The Cell Wall & the Gram Stain

1. Hans Christian Gram’s 1884 Stain

Hans Christian Gram, a Danish bacteriologist working in Berlin, reported in 1884 a staining procedure that distinguished what we now call Gram-positive (purple) from Gram-negative (pink) cells. Crystal violet binds the cytoplasm; iodine forms a large complex; ethanol decolourises Gram-negatives whose thin peptidoglycan cannot trap the dye, but does not decolourise Gram-positives whose thick wall holds it. Safranin then counter-stains the now-naked Gram-negatives pink.

The Gram stain remains the first test in the clinical microbiology lab: it predicts cell-wall structure, antibiotic susceptibility, and the toxin profile (LPS in Gram-negatives, teichoic acids and exotoxins in many Gram-positives).

2. Peptidoglycan Chemistry

Peptidoglycan is a glycopeptide polymer:

• Glycan strands — alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), β-1,4 linked.
• Stem peptide — a 5-residue peptide pendant from each MurNAc: L-Ala, D-Glu, (m)DAP or L-Lys, D-Ala, D-Ala.
• Cross-link — transpeptidase forms a peptide bond between the third residue (DAP/Lys) of one stem and the fourth (D-Ala) of another, releasing the terminal D-Ala.

The result is a giant covalent mesh that surrounds the cell. Gram-positives pile up ~40 layers (~30 nm thick); Gram-negatives have a single layer (~7 nm) sandwiched between two membranes. The use of D-amino acids and an unusual cross-link makes peptidoglycan an excellent antibiotic target — eukaryotes don’t have it.

3. Biosynthesis: Lipid II & the Penicillin-Binding Proteins

Peptidoglycan biosynthesis is a paradigmatic example of a multi-step membrane process:

1. Cytoplasmic stage: MurA–F enzymes build UDP-MurNAc-pentapeptide.
2. Membrane stage: MraY transfers the MurNAc-pentapeptide onto undecaprenyl phosphate, forming Lipid I; MurG adds GlcNAc to make Lipid II.
3. Lipid II is flipped across the membrane by MurJ.
4. Outside the membrane, transglycosylases polymerise the glycan strands and transpeptidases form the cross-links.

The transglycosylase + transpeptidase activities are housed in penicillin-binding proteins (PBPs). Penicillin and the other β-lactams covalently inhibit the transpeptidase active-site serine because their strained β-lactam ring mimics the D-Ala−D-Ala substrate. Vancomycin uses a different strategy: it binds the substrate D-Ala−D-Ala terminus and blocks both transglycosylation and transpeptidation.

4. Gram-Positive Specifics: Teichoic Acids

The thick Gram-positive wall is decorated with teichoic acids — polymers of glycerol-phosphate or ribitol-phosphate, anchored either to the membrane (lipoteichoic acids) or covalently to peptidoglycan (wall teichoic acids). They create a strongly anionic surface, mediate cation homeostasis (Mg²⁺ in particular), and are a major antigenic determinant. S. aureus wall teichoic acids are receptors for many phages and contribute to virulence.

5. Gram-Negative Specifics: The Outer Membrane & LPS

Gram-negatives have a second membrane outside the peptidoglycan. Its outer leaflet is dominated by lipopolysaccharide (LPS):

• Lipid A — the membrane anchor, ~6 fatty acids on a glucosamine disaccharide. The endotoxin: recognised by TLR4/MD-2 in mammals, drives septic shock.
• Core oligosaccharide — ~10 sugars including the unusual KDO (3-deoxy-D-manno-oct-2-ulosonic acid) and heptoses.
• O-antigen — a long, repetitive polysaccharide, highly variable between strains. The basis of Salmonella serotyping (~2500 serovars).

The outer membrane is studded with β-barrel porins (OmpF, OmpC) that allow size-selective passage of small molecules (~600 Da cutoff) and TonB-dependent active transporters for siderophores and vitamins.

6. Mycobacterium and the Atypical Walls

Mycobacterium tuberculosis and relatives have a compound wall that is technically Gram-positive (no outer membrane) but stains acid-fast (Ziehl-Neelsen) because of a thick layer of mycolic acids — long-chain (C60–C90), α-branched, β-hydroxylated fatty acids covalently attached to arabinogalactan. The result is a near-waxy hydrophobic barrier that repels most antibiotics, accounts for slow growth (24-hour generation time), and demands the specialised drug regimen for tuberculosis.

Mycoplasma goes the opposite way: no cell wall at all, a sterol-stabilised membrane, and total resistance to wall-targeting antibiotics.