Comparative Subproteome Analyses of Planktonic and Sessile Staphylococcus xylosus C2a: New Insight in Cell Physiology of a Coagulase-Negative Staphylococcus in Biofilm

Comparative Subproteome Analyses of Planktonic and Sessile

Staphylococcus xylosus C2a: New Insight in Cell Physiology of a

Coagulase-Negative Staphylococcus in Biofilm

Stella Planchon,†,# Mickael Desvaux,†,# Ingrid Chafsey,† Christophe Chambon,‡ Sabine Leroy,†

Michel Hebraud,†,‡ and Regine Talon*,†

INRA, UR454 Microbiologie, F-63122 Saint-Genes Champanelle, France, and INRA, Plate-Forme d’Explorationdu Metabolisme, Composante Proteomique, F-63122 Saint-Genes Champanelle, France

Received June 3, 2008

Staphylococcus xylosus is a Gram-positive bacterium found on the skin of mammals and frequentlyisolated from food plants and fermented cheese or meat. To gain further insight in protein determinantsinvolved in biofilm formation by this coagulase-negative Staphylococcus, a comparative proteomicanalysis between planktonic and sessile cells was performed. With the use of a protocol previouslydeveloped, protein patterns of the cytoplasmic and cell envelope fractions were compared by 2-DE.Following protein identification by MALDI-TOF mass spectrometry and bioinformatic analyses, thisstudy revealed differences in expression levels of 89 distinct proteins with 55 up-expressed and 34down-expressed proteins in biofilm compared to planktonic cells. Most proteins differentially expressedwere related to nitrogen and carbon metabolisms. Besides amino acid biosynthesis and proteintranslation, protein determinants related to protein secretion were up-expressed in biofilm, suggestinga more active protein trafficking in sessile cells. While up-expression of several enzymes involved inpentose phosphate and glycolytic pathways was observed in biofilm, connections with unexpectedmetabolic routes were further unravelled. Indeed, this proteomic analysis allowed identifying novelproteins that could be involved in a previously uncovered exopolysaccharide biosynthetic pathway inS. xylosus as well as several enzymes related to polyketide biosynthesis. This findings are particularlyrelevant considering exopolysaccharide production in S. xylosus is ica-independent contrary tocoagulase-negative model strain Staphylococcus epidermidis RP62A.

Keywords: biofilm formation • cell envelope proteins • coagulase-negative Staphylococcus • proteomicanalysis

Introduction

Staphylococcus xylosus is a low G+C Gram-positive bacte-rium belonging to the Firmicutes phylum, Bacilli class, Bacil-lales order, Staphylococcaceae family and Staphylococcus genus,which encloses pathogenic, opportunistic and saprophyticspecies.1 S. xylosus is frequently isolated from environmentalsources like soil but is considered as part of the normalmicrobiota of the skin in a variety of mammals includinghuman.1 Consequently, this species is a common contaminantof surfaces in food processing lines1 and it is also found infermented cheese or meat and some strains are used as starterculture in sausage manufacturing.2,3 While S. xylosus is gener-ally considered as saprophitic and even as technologicallypositive in food processing, unexpectedly some strains appear

involved in bacterial infections in animal (mastitis, dermatitis)and human (acute pyelonephritis, root canal infection, urinarytract infections)4-8 as well as resistant to antibiotics and metalsused in therapy or disinfection.9 In these different contexts,the ability of S. xylosus to form biofilm might be a distinctadvantage.

In a previous study, we have shown that strains of S. xylosusisolated from human skin and sausages were indeed able tocolonize hydrophilic and hydrophobic abiotic supports andformed multilayered biofilm where cells were embedded in anexopolysaccharide matrix.10 Such a matrix is common trait inbacterial biofilm.11 In Staphyloccocus species, the major com-ponent of the matrix is designated as polysaccharide intercel-lular adhesin (PIA) and its production is dependent upon theproteins encoded by the intercellular adhesion icaABCDlocus.12-14 In S. xylosus, however, the exopolysaccharide bio-synthesis appeared ica-independent and the metabolic path-way(s) involved remain unknown.10 Still, other surface com-ponents have been described as required for biofilm formationin Staphylococcus aureus and Staphylococcus epidermidis,namely, teichoic acids, autolysins and MSCRAMMs (Microbial

* To whom correspondence should be addressed. Dr. Regine Talon, INRA,UR454 Microbiologie, Qualite et Securite des Aliments, F-63122 Saint-GenesChampanelle, France. E-mail, [email protected]; fax, +33(0)-473-624581.

† INRA, UR454 Microbiologie.# These two authors contributed equally to this work.‡ INRA, Plate-Forme d’Exploration du Metabolisme, Composante Pro-

teomique.

10.1021/pr8004056 CCC: $40.75 2009 American Chemical Society Journal of Proteome Research 2009, 8, 1797–1809 1797Published on Web 03/02/2009

Surface Components Recognizing Adhesive Matrix Molecules),which are responsible for bacterial adhesion, whereas Aap(Accumulation associated protein),15 Bap (Biofilm associatedprotein) and the PIA are involved in cell aggregation-accumu-lation.14,16-19

While global approaches such as transcriptomics and/orproteomics have been applied to investigate the metabolismof S. aureus in biofilm,20,21 no data are available to date oncoagulase-negative staphylococci such as S. xylosus. Moreover,genomic data for this staphylococcal species are not as yetavailable, the physical and genetic map of S. xylosus C2a wasdone,22 sequencing of whole genome was achieved, but ge-nome assembly is still underway. With the use of a previouslydeveloped approach to investigate the cell envelope proteomeof S. xylosus,23 the present study aims at gaining further insightinto the physiology of biofilm formation following a compara-tive proteomic analysis between planktonic and sessile cellsin different subcellular compartments, essentially the cytoplasmand the bacterial cell envelope.

Materials and Methods

Bacterial Strain, Culture Conditions and Sampling. Thestrain S. xylosus C2a (University of Tubingen) was grown at 30°C for 48 h as previously described.10,23 For sessile cell growth,Petri dishes containing stainless steel discs were inoculated andincubated without shaking. After incubation, the stainless steeldiscs were first washed twice with sterile tryptone salt (TS) andadherent cells were further detached in 10 mL of TS in asonication bath as previously described.10 After centrifugation,the cell pellet was washed twice with Buffer 1 and thenresuspended in the same buffer to be stored frozen at -20 °Cuntil required.

Cell Envelope and Cytosoluble Proteins Extraction. The cellwall fraction (CW) and membrane fraction (MB) were obtainedas described by Planchon et al.23 Cytosoluble proteins wereseparated from membrane proteins by ultracentrifugation at200 000g for 30 min at 4 °C, thus, constituting the cytosoluble(CS) fraction. Proteins from CS fraction were precipitated with3 vol of acetone at -20 °C overnight. After a 13 000g centrifuga-tion for 40 min at 4 °C, the pellet was air-dried. Thesecytosoluble proteins were dissolved in Buffer 2 and stored at-80 °C until use.

Protein Separation by 2-DE and Identification by MALDI-TOF MS. Proteins from CS, MB and CW fractions were analyzedby 2-DE electrophoresis as previously described.23 The firstdimension by IEF was only performed at pH 4-7. For proteinsfrom CS fraction, IEF was carried out at 19 °C for a total of65 000 Vh. Second dimension was performed overnight in 12%acrylamide gels for proteins from CS fractions. Because of therelatively low quantity of biomass recovered from biofilms andconsequently the low quantity of extracted proteins for eachfraction, analytical gels were carried out with 60 µg of proteinsand silver stained according to Rabilloud et al.25 Six 2-DE gelswith samples from two different cultures were performed toevaluate the reproducibility of the methods. Proteins wereidentified from a semipreparative gel loaded with either 600µg of proteins from MB and CS fractions and stained withcolloidal Coomassie Blue26 or 200 µg of proteins from CWfraction and silver stained according to Yan et al.27

Following spot excision from the semipreparative gels,protein identification was performed as previously described.23

Briefly, the proteins were identified using mass spectrometricpeptide mapping data with Mascot (V. 21.3). Mascot scores

were obtained against the Staphylococcus database (102 020sequenses, July 2007) where scores greater than or equal to 63are significant (p < 0.05), including S. xylosus C2a genomicdatabase (2776 sequences, July 2007) where scores greater thanor equal to 47 are significant (p < 0.05).

2-DE Gel Image and Statistical Analyses. For the differentfractions CW, MB and CS, six 2-DE gels were performed withsamples from two independent cultures in each growth condi-tion, that is, planktonic (P) and biofilm (B). A total of 36 gelswere digitalized using a GS-700 imaging densitometer (Bio-Rad). The relative protein abundance between sessile andplanktonic cells was evaluated through the comparison of 2-DEgels by image analysis using the software Image Master 2DPlatinum (GE Healthcare, Upspsala, Sweden). The amount ofproteins loaded on each gel (60 µg) allowed most of the proteinspots to be within the linear range of the silver staining (0-60ng/spot) as described by Patton.28

To determine intraclass variation, gels were matched pair-wise in each class P and B, to create spot groups for eachfraction, that is (i) P-CW, (ii) B-CW, (iii) P-MB, (iv) B-MB, (v)P-CS and (vi) B-CS. Data were gathered in the group reportwhere each spot was assigned a relative value correspondingto spot volume. Spots present in at least N - 2 gels were takeninto account with N being the number of gels runs in eachcondition. The group report provided variations existing be-tween the same spot(s) present in all gels. Standard deviationsand means of variations were then determined in order tocalculate the coefficient of variation (CV). The CV was evaluatedin each class defined above.

Finally, gels belonging to the two classes P and B werematched for each fraction CW, MB and CS, that is (i) P-CW vsB-CW, (ii) P-MB vs B-MB, and (iii) P-CS vs B-CS. Spot valueswere expressed in percentage of volume in order to work onrelative abundances. In the class report, data were sorted bycenter of different spots group corresponding to the raw centraltendency as defined in Image Master 2D Platinum. Only spotgroups with a positive gap value were considered, the gap beingthe maximum difference between the two classes. The ratiobetween center of each class was determined for the selectedspots. An up- or down-expression was noticed if the ratio Rwas higher than 1 - (2 × CV) or less than 1 + (2 × CV),respectively. For protein spots differentially expressed, theKolmogorov-Smirnov two samples test was used in ImageMaster 2D Platinum. If the ratio R was higher than the valuegiven in the table of Kolmogorov-Smirnov test (p e 0.05), thenthe difference between the two samples was considered asstatistically significant.

