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

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

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 Walshaw and Woolfson, 2001, J. Mol. Biol.

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

E.coli K-12 strain collections 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

Keio & ASKA strains – growth kinetics

Keio strains - microscopy Aggregating cells Condensed nucleoid Phase contrastDAPI-stained DNA

Keio strains – flow cytometry Can the effects of gene deletion on cell size be identified using flow cytometry? BW25113 a b Keio 19 a bKeio 26 a b

ASKA strains - microscopy Highly condensed nucleoids Filamentous cells Misplaced nucleoids

ASKA strains – microscopy cont. Nucleoids disappearing? Cell lysis

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

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

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

Kenn Gerdes Elisa Galli Jan-Willem Veening Acknowledgements