A comprehensive two-dimensional map of cytosolic proteins ofBacillus subtilis

Knut Büttner1

Jörg Bernhardt1

Christian Scharf1

Roland Schmid2

Ulrike Mäder1

Christine Eymann1

Heike Antelmann1

Andrea Völker3

Uwe Völker3

Michael Hecker1

1Institut für Mikrobiologie,Ernst-Moritz-Arndt-UniversitätGreifswald, Germany

2Abteitung für Mikrobiologie,Universität OsnabrückGermany

3Laboratorium für Mikrobiologie,Philipps-Universität undMax-Planck-Institut fürterrestrische Mikrobiologie,Marburg, Germany

A comprehensive two-dimensional mapof cytosolic proteins of Bacillus subtilis

Proteomics relying on two-dimensional (2-D) gel electrophoresis of proteins followedby spot identification with mass spectrometry is an excellent experimental tool for phy-siological studies opening a new perspective for understanding overall cell physiology.This is the intriguing outcome of a method introduced by Klose and O’Farrell indepen-dently 25 years ago. Physiological proteomics requires a 2-D reference map on whichmost of the main proteins were identified. In this paper, we present such a referencemap with more than 300 entries for Bacillus subtilis proteins with an isoelectric point(pI) between 4 and 7. The most abundant proteins of exponentially growing cells werecompiled and shown to perform mainly housekeeping functions in glycolysis, tricar-boxylic acid cycle (TCC), amino acid biosynthesis and translation as well as proteinquality control. Furthermore, putative post-translational modifications were shown ata large scale, with 47 proteins in total forming more than one spot. In a few selectedcases evidence for phosphorylation of these proteins is presented. The proteome ana-lysis in the standard pI range was complemented by either stretching the mostcrowded regions in a narrow pH gradient 4.5–5.5, or by adding other fractions of thetotal B. subtilis proteome such as alkaline proteins as well as extracellular proteins. Abig challenge for future studies is to provide an experimental protocol covering thefraction of intrinsic membrane proteins that almost totally escaped detection by theexperimental procedure used in this study.*

Keywords: Bacillus subtilis / Master gel / Protein modifications / Mass spectrometry EL 4568

1 Introduction

About 4100 different open reading frames (ORFs) werepredicted from the genome sequence of Bacillus subtilispublished four years ago [1]. Already more than 25 yearsago, Klose [2] and O’Farrell [3] introduced the high-resolu-tion 2-D gel electrophoresis technique separating morethan 1000 different Escherichia coli proteins. The recentdevelopments in the sequencing of total genomes openedentirely new perspectives for proteome research allowingits application at a wide scale.

Almost 15 years ago we started to use the proteomicsapproach to get new information on the response ofB. subtilis cells to stress and starvation. A dramaticchange in the protein synthesis pattern in response tostress or starvation was found: various stress or starva-tion stimuli strongly induced the same large group of newproteins that should consequently be involved in theadaptation to the environmental constraints imposed tothe cell [4, 5]. But at that time, there was no information

on the function of proteins induced by stress or starvationstimuli. At the beginning of the Nineties we thereforestarted a joined effort with R. Schmid and K. Altendorf(Osnabrück) to identify stress proteins by N-terminal se-quencing [6, 7]. Later the sequencing of bacterial geno-mes has dramatically stimulated proteomics because itopened totally new perspectives of linking protein spotsto their genes aiming in the presentation of a comprehen-sive gene protein index. Such a 2-D protein referencemap is an excellent experimental tool for various physio-logical studies providing an overall picture of cell physiol-ogy as an entity. Using the 2-D gel electrophoresis intro-duced by Klose [2] and O’Farrell [3] in combination withthe quantitative evaluation of protein synthesis rates aswell as with the identification of protein spots by MALDI-MS we tried to uncover complex adaptational networks,because due to their relative simplicity the majority of pro-teins synthesized in growing, stressed or starved bacter-ial cells can be visualized [8–10].

However, some restriction of the 2-D gel electrophoresistechnique should be considered. Most studies onlydepict a subproteomic fraction, comprising in manycases only soluble, cytosolic proteins in the pH range of4–7 or 3–10. More alkaline or acidic proteins as well as

Correspondence: Dr. Michael Hecker, Ernst-Moritz-Arndt-Uni-versität Greifswald, Institut für Mikrobiologie, Friedrich-Ludwig-Jahn-Straße 15, D-17487 Greifswald, GermanyE-mail: [email protected]: +49-(0)-3834-864202

Abbreviation: TCC, tricarboxylic acid cycle* Dedicated to Joachim Klose, Berlin, at the occasion of his

65th birthday

2908 Electrophoresis 2001, 22, 2908–2935

ª WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001 0173-0835/01/1408–2908 $17.50+.50/0

membrane-bound or extracellular proteins have oftenbeen ignored so far. The dramatically developing DNAarray technologies, on the other hand, allow a semiquan-titative evaluation of the expression of almost all genes.For the analysis of global gene regulation, DNA arraytechniques will therefore at least partially replace proteo-mics because they are easier to handle and more com-prehensive in their results covering the entire genome.However, proteomics will maintain its great significanceand central position in functional genomics even in viewof the developing DNA array techniques, because notonly an intermediate but the final and therefore mostessential level of gene expression can be visualized,including substantial information on protein modification,protein stability or protein sorting. Nevertheless, to exploitthe full potential of proteomics, the visualization of thetotal proteome and not only of single subproteomic frac-tions is urgently necessary. A comprehensively annotatedproteome will provide a new experimental basis for phy-siological studies that aim in analyzing and understandingthe overall cell physiology. Because of their relatively lowcomplexity bacterial cells are exceptionally well-suitedmodel systems for the description of the entire proteomeof an organism, a realistic goal for future studies.

In this paper, some recent developments in proteomics ofB. subtilis are presented to provide a better experimentaltool for “physiological proteomics”. These studies involvea new 2-D reference gel map (pH 4–7) of B. subtilis withmore than 300 entries, complemented with alkaline orextracellular proteins, a list of protein modifications iden-tified so far as well as a list of the most abundant B. sub-tilis proteins.

2 Materials and methods

2.1 Strain and growth conditions

B. subtilis 168 was cultivated with vigorous agitation at37�C in a minimal medium as previously described [11].The master gel was prepared with a protein extract fromcells harvested during exponential growth. Bacteria wereexposed to various stresses and starvation conditions asdescribed earlier to facilitate the location and identifica-tion of protein spots particular to a certain growth condi-tion [8, 9].

2.2 Preparation of the protein extracts

Bacteria were harvested by centrifugation (8000�g,10 min, 4�C), washed with ice-cold TE buffer (10 mM Tris,pH 7.5, 1 mM EDTA) and finally resuspended in TE buffer.B. subtilis cells were disrupted by passage through a

French press and cell debris were removed by low spincentrifugation (12 000�g, 10 min, 4�C). After havingcleared the protein extract by an additional centrifugation(> 20 000�g, 30 min, 4�C), the protein concentration ofthe supernatant was determined with a commerciallyavailable kit (RotiNanoquant) according to Bradford [12].The protein solution was stored in frozen aliquots.

2.3 2-D PAGE

Isoelectric focusing was done with commercially avail-able IPG-strips (pH 4–7, pH 4.5–5.5; Amersham Pharma-cia Biotech, Uppsala, Sweden). Samples were loaded byrehydration for 24 h in a solution containing 8 M urea, 2 M

thiourea, 1% w/v CHAPS, 20 mM DTTand 0.5% v/v Phar-malyte 3–10. The isoelectric focusing was performed withthe Multiphor ll unit (Amersham Pharmacia Biotech)employing the following voltage profile: linear increasefrom 0 to 500 V for 500 Vh, 500 V for 2500 Vh, linearincrease from 500 to 3500 V for 10 000 Vh, and a finalphase of 3500 V for 35 000 Vh. After consecutive equili-bration of the gels in solutions containing DTT and iodoa-cetamide as suggested by Görg et al. [13], the separationin the second dimension was done in polyacrylamide gelsof 12.5% T and 2.6% C on the Investigator 2-D electro-phoresis system (Genomic Solutions, Chelmsford, MA,USA) with approximately 2 W per gel. Analytic and pre-parative gels were loaded with 50–80 and 400–800 �g ofcrude protein extract, respectively. Analytical gels werestained with silver nitrate according to Bloom et al. [14],and for staining of preparative gels PhastGel BlueRwas used as suggested by the manufacturer (AmershamPharmacia Biotech). Fluorescence was recorded with aStorm840 fluorescence imager after staining of the gelswith SyproRuby. After scanning, quantitative analysis ofthe 2-D PAGE images was performed with the Phoretix2D Advanced Software.

