Prokaryotes (Bacteria and Archaea) are single-celled organisms without a membrane-bound nucleus. They come in three basic shapes, have circular chromosomes, and are classified partly by their cell wall structure using the Gram stain. Archaea are more closely related to eukaryotes than to Bacteria, despite looking similar under a microscope.
Difficulty: Introductory | Prerequisites: None, though a basic understanding of cells is helpful.
This topic sits near the start of most introductory biology courses, right after the basics of cell structure. Prokaryotes are the oldest and most abundant organisms on Earth, and understanding their biology is the foundation for later topics in genetics, evolution, ecology, and microbiology. You need to know how prokaryotic cells are built before you can understand how they reproduce, transfer genes, and evolve antibiotic resistance. If you have not yet reviewed the general differences between prokaryotic and eukaryotic cells, do that first.
Prokaryote
An organism whose cells lack a membrane-bound nucleus and membrane-bound organelles. In simple terms, the DNA floats in the cytoplasm rather than sitting inside a nucleus.
Eukaryote
An organism whose cells contain a membrane-bound nucleus and organelles such as mitochondria and the endoplasmic reticulum. Think of it as the more compartmentalised type of cell.
Bacteria (domain)
One of the two prokaryotic domains. Bacteria have cell walls containing peptidoglycan and use ester-linked phospholipids in their membranes. This is the domain that includes familiar species such as E. coli and Streptococcus.
Archaea (domain)
The second prokaryotic domain. Archaea lack peptidoglycan in their cell walls and use ether-linked phospholipids. In simple terms, they look like bacteria under a microscope but are biochemically quite different, and more closely related to eukaryotes.
Eukarya (domain)
The domain containing all eukaryotic organisms: animals, plants, fungi, and protists.
Phylogenetic tree
A branching diagram showing the evolutionary relationships among groups of organisms, based on shared ancestry. Think of it as a family tree for species or domains.
Peptidoglycan
A polymer of sugars and amino acids that forms the mesh-like layer in bacterial cell walls. In simple terms, it is the rigid scaffolding that gives bacterial cells their shape.
Coccus (plural: cocci)
A spherical or round-shaped prokaryotic cell. Think of a tiny ball.
Bacillus (plural: bacilli)
A rod-shaped prokaryotic cell. Think of a capsule or cylinder.
Spirillum (plural: spirilla)
A spiral- or corkscrew-shaped prokaryotic cell.
Gram-positive
Bacteria with a thick peptidoglycan layer and no outer membrane. They retain the crystal violet dye during Gram staining and appear purple.
Gram-negative
Bacteria with a thin peptidoglycan layer sandwiched between an inner membrane and an outer membrane containing lipopolysaccharide. They lose the crystal violet dye during Gram staining and pick up the counterstain, appearing pink.
Lipopolysaccharide (LPS)
A large molecule found in the outer membrane of Gram-negative bacteria, composed of a lipid and a polysaccharide. It acts as a barrier to many antibiotics and can trigger immune responses in humans.
Periplasmic space
The compartment between the inner and outer membranes in Gram-negative bacteria. It contains enzymes that can degrade antibiotics and other foreign molecules.
Nucleoid
The region within a prokaryotic cell where the circular chromosome is concentrated. It is not enclosed by a membrane.
All life is classified into three domains: Bacteria, Archaea, and Eukarya. Both Bacteria and Archaea are prokaryotes, but they are not each other's closest relatives.
Archaea and Eukarya share a more recent common ancestor than either shares with Bacteria. This is the single most important phylogenetic point for the exam. On a phylogenetic tree, Bacteria branch off first, and Archaea and Eukarya form a sister group.
The three-domain system was proposed by Carl Woese in the late 1970s, based on ribosomal RNA (rRNA) sequence comparisons.
Before Woese's work, Archaea and Bacteria were grouped together as "prokaryotes" and treated as a single kingdom. Molecular evidence showed this grouping was misleading.
Key traits that distinguish the domains:
Bacteria: Peptidoglycan cell walls. Ester-linked membrane lipids. Circular chromosome. No nuclear membrane. 70S ribosomes.
Archaea: No peptidoglycan (pseudopeptidoglycan or other polymers instead). Ether-linked membrane lipids (sometimes branched). Circular chromosome. No nuclear membrane. 70S ribosomes, but many ribosomal proteins more similar to eukaryotic ones.
Eukarya: No peptidoglycan. Ester-linked membrane lipids. Linear chromosomes (multiple). Membrane-bound nucleus. 80S ribosomes.
