Transcript Transcriptome analysis of the TnrA regulon in Bacillus subtilis

Transcriptome analysis of the
TnrA regulon in Bacillus subtilis
Hanne Jarmer
Center 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
• 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
Glutamine synthetase
NH4+ + glutamate
glutamine
Access to nitrogen through
alternative pathways
gltAB
gabP
nasA
pucR
ykzB-ykoL
nasABCDEF
nrgAB
ansZ
ycs-kip
pucABCDE
glnR
glnA
GlnA Glutamine
Synthetase
GlnR
TnrA
tnrA
ureA
ureB
ureC
NH4/glutamate
wildtype
versus
wildtype
in different
media
Allantoin
Glutamate
Glutamine
TnrA
GlnA
wildtype
versus
mutant
in the same
medium
GlnR
NH2
Out
In
H
Adenine
N
N
H
N
N
H
Purine catabolism
H2O
Adeninedeaminase (adeC)
NH3
O
H
Hypoxanthine
N
HN
In
H
N
Out
TnrA
N
H
pbuG
O2
Xanthine dehydrogenase(pucABCDE)
O
Out
O
H
N
H
Xanthine
permease
(pbuX)
Guaninedeaminase(gde)
N
H
NH3
H2O
NH2
Guanine
N
HN
HN
In
O
N
H
N
N
H
pbuG
H Uric acid
N
O
N
H
CO2
2NH3 +CO2
Urease(ureABC)
PucR
H Allantoin
N
H2N
O
N
O
N H H
H
O2
Allantoinase(pucH)
Allantoin
permease
(pucI)
In
H2N
O
Allantoin
permease
(pucI)
Glyoxylic acid
O
OH
H2N
+
O
O
NH2
H
Pathway specific regulator
of purine catabolism
O
Out
H2O
Urea
O
In
Out
H2O2
O2
Uricase(pucLM)
Uricacid
permease
(pucJK)
Out
In
H
O2
Xanthinedehydrogenase(pucABCDE)
O
Out
O
Xanthine
N
HN
In
ALLANTOIN
H2O2
Ureidoglycolase (pucG)
CO2
Ureidoglycine
Ureidoglycolic acid
Allantoicacid
OH
O
OH
O
OH
Allantoate
Allantoate
NH2
HN
amidohydrolase(pucF) 2
amidohydrolase (pucF) H2N
N H N
H
H
O H O NH +CO
3
2
2
O
N H NH2 H O
2
H
NH3
O
N H OH
REFERENCE
SAMPLE
mRNA
mRNA
cDNA
cDNA
Cy5-cDNA
Cy3-cDNA
B. subtilis microarrays
• 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
NUMBERS:
2 different growth experiments
3 hybridizations (arrays) per culture
2 spots per gene on each array
2x3x2=
12 intensities for each gene
RESULT: 6 lists
Allantoin: 132
Glutamine:
60
Glutamate:
91
TnrA: 239
GlnA: 159
GlnR:
67
Extraction - Visualization
• 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
COFFEE BREAK - 10 MINUTES
GlnR
GlnA
TnrA
Opposite expression profile
Glutamine
Glutamate
Allantoin
– common TnrA regulated regulator?
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?
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
GlnR
GlnR
braB
GlnA
GlnA
TnrA
TnrA
Repressed
by TnrA ?
Glutamine
Glutamine
Repressed
Glutamate
by GlnR ?
Glutamate
Allantoin
Allantoin
pucA yrbD
ykoLyocR
tnrA bioA
pucR
yuiA nasA
yycB nrgA
yodF pucJ
gltA hisI
ykzB yyaF pucH ywoE gabP yhdG ycsF
Repressed by TnrA?
glnR
TnrA activated genes?
yrbD:
similar to sodium/proton-dependent
alanine carrier protein
yhdG:
similar to amino acid transporter
yyaF:
similar to GTP-binding protein
yrbD and yhdG have the TnrA box in a plausible position
Experimental investigations
TnrA repressed genes?
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-7oxononanoate aminotransferase
Experimental investigations
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
TnrA box
TnrA box
gatgtgaataaatataacaaaaaaagaagctgatttggtcaaggtaactaaattttttaaatattctttgaattaataggcctcttctatataatgatgacataagttgtaaacttttataacatcat
-35
-10
+1
g cggtacaaggtatgatatttatgttaccataacaaaatttaatagaaaattacgaaaaactagtattgacttctaatttttttaccatataataagatttgttcgtttcgtcatattatctgacaatta
TnrA box
TnrA box
braB
Verified new discoveries:
ureABC (urease operon) is activated by PucR
ACTIVITY OF yhdG
wildtype:
+N
–N
700
3.000
TnrA-mutant:
allantoin
glutamine
700
700
1.000
GlnA -mutant :
PucR -mutant :
yrbD and yhdG:
2.000
3.000
2.000
activated by TnrA
repressed 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
Acknowledgements
Novozymes Biotech Inc – Davis,
California:
Section of Molecular Microbiology DTU:
Randy Berka
Hans Henrik Saxild
Maria Tang
Lars Beier
Alan Sloma
Department of Microbiology –
Boston University:
Center for Biological Sequence
analysis – DTU:
Steen Knudsen
Susan Fisher
Chris Workman (software)
Lewis Wray, Jr.
Laurent Gautier (statistics)
Jaclyn Brandenburg
The array group (discussions)
Kristine Dahlin
Todays exercise
inferring regulatory networks
Experiment
Gene
nrgA
TnrA GlnA GlnR
purE
-1
1
1
-1
0
0
glnR
1
1
1
A
B
A
B
C
D
C
D