Transcript A Comparative Genomic Method for Computational

Comparative genomic analysis
of lipid biosynthesis and metabolism components
of the DnaA regulon
Megon Walker
Simon Kasif
Introduction
DnaA: Cellular Roles
1. Initiation of replication
– 4 oriC binding sites in E. coli
– strand separation
– conserved across bacteria
2. Transcription factor
–
–
–
–
footprinting assays
binds DNA selectively as a monomer
TTATNCACA binding site
transcriptional activity non-essential
3. Increases DNA supercoiling
– non-selective binding
Lodish et al., Molecular Cell Biology, 2000, Freeman: NY. p 459.
DnaA: Lipid biosynthesis
• Temperature sensitive dnaA transcription factor prohibitive
mutants at nonpermissive temperature
– period of generation of cell growth NOT prolonged
– altered lipid synthesis protein levels
• increased beta-ketoacyl synthase II (fadL)
• increased long-chain fatty acid transport protein (fabF)
– altered fatty acid composition of cell membrane phospholipids
• phosphatidylethanolamine (PE)
• phosphatidylglycerol (PG)
• How does DnaA regulate genes controlling the
phospholipid fatty acid composition and flagella
formation?
Suzuki, E. et al., Mol Microbiol, 1998. 28(1): p. 95-102.
Ohba, A. et al., FEBS Lett, 1997. 404(2-3): p. 125-8.
DnaA: Transcription Factor
• Goal
– DnaA regulon characterization via
identification of genes with DnaA binding
sites in promoter regions
– Repressor (dnaA, rpoH, uvrB, mioC, fadL)
– Activator (nrdAB, glpD, polA)
• Obstacles
– 9mers resembling binding site occur frequently
in E. coli genome
– not all sites matching consensus are actually
bound by DnaA (ftsAQ)
– some experimentally conserved binding sites
differ from consensus
– known DnaA regulated genes not functionally
related (replication, lipid synthesis, house
keeping genes)
Messer, W. et al., Mol Microbiol, 1997. 24(1): p. 1-6.
Comparative genomics
•
Haemophilus influenza genome completed in 1995, ~100 genomes
sequenced since
Availability of complete genomes of related bacteria allows
comparative analysis of regulatory patterns (gene number, content,
and order in groups of organisms)
Conservation of candidate DnaA binding sites across species is
additional evidence of regulatory functionality
If a regulator is conserved in several genomes
•
•
•
–
its regulon and binding sites in these genomes are conserved as well
–
–
true sites occur upstream of orthologous genes
false sites are scattered at random across the genome
Methods
Overview
Datasets of
transcription units,
upstream regions,
and orthology for
8 bacterial
genomes
Training set (12/9)
RegulonDB & literature
PATSER
Weight matrix
Threshold: μ-2σ2
E. coli sets of putative site scores above 6.0 bit cutoff in
noncoding regions (1031 Watson/1051 Crick). Performed for all
8 genomes.
Reference E. coli k12 transcription units sharing orthologous
members with TUs from 2+ genomes, all of which have upstream
DnaA binding sites in upstream regulatory regions (164/120)
Sets of 3+ DnaA-regulated, orthologous transcription units
containing at least 1 cross-species pair of binding sites displaying
conservation of sequence (2+ identical DnaA boxes) or location
(within 20 base pairs) (127/88)
Three Ortholog Selection Criteria
1.
Selection of genomes for comparative study
–
2.
3.
E. coli k12, H.influenzae, S. typhimurium Lt2, V. cholerae, P. aeruginosa, Y. pestis, B.
subtilis, B. halodurans
Transcription unit (TU) designation
–
open reading frames transcribed in the same direction
–
separated by less than 100 intergenic nucleotides
Pairwise identification of orthologs to genes in reference genomes:
–
–
–
reciprocal pairwise TBLASTN searches between all annotated genes of reference E.
coli k12 and the other 7 organisms
bidirectional best matches
lower similarity threshold 10-20
Altschul, S. et al., JMB,1990. 215: p 403-410.
PATSER: Position Weight Matrix Construction
Alignment matrix
dnaA TTATCCACA
mioC TTTTCCACA
rpoH TTATTCACA
TTATCCACA
uvrB TTATCCACT
TTATCCACA
nrdA TTATCCACA
TTATGCACT
polA TTATCCACA
dam TTCTCCACA
guaB TTATACAGA
fadL TTATACAAA
Hertz, G. et al, Comput Appl Biosci, 1990. 6(2): p. 81-92.
A
C
G
T
|
|
|
|
0
0
0
12
0
0
0
12
10
1
0
1
0
0
0
12
2
8
1
1
0
12
0
0
12
0
0
0
G
1
10
1
0
10
0
0
2
A
C
T
-2.56
-2.56
-2.56
1.16
-2.56
-2.56
-2.56
1.16
0.99
-0.80
-2.56
-1.09
-2.56
-2.56
-2.56
1.16
-0.51
1.12
-0.78
-1.09
-2.56
1.52
-2.56
-2.56
1.16
-2.56
-2.56
-2.56
-1.09
1.34
-0.78
-2.56
0.99
-2.56
-2.56
-0.52
Weight matrix
Training Set
Gene
Site Sequence
Position
Score
dnaA
ttatccaca
-211
10.59
mioC
tttttcaca
-302
6.31
rpoH
ttattcaca
-131
8.38
ttatccaca
-107
10.59
ttatccact
-419
9.09
ttatccaca
-405
10.59
ttatccaca
-162
10.59
ttatgcact
-150
7.19
polA
ttatccaca
-132
10.59
dam
ttctccaca
-30
8.81
guaB
ttatacaga
-45
6.84
fadL
ttatacaaa
-27
6.53
uvrB
nrdA
http://www.bio.cam.ac.uk/cgi-bin/seqlogo/logo.cgi
Salgado, H. et al., Nucleic Acids Res, 2001 Jan 1. 1(72-4).
