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.chromosome segregation | depletion forces | polymer physics | mother machine | optical trap

Summary 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 segre‐gation that becomes self‐enhanced when neigh‐bouring 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.

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SummaryBacterial membrane and nucleoids were stained concurrently by the lipophilic styryl dye FM 4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl) hexatrienyl)pyridinium dibromide] and 4Ј,6-diamidino-2-phenylindole (DAPI), respectively, and studied using fluorescence microscopy imaging. Observation of plasmolysed cells indicated that FM 4-64 stained the inner membrane preferentially. In live Escherichia coli pbpB cells and filaments, prepared on wet agar slabs, an FM 4-64 staining pattern developed in the form of dark bands. In dividing cells, the bands occurred mainly at the constriction sites and, in filaments, between partitioning nucleoids. The FM 4-64 pattern of dark bands in filaments was abolished after inhibiting protein synthesis with chloramphenicol. It is proposed that the staining patterns reflect putative membrane domains formed by DNA-membrane interactions and have functional implications in cell division.

The positioning of constrictions in Escherichia coli filaments pinching off anucleate cells was analyzed by fluorescence microscopy of dnaX(Ts), dnaX(Ts) sfiA, dnaA46(Ts), gyrA(Am) supF(Ts), and gyrB(Ts) mutants. In filaments with actively replicating nucleoids, constrictions were positioned close to the nucleoid, whereas in nonreplicating filaments, positioning of constrictions within the anucleate region was nearly random. We conclude that constriction positioning depends in an unknown way on nucleoid replication activity.Cell division in Escherichia coli involves the tight coordination in time and space of the processes of cell growth, DNA replication, and cell constriction. Concomitant with the increase in cell mass and DNA replication, the daughter chromosomes are segregated into the cell halves. After termination of DNA replication and completion of segregation, the cell is constricted between the nucleoids in the cell center. Constriction requires discontinuation of enzyme activity for cell wall synthesis at the constriction site (1,21). This may be achieved either by local activation of enzymes or by positioning of specific enzymes at the constriction site. Although termination of DNA replication has been suggested to signal the cell to initiate a constriction (11), the mechanism is still not well understood. So far, factors that determine the positioning of a constriction site have not been identified.According to the concept of zonal growth in bacterial cells (5,8), the site of constriction is determined by growth zones that occur in the lateral cell wall. It has also been suggested that "zones of adhesion" between the cell membrane and the peptidoglycan layer may be involved in the positioning of constrictions (16). Both ideas imply that constriction sites are predetermined within the cell envelope in such a way that the specific enzymes for constriction are concentrated at potential division sites, which are placed at regular distances from the cell pole. Experiments with cells carrying the temperature-sensitive DNA initiation mutation dnaA46(Ts) have been interpreted in terms of predetermined division sites (2). At the restrictive temperature, the SOS response and the related cell division inhibition are not induced in this mutant (for a review, see reference 12), which thus continues to divide, pinching off DNA-less cells. At 42°C, the dnaA46(Ts) mutant was reported to pinch off DNA-less cells of uniform length similar to the newborn cell length (5, 10), suggesting that the cells can measure the distance between constriction and pole.Contrary to the results of Hirota et al. (5), observations on other DNA-less cell-forming mutants suggest the absence of regularly spaced, predetermined constriction sites in the lateral cell wall. For instance, the dnaB mutation, by which DNA replication and constriction initiation are also uncoupled, produces DNA-less cells that are not uniform in size (7). Anucleate cells with a broad range of cell lengths are also pinched off from filaments of the dnaX(Ts) mutant r...