Bacteria

Bacteria

Part 1 of 4 — Prokaryotic Architecture & the Gram Stain

What a Prokaryote Does NOT Have

No nucleus (a single circular chromosome sits in the nucleoid), no membrane-bound organelles, no histone-wrapped chromatin (though DNA is supercoiled by gyrase), no 80S ribosomes. What it DOES have: 70S ribosomes, a plasma membrane WITHOUT cholesterol, often plasmids (small autonomous DNA circles), and in most species a peptidoglycan wall (Mycoplasma has none).

• Mycoplasma, lacking a wall, stabilizes its membrane with sterols taken from the host — the classic exception to "no cholesterol in prokaryotic membranes."

Transcription and translation are coupled — ribosomes load onto mRNA while RNA polymerase is still working, possible only because there is no nuclear envelope. Bacterial mRNA is often polycistronic (operons).

The Gram Stain Divides the Bacterial World

Peptidoglycan itself: chains of alternating NAG-NAM sugars, cross-linked by short peptides containing D-amino acids (a prokaryotic signature — human proteases cannot cut D-linkages). Lysozyme (tears, saliva) hydrolyzes the NAG-NAM glycosidic bond; beta-lactams block the cross-linking (transpeptidase) step. Acid-fast organisms (Mycobacterium) wrap the wall in waxy mycolic acids, which make them stain poorly with the Gram stain (an acid-fast stain is used instead).

Outside the Wall

• Capsule: polysaccharide slime layer; antiphagocytic — the major virulence factor of encapsulated organisms; the basis of polysaccharide vaccines.

• Capsules are visualized by negative staining.

• Flagella: rotary protein motors driven by the proton-motive force (not ATP directly); direction of rotation sets run-vs-tumble chemotaxis.
• Pili/fimbriae: adhesion to host surfaces; the special sex pilus initiates conjugation.
• Endospores (Bacillus, Clostridium): metabolically inert, dehydrated survival capsules — resistant to heat, desiccation, radiation; boiling does not reliably kill them, autoclaving (121 C, pressurized steam) does. A spore is a lifeboat, not a reproductive cell: one cell → one spore → one cell.

• The spore core is packed with calcium dipicolinate (dipicolinic acid + calcium).

Shapes and Arrangements

Cocci (spheres), bacilli (rods), and spiral forms (spirilla, spirochetes); division-plane geometry generates chains (strepto-) or clusters (staphylo-).

Spirochetes corkscrew using internal axial filaments (endoflagella).

Bacterial Structure & Gram Stain 🎯

Key Takeaways — Part 1

• Prokaryote kit: nucleoid + circular chromosome, plasmids, 70S ribosomes, coupled transcription-translation, polycistronic mRNA, no cholesterol in the membrane
• Gram-positive = thick peptidoglycan + teichoic acid (purple); gram-negative = thin wall + outer membrane with LPS/lipid A = endotoxin (pink)
• Peptidoglycan = NAG-NAM chains, D-amino-acid cross-links; lysozyme cuts sugars, beta-lactams block cross-linking; acid-fast = mycolic acids
• Capsule = antiphagocytic + vaccine target; flagella run on proton-motive force; pili adhere (sex pilus conjugates)
• Endospores (Bacillus, Clostridium) = dormant, dehydrated survival forms; boiling fails, autoclave to kill

• Low-yield extras: Mycoplasma (no wall) borrows host sterols for its membrane; capsules are seen by negative staining; the spore core holds calcium dipicolinate; spirochetes move by internal axial filaments

Worked Examples — Structure Logic

Question: Rank the vulnerability of a gram-positive coccus, a gram-negative rod, and a Mycoplasma to lysozyme in tears, and justify.

Solution:

1. Gram-positive: peptidoglycan is thick but EXPOSED at the surface — most vulnerable; lysozyme digestion leaves an osmotically fragile protoplast that lyses in hypotonic tears.
2. Gram-negative: has peptidoglycan, but the outer membrane shields it; lysozyme is largely blocked unless the outer membrane is first disrupted.
3. Mycoplasma: no peptidoglycan at all — nothing for lysozyme to cut; intrinsically insensitive.

MCAT Strategy: The same access logic governs lysozyme and vancomycin: possession of a target is necessary but not sufficient — the agent must reach it.

Question: Mutant pneumococci lacking one surface structure are readily engulfed by neutrophils in vitro and avirulent in mice, while the wild type resists phagocytosis and kills the mice. Injecting the purified structure alone into mice raises protective antibodies. Identify it.

