and double stranded)
    • the fact that shiga toxins are encoded in λ-like phage DNA & fluoroquinolone promotes the transcriptionally active form of phage genes --> more toxin when treated w/ this antibiotic
  83. How can the innate immune system trigger production of E. coli O157:H7 shiga toxins?
    • by causing DNA damage
    • neutrophils make H2O2 to damage bacterial DNA
    • the damage activates the RecA* protein --> causes O157:H7's λ-like phage repressors to be autoproteolyzed --> toxin gene expression
    • (*anything that induces an SOS response could potentially activate phage genes and any potential toxins)
  84. What are factors contributing to the spread of bacterial antibiotic resistance?
    • 1. antibiotics are given to animals in farming industry as prophylaxis --> they end up in our food & water
    • 2. excessively prescribed to treat symptoms
    • 3. noncompliance: 50% of patients stop taking antibiotic before the recommended dose has been taken

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  85. How does noncompliance enable resistant bacterial strains to become dominant?
    • natural selection
    • a patient takes prescribed antibiotics for a short while, but doesn't finish them
    • bacteria sensitive to the antibiotic are eliminated, but antibiotic resistant cells survive
    • when the patient feels better & stops taking the drug, remaining bacteria (sensitive & resistant) survive, and the immune system is overwhelmed trying to eliminate the problem by itself
    • finishing a prescription lowers the amount of bacteria the immune system needs to deal with --> allows it to focus on resistant bacteria because the majority of sensitive bacteria have been eliminated
  86. Phage Therapy
    the therapeutic use of bacteriophages to treat bacterial infections
  87. How could using a phage for antibiotic treatment be more beneficial than using antibiotics?
    • 1. it could potentially target & treat a specific pathogen but NOT wipe out gut flora
    • 2. applying one topically could potentially kill antibiotic resistant pathogens (eg. vancomycin resistant MRSA)
  88. Why would lysing gram-negative bacteria as a mode of phage therapy be dangerous?
    • because endotoxin would be released
    • solution: a phage that kills the gram-negative bacteria w/out causing lysis
Card Set:
MicroBio Phages 4/5/6
2013-10-16 20:44:45

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