Bioinformatic Analyses. Bioinformatic analyses were per-formed as previously described23 from Web-based servers orunder Unix-like environment and Sun Grid Engine (SGE) fromTopaze server homed at MIG (Mathematiques Informatique etGenomes) Research Unit (INRA, Jouy-en-Josas, France). Briefly,N-terminal signal peptides were predicted using SignalP v2.0and v3.0 using both neural network (NN) and hidden Markovmodel (HMM),30 Phobius,31 Signal-3 L,32 SOSUIsignal33 andPrediSi.34 Prediction of lipoproteins involved LipoP v1.0,35

DOLOP36 and also scanning for PS51257 profile and G + LPPpattern.37 Tat signal peptide prediction was performed fromTatP v1.038 and TatFind v1.4.39 Prediction of nonclassicalsecreted protein was performed from SecretomeP v2.0.38,40

Transmembrane domains (TMDs) were predicted using TM-pred v1.0,41 TMHMM v2.0,42 MEMSAT v3.0,43 HMMTOP v2.0,44

TopPred v2.045 and UMDHMMTMHP.46 These analyses were

research articles Planchon et al.

1798 Journal of Proteome Research • Vol. 8, No. 4, 2009

completed with prediction of subcellular localization of proteinsin Gram-positive bacteria, namely, PSORTb v2.0.447 and CELLOv2.5.48 Modular architecture of proteins was analyzed from (i)Pfam v20.0,49 using hidden Markov model (HMM),50 (ii) COGv1.051 using RPS-BLAST (Reverse Position-Specific BLAST) aspart of BLAST (Basic Local Alignment Search Tool) v2.2.16,52

(iii) SuperFamily v1.69 using HMM53 and (iv) Smart v4.054 usingRPS-BLAST. Proteins with no significant or unconclusivematches were further characterized following PSI-BLAST (Posi-tion-Specific Iterated BLAST)55 searches until convergence wasreached against UniProtKB v12.4.56

Results and Discussion

Protein Identification within Subproteomes. Bacterial cellswere fractionated into cytoplasmic (CS) and cell envelopeextracts following a previously developed protocol,23 wherefurther insight in the cell envelope was achieved by extractinga cell wall (CW) and a membrane (MB) fraction. Proteomicanalyses were performed by 2-DE with IEF in the pH range of4-7 (Figure 1). Proteins spots differentially expressed in biofilmversus planktonic cells of S. xylosus C2a were analyzed byMALDI-TOF mass spectrometry. From these subproteomes, 119protein spots displayed a significant difference in expressionlevels between planktonic and sessile mode of growth andcorresponded to 89 distinct proteins. Thirty-five proteins wereidentified within CS fraction, 47 in MB fraction and 13 in CWfraction, while 6 proteins were found common in two fractions(Tables 1 and 2).

To categorize these proteins relative to their predictedsubcellular localization, a rational bioinformatic approach wasapplied as previously described23 where data results from insilico analyses are given as Supporting Information (Table 1S)and results summarized in Table 2S. Three main final localiza-tions were considered, that is, cytoplasm (GO 0005737, 67proteins predicted), cell envelope (33 proteins) and extracellularmilieu (GO 0005576, 19 proteins). Cell envelope was decom-posed into cell wall (GO 0005618, 2 proteins) and membrane(GO 0005886, 31 proteins). Membrane was further separatedinto intrinsic to membrane (GO 0031226, 15 proteins), includingintegral to membrane (GO 0005887, 13 proteins) and anchoredto membrane (GO 0046658, 2 proteins), and extrinsic tomembrane (GO 0019897, 16 proteins), including protein com-plex (GO 0043234, 4 proteins) and internal side of membrane57

(GO 0031234, 12 proteins). Thirty proteins were also predictedas localized in an additional compartment.

Prediction of protein localization in some of these subcellularcompartments resulted from protein prediction in one of the8 protein categories considered (Supporting Information Table2S), that is, (i) cytosoluble (67 predicted), (ii) integral membraneprotein (IMP, 13 predicted), (iii) lipoprotein (2 predicted), (iv)subunit of membrane protein complex (4 predicted), (v)cytoplasmic protein interacting with membrane components(12 predicted), (vi) protein associated to cell wall by unknownmechanism (2 predicted), (vii) extracellular protein secreted viaunknown system (16 predicted) and (viii) extracellular proteinsecreted via Sec (3 predicted).57,58 Thirty proteins were alsopredicted as belonging to an additional category.

Out of the 7 proteins predicted with an N-terminal signalpeptide, 2 are finally predicted as lipoproteins, 2 as IMPs, 2 ascell wall associated proteins and one as extracellular (Support-ing Information Table 1S). On the example of type I signalpeptidase, though, it cannot be excluded that the metaldependent phosphohydrolase found in MB fraction and pre-

dicted as an extracellular protein secreted via Sec is actuallymembrane anchored via its predicted N-terminal signal pep-tide, which thus would be uncleaved with its H-domain servingas an R-helical transmembrane domain.59 Prediction of suchproteins, that is, single spanning type II IMPs, remains a majorpitfall in bioinformatic analysis since they are most oftenmispredicted as extracellular proteins with N-terminal cleavablesignal peptide.57,60 While known domains involved in proteinanchoring to cell wall could not be identified here,58 someproteins clearly described as cell wall associated by unknownmechanism were found, namely, immunodominant antigen A61

and MreC.62

As revealed by SecretomeP analysis, 16 proteins primarilypredicted as cytoplasmic were also predicted as secreted viaunknown secretion system as they neither exhibited N-terminalsignal peptide of any kind nor were predicted as substrates ofsecretion systems permitting translocation of proteins lackinga signal peptide in Gram-positive bacteria.63 While 6 of themwere here only found in CS fraction, 10 were indeed identifiedwithin the bacterial cell envelope, that is, either MB or CWfraction (Supporting Information Table 2S). Besides proteinsintrinsic to membrane (IMPs and lipoproteins), membrane-associated proteins here included those extrinsic to membrane,that is, subunits of membrane complex, and proteins interact-ing with membrane-bound components. Subunits of mem-brane complex included for example component of pyruvate/2-oxoglutarate dehydrogenase multienzyme complex64 orcomponents of F0-F1 ATP synthase.65 It can be stressed thatall cytoplasmic proteins interacting with membrane compo-nents are also considered as cytosoluble proteins. Indeed, suchproteins can interact more or less temporarily with membranecomponents, for example, ribosomal proteins with the Sectranslocon in the course of co-translational translocation.66

Finally, 86.2% (50/58) of proteins predicted as cell envelopelocalized and/or extracellular were indeed identified in the cellenvelope fractions, that is, MB and/or CW fractions (SupportingInformation Table 2S). Thirty-three out of 35 proteins (94.3%)predicted as cytoplasmic were as expected identified in the CSfraction.

Comparing biofilm to planktonic cells, 55 proteins were up-expressed (Table 1) and 34 proteins down-expressed (Table 2,Figure 1). Among the 55 up-expressed proteins, 25 were in CSfraction and 33 in the cell envelope fractions, including 2proteins also found in CS fraction. Among the 34 down-expressed proteins, 9 were in CS fraction and 27 in the cellenvelope fractions, with 2 proteins common to MB and CSfractions. These proteins differentially expressed in sessile cellscompared to planktonic cells were mainly associated withnitrogen and carbon metabolisms, essentially amino acidbiosynthesis and glycolysis/TCA cycle, respectively. Though,connections with unexpected metabolic pathways were hereunravelled.

Nitrogen Metabolism in S. xylosus Biofilm: From AminoAcids Biosynthesis to Protein Secretion. As indicated by cleardown-expression of urease (more than 10-fold lower for spotP1219) and undetectable level of glutamine synthetase (spotP689) compared to planktonic cells (Table 2), metabolismtaking place in sessile cells of S. xylosus is presumably notdealing with nitrogen starvation. Indeed, expression of theseenzymes is normally tightly regulated and activated in thepresence of poor nitrogen sources or during nitrogen limitation,as urease (EC:3.5.1.5) converts urea into ammonium and CO2

67

whereas glutamine synthetase (EC:1.4.1.13) is a critical and

Subproteome Analyses of Planktonic and Sessile S. xylosus C2a research articles

Journal of Proteome Research • Vol. 8, No. 4, 2009 1799

Figure 1. 2-DE of S. xylosus C2a proteins after 48 h of growth under planktonic conditions (A, C, E) and biofilm conditions (B, D, F). (Aand B) cytoplasmic proteins, (C and D) fraction enriched in membrane proteins, (E and F) fraction enriched in cell wall proteins. Proteinsextracts (60 µg) were separated by IEF over a linear pH gradient of 4-7 followed by a 10% SDS-PAGE for cell wall and membranesproteins and 12% for cytoplasmic proteins. Proteins were revealed with silver staining. The spot numbers are listed in Tables 1 and 2.Gel E originates from a previous investigation by Planchon et al.,23 where it then corresponded then to gel A in Figure 3. (Reprintedwith permission from ref 23. Copyright 2007 American Chemical Society.)



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9495

8243

gbS.

xylo

sus

C2a

4.1.

2.13

+(7

.0)

MB

/C-M

4.73

/4.7

532

.643

/35.

256

164

16/2

9B

961

Fru

cto

se-b

isp

ho

sph

ate

ald

ola

secl

ass

I94

9582

43gb

S.xy

losu

sC

2a4.

1.2.

13+

(3.4

)M

B/C

-M4.

73/4

.80

32.6

43/3

5.0

6696

22/1

95B

1342

Fru

cto

se-b

isp

ho

sph

ate

ald

ola

secl

ass

I94

9582

43gb

S.xy

losu

sC

2a4.

1.2.

13+

(40.

2)M

B/C

-M4.

73/4

.70

32.6

43/3

5.4

6014

018

/60

B43

6G

lyce

rald

ehyd

e-3-

ph

osp

hat

ed

ehyd

roge

nas

e73

6632

25re

fS.

sap

rop

hyt

icu

sA

TC

C15

305

1.2.

1.12

+C

S/C

4.89

/5.4

536

.203

/38.

726

759/

59

B44

0G

lyce

rald

ehyd

e-3-

ph

osp

hat

ed

ehyd

roge

nas

e73

6632

25re

fS.

sap

rop

hyt

icu

sA

TC

C15

305

1.2.

1.12

+C

S/C

4.89

/5.4

036

.203

/38.

129

8011

/74

B51

7P

ho

sph

ogl

uco

mu

tase

/P

ho

sph

om

ann

om

uta

seE

U47

5944

*gb

S.xy

losu

sC

2a5.

4.2.

2/5.

4.2.

8+

(6.3

)M

B/C

-M4.

96/5

.01

62.1

60/6

3.0

5015

324

/122

2.2.

Tri

carb

oxyl

icA

cid

(TC

A)

Cyc

leB

888

Cit

rate

syn

thas

eII

7366

2380

ref

S.sa

pro

ph

ytic

us

AT

CC

1530

52.

3.3.

1+

CS/

M5.

63/5

.70

42.3

75/4

4.3

2363

12/7

5

B86

7C

itra

tesy

nth

ase

II73

6623

80re

fS.

sap

rop

hyt

icu

sA

TC

C15

305

2.3.

3.1

+(2

.4)

MB

/M5.

