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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.

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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
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

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
project outline
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

E.coli K-12 strain collections
keio strains microscopy

Phase contrast

DAPI-stained DNA

Condensed nucleoid

Aggregating cells

Keio strains - microscopy
keio strains flow cytometry

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?

aska strains microscopy

Misplaced nucleoids

Filamentous cells

Highly condensed nucleoids

ASKA strains - microscopy
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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

conclusions
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
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
acknowledgements
Acknowledgements

Kenn Gerdes

Elisa Galli

Jan-Willem Veening