Bacterial DNA segregation by dynamic SopA polymers

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Bacterial DNA segregation by dynamic SopA polymers

• Grace E. Lim et al
• PNAS 2005

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abstract

• Many bacterial plasmids and chromosomes rely on
  ParA ATPase for proper positioning within the
  cell and for efficient segregation to daughter
  cells.
• The authors demonstrate that the F-plasmid
  partitioning protein SopA polymerizes into
  filaments in an ATP-dependent manner in vitro,
  and that the filaments elongate at a rate that is
  similar to that of plasmid separation in vivo.

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• The authors show that SopA is a dynamic protein
  within the cell, undergoing cycles of
  polymerization and depolymerization, and
  shuttling back and forth between nucleoprotein
  complexes that are composed of the SopB protein
  bound to SopC-containing plasmids.
• The dynamic behavior of SopA is critical for
  Sop-mediated plasmid DNA segregation .
• The authors also show that SopA colocalizes with
  SopB/SopC in the cell and that SopB/SopC
  nucleates the assembly of SopA and is required
  for its dynamic behavior .

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• The authors propose a mechanism in which plasmid
  separation is driven by the polymerization of
  SopA , and they speculate that the radial
  assembly of SopA polymers is responsible for
  positioning plasmids both before and after
  segregation.

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introduction

• Chromosome segregation in eukaryotes is mediated
  by dynamic proteins such as tubulin, which
  polymerize into filamentous structures that are
  capable of rapid reorganization by means of
  cycles of depolymerization and repolymerization.
• Although much less is understood about the
  mechanism of DNA segregation in bacteria, dynamic
  bacterial actin homologues have recently been
  shown to play an important role in the process.

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• One of the first DNA segregation systems to be
  identified in E. coli was the F plasmid Sop
  system, which consists of two protein
  components, SopA and SopB, and a cis-acting DNA
  sequence, sopC.
• Plasmids containing different ParA systems
  localize to distinct midcell positions and
  separate at different times, suggesting that
  different Par systems function independently from
  each other for both positioning and separation.

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• A distant ParA relative, ParF of plasmid TP228,
  was recently shown to assemble into polymers in
  vitro, suggesting that, as in the case of the
  actin-like ParM, polymerization might provide the
  driving force for plasmid segregation.

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Result 1

• Purified SopA Assembles into Polymers in Vitro.

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SopA was tagged at its N terminus with
hexahistidine and purified over a nickel affinity
gel column. When incubated with ATP, SopA
polymerized into long filaments, a process that
could be visualized in the fluorescence
microscope when the protein was stained with the
nonspecific stain Nile red.
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The distance between the ends of six growing
filaments were measured. The average elongation
rate for the six filaments is 0.18µm/min. These
rates are similar to those at which bacterial
plasmids and chromosomal DNA have been observed
to separate in vivo during segregation.
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• These findings suggest that SopA polymerization
  might be an integral part of the mechanism by
  which plasmid DNA is segregated within the cell.

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Result 2

• SopA-GFP Assembles into Polymers Within the Cell.

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When expressed in the presence of all three sop
components, SopA-GFP gave rise to a complex
pattern of intracellular fluorescence. Most cells
contained discrete foci (60 Fig. 2B) or a
nonuniform haze of fluorescence (35 Fig. 2 C
and D).
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• The haze typically took the form of a symmetric
  midcell peak that dropped off in intensity toward
  the two poles (Fig. 2C) or a quarter-cell peak
  with an asymmetric haze that extended toward the
  opposite pole (Fig. 2D). Some cells contained two
  foci connected by a central haze(Fig. 2E).

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The authors monitored these F cells with
time-lapse microscopy. In nearly all of the
cells, SopA-GFP oscillated between the
quarter-cell positions with a period of 20 min .
This oscillation might correspond to cycles of
SopA polymerization and depolymerization.
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Result 3

• SopA Colocalizes with SopB in the Cell.

The authors found that 94 of the SopA-GFP foci
were coincident with SopB-CFP foci. This finding
demonstrated that SopA-GFP does assemble at the
plasmid and that SopA and SopB likely interact at
the plasmid in vivo.
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Result 4

• SopB and sopC Nucleate SopA Assembly in Vivo.

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Result 5

• SopB Nucleates Assembly of SopA into Radial
  Asters in Vitro.

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Result 6

• Identification of a SopA Mutant Affecting
  Polymerization and Segregation.

In the course of constructing SopA-GFP, the
authors isolated a mutant (SopA1-GFP) that formed
long filaments in most cells even in the absence
of the other sop genes.
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• FRAP was therefore used to determine the rate at
  which subunits within the SopA1-GFP filaments
  exchanged with subunits from the cytoplasmic
  pool.

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SopA1 was able to trap the ordinarily dynamic
wild-type SopA into static filaments.
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A shift from dynamic to static SopA assembly
disrupts the function of the Sop plasmid
segregation system.
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New Model
Proposed mechanism for plasmid segregation as
mediated by the Sop system.
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Thanks!