title Center for Biological Sequence Analysis The Technical University of Denmark DTU Comparative Genomics of five Clostridium species Comparative Microbial

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Comparative Genomics of five Clostridium

species

Comparative Microbial Genomics Group

A DNA-Centric View

DIAGNOSIS, EPIDEMIOLOGY AND ANTIBIOTIC RESISTANCE OF THE GENUS CLOSTRIDIUM

17-18 October, 2003 - Parma, Italy

Adv. Bioinformatics Lekture, 21 November, 2003

OVERVIEW

1. Comparison of five Clostridium genomes

overview

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2. Local View: DNA Structures and Clostridium Promoters

3. Global View: oligomer bias in Clostridium genomes

Cp 100 genomes

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How do you compare more than 100 bacterial genomes?

Organism length (bp) Percent ATClostridium acetobutylicum ATCC 824 3,940,880 69.1Clostridium botulinum ATCC 3502 3,886,916 71.8Clostridium difficile 630X 4,290,252 70.9Clostridium perfringens 13 3,031,430 71.4Clostridium tetani E88 2,799,251 71.3 Clostridium genomes Average 3,589,746 70.9 Ave. of 150 Bact. genomes 3,323,702 52.7

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AT content

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AT content in 150 Sequenced Bacterial Chromosomes

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2

4

6

8

10

12

14

20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80

Percent AT

Number of Chromosomes

C. acetobutylicum

50% AT

C. botulinumC. difficileC. perfringensC. tetani

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AT content

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Global Direct Repeats in 150 Sequenced Bacterial Chromosomes

0

5

10

15

20

25

30

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Percent of Chromosome with Repeats > 80%

Number of chromosomes

C. perfringensC. tetani

C. botulinum

C. acetobutylicum

C. difficile

Global Repeats

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Global Direct Global Inverted4.2 2.32.8 1.3

6.4 5.03.5 2.83.8 3.14.14 2.94.13 2.9

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Global Inverted Repeats in 150 Sequenced Bacterial Chromosomes

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 8 9 10 11 12 13



C. botulinum

C. acetobutylicumC. perfringens

C. difficile

C. tetani

Local Inverted Repeats in 150 Sequenced Bacterial Chromosomes

0

5

10

15

20

25

30

35

40

0 3 6 9 12 15 18 21 24 27 30



C. difficileC. tetani

C. botulinumC. perfringens

C. acetobutylicum

Local Repeats

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Local Dir. Local Invert.11.4 8.017.9 12.3

15.9 10.116.7 12.116.1 10.115.6 10.528.3 6.14

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Local Direct Repeats in 150 Sequenced Bacterial Chromosomes

0

5

10

15

20

25

30

35

0 3 6 9 12 15 18 21 24 27 30 33



C. perfringensC. tetani

C. botulinum

C. acetobutylicum

C. difficile

Purine stretches

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pur stretchesYR stretches3.36 0.493.84 0.48

3.12 0.584.23 0.394.21 0.463.752 0.481.86 1.18

expect 1.0 expect 1.0

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purine stretches in 150 Sequenced Bacterial Chromosomes

0

10

20

30

40

50

60

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8

Fraction of purine stretches >= 10 bp


EXPECTED

C. acetobutylicumC. difficile C. perfringens

C. tetani

C. botulinum

Cp. rRNAs

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# Genes # rRNAs

3652 11

3524 10

3810 11

2693 10

2688 6

Organism length (bp) Percent ATClostridium acetobutylicum ATCC 824 3.940.880 69.1

Clostridium botulinum ATCC 3502 3.886.916 71.8

Clostridium difficile 630X 4.290.252 70.9

Clostridium perfringens 13 3.031.430 71.4

Clostridium tetani E88 2.799.251 71.3

Cp. protein

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All paralogs

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firmicutes

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C.tetani plasmid

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C.tet zoom

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Promoter structures

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Promoter Structural profile

-10 “TATA box”-35

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Campy pVir zoom

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Promoter structure


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-35

-10 TATA

J. Mol. Biol., 326:1361-1372, (2003). Parma, 2003

Translation start aligned


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J. Mol. Biol., 299:907-930, (2000).

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Promoter structures

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Promoter Structural profile

+1

CDS

DNA curvature,flexibility importanthere

meltsrigid

cruciform

-10-35

mRNACDS

’

-10-35

+1

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context


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Information Depends on Context…..

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sigmas

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Organism length (bp) Percent ATClostridium acetobutylicum ATCC 824 3.940.880 69.1

Clostridium botulinum ATCC 3502 3.886.916 71.8

Clostridium difficile 630X 4.290.252 70.9

Clostridium perfringens 13 3.031.430 71.4

Clostridium tetani E88 2.799.251 71.3

# sigma70s

11

11

11

9

17

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Sigma70s with colour


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Clostridium ’s

RpoH

C. perfringens

C. acetobutylicum

C. tetani

RpoD

RpoS

FliA

SpoII

spoI

Sig70 tree

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Proteobacteria

Firmicutes

Actinobacteria

70

ChlamydiaCyanobact.

