Ch 2Microbial Cell Structure (Bacteria + Archaea)
Peptidoglycan (PG, murein): polymer of NAG-NAM disaccharides cross-linked by tetrapeptides (L-Ala-D-Glu-mDAP-D-Ala in G−; L-Ala-D-Glu-L-Lys-D-Ala in G+ with cross-bridge). Lysozyme cleaves NAG-NAM β-1,4 bond. Penicillin binds D-Ala-D-Ala mimicry → inhibits transpeptidase → no cross-linking → cell lyses under turgor.
Gram-positive vs Gram-negative envelope:
- G+: thick PG (20–80 nm) outside plasma membrane. Teichoic + lipoteichoic acids embedded. NO outer membrane. NO periplasm proper. Stains purple (CV-I retained through ethanol decolorize).
- G−: thin PG (~10 nm) sandwiched between plasma membrane (inner) + outer membrane. Outer membrane outer leaflet = LPS (Lipid A endotoxin + core + O-antigen). Periplasm contains β-lactamases + binding proteins. Porins span outer membrane. Stains pink (decolorized by ethanol; safranin counterstains).
Archaea have NO peptidoglycan. Walls vary: pseudopeptidoglycan (NAG-NAT instead of NAG-NAM, lysozyme-resistant), or S-layer (paracrystalline 2D protein lattice), or no wall (Thermoplasma). Archaeal membranes use ether-linked isoprenoid alcohols instead of ester-linked fatty acids; sometimes monolayer (in hyperthermophiles). Confers extreme temperature/pH resistance.
Endospores (Bacillus, Clostridium): dormant heat/desiccation/chemical-resistant survival structures formed under starvation. Core dehydrated, packed with dipicolinic acid + Ca²⁺ (DPA-Ca²⁺) and SASPs (small acid-soluble proteins) protecting DNA. Multiple coats (cortex modified PG, spore coat proteins). Survive boiling (autoclave at 121°C 15 psi 15 min required to kill). Germinate when conditions favor.
Ch 4Microbial Metabolism — Energy
Glycolysis (Embden-Meyerhof-Parnas): glucose → 2 pyruvate, net 2 ATP (substrate-level phosphorylation) + 2 NADH. Most microbes share this pathway. Alternatives: Entner-Doudoroff (Pseudomonas, gives less ATP), pentose phosphate (NADPH for biosynthesis).
Pyruvate fates:
- Aerobic: pyruvate → acetyl-CoA → TCA cycle → CO₂ + 3 NADH + 1 FADH₂ + 1 GTP per acetyl-CoA. Then ETC + chemiosmosis → ~32-38 ATP/glucose total.
- Fermentation: organic e⁻ acceptor; substrate-level phosphorylation only. Lactate (Streptococcus, Lactobacillus), ethanol + CO₂ (Saccharomyces, Zymomonas), butyrate, propionate, mixed-acid (E. coli — distinguishable from Enterobacter via IMViC tests), 2,3-butanediol (Enterobacter — VP positive).
- Anaerobic respiration: ETC with non-O₂ acceptor — NO₃⁻ (denitrification → N₂), SO₄²⁻ (Desulfovibrio → H₂S), Fe³⁺ (Geobacter), CO₂ (methanogens → CH₄), fumarate.
The redox tower ranks acceptors by reduction potential. Higher = more energy yielded. O₂ (+820 mV) tops it. NO₃⁻ (+430 mV), Fe³⁺ (+200), SO₄²⁻ (−220), CO₂ (−250) descend. Microbes preferentially use the most positive available acceptor.
Chemolithotrophs use inorganic electron donors. Examples: Nitrosomonas (NH₃ → NO₂⁻), Nitrobacter (NO₂⁻ → NO₃⁻), Acidithiobacillus (S, S²⁻, Fe²⁺), hydrogenotrophic methanogens (H₂). Often autotrophic (CO₂ via Calvin cycle).
Phototrophs: oxygenic (cyanobacteria split H₂O → O₂ as byproduct + use 2 photosystems like plants), anoxygenic (purple/green sulfur bacteria use H₂S + 1 photosystem; no O₂ produced).
Ch 7Horizontal Gene Transfer (HGT)
Three HGT mechanisms:
- Generalized: random host DNA accidentally packaged during lytic assembly.
- Specialized: imprecise prophage excision picks up flanking host genes (λ phage gal/bio classic).
- F+ donor expresses sex pilus, contacts F− recipient.
- Mating bridge forms.
- Relaxase nicks oriT of F plasmid.
- Single strand transferred (5' first) to recipient.
- Both cells synthesize complementary strand.
- Recipient now F+. Hfr cells (F integrated into chromosome) transfer chromosomal genes too.
Restriction-modification defends against foreign DNA: methylase modifies host DNA at recognition sites; restriction enzyme cleaves unmethylated foreign DNA. Type II R enzymes are molecular biology workhorses.
