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

OverviewTraining set (12/9)RegulonDB & literature

Weight matrixThreshold: μ-2σ2

Datasets of transcription units, upstream regions, and orthology for 8 bacterial genomes

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)

PATSER

Three Ortholog Selection Criteria1. Selection of genomes for comparative study

– E. coli k12, H.influenzae, S. typhimurium Lt2, V. cholerae, P. aeruginosa, Y. pestis, B. subtilis, B. halodurans

2. Transcription unit (TU) designation– open reading frames transcribed in the same direction

– separated by less than 100 intergenic nucleotides

3. 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

dnaA TTATCCACAmioC TTTTCCACArpoH TTATTCACA TTATCCACAuvrB TTATCCACT TTATCCACAnrdA TTATCCACA TTATGCACTpolA TTATCCACAdam TTCTCCACAguaB TTATACAGAfadL TTATACAAA

A | 0 0 10 0 2 0 12 1 10

C | 0 0 1 0 8 12 0 10 0

G | 0 0 0 0 1 0 0 1 0

T | 12 12 1 12 1 0 0 0 2

A C G 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 -0.52Hertz, G. et al, Comput Appl Biosci, 1990. 6(2): p. 81-92.

Alignment matrix

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

uvrB ttatccact -419 9.09

ttatccaca -405 10.59

nrdA 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

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 synthesisCronan 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

• PlsC is involved in phospholipid biosynthesis preceding formation of cell membrane lipids PE and PG

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.

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 nonmotileCronan 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

• 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

• 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

Lodish et al., Molecular Cell Biology, 2000, Freeman: NY. p 459.

Acknowledgements

• 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.