Results
Putative dnaA regulon:
functional classifications
• Training Set (8/9)
• Lipid synthesis (6)
• Information Transfer: transcription, translation, DNA repair, ribosomal
assembly, nucleotide synthesis (13)
• Coenzyme Metabolism (4)
• Carbohydrate Transport & Metabolism (5)
• Amino Acid Transport & Metabolism (6)
• Energy Production and Conservation (6)
• Putative/hypothetical ORFs (42)
Tatusov, R. et al.. Nucleic Acids Res, 2001. 29(1): p. 22-8.
Riley, M. et al.. J Mol Biol, 1997. 268(5): p. 857-68.
acyl carrier protein (acpP)
acpP-fabF
Site Sequence
Position
Score
ecolik12
ttatacact
-54
7.45
paer
ttttccata
-17
6.09
vchol
tttttcaca
-77
6.30
stlt2
ttatacact
-54
7.55
ypes
ttatacact
-54
7.40
• AcpP is an acyl carrier protein
involved in lipid biosynthesis
• co-transcribed upstream of FabF
(fatty acid transport protein)
Cronan Jr, J.E. et al., Escherichia coli and Salmonella typhimurium:
cellular and molecular biology, F. Neidhardt, et al., Editors.1996,
American Society for Microbiology: Washington DC. p. 615.
carboxylase transferase (accD)
accD-folD-dedD
Site Sequence
Position
Score
ecolik12
ttatccaaa
-119
8.17
vchol
taatccaca
-72
6.91
stlt2
ttatccaaa
-113
8.26
• accD transcribes a subunit of
carboxylase transferase
• performs the initial step of fatty acid
synthesis
Cronan Jr, J.E. et al., Escherichia coli and Salmonella typhimurium:
cellular and molecular biology, F. Neidhardt, et al., Editors.1996,
American Society for Microbiology: Washington DC. p. 614.
PlsC (plsC)
plsC-sufI
Site Sequence
Position
Score
ecolik12
ttttccaga
-77
6.40
hinf
ttatgcaga
-162
6.40
stlt2
ttttccaga
-78
6.44
Cronan Jr, J.E. et al., Escherichia coli and Salmonella typhimurium:
cellular and molecular biology, F. Neidhardt, et al., Editors.1996,
American Society for Microbiology: Washington DC. p. 619.
• PlsC is involved in
phospholipid biosynthesis
preceding formation of cell
membrane lipids PE and PG
acyl carrier protein phosphodiesterase (acpD)
acpD
Site Sequence
Position
Score
ecolik12
ttattcaca
-54
8.38
bhal
ttatgcaaa
-379
6.09
paer
ttataaaca
-106
7.11
stlt2
ttatccgca
-424
6.91
stlt2
ttttccaga
-345
6.44
stlt2
ttattcaca
-62
8.48
ypes
ttatgcaga
-60
6.52
ypes
ttatccact
-444
9.10
• AcpD is classified as an acyl carrier protein phosphodiesterase
• highest scoring putative DnaA binding site
Psd (psd)
yjeQ-psd
Site Sequence
Position
Score
ecolik12
tgatccaca
-94
6.87
bhal
ttcttcaca
-209
6.89
hinf
tgatccaca
-323
7.01
hinf
ttatccaat
-100
6.26
vchol
ttattcaca
-94
8.38
• Psd catalyzes last step of PE synthesis
• psd knockouts nonmotile
Cronan Jr, J.E. et al., Escherichia coli and Salmonella typhimurium:
cellular and molecular biology, F. Neidhardt, et al., Editors.1996,
American Society for Microbiology: Washington DC. p. 620.
phosphatidylglycerophosphate
synthase (pgsA)
uvrY-uvrC-pgsA
Site Sequence
Position
Score
ecolik12
ttgtccaca
-130
7.04
stlt2
ttacccaca
-337
6.91
ypes
ttctccaga
-7
6.73
• pgsA encodes phosphatidylglycerophosphate synthase
• catalyzes the committed step of PG and CL
biosynthesis in E. coli
Cronan Jr, J.E. et al., Escherichia coli and Salmonella typhimurium:
cellular and molecular biology, F. Neidhardt, et al., Editors.1996,
American Society for Microbiology: Washington DC. p. 620.
Conclusion
Conclusion
• DnaA regulation may couple lipid cellular processes to DNA replication
– this may be accomplished by the transcription factor activity of DnaA upstream of
phospholipid biosynthesis genes fadL, acpP, fabF, accD, plsC, psd, and pgsA
– changes in expression of the phospholipid biosynthesis proteins alter the fatty acid
composition of the cell membrane
– interactions between DnaA protein and the membrane that modulate the activity of the
mutant DnaA
• The biological significance of the motifs
presented here will be verified
experimentally
– microarray analysis of temperature sensitive
dnaA mutants in a variety of bacteria
– chromatin immunoprecipitation studies to
verify true positive candidate binding sites
Lodish et al., Molecular Cell Biology, 2000, Freeman: NY. p 459.
Acknowledgements
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•
•
•
Simon Kasif (Bioinformatics, Boston University)
Alan Grossman (Microbiology, Massachusetts Institute of Technology)
Tohru Mizushima (Pharmaceutical Sciences, Kyushu University )
NSF (KDI) & GEM fellowship
Comparative genomic analysis of lipid biosynthesis and metabolism
components of the DnaA regulon. Genome Biology. In review.