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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