Molecular mechanisms of cell cycle control in Escherichia coli

1. Research project:
Molecular mechanisms of cell cycle control in Escherichia coli.

2. Group:
Kirsten Skarstad (PI), Anne Wahl (engineer), Solveig Fossum (postdoc), Line Johnsen (postdoc), Ingvild Flåtten (postdoc), Ingvild Odsbu (postdoc), Torsten Waldminghaus (postdoc), Lene Marie Skeie (PhD student), Caroline Stokke (PhD student), Henning Bråten (undergraduate), Heidi Evensen (undergraduate).

3. Home adress on the net:
http://radium.no/skarstad

4. Institution:
Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital Trust

5. Main aim of research group:
Cancer cells are characterized by loss of regulation, allowing them to go through the cell cycle in an uncontrolled fashion. The main aim of the project is to understand how the cell cycle is regulated in a simple model organism, the bacterium Escherichia coli.

6. Some important recent results (with a few key references):
At least three different control circuits operate to regulate initiation of chromosome replication to once and only once per cell cycle. First, a newly replicated origin is inactivated by a process termed sequestration which involves the SeqA protein. Second, the initiator protein, DnaA, is inactivated by the replisome and the newly discovered Hda protein as soon as replication has begun. And third, free DnaA protein is titrated away from the origin by replication of the datA site which binds exceptionally high amounts of DnaA. In addition, transcription from promoters near the origin affect initiation of replication both positively and negatively. DnaA interacts with RNA polymerase and by investigating the synchrony of initiation and the compatibility of origins it is possible to investigate the elements of replication control in vivo (Skarstad and Løbner-Olesen, EMBO J 22,140-50,2003). The presence of excess datA sites leads to underinitiation, instability of replication forks and delayed cell division (Morigen et al, Mol Microbiol 50,349-62,2003). Loss or mutation of the SeqA protein as well as its overexpression results, not only in aberrant sequestration, but also in altered chromosome organization and delayed cell division (Bach et al, EMBO J 22,315-23,2003; Fossum et al, Mol Microbiol 47,619-32,2003). The results indicate that the control of replication is closely tied to control of sister chromosome segregation and cell division. Construction of non-sequesterable origins showed that extra rounds of replication are flexibly accomodated whithin the cell cycle as long as the SeqA protein is intact (Bach and Skarstad, Mol Microbiol 51,1589-600, 2004). The SeqA protein is capable of organizing newly replicated DNA (Klungsøyr and Skarstad, Mol Microbiol 54, 123-31, 2004; Odsbu et al, Genes Cells 10, 1039-1049, 2005) and may play a role in cohesion of sister origins (Fossum et al, EMBO J 26:4514-4522, 2007) and organisation of replication forks (Morigen et al, Genes Cells 14, 643-657, 2009; Odsbu et al, PloS ONE, 4, e7617, 2009).

7. Methods in current use:
Cell cycle analysis by flow cytometry, immunofluorescence and GFP-based localization by microscopy. Strain construction by ET cloning and P1 transduction, plasmid construction by traditional cloning. Identification of genes, transcripts and proteins by PCR, Southern, Northern and Western. Protein purification. Studies of protein-protein interaction, protein-DNA interaction, replication and transcription in vitro.

8. Available equipment:
Institute of Cancer Research Core Facilities: FACS DIVA and LSRII Flow cytometers, DNA sequencing equipment, PhosphorImager, Ultracentrifuge.
Department of Cell Biology: Zeiss Axiovision and Leica fluorescence microscopes, ChemiGenius gel documentation equipment, PCR machines, scintillation counter, molecular biology- and cell cycle analysis-PC software.

9. Collaborators:
9.1 At the Institute for Cancer Research: Research groups studying cell cycle control (Erik Boye) and flow cytometry of microorganisms (Trond Stokke).
9.2 Other Norwegian collaborators: Research groups within the University of Oslo MLS MicroMN
9.3 Collaborators from other countries: E. Crooke, Georgetown University, Washington DC, A. Løbner-Olesen, Roskilde University, Denmark, L. Janniere, INRA, Jouy, France.

10. Is the group interested in joining a larger collaborative project at DNR: Yes

11. Tentative name of possible collaborative project(s):
Regulation of the cell cycle in eukaryotes, prokaryotes and archaea.

12: Some key search words:
Initiation of replication, cell cycle, chromosome segregation