Proteome signatures for stress and starvation inBacillus subtilis as revealed by a 2-D gel image...

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RESEARCHARTICLE

Proteome signatures for stress and starvationin Bacillus subtilis as revealed by a 2-D gel imagecolor coding approach

Le Thi Tam, Haike Antelmann, Christine Eymann, Dirk Albrecht, J�rg Bernhardtand Michael Hecker

Institut f�r Mikrobiologie, Ernst-Moritz-Arndt-Universit�t Greifswald, Greifswald, Germany

In this paper we have defined proteome signatures of Bacillus subtilis in response to heat, salt,peroxide, and superoxide stress as well as after starvation for ammonium, tryptophan, glucose,and phosphate using the 2-D gel-based approach. In total, 79 stress-induced and 155 starvation-induced marker proteins were identified including 50% that are not expressed in the vegetativeproteome. Fused proteome maps and a color coding approach have been used to define stress-specific regulons that are involved in specific adaptative functions (HrcA for heat, PerR and Furfor oxidative stress, RecA for peroxide, CymR and S-box for superoxide stress). In addition, star-vation-specific regulons are defined that are involved in the uptake or utilization of alternativenutrient sources (TnrA, sL/BkdR for ammonium; tryptophan-activated RNA-binding attenuationprotein for tryptophan; CcpA, CcpN, sL/AcoR for glucose; PhoPR for phosphate starvation). Thegeneral stress or starvation proteome signatures include the CtsR, Spx, sL/RocRn, sB, sH, CodY,sF, and sE regulons. Among these, the Spx-dependent oxidase NfrA was induced by all stressconditions indicating stress-induced protein damages. Finally, a subset of sH-dependent proteins(sporulation response regulator, YvyD, YtxH, YisK, YuxI, YpiB) and the CodY-dependent aspartylphosphatase RapA were defined as general starvation proteins that indicate the transition to sta-tionary phase caused by starvation.

Received: February 8, 2006Revised: April 26, 2006Accepted: May 11, 2006

Keywords:Bacillus subtilis / Color coding / Proteome signatures / Starvation / Stress

Proteomics 2006, 6, 0000–0000 1

1 Introduction

The proteome obtained by the high resolution 2-D proteinelectrophoresis reflects the physiological state of a cell [1, 2].From a physiological point of view there are two main pro-teomes in microorganisms – the proteomes of growing and

nongrowing cells. The proteome of growing cells provides astable “vegetative core proteome” with a variable portion thatis determined by definite growth conditions. In the vegeta-tive proteome of Bacillus subtilis 745 proteins were identifiedusing the 2-D gel-based approach. These include more than40% of the predicted vegetative proteomes in the standardpH range 4–7 and most of the proteins involved in the cen-tral metabolic pathways [3]. Gel-free proteome approachessuch as 2-D-LC and MS/MS (2-D LC-MS/MS) resulted in theidentification of 814 proteins in the vegetative proteome ofB. subtilis including 473 new proteins not detected using the2-D gel-based approach [4].

In contrast, “proteome signatures” reflect changes of theproteome in response to physiological changes of B. subtilis,such as after exposure to different stress or starvation condi-tions [1, 2]. Proteome signatures include all specifically and

Correspondence: Dr. Haike Antelmann, Institut f�r Mikrobiologie,Ernst-Moritz-Arndt-Universit�t Greifswald, F.-L.-Jahn-Str. 15, D-17487 Greifswald, GermanyE-mail: [email protected]: 149-3834-864202

Abbreviations: AcsA, acetyl-CoA synthetase; BMM, Belitskyminimal medium; Spo0A, sporulation response regulator; TRAP,tryptophan-activated RNA-binding attenuation protein

DOI 10.1002/pmic.200600100

Figs. 2, 3, 4, 5, 6, 7 and 8 = 4C

VCH Auftr.-Nr. 32745 Vers.: 6.06 1. Papierlauf Seite 1 – hg –S : / 3D / v c h / _ p ro t e om i c s / p r _ 0 6 _ 1 6 / p r _ 0 01 0 0 . 3 d Proteomics Montag, den 19. 06. 06, 12 Uhr 28

ª 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

2 L. Thi Tam et al. Proteomics 2006, 6, 0000–0000

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generally induced marker proteins that are representative forone stress or starvation condition. Specific marker proteinsare induced by one specific stimulus only whereas generalmarker proteins are induced by multiple stimuli. These pro-teome signatures are useful for the prediction of the physio-logical state of the cell. In several studies proteome sig-natures were established in B. subtilis for heat, cold, salt, oxi-dative, and disulfide stress as well as in response to glucose,phosphate, and amino acid starvation [5–12]. Proteome sig-natures include for example the PerR-dependent oxidativestress-specific proteins KatA, AhpC, and AhpF [12]; the Spx-dependent proteins NfrA, TrxA, and Tpx for disulfide stress[11]; the HrcA-dependent chaperones DnaK and GroEL forheat shock [2]; the carbon catabolite-controlled proteins MalAand AcoB for glucose starvation [6]; and the PhoPR-depend-ent proteins PstS and TuaD for phosphate starvation [5, 7]. Inaddition, proteome signatures were defined in response todifferent antibiotics which are useful to predict the mode ofaction and the target of new antibiotics or antimicrobialsubstances [13, 14].

In this paper, we have complemented the vegetativeproteome map of B. subtilis by the proteome map of B. sub-tilis in response to stress and starvation using the 2-D gel-based approach. For that purpose we have analyzed thecytoplasmic proteome of 35S-methionine-labeled B. subtiliscells in response to heat, salt, hydrogen peroxide, and para-quat stress as well as after ammonium, tryptophan, glucoseand phosphate starvation. The resulting proteome maps ofB. subtilis after stress and starvation comprises 201 markerproteins identified in the standard pH range 4–7 thatinclude 83 stress- and starvation-specific marker proteinswhich are absent in the vegetative proteome map and118 proteins which exhibit a basal expression level in grow-ing cells [3]. The 2-D gel images from different time pointsof all stress or starvation experiments were combined by animage fusion approach in a stress-or starvation-fused pro-teome map reflecting specific and general proteome sig-natures.

2 Materials and methods

2.1 Bacterial strains and culture conditions

Cells of B. subtilis 168 (trpC2) [15] were cultivated under vig-orous agitation at 377C in Belitsky minimal medium (BMM)as described previously [16]. The stress experiments wereperformed by the exposure of exponentially growing cells atan OD500 of 0.4–487C (for heat shock), 6% NaCl (for saltstress), 116 mM H2O2, or 100 mM paraquat (for oxidativestress) (Fig. 1a). Glucose starvation was provoked by cultiva-tion in BMMwithout citrate (BOC) and 0.05% w/v instead of0.2% glucose. For the ammonium, tryptophan, or phosphatestarvation experiments, cells were grown in BMM containing0.7 mM instead of 15 mM (NH4)2SO4, 4 mM instead of80 mM tryptophan, or 0.2 mM instead of 1 mM KH2PO4,

Figure 1. (a) Growth curves of B. subtilis 168 in response to heat,salt, hydrogen peroxide, and paraquat stress. B. subtilis wasgrown in minimal medium to an OD500 of 0.4, and labeled beforeand at different times after heat shock (5, 10, and 20 min) orexposure to salt, hydrogen peroxide, and paraquat stress (10, 20,and 30 min) as indicated by arrows (1, 2, 3). (b) Growth curves ofB. subtilis 168 in response to ammonium, tryptophan, glucoseand phosphate starvation. B. subtilis was grown under differentstarvation conditions and labeled during the exponential growthphase for control (co), at the transition phase (1) and 10, 30, and60 min after transition to stationary phase (2, 3, and 4) as indi-cated by arrows.

respectively. In each starvation experiment the stationaryphase was reached at an OD at 500 nm (OD500) of about 1(Fig. 1b).

2.2 Preparation of the cytoplasmicL-[35S]methionine-labeled protein fraction

Cells grown in minimal medium were pulse-labeled for5 min each with 10 mCi/mL of L-[35S]methionine at an OD500

of 0.4 (for control), at different times (5, 10, 20, and 30 min)



Proteomics 2006, 6, 0000–0000 Microbiology 3

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after stress exposure or during transition phase and 10, 30,and 60 min after transition to stationary phase caused byammonium, tryptophan, glucose, or phosphate starvation.L-[35S]methionine incorporation was stopped after 5 min byaddition of 1 mg/mL of chloramphenicol and an excess ofcold L-methionine (10 mM) on ice. Two independent experi-ments were performed for each stress or starvation condi-tion. The cells were disrupted by ultrasonication, and thesoluble protein fraction was separated from the cell debris bycentrifugation. Incorporation of L-[35S]methionine wasmeasured by precipitation of aliquots of protein extracts with10% TCA on filter papers, as described previously [17].

2.3 2-DE

The protein content was determined using the Bradfordassay [18], and 80 mg of the L-[35S]methionine-labeled proteinextract was separated by 2-DE (2-D PAGE) using the non-linear IPG in the pH range 4–7 (Amersham Biosciences) anda Multiphor II apparatus (Amersham Pharmacia Biotech) asdescribed previously [17]. The gels were stained with SyproRuby, dried on filter paper and exposed to phosphor screens(Molecular Dynamics, Sunnyvale, California) which wereread out with a Phosphor Imager SI instrument (MolecularDynamics) [17]. For identification of the proteins by MS,nonradioactive protein samples of 200 mg were separated bypreparative 2-D PAGE. The resulting 2-D gels were fixed in40% v/v ethanol, 10% v/v acetic acid and stained with Col-loidal CBB (Amersham Biosciences).

2.4 Quantitative image analysis

Quantitative image analysis was performed with the DECO-DON Delta2D software (http://www.decodon.com) which isbased on the dual channel image analysis technique pio-neered in 1999 [17]. Using this software, the 2-D gel imagesfrom stress and starvation experiments were aligned to areference image (control) by using a warp transformation. Toavoid incomplete groups of matching spots a fused 2-D gelwas created for spot detection. For preparing such a fusiongel, all 2-D gel images from each single stress or starvationexperiments were combined using the spot preserving“union fusion” algorithm of Delta2D [19]. Spot detection wasperformed in the fusion gel containing all spots present inany gel of one stress or starvation experiment according tothe automatically suggested parameters for backgroundsubtraction, average spot size, and spot sensitivity. Theresulting spot shapes were reviewed and manually edited inthe fusion gel if necessary. This reviewed spot mask serves asa spot detection consensus for all gel images of the stress orstarvation experiment, and was applied to the individual gelsto guide the spot detection and quantitation. This enablesspot quantitation in all gels at the same locations resulting in100% matching and in a reliable analysis of completeexpression profiles. Normalization was performed by calcu-lating the quantity of each single spot in percentage related

to the total spot quantity per gel. Proteins showing an induc-tion of at least two-fold compared to the control during theL-[35S]methionine pulse in any time point of the two inde-pendent stress or starvation experiments were designated asstress-or starvation-induced marker proteins that are listedand classified in Table 1.

