DNA segregation in prokaryotes
While the biochemical mechanisms underlying bacterial
DNA replication and repair have been elucidated in molecular detail,
comparatively little progress has been made toward understanding the
mechanism of bacterial chromosome segregation. Even less is known
about DNA segregation mechanisms in the Archaea, where genome sequencing
efforts have far outpaced molecular or cell biological studies of
chromosome replication and segregation. Our overall research goals
are to elucidate and compare DNA segregation mechanisms of phylogenetically
diverse prokaryotic species. Specifically, we seek to understand:
(1) the partitioning mechanisms of the broad host range antibiotic
resistance plasmids of Gram-negative bacteria such as Escherichia
coli and Pseudomonas aeruginosa; (2) the mechanisms by which plasmids
are partitioned into spores of the Gram-positive species Bacillus
anthracis and Bacillus subtilis; and (3) the partitioning mechanisms
of chromosomal and plasmid DNA in the extreme halophilic Archaea Haloferax
volcanii. To achieve these goals, we utilize a multidisciplinary approach,
with significant emphasis on developing and exploiting cell biological
methods to address detailed questions about the mechanisms governing
the dynamic behavior of DNA molecules within prokaryotic cells.
Segregation of Bacillus virulence plasmids during growth and
sporulation
The striking differences between strains of Bacillus
anthracis, Bacillus thuringiensis, and Bacillus cereus are encoded
by the unique plasmids each carries which render it either a pathogen
of mammals, insects, or an opportunistic pathogen. The infectious
particle of each of these agents is the spore, so the efficient packaging
of plasmid DNA into spores is a key step in the inheritance of pathogenicity.
However, sporulation presents a formidable challenge for the stable
inheritance of large, low copy number plasmids, since the asymmetrically-positioned
cell division event at the onset of sporulation produces daughter
cells that differ in volume by at least 10 fold. This large difference
in volume precludes random diffusion as a possible segregation mechanism
for the low copy number virulence plasmids, which nonetheless are
rarely lost from the population. Therefore, the stable inheritance
of these virulence plasmids demands an ability to be efficiently partitioned
during both vegetative growth and sporulation. Our goal is to identify
these partitioning systems by developing new tools for the study of
plasmid segregation during B. subtilis growth and sporulation, and
then apply these tools to other Bacillus species.
DNA replication and segregation in Haloferax volcanii
Very little is known about DNA segregation or cell
division in any species of Archaea. While most of the proteins involved
in DNA replication are either unique or homologous to eukaryotic proteins,
a report on the first identified replication origin (in Pyrococcus
abyssi) suggests that the mode of replication is bacterial, with bidirectional
replication initiating from a single origin. Interestingly, many Archaea
contain histones and histone-modifying enzymes such as acetyl transferases
like eukaryotes, however their cell division apparatus (where identified)
is distinctly bacterial, and relies upon the cell division protein
FtsZ. This blend of eukaryal and bacterial features allows interesting
questions with potentially profound evolutionary implications to be
addressed concerning the mechanisms of DNA replication and segregation.
For example, are the plasmid and chromosome segregation machineries
of Archaea similar to Bacteria or Eucarya, or are they entirely unique?
The extreme halophile Haloferax volcanii is one
of the few convenient model organisms which is easy to grow in the
laboratory and has well developed genetic tools. A complete cosmid
library has been constructed covering the entire genome and the genome
will be sequenced this year. We have recently begun an effort to understand
chromosome and plasmid segregation in H. volcanii.
We are currently addressing basic questions about
DNA replication and the H. volcanii cell cycle, such as: Where is
the chromosomal origin of replication and what proteins are required
for replication initiation? How is chromosome replication coordinated
with cell division? We will then address questions regarding chromosome
organization and segregation, including: What are the motor proteins
that drive chromosome segregation in Archaea? How is the chromosome
organized within the cells of H. volcanii, and what proteins are responsible
for maintaining or regulating this organization? H. volcanii naturally
contains four plasmids ranging in size from 6 Kb to 600Kb. How are
these plasmids partitioned into the daughter cells prior to cell division?
Do they depend upon members of the bacterial ParAB family for their
stable inheritance?
Segregation of antibiotic resistance plasmids in E. coli.
RK2 is representative of a large class of conjugative
plasmids that contribute to the spread of antibiotic resistance
among many human pathogens. Among the conjugal transfer plasmids,
RK2 is one of the most promiscuous, capable of transfer to nearly
all Gram-negative species, including E. coli, P. aeruginosa, Vibrio
cholerae, and Agrobacterium tumafaciens. In E. coli, RK2 is a multicopy
plasmid, with 10-15 copies per cell. For many years, each copy was
thought to be randomly distributed within the cell, and models for
the control of RK2 copy number were based upon the assumption that
both the plasmid molecules and the replication proteins freely diffused
in the cytoplasm. To test this idea, and to begin to determine the
mechanisms underlying faithful inheritance of this ubiquitous class
of antibiotic resistance plasmids, we used fluorescence in situ
hybridization (FISH) and GFP-tagging methods to determine the subcellular
location of RK2. In contrast to long held beliefs, RK2 plasmids
were found to be localized at midcell, forming clusters of approximately
5 plasmid molecules. Following duplication, these plasmid clusters
migrate with rapid kinetics to the 1/4 and 3/4 positions (the midpoints
of nascent daughter cells). Localization of the RK2 plasmid clusters
is regulated by the cell cycle and growth rate. Furthermore, nearly
identical localization was observed in P. aeruginosa and V. cholerae,
suggesting that RK2 employs an evolutionarily conserved mechanism
of segregation. Thus, RK2 provides a unique opportunity to study
a plasmid segregation machinery that is active in a variety of bacterial
species, thereby providing a unique tool for the identification
of evolutionarily conserved mechanisms of DNA segregation.
Lim, G., Derman, A., and Pogliano, J. (2005).
Bacterial DNA segregation by dynamic SopA polymers.
Proceedings of the National Academy of Sciences USA. 102:17658-17663.
Eric Becker, Nick C. Herrera, Felizza Gunderson, Alan Derman, Amber L. Dance, Jennifer Sims, Rachel Larsen and Joe Pogliano (2006).
DNA segregation by the bacterial actin AlfA during Bacillus subtilis growth and development
EMBO J. Dec 13; 25(24):5919-31.
