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Transcriptome analysis of the TnrA regulon in Bacillus subtilis. Hanne Jarmer Center for Biological Sequence analysis Technical University of Denmark. Bacillus subtilis. - PowerPoint PPT Presentation
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Transcriptome analysis of theTnrA regulon in Bacillus subtilis
Hanne JarmerCenter for Biological Sequence analysis
Technical University of Denmark
Bacillus subtilis• Bacillus means rod-shaped bacteria. The rod in the gut was Bacillus coli and the rod found in rotting hay was Bacillus subtilis
• Louis Pasteur used heat-attenuated Bacillus anthracis as the first anti-bacterial vaccine
• Subtilis means: highly sensitive – weak – mild (subtle)
• Soil bacteria typically found in association with plants
Bacillus subtilis• B. subtilis has been used for studying different systems: gene regulation, metabolism and differentiation
• The second most extensively studied bacteria
• The first to be transformed in the laboratory. This process was optimized by Anagnostopoulos and Spizizen in 1961
Bacillus subtilis
Bacillus subtilis• Fully sequenced and has around 4,225 predicted genes (SubtiList, R16.1)
• Non-hasardous
• Easy to grow and manipulate genetically
• Sporulates
• Secretes proteins
• Of extensive interest to industry
Bacillus subtilis
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
• PCR products from ~97% of all genes
• spotted onto poly-L-lysine coated glass microscope slides using a 16-pin microarrayer
• Hybridized to Cy3 and Cy5 labeled first-strand cDNAs
• Scanned
B. subtilis microarrays
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
GlnR
GlnA
TnrA
Glutamine
Glutamate
Allantoin
pucA yrbD nrgB nrgA pucJ hxlA ywpH pucL pucK ureC ureB gabP
ywfM yhdG dppA pucB nasA nasE ykoL tnrA ysnE cotZ guaD
Activated by TnrA or PucR?
Opposite expression profile
– common TnrA regulated regulator?
What is the function of these genes ?
yrbD: sodium/proton-dependent alanine carrier
hxlA: 3-hexulose-6-phosphate synthase
ywpH: single-strand DNA-binding protein
ywfM: unknown
yhdG: amino acid transporter
dppA: D-alanyl-aminopeptidase
ysnE: similar to acetyltransferase
cotZ: spore coat protein
TnrA box = TGT.A.......T.ACA
• Built a Weight Matrix from known sites
• searched the upstream regions
• sorted and extracted
• clustered and displayed
pucA yrbD nasA nrgA pucJ ykoL tnrA pucR
ykzB yyaF pucH ywoE gabP yhdG ycsF
GlnR
GlnA
TnrA
Glutamine
Glutamate
Allantoin
Repressed by TnrA ?
yuiA yycB yodF gltA hisI yocR bioA
Repressed by TnrA?
GlnR
GlnA
TnrA
Glutamine
Glutamate
Allantoin
braB glnR
Repressed by GlnR ?
TnrA activated genes?
yrbD and yhdG have the TnrA box in a plausible position
Experimental investigations
yrbD: similar to sodium/proton-dependent alanine carrier protein
yhdG: similar to amino acid transporter
yyaF: similar to GTP-binding protein
TnrA repressed genes?
Experimental investigations
yuiA: ??
yycB: similar to permease
yodF: similar to proline permease
hisI: phosphoribosyl-AMP cyclohydrolase/phosphoribosyl-ATP pyrophosphohydrolase
yocR: similar to sodium-dependent transporter
bioA: adenosylmethionine-8-amino-7-oxononanoate aminotransferase
GlnR repressed gene ?
braB: branched-chain amino acid transporter
Experimental investigations
A GlnR knockout with a braB-lacZ fusion do have
higher -galactosidase activity than the wildtype
.... and two putative binding sites
ureP3 gatgtgaataaatataacaaaaaaagaagctgatttggtcaaggtaactaaattttttaaatattctttgaattaataggcctcttctatataatgatgacataagttgtaaacttttataacatcat
g cggtacaaggtatgatatttatgttaccataacaaaatttaatagaaaattacgaaaaactagtattgacttctaatttttttaccatataataagatttgttcgtttcgtcatattatctgacaatta
braB
TnrA box
TnrA box
TnrA box
TnrA box
-35 -10 +1
Verified new discoveries:
ureABC (urease operon) is activated by PucR
ACTIVITY OF yhdG
+ N – N allantoin glutamine
wildtype: 700 3.000 700 700
TnrA-mutant: 1.000
GlnA -mutant : 2.000
PucR -mutant : 3.000 2.000
yrbD and yhdG: activated by TnrArepressed by PucR
...more verified new discoveries
• Compentence is induced in glutamate medium = one-step transformation procedure
• A TnrA mutant has reduced competence ...only 2% of wildtype level
• TnrA somehow regulates the development of competence
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
Redundant Parsimonious
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