2.5 Color coding

The fused images including either the different stress pro-teome maps (for all time points after heat, salt, hydrogenperoxide, and paraquat stress) or the different starvationproteome maps (for all times during the transition to sta-tionary phase after ammonium, tryptophan, glucose andphosphate starvation) and the corresponding controls werecombined using the union fusion approach of the Delta2Dsoftware to generate fused proteome maps for all stress orstarvation conditions (Figs. 2, 4). The induced marker pro-teins were color coded according to their expression profilesby the Delta2D software as indicated in the figure legends.

2.6 Protein identification by MALDI-TOF-TOF MS

Spot cutting from Colloidal Coomassie-stained 2-D gels,tryptic digestion of the proteins and spotting of the resultingpeptides onto MALDI-targets (Voyager DE-STR, PerSeptiveBiosystems) were performed using the Ettan Spot HandlingWorkstation (Amersham Biosciences, Uppsala, Sweden),according to the standard protocol described previously [3].The MALDI-TOF-TOF measurement of spotted peptidesolutions was carried out on a Proteome-Analyzer 4700(Applied Biosystems, Foster City, CA, USA) and proteinidentification was performed using the MASCOT searchengine (Matrix Science, London, UK) as described previously[3].

3 Results

3.1 Fused proteome maps for stress and starvationand color codes

Growing B. subtilis cells were exposed for 5–30 min to differ-ent stress conditions (heat, salt, hydrogen peroxide, andparaquat stress) that except for heat shock result in adecreased growth rate (Fig. 1a). The different starvationresponses were provoked by growing the cells with limitingamounts of ammonium, tryptophan, glucose, or phosphate,respectively, that result in the transition to stationary phase atan OD500 of 1.0 (Fig. 1b). Starved cells were labeled at thetransition phase and 10–60 min after transition to stationaryphase. The fused images combining either the differenttimes of all particular stress proteome maps or the differenttime points of all particular starvation proteome maps weregenerated using the union fusion approach of Delta2D. Allmarker proteins induced specifically by one stimulus or




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Table

1.Protein

synthes

isratiosofm

arke

rproteinsforstress

andstarva

tionin

B.s

ubtilis

a)

Gene

orop

eron

Protein

Regu

lonAm

moniu

mTryptophan

Glucose

Phosphate

Heat

Salt

H 2O 2

Paraquat

Function/sim

ilarityb

)

t010

3060

t010

3060

t010

3060

t010

3060

510

2010

2030

1020

3010

2030

Stress

proteo

mesign

atures

HrcA

regu

lon

HrcA

-grpE-dn

aKJ

GrpE

HrcA

4,23,6

1,0He

at-shock

protein

(activa

tiono

fDnaK)

DnaK

HrcA

4,93,3

1,6Cla

ssIh

eat-s

hock

protein

(mole

cular

chaperone)

GroE

SLGroE

LHrcA

19,1

19,1

7,8Cla

ssIh

eat-s

hock

protein

(chaperone)

GroE

SHrcA

3,2–

–Cla

ssIh

eat-s

hock

protein

(chaperone)

CtsR

regu

lon

ClpE*

CtsR

343,6

56,8

5,31,2

15,6

21,7

ATP-dependentC

lpprotease-lik

eClp

PCtsR,s

B6,6

7,61,7

2,45,9

6,8AT

P-dependentC

lpprotease

proteolyticsubunit

CtsR-m

csAB

-clp

C-radA

-yac

KClp

CCtsR,s

B23,4

16,8

1,519,2

22,2

36,5

Class

IIIstr

essr

esponse-related

ATPa

se

PerR

regu

lon

AhpC

FAh

pCPe

rR4,9

4,22,3

11,2

11,7

18,0

Alkylhydroperoxid

ereductase

(small

subunit)

AhpF

PerR

3,12,6

1,29,3

12,0

11,2

Alkylhydroperoxid

ereductase

(large

subunit)

KatA

PerR,s

E2,9

4,73,5

2,92,0

2,54,5

5,313,6

3,90,3

107,4

147,7

139,7

Vegetativec

atala

se1

MrgA

PerR

15,4

2,11,6

6,35,6

5,6Metalloregula

tionD

NA-bind

ingstr

ess

protein

Furr

egulon

DhbA

CEBF

DhbA

*Fur

10,4

1,70,9

8,513,8

9,7Ba

cillibactin

siderophore

biosynthesis

DhbC

*Fur

4,02,1

0,62,8

4,22,8

Bacillibactin

siderophore

biosynthesis

DhbE

*Fur

23,4

6,70,6

14,2

20,2

14,2

Bacillibactin

siderophore

biosynthesis

DhbB

*Fur

48,0

9,32,2

19,6

33,1

27,5

Bacillibactin

siderophore

biosynthesis

FeuA

lipo *

Fur

5,12,6

0,6AB

C-tra

nsporte

rbind

ingprotein

(ba-

cillibactin

ande

nterobactin

uptake)

YclQ

lipo

Fur

1,21,0

0,7AB

C-tra

nsporte

rbind

ingprotein

(siderophoreu

ptake)

YcgT

*Fur,Sp

x1,7

1,41,0

2,12,1

2,4sim

ilartot

hioredoxin

reductase

YfiYl

ipo*

Fur

1,51,1

0,8AB

Ctra

nsporte

rbind

ingprotein

(schiz

okinin,arthrobactinuptake)

YxeB

lipo *

Fur

13,6

1,91,7

12,2

12,3

9,0AB

Ctra

nsporte

rbind

ingprotein

(ferrioxam

ineuptake)

YwbLMN

YwbM

*Fur

2,90,9

1,03,6

7,46,8

Eleme

ntalFe

transport(ye

astFTS

3homo

log)

SOSregu

lon

RecA

RecA

/LexA

5,96,5

4,9MultifunctionalS

OSrepairregulator




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Table

1.Continued

Gene

orop

eron

Protein

Regu

lonAm

moniu

mTryptophan

Glucose

Phosphate

Heat

Salt

H 2O 2

Paraquat

Function/sim

ilarityb

)

t010

3060

t010

3060

t010

3060

t010

3060

510

2010

2030

1020

3010

2030

UvrBA

UvrB**

RecA

/LexA

2,62,9

2,3Excin

uclea

seAB

C(su

bunit

B)

CymRan

dS-bo

xregu

lons

CysK

CymR

(YrzC

)4,7

0,72,3

Cyste

inesynthetase

AMetE

S-bo

x2,8

1,93,2

Cobalam

in-ind

ependent

methion

inesynthase

YitJ

S-bo

x3,2

1,33,4

Similartoh

omocysteine

s-me

thyltransferase

YrrT-m

tn-yrhAB

YrhB

**CymR

(YrzC

)5,6

1,52,7

Cysta

thion

inegamm

a-lya

seYxeIJK

LMNO

PQYxeP

**CymR

(YrzC

)6,2

1,93,4

Peptida

se,M

20/M

25/M

40family

YxeK

**CymR

(YrzC

)2,0

1,72,2

Similartom

onooxygenase

Spxregu

lon

HemE

HYHe

mHSp

x5,0

2,61,1

2,02,0

2,0Ferro

chela

tase

IolABC

DEFG

HIJ

IolS

IolR,

Spx

2,82,5

0,53,0

2,93,6

1,82,1

0,90,9

Myo-in

ositolcatabolism

NfrA-ywc

HNfrA

Spx

3,20,8

1,30,6

1,23,2

2,30,7

0,73,9

5,94,2

FMN-contain

ingNA

DPH-linkedn

itro/

flavin

reductase

SodA

Spx,sB

5,87,7

1,73,3

4,23,8

Superoxid

edism

utase

Tpx

Spx

1,13,1

2,81,6

2,61,1

1,12,0

2,43,1

Thiol

peroxid

ase

TrxA

Spx,sB

2,42,5

2,7Thior

edoxin

YfjR

Spx

3,01,1

1,11,8

2,52,5

Dehydrogenasep

recursor

YhfIJ

KYh

fKSp

x2,7

––

1,53,0

3,2Nu

cleoside

-diph

osphate-sugar

epimerase

YjbCD

YjbC

Spx,sM

,sB

2,72,7

2,93,3

1,43,7

4,04,2

1,81,9

6,6Sim

ilartoN

-acetyltra

nsferase

YjbG

Spx

3,34,7

4,13,5

2,31,1

1,32,9

3,53,2

Oligo

endopeptida

seFh

omolo

gYp

wASp

x–

––

2,3Sim

ilartoc

arboxypeptida

seYq

iGSp

x3,5

2,01,3

2,30,9

0,72,8

1,61,7

ProbableNA

DH-dependent

flavin

oxido

reductase

YuaE

Spx,sE

,Cod

Y9,8

10,3

32,0

29,6

0,81,0

1,53,2

1,31,4

48,3

2,91,4

0,25,2

6,26,2

Unknow

nYu

gJSp

x2,3

0,90,9

3,82,7

3,2ProbableNA

DH-dependent

butanol

dehydrogenase1

TnrA

regu

lon

AsnZ

(yccC

)As

nZ**

TnrA1

7,63,6

6,07,1

Similartoa

sparagina

seCa

hTn

rA1

16,6

24,1

13,3

25,0

13,4

17,7

25,9

32,3

1,08,1

8,515,6

7,61,3

2,9Ce

phalo

sporinCdeacetyla

seDp

pABC

DEDp

pA**

TnrA1,C

odY

5,98,0

6,85,8

3,23,6

5,76,5

D-ala

nyl-a

minopeptida

seGlnR

AGlnA

TnrA2,G

lnR2,5

2,33,0

3,0Glutam

inesynthetase

OppA

BCDE

FOp

pAlipo

TnrA1

3,75,6

12,0

8,6Oligo

peptide

ABCtra

nsporte

r(bin

ding

protein

)Op

pDTn

rA1

––

–3,0

Oligo

peptide

ABCtra

nsporte

r(ATP

-bind

ingprotein

)Pu

cJKLM

PucL**

TnrA1,s

E ,PucR

4,15,1

26,7

18,3

Urica

se




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Table

1.Continued

Gene

orop

eron

Protein

Regu

lonAm

moniu

mTryptophan

Glucose

Phosphate

Heat

Salt

H 2O 2

Paraquat

Function/sim

ilarityb

)

t010

3060

t010

3060

t010

3060

t010

3060

510

2010

2030

1020

3010

2030

TasA

TasA

sTn

rA1,s

H2,1

3,62,8

2,6Translo

cation-dependenta

ntimicr

obial

sporec

omponent

UreA

BCUreC

TnrA1,C

odY,

sH,P

ucR

19,4

74,9

24,9

12,2

2,22,2

2,62,9

Urease

(alph

asubunit)

