Compaction and Segregation of DNA in .

Theoretical and experimental approaches have been applied to study the polymer physics underlying the compaction of DNA in the bacterial nucleoid. Knowledge of the compaction mechanism is necessary to obtain a mechanistic understanding of the segregation process of replicating chromosome arms (replichores) during the cell cycle. The first part of this review discusses light microscope observations demonstrating that the nucleoid has a lower refractive index and thus, a lower density than the cytoplasm.

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 cells, showing their gradual increase along with cell elongation.

Different Amounts of DNA in Newborn Cells of 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 will grow and divide after having added a constant size or length, , 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.

Does the eclipse limit bacterial nucleoid complexity and cell width?

Cell size of bacteria is related to 3 temporal parameters: chromosome replication time , period from replication-termination to subsequent division , and doubling time . Steady-state, bacillary cells grow exponentially by extending length only, but their constant width is larger at shorter 's or longer 's, in proportion to the number of chromosome replication positions (= /), at least in and . Extending by of result in continuous increase of , associated with rising , up to a limit before branching.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).