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Diversity & Classification

Prokaryotic Family Tree

We want you to know that prokaryotes are diverse and we’ve got the data to back it up. A great way to demonstrate diversity is by graphing a phylogenetic tree. A phylogenetic tree is kind of like a family tree, but for different species of living things. At the root of the tree is a shared ancestor and at the other end are all the relatives, both close and distant, of the current generation.

If you want to see a good example of a family tree, we recommend checking out the British royal family tree. This family tree traces the links from a common ancestor (in this case, Queen Victoria, who was born in 1819) through to her great great great great grandchildren, including Princes William and Harry.

The phylogenetic tree of life is similar to a family tree except instead of going back to a human, like Queen Victoria, it is rooted in a shared ancestor of all of the species. You may not look a lot like a bacterium, but you’re very distantly related to it. Of course any ancestor we shared with bacteria lived very, very, very long ago.

This is a diagram of the phylogenetic tree of life. It separates organisms based on their genomic sequences.

Image from here.

The root of the tree lies at the center bottom. This point represents the common ancestor of all living things. Modern organisms span the outer reaches of the tree from firmicutes, a type of bacteria, on the left to animals (like us!) on the right. This tree shows two important things:
  • Prokaryotes are really diverse
  • Archaea really are separate from bacteria and eukaryotes.
The diversity of organisms is represented by how much space they take up on the tree. Prokaryotes take up more space than eukaryotes, so they are more diverse.

Looking at the tree, you can see that archaea exist in a completely separate branch from bacteria. In fact, they are even grouped a little more closely with the eukaryotes than with the bacteria. This is because when it comes to the enzymes used for several of their most basic processes, notably the enzymes used for transcription and translation, archaea act more like eukaryotes than bacteria. Archaea and bacteria might look similar under a microscope (see images below), but, genetically, they’re made of different stuff.

In the first image below, bacterial cells appear next to a large plant cell. The archaea in the second image (image is not at the same scale!) look a lot like bacteria, but are a genetically distinct branch of life.

Prokaryotic Cell

In order to get into the differences in prokaryotic cells, we’d like to show you a schematic of one. It’s simple, really. You’ll note two membranes, a cell wall between them, and, finally some DNA. It doesn’t get much simpler than that. Actually, it does get a little simpler. Some prokaryotes don’t even have the outer membrane. We’ll get into that in a bit.

Differences in the cell wall affect how prokaryotes grow and how we classify them. In archaea, the cell wall, called the S-layer (for surface layer), is made of proteins. In bacteria the cell wall is made up of a material called peptidoglycan. Peptidoglycan molecules consist of a peptide (peptides are like really small proteins) linked with a glycan (aka a sugar).

Differences in prokaryotic cell walls also affect how other organisms respond to them. For example, the human immune system has specific receptors that recognize peptidoglycan, enabling it to detect bacteria. These receptors monitor the presence of bacteria in places where they should and shouldn’t be.

Gram Staining

Bacteria are divided into two major classes. This process is based on…cell walls (are you sensing these are important yet?), specifically the interaction of the cell walls with a specific stain. Hans Christian Gram, a Dane who worked in a German morgue, invented the stain. No relation to the sometimes-morbid Hans Christian Andersen, who wrote the Little Mermaid. Because the stain is named for a person, the first letter of Gram is always capitalized.

The Gram strain is still used as the first step in differentiating bacteria. The way it works is that a population of different bacteria is exposed to a specific dye called crystal violet. Initially, this dye colors all the bacteria. When the dye is washed away, the Gram-positive bacteria, which have thick cell walls, retain the dye. Bacteria that lose the dye are classified as Gram-stain negative, or, simply, Gram negative. A weaker stain, called a counter stain, is then added to visualize the Gram-negative bacteria.

The following images show the staining mentioned above. All bacteria are stained purple. They are then washed so only Gram-positive bacteria retain the dye. A second dye was then used that added a pink color to all bacteria. This enables us to see the Gram negatives, too. The panel at the top shows (pink) Gram-negative bacteria while the panel to the bottom shows (purple) Gram-positive bacteria.

Gram Negative

Gram Positive

Gram Positive and Gram Negative

Both Gram-negative and Gram-positive bacteria have similar cytoplasms and cytoplasmic membranes (also known as plasma membranes) comprised of phospholipid bilayers just like eukaryotic and archaeal cells. For more on the plasma membrane, see here. It’s the parts outside this membrane where Gram-negatives and Gram-positives especially differ. Their different outsides strongly affect how they interact with the world. Even at the microbial level, appearances matter.