Vpr

Vprs *

TnrA1,s

H9,3

21,8

12,6

7,7Mino

rextr

acellula

rserine

protease

YxbB

-A.yx

nB-

asnH

-yxaM

YxbB

**Tn

rA1,C

odY

9,511,2

10,7

12,4

6,26,8

8,610,0

2,34,8

3,17,8

Similartom

ethyltransfe

rase

sLRe

gulon(1

BkdR

,Roc

R,Ac

oR)

BkdR

-ptb-bcd

-buk-

Bcd

CodY

,sL ,B

kdR

9,012,3

14,4

7,9Leucine

dehydrogenase

-lpdV

-bkdAA

/AB/B

BkdA

ACo

dY,s

L ,Bk

dR10,0

6,01,3

0,9Branched-chainalp

ha-ketoa

ciddehydrogenaseE

1Su

bunit

(2-oxois

ovale

rate

dehydro-

genase

alpha

subunit)

LpdV

CodY

,sL ,B

kdR

5,84,6

1,01,2

Branched-chainalp

ha-ketoa

ciddehydrogenaseE

3Su

bunit

(dihy

drolipoam

idedehydrogenase)

RocA

BCRo

cACo

dY,s

L ,Roc

R,Cc

pA23,7

11,6

0,60,3

1,31,4

13,0

12,4

2,15,9

4,91,1

Pyrro

line-5c

arboxylated

ehydrogenase

RocD

EFRo

cDCo

dY,s

L ,Roc

R,Cc

pA3,6

3,82,5

1,20,8

1,16,7

3,9Ornithin

eaminotra

nsferase

RocD

EFRo

cFCo

dY,s

L ,Roc

R,Cc

pA4,9

2,50,9

0,70,8

0,41,7

5,3Argin

ase

RocG

CodY

,sL ,C

cpA

1,01,6

2,512,7

Glutam

ated

ehydrogenase

(majo

r)Ac

oABC

LAc

oA*

CodY

,sL ,A

coR

0,70,6

1,116,8

Acetoin

dehydrogenaseE

1com

p.(TPP

-dep.a

subunit)

AcoB

*Co

dY,s

L ,Aco

R0,7

0,50,3

14,0

Acetoin

dehydrogenaseE

1com

p.(TPP

-dep.b

subunit)

AcoC

*Co

dY,s

L ,Aco

R0,6

0,75,4

264,8

Acetoin

dehydrogenaseE

2com

ponent

(dihy

drolipoam

ideacetyltrans-

ferase)

AcoL*

CodY

,sL ,A

coR

1,00,8

1,022,7

Acetoin

dehydrogenaseE

3com

ponent

(dihy

drolipoam

idedehydrogenase)

TRAP

regu

lon

PabB

ACPa

bA**

TRAP

,RelA

38,4

35,7

32,5

22,8

Para-aminobenzoate

synthase

Glutam

ineam

idotra

nsferase

(subunit

B)TrpE

DCFB

ATrpE

**TR

AP25,0

22,8

31,6

31,8

Anthranilates

ynthase

TrpD

**TR

AP11,7

11,3

10,7

8,2An

thranilatep

hosphorib

osyltrans-

ferase




GALL

EYPR

OOF

Table

1.Continued

Gene

orop

eron

Protein

Regu

lonAm

moniu

mTryptophan

Glucose

Phosphate

Heat

Salt

H 2O 2

Paraquat

Function/sim

ilarityb

)

t010

3060

t010

3060

t010

3060

t010

3060

510

2010

2030

1020

3010

2030

TrpB

**TR

AP8,5

8,311,8

11,1

Tryptophan

synthase

(betas

ubunit)

TrpA

**TR

AP15,0

14,5

16,2

14,5

Tryptophan

synthase

(alph

asubunit)

CodY

regu

lon

AcsA

*Co

dY,C

cpA

5,06,4

9,35,3

1,14,5

13,4

21,8

1,63,7

5,75,4

Acetyl-Co

Asynthetase

AmhX

AmhX

CodY

4,24,2

3,73,4

3,42,6

3,83,8

0,41,0

9,1Am

idohydrola

seAp

pDFA

AppD

**Co

dY,R

elA,

CcpA

3,73,1

4,56,7

4,44,2

4,93,2

Oligo

peptide

ABCtra

nsporte

r(ATP

-bind

ingprotein

)Cit

BCo

dY,C

cpC,

CcpA

2,44,3

2,63,9

Aconitate

hydratase

Hag

CodY

0,74,1

4,02,1

Flagellin

protein

IlvD

CodY

1,61,9

2,62,8

Dihydroxy-aciddehydratase

RapA

-phrA

RapA

**Co

dY,R

elA,

CcpA

8,06,9

8,813,5

5,25,4

10,8

17,9

1,51,8

3,67,0

1,79,8

8,34,2

Response

regulator

asparta

tephosphatase

YurJ**

CodY

,TnrA2

2,63,2

1,10,4

19,3

19,3

13,3

9,3Multiples

ugar

ABCtra

nsporte

r(ATP

-bind

ingprotein

)Yu

rPON

ML

YurP

CodY

,TnrA2

3,84,3

3,41,4

1,72,3

2,11,3

Similartog

lutam

ine-fructose-6-

phosphatet

ransam

inase

YurO

lipo **

CodY

,TnrA2

4,36,7

6,72,2

9,810,1

10,9

6,1Sim

ilartom

ultiples

ugar-bind

ingprotein

YurL

CodY

,TnrA2

8,27,4

8,63,9

Similartor

ibokin

ase

YxbC

DYxbC

CodY

,RelA

,PerR6,5

8,99,5

11,0

1,54,0

3,67,8

3,81,5

1,1Sim

ilartou

nknown

protein

s

CcpA

regu

lon

BglPH

-yxiE

BglH*

CcpA

0,96,7

2,40,9

Beta-gluc

osida

seCit

Z-icd

-mdh

CitZ

CcpA

,Spx

3,34,0

6,95,4

3,42,8

3,44,0

0,91,8

1,62,9

Citrate

synthase

II(majo

r)GlpFK

GlpK

CcpA

,sE

0,87,0

2,811,9

Glycerolkin

ase

LicBC

AHLic

H*Cc

pA1,2

54,5

19,2

3,76-Ph

ospho-beta-gluc

osida

seMalA

MalA

CcpA

,Cod

Y0,9

1,57,9

57,3

6-Ph

ospho-alp

ha-gluc

osida

seMalL

MalL

**Cc

pA3,9

6,00,3

0,5Maltose-ind

uciblea

lpha-glu

cosid

ase

RbsRKD

ACB

RbsA

*Cc

pA1,2

15,3

5,424,9

RiboseA

BCtra

nsporte

rRb

sKCc

pA1,3

2,51,4

5,2Rib

okina

seYsiB

YsiB

CcpA

0,70,4

1,15,5

0,72,9

3,86,6

Similarto3

-hydroxbutyryl-Co

Adehydratase

YvdG

HIYvdG

lipo **

CcpA

0,90,9

0,411,3

Maltose/ma

ltodextr

in-bin

dingp

rotein

CcpN

regu

lon

GapB

GapB

*Cc

pN1,0

1,15,0

25,6

Glycerald

ehyde-3-phosphate

dehydrogenase

PckA

CcpN

1,20,5

1,117,2

Phosphoenolpy

ruvate

carboxykina

se

PhoP

Rregu

lon

TuaA

BCDE

FGH

TuaD

*Ph

oPR,

sF22,0

19,4

17,6

12,8

Biosynthesisof

teich

uronicacid




GALL

EYPR

OOF

Table

1.Continued

Gene

orop

eron

Protein

Regu

lonAm

moniu

mTryptophan

Glucose

Phosphate

Heat

Salt

H 2O 2

Paraquat

Function/sim

ilarityb

)

t010

3060

t010

3060

t010

3060

t010

3060

510

2010

2030

1020

3010

2030

TuaH

**Ph

oPR,

sF1,0

7,90,9

1,0Biosynthesisof

teich

uronicacid

PhoP

RPh

oPPh

oPR,

sE,C

cpA

1,93,2

3,84,4

Two-comp

onentresponser

egula

torfor

phosphater

egula

tion

PhoB

-ydh

FPh

oBs *

PhoP

R,sE

xAlkalinep

hosphatase

IIIPh

oDs *

PhoP

Rx

Alkalinep

hosphatase/phos-

phodies

terases

PstBA/BB

*Pho

PRx

PhosphateA

BCtra

nsporte

r(ATP

-bind

ingprotein

)PstSAC

/BA/BB

PstS

s *Ph

oPR

53,2

66,2

119,1

152,9

PhosphateA

BCtra

nsporte

r(phosphate

bindin

gprotein)