When drawing a phylogenetic tree for this course, place the root on the left, branch Bacteria off first, then split Archaea and Eukarya from each other. Annotate the branch points with the key distinguishing traits above.
Prokaryotes come in three basic shapes. Exams frequently ask you to identify them or match the name to the shape.
Coccus (cocci): Spherical. Examples include Staphylococcus and Streptococcus.
Bacillus (bacilli): Rod-shaped. Examples include E. coli and Bacillus subtilis.
Spirillum (spirilla): Spiral or corkscrew-shaped. Examples include Spirillum volutans and Treponema pallidum (technically a spirochete, a tighter spiral).
Arrangement patterns:
Cocci in particular are often described by how they cluster after cell division.
Diplo-: pairs (e.g. Diplococcus)
Strepto-: chains (e.g. Streptococcus)
Staphylo-: grape-like clusters (e.g. Staphylococcus)
Tetrad: groups of four
Sarcina: cubes of eight
Bacilli can also form chains (streptobacilli) but do not form the same variety of clusters that cocci do. Spiral-shaped cells are almost always found individually.
One of the clearest differences between prokaryotes and eukaryotes is how their genetic material is organised.
Bacteria and Archaea: Typically one circular chromosome, located in the nucleoid region (no membrane around it). The DNA is associated with histone-like proteins (in Bacteria) or true histones (in many Archaea) that help compact it. Many also carry plasmids, which are small, circular, self-replicating pieces of DNA separate from the main chromosome.
Eukaryotes: Multiple linear chromosomes, housed inside a membrane-bound nucleus. DNA is wrapped around histone proteins to form chromatin, which condenses into visible chromosomes during cell division.
Why this matters for reproduction:
Because prokaryotes have just one circular chromosome with fewer genes and no nuclear membrane to break down, they can replicate their DNA and divide far more quickly than eukaryotes. This is why bacterial generation times can be as short as 20 minutes, whereas eukaryotic cells typically take hours or days to divide.
Plasmids:
Plasmids are not essential for survival under normal conditions, but they often carry genes that provide advantages such as antibiotic resistance, toxin production, or the ability to metabolise unusual nutrients. Plasmids can be transferred between bacteria through horizontal gene transfer (covered in Part 2 of these notes).
The Gram stain is one of the most important techniques in microbiology. It divides bacteria into two major groups based on their cell wall structure, and the distinction has real consequences for antibiotic treatment.
The Gram staining procedure:
Apply crystal violet dye (purple) to a heat-fixed bacterial smear.
Add iodine, which forms a complex with the crystal violet inside the cell.
Wash with alcohol or acetone (the decolourisation step).
Apply safranin counterstain (pink/red).
Gram-positive bacteria:
Thick peptidoglycan layer (20 to 80 nm), sitting outside the plasma membrane.
No outer membrane.
The thick peptidoglycan traps the crystal violet-iodine complex during decolourisation, so the cells remain purple.
The single membrane and exposed peptidoglycan make these bacteria generally more susceptible to antibiotics such as penicillin, which targets peptidoglycan synthesis.
Gram-negative bacteria:
Thin peptidoglycan layer (5 to 10 nm), sandwiched between the inner (plasma) membrane and an outer membrane.
The outer membrane contains lipopolysaccharide (LPS).
The periplasmic space sits between the inner and outer membranes and contains enzymes that can break down antibiotics.
During decolourisation, the alcohol dissolves the outer membrane and washes the crystal violet out of the thin peptidoglycan layer. The cells then pick up the pink safranin counterstain.
Why Gram-negative bacteria are harder to kill with antibiotics:
The outer membrane acts as a physical barrier, blocking many large-molecule antibiotics from reaching their targets.
LPS adds an additional layer of protection.
Porins (channels in the outer membrane) are selective about what passes through.
Periplasmic enzymes (e.g. beta-lactamases) can degrade antibiotics before they reach the peptidoglycan.
Efflux pumps can actively pump antibiotics back out of the cell.
This is why Gram-negative infections (such as those caused by Pseudomonas aeruginosa or E. coli) are often more difficult to treat than Gram-positive infections.
The Gram stain is used in hospitals every day to guide initial antibiotic choice before full culture results come back. Knowing whether an infection is Gram-positive or Gram-negative narrows the field of effective drugs immediately. Archaeal extremophiles, meanwhile, are the source of heat-stable enzymes used in PCR (the technique behind COVID tests and forensic DNA analysis).
