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Conrad L Woldringh

Publications and source records attributed to Conrad L Woldringh.

10 recordsLinked to original sources
PubMed2006

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.

Chromosome Segregation↗
PubMed2005

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.

Bacterial Physiological Phenomena↗
PubMed2005

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.

Chromosomes, Bacterial↗
PubMed2005

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.

Algorithms↗
PubMed2003

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].

Bacterial Proteins↗
PubMed2002

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.

DNA Gyrase↗
PubMed2002

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.

Bacteria↗
PubMed1999

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.

Cell Division↗