Molecular Microbiology — Study Guide

U1 · Microorganisms & microbiology

Three domains

Bacteria, Archaea, Eukarya — Carl Woese's rRNA-based tree (1977). Archaea are sister to Eukarya, not Bacteria.

Koch's postulates

(1) Microbe present in disease, absent in healthy. (2) Isolated in pure culture. (3) Causes disease when introduced. (4) Re-isolated from new host. Modernized for non-culturable + viral pathogens.

Pasteur

Disproved spontaneous generation; pioneered fermentation, pasteurization, vaccines (rabies, anthrax).

Microbial sizes

Most bacteria 1-5 µm; viruses 20-300 nm; Mycoplasma ~0.3 µm; Thiomargarita up to 750 µm (visible!).

Gram stain

Crystal violet → iodine mordant → ethanol decolorize → safranin counter. Gram-positive retain purple (thick peptidoglycan); Gram-negative pink (thin PG + outer membrane).

U2 · Microbial cell structure

Peptidoglycan

Murein. Polymer of NAG-NAM cross-linked by tetrapeptides. Bacteria only — Archaea have pseudopeptidoglycan (NAG-NAT) or other walls.

Teichoic acids

Glycerol/ribitol-phosphate polymers anchored in Gram-positive cell wall; antigen + ion homeostasis.

LPS (lipopolysaccharide)

Gram-negative outer-membrane outer leaflet; Lipid A (endotoxin) + core + O-antigen. Triggers TLR4 / septic shock.

Periplasm

Gel-filled space between inner + outer membranes in Gram-negatives; site of secreted enzymes (β-lactamases).

Capsule / S-layer

Outer polysaccharide capsule (virulence factor; resists phagocytosis). S-layer = paracrystalline protein layer (especially Archaea).

Bacterial flagellum

Helical filament rotated by H⁺- or Na⁺-driven motor. Run-and-tumble chemotaxis. Right-handed helix in E. coli.

Pili / fimbriae

Surface filaments. Type IV pili for twitching motility + biofilm; F-pilus for conjugation.

Endospore

Dormant heat/chemical-resistant structure of Bacillus, Clostridium. Dipicolinic acid + Ca²⁺ in core. Triggered by starvation.

Archaeal membrane

Ether-linked isoprenoid lipids (vs ester-linked fatty acids in Bacteria/Eukarya); often monolayer in hyperthermophiles.

U3 · Microbial growth

Binary fission

One cell → two via FtsZ ring at midcell + septum formation.

Generation time

Time for population to double. E. coli ~20 min in rich media; M. tuberculosis ~24 hr.

Growth curve

Lag → exponential (log) → stationary → death. N = N₀ × 2^n.

Continuous culture (chemostat)

Steady-state growth at controllable rate by limiting one nutrient.

Optimal temperature classes

Psychrophile (<15°C), mesophile (20-45°C), thermophile (45-80°C), hyperthermophile (>80°C).

pH classes

Acidophile (<5), neutrophile (5-8), alkaliphile (>9). Stomach H. pylori = neutrophile that buffers via urease.

Oxygen classes

Obligate aerobe, facultative anaerobe (best in O₂ but tolerates), microaerophile (low O₂), aerotolerant anaerobe, obligate anaerobe.

Water activity (a_w)

Free water available for growth. Halophiles thrive at low a_w (high salt).

U4 · Metabolism — energy & biosynthesis

Catabolism vs anabolism

Catabolism breaks down to make energy (ATP) + reducing power (NADH/NADPH). Anabolism builds biomass.

Chemoorganotroph

Organic compounds = energy + carbon source. Most cultured bacteria.

Chemolithotroph

Inorganic e⁻ donors (H₂, NH₃, NO₂⁻, H₂S, Fe²⁺) for energy. CO₂ fixation for carbon (often).

Phototroph

Light-driven energy. Oxygenic (cyanobacteria, plants) split H₂O → O₂. Anoxygenic photosynthesis uses H₂S, etc.

Fermentation

Substrate-level phosphorylation only; organic e⁻ acceptor. Products: lactate, ethanol, butyrate, acetate, etc.

Aerobic respiration

Glycolysis + TCA + ETC; O₂ as terminal e⁻ acceptor → H₂O. Highest ATP yield.

Anaerobic respiration

ETC with non-O₂ acceptor: NO₃⁻ (denitrification), SO₄²⁻ (Desulfovibrio), Fe³⁺ (Geobacter), CO₂ (methanogens), fumarate.