63/6

.05

42.3

75/4

1.1

2364

9/71

B75

7Is

oci

trat

ed

ehyd

roge

nas

e73

6623

81re

fS.

sap

rop

hyt

icu

sA

TC

C15

305

1.1.

1.41

+(3

.3)

MB

/C4.

88/5

.00

46.3

44/4

4.5

2883

11/9

3



Tab

le1.

Co

nti

nu

ed

spo

tid

pro

tein

nam

eG

IaD

Bb

Stap

hyl

ococ

cus

stra

inE

Cc

bio

film

vsp

lan

kto

nic

cells

dfr

acti

on

/p

red

icti

on

ep

IT/p

IEf

MW

T/M

WE

gse

qu

ence

cove

rage

hM

asco

tsc

ore

ip

epti

des

mat

ch

B76

8Is

oci

trat

ed

ehyd

roge

nas

e73

6623

81re

fS.

sap

rop

hyt

icu

sA

TC

C15

305

1.1.

1.41

+(2

.5)

MB

/C4.

88/4

.85

46.3

44/4

3.9

2584

10/5

7

B77

1Is

oci

trat

ed

ehyd

roge

nas

e73

6623

81re

fS.

sap

rop

hyt

icu

sA

TC

C15

305

1.1.

1.41

+(2

.5)

MB

/C4.

88/4

.80

46.3

44/4

3.8

3082

14/7

4

B20

92-

oxo

glu

tara

ted

ehyd

roge

nas

eE

1co

mp

on

ent

7366

2634

ref

S.sa

pro

ph

ytic

us

AT

CC

1530

51.

2.4.

2+

(3.8

)M

B/M

5.34

/5.5

010

5.47

9/10

3.0

1664

10/8

4

B21

02-

oxo

glu

tara

ted

ehyd

roge

nas

eE

1co

mp

on

ent

7366

2634

ref

S.sa

pro

ph

ytic

us

AT

CC

1530

51.

2.4.

2+

(3.7

)M

B/M

5.34

/5.5

510

.547

9/10

3.0

2184

16/1

23

B42

5A

TP

syn

thas

eR

sub

un

it12

4007

217

spS.

sap

rop

hyt

icu

sA

TC

C15

305

3.6.

3.14

+(5

.6)

CW

/M4.

81/4

.80

54.5

20/5

9.0

2810

512

/83

2.3.

Fat

tyA

cid

and

Lip

idB

iosy

nth

etic

Pat

hw

ays

B11

703-

oxo

acyl

-acy

l-ca

rrie

rp

rote

inre

du

ctas

eB

EU

4759

40*

gbS.

xylo

sus

C2a

1.1.

1.10

0+

(2.6

)M

B/M

5.32

/5.4

525

.918

/23.

743

755/

37B

876

3-h

ydro

xy-3

-met

hyl

glu

tary

lC

oA

syn

thas

eE

U47

5906

*gb

S.xy

losu

sC

2a2.

3.3.

10+

(3.8

)M

B/M

4.56

/4.6

043

.458

/40.

342

8613

/66

B45

5A

cety

l-C

oA

carb

oxy

lase

Rsu

bu

nit

1500

1098

9gb

S.xy

losu

sC

2a6.

4.1.

2+

CS/

C5.

18/5

.40

35.4

60/3

7.6

4811

019

/91

2.4.

Exo

pol

ysac

char

ide

Bio

syn

thet

icP

ath

way

B11

18d

TD

P-g

luco

se4,

6-d

ehyd

rata

seE

U47

5925

*gb

S.xy

losu

sC

2a1.

1.1.

133

+(4

.6)

MB

/C-E

4.51

/4.5

023

.252

/27.

673

145

15/8

2

3.P

oly

keti

de

Seco

nd

ary

Met

abo

lite

Pat

hw

ayB

652

Po

lyke

tid

ecy

clas

e/d

ehyd

rase

EU

4759

21*

gbS.

xylo

sus

C2a

4.2.

1+

(2.8

)C

S/C

4.92

/5.1

20.8

54/2

0.6

3757

7/60

B88

7Is

op

ente

nyl

-dip

ho

sph

ate

δ-is

om

eras

eE

U47

5918

*gb

S.xy

losu

sC

2a5.

3.3.

2+

(2.5

)M

B/C

5.07

/5.1

038

.619

/39.

632

8111

/69

B78

0A

nti

bio

tic

bio

syn

thes

ism

on

oo

xyge

nas

eE

U47

5949

*gb

S.xy

losu

sC

2a4.

2.1

+(4

.0)

CS/

C-E

4.97

/5.3

012

.143

/10.

635

606/

45P

1109

Ino

sito

lm

on

op

ho

sph

atas

efa

mily

pro

tein

9495

8277

gbS.

xylo

sus

C2a

3.1.

3.25

+(4

.3)

MB

/C5.

46/5

.60

30.1

46/2

8.2

5214

19/

35

4.M

etab

oli

smo

fN

ucl

eoti

des

B34

7A

den

ylo

succ

inat

esy

nth

ase

EU

4759

14*

gbS.

xylo

sus

C2a

6.3.

4.4

+(2

.7)

CS/

C5.

19/5

.55

47.0

86/4

9.1

2157

6/61

B32

3P

oly

rib

on

ucl

eoti

de

nu

cleo

tid

yltr

ansf

eras

eE

U47

5938

*gb

S.xy

losu

sC

2a2.

7.7.

8+

(3.8

)M

B/C

-M4.

96/5

.15

76.6

86/7

9.1

2160

13/8

4B

709

Nu

cleo

sid

ed

iph

osp

hat

eki

nas

e73

6625

86re

fS.

sap

rop

hyt

icu

sA

TC

C15

305

2.7.

4.6

+(4

.7)

CS/

C5.

69/6

.40

16.7

42/1

7.3

5710

78/

39

B60

3A

den

ylat

eki

nas

eE

U47

5923

*gb

S.xy

losu

sC

2a2.

7.4.

3+

(2.9

)C

S/C

4.99

/5.3

024

.216

/23.

965

113

17/7

5B

606

Ad

enyl

ate

kin

ase

EU

4759

23*

gbS.

xylo

sus

C2a

2.7.

4.3

+C

S/C

4.99

/5.1

524

.216

/23.

868

101

16/9

3

5.M

etab

oli

smo

fC

oen

zym

esan

dP

rost

het

icG

rou

ps

B54

7D

ihyd

roxy

nap

hth

oic

acid

syn

thas

e73

6630

55re

fS.

sap

rop

hyt

icu

sA

TC

C15

305

4.1.

3.36

+(2

.5)

CS/

C5.

38/5

.70

30.3

90/2

6.8

3610

110

/58

6.T

ran

scri

pti

on

alR

egu

lato

rsB

738

Tra

nsc

rip

tio

nal

regu

lato

rM

arR

fam

ilyE

U47

5947

*gb

S.xy

losu

sC

2a+

CS/

C5.

55/5

.90

16.9

83/1

5.3

4865

9/70

B71

7A

nti

sigm

aB

fact

or

anta

gon

ist

7366

2120

ref

S.sa

pro

ph

ytic

us

AT

CC

1530

5+

(3.4

)C

S/C

4.52

/4.3

512

.184

/16.

769

648/

65

7.St

ress

Res

po

nse

Pro

tein

sB

805

Co

ldsh

ock

pro

tein

7366

3621

ref

S.sa

pro

ph

ytic

us

AT

CC

1530

5+

(2.3

)C

S/C

4.47

/4.5

57.

206/

7.1

8782

5/30

B62

9Su

per

oxi

de

dis

mu

tase

8977

980

emb

S.xy

losu

sC

2a1.

15.1

.1+

(2.8

)C

S/C

-E4.

88/5

.05

22.5

35/2

1.9

5380

6/66

8.U

nsp

ecifi

co

rU

nkn

ow

nF

un

ctio

nB

777

UP

F03

56p

rote

ino

fu

nkn

ow

nfu

nct

ion

DU

F14

47E

U47

5942

*gb

S.xy

losu

sC

2a+

CS/

C5.

34/4

.10

8.61

0/10

.869

574/

60

B10

58H

alo

acid

deh

alo

gen

ase-

like

hyd

rola

seE

U47

5924

*gb

S.xy

losu

sC

2a3.

6.3.

1+

(14.

0)M

B/C

4.97

/5.1

032

.446

/30.

751

135

12/6

7B

931

D-i

som

ersp

ecifi

c2-

hyd

roxy

acid

deh

ydro

gen

ase

EU

4759

22*

gbS.

xylo

sus

C2a

1.1.

1.27

2+

(4.2

)M

B/C

5.14

/5.3

034

.981

/37.

461

162

15/7

6

B83

4N

AD

H-d

epen

den

tfl

avin

oxi

do

red

uct

ase

EU

4759

45*

gbS.

xylo

sus

C2a

1.14

+(2

.2)

MB

/C5.

30/5

.60

42.3

98/4

2.3

6716

522

/108

9.C

ell

Surf

ace

Exp

ose

dP

rote

ins

B98

0A

BC

-typ

eM

n/Z

ntr

ansp

ort

syst

emM

n/Z

n-b

ind

ing

pro

tein

7366

3397

ref

S.sa

pro

ph

ytic

us

AT

CC

1530

5+

(13.

5)M

B/M

5.41

/5.4

534

.929

/35.

034

6810

/48



Tab

le1.

Co

nti

nu

ed

spo

tid

pro

tein

nam

eG

IaD

Bb

Stap

hyl

ococ

cus

stra

inE

Cc

bio

film

vsp

lan

kto

nic

cells

dfr

acti

on

/p

red

icti

on

ep

IT/p

IEf

MW

T/M

WE

gse

qu

ence

cove

rage

hM

asco

tsc

ore

ip

epti

des

mat

ch

B13

38A

BC

-typ

eM

n/Z

ntr

ansp

ort

syst

emM

n/Z

n-b

ind

ing

pro

tein

7366

3397

ref

S.sa

pro

ph

ytic

us

AT

CC

1530

5+

(3.7

)M

B/M

5.41

/5.2

534

.929

/35.

038

6614

/80

B97

8A

lkal

ine

sho

ckp

rote

in23

1500

1102

1gb

S.xy

losu

sC

2a+

(2.8

)C

W/C

-E4.

86/4

.90

18.5

63/1

9.1

5712

910

/25

B97

9A

lkal

ine

sho

ckp

rote

in23

1500

1102

1gb

S.xy

losu

sC

2a+

(2.9

)C

W/C

-E4.

86/4

.80

18.5

63/1

9.1

5910

511

/56

B81

3T

HiJ

/Pfp

Ip

rote

ase/

amid

ase

EU

4759

36*

gbS.

xylo

sus

C2a

+(1

8.5)

CW

/C-E

4.44

/4.4

523

.965

/28.

739

787/

41B

489

En

ola

se11

9369

404

spS.

sap

rop

hyt

icu

sA

TC

C15

305

4.2.

1.11

+(1

2.0)

CW

/M4.

