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