CRISPR-Cas adaptive immunity: bacteria + archaea retain "memory" of past phages as spacers in CRISPR array (acquired by Cas1+Cas2 during initial infection). On re-infection, crRNA-Cas9 complex scans foreign DNA for matches near PAM (NGG); if found, Cas9 nuclease cleaves both strands. Now repurposed for genome editing (Doudna + Charpentier 2020 Nobel).
Mobile genetic elements: insertion sequences (IS) — simplest transposons, transposase + inverted repeats. Composite transposons — flanked by IS elements, can carry resistance genes. Integrons — capture + express gene cassettes. Plasmids — extrachromosomal, often carry virulence + resistance. Conjugative plasmids have tra genes for transfer.
Ch 8Regulation of Gene Expression
An operon is a cluster of genes transcribed as one polycistronic mRNA from a single promoter. Common in bacteria; absent in eukaryotes (mostly).
- Glucose absent + lactose present → some lactose enters cell.
- β-galactosidase converts to allolactose (transglycosylation).
- Allolactose binds LacI repressor → falls off operator.
- Low glucose → high cAMP → CAP-cAMP forms.
- CAP-cAMP binds upstream of promoter → recruits RNA pol.
- lacZYA transcribed → β-gal + permease + transacetylase.
- Glucose present → low cAMP → poor CAP recruitment → weak transcription even with lactose (catabolite repression).
- Tryptophan abundant → binds TrpR repressor (Trp = corepressor).
- TrpR + Trp binds operator → blocks transcription.
- Plus ATTENUATION: leader peptide ribosome stalls at trp codons (low Trp) → mRNA forms anti-terminator hairpin → readthrough.
- High Trp → ribosome zooms through leader → mRNA forms terminator hairpin → premature stop.
σ factors: subunits of RNA pol that recognize specific promoter classes. E. coli has σ70 (housekeeping; recognizes -10 TATAAT + -35 TTGACA), σ32 RpoH (heat shock), σS RpoS (stationary phase), σ54 RpoN (nitrogen), σF/FliA (flagellar), σE RpoE (extracytoplasmic stress).
Two-component systems: membrane sensor histidine kinase autophosphorylates on stimulus → transfers phosphate to cytoplasmic response regulator → DNA binding → transcription. PhoP/PhoQ (Mg²⁺ sensing in Salmonella), EnvZ/OmpR (osmotic in E. coli).
Quorum sensing: cell-density-dependent gene regulation via diffusible autoinducers. Gram-negative use AHLs (LuxI synthase + LuxR receptor); Gram-positive use autoinducer peptides (AIPs) sensed by membrane HK. P. aeruginosa LasI/LasR + RhlI/RhlR control virulence + biofilm + alginate.
Riboswitches: 5'-UTR mRNA element binds metabolite directly → conformational change → premature termination or RBS occlusion. No protein needed. SAM, lysine, glycine, FMN, TPP, glucosamine-6-P riboswitches all known.
sRNAs: trans-acting small RNAs (often Hfq-dependent) bind target mRNA → block translation or recruit RNase E for decay. Major regulators of stress response.
Ch 13Pathogenesis — ROWEN FOCUS
Stages of pathogenesis: exposure → adhesion → invasion → infection → tissue damage → spread → exit. Successful pathogens accomplish each step.
Adhesion: pili (Type I, P, Type IV), fimbriae, surface adhesins bind specific host receptors. Determines tissue tropism. E. coli P-pili bind globoside on uroepithelium → UTI. V. cholerae TCP pilus binds intestinal mucin → cholera.
Toxins are major virulence factors:
- Exotoxins: secreted proteins, often A-B structure (B binds + delivers, A is enzymatic toxin moiety). Heat-labile, antibody-neutralizable (toxoid vaccines work).
- Diphtheria toxin: A subunit ADP-ribosylates EF-2 → blocks protein synthesis. Toxoid vaccine since 1923.
- Cholera toxin: A subunit ADP-ribosylates Gαs → adenylate cyclase locked on → cAMP↑ → CFTR opens → massive Cl⁻ + H₂O secreted → severe watery diarrhea ("rice water stool").
- Botulinum + tetanus toxins: zinc metalloproteases that cleave SNAREs (synaptobrevin, SNAP-25, syntaxin) → block neurotransmitter release. Botulinum at NMJ → flaccid paralysis. Tetanus at inhibitory interneurons → spastic paralysis.
- Shiga toxin: A subunit depurinates 28S rRNA → blocks translation. EHEC O157:H7.
- Pseudomonas exotoxin A: ADP-ribosylates EF-2 (same target as diphtheria).
- Endotoxin (LPS): structural component of Gram-negative outer membrane. Released on lysis. Lipid A → TLR4/MD2 → NF-κB → cytokine storm (TNFα, IL-1β, IL-6) → fever + vasodilation + capillary leak → septic shock + DIC. Heat-stable (autoclave doesn't destroy). Weakly immunogenic (no toxoid vaccines).