Solution:

1. Antiphagocytic + virulence-determining points to the capsule.
2. Purified capsular polysaccharide raising protective antibody is the principle behind pneumococcal vaccines: opsonizing anti-capsule IgG restores phagocytosis.
3. This mirrors Griffith's smooth (encapsulated, lethal) vs rough (unencapsulated, harmless) strains — the same molecule later starred in the transformation story.

MCAT Strategy: Capsule questions bundle three testable ideas: phagocytosis evasion, opsonization, and polysaccharide vaccine design.

Question: A drug collapses a bacterium's proton gradient without lowering cytoplasmic ATP (ATP is maintained by substrate-level phosphorylation). Predict effects on flagellar motility and on flagellin synthesis.

Solution:

1. The flagellar motor is driven directly by the proton-motive force, not ATP hydrolysis — motility stops immediately despite normal ATP.
2. Flagellin SYNTHESIS is ordinary translation, powered by GTP/ATP — it continues (until the PMF collapse has secondary effects).
3. Contrast with eukaryotic flagella (dynein ATPases, 9+2 microtubules): a favorite discrete comparison.

MCAT Strategy: Prokaryotic flagellum = PMF-driven rotary motor; eukaryotic flagellum = ATP-driven bending. Energy source is the discriminating experimental prediction.

Bacteria

Part 2 of 4 — Growth, Metabolism & Culture

Binary Fission and Exponential Math

Bacteria divide by binary fission: chromosome replication from a single origin, segregation, septum formation. Growth is exponential with a characteristic generation (doubling) time g:

$N = N_0 \times 2^{n}, \quad n = t/g$

Example: 100 cells of an organism with g = 20 min, grown 2 hours → n = 6 doublings → $100 \times 2^{6} = 6400$ cells. Log-scale plots turn exponential growth into a straight line — the slope IS the growth rate.

The Growth Curve (Closed Culture)

1. Lag — no division; cells synthesize enzymes suited to the new medium (adaptation, not dormancy).
2. Log (exponential) — constant doubling; cells most uniform and most susceptible to antibiotics that require growth (beta-lactams!).
3. Stationary — nutrients exhausted, wastes accumulate; division balances death. Endospore formation and many toxins/secondary metabolites appear here.
4. Death (decline) — exponential loss of viability.

Oxygen Relationships (Sort Any Organism from One Tube)

Why O$_2$ kills anaerobes: aerobic metabolism inevitably produces superoxide and peroxide; organisms lacking superoxide dismutase and catalase cannot neutralize them.

Nutritional Vocabulary

• Energy source: photo- (light) vs chemo- (chemical oxidation). Carbon source: auto- (CO$_2$) vs hetero- (organic molecules). Most pathogens are chemoheterotrophs.
• Obligate intracellular organisms (e.g., Rickettsia, Chlamydia) depend on host cells for ATP or other essential metabolites they cannot make themselves — they must be grown in host cells, never on agar alone.
• Media logic: selective media suppress competitors (grow only what you want); differential media make metabolic differences visible (e.g., lactose fermenters change an indicator's color); a medium can be both.
• Auxotrophs are mutants that lost a biosynthetic pathway and grow only when the end product is supplied — the workhorse of replica-plating genetics.

Growth & Metabolism 🎯

Key Takeaways — Part 2

• Growth math: $N = N_0 \times 2^{t/g}$; straight line on a log plot, slope = growth rate
• Curve: lag (adapt) → log (double; beta-lactams bite here) → stationary (spores, toxins) → death
• Oxygen classes by tube pattern: top only / everywhere-denser-top / even / bottom only / narrow subsurface band
• O$_2$ toxicity = ROS; defense = superoxide dismutase + catalase — obligate anaerobes lack them
• Chemoheterotrophs = most pathogens; obligate intracellular organisms (e.g., Rickettsia, Chlamydia) never grow on cell-free agar
• Selective suppresses, differential reveals; MacConkey is both (bile salts and crystal violet suppress gram-positives; lactose fermenters turn pink); auxotrophs need a supplied end product

Worked Examples — Growth Problems

Question: A culture grows from 2 times 10^3 to 1.28 times 10^5 cells in 3 hours of exponential growth. Find the generation time.

Solution:

1. Fold increase: 1.28 times 10^5 divided by 2 times 10^3 = 64 = $2^{6}$ → n = 6 doublings.
2. g = t/n = 180 min / 6 = 30 minutes.
3. Sanity check: powers of 2 worth memorizing — 2^5 = 32, 2^6 = 64, 2^10 = 1024 (approximately 10^3).

