Microbiology 3: Biology of Infectious Agents
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Prokaryotes = bacteria
o No nuclei or membrane-bound organelles
 Allows simultaneous synthesis of proteins and mRNA (translation coupled to
transcription)
o Do not endocytose
o Haploid (1 chromosome and extrachromosal plasmids)
Eukaryotes = fungi, protozoa, worms
o Diploid (23 chromosomes)
o Nuclear membrane separates translation and transcription
Problems of Living freely (unicellular organisms)
o Nutrition = feast or famine (intermittent food available)
 Efficiency and adaptability
o Occupancy = adhesive polysaccharides (create biofilm)
o Resistance = genetic strategies
Small size = high metabolic rate -> grow extremely fast
o Limited by diffusion (smaller less limited)
Bacteria have cytoplasmic membrane
o Some also have cell wall, outer membrane, flagella, pili, and capsule
 Serve as protection against environment and adherence
o Strongest antibody action to surface antigens
Gram staining -> crystal violet modified with potassium iodide, decolorized with alcohol and
counterstained with safranin
o Gram-positive = retain dye, purple
o Gram-negative = lose dye, red/pink
Acid-fast staining -> hot carbolfuchsin (red) decolrized with acid alcohol and counterstained with
methylene blue
o Acid-fast = remain red
o Others become blue
Gram-positive bacteria -> have thick cell wall composed of murein (aka peptidoglycan) and
teichoic acids (ribitol/glycerol + phosphodiester bonds)
o Murein = glycan chains (N-acetylglycosamine and N-acetylmuramic acid) cross-linked via
peptides
 Shapes into rods (bacilli), spheres (cocci), or helices (spirilla)
 Lysosyme + low-osmotic P = lysis
 Lysozyme + isosmotic medium = become spherical (spheroplasts)
o Withstand hydrophobic compounds (aka bile salts)
o Murein cross-linked between amino group of lysine and carboxyl group of D-alanine
Gram-negative bacteria -> outer membrane outside murein cell wall (bilayered), outer layer
contains lipopolysaccharide (LPS)
o LPS components:
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Lipid A (endotoxin): anchors LPS, disaccharides + fatty acids/phosphate groups
 Elicits fever and activates host defense mechanisms, shock and death
 Core: 2 sugars -> ketodeoxyoctanoic acid and heptose
 O antigen: long carb chain, exclude hydrophobic compounds
o Murein cross-linked between diaminopimelic acid and D-alanine
o Outer membrane has channels (porins) for passive diffusion of hydrophilic compounds
 Some necessary hydrophilic compounds (vit B12, oligosaccharides, iron
chelates) too large for porins -> use unique translocating proteins
o Periplasmic space/periplasm between outer and inner membrane
 Contains murein layer, degradative enzymes, binding proteins, β-lactamases
 Gram + secrete enzymes into the medium
o Outer membrane -> used by bacteriophages to attach but also confers antibiotic
resistance
Acid-Fast Bacteria -> tubercle bacillus, cell walls have waxes (long-chain hydrocarbons) with
murein, polysaccharides and lipids
o Impervious to harsh chemicals and avoid WBCs
o Grow very slowly (nutrient uptake limited by waxy coat)
Penicillins, cephalosporins and carbapenems (β-lactams) = inhibit murein synthesis
o Bactericidal and least toxic to humans
o In “tolerant” bacteria (deficient in autolysin), β-lactams are bacteriostatic
Murein building blocks aka N-acetylglucosamine (GlcNAc) or N-acetylmuramic acid made in
cytoplasm
o Transferred to lipid carrier and linked to disaccharides (inhibited by vancomysin)
 Bacitracin inhibits regeneration of lipid carrier
o Transpeptidation (cross-link via peptide bonds) between subterminal D-alanine and
lysine or diaminopimelic acid
 Terminal D-alanine cleaved away
 Penicillins and cephalosporins inhibit transpeptidase -> make penicilloyl-enzyme
complex (lethal)
Murein synthesis inhibitors lyse cell (continues to grow without stable wall)
o If cells not growing, not lysed
Mycoplasmas and archaea have no murein (not rigid and don’t have shape)
o Resistant to penicillin
o Mycoplasma pneumoniae have sterols in membrans
Anthrax bacillus have outside covering of S layer (tough)
Cytoplasmic membrane (CM) uptakes substrates (small compounds)
o Permeases (carrier proteins) facilitate entry of metabolites -> require E
