The Nucleoid
Chapter
12
DAVID E. PETTIJOHN
This chapter provides an overview of the structure of the bacterial nucleoid and certain of its biological implications. The intent is to provide a brief review of current understanding developed during the past two decades. This discussion is selective, and no attempt is made to comprehensively mention all relevant research. The descriptions focus on properties of the nucleoid of Escherichia coli, which is the most extensively studied one, but there is a limited comparison with properties of nucleoids of other bacteria. DNA-associated proteins that are believed to have a role in organizing DNA structure in the nucleoid are also discussed. In addition, there is a limited description of results suggesting possible effects of the nucleoid structure on protein-DNA interactions and gene expression. There is no discussion of the genomic sequencing of Escherichia coli, which is considered in part VI of this book.
Nucleoids in thin sections of bacterial cells have been imaged by electron microscopy for many years, but it was only clearly recognized in the 1980s that the observed structure can be seriously perturbed by commonly used procedures for fixation (24, 25; see chapter 4 of this volume). By using newer methods, cells that are rapidly frozen and cryosubstituted yield electron microscopic images of the nucleoid that show even and grainy distributions of the DNA (Fig. 1), unlike the aggregated or coarse fibrillar distributions seen with older methods. It should be noted, however, that the advantages claimed for cryosubstitution are not observed in every laboratory and that the ideal fixation methods are still somewhat controversial. By using the cryosubstitution methods, the nucleoid appears as an irregular dispersed structure occupying less than half the apparent intracellular space and is not confined into the tighter, more symmetrical shapes often observed after the older osmium fixation methods.
The nucleoids can be visualized in living bacterial cells at lower resolution by using several different methods for light microscopy (56). Fluorescence microscopy provides one useful approach for delineating the gross boundaries of the intracellular nucleoid (Fig. 2). A fluorescent probe like 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) readily enters cells of many E. coli strains, binds the DNA, and is nontoxic at concentrations that are useful for microscopy. The changes in the size and shape of nucleoids can be visualized from the associated fluorescent DAPI probe as the cells pass through their division cycles. The nucleoids are seen as variably shaped structures that occupy only a portion of the intracellular volume. However, it should be emphasized that what appears to be the boundary of the intracellular nucleoid visualized by this procedure probably does not depict the actual limits on DNA distribution within the cell. For example, isolated loops of DNA that probably extend beyond the apparent surface of the nucleoid would not be detected. Although less detail of the nucleoid structure is provided by fluorescence microscopy than by electron microscopy, there are advantages in that living cells can be studied and concerns about fixation or hydration artifacts are reduced. It should be noted that within the limits of resolution there are no inconsistencies between the sizes and shapes of the images obtained by fluorescence microscopy and electron microscopy.
Visualization of nucleoids by fluorescence microscopy as well as other methods for estimating the average nucleoid size, combined with knowledge of the average amount of DNA per cell, has permitted estimates of the average DNA packing density in the range 20 to 50 mg/ml (24, 25). This variation in packing density is partly attributable to variations in the size of the nucleoid that occur depending on the growth conditions of the bacteria or other reversible treatments, such as ionic shock or exposure to inhibitors of protein synthesis. The estimated DNA packing density in the nucleoid is similar to that estimated in the interphase nucleus of eukaryotes, and is much less than the 600- to 800-mg/ml figure for phage heads.
The methods developed over the past 20 years for isolating nucleoids depend on the introduction of stabilizing counterions during lysis of the bacteria. In this discussion, I emphasize properties of the isolated nucleoids which were also described in several earlier reviews (9, 37, 40) and discuss the short- and long-range structures of DNA in the nucleoid. Short-range DNA structure refers to interactions involving roughly 1,000 bp or less, while long-range structure involves extended organizations of the DNA that greatly exceed 1,000 bp.
