Microbio Bacteria Intro (1/2/3)

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Microbio Bacteria Intro (1/2/3)
2013-10-11 22:26:01

Exam 3
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  1. microbio 1
  2. bacteria size
    • single celled organisms that at ~ 1 micrometer in length or diameter
    • b/c they're so small they have a very large surface:volume ratio (over 20x that of animal cells)
    • makes possible the rapid transfer of nutrients and waste materials between the interior of the cell and its environment
    • results in a high metabolic rate & rapid growth
  3. Prokaryotic v. Eukaryotic
  4. What are the 3 main shapes of bacteria?
    • 1. cocci - spheres
    • 2. bacilli - rods (eg. C. difficile)
    • 3. spirilla - spirals
    • 4. spirochete - corkscrew
    • 5. vibrio - comma-shaped
  5. Clostridium difficile (C. difficile)
    • bacillus (rod-shaped)
    • spore-forming
    • gram-positive bacterium
    • strict anaerobe
    • natural auxotroph
    • as a human pathogen it causes diarrhea and pseudomembranous colitis
    • makes two large toxin proteins (TcdA, TcdB) that disrupt intestinal epithelium
  6. Gram stain
    stain made up of crystal violet
  7. gram positive bacteria
    • have a THICK peptidoglycan cell wall, but no outer membrane
    • when stained with gram stain --> purple as a result of crystal violet stuck in cell wall
    • tend to be less resistant to antibiotics due to LESS membrane protection
    • eg. Bacillus, Clostridium, Staphylococcus, Streptococcus, Enterococcus, Listeria

  8. gram negative bacteria
    • have two cell membranes, one on each side of a THIN layer of cell wall
    • tend to be more resistant to antibiotics as a result of DOUBLE plasma membrane
    • eg. E. coli, Salmonella, Shigella, Neisseria, Klebsiella, Vibrio, Pseudomonas

  9. Which drugs are more effective for which types of bacteria?
    • penicillin & rifampicin: much more effective in killing Gram-positive bacteria
    • streptomycin & tetracycline: work to efficiently kill Gram-negative bacteria
  10. cell wall (murein)
    • a structure found ONLY in bacteria made up most of peptidoglycan: polysaccharide chains of N- acetylmuramic acid (M) and N-acetylglucosamine (G) cross-linked by peptides create a rigid mesh-like structure
    • AAs that cross-link are species-specific and determine cell wall/bacteria shape
    • acts as a barrier to hydrophobic compounds
    • peptidoglycan synthesis = excellent antibiotic target
  11. lipotechoic acid
    • found only in the cell wall of gram positive bacteria and are vital to the wall's structural integrity
    • perhaps mediates some attachment to host cells
    • might alert immune system that a bacterial infection is happening
  12. How does penicillin work?
    • the polysaccharide chains that make up a bacterium's cell wall have peptide side-chains
    • in normal cell walls, two amino acids in the peptide side chains are cross-linked, providing structure and stability holding the pressure-filled bacterium together
    • these two AAs are D-alanine and L-lysine
    • penicillin mimics the peptide side-chain structure, and can be incorporated into a bacterium's cell wall HOWEVER it's structure prevents cross-linking between D-ala & L-lys
    • the bacterial wall is weakened and the cell explodes from its great internal pressure
  13. How long would it take for a single bacterium growing unchecked and dividing every 30 minutes (in exponential growth phase) to produce a mass as large as that of the Earth?
    2.5 days
  14. doubling time
    • the period of time required for cells in a microbial population to double in size, divide, and produce two new cells for each one that existed before
    • growth is exponential; during each doubling time the population increases by a factor of 2
  15. balanced growth
    • constant growth at a steady rate
    • an idealized state of unrestricted growth that is rarely, if ever, achieved in the real world
  16. Prototrophs
    bacteria that have NO requirements for organic compounds other than a simple carbon source (eg. sugar)
  17. Auxotrophs
    • bacteria with more complex nutritional requirements, typically requiring amino acids, vitamins, pyrimidines, or purines
    • are often missing biosynthesis pathways required to make compounds needed for growth
    • are often symbiotic or pathogenic bacteria whose host provides many nutrients (eg. haemophilus influenzae)
  18. What are the phases of bacterial growth?
    • 1. lag phase
    • 2. exponential growth phase
    • 3. stationary phase
    • 4. death
  19. lag phase
    • time it takes for a bacterial cell that HASN'T been growing to sense a nutrient-rich environment, use new nutrients to repair any cellular damage that may have occurred during non-growth period, & re-synthesize materials needed for unrestricted growth
    • time it lasts is dependent on how long the cells in question have been without nutrients
  20. exponential growth phase
    • when bacterial cells increases logarithmically at a rate determined by surrounding environment & species
    • only occurs for very short short periods in nature (realistically non-existent)