2.4 Protein identification

Identification was exclusively done with protein spots iso-lated from PhastGel BlueR stained gels. In this study, onlya limited number of proteins were identified by classicalN-terminal sequencing as previously described [15]. Themajority of the identifications were accomplished bymass spectrometry according to established protocols.Briefly, protein spots were excised from stained 2-D gels,destained, digested with trypsin (Promega, Madison, WI,USA) and peptides were extracted according to Otto et al.[16]. Occasionally, peptides were purified with C18-tipsaccording to the manufacturer’s instructions (Millipore,Bedford, MA, USA) and eluted with 50% acetonitrile-0.1% trifluoroacetic acid (v/v). Peptide solutions were

Electrophoresis 2001, 22, 2908–2935 2-D reference map of B. subtilis proteins 2909

Proteomicsan

d2-DE

mixed with an equal volume of saturated �-cyano-3-hydroxycinnamic acid solution in 50% acetonitrile-0.1%trifluoroacetic acid (v/v) and applied to a sample platefor MALDI-MS. Peptide masses were determined in thepositive ion reflector mode either in a Voyager DE RPor a Voyager-DE STR mass spectrometer (Applied Bio-systems, Foster City, CA, USA) with internal calibration.Mass accuracy was usually in the range between 10 and50 ppm. Peptide mass fingerprints were compared todatabases using the program MS-Fit (http://prospector.ucsf.edu). The searches considered oxidation of methio-nine, pyroglutamic acid formation at the N-terminal glut-amine and modification of cysteine by carbamidomethy-lation as well as partial cleavage leaving one internal clea-vage site.

3 Results

3.1 Theoretical protein map

Efforts to depict the entire proteome of an organismshould begin with a synthetic protein map simply derivedfrom the genome sequence (Fig. 1). This theoretical mapof a 2-D gel electropherogram of B. subtilis showed thetwo main pI regions also found in other bacteria [17–20].Approximately two thirds of the protein spots are locatedin the neutral or weak acidic region and the remainingthird in the more alkaline region.

3.2 Analysis of the standard pI region 4–7(master gel) and the narrow pI region 4.5–5.5

As shown by the synthetic protein map (Fig. 1), the pIregion 4–7 should represent the major part of the cytosolicproteome. For the proteome analysis presented here weutilized B. subtilis cells grown in a minimal medium forcingthose cells to produce all the enzymes involved in anabo-lism such as amino acid biosynthesis etc. Up to 1800 pro-tein spots were separated on the standard pI range (4–7)analytical gels (Fig. 2). Because some regions of this gel,especially the pI range 4.5–5.5, were overcrowded, high-abundant ones could bury low-abundance proteins. Nar-row pH gradient IPG-strips were utilized to obtain a betterresolution of such regions and an example of a narrow pIrange gel (pI 4.5–5.5) is displayed in Fig. 3. A much betterresolution particularly in the overcrowded region aroundthe main abundant protein EF-Tu is obvious. On the narrowpI range gel 66 protein spots could be identified by com-parison with the master gel (pI 4–7) with high confidenceand additional 67 were either re-identified or newly identi-fied by peptide mass finger printing. Thus far, only a fewlow-abundance proteins were identified because we

restricted the identification by mass spectrometry to pro-tein spots excised from Coomassie blue stained gels.Future studies will focus on the identification of more low-abundance proteins by using alternative detection sys-tems, such as fluorescence staining or alternative silverstaining protocols, which combine high sensitivity andcompatibility with mass spectrometry. Until now, 295 pro-teins were identified in both pI regions.

When the theoretical pI and Mr values were comparedwith those experimentally observed, in general a reason-able correlation was found, particularly for the pI values(Fig. 4). Clear deviations of theoretical and experimentallyobserved pI seem to reflect post-translational modifica-tions since multiple spots have been identified for at least30 of those (the deviating) proteins (see below and Fig. 9).In agreement with other studies, theoretically computedand experimentally observed Mr-values displayed greatervariation (Fig. 4). Dramatic deviations seem to have differ-ent reasons. In the case of MrgA, a dodecameric complexof the same monomer resists denaturation by 2-D gelelectrophoresis, whereas in the case of OdhB the differ-ence is due to proteolytic cleavage. YsnF can be takenas an example that illustrates the potential of proteomeanalysis in disclosing sequencing errors in genomesequences. We were able to identify a protein spot asYsnF, but a number of tryptic peptides clearly covered asequence extending beyond the N-terminus indicated inthe genome sequence. The potential open reading framewas preceded by an unusually long nontranslated regionand the combination of peptide mass finger printing andN-terminal sequencing revealed a sequencing error thatcaused a frame-shift in this region (data not shown).Finally, the recalculated theoretical pI and Mr valuesshowed a good correlation with those experimentallyobserved. Sequencing errors in the genome sequence ofB. subtilis were also discovered in the yqjl gene. However,in this case the extension of the amino acid sequence wasnot dramatic enough to allow detection in Fig. 4.

Using the fluorescence stain SyproRuby (BioRad, Rich-mond, CA, USA) it was also possible to determine therelative contribution of individual proteins to the total pro-tein visualized on the standard master gel (pI 4–7). Thisquantitative evaluation of the protein composition ofexponentially growing bacteria revealed that for most ofthe highly abundant proteins biochemical functions hadalready been assigned (Table 1). Those proteins performmainly housekeeping functions either as components ofthe translational apparatus, the glycolysis, the tricar-boxylic acid cycle (TCC) and the synthesis of amino acidsor in protein quality control such as the common chaper-ons or trigger factor (Table 1). Most of them show codonadaptation indices near or above 0.5 indicating that for

2910 K. Büttner et al. Electrophoresis 2001, 22, 2908–2935

Figure 1. Theoretical proteome of B. subtilis. The theoretical pI and the Mr of all potential ORFs of the B. subtilis genomesequence [1] were derived from the MICADO-database (http://locus.jouy.inra.fr/cgi-bin/genmic/madbase_home.pl).Assignments of potential membrane spanning domains were obtained from the B. subtilis section of the PEDANT-Systemcontaining the predictions for membrane spanning domains according to Persson and Argos [43] (http://pedant.gsf.de/ci-bin/wwwdbp.pl?Alias=Bsubtilis&Db=Bsubtilis&Title=Bsubtilis&Cmd=Mem). The number of membrane spanning do-mains (MSDs) is indicated by the color code on the right. The rectangles indicate the analytical windows of the 2-D gelelectrophoresis.

expression of the corresponding genes B. subtilis usescodons fitting to high-abundance tRNAs. The 100 mostabundant proteins account for approximately 60% of theprotein detectable on a gel of the pI interval from 4 to 7(Fig. 5). Additionally, it was shown that the direction oftranscription of these highly expressed genes in mostcases correlates to the direction of replication of theB. subtilis chromosome (Fig. 6).

To visualize the entire proteome, a physiological approachshould be taken into consideration because many genesare tightly controlled by environmental stimuli. We ana-

lyzed the 2-D protein pattern of B. subtilis cells in re-sponse to various stress or starvation stimuli such asheat, acid, oxidative or salt stress, or starvation for glu-cose, phosphate, oxygen or nitrogen [8–10, 21]. Manystress- or starvation-inducible proteins were alreadyadded to the vegetative proteins identified during expo-nential growth (Fig. 2). The improvement in the resolutionand reproducibility of gels resulting from the switch fromcarrier ampholyte gels to immobilized pH-gradient gelswill certainly facilitate the identification of additionalstress- or starvation-inducible proteins not yet identifiedin earlier studies [6, 8, 9].