55/4

.70

47.0

75/4

9.9

5520

516

/65

B74

6L-

lact

ate

deh

ydro

gen

ase

9495

8235

gbS.

xylo

sus

C2a

1.1.

1.27

+(2

.9)

CW

/M4.

88/5

.30

33.7

75/3

2.5

5013

811

/50

B74

8L-

lact

ate

deh

ydro

gen

ase

9495

8235

gbS.

xylo

sus

C2a

1.1.

1.27

+(3

.8)

CW

/M4.

88/5

.10

33.7

75/3

2.4

3910

610

/60

B61

7A

lan

ine

deh

ydro

gen

ase

7366

2366

ref

S.sa

pro

ph

ytic

us

AT

CC

1530

51.

4.1.

1+

(3.9

)C

W/C

5.02

/5.5

539

.903

/41.

043

113

11/6

0

B61

6A

lan

ine

deh

ydro

gen

ase

7366

2366

ref

S.sa

pro

ph

ytic

us

AT

CC

1530

51.

4.1.

1+

(5.1

)C

W/C

5.02

/5.5

039

.903

/41.

130

947/

36

B10

29P

hag

eca

psi

dp

rote

inE

U47

5941

*gb

S.xy

losu

sC

2a+

(2.1

)M

B/C

-E4.

55/4

.55

49.5

19/3

2.6

2066

8/57

B72

6A

rgin

ase

9346

3979

gbS.

xylo

sus

C2a

3.5.

3.1

+(3

.6)

CW

/C4.

88/5

.00

32.8

66/3

3.8

4611

410

/75

B65

9T

ran

scri

pti

on

alre

gula

tor

Ccp

A11

7768

5em

bS.

xylo

sus

C2a

+(7

.5)

CW

/C5.

09/5

.40

36.2

57/3

8.3

6413

815

/77

B98

4R

NA

liga

se73

6630

79re

fS.

sap

rop

hyt

icu

sA

TC

C15

305

6.5.

1.3

+(7

.6)

CW

/C5.

23/5

.40

19.4

34/1

8.7

3866

6/36

B48

230

Sri

bo

som

alp

rote

inS1

EU

4759

32*

gbS.

xylo

sus

C2a

+C

W/C

-M4.

46/4

.50

43.1

13/5

1.3

5115

117

/65

aG

enIn

foId

enti

fier

(GI)

.Ast

eris

k(*

)in

dic

ates

Gen

Ban

kte

mp

ora

ryas

sign

men

tn

um

ber

for

S.xy

losu

sC

2ap

rote

ins.

bD

ataB

ase

(DB

):G

enB

ank

(gb

),R

efSe

q(r

ef),

EM

BL

(em

b)

and

Swis

s-P

rot

(sp

).c

En

zym

eC

lass

ifica

tio

n(E

C).

dD

iffe

ren

cein

pro

tein

spo

tex

pre

ssio

nin

bio

film

com

par

edto

pla

nkt

on

icce

lls,

wh

ere+

(x)

mea

ns

x-fo

ldh

igh

erin

bio

film

;+

alo

ne

mea

ns

pro

tein

spo

tw

aso

nly

pre

sen

tin

bio

film

.e

Fra

ctio

n:

sub

cellu

lar

frac

tio

no

bta

ined

asd

escr

ibed

inM

ater

ials

and

Met

ho

ds:

cyto

solu

ble

(CS)

,m

emb

ran

e(M

B)

and

cell

wal

l(C

W)

frac

tio

ns.

MB

and

CW

rep

rese

nt

the

bac

teri

alce

llen

velo

pe.

Pre

dic

tio

n:

sub

cellu

lar

loca

lizat

ion

of

pro

tein

sp

red

icte

db

yb

ioin

form

atic

anal

yses

asd

escr

ibed

inth

eM

ater

ials

and

Met

ho

ds:

cyto

pla

sm(C

),m

emb

ran

e(M

),ce

llw

all

(W),

extr

acel

lula

rm

ilieu

(E),

cyto

pla

smm

emb

ran

e(C

-M),

cyto

pla

sm-e

xtra

cellu

lar

mili

eu(C

-E),

for

det

ails

see

Sup

po

rtin

gIn

form

atio

nT

able

1S.

fT

heo

riti

calp

I(p

IT)

isca

lcu

late

du

sin

gp

Kva

lues

of

amin

oac

ids

fro

mB

jellq

vist

refe

ren

ceta

ble

;ex

per

imen

tal

pI

(pIE

).g

Th

eori

tica

lm

ole

cula

rw

eigh

t(M

WT

)u

sin

gav

erag

em

ass

valu

eso

fam

ino

acid

s;ex

per

imen

tal

mo

lecu

lar

wei

ght

(MW

E),

MW

are

exp

ress

edin

kDa.

hE

xpre

ssed

asp

erce

nta

ge.

iM

asco

tsc

ore

sw

ere

ob

tain

edag

ain

stth

eSt

aph

yloc

occu

sd

atab

ase

(102

020

seq

uen

ces,

July

2007

)w

her

esc

ore

sgr

eate

rth

ano

req

ual

to63

are

sign

ifica

nt

(p<

0.05

),in

clu

din

gS.

xylo

sus

C2a

gen

om

icd

atab

ase

(277

6se

qu

ence

s,Ju

ly20

07)

wh

ere

sco

res

grea

ter

than

or

equ

alto

47ar

esi

gnifi

can

t(p

<0.

05).



Tab

le2.

Pro

tein

sId

enti

fied

by

MA

LDI-

TO

FM

ass

Sp

ectr

om

etry

Pre

sen

tin

Low

erA

mo

un

tin

Bio

film

Co

mp

ared

toP

lan

kto

nic

Cel

ls

spo

tid

pro

tein

nam

eG

IaD

Bb

Stap

hyl

ococ

cus

stra

inE

Cc

bio

film

vsp

lan

kto

nic

cells

dfr

acti

on

/p

red

icti

on

ep

IT/p

IEf

MW

T/M

WE

gse

qu

ence

cove

rage

hM

asco

tsc

ore

ip

epti

des

mat

ch

1.N

itro

gen

Met

abo

lism

1.1.

Nit

roge

nA

ssim

ilat

ion

and

Cat

abol

ism

P12

19U

reas

e�

sub

un

it41

0515

emb

S.xy

losu

sp

Ura

413.

5.1.

5-

(10.

4)M

B/C

5.29

/5.5

015

.432

/15.

490

100

15/2

26

P68

9G

luta

min

esy

nth

ase

EU

4759

37*

gbS.

xylo

sus

C2a

1.4.

1.13

-M

B/C

5.00

/5.8

050

.858

/50.

645

8417

/126

P40

7B

ran

ched

-ch

ain

amin

oac

idam

ino

tran

sfer

ase

9346

3971

gbS.

xylo

sus

C2a

2.6.

1.42

-(4

.3)

CS/

C-E

4.72

/4.8

040

.099

/41.

231

808/

74P

845

Pep

tid

ase

M20

D,

amid

oh

ydro

lase

AE

U47

5920

*gb

S.xy

losu

sC

2a3.

5.1.

14-

(7.0

)M

B/C

4.83

/4.8

042

.077

/40.

250

105

9/10

3P

764

Pep

tid

ase

M20

D,

amid

oh

ydro

lase

BE

U47

5913

*gb

S.xy

losu

sC

2a3.

5.1.

32-

(4.1

)M

B/C

4.99

/5.2

043

.697

/43.

731

668/

119

1.2.

Pro

tein

Tra

nsl

atio

n,

Mod

ifica

tion

and

Fol

din

gP

1146

30S

rib

oso

mal

pro

tein

S482

5816

22sp

S.sa

pro

ph

ytic

us

AT

CC

1530

5-

(2.6

)M

B/C

-M9.

69/5

.45

23.1

44/2

6.2

4563

8/12

8

P29

8T

ran

slat

ion

init

iati

on

fact

or

IF-2

EU

4759

39*

gbS.

xylo

sus

C2a

-(9

3.7)

MB

/C-M

5.05

/5.2

077

.187

/80.

020

658/

86P

299

Tra

nsl

atio

nin

itia

tio

nfa

cto

rIF

-2E

U47

5939

*gb

S.xy

losu

sC

2a-

MB

/C-M

5.05

/5.2

577

.187

/80.

021

6010

/125

2.C

arb

on

Met

abo

lism

2.1.

Pen

tose

Ph

osp

hat

ean

dG

lyco

lyti

cP

ath

way

sP

545

R-D

-1,4

-glu

cosi

das

e47

4177

emb

S.xy

losu

sC

2a3.

2.1.

20-

(2.4

)M

B/C

-E4.

63/4

.70

63.9

57/5

9.0

2681

9/85

P49

8E

no

lase

1193

6940

4sp

S.sa

pro

ph

ytic

us

AT

CC

1530

54.

2.1.

11-

(5.8

)M

B/M

4.55

/5.0

547

.075

/62.

955

233

17/4

7

P50

0E

no

lase

1193

6940

4sp

S.sa

pro

ph

ytic

us

AT

CC

1530

54.

2.1.

11-

(6.1

)M

B/M

4.55

/5.0

047

.075

/62.

950

203

16/5

3

P64

3E

no

lase

1193

6940

4sp

S.sa

pro

ph

ytic

us

AT

CC

1530

54.

2.1.

11-

(7.3

)M

B/M

4.55

/4.9

547

.075

/53.

548

190

15/5

9

2.2.

Tri

carb

oxyl

icA

cid

(TC

A)

Cyc

leP

417

Succ

inat

ed

ehyd

roge

nas

efl

avo

pro

tein

sub

un

it73

6629

56re

fS.

sap

rop

hyt

icu

sA

TC

C15

305

1.3.

99.1

-(2

.9)

MB

/M5.

25/5

.45

65.3

26/6

6.4

4218

525

/103

P42

0Su

ccin

ate

deh

ydro

gen

ase

flav

op

rote

insu

bu

nit

7366

2956

ref

S.sa

pro

ph

ytic

us

AT

CC

1530

51.

3.99

.1-

(4.0

)M

B/M

5.25

/5.4

065

.326

/66.

639

178

24/9

5

P92

7P

yru

vate

deh

ydro

gen

ase

E1

com

po

nen

t�-

sub

un

it73

6630

04re

fS.

sap

rop

hyt

icu

sA

TC

C15

305

1.2.

4.1

-M

B/M

4.62

/4.7

035

.238

/35.

539

100

10/5

6

2.3.

Fat

tyA

cid

and

Lip

idB

iosy

nth

etic

Pat

hw

ays

P50

2F

um

aryl

acet

oac

etat

eh

ydro

lase

9346

3984

gbS.

xylo

sus

C2a

3.7.

1.2

-(2

.4)

CS/

C-E

4.85

/4.8

032

.944

/33.