Substrate-level vs oxidative phosphorylation

SLP: phosphate from substrate to ADP (e.g., glycolysis step 7). Oxidative: ATP synthase using H⁺ gradient.

U5 · Metabolic diversity

Calvin cycle

RuBisCO fixes CO₂ in autotrophs. Found in cyanobacteria, plants, many lithotrophs.

Reverse TCA / 3-hydroxypropionate / Wood-Ljungdahl

Alternative CO₂ fixation pathways used by various Archaea + Bacteria. Wood-Ljungdahl in acetogens + methanogens.

Methanogenesis

Archaea-only (Euryarchaeota): CO₂ + H₂ → CH₄ + H₂O OR acetate → CO₂ + CH₄. Coenzyme F420, methyl-CoM.

Sulfate reduction

Desulfovibrio: SO₄²⁻ → H₂S using organic H donors. Marine sediments.

Nitrification

NH₃ → NO₂⁻ (Nitrosomonas) → NO₃⁻ (Nitrobacter). Two genera typically.

Denitrification

NO₃⁻ → NO₂⁻ → NO → N₂O → N₂. Anaerobic respiration; major N-cycle return flux.

Nitrogen fixation

N₂ → 2 NH₃ via nitrogenase (Mo-Fe protein). High ATP cost. Rhizobium + legumes; cyanobacteria; free-living.

U6 · Microbial genomes

Bacterial genome

Usually one circular chromosome in cytoplasm (nucleoid); supercoiled by gyrase + Topo I. ~few Mbp typically.

Plasmid

Extrachromosomal circular dsDNA; replicates independently. Carries optional genes (resistance, virulence, conjugation).

Origin of replication (oriC)

Site where replication initiates; DnaA binds DnaA boxes; helicase loads.

Replication enzymes

DnaB helicase, DnaG primase, DNA pol III holoenzyme (replicative), DNA pol I (Okazaki primer removal), ligase.

Topoisomerases

Type I single-strand cuts (Topo I); Type II double-strand cuts (DNA gyrase introduces neg supercoils, Topo IV decatenates daughters). Quinolone target.

Transposable elements

IS elements (insertion sequences) + composite transposons. Carry transposase + flanked by inverted repeats.

Pan-genome

Core genes shared across all strains + accessory genes in some. Highlights HGT.

U7 · Horizontal gene transfer

Transformation

Uptake of naked DNA from environment by competent cells (natural: Streptococcus pneumoniae, Bacillus subtilis; artificial: heat shock, electroporation).

Transduction

Phage-mediated DNA transfer. Generalized: any host DNA packaged. Specialized: prophage excision picks up flanking genes.

Conjugation

Cell-to-cell DNA transfer through pilus. F+ donor transfers F plasmid; Hfr integrates F into chromosome and transfers chromosomal genes.

Restriction-modification

Defense system: restriction enzyme cuts unmethylated foreign DNA; methylase protects host. Type II = molecular cloning workhorse.

CRISPR-Cas

Adaptive bacterial immunity. Spacers (records of past invaders) → crRNA + Cas9/12/13 → cleaves matching foreign DNA. Now genome-editing tool.

Site-specific recombination

Integrase between attP + attB (e.g., λ phage); recombines specific sites only.

U8 · Regulation of gene expression

Operon

Polycistronic mRNA from one promoter; coordinately regulated. Prokaryote-specific.

lac operon

Catabolic. Repressor (LacI) blocks transcription unless allolactose binds. CAP+cAMP activates when glucose low. Inducible operon.

trp operon

Anabolic. Repressor + tryptophan (corepressor) blocks transcription. Repressible operon. Also attenuation: leader peptide ribosome stalling.

Sigma (σ) factor

Subunit of bacterial RNA polymerase that recognizes specific promoter classes. E. coli: σ70 (housekeeping), σ32 (heat shock), σS (stationary), σ54 (N), σF (flagella), σE (extracytoplasmic stress).

Two-component system

Membrane sensor histidine kinase autophosphorylates; transfers phosphate to cytoplasmic response regulator → DNA binding. Bacteria-wide environmental sensing.

Quorum sensing

Cell-density-dependent gene regulation via diffusible autoinducers. AHLs (LuxI/LuxR) in Gram-negative; AIPs in Gram-positive. Controls biofilm + virulence.