Figure 2. Reference 2-D electrophoresis map of cytosolic B. subtilis proteins. The intracellular proteins of B. subtilis 168exponentially grown in minimal medium at 37�C were separated in a pH gradient 4–7 and stained with silver nitrate accord-ing to Bloom et al. [14]. The reference map was enlarged and divided into four sections in order to increase the resolution.Apparent molecular mass (�103) and pI scales are included. An overview of the entire gel is given in Fig. 7, submap B.Squares were used to indicate the position of proteins induced by stress or starvation and identified on gels prepared withthe corresponding protein extracts. If the protein could only be assigned to a small region but not to a precise position,ellipses were used instead of squares for labelling. Separations of protein extracts from stressed and starved cells can beviewed in “Sub2D – The two-dimensional protein index of Bacillus subtilis” (http://microbio2.biologie.uni-greifswald.de:8880/sub2d.htm). An extension of -F behind the gene name indicates that the protein spot corresponds to a fragment ofthe protein only. Detailed information onto each individual protein identified is included in the table in the appendix.


Figure 2. Caption see p. 2912






Figure 3. Reference map of cytosolic protein extracts of B. subtilis separated in a narrow pI gradient(4.5–5.5). The intracellular proteins of B. subtilis 168 exponentially grown in minimal medium at 37�Cwere separated in a pH gradient 4.5–5.5 and stained with silver nitrate according to Bloom et al. [14].Proteins identified by peptide mass finger printing are indicated with the following color code: spotslabelled with green color were identified in the narrow pI gradient gel only; protein spots indicated inblue color were identified in both the master gel (pI 4–7) and the narrow pI region; red labels weretransferred from the master gel (pI 4–7) to the narrow gel after visual comparison of the regional spotpattern. Apparent molecular mass (�103) and pI scales are included.

As a result of their particular induction pattern, these spotsthemselves could not be observed in exponentially growingcells and instead their corresponding locations were indi-cated on the master gel (squares and circles in Fig. 2). How-ever, information on all the proteins identified and examplesof gels prepared with protein extracts from cells harvestedduring various stresses and starvations may be accessedelectronically via the WWW by using “Sub2D – The two

dimensional protein index ofBacillus subtilis” (http://micro-bio2.biologie.uni-greifswald.de:8880/sub2d.htm). This da-tabase was established in accordance with the guidelinesfor the construction of federated web-based protein data-bases [22]. Thus far, the database contains 346 entries ofidentified proteins of B. subtilis and the gel images fromexponentially grown cells and those subjected to heat,ethanol and oxidative stress or phosphate starvation.


Table 1. Summary of the most abundant proteins in B.subtilis cytosolic extracts

Protein % Amounta) CAIb) Comment Functional class

EF-Tu 5.17 0.83 Three spots Translational apparatusRplL 2.70 0.79 Two spots Translational apparatusIIvC 2.21 0.57 Left Amino acid metabolismEF-G 1.91 0.73 Translational apparatusGroES 1.60 0.61GroEL 1.30 0.71 Two spots Protein quality controlHag 1.27 0.70 OtherMetC 0.89 0.49 Two spots Amino acid metabolismEno 1.23 0.74 GlycolysisCitC 1.21 0.60 TCCCitH 1.20 0.54 TCCGap 1.20 0.78 GlycolysisGlyA 1.17 0.59 Amino acid metabolismAroA 1.11 0.51 Left Amino acid metabolismSodA 1.07 0.65 Left OtherRpsF 0.91 0.74 Translational apparatusYkrT 0.91 0.45 Right Function unknownFbaA 0.89 0.74 GlycolysisTig 0.80 0.73 Protein quality controlPdhD 0.76 0.69 Left GlycolysisRplJ 0.75 0.71 Translational apparatusRpsB 0.73 0.76 Translational apparatusSerA 0.71 0.50 Amino acid metabolismPdhA 0.71 0.64 GlycolysisEF-Ts 0.70 0.83 Translational apparatusYjlD 0.67 0.67 Function unknownGlnA 0.58 0.62 Left Amino acid metabolismPdhB 0.57 0.69 GlycolysisSucC 0.54 0.50 TCCYdjL 0.49 0.56 Function unknownAhpC 0.48 0.82 Main spot OtherPdhC 0.47 0.68 GlycolysisTkt 0.47 0.58 OtherDnaK 0.45 0.69 Protein quality controlPgk 0.45 0.63 GlycolysisYwaA 0.44 0.49 Function unknownArgF 0.43 0.38 Amino acid metabolismYqjl 0.42 0.61 Function unknownRocD 0.42 0.46 Amino acid metabolismAtpA 0.40 0.55 OtherGuaB 0.40 0.56 Left OtherAsd 0.40 0.53 Amino acid metabolismYpfD 0.39 0.58 Function unknownLeuB 0.39 0.40 Amino acid metabolismYxjH 0.39 0.59 Function unknownPurB 0.39 0.54 Left OtherCysK 0.39 0.54 Amino acid metabolism

Table 1. Continued

Protein % Amounta) CAIb) Comment Functional class

??? 0.38 — Nonidentified proteinTrxA 0.37 0.71 Protein quality controlIlvD 0.37 0.48 Amino acid metabolismYwjH 0.37 0.52 Function unknown

a) Relative protein levels were quantified in percent oftotal protein content of a 2-D gel covering a pI rangeof 4 to 7. For detailed functions see appendix.

b) The codon adaptation index (CAI) was calculatedby the method of Sharp and Li [44]. Usually highlyexpressed proteins have CAI values greater than0.5, moderately expressed proteins have CAI valuesbetween 0.2 and 0.5, poorly expressed proteins haveCAI values between 0.1 and 0.2, while CAI valuesbelow 0.1 generally mean that the protein is expressedbelow detectable amounts, if expressed at all.

3.3 Towards the total proteome: analysis of thealkaline pI region and extracellular proteins

In order to depict the entire proteome alkaline proteinshave also to be considered as illustrated in Fig. 1 andFig. 7. However, optimization of the separation conditionsfor alkaline proteins required much more efforts than stan-dard pI range separations in the neutral or acidic pI range[23–25]. The analysis of the theoretical proteome of B.subtilis also revealed that most of the proteins with morethan two membrane-spanning domains are located in thealkaline region with a relatively high Mr (Fig. 1). However,among the 42 alkaline proteins identified so far (Fig. 7), nointrinsic membrane protein was found [25] confirming thedata of many laboratories that membrane proteins canonly occasionally be visualized with the current 2-D elec-trophoresis technology; especially proteins with multiplemembrane-spanning domains escape detection [26].This observation also gains support when the grand aver-age hydropathy (GRAVY) values [27] of all proteins poten-tially encoded by the B. subtilis genome sequence arecompared with those of the proteins identified on 2-Dgels thus far. This comparison revealed that the range ofGRAVY values was significantly different between theexperimentally identified proteins and the theoretical pro-teome, with proteins having a positive GRAVY value beingclearly underrepresented in the experimentally identifiedproteins (Fig. 8). Obviously strongly hydrophobic proteinsare preferentially lost during the 2-D gel electrophoresisand were consequently not detected.

Members of the genus Bacillus including B. subtilisare used for the industrial production of extracellularenzymes because of their high capacity for protein secre-tion. Therefore, extracellular proteins that are exemplarily


Figure 4. Comparison of the theoretical and experimental Mr and pI of proteins identified on the B. subtilis 2-D referencemap. The theoretical Mr and the pI were derived from the MICADO-database (http://locus.jouy.inra.fr/cgi-bin/genmic/mad-base_home.pl). (A) The migration distance of the spots from the start of the separating gel was plotted against the loga-rithms of the theoretical Mr. For clarity the horizontal axis displays scales for both the In-values of the Mr and the corre-sponding Mr.

shown in Fig. 7 for growing cells should be involved instudies that focus on the depiction of the total proteome.Extracellular proteins were already identified for phos-phate-starved B. subtilis cells [21] and a detailed analy-sis of the secretome of B. subtilis is in progress [28].