YdhF

s *Ph

oPR,

sE9,3

13,2

23,4

16,3

Lipoprotein

sBRe

gulon

Ctc

sB0,6

3,119,2

1,1x

4,2–

–19,2

22,2

36,5

Generalstre

ssprotein

Dps

sB2,4

1,61,7

3,50,6

3,02,3

0,61,1

1,81,7

2,02,8

1,91,2

9,19,6

7,1Stress-a

ndsta

rvation

-inducedD

NAbin

dingp

rotein

GsiB

sB1,5

3,12,8

2,40,5

0,257,8

5,51,2

1,63,9

2,350,4

43,8

21,2

1,123,6

37,6

Generalstre

ssprotein

GspA

*sB

0,51,1

9,11,7

1,54,1

3,53,4

19,4

15,4

2,50,5

5,72,0

Generalstre

ssprotein

GtaB

sB0,7

0,911,8

4,72,1

2,44,2

1,48,7

11,9

7,05,5

6,24,1

Glucosyla

tiono

fteic

hoicacid

KatE

sB0,8

0,57,9

2,7x

0,44,4

7,5Ca

talas

e2Na

dEsB

0,91,0

4,10,9

2,21,9

1,63,7

6,14,8

NADbio

synthesis

RsbR

STUV

W-

sigB-rsbX

RsbW

sB0,6

1,73,7

1,01,0

1,41,4

2,01,8

1,20,5

13,2

13,6

12,9

Anti-sfactor

ofsB

SigB

sB1,0

1,25,1

7,00,8

1,13,2

1,73,7

2,01,2

12,8

13,3

10,8

Generalstre

sssfactor

YcdFG

YcdF

sB2,3

––

0,83,6

4,7Sim

ilartog

lucose1

-dehydrogenase

YceC

DEFG

HYc

eCsW

,sB

10,7

11,0

12,3

Similartot

ellurium

resis

tancep

rotein

YceD

sW,s

B5,0

5,62,1

Similartot

ellurium

resis

tancep

rotein

YceH

sW,s

B2,1

1,51,6

2,55,3

6,9Sim

ilartotoxicanion

resis

tancep

rotein

YfhM

sW,s

B3,8

5,318,6

16,1

1,01,6

14,7

4,92,0

4,58,6

5,02,5

3,910,2

Similartoe

poxid

ehydrolas

eYd

aDEFG

YdaD

*sB

x0,4

13,4

4,0Ox

idoreductase

YdaE

*sB

9,39,1

8,38,7

10,2

5,28,8

6,00,4

3,68,7

1,41,8

3,25,4

2,7Probablesporec

oatp

olysaccharid

ebio

synthesis

protein

YdaG

sB0,8

1,04,6

0,7x

2,93,4

2,53,9

2,09,3

Generalstre

ssprotein

YdaP

YdaP

sB1,9

3,44,1

4,12,8

9,58,4

Similartop

yruvateo

xidase

YdaT

SYd

aT*

sB1,5

1,67,0

0,5Un

know

nYd

bD*

sBx

Similartom

anganese-containing

catalas

eYfkM

sB0,5

4,314,5

3,41,8

9,15,8

3,25,9

5,22,7

11,8

18,6

20,3

Generalstre

ssprotein

YfmA

-yflT

YflT

sB0,6

4,018,1

2,2x

Generalstre

ssprotein

YhdN

sB3,1

2,52,5

4,16,4

5,02,0

3,84,8

3,0Sim

ilartoa

ldo/ke

toreductase




GALL

EYPR

OOF

Table

1.Continued

Gene

orop

eron

Protein

Regu

lonAm

moniu

mTryptophan

Glucose

Phosphate

Heat

Salt

H 2O 2

Paraquat

Function/sim

ilarityb

)

t010

3060

t010

3060

t010

3060

t010

3060

510

2010

2030

1020

3010

2030

YkzA

sB0,3

1,622,1

2,1x

1,50,6

15,4

12,2

30,7

50,1

Similartoo

rganichydroperoxide

resis

tancep

rotein

YocK

*sB

0,51,3

12,6

1,8x

11,6

17,6

19,4

Similartog

eneralstr

essp

rotein

YvaA

sB5,1

4,42,6

Similartoo

xidoreductase

YsnF

sB0,4

2,615,3

7,42,2

1,78,5

27,4

22,3

3,76,5

4,89,7

31,6

Unknow

n

sHRe

gulon

FtsAZ

FtsA

sH,C

cpA

4,84,5

4,03,9

Require

dfor

septum

form

ation

durin

gsporula

tion

MinC

DMinD

sH5,9

8,06,8

5,83,2

3,65,7

6,51,0

2,93,0

5,3Ce

ll-divisio

ninhibitor(septum

placeme

nt)

RapG

-phrG

RapG

**sH

6,55,2

5,36,6

3,74,2

8,710,9

2,18,7

7,94,4

Response

regulator

asparta

tephos-

phatase

Spo0A

sH,R

elA,C

odY

6,76,2

7,46,3

3,62,5

4,03,0

0,91,0

0,92,0

1,72,9

3,04,0

Two-comp

onentresponser

egula

torfor

initiationo

fsporulation

SpoIIAA/AB

-sigF

SigF**

sH3,0

4,59,9

10,8

0,82,2

2,33,1

0,53,0

6,26,0

Sporula

tionf

orespore-specificsfactor

SpoV

GsH

,RelA

10,3

8,03,2

2,66,4

12,113,1

3,11,8

4,23,8

3,1Re

quire

dfor

sporec

ortexs

ynthesis

SpoV

S**

sH0,8

17,124,1

16,6

Require

dfor

dehydratation

ofthes

pore

core

YisK

sH3,5

2,81,4

0,74,7

4,84,5

3,30,7

1,24,6

17,3

1,84,2

4,12,7

5-Ox

o-1,2

,5-tricarboxilic

-3-pentenacid

decarboxyla

seYp

iABF

-qcrAB

CYp

iB**

sH,R

elA,S

px3,4

3,06,5

5,16,0

4,35,9

4,41,0

5,36,0

2,81,3

5,25,3

2,2Sim

ilartou

nknown

protein

sYtxG

HJYtxH

sH,R

elA,s

B5,5

4,94,3

4,96,4

7,46,4

5,60,7

2,95,6

1,11,5

2,03,9

2,12,6

1,81,2

6,18,6

6,8Sim

ilartog

eneralstr

essp

rotein

YuxI-

yukJ

YuxI*

*sH

27,9

25,2

21,1

28,5

2,32,1

2,72,8

0,80,5

2,73,1

2,116,5

52,0

38,8

Unknow

nYvyD

sH,R

elA,s

B46,6

13,0

3,22,5

76,0

62,7

50,5

36,2

0,65,5

30,5

2,78,1

22,2

21,3

2,93,6

––

16,8

1,14,0

2,26,5

9,0Sim

ilartos

54mo

dulating

factor

YwfBCD

EFG

YwfC

sH4,8

2,92,6

2,71,8

1,82,5

3,2Sim

ilartob

acilysin

biosynthesis

protein

bacA

sFRe

gulon

KatX*

sF,s

B–

––

2,6Majo

rcatala

seinspores

RsfA**

sF3,0

4,59,9

10,8

0,82,2

2,33,1

0,53,0

6,26,0

Regulator

oftra

nscription

ofsF-de-

pendentg

enes

SpoIIQ**

sF0,5

2,817,6

11,6

Require

dfor

comp

letion

ofengulfm

ent

SpoV

T**

sFcoo

Positive

andn

egative

regulator

ofsG

-dependent

genes

sERe

gulon

AsnO

**sE

coo

Asparagin

esynthetase

GlgB

CDAP

GlgA

**sE,s

H2,2

3,15,3

6,4Starch

(bacteria

lglyc

ogen)synthase

KamA

**sE

coo

0,96,6

10,3

31,6

lysine

2,3-aminomu

tase

PrkA

sE1,0

1,912,7

70,7

1,02,8

1,95,7

serin

eproteinkin

ase

SafA**

sE2,1

4,412,0

53,4

0,84,4

1,67,4

Morphogeneticprotein

associa

tedw

ithspoV

ID




GALL

EYPR

OOF

Table

1.Continued

Gene

orop

eron

Protein

Regu

lonAm

moniu

mTryptophan

Glucose

Phosphate

Heat

Salt

H 2O 2

Paraquat

Function/sim

ilarityb

)

t010

3060

t010

3060

t010

3060

t010

3060

510

2010

2030

1020

3010

2030

SpoIVA

sE1,2

0,819,4

128,4

0,73,3

0,93,2

Require

dfor

sporec

ortexforma

tion

andc

oata

ssem

blySp

oVR**

sE2,1

0,92,2

6,1Involve

dins

pore

corte

xsynthesis

SpoV

ID-ysxE

SpoV

ID**

sE,TnrA1

0,30,6

0,614,2

Require

dfor

assemb

lyofthes

pore

coat

YaaH

**sE,s

B1,3

3,326,5

104,9

Similartoc

orticalfra

gment-lytic

enzyme

YhbH

**sE

0,91,1

3,820,4

Putatives

tress

response

protein

YjbX**

sE0,8

0,71,4

10,9

0,92,7

2,65,3

1,31,5

5,83,9

Glutam

icacid-

richp

rotein

YybI**

sE9,4

2,58,4

7,4Un

know

n

Other

starva

tionor

stress

indu

cedproteins

AckA

AckA

Glu-

1,00,5

3,04,7

Acetatek

inase

AdeC

AdeC

RelA

3,65,4

5,93,3

Adenine

deam

inase

Ald

Ald

RelA,TnrA2

3,73,8

5,88,1

1,11,8

4,03,3

L-alan

inedehydrogenase

AroD

AroD

3,03,6

19,2

36,5

Shikima

te5-dehydrogenase

Dat

Dat

3,53,2

4,03,3

2,62,3

3,42,9

1,23,4

3,11,9

Probable

D-ala

ninea

minotra

nsferase

DegS

UDe

gS*

DegS

U7,6

5,86,2

5,5Tw

o-comp

.sensork

inase

for

degradative

enzyme

sDe

gUDe

gSU

4,84,9

6,04,6

Two-comp

.responser

egula

torfor

de-

gradative

enzyme

sDh

aSDh

aS2,6

1,07,7

7,90,9

2,22,2

6,00,6

2,67,2

7,4Aldehyde

dehydrogenase

EtfA**

0,62,1

10,1

18,9

Electront

ransferflav

oprotein(alph

asubunit)

EtfB**

0,84,0

13,440,9

Electront

ransferflav

oprotein(beta

subunit)

FabF

2,62,1

1,5Be

ta-ketoacyl-a

cylcarrie

rprotein

synthase

IIFolD

FolD

0,80,9

1,813,1

5,44,3

2,75,4

1,23,6

1,44,8

Methylen

etetrahydrofola

tedehydro-

genase

/HtpG

HtpG

3,35,1

3,90,5

8,45,9

6,6Cla

ssIII

heat-shock

protein

(mole

cular

chaperone)