Without counterions, the condensed state of the nucleoid DNA is lost as the DNA unfolds during or soon after lysis. A variety of simple monovalent or divalent salts and polyamines have been used as stabilizing counterions. In most studies, relatively high concentrations of simple salts (0.5 to 1.0 M) were required, but a polyamine such as spermidine is adequate for stabilization at as little as 1 to 2 mM. Many DNA-bound proteins become dissociated when high concentrations of simple salts are used but remain associated when nucleoids are stabilized by polyamines. One specialized method developed for Bacillus licheniformis allows the isolation of nucleoids at near physiological ionic strength without polyamines or detergents (49). Methods analogous to the E. coli procedures have also been applied to Bacillus subtilis (20). In all cases, the nucleoids were purified by sedimentation through sucrose or glycerol density gradients containing the stabilizing counterions. Typical sedimentation rates for nucleoids prepared in high salt are 1,500 to 2,200 S corrected for zero rotor speed. These values and other properties, to be discussed below, are similar for nucleoids from different bacterial species.
Nucleoids purified by the above procedures have been visualized by scanning electron microscopy (Fig. 3) and transmission electron microscopy (Fig. 4) after spreading on protein monolayers. Under the scanning electron microscope, the particles have the approximate size of those visualized in the cell and doublet structures such as those seen in exponentially growing cells are commonly observed. Thus, isolated nucleoids have a similar DNA packing density to that observed in vivo.
When the DNA of the nucleoids is spread for examination by transmission electron microscopy, the maximal observed extension of the DNA is much greater, suggesting that the state of compaction is changed greatly by the spreading conditions (Fig. 4). No free ends of the DNA are observed, as expected for an unbroken circular DNA. The DNA is seen to be organized in a series of loops and is supercoiled in plectonemic (interwound) supercoils.
Isolated nucleoids have a set of DNA-bound proteins and associated nascent RNA chains. Core RNA polymerase subunits are prominent proteins in nucleoids isolated in both low- and high-salt solutions or in the presence of stabilizing polyamines (9, 37, 40). RNA polymerase subunits are the major proteins of nucleoids isolated in the presence of 1.0 M NaCl, which dissociates many proteins that would otherwise be more abundant than the polymerase. These are active RNA polymerase molecules that are apparently trapped at intermediate positions in transcriptional units and have attached to them nascent RNA chains. When incubated in transcription runoff assays, the RNA chains that are made reflect the state of transcriptional regulation of the cells from which the nucleoids were isolated. For example, 40 to 50% of the newly labeled chains are rRNA sequences, the same proportion seen when exponentially growing cells were pulse-labeled (17, 40).
Nucleoids isolated at low salt concentration by using polyamines or after lysis in the cold contain large amounts of membrane proteins and the commonly known DNA-binding proteins such as protein HU. When such nucleoids are visualized by electron microscopy, a patch of nucleoid-associated membrane is frequently seen (Fig. 4).
When isolated nucleoids are incubated with protein denaturants such as the ionic detergent sodium dodecyl sulfate, the DNA unfolds. This transition is readily manifested by a dramatic increase in specific viscosity, decrease in sedimentation rate, increase in rotor speed dependence of the sedimentation coefficient, and appearance of the DNA under the electron microscope. These findings suggested that DNA-bound proteins are involved in maintaining the DNA in a condensed state. Early in this research, it was also observed in several laboratories that incubations with RNase resulted in unfolding of the DNA. However, nucleoids isolated from B. licheniformis by using a low salt concentration are less sensitive to RNase. Moreover, studies of chromosomal domains in living E. coli cells have indicated that nascent RNA chains are not critical to stabilizing chromosomal domains (the domain substructure of nucleoids is described below). Detailed studies of the RNA components of isolated nucleoids have revealed no unusual RNA chains involved in nucleoid stabilization (see references 39 and 40 for review). The suggestion has been made that the apparent stabilization may be due to a direct interaction such as an R-looping between nascent mRNA and DNA mediated by the negatively supercoiled DNA (39, 40). It is questionable if this RNA-DNA interaction is of biological significance.