Riboswitch

5'-UTR mRNA element binds metabolite directly → conformational change → premature termination or RBS occlusion. No protein needed.

sRNA / antisense RNA

Trans-encoded small RNAs (often Hfq-dependent) bind mRNA → block translation or recruit RNase E.

U9 · Viruses

Virus

Obligate intracellular acellular agent. Genome (DNA/RNA, ds/ss) + capsid (± envelope).

Capsid symmetries

Helical (TMV), icosahedral (T=1, 3, 7…), complex (T4 phage with head + tail).

Lytic cycle

Adsorption → penetration → replication → assembly → lysis (release).

Lysogenic cycle

Phage integrates (prophage) into host chromosome; replicates passively until induction (UV, etc.) → lytic cycle.

One-step growth curve

Plot of phage titer over time after synchronous infection; eclipse → latent → burst.

Retrovirus

+ssRNA → reverse transcriptase → dsDNA → integrase → provirus. HIV.

Baltimore classification

I dsDNA, II ssDNA, III dsRNA, IV +ssRNA, V −ssRNA, VI +ssRNA-RT (retro), VII dsDNA-RT (HBV).

CRISPR vs phages

Spacer acquisition from phage; later phages with matching protospacer cleaved.

U10 · Bacterial diversity

Proteobacteria

Largest, most diverse Gram-negative phylum. α (Rhizobium, Rickettsia), β, γ (E. coli, Pseudomonas, Vibrio), δ (Desulfovibrio, myxobacteria), ε (Helicobacter, Campylobacter).

Firmicutes

Gram-positive low-GC. Bacillus, Clostridium, Staphylococcus, Streptococcus, Lactobacillus.

Actinobacteria

Gram-positive high-GC. Mycobacterium (TB), Streptomyces (antibiotic source), Corynebacterium (diphtheria).

Cyanobacteria

Oxygenic photosynthesis; ancestor of chloroplasts. Heterocysts fix N₂ in some.

Spirochaetes

Long thin spirals with periplasmic flagella. Treponema (syphilis), Borrelia (Lyme), Leptospira.

Bacteroidetes

Anaerobic gut residents — gut microbiome dominant.

U11 · Archaea

Distinguishing features

Ether-linked isoprenoid membrane lipids; pseudopeptidoglycan or S-layer walls; unique RNA pol with 8-12 subunits (more like eukaryotic); histones in some.

Crenarchaeota

Mostly extreme thermophiles + acidophiles. Sulfolobus, Pyrococcus, Thermoproteus.

Euryarchaeota

Methanogens (Methanocaldococcus), extreme halophiles (Halobacterium), thermoacidophiles (Thermoplasma).

Thaumarchaeota

Ammonia oxidizers in marine + soil; major component of microbial N cycling.

Halophiles

Require >1.5 M NaCl. Use compatible solutes (K⁺, glycine betaine) for osmotic balance. Halobacterium uses bacteriorhodopsin proton pump.

Hyperthermophiles

Optimum T > 80°C. Pyrolobus fumarii grows at 113°C. Hydrothermal vents.

U12 · Microbial ecology

Microbiome

Microbial community in a defined habitat (gut, skin, soil, ocean). Studied via 16S rRNA + shotgun metagenomics.

16S rRNA

Universal phylogenetic marker; conserved + variable regions allow species ID without culture.

Biofilm

Surface-attached community in EPS matrix. Stages: attachment → microcolony → maturation → dispersal. Highly resistant to antibiotics + immune attack.

Quorum sensing in biofilms

AHL or AIP signals reach threshold density → coordinated gene expression (virulence, EPS, dispersion). Pseudomonas LasR + RhlR.

Carbon cycle role

Decomposers mineralize organic C → CO₂. Anaerobes ferment + methanogens produce CH₄ in wetlands + cattle.

Nitrogen cycle

Fixation (Rhizobium, cyanobacteria) → ammonification → nitrification → denitrification. Bacterial monopoly aside from lightning + Haber-Bosch.

Sulfur cycle

Sulfate reducers (Desulfovibrio) → H₂S; sulfide oxidizers (Beggiatoa, Thiobacillus) → SO₄²⁻.

Symbioses

Rumen microbiome digests cellulose; root nodules fix N₂; insect endosymbionts supply vitamins.