GcvT-gcvPA

BGc

vT–

––

6,4Am

inome

thyltransferase

GlmS

GlmS

Glu-

6,67,8

8,010,4

0,70,9

0,411,1

L-glutam

ine-D-fructose-6-phosphate

amido

transferase

Hom

Hom

3,13,4

3,92,5

Homo

serin

edehydrogenase

IspA*

*–

––

7,3Majo

rintracellular

serin

eprotease

Kbl*

Glu-

0,30,7

5,814,8

2-Am

ino-3-ketobutyrateC

oAligase

Men

BMen

B0,8

0,91,8

13,0

5,44,3

2,75,4

1,23,6

1,44,8

Dihydroxynapthoic

acidsynthetase

MurB

MurB

5,06,1

7,59,1

2,11,0

4,210,6

0,91,1

4,42,5

UDP-N-acetyle

nolpy

ruvoylg

lucosam

inereductase

PbpE

PbpE

wall

34,5

17,2

14,2

9,8Pe

nicillin-bin

dingp

rotein4




GALL

EYPR

OOF

Table

1.Continued

Gene

orop

eron

Protein

Regu

lonAm

moniu

mTryptophan

Glucose

Phosphate

Heat

Salt

H 2O 2

Paraquat

Function/sim

ilarityb

)

t010

3060

t010

3060

t010

3060

t010

3060

510

2010

2030

1020

3010

2030

PksB

CDE-ac

pK-pksF

PksC**

1,65,0

9,57,3

Involve

inpolyk

etide

synthesis

PtsG

HIPtsH

Glu-

0,52,0

9,11,0

3,6–

–Ph

osphocarrie

rproteinof

theP

Ts(HPr

protein

)Ya

aQ1,4

1,02,7

0,2Un

know

nYb

aL5,6

1,52,7

SimilartoA

TP-bind

ingMrp-lik

eprotein

YcgN

YcgN

3,219,8

15,7

9,65,3

5,94,3

8,10,7

1,628,2

4,0Sim

ilarto1

-pyrroline-5-carboxyla

tedehydrogenase

YhfE

1,12,0

2,91,3

Similartog

lucanase

YhjL*

*3,7

5,525,6

216,7

Sensorytransduction

pleiotropic

regulatoryp

rotein

YjcLK

YjcK**

2,62,9

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generally by multiple stimuli are labeled in these fused pro-teome maps for stress and starvation by a defined color codeof 15 different colors that indicates the induction profile. InFig. 2 we show the fused proteome map combining fourstress conditions: heat, salt, hydrogen peroxide, and paraquatstress. In Fig. 4 we show the fused proteomemap combiningfour starvation conditions: ammonium, tryptophan, phos-

phate, and glucose starvation. These fused images and colorcodes for different stress or starvation conditions can be usedas a tool to define stress/starvation-specific proteins inducedby one single stimulus or more general stress/starvationproteins induced by multiple stimuli. According to theirspecific color codes, the proteins can be classified into speci-fically or generally induced regulons (Figs. 3, 5 and Table 1).

Figure 2. Color-coded fused proteome map of B. subtilis exposed to heat, salt, hydrogen peroxide, and paraquat stress. The protein syn-thesis patterns (autoradiograms) of B. subtilis exposed to heat, salt, hydrogen peroxide, and paraquat stress were combined to generate afused stress proteome map of B. subtilis using the union image fusion approach of the Delta2D software. The induced marker proteinswere color coded according to their expression profiles. All spots induced specifically after one stress or generally also by other stressesare labeled in the fused proteome map by a defined color code that indicates the induction profile.




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Figure 3. The specific and general stress regulons in B. subtilis.Commonly shared (generally induced) and unique (specificallyinduced) stress regulons and proteins in the proteome of B. sub-tilis exposed to heat, salt, hydrogen peroxide, and paraquatstress according to the fused stress proteome map in Fig. 2. Thespecific and general stress regulons are underlined, and theencoded specific or general stress proteins are listed in paren-theses.

3.2 Stress proteome signatures

3.2.1 Proteome signature for heat shock (487C)

In total, 36 marker proteins are induced in the proteomemap after the temperature up-shift to 487C of which ClpEand GspA are not present in the vegetative proteome map(Fig. 2, Table 1). The heat shock response involves mainly theHrcA, sB, and CtsR regulons (Fig. 3) [20]. Since heat shockcauses protein damages, such as non-native disulfide bondformation, the Spx regulon that is required for thiol home-ostasis is induced [21].

The HrcA-dependent chaperones DnaK, GroEL, GroES,and GrpE are heat-specific marker proteins that are red-labeled in the fused stress proteome map (Fig. 2). Surpris-ingly, two proteins of the sB regulon (YceD and YvaA) arealso heat-specific marker proteins.

General marker proteins for heat and salt stress as well asstarvation for glucose and phosphate are 16 sB-dependentproteins (Ctc, Dps, GsiB, GspA, GtaB, NadE, RsbW, SigB,SodA, YcdF, YceH, YdaG, YfkM, YhdN, YkzA, YsnF) [22]. Inaddition, the CtsR-dependent chaperones and proteasesClpC, ClpP, and ClpE are general marker proteins for heatand salt stress. General Spx-dependent marker proteins forheat and oxidative stress include NfrA, IolS, YhfK, YjbG, andYqiG [21]. Finally, the class IV heat shock protein HtpG is ageneral marker protein for heat shock and phosphate starva-tion.

3.2.2 Proteome signature for salt stress (6% NaCl)

The response to salt stress provoked by the addition of6% NaCl is reflected by the induction of 33 marker proteinsof which ClpE, GspA, YocK, and YdaD are not present in thevegetative proteome map (Fig. 2, Table 1). These markerproteins can be classified according to the sB, CtsR, and Spxregulons (Fig. 3) [10, 21, 22].

Two pink-labeled marker proteins are specificallyinduced by salt stress, namely the sB-dependent telluriumresistance protein (YceC) and the fatty acid biosynthesis en-zyme (FabF). The general proteome signature for salt andheat stress includes the 16 above-mentioned sB-dependentmarker proteins. Other sB-dependent marker proteins YdaP,YdaD, KatE, YocK, and YfhM are induced generally by saltstress, phosphate, and glucose starvation [22]. The CtsR reg-ulon members ClpC, ClpE, and ClpP and the Spx-dependentproteins NfrA and YuaE are general marker proteins for saltand heat or oxidative stress. Finally, YurU involved in Fe-S-cluster assembly and the ABC-transporter ATP-binding pro-tein YurY belong to general marker proteins for salt stress,ammonium, or tryptophan starvation [10, 21, 22].

3.2.3 Proteome signature for oxidative stress(116 mMH2O2 and 100 mM paraquat)

The exposure of cells to 116 mM H2O2 or 100 mM paraquatresulted in the induction of 28 peroxide marker proteins or31 superoxide marker proteins including 14 proteins that areabsent in the vegetative proteome map (Fig. 2, Table 1).These marker proteins can be classified into the oxidativestress regulons PerR, Fur, and Spx as well as the oxygenradical specific SOS regulon and the sulfur limitation re-sponse (Fig. 3) [11, 21, 23, 24].

Marker proteins for peroxide and superoxide stress arethe light-orange labeled oxidative stress-specific PerR-de-pendent proteins KatA, AhpC, AhpF, and MrgA and sevenFur-dependent proteins including the siderophore bacilli-bactin biosynthesis enzymes (DhbA, DhbB, DhbC, andDhbE), the ferrioxamine ABC-transporter binding protein(YxeB), the Fe-transporter (YwbM), the thioredoxin reductase(YcgT), and three proteins belonging to Spx regulon (HemH,YfjR, YugJ) [21, 23]. These light-orange labeled oxidativestress-specific marker proteins are most strongly induced byboth reactive oxygen species and involved in specific adapta-tive functions to oxidative stress (Fig. 2). Surprisingly, threeorange-labeled ABC-transporter binding proteins of the Furregulon that are involved in the uptake of the siderophoresbacillibactin, enterobactin (FeuA), schizokinin, arthrobac-tin (YfiY), or unknown siderophores (YclQ) are specificallyinduced only after peroxides [23, 24].

The orange-labeled proteins UvrB and RecA of the DNAdamage inducible SOS regulon are induced specifically afterperoxide stress (Figs. 2, 3) [25]. In contrast, four yellow-labeled proteins involved in sulfur assimilation (YxeK, YxeP)and cysteine biosynthesis (CysK, YrhB) represent superoxide




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Figure 4. Color-coded fused proteome map of B. subtilis after ammonium, tryptophan, glucose and phosphate starvation. The proteinsynthesis patterns (autoradiograms) of B. subtilis in response to starvation for ammonium, tryptophan, glucose, and phosphate werecombined to generate a fused starvation proteome map of B. subtilis using the union image fusion approach of the Delta2D software. Theinduced marker proteins were color coded according to their expression profiles. All spots induced specifically after one starvation condi-tion or generally also by other starvation conditions are labeled in the fused proteome map by a defined color code that indicates theinduction profile.

specific marker proteins that are regulated by the cysteinemetabolism repressor CymR (YrzC). The superoxide specificmarker proteins MetE and YitJ are involved in methioninebiosynthesis and regulated by the S-box transcription anti-termination system (Figs. 2, 3) [11, 26]. Further superoxidespecific marker proteins are YbaL and YpsC.

General marker proteins for oxidative, heat or salt stressbelong to the Spx regulon including IolS, NfrA, Tpx, YhfK,YjbG, YqiG, and YuaE [21]. Of these the flavin mononucleo-tide-dependent NADPH oxidase NfrA was identified asglobal Spx-dependent protein induced by all four stress con-ditions.




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3.3 Starvation proteome signatures

3.3.1 Proteome signature for ammonium starvation(0.7 mM (NH4)2SO4)

The ammonium starvation proteome showed 84 inducedmarker proteins including 31 proteins that are not present inthe vegetative proteome map (Fig. 4, Table 1). These can beclassified according to the TnrA, sL/BkdR, sL/RocR, CodY,sB, sH, sF, and sE regulons (Fig. 5). Of major importanceare the red-labeled specific marker proteins of the TnrA andsL/BkdR regulons, which are known to be induced by nitro-gen starvation and involved in the utilization of alternativenitrogen sources such as proteins, peptides, purines,branched chain amino acids, and asparagine [27–30].