Uncertainties about the relevant RNA-DNA interactions also raise questions about the significance of the isolated nucleoids. The possibility cannot be excluded that the DNA structure in nucleoids has been reorganized during isolation. Dissociation of many DNA-bound proteins certainly occurs when nucleoids are prepared in high salt concentrations, and the partial restraint of DNA supercoils has therefore changed. However, several important parameters of nucleoid structure (DNA packing density, molecular weight of the DNA, specific linking number deficit, and domains of supercoiling) have been preserved during isolation. Thus, at a minimum, isolated nucleoids provide a useful model system.
Electron micrographs of isolated nucleoids show the DNA organized in a series of long loops, each of which is supercoiled (Fig. 4). Studies of the effect of ethidium bromide intercalation on compaction of isolated nucleoids demonstrated that the DNA of the nucleoids is negatively supercoiled with an estimated specific linking deficit of about σ = –0.05 (9, 40). This value is similar to that observed in closed circular DNAs of viruses, phages, and plasmids isolated from both prokaryotes and eukaryotes. It also differs by about 50% from estimates of the effective superhelical density measured in living bacteria (see below). Supercoiling of DNA is important for two reasons: (i) supercoils play a role in packaging the DNA; and (ii) supercoiled DNA is equilibrated with torsional strain in the double helix, which greatly influences important protein-DNA interactions.
In the studies mentioned above, nucleoids were prepared under conditions which lead to dissociation of major nucleoid proteins HU, IHF, and H-NS. These proteins are able to bend or coil DNA and/or have higher affinity for bent DNA. Thus, they provide mechanisms for distorting or introducing flexibility in the short- range structure of DNA in the nucleoid. There are enough of these proteins per E. coli cell to interact with the bulk of the DNA; for example there is sufficient HU to bind about 1 HU dimer per 300 to 400 bp. Because these proteins are known to restrain specific DNA topologies, including negative DNA supercoils and DNA bends, their binding to the DNA would reduce the effective superhelical density (or unrestrained superhelical density). Indeed, estimates of the unrestrained superhelical density of plasmids in vivo indicated that the effective superhelical density is about half of that of isolated plasmids with bound proteins removed (3, 21, 35, 38). Thus, it was proposed that the unrestrained superhelical density of nucleoid DNA would likewise be about half of the value determined for nucleoid DNA lacking bound HU, or about σ = –0.025. The unrestrained DNA supercoils in isolated nucleoids appear as plectonemic supercoils, with the major helical axis of one region of a DNA molecule coiled about the major axis of another part of the same molecule. Another kind of supercoiling (toroidal supercoiling) exists when the DNA is wrapped around an imaginary or real surface (as occurs in the nucleosome). Considerations of energetics (3) lead to the prediction that the DNA linking number deficit in plasmids or nucleoids would be expressed as plectonemic supercoils in vivo. This short-range topology has the effect of compacting the volume of the DNA.
The negative superhelical tension in the DNA also has a strong influence on many different protein-DNA interactions, including the interactions of promoters with RNA polymerase and the rates of transcription (4, 43, 44). To be maximally active, some promoters require negative superhelical tension and others are inhibited by it. Topoisomerases regulate the levels of this tension in the nucleoid and in other topologically closed DNAs (10). Topoisomerase I and DNA gyrase (a topoisomerase II) seem to have opposing effects, so that mutants with defective topoisomerase I have higher levels of DNA torsional tension and DNA gyrase mutants have lower levels. The interactions of RNA polymerase with DNA during transcription also lead to DNA supercoiling according to the twin domain model of Lui and Wang (28). Positive supercoils are generated ahead of the polymerase and negative supercoils are generated behind it; both of these are relaxed to appropriate levels by the activities of the topoisomerases.
