Cytological screening for novel cell division genes in Escherichia coli

1. Cytological screening for novel cell division genes in Escherichia coli Florian Szardenings Final Year Project Gerdes Lab, 2nd Floor Cookson Building Institute for Cell and Molecular Biosciences Newcastle University

2. Prokaryotic homologues to eukaryotic structural proteins • FtsZ – tubulin homologue • Formation of the division septum and constriction of the cell: Z-ring superstructure • MreB & Mbl – actin homologues • Essential for cell shape in non-spherical bacteria • Spatial organisation of the peptidoglycan synthesis machinery • Crescentin - intermediate filament homologue • Role in crescent shape of Caulobacter crescentus

3. Walshaw and Woolfson, 2001, J. Mol. Biol. Coiled coil proteins • Tertiary structure with a high coiled coil content • ZapB, TipN, Crescentin • Coiled coil: widespread oligomerisation motif found in most organisms • 2-5 alpha helices wound round one another • Key characteristic: heptad repeat • Important for protein-protein and protein-DNA interactions

4. Project outline • Coiled coils can be predicted based on sequence analysis using programs such as COILS • 24 candidate genes selected for screening • High predicted coiled coil content • All genes but 2 are of unknown function • Experimental approach • E.coli strains of ASKA and Keio collections used for overexpression and deletion studies • Growth assays, fluorescence microscopy and flow cytometry • BLAST & Pfam database searches

5. ASKA: overexpression Overexpression vector pCA24N Plasmid contains single cloned ORF ORF under control of IPTG-inducible promoter Kitagawa et al, 2005, DNA Research Keio: deletions Single gene deletions in all non-essential ORFs Baba et al, 2006, Molecular Systems Biology E.coli K-12 strain collections

6. Keio & ASKA strains – growth kinetics

7. Phase contrast DAPI-stained DNA Condensed nucleoid Aggregating cells Keio strains - microscopy

8. BW25113 a b Keio 19 a b Keio26a b Keio strains – flow cytometry Can the effects of gene deletion on cell size be identified using flow cytometry?

9. Misplaced nucleoids Filamentous cells Highly condensed nucleoids ASKA strains - microscopy

10. Nucleoids disappearing? Cell lysis ASKA strains – microscopy cont.

11. ASKA 13 • BLAST search yielded no results • Pfam indicates low sequence identity with small subunit of exonuclease family VII • ASKA 21 • BLAST search predicts a membrane fusion protein homologous to the haemolysin secretion protein D (HlyD) • Pfam: HlyD domain within the protein sequence BLAST and Pfam searches

12. Conclusions • ASKA & Keio collections • detect adverse effects of protein overexpression or deletion • identify potentially interesting genes for further analysis • Screening of many genes in parallel • 24 genes screened • Various effects – growth arrest, elongation, condensed or misplaced nucleoids, cell lysis, aggregating cells • 2 characterised already – ZapA and ZapB • BLAST & Pfam searches may yield information useful for further experimental approaches to clarify protein function • Flow cytometry has not been suitable for identifying effects on cell morphology caused by gene deletion – populations in the samples too heterogenous • Protein overexpression in ASKA strains might cause artefacts

13. Future Work • Reduce possibility of artefacts occurring in ASKA strains • Use lactose instead of IPTG • Low copy number vector • Creation of protein::GFP fusions for localisation studies • Growth assays using minimal media • Two-hybrid screening to identify possible protein-protein or protein-DNA interactions • Apply fixative to cell before microscopy

14. Acknowledgements Kenn Gerdes Elisa Galli Jan-Willem Veening