  21. What causes the exponential growth phase to end?
    • exhaustion of nutrients
    • build-up of toxic compounds
    • changes in the growth environment
  22. stationary phase
    • a period of NO growth and cellular adjustments
    • synthesis of many proteins to alter cells' physiologically and physically so they can survive unfavorable environmental conditions
    • some bacteria make antibiotics to try to kill neighboring bacteria and eat their remains
    • others form spores
    • eg. while stationary, C. difficile synthesizes toxin proteins or forms spores
  23. spores
    • when an bacterium becomes dormant to resist damaging environmental conditions or a lack of nutrients during it's stationary phase
    • allows sporulating bacteria to survive without nutrients or growth until better conditions are available
  24. death
    • characterized by a constant loss of viable cells
    • however as the bulk of the cells in a culture die, others survive by mutating and adjusting their ability to utilize the carcasses of the dead cells --> they remain viable
  25. Free-living Bacteria
    microorganisms that grow in the soil or water or on the surfaces of plants or animals
  26. Symbiosis
    mutually beneficial association between a microbe and its host
  27. Commensalism
    neither a microbe nor its host benefit or are harmed from co-habitation
  28. Parasitism/Pathogenesis
    association of a microbe with a host where the host is harmed or killed
  29. normal flora
    • all animals have co-habiting microorganisms (“normal flora”)
    • adults are made of ~10^13 human cells of 200 different types
    • humans have in/on them ~10^14 bacteria of 500 different species
    • bacteria inhabit the skin, mouth, nasopharynx, lungs and GI tract
    • normal flora broadly influences nutrition, the immune system, & protects against infection by other microorganisms
  30. Probiotics
    • live microorganisms that can provide health benefits to host
    • take advantage of beneficial effects of normal flora
    • eg. lactobacillus and bifidobacteria restore normal flora after antibiotic treatment
    • reduce diarrheal disease caused by viruses & bacteria
    • reducing recurrent bladder infections in catheterized patients
  31. How does normal flora protect against C. difficile
    • infection?
    • occupys niches C. difficile would otherwise take advantage of
    • produces anti-microbial peptides
    • metabolises compounds needed by C. difficile
  32. What are bacterias two mechanisms of generating ATP?
    • 1. respiration
    • 2. substrate-level phosphorylation
  33. respiration
    • production of ATP via electron transport chain coupled in the cytoplasmic membrane to the enzyme ATP synthase
    • electrons for ETC come from NADH or FADH
    • cells that use an electron transport cytochrome chain to generate ATP are said to be respiring
  34. aerobic respiration
    bacteria w/ an ETC that use oxygen as the terminal electron acceptor
  35. anaerobic respiration
    bacteria w/ an ETC that use alternative electron acceptors, such as fumarate or nitrate
  36. facultative anaerobes
    can carry out either aerobic or anaerobic respiration, depending on whether oxygen is present or not
  37. obligate anaerobes
    can only produce energy using alternate ETC electron acceptors; die in the presence of oxygen
  38. substrate level phosphorylation
    • generation of a highly reactive phosphate bond as a metabolic intermediate and then transfer of that bond to ADP, generating ATP
    • cells that oxidize a carbon source to generate ATP by substrate-level phosphorylation are said to be fermenting
    • fermentation can be carried out aerobically and anaerobically only in bacteria & some yeasts
  39. Spore formation
    • dormant forms of certain bacterial species (almost always Gram-positives), such as Clostridium and Bacillus, that are extremely resistant to heat, chemicals, radiation and antibiotics
    • their low water content allows survival under harsh environmental conditions
    • commitment of a cell to form a spore can be triggered by adverse environmental conditions, especially nutritional limitation
  40. What triggers spore formation?
    • sporulation is triggered by nutritional limitation
    • examples include pseudomembranous colitis, botulism, tetanus, anthrax, and some types of food poisoning
  41. C. difficile Spore Formation
    • spore is the C. difficile cell form that initiates infection
    • in order to cause an infection, C. difficile spores have to germinate in the GI tract and become growing cells again
  42. microbio 2
  43. TcdA and TcdB
    • C. difficile genes that encode it's two toxins, toxin A & toxin B
    • are encoded in a large gene cluster called the pathogenicity locus (PaLoc)
    • can be transcribed from multiple promoter sites ONLY when the cells are in the stationary phase (due to nutritional limitation/no glucose in environment)
  44. holin protein
    C. difficile gene located between its two toxin TcdA & TcdB genes that punches holes in the cell membrane, allowing the toxin proteins to escape from the cell
  45. TcdR and TcdC
    two regulatory genes that bracket the PaLoc (pathogenicity locus) in C. difficile

