Genome Packaging in Prokaryotes: the Circular Chromosome of E. coli

Most students learn at an early age that organisms can be broadly divided into two types: prokaryotes and eukaryotes. In primary school, children are taught that the main difference between these organisms is that eukaryotic cells contain membrane-bound organelles, such as the nucleus, while prokaryotic cells do not. There is much more to the story, however, particularly with regard to chromosomal structure and organization.

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?

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.

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).

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.

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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