3.4 Analysis of modifications

47 proteins were found as multiple spots (in total 109), inmost cases in two forms (36 proteins). Eight proteins witha high molecular mass were found to form protein chains


Figure 4. (B) The theoretical pI was plotted against the migration distance of the spot center from the basic end of the IPG-strip (pH-range 4–7). The spots corresponding to PhoD, YqqJ, and YqqK are located at the basic end since their pI is higherthen 7.

with differing pI. Multiple spots can be attributed eitherto post-translational modifications or to artificial chemi-cal modifications occurring during protein preparation.A survey on multiple protein spots that were observedin this study is shown in Fig. 9. Spots of apparently dif-ferent molecular mass were found for only a few pro-

teins. MrgA is an interesting example for oligomerization:in addition to the monomer, a 12-mer of almost identicalpI, which was resistant to denaturation during samplepreparation and electrophoresis, was located in the high-molecular-mass region of the 2-D gels (see Figs. 2, 4,and 9; [10]).


Figure 5. Schematic presentation of the protein abun-dance profile of exponentially growing B. subtilis. Therelative intensity of each protein was calculated by com-puter analysis of fluorescence-stained gels. Proteins werecombined in blocks of ten by descending intensity andthe relative contribution of each block to the total proteincontent on the gel was plotted. The horizontal axis con-tains two scales displaying the sum of proteins taken inconsideration (in blocks of ten) and the summarized(accumulated) relative quantity, respectively. The calcula-tion was generated from the averages of four SyproRuby-stained 2-D gels prepared with crude protein extractsfrom exponentially growing B. subtilis cells.

Figure 6. Graphical presentation of the chromosomallocation of the genes encoding the 50 most abundantproteins of exponentially growing B. subtilis. Gene namesare given on a circle representing the B. subtilis chromo-some. The direction of both transcription and replicationis color-coded (blue, clockwise; red, counter-clockwise).Brackets summarize genes organized in transcriptionalunits (operons). p’s and t’s indicate promoters or termina-ters, respectively.

Processing products with an alteration in mass as well asin charge were shown to exist for GroEL, Pgk, RpsB, TufA,and OdhB. Most multiple spots were the results of differ-ent pI values that might indicate functionally relevantpost-translational modifications such as phosphorylation.Detailed studies will be required to disclose the type ofmodification for each single protein. For a few proteins,such as CitC, ClpC, GroEL, GsiB, GuaB, PrkA, and YkrS,phosphorylation did indeed seem to occur. The acidicforms of these proteins displayed in MALDI-MS a massincrease of 80 Da provoked by potential phosphorylationthat was not found for the corresponding peptide of themore basic spot (data not shown).

For another example, the antagonist protein RsbV, thephosphorylation has already been proven to have func-tional relevance [29–31]. Only the nonphosphorylatedform can bind to its partner the anti-sigma factor RsbW[29, 31, 32] and thereby trigger expression of the generalstress regulon. Phosphorylation of this protein at a serine-leads to its inactivation [31, 32]. The occurrence of thephosphorylated and nonphosphorylated forms of the ser-ine-containing peptide has been confirmed by peptidemass finger printing in this study (Fig. 9 and Völker et al.,unpublished data).

Another post-translational modification of proteins is theremoval of the start methionine by N-terminal methionyla-minopeptidase. The analysis of the B. subtilis genomerevealed the presence of two paraloguous genes probablycoding for proteins with corresponding enzymatic activity[1]. We analyzed the 83 N-terminal sequences determinedin this and previous work [6–9, 15, 33] for the occurrence ofthe N-terminal methionine in the mature protein and triedto correlate the presence or absence of the N-terminalmethionine to the physicochemical properties of the sub-sequent amino acids (Fig. 10). More than one-third ofthe N-termini determined still contained their start methio-nine. In agreement with studies inE. coli [34] andSynecho-cystis sp. [35] cleavage seemed to be influenced by thebulkiness of the side chain of the penultimate amino acidresidue. If the N-terminal methionine was followed byamino acids with small side chains, such as alanine,serine, threonine, glycine, proline, or valine, methionyl-aminopeptidase seemed to be particularly active, resultingin almost all cases in removal of theN-terminal methionine.On the contrary, bulkier amino acid residues in the secondposition appeared to prevent cleavage.

4 Discussion

The majority of proteins synthesized in a cell population ata single moment under a definite physiological conditioncan be visualized if not only cytoplasmic but also extra-


Figure 7. Towards the total proteome of B. subtilis – combination of subproteomes. Because proteins are localized indifferent compartments, the total proteome has to be assembled from subproteomes. The figure illustrates the proteinfractions separated on the master gel (submap B), the pI section 7–12 of a wide pH-gradient from 4–12 that facilitatesseparation of alkaline proteins (submap A), as well as a survey of the extracellular proteins in the pI range 3–10 (submapC). Apparent molecular mass (�103) and pI scales are included.

Figure 8. Comparison of the hydropathy of all proteinspredicted from the B. subtilis genome sequence and ofthe proteins identified on 2-D gels. The grand averagehydropathy values of all proteins calculated accordingto Kyte and Doolittle [27] were obtained from the toolProtParam from ExPASy at http://www.expasy.ch/tools/protparam.html. The upper part of the figure displays thehydropathy distribution of all predicted B. subtilis proteinswhereas the lower part shows the hydropathy distributionof all proteins identified on the 2-D gels thus far.

cellular proteins and alkaline cytoplasmic and extracellu-lar proteins are considered. This is the intriguing outcomeof a method described more than 25 years ago by Klose[2] and O’Farrell [3]. Klose’s pioneering work provided afoundation for today’s broad application of proteomicsfor analyzing global pattern and changes of gene expres-sion, which opens totally new perspectives to understandcell physiology as an entity [36]. However, the potentialof this powerful technique can only be exploited if thegenome sequence is available providing the experimentalbasis for high-throughput protein identification by MALDI-MS. With an increasing number of identified proteinsmore substantial and profound conclusions consideringdifferent aspects of bacterial physiology can be drawn.To give one example: a global and new understanding ofglycolysis and TCC regulation in B. subtilis was feasibleafter all enzymes involved in TCC and glycolysis hadbeen identified on the 2-D protein gels [37]. Therefore,the final goal of those studies will be the identification ofalmost all proteins of B. subtilis aiming to define a pro-teome as comprehensive as possible.

The master gel of exponentially grown B. subtilis cells(pI 4–7, including pI 4.5–5.5) presented in this paper con-tains 295 identified proteins. With this master gel we


Figure 9. Overview of the pro-teins that were associated withmultiple spots. The number ofdifferent spots of the proteinsand the character of their modi-fication (C, charge only; M,mass only; C-M, charge andmass) are indicated in the tableon the left. Proteins that areunderlined were selected forthe display of the 2-D gelregions containing the multiplespots in the right part of the fig-ure.

extend a protein map based on a joint effort of Europeanand Japanese groups and published by Bernhardt et al.[38]. Furthermore, 51 proteins were identified on wide pIrange gels including the alkaline region [25]. However,there are still many non-identified proteins in all theseareas, in most cases low-abundance proteins that haveto be identified in future studies. At least 233 of the iden-tified proteins can be assigned to different branches ofcell physiology, the remaining 113 correspond to pre-dicted gene products with still unknown function whoseexistence has been experimentally proven in this study.About 70 proteins were identified in the extracellularregion [21, 28]. In total more than 420 proteins wereidentified in growing cells of B. subtilis. If one assumesthat one-half to two-thirds of the 4100 B. subtilis genesmight be expressed in growing cells, almost one-fourthof the synthesized proteins have been identified so far.In addition to the low-abundance proteins still waitingfor their identification, the fraction of membrane proteinsalmost completely escaped detection by our approach,because no experimental protocol is available thus farfor separating the entire set of intrinsic membrane pro-


Figure 10. Quantitative evaluation of the removal of the N-terminal methionine. Eighty-three N-terminalamino acid sequences determined in this and previous work [6–9, 15, 33] were analyzed for the occur-rence of the N-terminal methionine in the mature protein. For both classes, with and without N-terminalmethionine, the frequency of the amino acid following theN-terminal methionine is displayed.

teins. From the theoretical proteome we learned thatalmost one-fourth of the total proteins belongs to this stilluncharacterized group (this work and [39, 40]. Futurestudies will focus on the systematic identification of low-abundance proteins to accomplish a more comprehen-sive proteomic map.