MCAT Strategy: Convert the fold-change to a power of 2 first; the arithmetic is designed to come out clean.

Question: Mutagenized colonies are replica-plated onto minimal medium and minimal + histidine. Colony 7 grows only on the histidine plate. Classify colony 7 and describe the biochemical lesion.

Solution:

1. Growth on supplemented but not minimal medium = histidine auxotroph (his-minus).
2. Some enzyme in the histidine biosynthesis pathway is inactivated by mutation; the cell now depends on imported histidine.
3. Wild-type (prototroph) grows on both plates. Cross-feeding tests between different his-minus mutants can even order the pathway's steps.

MCAT Strategy: Minimal medium asks 'can you build everything yourself?' — a no answer plus rescue by one compound pinpoints the broken pathway.

Question: A food-poisoning organism produces its toxin only upon nutrient depletion, and a sporulating pathogen becomes transmissible only in old cultures. Connect both facts to the growth curve.

Solution:

1. Both toxin production (a secondary metabolite) and sporulation are STATIONARY-phase programs, triggered by starvation signaling — not expressed during happy log growth.
2. Logic: when conditions collapse, invest in survival (spore) or in aggressively harvesting resources from a host (toxin).
3. Experimental corollary: harvesting a culture in log phase yields little toxin; the same culture 12 hours later can be highly toxigenic.

MCAT Strategy: Map any 'appears late in culture' observation to stationary-phase stress responses.

Bacteria

Part 3 of 4 — Bacterial Genetics: Transformation, Transduction & Conjugation

Vertical vs Horizontal

Binary fission copies genes vertically (parent to daughter, clonal). Bacteria have no meiosis and no true sexual reproduction — yet they shuffle genes horizontally through three mechanisms. Distinguishing them experimentally is a classic MCAT move:

Transformation

Competent cells import naked DNA fragments released by lysed neighbors and recombine them into the chromosome. Griffith's experiment (heat-killed smooth + live rough pneumococci → live smooth) discovered it; Avery-MacLeod-McCarty proved the transforming principle was DNA (DNase, but not protease or RNase, abolished transformation). Lab transformation with plasmids is the backbone of molecular cloning.

Lab methods that make cells take up plasmids: CaCl$_2$ treatment with heat shock, or electroporation.

Transduction (Phage-Mediated)

• Generalized: during LYTIC packaging, a capsid accidentally stuffs a random fragment of host chromosomal DNA. Any gene can move, at low frequency.
• Specialized: a LYSOGENIC prophage excises imprecisely, dragging along only the host genes that flanked its integration site. Only specific genes move.
• Lysogenic conversion: the intact prophage itself donates new genes — diphtheria, cholera, and Shiga-like toxins, and some botulinum toxins, are phage-encoded. (Full phage life cycles are covered in the Viruses lesson.)

Conjugation

An F$^+$ cell (carrying the fertility plasmid) builds a sex pilus, contacts an F$^-$ cell, and transfers a single strand of the F plasmid while both cells synthesize the complementary strands; both cells end up F$^+$.

The transfer runs by rolling-circle replication.

Variations:

• Hfr: F integrates into the chromosome. Mating now drags CHROMOSOMAL genes across in map order; the bridge usually breaks before the full F sequence transfers, so the recipient stays F$^-$. Interrupted-mating experiments (genes appearing over time) built the first chromosome maps.
• F': imprecise excision of an integrated F carries adjacent chromosomal genes on the plasmid (analogous logic to specialized transduction).
• R plasmids use the same machinery to spread multidrug resistance, even between species.

Mobile Elements & Operon Speed

Transposons ("jumping genes") hop between chromosome and plasmids, ferrying resistance cassettes onto conjugative vehicles. And remember why bacteria adapt so fast: genes sit in operons under single promoters (the lac operon logic), so one regulatory event retunes a whole pathway — combine that with horizontal transfer and generation times in minutes, and populations remodel their genomes on human timescales.

