The Bacterial Nucleoid: From Electron Microscopy to Polymer Physics-A Personal Recollection.

In the 1960s, electron microscopy did not provide a clear answer regarding the compact or dispersed organization of the bacterial nucleoid. This was due to the necessary preparation steps of fixation and dehydration (for embedding) and freezing (for freeze-fracturing). Nevertheless, it was possible to measure the lengths of nucleoids in thin sections of slow-growing Escherichia coli cells, showing their gradual increase along with cell elongation. Later, through application of the so-called agar filtration method for electron microscopy, we were able to perform accurate measurements of cell size and shape. The introduction of confocal and fluorescence light microscopy enabled measurements of size and position of the bacterial nucleoid in living cells, inducing the concepts of "nucleoid occlusion" for localizing cell division and of "transertion" for the final step of nucleoid segregation. The question of why the DNA does not spread throughout the cytoplasm was approached by applying polymer-physical concepts of interactions between DNA and proteins. This gave a mechanistic insight in the depletion of proteins from the nucleoid, in accordance with its low refractive index observed by phase-contrast microscopy. Although in most bacterial species, the widely conserved proteins of the ParABS-system play a role in directing the segregation of newly replicated DNA strands, the basis for the separation and opposing movement of the chromosome arms was proposed to lie in preventing intermingling of nascent daughter strands already in the early replication bubble. E. coli, lacking the ParABS system, may be suitable for investigating this basic mechanism of DNA strand separation and segregation.

Different Amounts of DNA in Newborn Cells of Escherichia coli Preclude a Role for the Chromosome in Size Control According to the "Adder" Model.

According to the recently-revived adder model for cell size control, newborn cells of Escherichia coli will grow and divide after having added a constant size or length, ΔL, irrespective of their size at birth. Assuming exponential elongation, this implies that large newborns will divide earlier than small ones. The molecular basis for the constant size increment is still unknown. As DNA replication and cell growth are coordinated, the constant ΔL could be based on duplication of an equal amount of DNA, ΔG, present in newborn cells. To test this idea, we measured amounts of DNA and lengths of nucleoids in DAPI-stained cells growing in batch culture at slow and fast rates. Deeply-constricted cells were divided in two subpopulations of longer and shorter lengths than average; these were considered to represent large and small prospective daughter cells, respectively. While at slow growth, large and small prospective daughter cells contained similar amounts of DNA, fast growing cells with multiforked replicating chromosomes, showed a significantly higher amount of DNA (20%) in the larger cells. This observation precludes the hypothesis that ΔL is based on the synthesis of a constant ΔG. Growth curves were constructed for siblings generated by asymmetric division and growing according to the adder model. Under the assumption that all cells at the same growth rate exhibit the same time between initiation of DNA replication and cell division (i.e., constant C+D-period), the constructions predict that initiation occurs at different sizes (Li) and that, at fast growth, large newborn cells transiently contain more DNA than small newborns, in accordance with the observations. Because the state of segregation, measured as the distance between separated nucleoids, was found to be more advanced in larger deeply-constricted cells, we propose that in larger newborns nucleoid separation occurs faster and at a shorter length, allowing them to divide earlier. We propose a composite model in which both differential initiation and segregation leads to an adder-like behavior of large and small newborn cells.

Does the eclipse limit bacterial nucleoid complexity and cell width?

Cell size of bacteria M is related to 3 temporal parameters: chromosome replication time C, period from replication-termination to subsequent division D, and doubling time τ. Steady-state, bacillary cells grow exponentially by extending length L only, but their constant width W is larger at shorter τ's or longer C's, in proportion to the number of chromosome replication positions n (= C/τ), at least in Escherichia coli and Salmonella typhimurium. Extending C by thymine limitation of fast-growing thyA mutants result in continuous increase of M, associated with rising W, up to a limit before branching. A set of such puzzling observations is qualitatively consistent with the view that the actual cell mass (or volume) at the time of replication-initiation Mi (or Vi), usually relatively constant in growth at varying τ's, rises with time under thymine limitation of fast-growing, thymine-requiring E. coli strains. The hypothesis will be tested that presumes existence of a minimal distance lmin between successive moving replisomes, translated into the time needed for a replisome to reach lmin before a new replication-initiation at oriC is allowed, termed Eclipse E. Preliminary analysis of currently available data is inconsistent with a constant E under all conditions, hence other explanations and ways to test them are proposed in an attempt to elucidate these and other results. The complex hypothesis takes into account much of what is currently known about Bacterial Physiology: the relationships between cell dimensions, growth and cycle parameters, particularly nucleoid structure, replication and position, and the mode of peptidoglycan biosynthesis. Further experiments are mentioned that are necessary to test the discussed ideas and hypotheses.