Secretion systems deliver effectors:
- T3SS (Type III) — needle injectisome (flagellum-related), delivers effector proteins directly into host cell cytosol. Yersinia (Yop effectors), Salmonella (Sip), Shigella (Ipa), Pseudomonas (ExoS/T/U/Y), EPEC. ROWEN SPECIALTY.
- T4SS (Type IV) — conjugation-derived; transfers DNA OR proteins. H. pylori CagA injection → gastric cancer association. Agrobacterium T-DNA → plant tumors. Legionella Dot/Icm → intracellular survival.
- T6SS (Type VI) — phage-tail-derived "harpoon" injects toxins into adjacent cells (often competing bacteria — interbacterial competition). Pseudomonas, Vibrio, Burkholderia.
Pseudomonas aeruginosa — Rowen's organism in detail:
Gram-negative rod, motile via flagella + Type IV pili (twitching), opportunistic pathogen of immunocompromised + CF patients. Intrinsically multidrug-resistant via efflux pumps (MexAB-OprM, etc.) + low outer membrane permeability + chromosomal AmpC β-lactamase.
- Initial colonization in CF airway (defective CFTR → thick mucus → impaired clearance → bacterial niche).
- Selective pressure (oxidative stress, antibiotic exposure, dehydration) selects for mutants in mucA.
- mucA encodes anti-σ factor that sequesters σ22 (AlgT/U) at inner membrane.
- mucA loss-of-function → σ22 freed → activates algD operon.
- algD operon encodes alginate biosynthesis enzymes (acetylated polysaccharide of mannuronate + guluronate).
- Mucoid phenotype: alginate-encased biofilm. Resists phagocytosis, antibiotics, immune clearance.
- Biofilm-embedded P. aeruginosa is 100–1000× more antibiotic-tolerant than planktonic — slow growth + persisters + EPS diffusion barrier.
- Chronic colonization → progressive lung damage → CF mortality.
Quorum sensing in P. aeruginosa: hierarchical AHL system. LasI synthesizes 3-oxo-C12-HSL → LasR receptor activates virulence genes (lasA, lasB, lasI). RhlI synthesizes C4-HSL → RhlR activates rhamnolipids + pyocyanin + biofilm genes. PQS (Pseudomonas quinolone signal) integrates further. Anti-QS therapeutics in development.
Ch 14Antimicrobials + Resistance
Antibiotic classes by target:
- Cell wall: β-lactams (penicillins, cephalosporins, carbapenems, monobactams) inhibit transpeptidase (PBP). Vancomycin binds D-Ala-D-Ala. Bacitracin blocks lipid carrier. Bactericidal.
- 30S ribosome: aminoglycosides (streptomycin, gentamicin, tobramycin — bactericidal, mistranslation). Tetracyclines (block tRNA entry, bacteriostatic).
- 50S ribosome: macrolides (erythromycin, azithromycin — block exit tunnel, static). Chloramphenicol (peptidyl transferase, static). Lincosamides. Oxazolidinones (linezolid — vs MRSA, VRE).
- DNA replication: fluoroquinolones (ciprofloxacin, levofloxacin) inhibit DNA gyrase + Topo IV. Bactericidal.
- RNA pol: rifampin binds RpoB. Bactericidal. Single-step mutational resistance — used in combo for TB.
- Folate synthesis: sulfonamides (DHPS) + trimethoprim (DHFR) — sequential blockade synergistic (TMP-SMX).
- Membrane: daptomycin (lipopeptide; depolarizes Gram-positive membrane). Polymyxins (Lipid A binding; reserve drug).
Resistance mechanisms:
- β-lactamases hydrolyze β-lactam ring.
- ESBLs (extended-spectrum) cleave 3rd-gen cephalosporins.
- Carbapenemases: KPC (class A serine), NDM-1 (metallo), OXA-48. Critical priority pathogens.
- Aminoglycoside-modifying enzymes (acetyltransferases, phosphorylases, adenylyltransferases).
- MRSA: mecA → PBP2a (low β-lactam affinity). Treatment: vanco, daptomycin, linezolid, ceftaroline.
- VRE: vanA/vanB → D-Ala-D-Lac instead of D-Ala-D-Ala → ~1000× lower vanco affinity.
- Quinolone R: gyrA + parC mutations.
- Rifampin R: rpoB mutations.
- Macrolide R: 23S rRNA methylation (erm genes) or efflux (mef).
WHO Priority Pathogens (2024): CRITICAL — Acinetobacter baumannii (carbapenem-R), Pseudomonas (carbapenem-R), Enterobacterales (carbapenem-R, 3rd-gen ceph-R). HIGH — VRE, MRSA, vanco-R Staph, Salmonella + Shigella (fluoroquinolone-R), N. gonorrhoeae (multidrug-R).
MIC = Minimum Inhibitory Concentration: lowest [drug] that prevents visible growth. Standardized via broth microdilution or E-test. Used to define susceptible/intermediate/resistant.
Antibiotic stewardship: optimize antibiotic use to reduce resistance + adverse events. Strategies: shortest effective course, narrowest spectrum, IV→PO conversion, prospective audit + feedback.