  46. bacterial transcription
    • the chemical process by which RNA is made from a DNA template
    • catalyzed by DNA-dependent RNA polymerase
    • is template-dependent and asymmetric with respect to the DNA template
    • the DNA template is read 3’-->5’ so that the RNA template can be made 5’-->3’
  47. unit of transcription
    a sequence of DNA bases transcribed to give a single, discrete complementary RNA
  48. promoter site
    • a special region of DNA where RNA polymerase binds to initiate transcription
    • usually found 10-100 base pairs upstream (before) of the beginning of the coding region
    • "-35" and "-10" regions are two key elements of most promoters
  49. termination site
    sequence within the DNA or RNA product at which RNA synthesis is signaled to stop
  50. holoenzyme
    • the bacterial DNA-dependent RNA polymerase made up of these subunits: α2ββ'σ
    • β subunit: has catalytic activity (polymerizing), binds substrates, holds subunit (DNA strand) onto RNA pol
    • β' subunit: allows RNA pol to bind/stay bound to DNA
    • α subunit: interacts w/ regulatory proteins
    • σ subunit: recognizes gene promoter region; RELEASED after transcription has been initiated
  51. core enzyme
    • α2ββ': the holoenzyme missing the σ (sigma) subunit
    • serves to elongate transcripts
    • the core enzyme is the same in all bacteria σ factor that differs and recognizes an individual promoter site