Students often think Bacteria and Archaea are closely related because both are prokaryotes. They are not. Archaea are more closely related to Eukarya. The word "prokaryote" describes a cell plan (no nucleus), not a close evolutionary relationship.
Students often assume Gram-negative means "more dangerous." It means harder to treat with certain antibiotics, not necessarily more virulent. Plenty of severe diseases are caused by Gram-positive bacteria (e.g. MRSA, Clostridium difficile).
Students frequently mix up the colours: Gram-positive = purple, Gram-negative = pink. A mnemonic: "positive" starts with P, purple starts with P.
Students sometimes think the nucleoid is a membrane-bound structure. It is not. It is simply the region where the chromosome is concentrated.
⚠️ Be prepared to draw and annotate a three-domain phylogenetic tree, placing Archaea closer to Eukarya than to Bacteria.
⚠️ Know the Gram staining steps in order and be able to explain why Gram-positive cells appear purple and Gram-negative cells appear pink.
⚠️ Be able to explain at least three reasons why Gram-negative bacteria are more resistant to antibiotics.
⚠️ Know the three prokaryotic shapes and be able to match common organisms to their morphology.
⚠️ Distinguish between the nucleoid and a true nucleus.
True or false: Archaea and Bacteria are more closely related to each other than either is to Eukarya. (False. Archaea and Eukarya are sister groups.)
Fill in the blank: A spherical bacterium is called a ________. (Coccus.)
True or false: Gram-positive bacteria have an outer membrane. (False. Only Gram-negative bacteria have an outer membrane.)
Fill in the blank: The rigid scaffolding in bacterial cell walls is made of ________. (Peptidoglycan.)
True or false: Prokaryotic chromosomes are typically linear. (False. They are typically circular.)
Q: Name the three domains of life and state which two are prokaryotic.
A: Bacteria, Archaea, and Eukarya. Bacteria and Archaea are prokaryotic.
Q: On a phylogenetic tree, which domain is more closely related to Eukarya, Bacteria or Archaea? What evidence supports this?
A: Archaea. Evidence comes from ribosomal RNA sequence comparisons (Woese, late 1970s), which showed that archaeal rRNA sequences are more similar to eukaryotic sequences than to bacterial ones. Several molecular features, including similar RNA polymerases and histone-like proteins, also support this grouping.
Q: Describe the structural difference between Gram-positive and Gram-negative cell walls and explain how this affects the Gram staining result.
A: Gram-positive bacteria have a thick peptidoglycan layer (20 to 80 nm) and no outer membrane. The thick peptidoglycan retains the crystal violet-iodine complex during decolourisation, so the cells stain purple. Gram-negative bacteria have a thin peptidoglycan layer (5 to 10 nm) between an inner membrane and an outer membrane containing LPS. The thin peptidoglycan cannot retain the dye after the outer membrane is dissolved by alcohol, so the cells lose the purple dye and pick up the pink safranin counterstain.
Q: Give three reasons why Gram-negative bacteria are generally more resistant to antibiotics.
A: (1) The outer membrane acts as a physical barrier to many antibiotics. (2) Porins in the outer membrane are selective, limiting entry. (3) Beta-lactamases and other enzymes in the periplasmic space can degrade antibiotics before they reach their targets. (Efflux pumps are a valid fourth reason.)
Q: What is the shape of a bacterium described as a "streptobacillus"?
A: Rod-shaped cells arranged in chains. "Strepto-" means chains, "bacillus" means rod-shaped.
This material connects directly to prokaryotic genetics and reproduction (Part 2 of these notes), where the simple chromosome structure covered here explains why prokaryotes reproduce so quickly and evolve so fast. The Gram stain and cell wall differences also feed into pharmacology and medical microbiology, where antibiotic selection depends on knowing the target organism's wall type. The three-domain phylogenetic tree comes back in any discussion of evolution, taxonomy, or the origin of eukaryotic cells (endosymbiotic theory).
Prokaryote, prokaryotic cell, Bacteria domain, Archaea domain, Eukarya, eukaryote vs prokaryote, three domains of life, Carl Woese, rRNA phylogeny, phylogenetic tree, peptidoglycan, murein, cell wall bacteria, coccus, bacillus, spirillum, spirochete, Gram stain, Gram-positive, Gram-negative, crystal violet, safranin, lipopolysaccharide, LPS, outer membrane, periplasmic space, beta-lactamase, efflux pump, porin, nucleoid, circular chromosome, plasmid, histone-like proteins, BIO 101, intro biology, microbiology basics