Gene Transfer Mechanisms 🎯

Key Takeaways — Part 3

• Three horizontal routes, three diagnostics: DNase kills transformation; contact required = conjugation; phage required = transduction
• Griffith found transformation; Avery proved DNA (DNase abolished it)
• Transduction: generalized = random fragment, lytic mispackaging; specialized = flanking genes, sloppy prophage excision; lysogenic conversion = the phage's own toxin genes
• Conjugation: F$^+$ x F$^-$ → both F$^+$; Hfr transfers chromosome in map order, recipient stays F$^-$; F' carries hitchhiker genes
• Transposons move genes BETWEEN replicons inside a cell; plasmids move them BETWEEN cells; operons make regulation fast
• No meiosis anywhere — bacterial 'sex' is unidirectional donation

• Low-yield extras: lab transformation uses CaCl$_2$ with heat shock or electroporation; F transfer runs by rolling-circle replication

Worked Examples — Transfer Experiments

Question: Strain A (streptomycin-resistant) transfers resistance to strain B in mixed culture. Design a three-condition experiment to determine the mechanism, and give the expected results for conjugation.

Solution:

1. Condition 1 — add DNase to the co-culture: conjugation unaffected (DNA travels through the pilus, protected).
2. Condition 2 — separate strains with a 0.2 micron filter (U-tube): conjugation ABOLISHED (contact required); transformation and transduction persist.
3. Condition 3 — use cell-free filtrate of strain A on strain B: conjugation gives NO transfer (a filtrate has no cells); transduction would (phage particles pass filters).
4. Conjugation signature: transfer in mixed culture only, DNase-resistant, filter-blocked, filtrate-negative.

MCAT Strategy: Memorize the 3x3 grid of mechanism vs. DNase/filter/filtrate — nearly every genetics-transfer passage is one cell of that grid.

Question: In an Hfr cross, recipients acquire azi-r at 9 min, ton-r at 11 min, lac+ at 18 min, gal+ at 25 min. A different Hfr strain (same species, different F integration site) transfers gal+ first. Reconcile, and state what the times give you.

Solution:

1. Entry times are proportional to each gene's DISTANCE from the F integration point (the transfer origin) — the time axis is literally a map axis (minutes became the E. coli map unit).
2. A different integration site (or orientation) changes the starting point and direction, so gene ORDER of entry changes, but the relative SPACING between genes is consistent.
3. Combining several Hfr strains' data yields one circular chromosome map — an early proof of chromosome circularity.

MCAT Strategy: Time-of-entry = genetic distance; different Hfr strains = different clock-start positions on the same circle.

Question: An F' lac+ cell mates with an F-minus lac-minus recipient. What is the recipient's genotype and phenotype afterward, and how does this differ from an Hfr cross transferring lac+?

Solution:

1. F' transfer moves the whole plasmid efficiently: the recipient becomes F' lac+ / lac-minus — a partial diploid (merodiploid) carrying two lac copies, and it can now donate too.
2. Phenotype: Lac+ (the plasmid copy complements), and merodiploids are the tool that defined cis vs trans action in the lac operon (e.g., lacI works in trans, operator only in cis).
3. Hfr contrast: chromosomal lac+ arrives as a linear fragment that must RECOMBINE to be kept, recipient stays F-minus, and no stable diploid forms.

MCAT Strategy: F' = stable partial diploid (complementation tests); Hfr = recombination required. When a question builds cis/trans logic, an F' merodiploid is hiding in it.

Bacteria

Part 4 of 4 — Pathogenesis, Toxins & MCAT Integration

From Colonization to Disease

Infection proceeds through recognizable stages, each with a molecular tool: adhesion (pili, adhesins binding host receptors) → colonization/invasion (invasins, degradative enzymes that break down host tissue) → immune evasion (capsule, IgA proteases, antigenic variation, intracellular hiding) → damage (toxins, or the host's own inflammation). Not all colonists cause disease: normal flora occupy niches, compete for nutrients, and even prime immunity — broad-spectrum antibiotics that clear them open the door to opportunists (C. difficile colitis, Candida overgrowth).

Named spreading enzymes: hyaluronidase and collagenase digest connective-tissue matrix.

Exotoxin vs Endotoxin — the Highest-Yield Table in Microbiology

The A-B Toxin Architecture

Many exotoxins are two-part machines: the B (binding) subunit docks onto a specific host receptor and delivers the A (active) subunit, an intracellular enzyme. Classic catalytic strategies:

• ADP-ribosylation: diphtheria toxin ADP-ribosylates elongation factor EF-2, halting host translation; cholera toxin ADP-ribosylates a G$_s$ protein, locking adenylyl cyclase ON → cAMP floods the enterocyte → massive Cl$^-$/water secretion (watery diarrhea with an intact epithelium).
• Protease: tetanus and botulinum toxins cleave SNARE proteins. Tetanus acts on INHIBITORY interneurons → spastic paralysis; botulinum blocks acetylcholine release at the neuromuscular junction → flaccid paralysis. Opposite phenotypes, same molecular step (blocked vesicle fusion) in different neurons.
• Superantigens (toxic shock syndrome toxin): crosslink MHC II to T-cell receptors NONSPECIFICALLY, activating up to 20 percent of all T cells at once — a cytokine storm without any antigen specificity.