  52. σ subunit critical properties (3)
    • causes very STABLE binding at promoter regions
    • REDUCES binding at non-promoter regions of DNA
    • by removing itself, it allows elongation of the transcript
  53. What are two types of transcription termination?
    • factor-INDEPENDENT termination: a sequence encoded in the DNA that when transcribed into RNA results in a stem-loop structure, ending transcription
    • factor-DEPENDENT termination: requires an accessory protein, eg. ρ (rho) factor
  54. ρ (rho) factor
    accessory protein that serves to terminate DNA transcription, specifically in factor-dependent termination
  55. What is the half-life of mRNA?
    ~2 min
  56. Which subunit of RNA pol (holoenzyme/core enzyme) do inhibitors of RNA synthesis bind to, to prevent transcription?
    • the β subunit
    • it's responsible for catalytic/polymerization activity that binds substrates & holds σ subunit to RNA pol for transcription
  57. What are the names of two drugs that inhibit RNA synthesis?
    • 1. Rifamycin (-rifampicin): blocks elongation
    • 2. Lipiarmycin: blocks initiation
  58. Rifamycins
    • drugs that bind to the β subunit of RNA polymerase & block the first translocation after initiation
    • used in treatment of tuberculosis
    • a related compound, rifaximin (a related compound) used as adjunct therapy for C. difficile infection
    • most common drug of this class = rifampicin
  59. Lipiarmycins
    • drugs that bind to the β subunit and block initiation at the point of DNA binding
    • fidaxomicin is the only member of this family used therapeutically to treat C. difficile infections
  60. TcdR
    • gene which encodes a σ subunit that recognizes the TcdA and TcdB promoter and activates transcription of the toxin producing genes
    • a positive regulator gene that primarily controls C. diff toxin production
    • (toxin gene promoters aren't recognized by major σ subunit that directs transcription of MOST bacterial growth genes)
  61. CodY
    • repressor protein that inhibits transcription of the TcdR gene during a given exponential growth phase
    • binds to the TcdR promoter region & prevents RNA pol from binding to the promoter/transcribing gene
  62. What enables CodY to bind to and block the TcdR promotor?
    • CodY can only block TcdR transcription if it interacts with certain amino acids, specifically leucine, isoleucine & valine (VILe)
    • the amino acids are indicative of a rich medium suitable for growth, therefore toxin production is inhibited (only occurs in stationary phase)

  63. What is the mechanims by which bacteria lose the ability to repress TcdR expression?
    • when cells experience nutrient limitation, the intracellular concentration of amino acids decrease (such as leucine, isoleucine & valine)
    • without 1 of those 3 AAs, CodY loses its ability to bind to the promotor region of TcdR, which can subsequently be transcribed
  64. TcdC
    • a gene that encodes an inhibitor of TcdR, i.e. an ANTI-sigma factor
    • only present during during cell growth phase
  65. 3rd mechanism controlling toxin production
    toxin genes are completely repressed when glucose is present, even if cells are in stationary phase
  66. microbio 3
  67. What is the primary cause of death among cystic fibrosis patients?
    bacterial infection - pseudomonas aeruginosa
  68. pseudomonas aeruginosa
    • gram-negative, highly motile, alginate-producing, rod-shaped bacterium
    • primary cause of infection in cancer patients, burn victims, and major cause of death in those with cystic fibrosis
    • has 3 major virulence factors [toxins]: LPS (endotoxin), exotoxin A, & exoenzyme S
  69. periplasmic space
    • lies between the cytoplasmic membrane and the outer membrane of gram-negative bacteria & includes the cell wall
    • found only in Gram-negative bacteria (gram-positive bacteria have no outer membrane)
    • contains:
    • hydrolytic enzymes (eg. alkaline phosphatase & ribonuclease) that initiate food molecule breakdown of
    • specific chemoreceptors that measure concentrations of foods or poisons and direct movement
    • a variety of chaperone and transport proteins
    • periplasmic polysaccharides + other small molecules buffer the cell from changing osmotic and ionic environments, preserving a uniform internal environment needed for bacteria growth + viability
  70. outer membrane
    • bilayer with a typical phospholipid inner layer and a lipopolysaccharide (LPS) outer layer
    • 1. forms the outer limit of the periplasm
    • 2. presents an outer surface w/ a strong negative charge (important for evading host defenses, eg. phagocytosis)
    • 3. LPS = barrier to hydrophobic compounds, phospholipid bilayer = barrier to hydrophilic compounds
  71. a bacterium's outer membrane layer of LPS is hydro______, while its outer membrane layer of phospholipid is hydro______
    • LPS = hydrophilic
    • phospholipid = hydrophobic
  72. What are the only two things that can diffuse across a gram-negative bacteria's outer membrane?
    • only water & a few gases can diffuse across the outer membrane due to it's phospholipid and LPS makeup
    • excluded compounds: host defense agents like lysozyme, bile salts, and digestive enzymes, & variety of antibiotics
  73. porins
    • hydrophilic protein pores in the outer membrane of gram-negative bacteria that allow nutrients sized 700 Da or less to enter the periplasmic space
    • most toxic compounds are too large to diffuse through the outer membrane's pores
  74. Why are gram-positive cells 1000x more sensitive to rifampicin than gram-negative cells?
    because the drug cannot readily pass through the outer membrane of gram-negative bacteria; it doesn't readily fit through the porins
  75. Endotoxin/LPS/O-Ag
    • the lipid A component of LPS (in gram-neg outer membrane)
    • it's harmful to humans and produces such effects as fever, changes in white blood cell count, diarrhea, shock, prostration (extreme weakness), & death
    • only released when the bacterium dies
    • endotoxin-mediated damage increases when lots of gram-negative bacteria in the blood are killed by antibiotics or host defenses --> septic shock can result

