Much of what is known about prokaryotic chromosome structure was derived from studies

of Escherichia coli, a bacterium that lives in the human colon and is commonly used in laboratory
cloning experiments. In the 1950s and 1960s, this bacterium became the model organism of choice
for prokaryotic research when a group of scientists used phase-contrast microscopy
and autoradiography to show that the essential genes of E. coli are encoded on a single circular
chromosome packaged within the cell nucleoid (Mason & Powelson, 1956; Cairns, 1963).

Prokaryotic cells do not contain nuclei or other membrane-bound organelles. In fact, the word
"prokaryote" literally means "before the nucleus." The nucleoid is simply the area of a prokaryotic
cell in which the chromosomal DNA is located. This arrangement is not as simple as it sounds,
however, especially considering that the E. coli chromosome is several orders of magnitude larger
than the cell itself. So, if bacterial chromosomes are so huge, how can they fit comfortably inside a
cell—much less in one small corner of the cell?

DNA Supercoiling

The answer to this question lies in DNA packaging. Whereas eukaryotes wrap their DNA around
proteins called histones to help package the DNA into smaller spaces, most prokaryotes do not have
histones (with the exception of those species in the domain Archaea). Thus, one way prokaryotes
compress their DNA into smaller spaces is through supercoiling (Figure 1). Imagine twisting a rubber
band so that it forms tiny coils. Now twist it even further, so that the original coils fold over one
another and form a condensed ball. When this type of twisting happens to a bacterial genome, it is
known as supercoiling. Genomes can be negatively supercoiled, meaning that the DNA is twisted in
the opposite direction of the double helix, or positively supercoiled, meaning that the DNA is twisted
in the same direction as the double helix. Most bacterial genomes are negatively supercoiled during
normal growth.

Proteins Involved in Supercoiling

Figure 1

During the 1980s and 1990s, researchers discovered that multiple proteins act together to fold and
condense prokaryotic DNA. In particular, one protein called HU, which is the most abundant protein
in the nucleoid, works with an enzyme called topoisomerase I to bind DNA and introduce sharp
bends in the chromosome, generating the tension necessary for negative supercoiling. Recent
studies have also shown that other proteins, including integration host factor (IHF), can bind to
specific sequences within the genome and introduce additional bends (Rice et al., 1996). The folded
DNA is then organized into a variety of conformations (Sinden & Pettijohn, 1981) that are supercoiled
and wound around tetramers of the HU protein, much like eukaryotic chromosomes are wrapped
around histones (Murphy & Zimmerman, 1997).

Once the prokaryotic genome has been condensed, DNA topoisomerase I, DNA gyrase, and other
proteins help maintain the supercoils. One of these maintenance proteins, H-NS, plays an active role
in transcription by modulating the expression of the genes involved in the response to environmental
stimuli. Another maintenance protein, factor for inversion stimulation (FIS), is abundant during
exponential growth and regulates the expression of more than 231 genes, including DNA
topoisomerase I (Bradley et al., 2007).

Accessing Supercoiled Genes

Supercoiling explains how chromosomes fit into a small corner of the cell, but how do the proteins
involved in replication and transcription access the thousands of genes in prokaryotic chromosomes
when everything is packaged together so tightly? It has been determined that prokaryotic DNA
replication occurs at a rate of 1,000 nucleotides per second, and prokaryotic transcription occurs at a
rate of about 40 nucleotides per second (Lewin, 2007), so bacteria must have highly efficient
methods of accessing their DNA strands. But how?

Researchers have noted that the nucleoid usually appears as an irregularly shaped mass within the
prokaryotic cell, but it becomes spherical when the cell is treated with chemicals to inhibit
transcription or translation. Moreover, during transcription, small regions of the chromosome can be
seen to project from the nucleoid into the cytoplasm (i.e., the interior of the cell), where they
unwind and associate with ribosomes, thus allowing easy access by various transcriptional proteins
(Dürrenberger et al., 1988). These projections are thought to explain the mysterious shape of
nucleoids during active growth. When transcription is inhibited, however, the projections retreat into
the nucleoid, forming the aforementioned spherical shape.

