transcriptome analysis of the tnra regulon in bacillus subtilis hanne jarmer center for biological...

Transcriptome analysis of theTnrA regulon in Bacillus subtilis

Hanne JarmerCenter for Biological Sequence analysis

Technical University of Denmark

NH4+ + glutamate glutamine

Glutamine synthetase

tnrA

TnrA

GlnRGlnA - Glutamine Synthetase

ureA ureB ureC

glnR glnA

Access to nitrogen through alternative pathways

gabPnasApucRykzB-ykoLnasABCDEFnrgABansZycs-kippucABCDE

gltAB

wildtypeversus

mutantin the samemedium

Allantoin

NH4/glutamate

Glutamate

TnrA

wildtype versus

wildtypein differentmedia

Glutamine

GlnA GlnR

Pathway specific regulator of purine catabolism

TnrA

H2O

NH3

H

HH

N

N

N

N

NH2

Adenine deaminase (adeC)

N

HN

N

N

O

H

HH

Xanthine dehydrogenase (pucABCDE)

H2O2

O2

Adenine

Hypoxanthine

Xanthine

H

H

N

HN

N

N

O

OH

Out In

Xanthine permease (pbuX)

Uric acid permease (pucJK)

InOut

HN

HN

N

N

O

O

O H

Uric acid

O2

H2O2

Xanthine dehydrogenase (pucABCDE)

H

Guanine deaminase (gde)

NH3 H2O

OutIn

pbuGN

HN

N

N

O

NH2

H

H

Guanine

pbuG

In Out

H

Uricase (pucLM)

CO2

O2

Allantoin

HH

Out In

Allantoinpermease (pucI)

HN

H2N

N

NO

O

O

H H

Allantoic acid

O2

CO2

Allantoinase (pucH)

N

H2NO

O

OH

N

NH2

O H2O NH3+CO2

Allantoateamidohydrolase (pucF)

N

H2N

NH2

O

O

OHUreidoglycine

HH

Allantoateamidohydrolase (pucF)

NH3H2O

Ureidoglycolic acid

N

H2N

OH

O

O

OH

H

Ureidoglycolase (pucG)

H2N

O NH2H

O OH

O

+

Urea Glyoxylic acid

Urease (ureABC)

2NH3 + CO2

H2O

H

InOut

Allantoinpermease (pucI)

InOut

Purine catabolism

PucR

ALLANTOIN

mRNAmRNA

cDNAcDNA

Cy3-cDNACy5-cDNA

SAMPLE REFERENCE

NUMBERS:

3 hybridizations (arrays) per culture

2 spots per gene on each array

2 different growth experiments

2 x 3 x 2 = 12 intensities for each gene

Allantoin: 132

Glutamine: 60

Glutamate: 91

TnrA: 239

GlnA: 159

GlnR: 67

RESULT: 6 lists

• Extraction: The gene should be significantly affected in at least two of the six experiments (except “Glutamine”)

• List of ~100 genes highly likely to be involved in the TnrA-GlnA system

• Visualization: ClustArray

Extraction - Visualization

Novozymes Biotech Inc – Davis, California:

Randy Berka

Maria Tang

Alan Sloma

Department of Microbiology – Boston University:

Susan Fisher

Lewis Wray, Jr.

Jaclyn Brandenburg

Section of Molecular Microbiology - DTU:

Hans Henrik Saxild

Lars Beier

Center for Biological Sequence analysis – DTU:

Steen Knudsen

Chris Workman (software)

Laurent Gautier (statistics)

The array group (discussions)

Kristine Dahlin

Acknowledgements

Todays exerciseinferring regulatory networks

Gene

Experiment

TnrA GlnA GlnR

-1 1 0nrgA

1 -1 0

1 1 1glnR

purE

A B

DC

A B

DC