I noted above that different prokaryotic proteins can restrain negative DNA supercoils in vitro or organize other topologies that compact the DNA. I also described evidence showing that negative DNA supercoils exist in vivo and that some, but not all, of these supercoils are restrained and therefore do not contribute to the effective superhelical density in vivo. It is not known which, if any, of the known proteins are responsible in vivo for the primary restraint of DNA supercoils. It is also not clear if the restraint occurs from the wrapping of toroidal left-handed coils of DNA in nucleosome-like structures or if the restraint occurs through stabilizing nodes where DNA double helices pass over each other in plectonemic supercoils. In either case, however, the restraint would have the effect of organizing the short-range structure of the nucleoid DNA and therefore have the effect of compacting it. Moreover, recent studies have shown that mutant E. coli cells that lack HU protein have plasmids with reduced superhelical density, suggesting that HU protein may actually restrain negative DNA supercoils in vivo (22, 60). Thus, these protein-DNA interactions appear to play a role in organizing short-range DNA structure in the nucleoid. In support of this general idea, it was recently shown that a histone homolog introduced into E. coli cells leads to nucleoid compaction (2).
The unrestrained superhelical tension present in the nucleoid DNA can be relaxed when swivels, such as single-strand breaks, are introduced into the DNA in vitro or in vivo. It was first observed by Worcel and Burgi (57) that many more than one single-strand DNA break per nucleoid equivalent of DNA was required to relax all the superhelical tension in isolated nucleoids. This implied that, at least in the isolated nucleoids, the DNA is organized into long-range units such as loops which prevent relaxation from one chromosomal domain from being propagated into another. A domain of supercoiling was defined as a region of a DNA molecule bounded by topological constraints on the rotation of the double helix. Subsequent studies designed to define the domain substructure of chromosomes in vivo relied on psoralen photoaffinity probes to quantify the unrestrained superhelical tension in nucleoids of living cells. These experiments showed that at least one single-strand DNA break per 100,000 bp of DNA, introduced in vivo by gamma irradiation, was required to relax the superhelical tension (48). It was estimated that the average size of a domain in the E. coli nucleoid was about 100 kbp (see Fig. 5 for a model). It is of interest that similar sized domains have been measured in chromosomes from diverse biological sources including eukaryotes (for a review, see reference 1).
The concept of a domain substructure of nucleoids has biological significance, because it ensures that topological changes in one domain need not affect the entire chromosome. It becomes possible to repair, replicate, or recombine DNA in one domain, all of which involve changes in DNA topology, without influencing DNA organization in other domains of the same chromosome. It also becomes possible in principle to regulate superhelical tension independently in different domains and thus to regulate coordinately banks of genes in a single domain—although this mechanism has not been demonstrated. It should be emphasized that this model does not require that the sites of domains be fixed or even long-lived. Because the domain is so large, nearby genes would almost always be in the same domain, even if the boundaries defining domains were continuously in flux.
The domain substructure of chromosomes also provides a basis for organizing the long-range DNA structure in the nucleoid. If the domains are topologically equivalent to loops, as appears from the electron micrographs of isolated nucleoids, they have the effect of achieving long-range compaction of the nucleoid DNA into an organized structure. For example, the fully extended circular chromosome of E. coli would have a diameter of about 430 μm, but when organized into 50 fully extended equal-sized loops, it would be about 17 μm in maximal dimension. Thus, the short-range order need provide only a 30-fold condensation of the loop length to compact the DNA into a nucleoid-sized structure.
Binding of DNA to the bacterial cytoplasmic membrane has been extensively studied and is reviewed in other chapters of this volume. In particular, the binding of the replicative origin to the cell membrane has been investigated in some detail (see, for example, reference 33). It was proposed some years ago that not only direct membrane-DNA interactions but also binding of nascent membrane proteins to the membrane could provide topological restraints on the DNA of the nucleoid (26). Indeed, recent results have shown that membrane-associated nascent proteins with their attached cotranscribed mRNA can act to anchor the associated active RNA polymerase and therefore restrict rotation of the bound DNA template (29). This anchored site of transcription therefore has the potential of defining the boundary of a chromosomal domain.