Furthermore, it should be stressed that the depiction ofthe entire proteome can most likely not solely be accom-plished by a continuous improvement and refinement ofthe analytical tools. A physiological approach may havethe same significance, frequently ignored in biochemicalstudies. From a physiological point of view a considerablepart of the genome remains more or less silent in expo-nentially growing cells because particular environmentalstimuli are necessary for its expression. The correspond-ing stimuli have to be imposed to the bacteria in order toallow detection and identification of these stress- or star-vation-inducible proteins on the 2-D gels. Examples forsuch genes controlled by environmental stimuli includethe more than 150 �B-dependent genes (Petersohn etal., unpublished data), about 100 or more genes of thepho regulon [21] or of the oxy stimulon (Scharf et al.,unpublished data) or more than 200 required for sporula-tion, all of which are more or less silent in growing cells. Afew proteins induced by stress or starvation stimuli thatare totally absent or show only a basal level expressionin growing cells have already been included into the mas-ter gel. Those physiological studies are not only essentialsteps on the way towards uncovering the total proteomebut are also crucial for understanding regulation of geneexpression at a large scale [8, 21, 41, 42].

One of the advantages of proteomics compared to DNAchip technologies is the fact that post-translational mod-ifications of proteins frequently required for their biologi-

cal activity can be visualized. A list of proteins occurring inmultiple protein spots is presented in the paper. At leastsome of them are probably the result of post-translationalmodification. A comprehensive analysis of the phospho-proteome of B. subtilis (in cooperation with S. Seror, Paris)is in progress. These studies on post-translational modifi-cation of proteins will also profit from our efforts to extendthe map of identified proteins.

This investigation received financial support from the Eu-ropean Community (project BI04-CT95-0278), the Deut-sche Forschungsgemeinschaft (He1887/6-1/Vö6229/3-1),the Bundesministerium für Bildung, Wissenschaft, For-schung und Technologie (01GG9833/9), the Bildungs-ministerium of the country Mecklenburg-Vorpommern(project 98 022 90-2000) and the Fonds of ChemicalIndustry of Germany. We thank Peter Jungblut andMichael Karas for advice in MALDI-MS.

Received January 23, 2001

5 References

[1] Kunst, F., et al., Nature 1997, 390, 249–256.[2] Klose, J., Humangenetik 1975, 26, 231–243.[3] O’Farrell, P. H., J. Biol. Chem. 1975, 250, 4007–4021.[4] Richter, A., Hecker, M., FEMS Microbiol. Lett. 1986, 36, 69–

71.[5] Hecker, M., Richter, A., Schroeter, A., Wölfel, L., Mach, F.,

Z. Naturforsch. 1987, 42, 941–947.[6] Völker, U., Engelmann, S., Maul, B., Riethdorf, S., Völker, A.,

Schmid, R., Mach, H., Hecker, M., Microbiology 1994, 140,741–752.

[7] Völker, U., Mach, H., Schmid, R., Hecker, H., J. Gen. Micro-biol. 1992, 138, 2125–2135.

[8] Antelmann, H., Bernhardt, J., Schmid, R., Mach, H., Völker,U., Hecker, M., Electrophoresis 1997, 18, 1451–1463.


[9] Bernhardt, J., Völker, U., Völker, A., Antelmann, H., Schmid,R., Mach, H., Hecker, M., Microbiology 1997, 143, 999–1017.

[10] Bernhardt, J., Büttner, K., Scharf, C., Hecker, M., Electro-phoresis 1999, 20, 2225–2240.

[11] Stülke, J., Hanschke, R., Hecker, M., J. Gen. Microbiol.1993, 139, 2041–2045.

[12] Bradford, M. M., Anal. Biochem. 1976, 72, 248–254.[13] Görg, A., Boguth, G., Obermaier, C., Posch, A., Weiss, W.,

Electrophoresis 1995, 16, 1079–1086.[14] Bloom, H., Beier, H., Gross, H., Electrophoresis 1987, 8, 93–

99.[15] Schmid, R., Bernhardt, J., Antelmann, H., Völker, A., Mach,

H., Völker, U., Hecker, M., Microbiology 1997, 143, 991–998.[16] Otto, A., Thiede, B., Müller, E. C., Scheler, C., Wittmann-Lie-

bold, B., Jungblut, P., Electrophoresis 1996, 17, 1643–1650.[17] VanBogelen, R. A., Abshire, K. Z., Moldover, B., Olson, E. R.,

Neidhardt, F. C., Electrophoresis 1997, 18, 1243–1251.[18] Link, A. J., Robison, K., Church, G. M., Electrophoresis

1997, 18, 1259–1313.[19] Link, A. J., Hays, L. G., Carmack, E. B., Yates III, J. R.,

Electrophoresis 1997, 18, 1314–1334.[20] Wasinger, V. C., Bjellqvist, B., Humphery-Smith, I., Electro-

phoresis 1997, 18, 1373–1383.[21] Antelmann, H., Scharf, C., Hecker, M., J. Bacteriol. 2000,

182, 4478–4490.[22] Appel, R. D., Bairoch, A., Sanchez, J. C., Vargas, J. R.,

Golaz, 0., Pasquali, C., Hochstrasser, D. F., Electrophoresis1996, 17, 540–546.

[23] Görg, A., Boguth, G., Obermaier, C., Weiss, W., Electro-phoresis 1998, 19, 1516–1519.

[24] Görg, A., Obermaier, C., Boguth, G., Csordas, A., Diaz, J. J.,Madjar, J. J., Electrophoresis 1997, 18, 328–337.

[25] Ohlmeier, S., Scharf, C., Hecker, M., Electrophoresis 2000,21, 3701–3709.

[26] Santoni, V., Molloy, M., Rabilloud, T., Electrophoresis 2000,21, 1054–1070.

[27] Kyte, J., Doolittle, R. F., J. Mol. Biol. 1982, 157, 105–132.[28] Antelmann, H., Tjalsma, H., Voigt, B., Ohlmeier, S., Bron, S.,

vanDijl, J. M., Hecker, M., Genom. Res., in press.[29] Dufour, A., Haldenwang, W. G., J. Bacteriol. 1994, 176,

1813–1820.[30] Völker, U., Völker, A., Haldenwang, W. G., J. Bacteriol. 1996,

178, 5456–5463.[31] Yang, X. F., Kang, C. M., Brody, M. S., Price, C. W., Genes

Dev. 1996, 10, 2265–2275.[32] Alper, S., Dufour, A., Garsin, D. A., Duncan, L., Losick, R.,

J. Mol. Biol. 1996, 260, 165–177.[33] Graumann, P., Schröder, K., Schmid, R., Marahiel, M. A.,

J. Bacteriol. 1996, 178, 4611–4619.[34] Hirel, P. H., Schmitter, M. J., Dessen, P., Fayat, G., Blanquet,

S., Proc. Natl. Acad Sci. USA 1989, 86, 8247–8251.[35] Sazuka, T., Yamaguchi, M., Ohara, O., Electrophoresis 1999,

20, 2160–2171.[36] VanBogelen, R. A., Schiller, E. E., Thomas, J. D., Neidhardt,

F. C., Electrophoresis 1999, 20, 2149–2159.[37] Tobisch, S., Zühlke, D., Bernhardt, J., Stülke, J., Hecker, M.,

J. Bacteriol. 1999, 181, 6996–7004.[38] Bernhardt, J., in: Schumann, W., Ehrlich, D., Ogasawara, N.

(Eds.), Functional Analysis of Bacterial Genes: A PracticalManual, John Wiley & Sons, Chichester 2001, pp. 63–75.

[39] Boyd, D., Schierle, C., Beckwith, J., Prot. Sci. 1998, 7, 201–205.