Compaction of isolated Escherichia coli nucleoids: Polymer and H-NS protein synergetics.

Escherichia coli nucleoids were compacted by the inert polymer polyethylene glycol (PEG) in the presence of the H-NS protein. The protein by itself appears to have little impact on the size of the nucleoids as determined by fluorescent microscopy. However, it has a significant impact on the nucleoidal collapse by PEG. This is quantitatively explained by assuming the H-NS protein enhances the effective diameter of the DNA helix leading to an increase in the depletion forces induced by the PEG. Ultimately, however, the free energy of the nucleoid itself turns out to be independent of the H-NS concentration. This is because the enhancement of the supercoil excluded volume is negligible. The experiments on the nucleoids are corroborated by dynamic light scattering and EMSA analyses performed on DNA plasmids in the presence of PEG and H-NS.

Chromosome replication, cell growth, division and shape: a personal perspective.

The origins of Molecular Biology and Bacterial Physiology are reviewed, from our personal standpoints, emphasizing the coupling between bacterial growth, chromosome replication and cell division, dimensions and shape. Current knowledge is discussed with historical perspective, summarizing past and present achievements and enlightening ideas for future studies. An interactive simulation program of the bacterial cell division cycle (BCD), described as "The Central Dogma in Bacteriology," is briefly represented. The coupled process of transcription/translation of genes encoding membrane proteins and insertion into the membrane (so-called transertion) is invoked as the functional relationship between the only two unique macromolecules in the cell, DNA and peptidoglycan embodying the nucleoid and the sacculus respectively. We envision that the total amount of DNA associated with the replication terminus, so called "nucleoid complexity," is directly related to cell size and shape through the transertion process. Accordingly, the primary signal for cell division transmitted by DNA dynamics (replication, transcription and segregation) to the peptidoglycan biosynthetic machinery is of a physico-chemical nature, e.g., stress in the plasma membrane, relieving nucleoid occlusion in the cell's center hence enabling the divisome to assemble and function between segregated daughter nucleoids.

Segregation of chromosome arms in growing and non-growing Escherichia coli cells.

In slow-growing Escherichia coli cells the chromosome is organized with its left (L) and right (R) arms lying separated in opposite halves of the nucleoid and with the origin (O) in-between, giving the pattern L-O-R. During replication one of the arms has to pass the other to obtain the same organization in the daughter cells: L-O-R L-O-R. To determine the movement of arms during segregation six strains were constructed carrying three colored loci: the left and right arms were labeled with red and cyan fluorescent-proteins, respectively, on loci symmetrically positioned at different distances from the central origin, which was labeled with green-fluorescent protein. In non-replicating cells with the predominant spot pattern L-O-R, initiation of replication first resulted in a L-O-O-R pattern, soon changing to O-L-R-O. After replication of the arms the predominant spot patterns were, L-O-R L-O-R, O-R-L R-O-L or O-L-R L-O-R indicating that one or both arms passed an origin and the other arm. To study the driving force for these movements cell growth was inhibited with rifampicin allowing run-off DNA synthesis. Similar spot patterns were obtained in growing and non-growing cells, indicating that the movement of arms is not a growth-sustained process, but may result from DNA synthesis itself. The distances between loci on different arms (LR-distances) and between duplicated loci (LL- or RR-distances) as a function of their distance from the origin, indicate that in slow-growing cells DNA is organized according to the so-called sausage model and not according to the doughnut model.

Physical manipulation of the Escherichia coli chromosome reveals its soft nature.

Replicating bacterial chromosomes continuously demix from each other and segregate within a compact volume inside the cell called the nucleoid. Although many proteins involved in this process have been identified, the nature of the global forces that shape and segregate the chromosomes has remained unclear because of limited knowledge of the micromechanical properties of the chromosome. In this work, we demonstrate experimentally the fundamentally soft nature of the bacterial chromosome and the entropic forces that can compact it in a crowded intracellular environment. We developed a unique "micropiston" and measured the force-compression behavior of single Escherichia coli chromosomes in confinement. Our data show that forces on the order of 100 pN and free energies on the order of 10(5) k(B)T are sufficient to compress the chromosome to its in vivo size. For comparison, the pressure required to hold the chromosome at this size is a thousand-fold smaller than the surrounding turgor pressure inside the cell. Furthermore, by manipulation of molecular crowding conditions (entropic forces), we were able to observe in real time fast (approximately 10 s), abrupt, reversible, and repeatable compaction-decompaction cycles of individual chromosomes in confinement. In contrast, we observed much slower dissociation kinetics of a histone-like protein HU from the whole chromosome during its in vivo to in vitro transition. These results for the first time provide quantitative, experimental support for a physical model in which the bacterial chromosome behaves as a loaded entropic spring in vivo.

Characterization of Escherichia coli nucleoids released by osmotic shock.