  76. adhesins
    • general term for proteins or other molecules on bacterial surfaces that allow then to adherence to OTHER cells or surfaces
    • eg. pili
  77. pili/fimbriae
    • straight, hair-like appendages that project out from cell surface and stick to both biological and non-biological surfaces
    • bind to specific cell receptor molecules, usually sugars
    • eg. uropathogenic E. coli can have 100 to 300 pili that stick to bladder walls and facilitate biofilm formation
  78. flagella
    • used primarily for locomotion (to find food or evade danger) but can ALSO be used for adhesion
    • bacterium may have one, several, or many flagella per cell
    • eg. they're critical for P. aeruginosa infection as they allow the bacteria to swim through the mucous layer and adhere/attack intestinal epithelium
  79. How is flagellar rotation regulated?
    • by a “two-component” sensory system
    • 1. a membrane-bound sensor histidine kinase detects specific molecules in the environment --> autophosphorylates
    • 2. a cytoplasmic response regulator activated (phosphorylated) by the KINASE causes flagellar motor to turn clockwise (tumbling movement) or counterclockwise (directed movement)
  80. What are three potentially adhesive structures of bacteria?
    • 1. pili/fimbriae
    • 2. flagella
    • 3. capsules
  81. capsules
    • a slimy, loose network of polymers surrounding cell that can be made up of polysaccharides, or proteins, or both
    • principally function to protect a cell: thicker the capsule, the more the protection
    • capsules are anti-phagocytic, protecting some pathogens from being engulfed by host immune cells
    • important capsulated pathogens: P. aeruginosa, streptococcus pneumoniae, haemophilus influenzae, & neisseria meningitidis
  82. streptococcus often have _______ acid in their capsules while neisseria often have _______ acid
    streptococcus often have hyaluronic acid in their capsules, while neisseria often have sialic acid
  83. Benefits of capsules:
    • 1. keeps the cells from drying out quickly
    • 2. help cells adhere to a surface where growth conditions are favorable
    • mutant bacteria unable to make a capsule are EASILY eaten by phagocytes = harmless
  84. Encapsulated bacteria are often capable of causing which two diseases?
    septicemia and meningitis
  85. Does pseudomonas aeruginosa produce a capsule? If so what kind?
    • produces extracellular polysaccharide ==> alginate
    • called “mucoid”, which is a term for bacteria that produces large amounts of alginate
    • alginate capsule protects P. aeruginosa against host defenses & antibiotics
  86. What controls the production of alginate in pseudomonas aeruginosa?
    AlgU: gene that encodes a sigma factor which activates [brings together the core enzyme] alginate gene transcription (analogous to TcdR in C. difficile)
  87. MucA
    • protein located in the periplasm that normally holds onto AlgU, preventing it from moving to the alginate gene promotor and activating transcription
    • when cells sense cell membrane wall stress however, proteases degrade MucA and AlgU is free