While an anchoring mechanism for defining the boundaries of domains in nucleoids has yet to be demonstrated, it has been repeatedly observed in plasmids carrying genes specifying membrane proteins (8, 29, 42). Since an anchored RNA polymerase cannot rotate around its DNA template during transcription, the energetics of transcription drive the rotation of DNA. In cells deficient for topoisomerase I, the rotation of the DNA double helix forces plasmids to become hypernegatively supercoiled only when genes coding for the anchoring proteins are actively transcribed. These findings led to the proposal that similar interactions in the nucleoid at the sites of genes specifying membrane proteins could result in the segregation of the chromosome into topological domains. This type of interaction would create localized domains in the nucleoid. However, prior results (37, 40) suggest that the ∼100-kbp topological domains of nucleoids measured in vivo are independent of nascent membrane proteins and their associated mRNA chains. The elimination of nascent mRNA by treatment of the cells with rifampin did not lead to a significant decrease in the number of domains per nucleoid (48). This implies that at least some of the restraints on the rotation of nucleoid DNA are different from those in plasmids and are independent of the above anchoring mechanism.
It was first demonstrated in the early 1970s that high concentrations of neutral nonbinding polymers will collapse DNA into a compact structure (27). This structural transition seems to result from collisions between the DNA and the nonbinding polymer. As the collisions begin to compete with water-DNA interactions, the DNA changes structure to a form which reduces the "crowding" interactions with the polymer in a process somewhat analogous to a hydrophobic interaction. Initial studies used polyethylene glycol as the crowding polymer to induce the so-called psi structure of DNA, but later studies showed that other polymers work as well. It has been thought that this phenomenon could play a role in maintaining the compact state of DNA in nucleoids and that in living cells the high concentration of non-DNA- binding proteins in the bacterial cytoplasm could crowd the nucleoid DNA into its compact state.
Recent studies have renewed interest in this possibility (62, 63). It was demonstrated that DNA condensed by crowding reacted efficiently with DNA-active proteins and enzymes. For example, the rates of ligation of the cohesive ends of lambda DNA by DNA ligase were stimulated when the DNA was condensed by macromolecular crowding agents. Moreover, it was found that the HU protein at very high nonphysiological concentrations can condense DNA but that in the presence of modest concentrations of polyethylene glycol, HU concentrations similar to that existing in the cell are sufficient to induce the condensation (30). It was proposed that the combined effects of crowding by cytoplasmic and DNA-bound proteins such as HU and H-NS may induce the compact state of DNA in nucleoids. While it is not expected that crowding transitions would provide an organized long- and short-range structure to the nucleoid DNA, it seems likely that these excluded volume effects play some role in the final state of compaction of the nucleoid.
The HU protein has long been considered a candidate for the protein that restrains DNA supercoils in nucleoids, because (i) it is the major DNA-binding protein known, (ii) it restrains negative DNA supercoils in vitro, and (iii) like histones that restrain negative supercoils in eukaryotes, it has little sequence specificity (for reviews, see references 11, 19, 36, and 46). However, recent results indicate that certain features of the HU-DNA interaction make it more analogous functionally to the eukaryotic HMG-1 and HMG-2 proteins (34, 41). Like the HMG-1 and HMG-2 proteins, HU bends DNA and binds preferentially to bent or kinked DNA and to DNA in cruciform structures or four-way junctions which at their strand intersections can resemble bent DNA (41, 53). The preferred binding to bent DNA could also explain the recent observation that HU prefers to bind to DNA sequences containing dA tracks (53). The preference for binding bent or angular DNA in four-way junctions also suggests a possible mechanism for restraining DNA supercoils, since crossover points (or nodes) in plectonemic supercoiled DNA can roughly resemble four-way junctions. However, the pattern of DNase protection of DNA complex with HU also indicates a possible wrapping of the DNA on the HU (6). At near saturation, HU binds DNA fragments with a stoichiometry of about one HU dimer per 9 bp; however, there is not enough HU per nucleoid to achieve this stoichiometry in vivo, except perhaps at local sites of preferred binding.