739

869/

98P

378

Dih

ydro

lipo

amid

eS-

acet

yltr

ansf

eras

eco

mp

on

ent

of

pyr

uva

ted

ehyd

roge

nas

eco

mp

lex

E2

7366

3003

ref

S.sa

pro

ph

ytic

us

AT

CC

1530

52.

3.1.

12-

(14.

3)M

B/M

4.76

/4.8

046

.588

/70.

130

9812

/40

P38

2D

ihyd

rolip

oam

ide

S-ac

etyl

tran

sfer

ase

com

po

nen

to

fp

yru

vate

deh

ydro

gen

ase

com

ple

xE

2

7366

3003

ref

S.sa

pro

ph

ytic

us

AT

CC

1530

52.

3.1.

12-

(5.1

)M

B/M

4.76

/4.9

046

.588

/70.

030

8810

/62

P35

6D

ihyd

rolip

oam

ide

S-ac

etyl

tran

sfer

ase

com

po

nen

to

fp

yru

vate

deh

ydro

gen

ase

com

ple

xE

2

7366

3003

ref

S.sa

pro

ph

ytic

us

AT

CC

1530

52.

3.1.

12-

(52.

2)M

B/M

4.76

/4.8

046

.588

/72.

430

106

12/4

1

P34

0D

ihyd

rolip

oam

ide

deh

ydro

gen

ase

1500

1103

5re

fS.

sap

rop

hyt

icu

sA

TC

C15

305

1.8.

1.4

-M

B/M

4.80

/5.3

549

.510

/75.

332

105

11/8

1

P38

4A

ceto

acet

ate

syn

thas

e,la

rge

sub

un

it,

bio

syn

thet

icty

pe

EU

4759

07*

gbS.

xylo

sus

C2a

2.2.

1.6

-M

B/M

5.65

/5.0

563

.981

/70.

623

499/

66



Tab

le2.

Co

nti

nu

ed

spo

tid

pro

tein

nam

eG

IaD

Bb

Stap

hyl

ococ

cus

stra

inE

Cc

bio

film

vsp

lan

kto

nic

cells

dfr

acti

on

/p

red

icti

on

ep

IT/p

IEf

MW

T/M

WE

gse

qu

ence

cove

rage

hM

asco

tsc

ore

ip

epti

des

mat

ch

P25

6N

AD

bin

din

g3-

hyd

roxy

acyl

-Co

Ad

ehyd

roge

nas

eE

U47

5912

*gb

S.xy

losu

sC

2a1.

1.1.

35-

MB

/M5.

34/5

.40

84.3

53/1

00.5

3415

222

/126

P11

013-

oxo

acyl

-acy

l-ca

rrie

rp

rote

inre

du

ctas

eA

EU

4759

08*

gbS.

xylo

sus

C2a

1.1.

1.10

0-

MB

/M4.

87/4

.90

29.1

60/2

8.6

4059

9/11

1P

545

3-o

xoac

yl-a

cyl-

carr

ier

pro

tein

red

uct

ase

AE

U47

5908

*gb

S.xy

losu

sC

2a1.

1.1.

100

-(3

.0)

CS/

M4.

87/4

.95

29.1

60/2

7.3

4612

011

/64

P54

83-

oxo

acyl

-acy

l-ca

rrie

rp

rote

inre

du

ctas

eA

EU

4759

08*

gbS.

xylo

sus

C2a

1.1.

1.10

0-

(3.8

)C

S/M

4.87

/4.8

529

.160

/27.

225

746/

36

2.4.

Exo

pol

ysac

char

ide

Bio

syn

thet

icP

ath

way

P54

9d

TD

P-g

luco

se4,

6-d

ehyd

rata

seE

U47

5925

*gb

S.xy

losu

sC

2a1.

1.1.

133

-(3

.1)

CS/

C-E

4.51

/4.5

023

.252

/26.

659

152

14/5

4P

557

dT

DP

-glu

cose

4,6-

deh

ydra

tase

EU

4759

25*

gbS.

xylo

sus

C2a

1.1.

1.13

3-

(3.7

)C

S/C

-E4.

51/4

.45

23.2

52/2

5.7

5812

611

/69

P43

5Lu

cife

rase

-lik

em

on

oo

xyge

nas

eLu

xAh

om

olo

gue

EU

4759

16*

gbS.

xylo

sus

C2a

1.14

.14.

3-

(2.3

)C

S/C

5.58

/6.0

039

.281

/38.

528

7911

/85

3.P

oly

keti

de

Seco

nd

ary

Met

abo

lite

Pat

hw

ayP

1109

Ino

sito

lm

on

op

ho

sph

atas

efa

mily

pro

tein

9495

8277

gbS.

xylo

sus

C2a

3.1.

3.25

+(4

.3)

MB

/C5.

46/5

.60

30.1

46/2

8.2

5214

19/

35

4.M

etab

oli

smo

fN

ucl

eoti

des

P30

3R

ibo

nu

cleo

tid

ere

du

ctas

eR

sub

un

it73

6632

95re

fS.

sap

rop

hyt

icu

sA

TC

C15

305

1.17

.4.1

-(4

.3)

MB

/C-M

5.22

/5.4

580

.101

/78.

424

8116

/121

P55

2B

ifu

nct

ion

alp

uri

ne

bio

syn

thes

isp

rote

inP

urH

EU

4759

43*

gbS.

xylo

sus

C2a

2.1.

2.3

-(1

9.9)

MB

/C-M

5.08

/5.3

053

.900

/58.

538

117

13/8

5P

895

Rib

ofl

avin

bio

syn

thes

isp

rote

inE

U47

5929

*gb

S.xy

losu

sC

2a1.

1.1.

193

-M

B/C

-M5.

74/5

.95

38.6

72/3

7.5

6419

519

/86

5.M

etab

oli

smo

fC

oen

zym

esan

dP

rost

het

icG

rou

ps

P92

6N

H3-

dep

end

ent

NA

D+

syn

thet

ase

EU

4759

27*

gbS.

xylo

sus

C2a

6.3.

1.5

-(2

.3)

MB

/C-E

5.51

/5.7

030

.557

/35.

548

133

10/5

0P

880

Dih

ydro

fola

tere

du

ctas

eE

U47

5934

*gb

S.xy

losu

sC

2a1.

5.1.

3-

(6.8

)C

S/C

6.11

/6.5

518

.436

/19.

458

106

8/60

7.St

ress

Res

po

nse

Pro

tein

sP

682

Un

iver

sal

stre

ssp

rote

inU

spA

ho

mo

logu

eE

U47

5915

*gb

S.xy

losu

sC

2a-

(3.5

)C

S/C

-E4.

90/5

.10

1595

1/19

.042

857/

38P

1238

Un

iver

sal

stre

ssp

rote

inU

spA

ho

mo

logu

eE

U47

5915

*gb

S.xy

losu

sC

2a-

(4.1

)M

B/C

-E4.

90/5

.00

15.9

51/7

.750

9411

/71

8.U

nsp

ecifi

co

rU

nkn

ow

nF

un

ctio

nP

524

Oxi

do

red

uct

ase,

ald

o/k

eto

red

uct

ase

EU

4759

28*

gbS.

xylo

sus

C2a

1.1.

1.27

4-

(3.1

)C

S/C

4.61

/4.7

531

.413

/30.

125

636/

56P

1207

AlG

2-lik

ep

rote

inE

U47

5910

*gb

S.xy

losu

sC

2a-

(3.8

)M

B/M

6.85

/5.9

023

.481

/19.

646

529/

233

P87

9A

cety

ltr

ansf

eras

eA

(GN

AT

)fa

mily

EU

4759

17*

gbS.

xylo

sus

C2a

2.3

-(2

9.5)

CS/

C-E

5.37

/5.7

019

.721

/19.

053

104

9/59

P69

1A

cety

ltr

ansf

eras

eB

(GN

AT

)fa

mily

EU

4759

48*

gbS.

xylo

sus

C2a

2.3

-(3

3.8)

CS/

C5.

26/5

.60

18.5

27/1

8.5

6111

212

/59

P45

7M

etal

dep

end

ent

ph

osp

ho

hyd

rola

se73

6627

88re

fS.

sap

rop

hyt

icu

sA

TC

C15

305

3.1.

3.5

-M

B/E

5.20

/5.2

558

.305

/65.

132

6414

/103

9.C

ell

Surf

ace

Exp

ose

dP

rote

ins

P82

7C

ell-

shap

ed

eter

min

ing

pro

tein

Mre

C15

0011

019

gbS.

xylo

sus

C2a

-C

W/C

-E6.

92/5

.65

31.0

89/2

6.5

4389

9/82

P80

0Im

mu

no

do

min

ant

anti

gen

A94

9582

55gb

S.xy

losu

sC

2a-

(6.5

)C

W/W

5.94

/5.3

025

.379

/27.

934

709/

61P

338

Cat

alas

eC

EU

4759

09*

gbS.

xylo

sus

C2a

1.11

.1.6

-(8

.9)

MB

/C-E

5.16

/5.2

575

.488

/75.

143

149

26/1

02P

328

Cat

alas

eC

EU

4759

09*

gbS.

xylo

sus

C2a

1.11

.1.6

-M

B/C

-E5.

16/5

.30

75.4

88/7

6.2

3414

318

/106

P11

19T

ran

scri

pti

on

ple

iotr

op

icre

pre

sso

rC

od

Y73

6628

22re

fS.

sap

rop

hyt

icu

sA

TC

C15

305

-(2

.3)

MB

/C5.

44/6

.40

28.5

43/2

7.7

5214

512

/28

P48

7G

luco

se-6

-ph

osp

hat

eis

om

eras

e91

2066

99sp

S.sa

pro

ph

ytic

us

AT

CC

1530

55.

3.1.

9-

(3.1

)C

W/M

4.84

/4.8

049

.796

/48.

439

135

13/8

0

aG

enIn

foId

enti

fier

(GI)

.A

ster

isk

(*)

ind

icat

esG

enB

ank

tem

po

rary

assi

gnm

ent

nu

mb

erfo

rS.

xylo

sus

C2a

pro

tein

s.b

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efficient enzyme in NH4+ assimilation especially at low con-

centration.68 Moreover, branched chain amino-transferaseinvolved in catabolism of valine, leucine and isoleucine wasdown-expressed (spot P407) in S. xylosus growing in biofilmcompared to planktonic cells (Table 2). In Bacillus subtilis,ureABC encoding urease and branched chained amino acidsdegradative operon are well-known as down-expressed inconditions of nitrogen excess and controlled by multipleregulatory factors, for example, CodY, GlnR or TnrA.68 It canbe noticed that a homologue to NifU (3.0 × 10-31 e E-valueswith convergence reached after 5 iterations with PSI-BLAST),that is, an enzyme of NIF (nitrogen fixation) system requiredfor the formation of metalloclusters of nitrogenase69 andannotated as an hypothetical protein in Staphylococcus speciesis up-expressed in S. xylosus biofilm (Table 1, spot B789).Though, as this protein is also involved in the maturation ofother FeS proteins, it can be found in organisms that do notfix atmospheric nitrogen at all.70 Fixation of N2 by S. xylosushas never been reported and would require further experimen-tal investigation.