  88. What happens over time to a person with CF infected by P. aeruginosa?
    • the bacteria produce relatively little alginate in the beginning of an infection, however over time, mutant bacteria that produce HUGE amounts of alginate (described as mucoid mutants) take over the population, worsening the infection
    • * these mutants typically have a mutation in mucA
  89. Why aren’t antibiotics effective in eliminating P. aeruginosa?
    • airway mucus is viscous, difficult for antibiotics to move through
    • presence of a mucoidy (alginate covering) makes it difficult for antibiotics to penetrate
    • biofilm formation - another difficulty for antibodies to get past
  90. Biofilms
    • differentiated mushroom/column-like structures of bacteria w/ liquid-filled cavities around them that can form on a wide variety of surfaces
    • may contain a one or multiple types of bacteria
  91. Steps of Biofilm formation
    • planktonic bacteria find a viable surface using chemotaxis and 'motilate' there using pili &/or flagellum
    • 1. bacteria reversibly attach - flagellum/pili are present
    • 2. cells irreversibly attach to the surface; their flagellum/pili are retracted
    • 3. cells grow and duplicate to form a microcolony - a capsule (extracellular polysaccharide) is synthesized (eg. Alginate)
    • 4. biofilm cells prepare for being released by expressing flagellum or pili again
    • 5. bacteria are released from the biofilm and become planktonic again (+ flagellum/pili)

  92. How do bacteria know when to form a biofilm?
    using chemotaxis to determine whether there's an available nutrient source and intracellular signaling to sense if they have a lot of neighbors
  93. Quorum Sensing
    sensing the concentration of a compound that is secreted by cells
  94. intracellular signaling
    the ability for bacteria to signal to their neighbors (of the same or different species) about the environment & the relative population density of each type of bacteria present
  95. Bacterial cells that are unable to form a biofilm are defective in what process?
    INTRAcellular cell-cell signaling, or quorum-sensing
  96. What do gram-negative and gram-positive bacteria respectively use as signaling compounds?
    • gram-negative: acylated homoserine lactones (the more complicated bacteria use the more complicated signaling compound)
    • gram-positives: peptides (Positives use Peptides)
  97. By what mechanism does the bacteria Salmonella enterica decide whether to turn on its pathogenic functions?
    • intracellular signaling - individual bacterium send out a signal molecule, then monitor how strong that signal is in the environment
    • a large population of S. enterica in the gut will result in a high level of signal returned; this initiates the pathogenic pathway
    • if the cell receives little or no signal in return, it won't turn on pathogenic functions & will remain harmless
  98. acylated homoserine lactones
    • the primary species-SPECIFIC signaling molecule for gram-negative bacteria
    • e/a species produces a homoserine lactone w/ a DISTINCTIVE acyl (fatty acid) group
  99. peptide signaling
    • used by gram-positive bacteria, usually short peptides (5-15 amino acids in length)
    • are produced intracellularly as longer precursors then shortened during secretion and by extracellular proteolysis
    • taken up by producer or other cells nearby using peptide transporters
  100. How do both acylated homoserine lactones (-) AND peptides (+) often produce signals?
    via two-component regulatory systems
  101. Two-component regulatory systems
    • commonly used mechanisms in bacteria for sensing and responding to environmental signals
    • 1. sensor kinase: usually traverses cytoplasmic membrane & detects specific environmental signals
    • 2. recognition of a specific signal outside the membrane results in conformational change of sensor kinase so that it phosphorylates the part of itself in the cytoplasm
    • 3. newly acquired phosphate group is then transferred to response regulator (RR) whose activity is altered as a result of the phosphorylation
    • 4. phosphorylated response regulator usually goes on to act as a transcription factor, either activating or repressing transcription
  102. What else can the bacterial phosphorylation cascade be used for besides transcription regulation?
    the phosphorylation cascade can also be used by the chemotaxis-motility system to regulate the flagellar motor
  103. What are some potential novel targets for anti-infectives (eg. for P. aeruginosa)?
    look for disrupting special properties such as alginate polysaccharide coating formation, pili formation, and/or any signal transduction mechanism