The specific proteome signature for ammonium starva-tion is reflected by seven red-labeled TnrA-regulated proteinsincluding the asparaginase (AsnZ), the glutamine syntheta-se (GlnA), the uricase (PucL), the oligopeptide-transporters(OppA and OppD), the extracellular serine protease (Vpr),and the translocation-dependent antimicrobial spore com-ponent (TasA) (Figs. 4, 5 and Table 1) [28, 29]. In addition,the red-labeled sL/BkdR-dependent proteins (Bcd, LpdV, andBkdAA) represent specific marker proteins for ammoniumstarvation that are involved in the catabolism of branchedchain amino acids (isoleucine and valine) (Fig. 5) [30]. Otherspecific marker proteins are AroD, CitB, GcvT, IspA, YqeH,YvaB, and LiaH; the sF-dependent sporulation proteinsKatX, SpoIIQ, and SpoVT; and the sE-dependent sporulationproteins AsnO, SpoVR, SpoVID, YaaH, and YhbH [31, 32].

Figure 5. The specific and general starvation regulons in B. sub-tilis. Commonly shared (generally induced) and unique (specifi-cally induced) starvation regulons and proteins in the proteomeof B. subtilis after ammonium, tryptophan, phosphate, and glu-cose starvation according to the fused starvation proteome mapin Fig. 4. The specific and general starvation regulons are under-lined and the encoded specific or general starvation proteins arelisted in parentheses.

The general proteome signature for ammonium andtryptophan or phosphate starvation is represented by11 CodY-dependent proteins including the dipeptide- andoligopeptide-transporters (AppD, DppA), the urease (UreC),the acetyl-CoA synthetase (AcsA), the aminohy-drolase (AmhX), the sugar metabolism system (YurJ, YurO,YurP), the Spo0F,P aspartyl phosphatase (RapA), and theproteins of unknown functions (YxbB and YxbC) [33, 34]. Inaddition to AcsA, the sL/RocR-dependent arginine and orni-thine catabolic proteins (RocA, RocD, RocF), the citrate syn-thase (CitZ), and the 3-hydroxbutyryl-CoA dehydratase (YsiB)are also under CcpA-dependent glucose repression con-sistent with their general induction in response to ammo-nium and glucose starvation [5, 35–37]. Further generalmarker proteins for ammonium and glucose starvationinclude four sB-dependent proteins (Dps, SigB, YdaE, YfhM)[22] and six sE-dependent sporulation proteins (YuaE, KamA,PrkA, SafA, SpoIVA, YjbX) [31, 32].

In addition, 11 marker proteins of the transition phasesH regulon are generally induced by ammonium and tryp-tophan starvation including proteins involved in cell divi-sion (MinD), the sporulation response regulator (Spo0A),the response regulator aspartyl phosphatase (RapG), thesporulation protein (SpoVG), the sB- and sH-controlled pro-teins (YvyD and YtxH), the 5-oxo-1,2,5-tricarboxilic-3-pentenacid decarboxylase (YisK), the sF transcription factor, and theproteins of unknown functions (YpiB, YuxI, and YwfC) [38–40]. Interestingly, a subset of six sH-dependent proteins(Spo0A, YvyD, YtxH, YpiB, YisK, YuxI) and the CodY-de-pendent RapA are induced by all four starvation conditionsand these proteins are defined as general starvation proteins.

3.3.2 Proteome signature for tryptophan starvation(4 mM tryptophan)

In response to tryptophan starvation we identified 47 markerproteins including 20 proteins that are not present in thevegetative proteome map (Fig. 4, Table 1). These markerproteins can be classified according to the tryptophan-acti-vated RNA-binding attenuation protein (TRAP), CodY, sB,sE, and sH regulons (Fig. 5).

Specifically and most strongly induced are five pink-labeled tryptophan biosynthetic enzymes TrpA, TrpB,TrpD, TrpE, and PabA (TrpG) that are regulated by theTRAP (Fig. 5) [41, 42]. Further tryptophan starvation-spe-cific marker proteins include two CodY-dependent pro-teins (the dihydroxy-acid dehydratase IlvD and the riboki-nase YurL), the sE-dependent sporulation protein (YybI),and two sH-dependent proteins (FtsA and SpoVS); thehomoserine dehydrogenase (Hom), the adenine deamina-se (AdeC), and the two component system regulatingextracellular enzyme synthesis, competence, and motility(DegS and DegU).

General marker proteins for tryptophan and ammoniumstarvation belong to the CodY- and sH-regulons as written inSection 3.3.1. In addition, the sB-dependent proteins GsiB




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and YdaE as well as the sE-dependent catalase KatA are gen-eral marker proteins for tryptophan and other starvationconditions.

3.3.3 Proteome signatures for glucose starvation(0.05% glucose)

The glucose starvation proteome signature revealed theinduction of 65 marker proteins of which 26 proteins are notpresent in the vegetative proteome map (Fig. 4, Table 1).These include the most strongly induced 22 carbon catabo-lite-controlled proteins and members of the sB, sF, and sE

regulons (Fig. 5) [6, 36, 37, 43, 44].Specifically induced in response to glucose starvation are

nine orange-labeled CcpA-dependent carbon catabolite-con-trolled proteins involved in utilization of alternative carbonsources such as aryl-b-glucosides (BglH), lichenan (LicH),glycerol (GlpK), ribose (RbsA and RbsK), maltose (MalA,MalL, and YvdG), and glutamate (RocG). In addition, theorange-labeled sL/AcoR-dependent proteins for utilization ofacetoin (AcoA, AcoB, AcoC, and AcoL) are glucose starvation-specific marker proteins (Fig. 5) [6, 36, 37]. The CcpN-regu-lated proteins involved in gluconeogenesis including theglyceraldehyde-3-phosphate dehydrogenase (GapB) and thephosphoenolpyruvate carboxykinase (PckA) are also specifi-cally induced in response to glucose starvation (Fig. 5) [44].Finally, the acetate kinase (AckA), the 2-amino-3-ketobuty-rate CoA ligase (Kbl), and the electron transfer flavoproteinsubunits (EtfA and EtfB) were identified as glucose starva-tion-specific marker proteins [6, 37].

The general proteome signature for glucose and ammo-nium or tryptophan starvation includes six further CcpA-controlled proteins (AcsA, CitZ, RocA, RocD, RocF, andYsiB). In addition, 17 sB-dependent proteins (GsiB, GspA,GtaB, Dps, RsbW, SigB, YfkM, YsnF, Ctc, KatE, YdaE, YdaG,YflT, NadE, YfhM, YocK, YkzA) are induced in response toglucose and phosphate starvation as well as by heat and saltstress [22]. The six sH-dependent general starvation proteins(Spo0A, YisK, YpiB, YuxI, YvyD, and YtxH) and RapA aregeneral marker proteins for all starvation conditions [38–40].Finally, the sF-dependent RsfA and six sE-dependent spor-ulation proteins are general marker proteins for glucose andammonium starvation as referred in Section 3.3.4.ncheckchangen [31, 32].

3.3.4 Proteome signature for phosphate starvation(0.2 mM KH2PO4)

The proteome signature in response to phosphate starvationrevealed the induction of 62 marker proteins of which 27 areabsent in the vegetative proteome map (Fig. 4, Table 1).These marker proteins can be classified according to thephosphate starvation-specific PhoPR regulon as well as thegenerally induced sB, CodY, and sH regulons (Fig. 5).

Eight yellow-labeled PhoPR-dependent proteins arespecifically and most strongly induced in response to phos-phate starvation including the alkaline phosphatase (PhoB),the phosphodiesterase (PhoD), the response reg-ulator (PhoP), the phosphate-specific ABC-transporter bind-ing protein (PstS), the ATP binding protein of the phosphate-transporter (PstBB/BA), the lipoprotein YdhF, and the tei-churonic acid biosynthesis proteins TuaD and TuaH (Fig. 5)[5, 7, 45, 46]. In addition, PksC involved in polyketide syn-thesis, the putative regulator YhjL, the glucanase YhfE, thetransaldolase YwjH, the sH-dependent glycogen synthaseGlgA, the sD-dependent flagellin Hag, YaaQ and the sB-de-pendent proteins YdaT and YdbD can be regarded as phos-phate starvation-specific marker proteins [5].

General CodY-dependent marker proteins for phosphateand ammonium starvation include AcsA, YxbB, YxbC, andRapA [33, 34]. In addition, 17 sB-dependent proteins aregeneral marker proteins for phosphate and glucose starva-tion (GsiB, GspA, GtaB, Dps, RsbW, YfkM, YsnF, Ctc, KatE,YdaG, YflT, YocK, YkzA, YfhM, YdaE) or phosphate starva-tion and salt stress (YdaD, YdaP) [22]. Four sH-dependentproteins are induced by phosphate and ammonium starva-tion (MinD, RapG, SpoVG, SigF) in addition to the six sH-dependent general starvation proteins (Spo0A, YisK, YpiB,YuxI, YvyD, and YtxH) [38–40]. The sE-dependent YjbX andthe sF-dependent RsfA are general marker proteins forphosphate, glucose, and ammonium starvation and HtpGoverlaps between heat shock and phosphate starvation.

4 Discussion

4.1 The proteome of B. subtilis in response to stressand starvation

Soil-dwelling bacteria, such as B. subtilis have developed avery complex adaptational network to overcome changes intheir environment provoked by different stress and starva-tion conditions. Based on previous genome-wide expressionprofiling with mutants in the central regulatory genes (e.g.,alternative sigma factors, transcriptional activators, andrepressors) the stimulons that are induced by the specificstress or starvation conditions are classified in their respec-tive regulons. However, these analyses have not only definedspecifically induced regulons, but also enabled the globalanalysis to define generally induced regulons that respond todifferent stimuli. The proteome analysis is a powerful tool tovisualize the panorama view of the physiological state of thecell. A set of marker proteins is induced in response tochanges in the environment that was defined as proteomesignature indicative for specific stress or starvation condi-tions [1, 2].