One class of preferred binding site for HU is at regulatory regions of certain genes where HU can bind cooperatively with regulatory proteins. Studies of this process demonstrated that HU can serve as an auxiliary protein that cooperatively facilitates the binding of the lac repressor and the catabolite activator protein (14). The available evidence suggests that HU facilitates short-range structural transitions (such as DNA bends) in the target sequence recognized by the regulatory protein. HU can also function in facilitating other types of protein-DNA interactions including those involved in site-specific recombination and the initiation of DNA replication at oriC (for reviews, see references 11 and 36). These interactions are also believed to occur via structural deformations in the DNA facilitated by the HU.
The integration host factor (IHF) protein has 30% amino acid sequence identity with HU (11); however, the DNA-binding arms of IHF have homologies with the eukaryotic TATA-binding factor or TFIID (32). Like HU, IHF facilitates DNA bending, but unlike HU, it recognizes a specific DNA sequence. For example, IHF binds at the att site of the E. coli chromosome, specifically bends the DNA at that site, and thereby facilitates the interaction of other proteins bound on each side of the bend to promote site-specific recombination of lambda DNA (15, 31, 55). The requirement for an att site can be substituted by a sequence that has an intrinsic DNA bend, in which case IHF is no longer required to facilitate recombination (18). IHF also is involved in the regulation of different genes (usually as a positive regulator) through its specific interaction near other promoters. In some cases, HU and IHF can substitute for one another in in vitro assays, and in some cases, they may also complement mutations in one another.
The H-NS protein is localized in the nucleoid (13), and because it can compact DNA (51), it is apparently also involved in organizing short-range DNA structure. H-NS shows little sequence specificity but, like HU, binds preferentially to bent DNA (5, 58). Mutations in hns affect the expression of many different genes of apparently unrelated function such as bgY, drdX, and osmZ (the gene coding for H-NS itself), and the transcriptional regulation of many other unidentified genes is also affected by the hns deletion mutations (61). Two different models were proposed to explain the action of H-NS. Hulton et al. (23) obtained evidence that the H-NS changes DNA topology and proposed that it affects transcription by modulating levels of negative DNA supercoiling required for transcription. Ueguchi and Mizuno (54) obtained evidence that H-NS functions directly as a transcriptional repressor and interferes with the formation of open promoters.
Genetic studies of E. coli suggest that these nucleoid-associated proteins may have partly overlapping functions (for reviews, see references 11, 36, and 46). Deletion of the hupA and hupB genes coding for HU is not lethal, although these mutants grow slowly, make frequent errors in nucleoid segregation, and are defective in several functions including propagation of certain plasmids and phages. Likewise, mutations that render mutants unable to make functional H-NS or IHF are not lethal. However a triple mutant lacking all three proteins could not be constructed, suggesting that the proteins share a common function (60).
It has been known for many years that no ribosomes can be seen in electron micrographs of nucleoids, although particles of ribosome size are readily visible in the cytoplasm. Indeed, a ribosome-free region of the cell is a commonly used criterion for defining the boundary of the nucleoid (24). Since in bacteria transcription and translation are probably always closely coupled, it seems that little transcription could occur on DNA sequences located deep in the nucleoid. It has therefore been considered likely that transcriptionally active DNA is located near the nucleoid surface or on DNA loops extending from the nucleoid. Early attempts to localize sites of transcription in the nucleoid used electron microscopic autoradiography of sections of bacteria having nascent mRNA pulse-labeled (45). Although the size of autoradiographic tracks limited resolution, quantitative analysis led to the conclusion that transcription occurs on the nucleoid surface.