[40] Tjalsma, H., Bolhuis, A., Jongbloed, J. D., Bron, S., van Dijl,J. M., Microbiol. Mol. Biol. Rev. 2000, 64, 515–547.

[41] Hecker, M., Schumann, W., Völker, U., Mol. Microbiol. 1996,19, 417–428.

[42] Hecker, M., Völker, U., Mol. Microbiol. 1998, 29, 1129–1136.[43] Persson, B., Argos, P., J. Mol. Biol. 1994, 237, 182–192.[44] Sharp, P. M., Li, W. H., Nucleic Acids Res. 1987, 15, 1281–

1295.


6 Appendix

Table 1. Summary of B. subtilis proteins on 2-D gels

No. Gene product, homology, EC code, synonyms

1 AcoA bg12558 Acetoin dehydrogenase E1 component(TPP-dependent alpha subunit); syn YfjK

35 912 4.91 B3,A2 MS C

2 AcoB bg12559 Acetoin dehydrogenase E1 component(TPP-dependent alpha subunit); syn YfjK

36 689 4.40 B4 MS

3 AcoC bg12560 Acetoin dehydrogenase E2 component(dihydrolipoamide acetyltransferase); syn Yfjl

42 725 6.52 A1 MS C

4 AcoL bg12561 Acetoin dehydrogenase E3 component(dihydrolipoamide dehydrogenase); syn YfjH

48 689 5.27 A2 MS

5 AcsA bg10370 Acetyl CoA synthetase (EC 6.2.1.1) 64 717 5.55 A2 MS C6 Adk bg10446 Adenylate kinase (EC 2.7.4.3) 23 972 4.45 C4, n MS; N7 AhpC bg11385 Alkyl hydroperoxide reductase (small subunit)

(EC 1.6.4.-)20 482 4.28 C4 MS CM

8 AhpF bg11204 Alkyl hydroperoxide reductase (large subunit)(EC 1.6.99.3)

54 705 4.71 A3, n MS; N C

9 Ald bg10468 L-Alanine dehydrogenase (EC 1.4.1.1) 39 525 5.12 B2, n MS MS10 AmhX bg11789 Amidohydrolase (EC 3.5.1.-) 42 370 6.12 B2 MS11 AnsA bg10300 L-Asparaginase (EC 3.5.1.1) 36 298 4.45 B4 MS12 AnsB bg10301 L-Asparatase (EC 4.3.1.1) 52 386 5.69 A2 MS13 ArgB bg10193 N-Acetylglutamate 5-phosphotransferase

(EC 2.7.2.8)27 574 6.21 C1 MS

14 ArgC bg10191 N-Acetylgluamate gamma semialdehydedehydrogenase (EC 1.2.1.38)

37 917 5.17 B3, n MS MS

15 ArgD bg10194 N-Acetylornithine aminotransferase(EC 2.6.1.11)

40 735 5.93 B1 MS

16 ArgF bg10197 Ornithine carbamoyltransferase(EC 2.1.3.3)

34 509 4.96 B3, n MS MS

17 ArgG bg12570 Argininosuccinate synthase(EC 6.3.4.5)

44 657 5.03 A/B3, n MS MS C

18 ArgH bg12571 Argininosuccinate lyase (EC 4.3.2.1) 51 762 4.86 A/B3, n MS MS19 AroA bg10375 3-Deoxy-D-arabino-heptulosonate-7-phosphate

synthase (EC 4.1.2.15) and chorismatemutase (EC 5.4.99.5)

39 382 5.34 B2 MS C

20 AroB bg10285 3-Dehydroquinate synthase (EC 4.6.1.3) 40 658 6.94 a MS21 AroE bg10294 5-Enolpyruvoylshikimate-3-phosphate

(EC 2.5.1.19)45 078 6.34 a MS

22 AroF bg10284 5-Enolpyruvoylshikimate-3-phosphatephospholyase, chorismate synthease(EC 4.6.1.4)

39 812 6.58 B1 MS

23 Asd bg10783 Aspartate semialdehyde dehydrogenase(EC 1.2.1.11)

37 690 4.97 B3, n MS MS

24 AsnS bg10958 Asparaginyl-tRNA synthetase (EC 6.1.1.22) 48 920 5.12 A2 MS25 AspB bg11513 Aspartate aminotransferase (EC 2.6.1.1) 42 928 5.30 B2 MS26 AtpA bg10819 ATP synthase alpha chain (EC 3.6.1.34) 54 432 5.04 A3, n MS27 AtpC bg10822 ATP synthase epsilon chain (EC 3.6.1.34) 14 069 5.26 D2 MS; N28 AtpD bg10821 ATP synthase beta chain (EC 3.6.1.34) 51 255 4.61 A3, n MS MS29 AtpG bg10820 ATP synthase (subunit gamma) (EC 3.6.1.34) 31 502 6.64 a MS

Proteinname

accordingto

SubtiLista)

Accessionnumber

inSu

btiList

TheoreticalM

r

Theoreticalpl

Gridsquare

b)

4.0–

7.0c

)

4.0–

12.0

c)

4.5–

5.5c

)

Charge-/mass–

modificationd

)


6 Appendix. Continued


30 Bcd bg11723 Leucine dehydrogenase in branched-chainfatty acid biosynthesis, NADH-dependentdecarboxylase inhibitor; syn Bkd

39 833 4.94 n MS

31 Bex bg11188 GTP binding protein; similar to and cancomplement Era, an essential smallG-protein in E. coli

33 922 8.38 a MS

32 BglH bg10935 Beta glucosidase (EC 3.2.1.21) 54 497 4.96 A3 MS33 BioA bg11524 Adenosylmethionine-8-aminio-7-oxononanoate

aminotransferase (EC 2.6.1.62)49 946 5.32 B2 MS

34 CarA gb10195 Carbamoyl-phosphate transferase-arginine(subunit A) (EC 6.3.5.5); syn Cpa

38 901 6.21 B1 MS

35 CarB bg10196 Carbamoyl-phosphate transferase-arginine(subunit B) (EC 6.3.5.5); syn Cpa

112 447 4.73 A3, n MS

36 CcpA bg10376 Transcriptional regulator of carbon cataboliterepression

36 784 5.06 B2 MS

37 CheV bg10823 Modulation of CheA activity in responseto attractions

34 479 4.62 B3, n MS MS

38 CitB bg10478 Aconitate hydratase (EC 4.2.1.3) 99 136 4.90 A3, n MS39 CitC bg10856 Isocitrate dehydrogenase (EC 1.1.1.42) 46 255 4.83 B3, n MS40 CitG bg10384 Fumarate hydratase (EC 4.2.1.2) 50 365 5.47 B2 MS41 CitH bg11386 Malate dehydrogenase (EC 1.1.1.37) 33 490 4.73 B3, n MS; N42 CitZ bg10855 Citrate synthase II (EC 4.1.3.7) 41 569 5.45 B2 MS43 ClpC bg10148 ATPase subunit of Clp protease; syn MecB 89 927 5.75 A1, A2 MS; N C44 ClpE bg12578 ATP dependent Clp protease like; syn YkvH 77 722 5.14 A2, A3 MS C45 ClpP bg19016 Proteolytic subunit of Clp protease

