Characterizing ParA2, an essential protein for Vibrio cholerae chromosome segregation.
Partitioning, or par, loci were initially identified on low copy plasmids as elements
essential for plasmid segregation and inheritance. However, it is now apparent that many
bacterial chromosomes harbor par loci as well. The role of chromosomal par loci remains
incompletely understood, but their homology to plasmid par loci has led to speculation
that chromosomal par loci may play ... read morea key role in mediating chromosome segregation. The
genome of Vibrio cholerae, the causative agent of cholera, is divided between two
chromosomes, each of which encodes its own par locus. Prior studies have shown that the
parAB2 locus is essential for the proper segregation of chromosome II. In order to begin
to elucidate the molecular mechanisms by which parAB2 mediates V. cholerae chromosome II
segregation, I focused on ParA2, the protein thought to drive the partitioning process.
In vitro, I found that ParA2 shares biochemical characteristics with other members of
the ParA family of partitioning proteins. However, unlike most ParA family proteins,
which polymerize in the presence of ATP, ParA2 only formed higher ordered filamentous
structures on DNA. Three-dimensional reconstructions of these filaments revealed that
ParA2 binds DNA to form unusual left-handed nucleoprotein filaments that are bipolar and
symmetric. These filaments do not distort the DNA helix bound in the interior.
Subsequent in vivo studies using ParA2 fluorescent protein fusions showed that this
partitioning protein is dynamic and nucleoid associated. Strikingly, ParA2 is often
found between a ParB2-parS2 centromere complex and the cell pole towards which that
complex is moving. Together, my findings suggest that ParA2 forms bipolar filaments over
the nucleoid and utilizes a pulling mechanism to mediate partitioning of chromosome
Thesis (Ph.D.)--Tufts University, 2011.
Submitted to the Dept. of Molecular Microbiology.
Advisor: Matthew Waldor.
Committee: Ekaterina Heldwein, Andrew Wright, Linc Sonenshein, Ralph Isberg, and David Rudner.
Keyword: Microbiology.read less