Cytological screening for novel cell division genes in Escherichia coli

Florian SzardeningsFinal Year Project

Gerdes Lab, 2nd Floor Cookson BuildingInstitute 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

ASKA 13 I

0

0,5

1

1,5

2

2,5

3

3,5

30 90 180

240

330

390

450

time after inoculation (min)

OD

450

ASKA 10 I

0

0,5

1

1,5

2

2,5

3

3,5

time after inoculation (min)

ASKA 16 I

0

0,5

1

1,5

2

2,5

3

3,5

4

time after inoculation (min)

Growth Keio 10-20 & BW25113 (WT)

0,0000

1,0000

2,0000

3,0000

4,0000

5,0000

6,0000

7,0000

time after inoculation (min)

OD

450

10

12

13

14

15

16

17

18

19

20

WT

Keio strains - microscopy

Aggregating cellsCondensed nucleoid

Phase contrast DAPI-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 b Keio 26 a b

ASKA strains - microscopy

Highly condensed nucleoidsFilamentous 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