Bacterial Classification, Structure, and Replication

Differences Between Eukaryotes and Prokaryotes

Prokaryotes (bacteria and archae) lack a membrane-bound nucleus and internal organelles, distinguishing them from eukaryotic cells (animals, plants, fungi). Typical bacterial cells are approximately $1\,\mu m$ in diameter, compared to animal cells ranging from $7\,\mu m$ to several feet. Bacterial genomes, such as that of Escherichia coli, generally consist of a single circular double-stranded DNA molecule containing about $5 \times 10^6$ base pairs ($5000\,kb$) spanning $1.3\,mm$ in length, whereas human eukaryotic genomes contain $2.9 \times 10^9$ base pairs across $23$ chromosome pairs spanning $990\,mm$. Prokaryotes utilize $70S$ ribosomes (composed of $50S$ and $30S$ subunits) rather than eukaryotic $80S$ ribosomes (composed of $60S$ and $40S$ subunits). Most bacteria possess a peptidoglycan cell wall surrounding their membranes, enabling survival in low osmotic pressure and extreme environments.

Bacterial Classification and Identification

Bacteria are classified by microscopic morphology, metabolic characteristics, antigenicity, and genetic sequencing. Microscopic evaluation relies on cell shape (spherical cocci, rod-shaped bacilli, spiral spirilla, or branched filaments), cellular arrangement (such as Staphylococcus clusters or Streptococcus diplococci), and Gram staining properties. Gram-positive bacteria trap crystal violet within a thick, cross-linked peptidoglycan meshwork and appear purple ("P-PURPLE-POSITIVE"). Gram-negative bacteria have a thin peptidoglycan layer that loses crystal violet upon decolorization and are counterstained red with safranin in less than $10\,\text{minutes}$. Mycobacteria, which have a waxy outer coat, require acid-fast staining, while mycoplasmas lack peptidoglycan completely. Further differentiation employs metabolic profiling, serotyping using specific antibodies against antigens (used for Treponema pallidum, Francisella, E. coli O157:H7, and Streptococcus pyogenes), and nucleic acid techniques such as ribosomal DNA sequence analysis or polymerase chain reaction.

Bacterial Envelopes and External Structures

The bacterial cytoplasm contains the DNA nucleoid and extrachromosomal plasmids without histones or nucleosomes, permitting coupled transcription and translation. The cytoplasmic membrane is a sterol-free lipid bilayer responsible for electron transport, energy generation, and transport, lined with actin-like protein filaments determining cell shape. Gram-positive cell walls feature a thick peptidoglycan layer ($150$ to $500\,\text{\AA}$) containing teichoic and lipoteichoic acids, along with surface proteins like streptococcal M protein or staphylococcal R protein; lysozyme digestion yields osmotically fragile protoplasts. Gram-negative cell walls contain a thin peptidoglycan layer ($5\%$ to $10\%$ of cell wall weight) inside a periplasmic space rich in transport proteins and hydrolytic or virulence enzymes (such as collagenases, hyaluronidases, and $\beta$-lactamases). The gram-negative outer membrane is an asymmetric bilayer containing phospholipids in the inner leaflet and lipopolysaccharide (LPS, or endotoxin) in the outer leaflet, joined to peptidoglycan via lipoproteins and traversed by porin channels permitting passage of molecules under $700\,Da$. LPS consists of lipid A (the active endotoxin component), a core polysaccharide containing $2\text{-keto-}3\text{-deoxy-octanoate}$, and a variable O antigen. External bacterial structures include polysaccharide or polypeptide capsules (such as Bacillus anthracis polypeptide capsule) that confer antiphagocytic protection, proteinaceous flagella ($15$ to $20\,nm$ diameter) driven by membrane potential for chemotaxis, and peritrichous fimbriae or pili ($3$ to $8\,nm$ diameter, up to $15$ to $20\,\mu m$ length) that facilitate tissue adherence or plasmid transfer via F pili.