Nucleoids were isolated by osmotic shock from Escherichia coli spheroplasts at relatively low salt concentrations and in the absence of detergents. Sucrose-protected cells, made osmotically sensitive by growth in the presence of ampicillin or by digestion with low lysozyme concentrations (50-5 µg/ml), were shocked by 100-fold dilution of the sucrose buffer. Liberated nucleoids stained with 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI), the dimeric cyanine dye TOTO-1, or fluorescent DNA-binding protein appeared as cloud-like structures, in the absence of phase contrast. Because UV-irradiation disrupted the DAPI-stained nucleoids within 5-10 s, they were imaged by time-lapse microscopy with exposure times less than 2 s. The volume of nucleoids isolated from ampicillin- or low-lysozyme spheroplasts and minimally exposed to UV (<2 s) was on average ∼42 µm(3). Lysozyme at concentrations above 1 µg/ml in the lysate compacted the nucleoids. Treatment with protease E or K (20-200 µg/ml) and sodium dodecyl sulfate (SDS; 0.001-0.01%) caused a twofold volume increase and showed a granular nucleoid at the earliest UV-exposure; the expansion could be reversed with 50 µM ethidium bromide, but not with chloroquine. While DNase (1 µg/ml) caused a rapid disruption of the nucleoids, RNase (0.1-400 µg/ml) had no effect. DAPI-stained nucleoids treated with protease, SDS or DNase consisted of granular substructures at the earliest exposure similar to UV-disrupted nucleoids obtained after prolonged (>4 s) UV irradiation. We interpret the measured volume in terms of a physical model of the nucleoid viewed as a branched DNA supercoil crosslinked by adhering proteins into a homogeneous network.

Structural and physical aspects of bacterial chromosome segregation.

Microscopic observations on the bacterial nucleoid suggest that the chromosome occurs in the cell as a compact nucleoid phase separate from the cytoplasm. Physical theory likewise predicts a phase separation, taking into consideration DNA supercoiling, nucleoid-binding proteins, and excluded-volume interactions between DNA and cytoplasmic proteins. Specific DNA loci, visualized as oriC-GFP spots in the densely packed nucleoid, exhibit a very low diffusion coefficient indicating that they are virtually immobile and may primarily be moved by overall length growth. Such gradual movement could be effectuated by replication, transertion (combined transcription, translation, and insertion of proteins), and actin- (MreB) directed surface synthesis. Differences in the movement and positioning of gene loci between Escherichia coli and Caulobacter crescentus are discussed. We propose that a low diffusion coefficient could explain the linear positioning of genes in the nucleoid and that differential transcriptional activity could induce different mobilities between either replichores (E. coli) or daughter strands (C. crescentus). The transertion process, possibly in combination with MreB cytoskeletal tracks, could overcome the compaction forces and move specific chromosomal regions and the nucleoid as a whole without invoking a dedicated mechanism.

Single-particle tracking of oriC-GFP fluorescent spots during chromosome segregation in Escherichia coli.

DNA regions close to the origin of replication were visualized by the green fluorescent protein (GFP)-Lac repressor/lac operator system. The number of oriC-GFP fluorescent spots per cell and per nucleoid in batch-cultured cells corresponded to the theoretical DNA replication pattern. A similar pattern was observed in cells growing on microscope slides used for time-lapse experiments. The trajectories of 124 oriC-GFP spots were monitored by time-lapse microscopy of 31 cells at time intervals of 1, 2, and 3 min. Spot positions were determined along the short and long axis of cells. The lengthwise movement of spots was corrected for cell elongation. The step sizes of the spots showed a Gaussian distribution with a standard deviation of approximately 110 nm. Plots of the mean square displacement versus time indicated a free diffusion regime for spot movement along the long axis of the cell, with a diffusion coefficient of 4.3+/-2.6x10(-5) microm2/s. Spot movement along the short axis showed confinement in a region of the diameter of the nucleoid ( approximately 800 nm) with an effective diffusion coefficient of 2.9+/-1.7x10(-5) microm2/s. Confidence levels for the mean square displacement analysis were obtained from numerical simulations. We conclude from the analysis that within the experimental accuracy--the limits of which are indicated and discussed--there is no evidence that spot segregation requires any other mechanism than that of cell (length) growth.

Is Escherichia coli getting old?

Whether or not bacteria divide symmetrically, the inheritance of cell poles is always asymmetrical. Because each cell carries an old and a new pole, its daughters will not be the same. Tracking poles of cells and measuring their lengths and doubling times in micro-colonies, Stewart et al.1 observed that growth rate diminished in cells inheriting old poles and concluded that these cells are susceptible to aging. Here, their results are compared with studies on the variabilities of length and age at division. It is argued that the decreased growth rate in old pole cells falls within the expected variation and may therefore be sufficiently far from a catastrophe-like cell death through aging.