(EC 3.4.21.92)21 536 5.01 C3, n MS; N

46 CodY bg10968 Pleiotropic transcriptional repressor 28 862 4.75 B3 MS47 ComGA bg10483 Required for exogenous DNA-binding 40 301 8.89 a MS48 ComK bg11059 Competence transcription factor (CTF) 22 284 7.67 a MS49 CspB bg10824 Cold shock protein B 7 229 4.34 D4 N C50 Ctc bg10115 Member of the family of L25P ribosomal

proteins21 910 4.22 B4 MS

51 CtrA bg10410 CTP synthetase (EC 6.3.4.2); syn PyrG 59 548 5.16 A2, n MS52 CysK bg10136 Cysteine synthetase A (EC 4.2.99.8) 32 666 5.49 B2 MS53 DapA bg10785 Dihydrodipicolinate synthase (EC 4.2.1.52) 30 889 4.93 B3 MS; N54 DhbA bg11019 2,3-Dihydro-2,3-dihydroxybenzoate

dehydrogenase (EC 1.3.1.28)27 343 5.31 C2 MS

55 DhbB bg11241 Dihydroxybenzoate synthase (EC 3.3.2.1)siderophore

34 952 4.43 B4 MS

56 DhbC bg11242 Isochorismate synthase (EC 5.4.99.6)siderophore

43 286 5.17 B2, n MS

57 DivlVA bg11835 Required for initiation of cell division andseptum formation

19 197 4.85 C3, n MS; N MS

58 DltA bg10551 D-Alanyl-D-alanine carrier protein ligase(EC 6.3.2.–)

55 641 4.93 A3 MS

59 DnaJ bg10665 Heat shock protein, activation of DnaK 40 689 7.92 a MS60 DnaK bg10664 Chaperone 65 826 4.57 A3, n MS

Proteinname

accordingto

SubtiLista)

Accessionnumber

inSu

btiList

TheoreticalM

r

Theoreticalpl

Gridsquare

b)

4.0–

7.0c

)

4.0–

12.0

c)

4.5–

5.5c

)

Charge-/mass–

modificationd

)




61 DppA bg10842 Dipeptide ABC transporter (sporulation) 30 091 5.19 B3 MS62 Dps bg12584 DNA-protecting protein confers unspecific

oxidative stress resistance16 451 4.44 D3 MS; N

63 EF-G bg11939 Elongation factor of translation 76 360 4.62 A3, n MS MS C64 EF-Ts bg19025 Elongation factor of translation 32 200 4.98 B3, n MS65 EF-Tu bg11056 Elongation factor of translation 43 432 4.72 B3, D2, n MS MS CM66 Eno bg10899 Enolase (EC 4.2.1.11) 46 418 4.49 B3, B4, n MS; N C67 FabD bg11836 Malonyl CoA-acyl carrier protein transacylase

(EC 2.3.1.39) fatty acid synthesis33 880 4.51 B3, n MS

68 FabG bg11535 3-Ketoacyl-acyl carrier reductase(EC 1.1.1.100) fatty acid synthesis;syn YlpF

26 132 8.09 a MS

69 FbaA bg10412 Fructose-1,6-bisphosphate alsolase(EC 4.1.2.13)

30 248 5.03 B3, n MS; N MS

70 Ffh bg10830 Signal recognition particle 49 376 9.42 a MS71 FolD bg11711 Methylenetetrahydrofolate dehydrogenase

(EC 1.5.1.5) and methenyltetrahydrofolatecyclohydrolase (EC 3.5.4.9)

30 534 5.60 B2 MS

72 Frr bg12587 Ribosome recycling factor 20 621 5.33 C2 MS; N73 FtsA bg10231 Required for septum formation during

sporulation47 940 5.09 n MS

74 FtsE bg12590 Cell division ATP binding-protein 25 472 8.42 a MS75 FtsZ bg10232 Cell division initiation protein 40 195 4.81 B3, n MS76 Gap bg10827 Glyceraldehyde-3-phosphate dehydrogenase

(EC 1.2.1.12)35 676 5.03 B3, n MS

77 GapB bg12592 Glyceraldehyde-3-phosphate dehydrogenase(EC 1.2.1.12)

37 320 6.45 B1 MS

78 GcaD bg10113 UDP-N-acetylglucosamine pyrophosphorylase(EC 2.7.7.23)

49 290 5.65 A2 MS

79 GlmS bg10948 L-Glutamine-D-fructose-6-phosphate amidotransferase (EC 2.6.1.16)

65 163 4.8 A3 MS

80 GlnA bg10425 Glutamine synthetase (EC 6.3.1.2) 50 113 4.87 A3, n MS MS C81 GltB bg12594 Glutamate synthase (small subunit) (EC 1.4.1.13) 54 780 7.7 a MS82 GltX bg10154 Glutamyl-tRNA synthetase (EC 6.1.1.17) 55 555 5.02 A3 MS83 GlvA bg11839 6-Phospho alpha glucosidase (EC 3.2.1.122) 50 384 4.74 A3 MS84 GlyA bg10944 Serine hydroxymethyl transferase (EC 2.1.2.1) 45 328 5.48 B2 MS; N85 GlyS bg11658 Glycyl-tRNA-synthetase beta chain (EC 6.1.1.14) 76 051 4.97 A3, n MS MS86 GreA bg19019 Transcription elongation factor 17 129 4.48 C3, n MS; N87 GroEL bg10324 Chaperone 57 252 4.53 A3, A4, n MS; N CM88 GroES bg10422 Chaperone 11 664 4.78 D3 MS89 GrpE bg10663 Chaperone 21 537 4.26 B4 MS90 GsiB bg10826 General stress protein, similar to plant

desiccation proteins13 657 5.15 D3 MS; N C

91 GspA bg10558 General stress protein, similar to glycosyland galactosyl transferases

33 368 5.09 B3 MS; N C

92 GtaB bg10402 UDP-glucose pyrophosphorylase (EC 2.7.7.9.) 32 917 4.91 B3 MS; N C93 GuaA bg10647 GMP synthethase (EC 6.3.5.2) 57 782 4.75 A3, n MS MS

Proteinname

accordingto

SubtiLista)

Accessionnumber

inSu

btiList

TheoreticalM

r

Theoreticalpl

Gridsquare

b)

4.0–

7.0c

)

4.0–

12.0

c)

4.5–

5.5c

)

Charge-/mass–

modificationd

)




94 GuaB bg10073 Inosine-5’-monophosphate dehydrogenase(EC 1.1.1.205)

52 824 6.17 A1 MS C

95 GudB bg11435 Glutamate dehydrogenase, expressed underall growth conditions but intrinsically nactive

47 048 5.58 B2 MS

96 Hag bg10655 Flagellin 32 472 4.78 B3, n MS97 HisA bg12597 Phophoribosylformimino-5-aminoimidazole

carboxamide ribotide isomerase(EC 5.3.1.16)

26 379 4.84 C3, n MS MS

98 HisB bg12598 Imidazoleglycerol-phosphate dehydratase(EC 4.2.1.19)

21 403 5.6 C2 MS

99 HisD bg12599 Histidinol dehydrogenase (EC 1.1.1.23) 46 094 4.78 A3, n MS MS100 Hisl bg12603 Phosphoribosyl AMP cyclohydrolase

(EC 3.5.4.19) and phosphoribosyl ATPpyrophosphohydrolase (EC 3.6.1.31)

23 754 4.63 C3, n MS

101 HtpG bg11359 Class IV heat-shock protein; syn YxbB 72 078 4.67 A3 MS102 HutU bg10668 Urocanase (EC 4.2.1.49) 60 428 5.66 A2 MS103 IlvA bg10673 Threonine dehydratase (EC 4.2.1.16) 46 490 5.54 A/B2 MS104 IlvB bg10670 Acetolactase synthase (large subunit)

(EC 4.1.3.18)62 417 5.19 A2, n MS MS C

105 IlvC bg10672 Ketol-acid reductoisomerase (EC 1.1.1.86) 37 302 5.37 B2, n MS; N C106 IlvD bg11532 Dihydroxy-acid dehydratase (EC 4.2.1.9) 59 376 5.31 A2, n MS; N107 IlvN bg10671 Acetolactate synthase (acetohydroxy-acid

synthase) (small subunit) (EC 4.1.3.18)19 309 10.3 a MS

108 InfC bg11944 Translation initiation factor IF3 19 588 10.3 a MS109 IolA bg11117 Methylmalonate-semialdehyde dehydrogenase

(EC 1.2.1.27)53 285 5.14 A2, n MS

110 IolH bg11123 Myoinositol catabolism 33 370 4.99 B3 MS111 IolS bg11363 Similar to auxin-induced protein of common

tobacco, similar to oxidoreductases;syn YxbF

35 014 5.38 B2 MS

112 KatA bg10849 Catalase 1 (EC 1.11.1.6) 54 567 6.15 A1 MS; N113 KatB bg11102 Catalase 2 (EC 1.11.1.6); syn KatE 77 309 5.85 A1 MS; N114 KatX bg11945 Major catalase in spores 62 192 5.34 A2 MS115 Kbl bg12610 2-Amino-3-ketobutyrate CoA ligase

