1. Bacterial Cell Division: Mechanism and Regulation Bacillus subtilis as a model Cell division in a pathogen eg Staphylococcus aureus Antibacterials: new antibiotic targets and natural antibiotics Associate Professor Liz Harry Institute for the Biotechnology of Infectious Diseases (IBID), University of Technology, Sydney

2. Bacillus subtilis A New Assembly Pathway For The Cytokinetic Z Ring

3. Bacterial Cell Division

4. The Importance of FtsZ First to division site Required for assembly of division complex Highly conserved Target of cell division inhibitors Prokaryotic precursor of tubulin Antibiotic target

5. The FtsZ Protein

6. Z Ring Assembly Model Ability of FtsZ to polymerize and availability of a midcell site are important NS

7. Investigation of ‘Background’ FtsZ Staining

8. FtsZ Localization: Vegetative Cells Immunofluorescence 5 μm

9. Deconvolution Wild type, vegetative cells. IFM

10. cells with just an FtsZ helix n = 124 cells with a helix and a Z ring n = 216 Cell Cycle Relationship 5.1 ± 0.07 µm 3.9 ± 0.05 µm Does the formation of this FtsZ helix require prior Z ring formation or can it form de novo?

11. Spore Outgrowth in B. subtilis No complications from previous cell cycle events Good synchrony

12. Outgrown Spores (IFM) The Z helix can form independently of the Z ring Time (min) Cell Length (µm) Helix Only (%) Helix and Ring (%) 180 3.2 ± 0.07 (n = 133) 8218 210 3.6 ± 0.10 (n=121) 7525 270 3.7 ± 0.09 (n=183) 5743

13. The Z Helix in Live Cells Using FtsZ-YFP Spore outgrowth 210` 180 min 210` 240` 270 min Spore coat

14. The Z Helix Is Highly Dynamic Time-lapse imaging of outgrowing spores containing an inducible copy of FtsZ-YFP Images taken every 90 sec over 1 hour

15. FtsZ Helix-to-Ring Transition Images taken every 90 sec over 2 hours Time-lapse imaging of outgrowing spores containing an inducible copy of FtsZ-YFP

16. Time Lapse Images 1 11 71 61 15 33 19 77100 90 * Outgrowing spores

17. Cell-Cycle Regulated Transition Clear conversion of an FtsZ helix to ring was observed in 19 distinct cells A heated stage was required The average cell length at which a Z ring formed was 4.6 ± 0.1 µm

18. Time Position Position of Z Structures Within The Cell

19. What About FtsA? In B. subtilis FtsA is required for efficient Z ring formation In B. subtilis FtsA interacts directly with FtsZ prior to Z ring formation FtsA connects FtsZ to the inner membrane

20. FtsA Also Forms a Helix! Wild type vegetative cells-IFM

21. The Old Model NS

22. New Model For Z Ring Formation

23. The Big Questions What’s the trigger? Role of Z ring regulation systems (Min, nucleoid occlusion, chromosome replication) and FtsZ modulator proteins? Does the helix have another cytoskeletal/cell growth role? Is the helix a true intermediate? Does the ring arise by remodelling of the helix?

24. The Min System MinCD inhibitor Acts at the level of Z ring assembly

25. The Min System MinCD inhibitor Acts at the level of Z ring assembly

26. Nucleoid Occlusion

27. Dr Margaret Migocki Ms Phoebe Peters Thanks…… ARC Discovery ARC Network for Fluorescence (FABLS) Carola Thoni (Leica Microsystems) Arne Müller (Carl Zeiss)