Modes of transport across CM:
o Facilitated diffusion -> down concentration gradient (ex. glycerol)
o Group translocation -> aka phosphorylation-linked transport; substances chemically
altered (ex. glucose)
 E.coli uses this pathway for sugar transport (strictly aerobic bacteria don’t use)
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Active transport -> E used to concentrate substance intracellularly (ex. lactose)
 E from proton motive force (extrusion of protons from cell) couples
energetically unfavorable transport with energetically facorable proton effluc =
symport
 Dominant transport mechanism
o All use specific binding proteins and actual transport is from permeases (high affinity
outside and low affinity inside cell)
Iron is essential for bacterial growth -> uptake it via excreting chelating compounds
(siderophores)
Cytochromes and oxidative metabolism are in CM along with nascent proteins
Uptake of DNA by bacteria depend on proton motive force
Genome of bacteria = single circular chromosome
o DNA coiled into nucleoid (twisted into supercoils)
o DNA gyrase introduces supercoils and topoisomerase I relaxes them (nicks DNA)
Replication of DNA is bidirectional
o Starts at replicative origin
Rate of DNA polymerase movement (aka replication) is nearly independent of the growth rate
o Replication is regulated by frequency of initiation of DNA synthesis
Metronidazole = modified to active form by bacteria (DNA replication inhibitor)
o Nitro group partially reduced to render active (by anaerobic bacteria)
 Incorporated into DNA = lethal synthesis (unstable)
Nalidixic acid = inhibits DNA gyrase, bactericidal
Fluoroquinolones = interfere with DNA gyrase or topoisomerase (dsDNA breaks)
Bacterial ribosomes have smaller subunits and RNA than eukaryotes
o 21 proteins (small) and 35 proteins (large) subunits
Protein synthesis = principal biosynthetic activity of rapidly growing bacteria
o RNA is made at rate proportional to number of RNA polymerase molecules engaged in
transcription
o Protein synthesis proportional to cellular concentration of ribosomes
 Regulated by frequency of initiation
Rifampin = inhibitor of bacterial transcription; acts at ignition step (binds to free RNA
polymerases)
o Used in tuberculosis and leprosy
o Mammalian RNA polymerases don’t bind rifampin
Inhibitors of protein synthesis = bind to small or large ribosomal subunit
o Chloramphenicol and macrolides (erythromycin) = block formation of peptide bonds
(bind near tRNA bind site on large subunit)
 Action is reversible, bacteriostatic
o Aminoglycosides (streptomycin, gentamicin, neomycin) = bactericidal, irreversible,
bind 30S subunit
 Enhances 30S and 50S interaction (Free 70S particles)
 Inhibits elongation of peptide chains
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o Linezolid = blocks assembly of initiation complex
o Tetracycline = inhibits tRNA binding
Some bacteria have capsules, flagella and pili
o Capsule = slimy outer coat difficult for WBC phagocytoses, high MW polysaccharides
 Pneumococci, meningococci and bacteria likely to encounter phagocytes
encapsulated
o Flagella = long, helical filaments for motility (1 or many)
 Peritrichous bacteria has > 1 flagella
o Pili (aka fimbriae) = attach bacteria to cells and sufaces
 F pilus = sex pilus of E. coli required for colonization and conjucation
Flagella used for chemotaxis
o Spin around from point of attachment
o Each flagellum has counterclockwise helical pitch
 If >1, bundles form and beat together -> swim in straight line
 If rotate clockwise, bacteria tumble randomly
o Without attractants or repellants, alternate between swimming and tumbling
 Attractant -> more swimming
 Repellants -> swimming stops quickly
Microbes are attracted to specific tissues (tissue tropism) -> attachment to specific receptors
o Use pili to attach
o Bacteria that conjugate have sex pili (longer and link donor and recipient)
Gonoccoci code for pilin (form pili) -> each version elicits different antibody
o Allow for quick avoidance of immune system (shift from one pilin to another)
o Pilin gene components: 1 constant portion (not antigenic) and 1 variable region (highly
antigenic)
Salmonella change expression of genes encoding flagella = phase variation
