Harvard University - Department of Molecular & Cellular Biology

HOW DOES E. COLI SEGREGATE ITS SISTERS WITHOUT A SPINDLE? [KLECKNER LAB]

by Beth Weiner and Nancy Kleckner

April 26th, 2013

(l to r) Guillaume Witz, Mara Prentiss, Nancy Kleckner, Beth Weiner, and Jay K. Fisher

The E. coli chromosome is a single circular unit which, if stretched out, would be 150 millimeters long that is somehow contained within a cylindrical cell of micron-scale dimensions.  Critical unanswered questions for bacterial researchers are: (i) whether/how the chromosome is organized; and (ii) how the genome is replicated and segregated into two separate domains in a coordinated and timely manner.  Sister segregation is particularly mysterious since bacteria lack a eukaryotic-like spindle apparatus. Fisher et al.  address these issues by high resolution 4D imaging of the E.coli nucleoid throughout the cell cycle.  Their observations reveal a defined organization, short- and long-time scale dynamics and specific features that point to a unique mechanical model for bacterial sister segregation.  This model potentially has broad implications for all types of bacteria and for sister segregation during evolving life.

Previous studies of nucleoid organization and structure have been limited by technical constraints. Jay Fisher and his colleagues developed an imaging method that combines high spatial resolution and high temporal resolution. Bacteria whose nucleoids were defined by at fluorescently tagged version of the general nucleoid-associated protein HU were inserted into microfluidic channels that ‘hugged’ them in such a way as to keep them immobile while permitting constant flow of growth medium. Nucleoids could thus be imaged in three-dimensions by epi-fluorescence microscopy, with Z-stacks acquired in less than 2 sec and with intervals between each stack as short as 5 sec.  Additional markers permitted the definition of nucleoid state and dynamics at defined times throughout a previously well-characterized cell cycle.

This analysis reveals that E.coli nucleoids are well-defined ellipsoids that are deformed into a variety of helix-like forms. Three basic features emerge. (i) Nucleoid density efficiently coalesces into longitudinal bundles, giving a stiff, low DNA density ellipsoid. (ii) This ellipsoid is radially confined within the cell cylinder.  Radial confinement gives the helical shape.  Moreover, the tendency for minimization of radial confinement drives and directs global nucleoid dynamics, including sister segregation.  (iii) Longitudinal density waves flux back and forth along the nucleoid, over short (5 sec) and long (10 min) enhancing internal nucleoid mobility.  Furthermore, sisters separate end-to-end in sequential discontinuous pulses. Pulses occur at 20min intervals, at defined cell cycle times.  This progression is mediated by sequential installation and release of programmed tethers, implying cyclic accumulation and relief of intra-nucleoid mechanical stress. The observed effects could comprise chromosome-based cell cycle engine.


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