In this study, the proteome signatures of B. subtilis fordifferent stress and starvation conditions could be completedby 45 new marker proteins not detected in the previousseparate proteome analyses for heat, salt, oxidative stress, or




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Figure 6. The vegetative proteome map of B. subtilis including all newly induced marker proteins after stress and starvation The proteinsynthesis pattern (autoradiogram) of B. subtilis during vegetative growth conditions. All 83 red-labeled proteins are induced by stress orstarvation only and not present in the vegetative proteome map.

starvation for glucose and phosphate (Figs. 6–8) [2, 5–7, 10,12]. In total, 201 stress- or starvation-induced proteins havebeen identified, including 83 proteins that are absent in thevegetative proteomemap (Figs. 6–8). Heat, salt, and oxidativestress caused the induction of 79 marker proteins including18 new proteins not present in the vegetative proteome map.In response to ammonium, tryptophan, glucose, and phos-phate starvation 155 marker proteins are induced including68 new proteins not detected in the vegetative proteome map(Figs. 6–8). As novel proteome signatures for B. subtilis, theresponses to ammonium and tryptophan starvation were

defined including 32 newly identified marker proteins.There are 68 new starvation proteins compared to only18 new stress proteins. This indicates that the starvationproteins are not expressed during vegetative growth condi-tions in contrast to the stress proteins that show a basalexpression level in vegetative growing cells. The total num-ber of 83 new proteins seems probably low since in mostcases more than 100 genes are induced in response to onestress or starvation condition at the transcriptome level.However, we have to consider that some induced markerproteins could not be identified in coomassie-stained 2-D




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Figure 7. The fused proteome map of all stress and starvation conditions of B. subtilis including all induced marker proteins. The proteinsynthesis patterns (autoradiograms) of B. subtilis in response to heat, salt, hydrogen peroxide, and paraquat stress as well as after star-vation for ammonium, tryptophan, glucose, and phosphate were combined to generate a fused global proteome map of B. subtilis cellsafter stress and starvation using an image fusion approach of the Delta2D software. All 201 labeled proteins are induced by stress or star-vation. Among these the 83 proteins that are not present in the vegetative proteome map are marked with red labels. Some induced pro-teins which were identified in previous proteome analyses but not in the present study were marked with circles.

gels since these are synthesized at increased levels afterlabelling with 35S-methionine but do not accumulate in theproteome. Furthermore, the gene products for manyinduced genes that have been identified in the tran-scriptome analysis escape the 2-D gel based proteomeapproach since these are hydrophobic transmembrane pro-teins or alkaline proteins.

4.2 Functional characterization of stress proteins

The catalog of stress and starvation proteome signatures mayhelp to define the mode of action of unknown anti-microbials, antibiotics, and xenobiotics or to analyze thephysiological changes and kind of limitation in complexnondefined media, for example, during an industrial




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monly shared and unique proteins in the vegetative proteomemap of B. subtilis (Fig. 6) as well as in the fused stress and star-vation proteome maps (Figs. 2, 4).

fermentation process [13, 14, 47, 48]. Using the novel gel-based approach of fused proteome maps and color codes forall stress or starvation conditions it was possible to visualizefor the first time all specifically and generally induced reg-ulons in one single image for stress (Figs. 2, 3) and starvation(Figs. 4, 5), respectively. For example, the heat shock sig-nature is indicated by the induction of the heat-specificHrcA-dependent chaperones and the general induction ofthe sB, CtsR, and Spx regulons (Fig. 3) [20]. The treatmentwith hydrogen peroxide and paraquat resulted in the oxida-tive stress-specific PerR and Fur regulons as well as in theSpx regulon as general indicator for protein damages by non-native disulfide bond formation (Fig. 3) [12, 21, 24]. In con-trast, the SOS regulon induction is specific for DNA dama-ges caused by peroxides only. The CymR regulon that reg-ulates cysteine metabolism and the S-box regulon involved inmethionine biosynthesis were induced in response to super-oxides only (Fig. 3) [12, 24–26]. These specific and generalstress regulons are strongly expressed after 10 min of stressexposure and this expression proceeds until the 20–30 mintime points (Table 1). One exception is the heat shock re-sponse resulting in a rapid increase of protein synthesis after5 min which drops after 10 min.

The specific adaptative functions of the stress-specificregulons (e.g., HrcA, PerR) are to confer specific resistancemechanisms against the stress and thereby enable the cell(i) to neutralize the stressor, (ii) to adapt to the specific stres-sor, or (iii) to repair damages caused by the stressor [2]. Incontrast, the sB-dependent general stress regulon encodesproteins with different functions conferring a multiple,nonspecific and preventive stress resistance to nongrowingB. subtilis cells in anticipation of future stress possiblyencountered during long-term stationary growth stages [2,49]. Induction of the general stress response by one stressaffords significant cross-protection against other stresses [2].It was further demonstrated that sB is required for growthand stationary phase survival at low temperatures [50].

Interestingly, the flavin mononucleotide-dependentNADPH oxidase NfrA is one representative of the Spx reg-ulon that is generally induced by all analyzed stresses indi-cating protein damage by non-native disulfide formation inresponse to heat, salt and oxidative stress (Fig. 3). In Staphy-lococcus aureus NfrA has been shown to be involved in themaintainance of the thiol-disulfide balance under oxidativestress conditions [51]. Since the color coding approach iden-tified NfrA as a broader general stress protein it will beinteresting to analyze the survival of a B. subtilisNfrAmutantunder different stress conditions.

4.3 Functional characterization of starvation proteins

For the first time, a fused proteome map for ammonium,tryptophan, glucose and phosphate starvation was createdthat provided the tool to define specifically induced starva-tion regulons and more generally induced transition phaseregulons (Figs. 4, 5). The induction of the TnrA- and sL/BkdR-dependent catabolic enzymes for alternative nitrogensources indicates an ammonium starvation-specific pro-teome signature (Fig. 5) [27–30]. In contrast, the induction ofthe TRAP-regulated tryptophan biosynthesis enzymes is thespecific proteome signature for tryptophan starvation (Fig. 5)[41, 42]. In response to glucose starvation several carbon cat-abolite-controlled marker proteins are specifically activatedin the absence of glucose and repressed in the presence ofglucose by CcpA, CcpN, and AcoR (Fig. 5) [36, 37, 44].Finally, the induction of the PhoPR regulon is the specificproteome signature for phosphate starvation (Fig. 5) [5, 7, 45,46]. These specific starvation regulons such as TnrA, TRAP,CcpA, or PhoPR were shown to be specifically involved in theuptake and utilization of alternative nutrient sources in re-sponse to the specific nutrient limitation. Consequently, thespecific starvation response is strongly induced during thetransition phase and remains constant until 60 min aftertransition to stationary phase (Table 1).

The general proteome signature for different kinds ofnutrient limitation is mediated by the CodY, sB, and sH

transition phase regulons (Fig. 5). These general starvationregulons are required for the adaptation of the cell to post-exponential stationary phase processes such as survivalunder nongrowing conditions, competence, or sporulation.The induction of the CodY regulon after ammonium andtryptophan starvation is reflective for the reduced growth rateresulting in the drop of the GTP level and consequentlyCodY derepression [33, 34]. The CodY regulon encodes pro-teins that allow broader adaptation to nutrient depletionincluding extracellular degradative enzymes, transporterproteins, catabolic enzymes, factors involved in geneticcompetence, antibiotic synthesis pathways, chemotaxis pro-teins, and sporulation proteins [33]. In addition, the sB reg-ulon is the general proteome signature for glucose andphosphate starvation and required for the stationary phasesurvival upon starvation [2, 49].




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As a novel finding, the color-coded fused proteome mapfor starvation defined six sH-dependent proteins YvyD, YtxH,YisK, YpiB, Spo0A, YuxI and the CodY-dependent RapA asgeneral starvation proteins indicating the transition phase inresponse to nutrient limitation (Fig. 5). SigmaH directs thetranscription of several genes that function in the transitionfrom exponential growth to stationary phase, including theinitiation of spore formation and genetic competence [38]. Inaddition, sH controls many genes that are required for gen-eral adaptation to nutrient depletion. These genes areinvolved in transport, generation of potential nutrientsources, cell wall metabolism, proteolysis and cytochromebiogenesis [38]. It has been shown that both sB and sH con-tribute to stationary phase survival under acidic and alkalineconditions and this effect was independent from the simpleloss of sporulation ability [49].

4.4 Regulatory mechanisms of specific and generalstress/starvation proteins

Since most of the proteome signatures can be explained byknown regulators,wehave classified the stress- and starvation-induced proteins into specific and general regulons. The spe-cific stress or starvation proteins are controlled by one singleregulator (e.g., HrcA, PerR, TnrA, TRAP, CcpA, or PhoP). Incontrast, general starvation proteins can be controlled by sin-gle general regulators (sB, sH, or CodY), by multiple generalregulators (e.g., YvyD and YtxH by sB/sH) or by overlappingspecific and general regulators (e.g., DppA and YxbB by TnrA/CodY; UreC by TnrA/CodY/sH; AcsA by CcpA/CodY; RocADFby CcpA/sL/RocR/CodY). In addition, not all sB-dependentproteins are induced generally by the known conditions forsB

activation (e.g., heat, salt stress; phosphate and glucose starva-tion) (Table 1, Figs. 3, 5). Surprisingly, the sB-dependent pro-teins YceD and YvaA are induced specifically after heat shock,YceC specifically after salt stress, and YdbD and YdaT specifi-cally after phosphate starvation (Fig. 3). These differences inthe induction of individual sB regulon members might beexplained by additional promoters (e.g.,sW for YceC), differentpromoter strength, RNAor protein stability. In addition, somesB-dependent proteins might not be detected since theseoverlap with other proteins in the 2-D gel.

4.5 Functional characterization of the sH-dependentgeneral starvation proteins

Among the general starvation proteins Spo0A and RapAcounteract in the initiation of sporulation as last resort re-sponse to starvation. The process of sporulation is governedby a multicomponent phosphorelay that results in phospho-rylation of the key Spo0A [52, 53]. A certain threshold level ofSpo0A,P is necessary to initiate sporulation which is deter-mined by the phosphorelay [54]. The RapA phosphatasedephosphorylates the phosphorelay response regulatorSpo0F,P and consequently reduces the level of Spo0A,P[52, 53]. The coincidental induction of the counteracting

Spo0A and RapA proteins as general proteome signature forstarvation indicates that sporulation is a process with a bi-stable outcome, some cells initiate sporulation while othersdo not [54, 55]. It was demonstrated that the main function ofRapA is to maintain the bistable sporulation gene expres-sion, RapA is repressed in sporulating cells and still accu-mulated in nonsporulating cells [55]. The induction of the sF

and sE early sporulation regulons during the stationaryphase in response to nitrogen and glucose starvation reflectsthat some cells have initiated the sporulation process sincethey could not overcome the nutrient limitation.

