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The aim of our research is to understand temporally and spacially regulated
events during the procaryotic cell cycle. Our special focus is on chromosome
organization and separation of sister chromosomes during DNA replication, as
well as coordination of segregation with cell division.
Bacterial chromosomes are know to have a preferred arragement. Chromosome
replication origins localize towards the cell poles during most of the cell
cycle, while terminus regions are positioned at mid cell. Individual regions on
the chromosome can be visualized with GFP and a tandem array of lactose
operators integrated at any site in the chromosome. At one time during the cell
cycle, chromosome regions are separated in a dynamic fashion (see
movie of GFP tagged origins), as shown in the
following image, where terminus regions are tagged with GFP (left column), and septa between
cells are indicated by white lines (right column Nomarski DIC). Numbers indicate minutes of image
acquisition.
movie to this figure
A major player in chromosome arrangement and segregation is
SMC, a
DNA-binding ATPase that is able to supercoil DNA. Both ends of SMC are thought
to bind to DNA, and to introduce writhe. We are studying SMC biochemically and
genetically.
We have found that SMC forms a complex with two conserved prokaryotic
proteins, ScpA and ScpB. In the absence of any of the three, localization of the
complex is lost. We purified and have analysed all three proteins. ScpA and ScpB
bind to the SMC head domains, and SMC binds to DNA in an unusual manner,
probably as a ring structure.
In Bacillus subtilis, SMC localizes at the edge of nucleoids that
contain the chromosomes, in a cell cycle dependend manner. We are investigating
what determines the specific subcellular localization of SMC. Image below:
SMC-YFP in green, membranes are stained in red (left panel), DNA stained in blue
(right panel).
Study of the role of actin like proteins in bacteria
Actin like proteins MreB and Mbl move along helical tracks
underneath the cell membrane of B. subtilis cells. They are essential for
rod cell shape, as well as for efficient chromosome segregation. We are studying
their role and function during the cell cycle.
Study of DNA double strand break repair in bacteria
We have found that defined repair centers exist in bacteria in
which double strand breaks (DSBs) in DNA are repaired, using homologous
recombination. RecN, O, F, and R proteins are differentially recruited into the
repair centers. Several other proteins are recruited to RCs in a defined
choreography. Our aim is to understand the sequential order of repair events
and to understand repair in the three dimensional context of the cell.
RecN-YFP after induction of breaks
GFP-RecA threads after induction of breaks
A DNA uptake machinery in bacteria
Competence
describes the physiological state in which bacterial cells
from various different species take up DNA from their
environment and incorporate the DNA into their chromosome via
homologous recombination (HR). This way, the cell can gain
novel genetic information, e.g. resistance genes, which is a
clinically relevant problem. We have found that two DNA
recombination proteins, RecA and RecN, accumulate at a single
cell pole in competent Bacillus subtilis cells, where
the DNA uptake machinery is located. RecN is an ATP dependent
ssDNA-binding protein that binds to incoming ssDNA. From this
pole, RecA forms transient fast-growing filaments that extend
from the pole and appear to transport the incoming DNA to the
homologous region on the chromosome. Thus, competence is a
spatially highly organized process, in which DNA takes a
defined path from one pole to the chromosome.
assembly of RecA at a single cell pole at the
onset of competence, 1 min intervals
GFP-RecA thread are highly dynamic structures, 1 min intervals
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