Gram-Negative Bacteria – Comprehensive Study Notes

Gram-Negative Cell Wall & Gram Stain

• Thin peptidoglycan layer ≈ $10\%$ of cell wall mass.

• Second (outer) membrane:

  • Contains lipopolysaccharide (LPS) → endotoxin.

  • Provides additional permeability barrier & contributes to pink color after Gram stain (crystal-violet does not remain).

• Medical relevance of LPS:

  • Lipid A moiety triggers strong innate immune responses (fever, hypotension, septic shock).

  • O-antigen used for serotyping (e.g., E. coli O157:H7).

Phylogenetic Overview: Proteobacteria vs. Non-Proteobacteria

• Staining pattern reflects wall architecture, not ancestry.

• 16S rRNA gene sequencing divides Gram-negatives into two supergroups:

  • Proteobacteria – monophyletic megaclade (5 classes: $\alpha,\;\beta,\;\gamma,\;\delta,\;\varepsilon$).

  • Non-proteobacteria – multiple independent clades (Thermus-Deinococcus, Cyanobacteria, Bacteroidetes, etc.).

• Evolutionary note:

  • Endosymbiotic origin of mitochondria traced to an $\alpha$-proteobacterial ancestor.

  • Chloroplasts originated from a cyanobacterial (non-proteo) lineage.

Phylum Proteobacteria – General Traits

• Largest coherent bacterial group by 16S similarity.

• Share classic Gram-negative envelope, yet occupy diverse niches:

  • Soil, water, plant roots, animal intestines, hospital plumbing.

  • Lifestyles: free-living, mutualists, predators, primary pathogens, opportunists.

• Ecological & clinical importance:

  • Nitrogen cycles, wastewater treatment, bioremediation.

  • Major human, animal, and plant pathogens.

$\alpha$-Proteobacteria

• Rickettsia spp.

  • Tiny obligate intracellular parasites; reside in cytosol of phagocytes.

  • Auxotrophs: lack glycolysis, but oxidize host glucose ⇒ ATP.

  • R. rickettsii → Rocky Mountain Spotted Fever (tick-borne). Endothelial infection → capillary leakage & rash.

  • R. prowazekii → Epidemic typhus (body louse, fleas).

• Family Rhizobiaceae

  • Rhizobium sp.: root nodule symbionts of legumes; nitrogen fixation (N$<em>2 \rightarrow$ NH$</em>3$) → critical to agriculture.

  • Agrobacterium tumefaciens: Ti plasmid induces crown-gall tumors; Ti plasmid exploited in plant genetic engineering; does not fix N.

$\beta$-Proteobacteria

• Neisseria (diplococci)

  • N. gonorrhoeae → gonorrhea; often self-limiting in men, but severe sequelae in women (PID, infertility).

  • N. meningitidis → meningococcal meningitis; ≥$10\%$ healthy carriage.

• Burkholderia cepacia complex – opportunistic; lethal to cystic-fibrosis patients.

• Bordetella

  • B. pertussis: whooping cough; adult carriers $2!–!5\%$; infant mortality 10-20/yr (US).

  • B. bronchiseptica: kennel cough in dogs.

$\gamma$-Proteobacteria (largest class)

Enterobacteriaceae – “Enterics”

• Gram-negative facultative rods; natural habitat = vertebrate intestine.

• Categories:

  • Opportunists (e.g., Klebsiella, Enterobacter, Citrobacter).

  • Primary pathogens: Yersinia, Salmonella, Shigella, certain E. coli serotypes.

• Hygiene indicators: E. coli, coliforms → easy detection signals fecal contamination of food/water.

Spotlight Pathogens & Outbreaks

• Yersinia pestis – bubonic & pneumonic plague; still causes ~7 U.S. cases/yr (Arizona case, first death since 2007).

• E. coli O157:H7

  • Carries Shiga toxin 2 via lysogenic bacteriophage.

  • Mechanism: binds Gb3, inactivates $60S$ ribosome → cell death, hemorrhagic colitis, possible hemolytic-uremic syndrome (HUS) in $2!–!7\%$.

  • U.S. burden: $63!\text{–}!73{,}000$ infections, >2,000 hospitalizations, ~ 60 deaths/yr.

  • Notable outbreaks: 1993 Jack-in-the-Box, 2006 spinach, 2009 Nestlé cookie dough (flour vector), 2016 General Mills flour, 2024 McDonald’s burgers.

• Salmonella enterica

  • Food vehicles beyond poultry: cucumbers (551 illnesses 2024, 69 in 2025), eggs (1.7 million recalled 2025).

• Shigella (dysentery)

  • Fecal-oral, highly contagious (ID$_{50} \approx 10!–!100$ cells).

  • Emerging antibiotic resistance in WA/OR; Trader Joe’s dip recall.

Vibrionaceae

• Marine & brackish habitats (water, sediments, shellfish).

• Vibrio cholerae

  • Epidemic serogroups O1, O139; infection requires lysogenic phage with ctx toxin gene.

  • Haiti 2010 earthquake → largest outbreak in a century (≈10,000 deaths) traced to UN aid workers (Nepal strain) contaminating river.

  • Symptom hallmark: “rice-water stool”, severe dehydration.