Because there is no nuclear membrane to separate prokaryotic DNA from the ribosomes within the
cytoplasm, transcription and translation occur simultaneously in these organisms. This is strikingly
different from eukaryotic chromosomes, which are confined to the membrane-bound nucleus during
most of the cell cycle. In eukaryotes, transcription must be completed in the nucleus before the
newly synthesized mRNA molecules can be transported to the cytoplasm to undergo translation into
proteins.

Variations in Prokaryotic Genome Structure

 Figure 2: Deer tick.

This black-legged tick (Ixodes sp.) carries the bacterium (Borrelia sp.) that causes Lyme disease.

Jim Gathany and William L. Nicholson, Ph.D/CDC.

Recently, it has become apparent that one size does not fit all when it comes to prokaryotic
chromosome structure. While most prokaryotes, like E. coli, contain a single circular
DNA molecule that makes up their entire genome, recent studies have indicated that some
prokaryotes contain as many as four linear or circular chromosomes. For example, Vibrio cholerae,
the bacteria that causes cholera, contains two circular chromosomes. One of these chromosomes
contains the genes involved in metabolism and virulence, while the other contains the remaining
essential genes (Trucksis et al., 1998). An even more extreme example is provided by Borrelia
burgdorferi, the bacterium that causes Lyme disease. This organism is transmitted through the bite of
deer ticks (Figure 2), and it contains up to 11 copies of a single linear chromosome (Ferdows &
Barbour, 1989). Unlike E. coli, Borrelia cannot supercoil its linear chromosomes into a tight ball within
the nucleoid; rather, these strands are diffused throughout the cell (Hinnebusch & Bendich, 1997).

Other organisms, such as Bacillus subtilis, form nucleoids that closely resemble those of E. coli, but
they use different architectural proteins to do so. Furthermore, the DNA molecules of Archaea, a
taxonomic domain composed of single-celled, nonbacterial prokaryotes that share many similarities
with eukaryotes, can be negatively supercoiled, positively supercoiled, or not supercoiled at all. It is
important to note that archaeans are the only group of prokaryotes that use eukaryote-like histones,
rather than the architectural proteins described above, to condense their DNA molecules
(Sandman et al., 1990). The acquisition of histones by archaeans is thought to have paved the way
for the evolution of larger and more complex eukaryotic cells (Minsky et al., 1997).

Other DNA Differences Between Prokaryotes and Eukaryotes

Most prokaryotes reproduce asexually and are haploid, meaning that only a single copy of
each gene is present. This makes it relatively easy to generate mutations in the lab and study the
resulting phenotypes. By contrast, eukaryotes that reproduce sexually generally contain multiple
chromosomes and are said to be diploid, because two copies of each gene exist—with one copy
coming from each of an organism's parents.

Yet another difference between prokaryotes and eukaryotes is that prokaryotic cells often contain
one or more plasmids (i.e., extrachromosomal DNA molecules that are either linear or circular).
These pieces of DNA differ from chromosomes in that they are typically smaller and encode
nonessential genes, such as those that aid growth in specific conditions or encode antibiotic
resistance. Borrelia, for instance, contains more than 20 circular and linear plasmids that encode
genes responsible for infecting ticks and humans (Fraser et al., 1997). Plasmids are often much
smaller than chromosomes (i.e., less than 1,500 kilobases), and they replicate independently of the
rest of the genome. However, some plasmids are capable of integrating into chromosomes or moving
from cell to cell.

Perhaps due to the space constraints of packing so many essential genes onto a single chromosome,
prokaryotes can be highly efficient in terms of genomic organization. Very little space is left between
prokaryotic genes. As a result, noncoding sequences account for an average of 12% of the
prokaryotic genome, as opposed to upwards of 98% of the genetic material in eukaryotes (Ahnert et
al., 2008). Furthermore, unlike eukaryotic chromosomes, most prokaryotic genomes are organized
into polycistronic operons, or clusters of more than one coding region attached to a single promoter,
separated by only a few base pairs. The proteins encoded by each operon often collaborate on a
single task, such as the metabolism of a sugar into by-products that can be used for energy (Figure 3).