Integration: Reading a Pathogenesis Passage

Ask in order: (1) Which stage of infection is the experiment probing — adhesion, evasion, or damage? (2) Is the damage molecule a secreted protein (mutable, vaccinable, neutralizable by antitoxin) or structural LPS? (3) Where does the gene live — chromosome, plasmid, or prophage — and what does that predict about spread (Part 3) and about antibiotic strategy (the Antimicrobials lesson)? Bacterial vignettes are integration tests: structure (Part 1) sets drug access, growth phase (Part 2) sets drug timing, genetics (Part 3) sets resistance spread, and virulence factors (this part) set the clinical picture.

Pathogenesis & Toxins 🎯

Key Takeaways — Part 4

• Infection stages: adhere (pili) → invade (enzymes) → evade (capsule, IgA protease, hiding) → damage (toxins or inflammation)
• Exotoxin = secreted protein, both Gram types, heat-labile, hyperpotent, toxoid-vaccinable, often plasmid/phage-encoded
• Endotoxin = lipid A of LPS, gram-negative only, released on lysis, heat-stable, septic shock
• A-B architecture: B binds, A acts — ADP-ribosylation (diphtheria: EF-2; cholera: G$_s$ → cAMP), SNARE proteases (tetanus spastic vs botulinum flaccid)
• Superantigens crosslink MHC II-TCR nonspecifically → cytokine storm
• Normal flora are a defense; clearing them invites C. difficile — antibiotics vacate niches, they do not create pathogens

• Low-yield extras: hyaluronidase and collagenase are the named tissue-spreading enzymes

Worked Examples — Pathogenesis Integration

Question: A patient with gram-negative bacteremia deteriorates in the hours after the first bactericidal antibiotic dose. Propose the mechanism.

Solution:

1. Endotoxin is a structural membrane component, released chiefly when cells LYSE.
2. A cidal drug lysing a large bacterial load dumps a bolus of LPS at once; lipid A activates TLR4 on macrophages → TNF/IL-1 surge → vasodilation and capillary leak.
3. The bacteria are dying, but the inflammatory cargo peaks — killing the organism and neutralizing its products are separate problems.

MCAT Strategy: Endotoxin release scales with lysis, not with bacterial viability; expect questions pairing antibiotic mechanism (lysis vs static) with endotoxin kinetics.

Question: Purified subunit A of a toxin, added to intact cells, does nothing; microinjected into the cytoplasm, it halts protein synthesis. Purified subunit B binds cells but is harmless. Antibodies against which subunit best protect intact animals, and why?

Solution:

1. A is the catalytic warhead (here, EF-2 ADP-ribosylation, diphtheria-style) but cannot ENTER without help; B is the delivery system.
2. Anti-B antibodies block receptor binding — the toxin never gets in; this is usually the most protective neutralization target on the intact toxin.
3. The microinjection bypass proves the barrier is entry, not catalysis — the classic logic for all A-B toxins.

MCAT Strategy: For A-B toxins: activity requires BOTH binding and catalysis; each subunit alone is a null, and neutralizing antibodies work at the binding step.

Question: An encapsulated, lactose-fermenting gram-negative rod causes a UTI. It carries a conjugative R plasmid with a beta-lactamase gene, and biofilm forms on the patient's catheter. Name the concept from each lesson part that predicts a treatment obstacle.

Solution:

1. Part 1 (structure): gram-negative outer membrane restricts drug entry; capsule fights phagocytosis; LPS threatens sepsis on lysis.
2. Part 2 (growth): biofilm cells sit near stationary phase — beta-lactams, which need growth, underperform; the catheter likely must come out.
3. Part 3 (genetics): the R plasmid can conjugate resistance to neighboring flora — isolation and drug choice matter beyond this one strain.
4. Part 4 (pathogenesis): beta-lactamase = 'destroy the drug'; pair the beta-lactam with an inhibitor (clavulanate) or switch class.

MCAT Strategy: This layering — envelope, growth phase, gene mobility, virulence factor — is exactly how the MCAT builds multi-paragraph microbiology passages. Practice naming which layer each experimental detail addresses.