Gram-positive bacteria have a thick cell wall that surrounds their cytoplasmic membrane. In some species the wall is coated with polysaccharides in a final layer called the capsule. The capsule is slippery, helping bacteria avoid predators. It also blocks some viruses and detergents from harming the bacteria.

Gram-negative bacteria have an extra membrane, called the outer membrane, that surrounds their cell wall. The inner face of the outer membrane is composed of phospholipids, like the inner membrane. The outer face is composed of a special material called lipopolysaccharide (LPS). LPS is part lipid, like phospholipids, but, as you might have guessed, has a long polysaccharide (sugar) component as well. The polysaccharide part protrudes outside the cell. Some Gram-negative strains have capsules too.

The existence of an outer membrane means that Gram-negative bacteria have an extra cellular compartment between the two membranes. This region is called the periplasm. The periplasm is kind of like a giant cellular foyer or "airlock" that surrounds the cytoplasm. The cells control which chemicals and nutrients are allowed to cross each of their membranes.

The Gram-negative bacterial cell wall is located in the periplasm. It is thinner than the Gram-positive cell wall. Gram-negative bacteria are okay with this because they get extra stability from LPS in their outer membrane.

The images below demonstrate these features.


Prokaryotes, both bacteria and archaea, take on some inventive shapes or morphologies. The shapes depend on the way their cell walls are constructed. The most common shapes are:
  • Rod-shaped (Bacillus)
  • Spherical (Coccus)
  • Spiral-shaped (Spirilla are rigid; Spirochetes are flexible)
Warning! Bacillus is both a bacterial shape AND a bacterial genus. Lots of bacteria outside of the genus Bacillus are bacillus-shaped. For example, E. coli, which is in the genus Escherichia, is bacillus-shaped. We’re happy to report that species in the genus Bacillus are also bacillus shaped. Otherwise that could have been very confusing…

The images below are presented in false color in order to help distinguish the bacteria from the background. From top to bottom, they are Spheres, Rods, Spirals, and finally Curved Rods. We threw the curved rods (also called comma-shaped) in there just to point out that variations on the three major shapes are common.



Curved Rod (Vibrio)


DNA-based Prokaryotic Classification

Investigating a bacterium’s morphology can be useful for understanding and treating infections. For example, if you think someone might have the disease cholera, but they don’t have cells that look like the bacteria that cause cholera, you might want to try a different treatment.

Prokaryotes can be more difficult to classify than multicellular eukaryotes. If you’re trying to tell if a horse and a zebra are different organisms, you can consider their stripes, their bone structure, and if they can produce fertile offspring. The answer to that last one, by the way, is "generally no". Prokaryotes do not have as many obvious features to distinguish one from another, so scientists have figured out other ways of classifying them.


One way that prokaryotes are classified is by determining their serotypes. Serotype is determined by testing which antibodies (generally from an animal, like antibodies from a rabbit immune system) interact with a given sample. American microbiologist Rebecca Lancefield developed the serotype classification system in 1933.

Different antibodies recognize different parts of molecules on the outsides of the prokaryotic cells. A major antigen responsible for serotyping in Gram-negative bacteria is the O-antigen, the polysaccharide component of LPS. While serotyping antibodies often recognize the outer regions of cells, they also respond to virulence factors (called exotoxins) that are released by pathogenic bacteria. Pathogenic bacteria are bacteria that can cause disease. We’ll talk more about pathogens and exotoxins later.

Serotyping can be very sensitive and even identify different classes of bacteria of the same species, called strains. For example, the species Vibrio cholerae, which causes the disease cholera, has more than 200 serotypes.

DNA Barcodes

Another method, that is increasingly used, is DNA sequencing. DNA sequencing can be used in one of two ways. In the first option, an entire bacterial genome sequence can be determined and aligned with existing sequences for similarity studies. A more time (and money!) sensitive technique involves only sequencing a specific region of the genome known to be variable across species. Such regions are sometimes called "DNA barcodes" since they are unique to a given species. The DNA barcode sequence can be used like real-life barcodes to look up the organism attached to it.

Brain Snack

DNA barcoding was used by high school students in New York to investigate if fish used for sushi were labeled correctly. In a big news story dubbed "Sushigate", the students, with the help of university researchers, found that 25% of fish were mislabeled. Check it out here.
The work pioneered by these high school students continues. A more recent investigation in Los Angeles showed that over half of seafood there was mislabeled!

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