2-18 The cell DNA is organized into a nucleoid

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• Prokaryotic DNA is organized into a DNA-protein complex called the nucleoid.
• The chromosome is much longer than the typical prokaryote and must be compacted to fit inside the cell. This is accomplished by supercoiling the DNA (adding twists) and complexing it with DNA-binding proteins.

The nucleoid is a bit difficult to define because unlike eukaryotes with their nuclei, prokaryotic chromosomes are not confined to a specific organelle. The nucleoid is sometimes referred to as the region of the bacterial cell that contains the chromosome, but that suggests there is a specific region in any cell where the chromosomes is always found, and this is probably not true. A better definition might be that the nucleoid is the region of the cell that is currently occupied by the DNA-protein complex that makes up the chromosome. So what do we know about prokaryotic chromosomes? What does a typical chromosome look like?

It is a little awkward to talk about "typical" anything in prokaryotes, since there is always such a large range for any given property. In the case of prokaryotic chromosomes, the smallest known is about 160 kB, while the largest is about 10,000 kB. E. coli is certainly well-studied and might well be considered typical, with strain K12 having a chromosome of 4,639,221 bp containing 4405 genes. The chromosome for this bacterium is circular and this is a common arrangement, but there are a number of species with linear chromosomes. If stretched out, this material would be about 1400 µm long or about 1/16^th of an inch. The E. coli cell is about 1-5 µm in length so it is clear that an amazing degree of packing is necessary to fit the chromosome inside its tiny host. The nucleoid occupies about half of the bacterial cytoplasm and has a density of 20-50 mg/ml, similar to what is observed for the nucleus of a non-dividing eukaryotic cell. The following description of the nucleoid refers specifically to that of E. coli, but is probably generally similar to that of other prokaryotes. Figure 2-26 shows the nucleoid of E. coli.

The nucleoid is composed of DNA in association with a number of DNA-binding proteins that help it maintain its structure. The protein HU non-specifically binds to DNA and bends it, with the DNA apparently wrapping around the HU protein. Another protein found on DNA is IHF, whose sequence is reminiscent of that of HU and is therefore evolutionarily related. It also facilitates bending of DNA, but it does so by binding to specific DNA sites. A third protein, H-NS, binds to DNA non-specifically and is apparently involved in compacting DNA structure. It is found throughout the nucleoid and is likely the major DNA-binding protein that organizes the chromosome. The nucleoid also contains a large amount of RNA polymerase and RNA, as well as small amounts of many different proteins that regulate the expression of specific genes. These seem not to perform any structural role, but reflect the importance of RNA transcription in the nucleoid in growing cells.

The DNA double helix typically has a bit of twisting tension in the opposite direction of the helix ladder (the twists typically "unwind" the left-handed DNA helix). Negative supercoiling, as it is called, is produced by the action of enzymes termed topoisomerases. Figure 2-27 shows supercoiling of the DNA. This negative supercoiling makes it slightly easier to separate the two strands of the double helix, as must be done to start transcription and replication.

As shown in Figure 2-28, the chromosome is further folded into 50 or so loops of about 100,000 base pairs. These domains supercoil independently and indeed, even small sections within the same loop can transiently have different degrees of supercoiling. Because supercoiling changes fairly rapidly, it has been extremely difficult to study in living cells. Nevertheless, it is clear that a variety of environmental effects and even the act of transcription itself can affect the supercoiling of a region. In turn, the specific supercoiling of a region can affect the ability of the cell to express genes in that region. Because supercoiling is affected by so many things and in turn can affect expression of much of the genome, it will be important for microbiologists to develop a better understanding of this phenomenon in the future.

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