Nutritional requirements:
o Photosynthetic/chemosynthetic = subsist on CO2 and minerals
o Organisms the need preformed organic compounds = pathogenic microbes
 E. coli need glucose; others need vitamins, AAs, neuclotides, etc
Oxygen requirements:
o Strict aerobes = must have O2 (tubercle bacillus)
 Perform respiration only (final e- acceptor = molecular oxygen)
o Strict/obligate anaerobes = cannot grow in O2 (botulism and tetanus)
 Carry out fermentation (final e- acceptor = organic molecule [pyruvate, acetyl
coA])
 Anaerobic respirers carry out respiration using nitrate or sulfate as final eacceptor
o Facultative anaerobes = grow regardless of O2 (E. coli and intestinal bacteria)
 Perform either respiration or fermentation depending on O2 status
Respiration yields more E than fermentation
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E. coli can use 30 substances for E while Pseudomonas can use hundreds (omnipresent in water
supply and soil)
Minimal media = water solutions of glucose, ammonia, phosphate, sulfate and other minerals
o Bacteria that can use available compounds survive
Nutrient broth (rich medium) = meat extract and soluble complex proteins
o Nutritionally fastidious organisms require complex media (staphylococci, streptococci)
Add agar to either nutrient broth or minimal media = solid media
Chlamydia = obligate intracellular parasites (only replicate inside host not on media)
Treponema pallidum (syphilis) or Mycobacterium leprae (leprosy) should but don’t grow on
media
Generation/doubling time = time it takes for bacterium to become 2
Total count = body count of bacteria present whether living or dead
o Estimate by measuring property proportional to # present (like turbidity)
Colony count = living/viable bacteria
o Number of colonies x dilution factor = colony-forming units (CFUs)
 If bacteria grow in clups or chains, CFUs is underestimate of total bacteria
Law of growth: Nt = N0ekt for balanced growth
o Describes a geometric progression or decay with time of radioactive isotope
Bacteria grow to certain density and exhaust nutrients or have toxic metabolites (stop growth)
o Aerobic bacteria -> crowding exhausts O2
o Anaerobes -> toxic metabolites = H2O2 or acids
o Stage of culture where growth stops = stationary phase
Exponential growth allows small bacteria to rapidly initiate infection
o Ex. acute bacterial meningitis in child
Bacteria don’t cease all metabolic activity at stationary phase -> continue synthetic activities for
adaptation
o Can use ribosomes (that aren’t being used) as source of AAs
Bacteria exposed to injuries -> proteins repaire damage = SOS response
o Protective response turned on when starved
Even when not growing bacteria can still damage host
o Elicit immune response
o Produce toxins in stationary phase (to replenish nutrients)
Cessation of growth may initiate sporulation -> produce spores resistant to chemical and
physical insult
o Mother cell lyses releasing lots of toxins (ex. tetanus, gas gangrene)
Metabolic efficiency = metabolic parsimony (bacteria don’t make constituents they can’t use)
o E. coli, when supplied with leucine, will instantly stop making leucine and rely on
exogenous supply
 First enzyme in leucine synthesis path allosterically inhibited by presence of
leucine = feedback or end-product inhibition
 Synthesis of enzymes of leucine path turned off
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Genes for path strung together in operon -> transcription turned on/off
by single regulatory switch = promotor (where RNA polymerase binds)
 Operons regulated by attenuation -> RNA pol encounters attenuator (in
presence of leucine) and transcription terminated -> stop synthesis of
enzymes
o Bacteria using lactose need to make β-galactosidase but without lactose, stop making it
 Also, operator (past promoter) binds repressor (when lactose is absent) -> stops
transcription of β-galactosidase
 β-galactosidase = inducible enzyme (made on demand); lactose = inducer
 Constitutive enzymes made at all times
Regulation of gene expression comes at E cost
o Regulation by attenuation depends on mRNA synthesis not used if enzymes of the
operon not made
o Using repressor path requires constitutive synthesis of protein repressor molecules