Several enzymes taking steps in amino acids biosynthesiswere up-expressed in S. xylosus biofilm (Table 1). They covera wide range of amino acid categories from aliphatic, polar toaromatic, as most of these enzymes are involved in severalamino acid biosynthetic pathways, for example, the aspartateamino transferase (EC:2.6.1.1), urocanate hydratase (EC:4.2.1.49) or dihydrodipicolinate reductase (EC:1.3.1.26). Con-trary to most Staphylococcus species, S. xylosus C2a is pro-totrophic for all amino acids.71 It is worth noticing that a DHAP(3-deoxy-D-arabino-heptulosonate 7-phosphate; EC:2.5.1.54)synthetase is highly expressed in S. xylosus biofilm (more than20-fold higher for spots B320 and B322) compared to planktoniccells (Table 1). This enzyme involved in phenylalanine, tyrosineand tryptophane biosynthesis, also exhibited in its N-proximalregion a chorismate mutase domain (EC 5.4.99.5; PF01817:E-value ) 2.7 × 10-29), which catalyzes the first of 7 steps inthe biosynthesis of chorismate.72

In correlation with active amino acid anabolism in S. xylosusbiofilm, tyrosyl-tRNA synthetase, required for specific attach-ment of tyrosine to the 3′ end of tRNATyr for subsequentincorporation of this aromatic amino acid in polypeptidicchain, was also found up-expressed (Table 1, spot B357).Expression level of ribosomal proteins was also enhanced, andconsidering 30S ribosomal protein S2 in particular (spot B950),its level is 35-fold higher in the MB fraction of sessile comparedto planktonic cells of S. xylosus, which suggests ribosomeassociation with Sec translocon in the course of co-translationaltranslocation in SRP (signal recognition particle)-dependentmanner.66 Furthermore, signal peptidase of Type I involved inmaturation of translocated proteins by cleavage of their N-terminal signal peptide was also up-expressed in MB fractionof sessile cells (Table 1, spot B1208). Bearing in mind that themechanisms of protein folding and secretion are closelyintertwined,73 higher levels of several key proteins involved infolding process, namely, peptidyl-prolyl isomerase (spot B617)assisted by chaperones DnaK (spot B394) and GroES (spotB739), were also found. It is worth noting that DnaK and GroESare also induced by stress conditions encountered in biofilm.74-76

On the contrary, translation initiation factor IF-2 was stronglydown-expressed (more than 90-fold lower for spots P298 andP299) in S. xylosus biofilm (Table 2). Actually, the activity ofIF-2 is related to secretion since mutation can restore asecretion defect and somehow compensated for by a reduction

in the translation initiation rate.77 Taken together, these resultsstrongly suggest protein trafficking is more active in sessile thanin planktonic cells of S. xylosus.

Carbon Metabolism in S. xylosus Biofilm: From Carbo-hydrate Catabolism to Exopolysaccharide Biosynthesis. Asindicated by up-expression of two key enzymes of the pentosephosphate cycle (Table 1), namely, glucose-1-phosphate de-hydrogenase (G1PDH; EC:1.1.1.47; spot B577) and transaldolase(EC:2.2.1.2; spots B1153 and B1154), respectively, this routeseems to constitute the first steps of carbohydrate catabolismin sessile cells of S. xylosus. While G1PDH belongs to a non-PTS glucose uptake system in S. xylosus,78 transaldolaseproduces fructose 6-phosphate that connects pentose phos-phate pathway with glycolysis. From there, key enzymes of theglycolysis were highly expressed in biofilm, namely, fructose-bisphosphate aldolase (EC:4.1.2.13; spots B960, B961 andB1342) and glyceraldehyde-3-phosphate dehydrogenase (EC:1.2.1.12; spots B436 and B440) (Table 1). At first sight, presenceof some of these glycolytic enzymes in the MB fraction seemsawkward, but this well-known phenomenon was also suggestedto be related to some kind of post-translational regulation.79

Downstream, several enzymes of the tricarboxylic acid cycle(TCA) were up-expressed in S. xylosus biofilm (Table 1, spotsB209, B210, B757, B768, B771, B867 and B888).

Interestingly, phosphoglucomutase (PGM; EC:5.4.2.2), whichis involved in both pentose phosphate and glycolytic pathwayswhere it converts glucose 1-phosphate into glucose 6-phos-phate, was up-expressed (more than 5-fold higher for spotB517) in S. xylosus biofilm compared to planktonic cells (Table1). This enzyme belongs to the phosphoglucomutase/phos-phomannomutase family (EC:5.4.2.2/5.4.2.8; PF02878: E-value) 1.1 × 10-54; COG1109: E-value ) 2.0 × 10-74) and thus mightalso be involved in conversion of mannose 1-phosphate intomannose 6-phosphate. In several bacterial species, PGM isactually bifunctional and is required for formation of variousexopolysaccharides (EPS).80 Indeed, as a rigid node at least inseveral Gram-positive bacteria,81,82 PGM is controlling andpartitioning carbon flux toward central metabolism and EPSbiosynthesis pathways. In relation with EPS biosynthesis, aprotein annotated as hypothetical in Staphylococcus species butexhibiting clear homology with dTDP-glucose 4,6-dehydratase(EC:4.2.1.46; 8.0 × 10-86 e E-values with convergence reachedafter 27 iterations with PSI-BLAST) and displaying a dTDP-4-dehydrorhamnose reductase domain (EC:1.1.1.133; PF04321:E-value ) 9.4 × 10-5) responsible for binding a sugar nucleotideand synthesis of dTDP-rhamnose was up-expressed in S.xylosus biofilm (Table 1, spot B1118). As part of nucleotidesugar metabolism, this protein could participate in bacterialsurface polysaccharide production in sessile cells of S. xylosus.Indeed, in Streptococcus thermophilus, it appeared that activi-ties of enzymes involved in glycolysis, sugar nucleotide and EPSbiosyntheses were strongly correlated, in particular PGM anddTDP-4-dehydrorhamnose reductase.80,83

Furthermore, an homologue to a LuxA-like protein fromAcidithiobacillus ferroxidans (4.0 × 10-43 e E-values withconvergence reached after 6 iterations with PSI-BLAST) wasidentified. As defined in A. ferroxidans,84 LuxA-like from S.xylosus belongs to COG2141 corresponding to a protein familyof coenzyme F420-dependent N5,N10-methylene tetrahy-dromethanopterin and related flavin-dependent oxidoreduc-tase (E-value ) 7.0 × 10-37) and contains a Pfam domaincharacteristic of bacterial luciferase-like monooxygenase(PF00296: E-value ) 1.4 × 10-25). Role of such LuxA-like protein



remains speculative but was suggested as related to regulationof EPS biosynthesis as it is part of the gal operon together withpgm, though such genetic organization is not conserved fromone bacterial species to another.84 This LuxA-like protein washere identified as down-expressed in biofilm compared toplanktonic cells (Table 2, spot P435). Altogether, this proteomicanalysis allowed us to identify novel proteins that could beinvolved in a previously uncovered EPS biosynthetic pathwayin S. xylosus. Indeed, in a former study, we have shown that S.xylosus synthesized EPS during its growth in biofilm whichsynthesis was ica-independent and the composition of this EPSwas unknown.10

Polyketide Secondary Metabolite Pathway in SessileCells of S. xylosus. From the glucose 1-phosphate hub, it canbe further noticed that carbon flux can be directed toward theformation of polyketide sugar units, via the activity dTDP-4-dehydrorhamnose reductase also involved in EPS bio-synthesis.85,86 While several enzymes of fatty acid/lipid me-tabolism were down-expressed (Table 2), one of them wasparticularly highly expressed in S. xylosus biofilm as it wasundetectable in planktonic cells, namely, acetyl-CoA carboxy-lase (EC:6.4.1.2; Table 1, spot B455). Actually, this enzymeallows the conversion of acetyl-CoA into malonyl-CoA, whichare both essential building blocks for biosynthesis of polyketideas polyketide synthases condense them to form longer chainsof carbon.87,88 Moreover, among up-expressed proteins in CSfraction (Table 1), protein directly involved in polyketidebiosynthetic pathway could be identified,89 that is, (i) thebifunctional enzyme DAHP synthetase/chorismate mutase(spots B320 and B322) involved in aromatic amino acidbiosynthesis but also required for producing starter units inpolyketide assembly,72,90 (ii) a protein annotated as hypotheti-cal in Staphylococcus species but here identified as homologousto polyketide cyclase/dehydrase (EC:4.2.1; 4.0 × 10-26 e E-values after 27 iterations with PSI-BLAST; spot B652), which isan enzyme necessary for assembly and cyclization of thepolyketide chains,91 and (iii) an orthologue to antibiotic bio-synthesis monooxygenase (EC:4.2.1; 8.0 × 10-18e E-values withconvergence reached after 17 iterations with PSI-BLAST)exhibiting such a domain (PF03992: E-value ) 1.1 × 10-9;COG2329: E-value ) 2.0 × 10-13; spot B780), which is anenzyme catalyzing oxygenations of polyketide biosyntheticintermediates without the need for prosthetic groups, metalions or cofactors normally associated with activation of mo-lecular oxygen.92 It can also be noticed that up-expressedinositol monophosphatase (spot P1109) generates myoinositol,a key compound in biosynthesis of several aminoglycosideantibiotics.93 Taken together, these results indicate that severalenzymes related to polyketide biosynthesis are up-expressedin S. xylosus biofilm, which suggests that such pathways arepossibly required for sedentary lifestyle.