U13 · Pathogenesis — Rowen focus

Virulence factor

Microbial product that contributes to disease: toxins, capsules, adhesins, secretion systems, immune evasion.

Adhesion + colonization

Pili, fimbriae, surface adhesins bind host receptors. E. coli P-pili to UTI; Vibrio cholerae TCP to gut.

Exotoxins

Secreted proteins. Diphtheria (ADP-ribosylates EF-2), cholera (constitutive Gαs cAMP), botulinum + tetanus (cleave SNAREs), Shiga (depurinates 28S rRNA).

Endotoxin (LPS)

Lipid A of Gram-negative outer membrane. Released on cell lysis; TLR4 → cytokine storm → septic shock.

Type III secretion system (T3SS) [Rowen specialty]

Needle-like injectisome that delivers effectors directly into host cell cytosol. Yersinia, Salmonella, Pseudomonas aeruginosa, EPEC.

Type IV secretion (T4SS)

Conjugation-related secretion; Helicobacter CagA, Agrobacterium T-DNA, Legionella Dot/Icm.

Type VI secretion (T6SS)

Bacteriophage-derived; injects toxins into competing bacteria + sometimes host.

Pseudomonas aeruginosa [Rowen's organism]

Opportunistic pathogen; biofilms in CF lungs. Mucoid conversion (alginate overproduction) marks chronic infection. Multidrug resistance via efflux + porin loss + β-lactamases.

Mucoid conversion in P. aeruginosa

Mutation in mucA (anti-σ factor) releases σ22 (AlgT/U) → activates alginate biosynthesis genes → mucoid phenotype. Hallmark of CF lung adaptation.

Biofilm resistance

Slow growth, persister cells, EPS diffusion barrier, altered gene expression, quorum sensing → 100-1000× more antibiotic-tolerant.

U14 · Antimicrobials & resistance

Cell wall inhibitors

β-lactams (penicillin, cephalosporin, carbapenem) inhibit transpeptidase / PBPs. Vancomycin binds D-Ala-D-Ala. Bactericidal.

Protein synthesis inhibitors

30S: aminoglycosides (streptomycin, gentamicin), tetracyclines. 50S: macrolides (erythromycin), chloramphenicol, lincosamides, oxazolidinones (linezolid).

Nucleic acid inhibitors

Quinolones (ciprofloxacin) inhibit gyrase/Topo IV. Rifampin binds RpoB → blocks transcription. Metronidazole damages DNA in anaerobes.

Folate antagonists

Sulfonamides + trimethoprim block folate synthesis (sequential steps); synergistic combo (TMP-SMX).

MIC

Minimum inhibitory concentration: lowest [drug] that prevents visible growth. Standard antibiotic susceptibility metric.

Resistance mechanisms

(1) Enzymatic inactivation (β-lactamase, AME). (2) Efflux pumps (tetracycline, fluoroquinolone). (3) Target modification (PBP2a in MRSA, ribosomal methylation, DNA gyrase mutation). (4) Reduced uptake (porin loss).

MRSA, VRE, ESBL, CRE

MRSA = methicillin-resistant S. aureus (mecA → PBP2a). VRE = vancomycin-resistant Enterococcus (D-Ala-D-Lac). ESBL = extended-spectrum β-lactamase. CRE = carbapenem-resistant Enterobacterales (KPC, NDM-1).

Horizontal spread of resistance

Plasmids, integrons, transposons carry multiple resistance genes — co-selection drives MDR.

U15 · Applied / clinical microbiology

Industrial fermentation

Lactic acid (yogurt, sauerkraut), ethanol (beer, fuel), penicillin (Penicillium), insulin (recombinant E. coli), citric acid (Aspergillus).

Bioremediation

Microbes degrade pollutants: Pseudomonas on hydrocarbons, Geobacter on uranium, Dehalococcoides on chlorinated solvents.

Vaccine types

Live attenuated (MMR, OPV), inactivated (IPV, flu), subunit (HBV), toxoid (DT, TT), conjugate (Hib, pneumococcal), mRNA (COVID).

Sterilization vs disinfection

Sterilization eliminates ALL microbes (autoclave 121°C 15 psi 15 min). Disinfection reduces pathogens on inanimate surfaces. Antisepsis on living tissue.

Lab diagnosis

Culture + Gram stain + biochemical tests + MALDI-TOF + 16S rRNA + PCR. Rapid Ag/Ab tests for select pathogens.