• Vibrio vulnificus

  • Wound exposure → necrotizing fasciitis; ingestion (raw oysters) → fulminant septicemia; common in warm Florida waters.

Pseudomonadaceae

• Pseudomonas aeruginosa

  • Environmental ubiquity; produces blue-green pigments (pyocyanin/pyoverdin) & pyorubin.

  • #4 nosocomial pathogen; notorious for multidrug resistance; thrives in burn units; selected by triclosan.

Legionellales

• Legionella pneumophila

  • Aerosols from hot tubs, cooling towers, plumbing; causes Legionnaires’ disease (severe atypical pneumonia; GI symptoms possible).

$\delta$-Proteobacteria

• Mostly aerobic; some predators.

• Myxobacteria

  • Social gliding swarms (“wolf packs”); secrete lytic enzymes; form myxospores.

  • Source of novel antibiotics & anti-cancer agents.

• Bdellovibrio bacteriovorus

  • Obligate predator of Gram-negatives; enters periplasm, consumes host; dubbed a “living antibiotic”; regulates bacterial populations.

$\varepsilon$-Proteobacteria

• Gastrointestinal symbionts & pathogens.

• Campylobacter jejuni

  • Common on retail poultry; invades duodenum → bloody enteritis.

• Helicobacter pylori

  • ~$50\%$ global carriage; linked to peptic ulcers, chronic gastritis, gastric cancer.

  • Virulence: urease, flagella, CagA oncoprotein (injected via type IV secretion).

Non-Proteobacteria Highlights

Thermus–Deinococcus Group

• Gram-positive stain yet double membrane (no LPS).

• Thermus aquaticus: hyperthermophile; source of Taq polymerase (PCR revolution).

• Deinococcus radiodurans: tolerates massive desiccation & $\gamma$-radiation; reassembles shattered genome within 12-24 h.

Aquificae & Thermotogae

• Ancient hyperthermophiles; extensive archaeal gene exchange.

• Aquifex: oxidizes $H<em>2 \rightarrow H</em>2O$ (“water maker”); naturally competent for diverse DNA uptake.

• Thermotoga: sheath-like “toga” outer envelope.

Photosynthetic Non-proteos

• Chlorobi (Green sulfur bacteria) – anoxygenic; bacteriochlorophyll absorbs 800-1000 nm; cyclic photophosphorylation.

• Cyanobacteria – oxygenic “blue-green algae”

  • Chlorophyll a in thylakoids; RuBisCO for $CO_2$ fixation.

  • Major marine carbon & nitrogen fixers; some produce neurotoxic cyanotoxins.

  • Chronic exposure (e.g., Florida 2018 dual bloom with Karenia brevis) linked to neurodegenerative diseases.

Chlamydiae

• Obligate intracellular; no peptidoglycan but have LPS.

• Biphasic life cycle:

  • Elementary body (EB) – small, infectious, environmentally hardy.

  • Reticulate body (RB) – large, non-infectious, replicative inside host epithelium.

• Chlamydia trachomatis: most reported bacterial STD; 75% women & 50% men asymptomatic; also causes trachoma blindness.

• Associations: myocarditis, atherosclerosis, lymphoma, infertility.

Spirochaetes

• Helical cells with internal endoflagella (axial filaments).

• Borrelia burgdorferi – Lyme disease (deer tick): erythema migrans (bull’s-eye rash), facial palsy, arthritis, neuropathy.

• Treponema pallidum – syphilis: primary painless chancre → secondary palm/sole rash → latent → tertiary complications; U.S. resurg mence (20% rise 2023; congenital cases up 10× since 2012).

Bacteroidetes (Genus Bacteroides)

• Strict anaerobes; dominate intestines & tonsillar crypts; colonize infant gut early.

• Opportunistic if escaping gut (e.g., ruptured appendix → peritonitis); harbor numerous antibiotic-resistance genes.

Practical & Epidemiological Notes

• Produce washing: avoid soap; rinse under running water with friction; scrub without breaking skin; blot dry. Baking-soda soak (1 tsp/2 cups, 12 min) removes pesticides more effectively than commercial veggie washes. John Snow (1854): used spatial evidence to link Broad St. pump to cholera → birth of modern epidemiology; also pioneered surgical ether/chloroform.

• Nosocomial threats: Pseudomonas aeruginosa among top 5; thrives in disinfectant-rich settings.

Exam Review / Quick-Fire Questions

• Major hygiene indicator organism? → Escherichia coli (fecal coliform standard).

• Common nosocomial Gram-negative? → Pseudomonas aeruginosa.

• “Living antibiotic” predator? → Bdellovibrio bacteriovorus ($\delta$-proteobacterium).

• Bacterial “wolf-packs”? → Myxobacteria.

• Stomach-ulcer agent? → Helicobacter pylori.

• Infectious form of Chlamydia? → Elementary Body (EB).

• Lyme disease pathogen + key symptom set? → Borrelia burgdorferi; bull’s-eye rash, joint inflammation, neuro signs.

• Syphilis agent & primary lesion? → Treponema pallidum; painless chancre.

• Vibrio species requiring phage-encoded toxin for epidemics? → Vibrio cholerae (ctx).

• Enteric with phage-encoded Shiga toxin? → E. coli O157:H7.