Later studies used immunogold staining of antibodies directed at either topoisomerase I or RNA polymerase (12, 24). Analysis of thin sections of E. coli cells indicated that both of these proteins are localized primarily on the nucleoid surface. The surface location of RNA polymerase and topoisomerase I (which functions to relax negative DNA supercoils generated during transcription) is compatible with the suggested location of transcription in the surface of the nucleoid. If transcription is indeed localized in this manner, it would require that the nucleoid DNA have a dynamic organization, so that all genes can potentially cycle from the interior onto the surface or into an extruded loop. A dynamic organization was also suggested from earlier studies of nucleoid structure (37, 50).
Immunogold staining of the HU protein also revealed that HU is localized at the nucleoid surface (12). By contrast, similar studies of H-NS showed that this protein is distributed throughout the nucleoid (13), which suggested that H-NS is the major nucleoid protein involved in packaging DNA of the nucleoid interior. These researchers suggested that HU might not be involved in DNA packaging and instead may be primarily bound to nascent RNA at or near the surface. However, immunofluorescence studies of fluorescently tagged HU protein introduced into permeabilized E. coli cells (as in Fig. 2) showed that the added HU bound throughout the nucleoid (47). Thus, it appears that there is no intrinsic restriction on the binding of HU within the nucleoid interior. The significance of these studies to the organization of nucleoid structure is not yet clear, and further research is required to elaborate details of the roles of the major nucleoid proteins.
Fluorescein-tagged proteins that do not bind to DNA (albumin, insulin, etc.) remained primarily in the cytoplasm of permeabilized cells, and little could be detected within the nucleoid volume (Fig. 2). At the concentrations of DNA that exist in the nucleoid, DNA occupies less than 5% of the nucleoid volume, yet proteins that do not bind DNA enter this volume negligibly, if at all. On the other hand, DNA-binding proteins such as HU enter the nucleoid volume quickly, implying a rapid DNA-to-DNA exchange mechanism not available to non-DNA-binding proteins.
The protein-DNA interactions that are responsible for organizing long- and short-range DNA structure in the nucleoid are yet to be clearly elucidated. By analogy with eukaryotic chromosomes, it has been proposed that a topoisomerase II (DNA gyrase) may play a role in restraining DNA loops and defining chromosomal domains (9). This possibility has been reexamined by using pulsed-field gel electrophoresis to resolve large DNA fragments of the nucleoid cut in vivo at sites where DNA gyrase is bound (7, 50). It was found that sites are grouped in 5- to 10-kb clusters that are mostly 50 to 100 kb apart. Thus, the average separation is similar to the average domain size in E. coli, which led to the speculation that DNA gyrase may play a role in defining chromosomal domains. A model like this requires that the nucleoid DNA have regular repeated DNA sequences that are recognized by the proteins that define the chromosomal domains. The REP (or PU) sequences are candidate recognition elements (16, 52). They are extragenic palindromic sequences of roughly 30 bp repeated hundreds of times in the chromosomes of both E. coli and S. typhimurium. The sequences form about 0.5% of the total chromosomal DNA and are distributed throughout the chromosome. Although several different functions have been proposed for REP, it is known that DNA gyrase specifically recognizes REP and that the recognition is stimulated by HU protein (59). Although the required specificity is there to support the above model, the situation is still uncertain. For example, there are many more REP sites than bound DNA gyrase complexes in vivo and the sites that are bound by gyrase are strongly influenced by nearby transcription (7). Further research will be required to develop the model.
While it is apparent that much has been learned about the nucleoid, it is also evident that the fundamental interactions organizing the structure of DNA in the nucleoid are still to be clearly defined. The available information reviewed above indicates that the final story will involve a set of interactions. The different DNA-bending proteins which are involved in organizing short-range structure seem to have partly overlapping functions, and there are several different players in this system. The molecular interactions organizing long-range structure are even less clear but seem to involve multiple kinds of interactions. The eventual solution of these intriguing mysteries about nucleoid structure will certainly stimulate our overall understanding of the storage and expression of genetic information.
This work was supported by research grant GM 18243 from the U.S. National Institutes of Health. I also acknowledge support from the Lucille P. Markey Charitable Trust.
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