Restricted diffusion of DNA segments within the isolated Escherichia coli nucleoid.

To study the dynamics and organization of the DNA within isolated Escherichia coli nucleoids, we track the movement of a specific DNA region. Labeling of such a region is achieved using the Lac-O/Lac-I system. The Lac repressor-GFP fusion protein binds to the DNA section where tandem repeats of the Lac operator are inserted, which allows us to monitor the motion of the DNA. The movement of such a GFP spot is followed at 48 ms temporal resolution during 12s. The spots are found to diffuse within a confined space, so that the nucleoid appears to behave like a viscoelastic network. The distribution of the "particle" position in time can be fitted to a Gaussian function indicating that the motion of the particle is Brownian. An average self-diffusion constant Ds=0.12 microm(2) s-1 is derived via the time auto-correlation functions of the displacement and is compatible with the collective diffusion coefficient measured previously by dynamic light scattering. Restriction of a DNA sequence to a small region of the nucleoid is tentatively related to the existence of so-called supercoiling domains.

Localizing cell division in spherical Escherichia coli by nucleoid occlusion.

The spatial relationship between FtsZ localization and nucleoid segregation was followed in Escherichia coli thyA cells, made spheroidal by brief exposure to mecillinam and after manipulating chromosome replication time using changes ('steps') in thymine concentration [Zaritsky et al., Microbiology 145 (1999) 1015-1022]. In such cells, fluorescent FtsZ-GFP arcs did not overlap the DAPI-stained nucleoids. It is concluded that FtsZ rings are deposited between segregating nucleoids, consistent with the nucleoid occlusion model [Woldringh et al., J. Bacteriol. 176 (1994) 6030-6038].

DNA supercoiling by gyrase is linked to nucleoid compaction.

The genes of E. coli are located on a circular chromosome of 4.6 million basepairs. This 1.6 mm long molecule is compressed into a nucleoid to fit inside the 1-2 microm cell in a functional format. To examine the role of DNA supercoiling as nucleoid compaction force we modulated the activity of DNA gyrase by electronic, genetic, and chemical means. A model based on physical properties of DNA and other cell components predicts that relaxation of supercoiling expands the nucleoid. Nucleoid size did not increase after reduction of DNA gyrase activity by genetic or chemical means, but nucleoids did expand upon chemical inhibition of gyrase in chloramphenicol-treated cells, indicating that supercoiling may help to compress the genome.

The role of co-transcriptional translation and protein translocation (transertion) in bacterial chromosome segregation.

Many recent reviews in the field of bacterial chromosome segregation propose that newly replicated DNA is actively separated by the functioning of specific proteins. This view is primarily based on an interpretation of the position of fluorescently labelled DNA regions and proteins in analogy to the active segregation mechanism in eukaryotic cells, i.e. to mitosis. So far, physical aspects of DNA organization such as the diffusional movement of DNA supercoil segments and their interaction with soluble proteins, leading to a phase separation between cytoplasm and nucleoid, have received relatively little attention. Here, a quite different view is described taking into account DNA-protein interactions, the large variation in the cellular position of fluorescent foci and the compaction and fusion of segregated nucleoids upon inhibition of RNA or protein synthesis. It is proposed that the random diffusion of DNA supercoil segments is transiently constrained by the process of co- transcriptional translation and translocation (transertion) of membrane proteins. After initiation of DNA replication, a bias in the positioning of transertion areas creates a bidirectionality in chromosome segregation that becomes self-enhanced when neighbouring genes on the same daughter chromosome are expressed. This transertion-mediated segregation model is applicable to multifork replication during rapid growth and to multiple chromosomes and plasmids that occur in many bacteria.

Varying division planes of secondary constrictions in spheroidal Escherichia coli cells.

Planes of successive divisions in Escherichia coli have been proposed to be either parallel or perpendicular to each other, restricted to one or two dimensions. To test the hypothesis that divisions can occur in planes alternating in three dimensions, a method was developed to generate cells with secondary constrictions during growth in suspension. The method involves a combination of thymine limitation (to manipulate chromosome replication rate) and mecillinam treatment (to inhibit penicillin-binding protein 2). The former modifies timing of terminations, the latter results in spheroidal cells. Such cells displayed secondary constrictions after adding deoxyguanosine (accelerating replication rate), thus temporarily enhancing division signals. The successive constrictions were seen to develop in planes that were tilted relative to each other, and in positions related to those of the nucleoids, visualized by staining with DAPI (4',6-diamidino-2-phenylindole dihydrochloride hydrate). Visualizing cell envelopes with FM 4-64 by confocal scanning laser microscopy supported the conclusion that planes of successive divisions can alternate in three dimensions.