Transcript BLAST and Pfam searches

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 proteinprotein 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
Keio: deletions
- Overexpression vector
pCA24N
- Plasmid contains single
cloned ORF
- ORF under control of
IPTG-inducible promoter
- Single gene deletions in
all non-essential ORFs
Kitagawa et al, 2005, DNA Research
Baba et al, 2006, Molecular Systems
Biology
Keio & ASKA strains – growth kinetics
Growth Keio 10-20 & BW25113 (WT)
ASKA 13 I
7,0000
3,5
6,0000
3
10
5,0000
12
2,5
14
2 4,0000
OD450
15
16
17
1,5 3,0000
18
19
1
20
2,0000
WT
0,5
1,0000
time after inoculation (min)
45
0
45
0
33
0
27
0
21
0
39
0
39
0
33
0
24
0
18
0
tim e after inoculation (m in)
15
0
30
0,0000
90
90
0
30
OD450
13
Keio strains - microscopy
Phase contrast
Condensed nucleoid
DAPI-stained DNA
Aggregating cells
Keio strains – flow cytometry
Can the effects of gene deletion on cell size be
identified using flow cytometry?
BW25113
Keio 19
a
a
b
b
Keio 26
a
b
ASKA strains - microscopy
Misplaced nucleoids
Filamentous cells
Highly condensed nucleoids
ASKA strains – microscopy cont.
Nucleoids disappearing?
Cell lysis
BLAST and Pfam searches
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
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
Acknowledgements
Kenn Gerdes
Elisa Galli
Jan-Willem Veening