Another interesting general starvation protein is the YvyDprotein that is similar to s54 modulation factors. It has beenshown inE. coli that theYvyD-homologousYfiAproteinbinds tothe 30S subunits of the ribosomes, inhibits the 100S ribosomeformation and reduces translation errors [56]. The B. subtilisYtxH protein shares some similarities to plant desiccation-related proteins [39]. The functions of the sB/sH-dependentproteins YvyD and YtxH during the starvation response inB. subtilis remains to be elucidated [39, 40]. In addition, thegeneral starvation proteins YvyD, YtxH, Spo0A, YpiB, andRapAproteins are under positive stringent control [8]. Thus, it mightbe that also YuxI and YisK are controlled by RelA in addition tosH. It is currently unknown whether these sH-dependent gen-eral starvation proteins contribute to the stationary phase survi-val which has been demonstrated previously forsB andsH [49].Thus, the fused starvation proteome map and color codeapproachprovide important leads for future research onproteinfunctions of the novel identified general starvation proteinsduring stationary phase survival inB. subtilis.

4.6 Outlook

In summary, this study can be regarded as one step towardsthe description of the complete cytoplasmic proteome ofB. subtilis, which includes a physiological approach by thedescription of proteome signatures in response to stress andstarvation. In total, 1301 cytoplasmic proteins are currentlyidentified in the proteome of B. subtilis during the growthand in response to stress and starvation using gel-based andgel-free proteome approaches [3, 4]. In addition, proteinquantification in response to heat shock revealed that heatshock proteins were up-regulated at similar levels using thegel-based approach and the gel-free ITRAQ� labelling tech-nology [4]. Future studies will be aimed on the definition ofthe complete proteome of B. subtilis cells after stress andstarvation using gel-free proteome approaches to identifyand quantify those stress- and starvation-induced proteinsthat escaped the gel-based proteome approach.

We thank the Decodon company for support with the DecodonDelta2D software. This work was supported by a scholarship of the“Ministry of Education and Training of Viet Nam” (MOET) toL.T.T., and grants from the Deutsche Forschungsgemeinschaft, theBundesministerium f�r Bildung und Forschung, the Fonds derChemischen Industrie, the Bildungsministerium of the country




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Mecklenburg-Vorpommern (EMAU 0202120), European Uniongrants (LSHG-CT-2004-503468), and Genencor International(Palo Alto, California, USA) to M.H.

5 References

[1] VanBogelen, R. A., Schiller, E. E., Thomas, J. D., Neidhardt, F.C., Electrophoresis 1999, 20, 2149–2159.

[2] Hecker, M., V�lker, U., Proteomics 2004, 4, 3727–3750.

[3] Eymann, C., Dreisbach, A., Albrecht, D., Bernhardt, J. et al.,Proteomics 2004, 4, 2849–2876.

[4] Wolff, S., Otto, A., Albrecht, D., Stahl Zeng, J. et al.,Mol. Cell.Proteomics 2006 (in press)nupdaten.

[5] Antelmann, H., Scharf, C., Hecker, M., J. Bacteriol. 2000, 182,4478–4490.

[6] Bernhardt, J., Weibezahn, J., Scharf, C., Hecker, M., GenomeRes. 2003, 13, 224–237.

[7] Eymann, C., Mach, H., Harwood, C. R., Hecker, M., Micro-biology 1996, 142, 3163–3170.

[8] Eymann, C., Homuth, G., Scharf, C., Hecker, M., J. Bacteriol.2002, 184, 2500–2520.

[9] Graumann, P. L., Marahiel, M. A., Arch. Microbiol. 1999, 171,135–138.

[10] H�per, D., Bernhardt, J., Hecker, M., Proteomics 2006, 6,1550–1562.

[11] Leichert, L. I., Scharf, C., Hecker, M., J. Bacteriol. 2003, 185,1967–1975.

[12] Mostertz, J., Scharf, C., Hecker, M., Homuth, G., Micro-biology 2004, 150, 497–512.

[13] Bandow, J. E., Br�tz, H., Leichert, L. I., Labischinski, H.,Hecker, M., Antimicrob. Agents Chemother. 2003, 47, 948–955.

[14] Sender, U., Bandow, J., Engelmann, S., Lindequist, U.,Hecker, M., Pharmazie 2004, 59, 65–70.

[15] Anagnostopoulos, C., Spizizen, J., J. Bacteriol. 1961, 81,741–746.

[16] St�lke, J., Hanschke, R., Hecker, M., J. Gen. Microbiol. 1993,139, 2041–2045.

[17] Bernhardt, J., B�ttner, K., Scharf, C., Hecker, M., Electropho-resis 1999, 20, 2225–2240.

[18] Bradford, M. M., Anal. Biochem. 1976, 72, 248–254.

[19] Luhn, S., Berth, M., Hecker, M., Bernhardt, J., Proteomics2003, 3, 1117–1127.

[20] Hecker, M., V�lker, U., Adv. Microb. Physiol. 2001, 44, 35–91.

[21] Nakano, S., Kuster-Schock, E., Grossman, A. D., Zuber, P.,Proc. Natl. Acad. Sci. USA 2003, 100, 13603–13608.

[22] Petersohn, A., Brigulla, M., Haas, S., Hoheisel, J. D. et al., J.Bacteriol. 2001, 183, 5617–5631.

[23] Ollinger, J., Song, K.-B., Antelmann, H., Hecker, M., Hel-mann, J. D., J. Bacteriol. 2006, 188, 3664–3673.

[24] Helmann, J. D., Wu, M. F., Gaballa, A., Kobel, P. A. et al., J.Bacteriol. 2003, 185, 243–253.

[25] Fernandez, S., Ayora, S., Alonso, J. C., Res. Microbiol. 2000,151, 481–486.

[26] Even, S., Burguiere, P., Auger, S., Soutourina, O. et al., J.Bacteriol. 2006, 188, 2184–2197.

[27] Fisher, S. H., Mol. Microbiol. 1999, 32, 223–232.

[28] Wray, L. V., Jr., Ferson, A. E., Rohrer, K., Fisher, S. H., Proc.Natl. Acad. Sci. USA 1996, 93, 8841–8845.

[29] Yoshida, K., Yamaguchi, H., Kinehara, M., Ohki, Y. H. et al.,Mol. Microbiol. 2003, 49, 157–165.

[30] Debarbouille, M., Gardan, R., Arnaud, M., Rapoport, G., J.Bacteriol. 1999, 181, 2059–2066.

[31] Eichenberger, P., Jensen, S. T., Conlon, E. M., Van Ooij, C. etal., J. Mol. Biol. 2003, 327, 945–972.

[32] Feucht, A., Evans, L., Errington, J., Microbiology 2003, 149,3023–3034.

[33] Molle, V., Nakaura, Y., Shivers, R. P., Yamaguchi, H. et al., J.Bacteriol. 2003, 185, 1911–1922.

[34] Ratnayake-Lecamwasam, M., Serror, P., Wong, K. W.,Sonenshein, A. L., Genes Dev. 2001, 15, 1093–103.

[35] Gardan, R., Rapoport, G., Debarbouille, M., Mol. Microbiol.1997, 24, 825–837.

[36] Yoshida, K., Kobayashi, K., Miwa, Y., Kang, C. M. et al.,Nucleic Acids Res. 2001, 29, 683–692.

[37] Koburger, T., Weibezahn, J., Bernhardt, J., Homuth, G. et al.,Mol. Genet Genomics. 2005, 274, 1–12.

[38] Britton, R. A., Eichenberger, P., Gonzalez-Pastor, J. E., Faw-cett, P. et al., J. Bacteriol. 2002, 184, 4881–4890.

[39] Var�n, D., Brody, M. S., Price, C. W.,Mol. Microbiol. 1996, 20,339–350.

[40] Drzewiecki, K., Eymann, C., Mittenhuber, G., Hecker, M., J.Bacteriol. 1998, 180, 6674–6680.

[41] Babitzke, P., Gollnick, P., J. Bacteriol. 2001, 183, 5795–5802.

[42] Gollnick, P., Babitzke, P., Antson, A., Yanofsky, C., Annu. Rev.Genet. 2005, 39, 47–68.

[43] Blencke, H. M., Homuth, G., Ludwig, H., M�der, U. et al.,Metab. Eng. 2003, 5, 133–149.

[44] Servant, P., Le Coq, D., Aymerich, S., Mol. Microbiol. 2005,55, 1435–1451.

[45] Allenby, N. E., O’Connor, N., Pragai, Z., Ward, A. C. et al., J.Bacteriol. 2005, 187, 8063–8080.

[46] Hulett, F. M., in: Sonenshein, A. L., Hoch, J. A., Losick, R.(Eds.), Bacillus Subtilis and its Closest Relatives: FromGenes to Cells, ASM press, Washington DC 2002, pp. 193–201.

[47] Antelmann, H., Sapolsky, R., Miller, B., Ferrari, E. et al., Pro-teomics 2004, 4, 2408–2424.

[48] Voigt, B., Schweder, T., Becher, D., Ehrenreich, A. et al., Pro-teomics 2004, 4, 1465–90.

[49] Gaidenko, T. A., Price, C. W., J. Bacteriol. 1998, 180, 3730–3733.

[50] Mendez, M. B., Orsaria, L. M., Philippe, V., Pedrido, M. E.,Grau, R. R., J. Bacteriol. 2004, 186, 989–1000.

[51] Streker, K., Freiberg, C., Labischinski, H., Hacker, J., Ohlsen,K., J. Bacteriol. 2005, 187, 2249–2256.

[52] Perego, M., Hoch, J. A., in: Sonenshein, A. L., Hoch, J. A.,Losick, R. (Eds.), Bacillus Subtilis and its Closest Relatives:From Genes to Cells, ASM press, Washington DC 2002,pp. 473–481.

[53] Sonenshein, L., Curr. Opin. Microbiol. 2000, 3, 561–566.

[54] Fujita, M., Gonzalez-Pastor, J. E., Losick, R., J. Bacteriol.2005, 187, 1357–1368.

[55] Veening, J.-W., Harmon, L.W., Kuipers, O. P.,Mol. Microbiol.2005, 56, 1481–1494.

[56] Ueta, M., Yoshida, H., Wada, C., Baba, T. et al., Genes Cells.2005, 10, 1103–1112.



Proteome signatures for stress and starvation inBacillus subtilis as revealed by a 2-D gel image...

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