Cell Surface Exposed Proteins. While immunodominantantigen A and MreC were as expected sublocalized in the cellwall,23 they were both down-expressed in S. xylosus biofilm(Table 2, spots P800 and P827, respectively). Adding lipoproteinbinding protein from ABC-type Mn/Zn transport system (Table1, spots B980 and B1338), only three proteins differentiallyexpressed were originally predicted and found as surfaceexposed in S. xylosus. Though, some cytoplasmic proteins,namely, alkaline shock protein 23 (spots B978, B979), THiJ/PfpI protease/amidase (spots B813) and catalase C (P328, P338),were also predicted as translocated across the cytoplasmicmembrane via unknown system(s), which could thus explain

their localization in the bacterial cell envelope (Tables 1, 2 andSupporting Information 1S, 2S). Concerning catalase C, formerstudies reported the presence of two catalases in S. xylosus C2a,namely, KatA and KatB.23,94 The present study allowed uncov-ering a third catalase KatC, which exhibits similarity withcatalase KatE (E-value ) 0.0) from B. subtilis.95 Despite theabsence of a signal sequence and typical motifs required forcell-surface display,58 the attachment to bacterial cell surfacesof enzymes primarily cytoplasmic seems awkward at first sightbut is commonly reported from various proteomic investiga-tions in Gram-positive bacteria, including staphylococci.96 Suchproteins could further moonlight on the bacterial cell-surface,for example, S. aureus enolase exhibits there efficient plasmi-nogen-binding activity.97 Alternatively, some of these proteinscould be related to cell wall biogenesis such as lactate dehy-drogenase (spots B746 and B748) as described in Lactobacillusplantarum98 or alanine dehydrogenase (spots B616 and B617)as reported in Mycobacterium tuberculosis.99 Interestingly, axenologue to a phage capside protein (PF05065: E-value ) 7.8× 10-88; spot B1029) was up-expressed in sessile cells of S.xylosus. Phage-release is a frequent and normal event inbiofilms of S. aureus where resulting lysis of cells wouldpromote persistence and survival of the remaining cells, as theygain a nutrient reservoir.100 As lysogeny regulation is relatedto bacterial competence and virulence in S. aureus, it is thentempting to liken this with allolysis, another apoptotic butnonlysogenic mechanism, which was first described in Strep-tococcus pneumoniae, where competent cells triggered lysis ofnoncompetent cells in a tightly control process involvingbacteriocins.101 Besides genetic and nutrient exchange, allolysiswould explain the cell surface association of primarily cyto-plasmic proteins, which have no obvious mechanism of secre-tion but would instead be scavenged from apoptosed cells;102

some of these proteins could further moonlight on S. xylosuscell surface.103

Conclusions

With up-expression of some proteins involved in amino acidsmetabolism, translation and secretion, nitrogen metabolismappeared as quite active in sessile cells of S. xylosus. Asdescribed in B. subtilis,104 some secreted proteins could havea role in the formation of S. xylosus biofilm, which wouldrequire further investigations of the exoproteome. In addition,several primarily cytoplasmic proteins found on the bacterialcell surface might moonlight and have adhesive propertiesplaying a role in the formation of biofilm. For example,plasminogen binding activity for enolase has never beeninvestigated in coagulase-negative staphylococci, whereas it hasbeen clearly described in several Gram-positive bacteria in-cluding S. aureus.97

Besides active carbohydrate catabolism involving pentosephosphate and glycolytic pathways, this proteomic analysisrevealed up-expression of several enzymes related to EPSbiosynthesis in S. xylosus biofilm when compared to planktoniccells. This biosynthetic route could be somehow related to theLeloir pathway as described in Acidithiobacillus ferrooxidans,as two proteins here identified exhibited homology withproteins encoded by rfbBD and luxA genes involved in EPSformation in this microorganism.84 While formation of EPS hasclearly been reported in S. xylosus,10 the absence of ica genesas described in S. epidermidis RP62A, which is used as aparadigm for coagulase-negative Staphylococcus,105 was quitepuzzling. Present findings provide good basis to further inves-



tigate and unravel the metabolic pathway involved in thebiosynthesis of EPS in an ica-independent manner in S. xylosus.Furthermore, our investigation is the first to highlight apreviously overlooked aspect of cell physiology of Staphylo-coccus species, namely, the biosynthesis of polyketide second-ary metabolites. Interestingly, the lipopeptide surfactin whoseformation required polyketide synthetases, shows weak anti-biotic but strong surfactant properties and is specificallyrequired for biofilm formation in B. subtilis.106 The presentfindings should also promote further investigations in thatdirection to determine how this polyketide pathway is relatedto S. xylosus biofilm formation.

Acknowledgment. This work was supported in partby INRA. Stella Planchon is a Ph.D. research fellow grantedby the French Minister of National Education and Research(MENR). The authors thank David S. Holmes (Center forBioinformatics and Genome Biology, Millenium Institute ofFundamental and Applied Biology, Life Science Foundation,Andres Bello University, Santiago, Chile) for providing themwith genome information on A. ferrooxidans, in particularcoordinates of luxA. Excellent technical assistance of NicoleGarrel and Brigitte Duclos as well as Sibille Farrer for MSanalyses is also acknowledged. The authors further thankanonymous reviewers for constructive comments.

Supporting Information Available: Summary of the89 proteins differentially expressed and identified from pro-teomic analysis in the different cell fractions in relation to thefinal subcellular localization predictions as well as predictedprotein categories by a rational bioinformatic approach. Thismaterial is available free of charge via the Internet at http://pubs.acs.org.

References(1) Kloos, W. E.; Schleifer, K. H. In Bergey’s Manual of Systematic

Bacteriology; Sneath, P. H. A., Mair, N. S., Sharpe, M. E., Holt,J. G., Eds.; The Williams Wilkins Co.: Baltimore, MA, 1986; pp1013-1035.

(2) Irlinger, F. Int. J. Food Microbiol. 2008, 126 (3), 311–315.(3) Talon, R.; Leroy-Setrin, S.; Fadda, S. In Research Advances in

Quality of Meat and Meat Products; Fidel, Toldra, Ed.; ResearchSignpost: India, 2002; pp 175-191.

(4) Esper, R. C.; Morales, M. T.; Ruiz, S. S. Rev. Med. Hosp. Gen. Mex.2000, 63, 107–111.

(5) Siqueira, J. F. J.; Lima, K. C. Aust. Endod. J: J. Aust. Soc. Endod.Inc. 2002, 28, 61–63.

(6) Almeida, R. A.; Oliver, S. P. Microb. Pathog. 2001, 31, 205–212.(7) Cucarella, C.; Tormo, M. A.; Ubeda, C.; Trotonda, M. P.; Monzon,

M.; Peris, C.; Amorena, B.; Lasa, I.; Penades, J. R. Infect. Immun.2004, 72, 2177–2185.

(8) Won, Y. S.; Kwon, H. J.; Oh, G. T.; Kim, B. H.; Lee, C. H.; Park,Y. H.; Hyun, B. H.; Choi, Y. K. Microbiol. Immunol. 2002, 46, 629–632.

(9) Ug, A.; Ceylan, O. Arch. Med. Res. 2003, 34, 130–136.(10) Planchon, S.; Gaillard-Martinie, B.; Dordet-Frisoni, E.; Bellon-

Fontaine, M. N.; Leroy, S.; Labadie, J.; Hebraud, M.; Talon, R.Int. J. Food Microbiol. 2006, 109, 88–96.

(11) Donlan, R. M. Emerg. Infect. Dis. 2002, 8, 881–890.(12) Heilmann, C.; Schweitzer, O.; Gerke, C.; Vanittanakom, N.; Mack,

D.; Gotz, F. Mol. Microbiol. 1996, 20, 1083–1091.(13) Mack, D.; Fischer, W.; Krokotsch, A.; Leopold, K.; Hartmann, R.;

Egge, H.; Laufs, R. J. Bacteriol. 1996, 178, 175–183.(14) Cramton, S. E.; Gerke, C.; Schnell, N. F.; Nichols, W. W.; Gotz, F.

Infect. Immun. 1999, 67, 5427–5433.(15) Foster, T. J.; Hook, M. Trends Microbiol. 1998, 6, 484–488.(16) Heilmann, C.; Hussain, M.; Peters, G.; Gotz, F. Mol. Microbiol.

1997, 24, 1013–1024.(17) Hussain, M.; Herrmann, M.; von Eiff, C.; Perdreau-Remington,

F.; Peters, G. Infect. Immun. 1997, 65, 519–524.(18) Cucarella, C.; Solano, C.; Valle, J.; Amorena, B.; Lasa, I.; Penades,

J. R. J. Bacteriol. 2001, 183, 2888–2896.

(19) Gross, M.; Cramton, S. E.; Gotz, F.; Peschel, A. Infect. Immun.2001, 69, 3423–3426.

(20) Beenken, K. E.; Dunman, P. M.; McAleese, F.; Macapagal, D.;Murphy, E.; Projan, S. J.; Blevins, J. S.; Smeltzer, M. S. J. Bacteriol.2004, 186, 4665–4684.

(21) Resch, A.; Rosenstein, R.; Nerz, C.; Gotz, F. Appl. Environ.Microbiol. 2005, 71, 2663–2676.

(22) Dordet-Frisoni, E.; Talon, R.; Leroy, S. FEMS Microbiol. Lett. 2007,266, 184–193.

(23) Planchon, S.; Chambon, C.; Desvaux, M.; Chafsey, I.; Leroy, S.;Talon, R.; Hebraud, M. J. Proteome Res. 2007, 6, 3566–3580.

(24) Mastro, R.; Hall, M. Anal. Biochem. 1999, 273, 313–315.(25) Rabilloud, T.; Blisnick, T.; Heller, M.; Luche, S.; Aebersold, R.;

Lunardi, J.; Braun-Breton, C. Electrophoresis 1999, 20, 3603–3610.(26) Neuhoff, V.; Arold, N.; Taube, D.; Ehrhardt, W. Electrophoresis

1988, 9, 255–262.(27) Yan, J. X.; Wait, R.; Berkelman, T.; Harry, R. A.; Westbrook, J. A.;

Wheeler, C. H.; Dunn, M. J. Electrophoresis 2000, 21, 3666–3672.(28) Patton, W. F. Electrophoresis 2000, 21, 1123–1144.(29) Bryson, K.; Loux, V.; Bossy, R.; Nicolas, P.; Chaillou, S.; van de

Guchte, M.; Penaud, S.; Maguin, E.; Hoebeke, M.; Bessieres, P.;Gibrat, J. F. Nucleic Acids Res. 2006, 34, 3533–3545.

(30) Bendtsen, J. D.; Nielsen, H.; von Heijne, G.; Brunak, S. J. Mol.Biol. 2004, 340, 783–795.

(31) Kall, L.; Krogh, A.; Sonnhammer, E. L. J. Mol. Biol. 2004, 338, 1027–1036.

(32) Shen, H. B.; Chou, K. C. Biochem. Biophys. Res. Commun. 2007,363, 297–303.

(33) Gomi, M.; Sonoyama, M.; Mitaku, S. Chem-Bio Info. J. 2004, 4,142–147.

(34) Hiller, K.; Grote, A.; Scheer, M.; Munch, R.; Jahn, D. Nucleic AcidsRes. 2004, 32, W375-W379.

(35) Juncker, A. S.; Willenbrock, H.; Von Heijne, G.; Brunak, S.; Nielsen,H.; Krogh, A. Protein Sci. 2003, 12, 1652–1662.

(36) Madan Babu, M.; Sankaran, K. Bioinformatics 2002, 18, 641–643.(37) Sutcliffe, I. C.; Harrington, D. J. Microbiology 2002, 148, 2065–

2077.(38) Bendtsen, J. D.; Nielsen, H.; Widdick, D.; Palmer, T.; Brunak, S.

BMC Bioinf. 2005, 6, 167.(39) Rose, R. W.; Bruser, T.; Kissinger, J. C.; Pohlschroder, M. Mol.

Microbiol. 2002, 45, 943–950.(40) Bendtsen, J. D.; Kiemer, L.; Fausboll, A.; Brunak, S. BMC Microbiol.