Peptidoglycan Synthesis

Peptidoglycan consists of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) disaccharide chains cross-linked by tetrapeptide chains attached to MurNAc. Precursors are synthesized inside the cytoplasm as UDP-MurNAc-pentapeptide ending in a D-alanine-D-alanine dipeptide. This building block is attached to the membrane-bound undecaprenol phosphate (bactoprenol, a $C_{55}$ isoprenoid) carrier, joined to GlcNAc, and translocated across the plasma membrane. Transglycosylases attach the disaccharide to the existing glycan chain, and membrane-bound transpeptidases (penicillin-binding proteins or PBPs) cross-link the peptide chains by exchanging a peptide bond between the diamino amino acid at position three (such as lysine or diaminopimelic acid) and the D-alanine at position four, releasing the terminal D-alanine. Antibiotics like $\beta$-lactams, vancomycin, and bacitracin inhibit distinct steps in this assembly pathway, promoting autolysin-mediated cell wall degradation and cell lysis during bacterial growth.

Endospore Formation and Germination

Certain gram-positive soil bacteria, specifically within the genera Bacillus and Clostridium, can form dormant endospores under nutrient starvation. Endospores possess a condensed core containing a complete chromosome copy, essential ribosomes, and high concentrations of calcium bound to dipicolinic acid, enclosed by an inner membrane, two peptidoglycan layers (the cortex), and a thick keratin-like protein coat. Sporogenesis requires $6$ to $8\,\text{hours}$, yielding structures extremely resistant to heat, desiccation, radiation, and chemical disinfectants. Germination into an active vegetative cell takes approximately $90\,\text{minutes}$ following coat disruption by mechanical stress or heat and exposure to water and specific nutrient triggers such as alanine.

Questions & Discussion

Regarding how prokaryotic and eukaryotic differences affect infection and treatment, the smaller prokaryotic size enables penetration into tighter tissue spaces, while the lack of a nuclear membrane couples transcription and translation. Topoisomerases maintaining single circular chromosomes serve as targets for quinolones, and haploid genomes mean single mutations directly alter phenotypes. Bacterial $70S$ ribosomes offer selective targets for antibiotics compared to eukaryotic $80S$ ribosomes, and peptidoglycan cell walls provide osmotic protection while offering target sites for $\beta$-lactams, vancomycin, and bacitracin.

Regarding gram-positive and gram-negative wall differences, the thick gram-positive peptidoglycan traps crystal violet stain, whereas the thin gram-negative peptidoglycan requires safranin counterstaining. Gram-negative LPS acts as a potent endotoxin triggering fever and sepsis via host receptor activation, while the outer membrane acts as a permeability barrier against complement and antibiotics.

Virulence factors protecting against immune responses include peptidoglycan and O antigen (restricting complement access), capsules (preventing phagocytosis and antibody binding), and surface proteins like Staphylococcus protein A or Streptococcus M protein. Factors eliciting toxic responses include LPS endotoxin and teichoic acids.

When peptidoglycan synthesis is inhibited during active cell growth, ongoing autolysin activity degrades the cell wall, causing cell lysis; UDP-MurNAc-pentapeptide precursors accumulate internally when downstream cross-linking or bactoprenol recycling is blocked.

Spores exhibit environmental resistance because they are desiccated, metabolically inactive, and encased in thick peptidoglycan and keratin-like protein coats.

To selectively eliminate gram-positive bacteria from a bacterial mixture, lysozyme or mild detergent treatments are effective; lysozyme degrades exposed gram-positive peptidoglycan while the gram-negative outer membrane acts as a barrier against it, and mild detergents affect gram-positive membranes more strongly. Ethylenediaminetetraacetic acid (EDTA) selectively disrupts the gram-negative outer membrane via cation chelation, ampicillin affects both bacterial types through porin channels, and transpeptidase treatment has no effect.