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.
Elmore
Steven
S
Section Molecular Cytology, Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, The Netherlands.
Müller
Michiel
M
Vischer
Norbert
N
Odijk
Theo
T
Woldringh
Conrad L
CL
D000465:Algorithms; D020090:Chromosome Segregation; D002876:Chromosomes, Bacterial; D004261:DNA Replication; D004269:DNA, Bacterial; D004058:Diffusion; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000502:physiology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D007211:Indoles; D007763:Lac Operon; D018715:Microscopy, Video; D051738:Origin Recognition Complex; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D007211:Indoles; C114650:OriC chromosomal replication origin; D051738:Origin Recognition Complex; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C007293:DAPI