Figure 3: The prokaryotic lac operon.

Three structural genes code for proteins involved in lactose import and metabolism in bacteria. The
genes are organized together in a cluster called the lac operon.

© 2013 Nature Education All rights reserved.

Figure Detail

The organization of prokaryotic DNA therefore differs from that of eukaryotes in several important
ways. The most notable difference is the condensation process that prokaryotic DNA molecules
undergo in order to fit inside relatively small cells. Other differences, while not as dramatic, are
summarized in Table 1.

Table 1: Prokaryotic versus Eukaryotic Chromosomes

Prokaryotic Chromosomes Eukaryotic Chromosomes

 Many prokaryotes contain a single  Eukaryotes contain multiple linear

circular chromosome. chromosomes.

 Prokaryotic chromosomes are  Eukaryotic chromosomes are

condensed in the nucleoid via DNA condensed in a membrane-bound
supercoiling and the binding of nucleus via histones.
various architectural proteins.
 In eukaryotes, transcription occurs in
 Because prokaryotic DNA can interact the nucleus, and translation occurs in
with the cytoplasm, transcription and the cytoplasm.
translation occur simultaneously.
 Most eukaryotes contain two copies
 Most prokaryotes contain only one of each gene (i.e., they are diploid).
copy of each gene (i.e., they are
 Some eukaryotic genomes are
haploid).
organized into operons, but most are
 Nonessential prokaryotic genes are
 commonly encoded on not.
extrachromosomal plasmids.
 Extrachromosomal plasmids are not
 Prokaryotic genomes are efficient commonly present in eukaryotes.
and compact, containing
 Eukaryotes contain large amounts of
little repetitive DNA.
noncoding and repetitive DNA.

References and Recommended Reading

Abbott, A. Lyme disease: Uphill struggle. Nature 439, 524–525 (2006) doi:10.1038/439524a (link to
article)

Ahnert, S. E., et al. How much non-coding DNA do eukaryotes require? Journal of Theoretical
Biology 252, 587–592 (2008)

Bendich, A. J., & Drlica, K. Prokaryotic and eukaryotic chromosomes: What's the
difference? Bioessays 22, 481–486 (2000)

Bradley, M. D., et al. Effects of Fis on Escherichia coli gene expression during different growth
stages. Microbiology 153, 2922–2940 (2007)

Cairns, J. The chromosome of Escherichia coli. Cold Spring Harbor Symposia on Quantitative
Biology 28, 43–46 (1963)

Dürrenberger, M., et al. Intracellular location of the histonelike protein HU in Escherichia coli. Journal
of Bacteriology 170, 4757–1768 (1988)

Endy, D., & Brent, R. Modelling cellular behaviour. Nature 409, 391–395 (2001)
doi:10.1038/35053181 (link to article)

Ferdows, M. S., & Barbour, A. G. Megabase-sized linear DNA in the bacterium Borrelia burgdorferi,
the Lyme disease agent. Proceedings of the National Academy of Sciences 86, 5969–5973 (1989)

Fraser, C. M., et al. Genomic sequence of a Lyme disease spirochaete, Borrelia

burgdorferi. Nature 390, 580–586 (1997) doi:10.1038/37551 (link to article)

Hinnebusch, B. J., & Bendich, A. J. The bacterial nucleoid visualized by fluorescence microscopy of
cells lysed within agarose: Comparison of Escherichia coli and spirochetes of the
genus Borrelia. Journal of Bacteriology 179, 2228–2237 (1997)

Jacob, F., & Monod, J. Genetic regulatory mechanisms in the synthesis of proteins. Journal of
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Mason, D. J., & Powelson, D. M. Nuclear division as observed in live bacteria by a new
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Minsky, A., et al. Nucleosomes: A solution to a crowded intracellular environment. Journal of

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Murphy, L. D., & Zimmerman, S. B. Isolation and characterization of spermidine nucleoids

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