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