2005, 5, 58.(41) Hofmann, K.; Stoffel, W. Biol. Chem. Hoppe-Seyler 1993, 374,

166–170.(42) Sonnhammer, E. L.; von Heijne, G.; Krogh, A. Proc. Int. Conf.

Intell. Syst. Mol. Biol. 1998, 6, 175–182.(43) Jones, D. T. Bioinformatics 2007, 23, 538–544.(44) Tusnady, G. E.; Simon, I. Bioinformatics 2001, 17, 849–850.(45) Claros, M. G.; von Heijne, G. Comput. Appl. Biosci. 1994, 10, 685–

686.(46) Zhou, H.; Zhou, Y. Protein Sci. 2003, 12, 1547–1555.(47) Gardy, J. L.; Laird, M. R.; Chen, F.; Rey, S.; Walsh, C. J.; Ester, M.;

Brinkman, F. S. Bioinformatics 2005, 21, 617–623.(48) Yu, C. S.; Lin, C. J.; Hwang, J. K. Protein Sci. 2004, 13, 1402–1406.(49) Bateman, A.; Coin, L.; Durbin, R.; Finn, R. D.; Hollich, V.; Griffiths-

Jones, S.; Khanna, A.; Marshall, M.; Moxon, S.; Sonnhammer, E. L.;Studholme, D. J.; Yeats, C.; Eddy, S. R. Nucleic Acids Res. 2004,32, D138-D141.

(50) Eddy, S. R. Curr. Opin. Struct. Biol. 1996, 6, 361–365.(51) Tatusov, R. L.; Fedorova, N. D.; Jackson, J. D.; Jacobs, A. R.;

Kiryutin, B.; Koonin, E. V.; Krylov, D. M.; Mazumder, R.; Mekhe-dov, S. L.; Nikolskaya, A. N.; Rao, B. S.; Smirnov, S.; Sverdlov,A. V.; Vasudevan, S.; Wolf, Y. I.; Yin, J. J.; Natale, D. A. BMC Bioinf.2003, 4, 41.

(52) Altschul, S. F.; Gish, W.; Miller, W.; Myers, E. W.; Lipman, D. J. J.Mol. Biol. 1990, 215, 403–410.

(53) Wilson, D.; Madera, M.; Vogel, C.; Chothia, C.; Gough, J. NucleicAcids Res. 2007, 35, D308-D313.

(54) Schultz, J.; Milpetz, F.; Bork, P.; Ponting, C. P. Proc. Natl. Acad.Sci. U.S.A. 1998, 95, 5857–5864.

(55) Altschul, S. F.; Madden, T. L.; Shaffer, A. A.; Zhang, J.; Zhang, Z.;Miller, W.; Lipman, D. J. Nucleic Acids Res. 1997, 25, 3389–3402.

(56) Wu, C. H.; Apweiler, R.; Bairoch, A.; Natale, D. A.; Barker, W. C.;Boeckmann, B.; Ferro, S.; Gasteiger, E.; Huang, H.; Lopez, R.;Magrane, M.; Martin, M. J.; Mazumder, R.; O’Donovan, C.;Redaschi, N.; Suzek, B. Nucleic Acids Res. 2006, 34, D187-D191.

(57) Desvaux, M.; Hebraud, M. In Handbook of Listeria monocytogenes;Dongyou L. , Ed.; CRC Press, Taylor and Francis Group: BocaRaton, FL, 2008.



(58) Desvaux, M.; Dumas, E.; Chafsey, I.; Hebraud, M. FEMS Microbiol.Lett. 2006, 256, 1–15.

(59) Molloy, M. P.; Herbert, B. R.; Walsh, B. J.; Tyler, M. I.; Traini, M.;Sanchez, J. C.; Hochstrasser, D. F.; Williams, K. L.; Gooley, A. A.Electrophoresis 1998, 19, 837–844.

(60) Tjalsma, H. Proteomics 2007, 7, 73–81.(61) Sakata, N.; Terakubo, S.; Mukai, T. Curr. Microbiol. 2005, 50, 47–

51.(62) Stewart, G. C. Mol. Microbiol. 2005, 57, 1177–1181.(63) Desvaux, M.; Hebraud, M. FEMS Microbiol. Rev. 2006, 30, 774–

805.(64) Vilhelmsson, O.; Miller, K. J. Appl. Environ. Microbiol. 2002, 68,

2353–2358.(65) Muller, V.; Gruber, G. Cell. Mol. Life Sci. 2003, 60, 474–494.(66) Valent, Q. A.; Scotti, P. A.; High, S.; de Gier, J. W.; von Heijne, G.;

Lentzen, G.; Wintermeyer, W.; Oudega, B.; Luirink, J. EMBO J.1998, 17, 2504–2512.

(67) Mobley, H. L.; Island, M. D.; Hausinger, R. P. Microbiol. Rev. 1995,59, 451–480.

(68) Fisher, S. H. Mol. Microbiol. 1999, 32, 223–232.(69) Dos Santos, P. C.; Smith, A. D.; Frazzon, J.; Cash, V. L.; Johnson,

M. K.; Dean, D. R. J. Biol. Chem. 2004, 279, 19705–19711.(70) Hwang, D. M.; Dempsey, A.; Tan, K. T.; Liew, C. C. J. Mol. Evol.

1996, 43, 536–540.(71) Fiegler, H.; Bruckner, R. FEMS Microbiol. Lett. 1997, 148, 181–

187.(72) Bentley, R. Crit. Rev. Biochem. Mol. Biol. 1990, 25, 307–384.(73) Li, W.; Zhou, X.; Lu, P. Res. Microbiol. 2004, 155, 605–610.(74) Bergmann, S.; Rohde, M.; Hammerschmidt, S. Infect. Immun.

2004, 72, 2416–2419.(75) Antelmann, H.; Williams, R. C.; Miethke, M.; Wipat, A.; Albrecht,

D.; Harwood, C. R.; Hecker, M. Proteomics 2005, 5, 3684–3695.(76) Maira-Litran, T.; Allison, D. G.; Gilbert, P. J. Appl. Microbiol. 2000,

88, 243–247.(77) Laursen, B. S.; Siwanowicz, I.; Larigauderie, G.; Hedegaard, J.; Ito,

K.; Nakamura, Y.; Kenney, J. M.; Mortensen, K. K.; Sperling-Petersen, H. U. J. Mol. Biol. 2003, 326, 543–551.

(78) Fiegler, H.; Bassias, J.; Jankovic, I.; Bruckner, R. J. Bacteriol. 1999,181, 4929–4936.

(79) Gutowicz, J.; Terlecki, G. Cell Mol. Biol. Lett. 2003, 8, 667–680.(80) Levander, F.; Radstrom, P. Appl. Environ. Microbiol. 2001, 67,

2734–2738.(81) Degeest, B.; de Vuyst, L. Appl. Environ. Microbiol. 2000, 66, 3519–

3527.(82) Desvaux, M.; Guedon, E.; Petitdemange, H. Appl. Environ.

Microbiol. 2001, 67, 3837–3845.(83) Levander, F.; Svensson, M.; Radstrom, P. Appl. Environ. Microbiol.

2002, 68, 784–790.

(84) Barreto, M.; Jedlicki, E.; Holmes, D. S. Appl. Environ. Microbiol.2005, 71, 2902–2909.

(85) Nedal, A.; Zotchev, S. B. Appl. Microbiol. Biotechnol. 2004, 64,7–15.

(86) Trefzer, A.; Salas, J. A.; Bechthold, A. Nat. Prod. Rep. 1999, 16,283–299.

(87) Hopwood, D. A. PLoS Biol. 2004, 2, E35.(88) Schneider, G. Curr. Opin. Struct. Biol. 2005, 15, 629–636.(89) Walsh, C. T. Science 2004, 303, 1805–1810.(90) Gosset, G.; Bonner, C. A.; Jensen, R. A. J. Bacteriol. 2001, 183,

4061–4070.(91) McDaniel, R.; Ebert-Khosla, S.; Hopwood, D. A.; Khosla, C. J. Am.

Chem. Soc. 1994, 116, 10855–10859.(92) Sciara, G.; Kendrew, S. G.; Miele, A. E.; Marsh, N. G.; Federici, L.;

Malatesta, F.; Schimperna, G.; Savino, C.; Vallone, B. EMBO J.2003, 22, 205–215.

(93) Flatt, P. M.; Mahmud, T. Nat. Prod. Rep. 2007, 24, 358–392.(94) Barriere, C.; Bruckner, R.; Centeno, D.; Talon, R. FEMS Microbiol.

Lett. 2002, 216, 277–283.(95) Engelmann, S.; Lindner, C.; Hecker, M. J. Bacteriol. 1995, 177,

5598–5605.(96) Gatlin, C. L.; Pieper, R.; Huang, S. T.; Mongodin, E.; Gebregeorgis,

E.; Parmar, P. P.; Clark, D. J.; Alami, H.; Papazisi, L.; Fleischmann,R. D.; Gill, S. R.; Peterson, S. N. Proteomics 2006, 6, 1530–1549.

(97) Antikainen, J.; Kuparinen, V.; Lahteenmaki, K.; Korhonen, T. K.FEMS Immunol. Med. Microbiol. 2007, 51, 526–534.

(98) Goffin, P.; Deghorain, M.; Mainardi, J. L.; Tytgat, I.; Champomier-Verges, M. C.; Kleerebezem, M.; Hols, P. J. Bacteriol. 2005, 187,6750–6761.

(99) Andersen, A. B.; Andersen, P.; Ljungqvist, L. Infect. Immun. 1992,60, 2317–2323.

(100) Resch, A.; Fehrenbacher, B.; Eisele, K.; Schaller, M.; Gotz, F. FEMSMicrobiol. Lett. 2005, 252, 89–96.

(101) Guiral, S.; Mitchell, T. J.; Martin, B.; Claverys, J. P. Proc. Natl. Acad.Sci. U.S.A. 2005, 102, 8710–8715.

(102) Bergmann, S.; Rohde, M.; Chhatwal, G. S.; Hammerschmidt, S.Mol. Microbiol. 2001, 40, 1273–1287.

(103) Jeffery, C. J. Trends Biochem. Sci. 1999, 24, 8–11.(104) Hamon, M. A.; Stanley, N. R.; Britton, R. A.; Grossman, A. D.;

Lazazzera, B. A. Mol. Microbiol. 2004, 52, 847–860.(105) Conlon, K. M.; Humphreys, H.; O’Gara, J. P. J. Bacteriol. 2002,

184, 4400–4408.(106) Hofemeister, J.; Conrad, B.; Adler, B.; Hofemeister, B.; Feesche,

J.; Kucheryava, N.; Steinborn, G.; Franke, P.; Grammel, N.;Zwintscher, A.; Leenders, F.; Hitzeroth, G.; Vater, J. Mol. Genet.Genomics 2004, 272, 363–378.

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