“other phyla”

RpoDRpoS

“RpoD like”

sigEsporulationiniation

RpoH

sigFsporulation

sigGsporulation sigH

RpoFflagella

sigmas

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Environmental influence

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’

-10-35

DNA curvature,flexibility importanthere

+1 50 mM salt

20 deg C

’

-10-35 +1

300 mM salt

37 deg C

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Context 2


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Information Depends on Context…..

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C. acetobutylicum

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C. botulinum

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C.difficile Atlas

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C.tetani genome

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Toxin tetanospasmin

• Anaerobic conditions allow germination of spores and production of toxins.

• The tetanus toxin is a 150 KDa protein, which is cleaved extra cellular, to a 100 Kda and a 50 Kda, that are binded with a sulfur bridge.

• Toxin binds in central nervous system

• Interferes with neurotransmitter release to block inhibitor impulses.

• Leads to unopposed muscle contraction and spasm.

C.perfringens

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C.perf. skew

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Oligomer skew in C. perfringens

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 0.5 1 1.5 2 2.5 3 3.5

Millions

Millions

chromosomal position (Mbp)

information content (bits)

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oligomers

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LEADING STRAND, Oligomer bias (up to 8mers)Origin position: 0 Strand difference(bits): 4105557Oligo bitsG 92292GA 81583AG 64095AGA 60700GG 46169GAG 34200GAA 33918A 31417GGA 31259AA 25862AGG 25340AAG 24943AGAG 24464AGAA 23535AAGA 23005GAAA 18825GAGA 18448ATG 18129GAT 17623GGG 17200

LAGGING STRAND, Oligomer biasTerminus position: 1361000Strand difference(bits): 3977053Oligo bitsC 93908TC 82712CT 62349TCT 57477CC 47113TTC 35786CTC 32995TCC 32250T 29779CCT 24636TT 23842TTCT 23751CTCT 22962CTT 22661TCTT 21517TTTC 19334CCC 18554CAT 18537TCTC 17833ATC 17657

C. Perf skew2

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Oligomer skew in C. perfringens

-4

-3

-2

-1

0

1

2

3

4

5

0 0.5 1 1.5 2 2.5 3 3.5

Millions

Millions

Chromosome Position (bp)

Information Content (bits)

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Organisms S/N

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Organism Signal NoiseClostridium perfringens 43.792.284Clostridium difficile 43.053.789Clostridium perfringens 42.403.666Clostridium botulinum 42.239.771Clostridium acetobutylicum 39.364.732Bacillus cereus 31.536.141Bacillus anthracis 30.115.576Thermoanaerobacter tengcongensis 24.539.893Xylella fastidiosa 21.905.088Enterococcus faecalis 17.899.285Staphylococcus aureus 16.446.595Staphylococcus aureus 16.043.397Oceanobacillus iheyensis 15.185.416Fusobacterium nucleatum 15.164.722Blochmannia floridanus 14.386.685Listeria innocua 13.880.720Borrelia burgdorferi 13.805.045Xylella fastidiosa 13.778.823Bacteroides thetaiotaomicron 13.755.678Bacillus halodurans 13.098.141Bacteroides fragilis 12.662.606Lactobacillus plantarum 12.521.367Listeria monocytogenes 12.283.482Lactococcus lactis 11.556.365Staphylococcus epidermidis 11.211.357Bacillus subtilis 11.103.547Streptococcus mutans 9.348.241Streptococcus agalactiae 9.181.368Photorhabdus luminescens 9.075.427Streptococcus pneumoniae TIGR4 9.073.648Streptococcus pneumoniae R6 8.659.665Streptococcus agalactiae 8.652.586Mycoplasma penetrans 8.483.056Mycobacterium leprae 8.273.044Campylobacter jejuni 7.619.615Streptococcus pyrogenes 7.611.987Treponema pallidum 7.599.792Erwinia carotovora 7.545.523Streptococcus pyrogenes 7.089.889Mycoplasma gallisepticum 6.752.566Shewanella oneidensis 6.710.673Pseudomonas aeruginosa 6.699.858Rhodospirellula baltica 6.652.206Prochlorococcus marinus 6.465.620Wolinella succinogenes 5.294.449

Signal/Noise plot

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S/N values for 612 chromosmes/plasmids

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100

200

300

400

500

600

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00

Bin

Frequency

Clostridiumacetobutylicum

Bin Frequency------------------5.00 56010.00 2415.00 1220.00 525.00 330.00 035.00 240.00 145.00 4

Clostridium x4

B. cereusB. anthracis

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Summary

1. DNA sequence DNA structure Function

summary


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2. Clostridium difficile is more likely to be variable.

3. Clostridium DNA is different!

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Acknowledgements


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Acknowledgements• Jakob Bondo• Jimmy Hoffmann Hansen• Paiman Khorsand-Jamal • Anne Egholm Pedersen• Maria Seier Petersen• Peter Hallin

[email protected]

The Danish Research Foundation

www.cbs.dtu.dk • Hans-Henrik Stærfeldt• Lars Juhl Jensen• Carsten Friis• Marie Skovgaard • Peder Worning• Thomas Scheritz Pontén• Søren Brunak

Parma, 2003

Documents

title Center for Biological Sequence Analysis The Technical University of Denmark DTU Comparative Genomics of five Clostridium species Comparative Microbial