(glycine acetyl transferase) (EC 2.3.1.29)43 124 6.25 B1 MS

116 LeuA bg11948 2-Isopropylmalate synthase (EC 4.1.3.12) 56 742 5.66 A2 MS; N C117 LeuB bg10675 3-Isopropylmalate dehydrogenase

(EC 1.1.1.85)39 787 4.74 B3, n MS MS

118 LeuC bg11949 3-Isopropylmalate dehydratase (large subunit)(EC 4.2.1.33)

52 225 6.13 B1 MS;N

119 LeuD bg11950 3-Isopropylmalate dehydratase (small subunit)(EC 4.2.1.33)

22 876 4.58 C3, n MS MS

120 LonA bg10338 Class IV heat-shock ATP-dependent Lonprotease(EC 3.4.21.53)

86 418 4.64 B3, n MS MS

121 LysC � bg10350 Aspartokinase II (alpha subunit) (E 2.7.2.4)(LysC aa 1–408)

43 646 4.64 C3, n MS

Proteinname

accordingto

SubtiLista)

Accessionnumber

inSu

btiList

TheoreticalM

r

Theoreticalpl

Gridsquare

b)

4.0–

7.0c

)

4.0–

12.0

c)

4.5–

5.5c

)

Charge-/mass–

modificationd

)




122 LysC � bg10191 Aspartokinase II (alpha subunit) (E 2.7.2.4)(LysC aa 246–408)

17 726 4.78 C3, n MS

123 LysS bg10144 Lysyl-tRNA synthetase (EC 6.1.1.6) 57 370 5.03 A3 MS124 LytC bg10407 N-Acetylmuramoyl-L-alanine amidase

(major autolysin) (EC 3.5.l1.28)52 458 10.1 a MS

125 Mbl bg10916 MreB-like protein, similar to MreBmorphogene of E. coli

33 690 6.16 B2 MS

126 MetC bg12616 Cobalamine-independent methionine synthase 86 641 4.84 A3, n MS MS C127 MetK bg11840 S-Adenosylmethionine synthetase (EC 2.5.1.6) 43 882 5.03 A3, n MS; N128 MrgA bg10864 DNA binding protein 17 190 4.60 D3, A3 MS; N M129 MtrA bg10277 GTP cyclohydrolase I (EC 3.5.4.16) 21 074 6.34 C1 MS130 MurA bg11955 UDP-N-acetylglucosamine 1-carboxyvinyl

transferase (EC 2.5.1.7)46 537 5.45 B2 MS

131 MurE bg10223 UPD-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase (EC 6.3.2.13)

54 157 5.56 A2 MS

132 MurG bg10227 UPD-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide)pyrophosphoryl-undecaprenolN-acetylglucosamine transferase(EC 2.7.8.13)

39 820 9.57 a MS

133 NadE bg10694 NH3 dependent NAD* synthetase (EC 6.3.5.1) 30 244 4.89 B3, n MS; N134 NdK bg10282 Nucleoside diphosphate kinase (EC 2.7.4.6) 16 816 5.57 D2 MS; N135 OdhA bg10272 2-Oxoglutarate dehydrogenase (E1 subunit)

(EC 1.2.4.2)105 093 5.84 A2 MS

136 OdhB bg10273 2-Oxoglutarate dehydrogenase (E2 subunit)(EC 2.3.1.61)

45 827 4.86 A3, C2 NS M

137 OpuCA bg12637 ATPase, part of the osmoprotectanttransporter

43 088 6.55 a MS

138 PanB bg11519 Ketopantoate hydroxymethyl transferase(EC 2.1.2.11)

29 607 5.28 C2, n MS

139 PdhA bg10207 Pyruvate dehydrogenase (E1 alpha subunit)(EC 1.2.4.1)

41390 5.84 B1 MS; N

140 PdhB bg10208 Pyruvate dehydrogenase (E1 beta subunit)(EC 1.2.4.1)

35 320 4.55 B3, n MS

141 PdhC bg10209 Pyruvate dehydrogenase (dihydrolipoamideacetyltransferase E2 subunit) (EC 2.3.1.12)

47 376 4.86 A3, n MS

142 PdhD bg10210 Dihydrolipoamide dehydrogenase E3 subunitof both pyruvate dehydrogenase and2-oxoglutarate dehydrogenase complexes(EC 1.8.1.4)

49 567 4.76 A3, n MS MS C

143 Pfk bg12644 6-Phosphofructokinase (EC 2.7.1.11) 34 100 6.14 B1 MS; N144 Pgk bg11062 Phosphoglycerate kinase (EC 2.7.2.3) 42 030 4.77 B3, n MS M145 Pgm bg10898 Phosphoglycerate mutase (EC 5.4.2.1) 56 140 5.21 A2, n MS146 PheT bg10875 Phenylalanyl-tRNA-synthetase beta chain

(EC 6.1.1.20)87 756 4.86 A3, n MS

147 PhoB bg10697 Alkaline phosphatase III (EC 3.1.3.1) 50 358 5.90 B2 MS148 PhoD bg11174 Phosphodiesterase/alkaline phosphatase D

(EC 3.1.4.1)62 657 8.39 A1, a MS MS

Proteinname

accordingto

SubtiLista)

Accessionnumber

inSu

btiList

TheoreticalM

r

Theoreticalpl

Gridsquare

b)

4.0–

7.0c

)

4.0–

12.0

c)

4.5–

5.5c

)

Charge-/mass–

modificationd

)




149 PlsX bg11843 Involved in fatty acid/phospholipid synthesis;syn YlpD

35 607 5.49 B2 MS

150 Pnp bg11330 Purine nucleoside phosphorylase (EC 2.4.2.1) 28 976 4.86 C3 MS151 PnpA bg11491 Polynucleotide phosphorylase (PNPase)

(EC 2.7.7.8)77 280 4.89 A3, n MS

152 PpiB bg10512 Peptidyl-prolyl isomerase (EC 5.2.1.8) 15 115 5.47 D2 MS; N C153 PrkA bg10804 Serine protein kinase 72 707 5.46 A2 MS C154 ProS bg12659 Prolyl-tRNA synthetase 63 131 5.01 A3, n MS MS155 Prs bg10114 Phosphoribosyl pyrophosphate synthetase

(EC 2.7.6.1); syn KprS34 714 5.90 B1 MS

156 Pta bg10634 Phosphate acetyltransferase (EC 2.3.1.8) 34 636 4.65 B3, n MS157 PtsH bg10200 Phosphocarrier protein of phosphotransferase

system9 051 4.58 D3, n MS

158 Ptsl bg10201 Phosphotransferase system (PTS) enzyme I(EC 2.7.3.9)

60 713 4.62 A3 MS

159 PurA bg10002 Adenylosuccinate synthetase (EC 6.3.4.4) 47 509 5.46 A2 MS160 PurB bg10702 Adenylosuccinate lyase (EC 4.3.2.2) 49 320 5.82 B1, B2 MS C161 PurC bg10703 Phosphoribosyl aminoimidazole-

succinocarboxamide synthetase (EC 6.3.2.6)27 311 4.88 B3, n MS

162 PurD bg10711 Phosphoribosyl glycinamide synthetase(EC 6.3.4.13)

45 118 4.60 B3, n MS MS

163 PurF bg10707 Glutamine phosphoribosyl pyrophosphateamidotransferase (EC 2.4.2.14); syn PurB

51 453 5.87 A1 MS

164 PurH bg10710 Phosphoribosyl aminoimidazolecarboxamideformyltransferase (EC 2.1.2.3) and IMPcyclohydrolase (EC 3.5.4.10)

55 571 5.13 A2, A3, n MS C

165 PurQ bg10706 Phosphoribosyl formylglycinamidinesynthetase I (EC 6.3.5.3)

80 105 4.55 A3, n MS

166 PycA bg12660 Pyruvate carboxylase (EC 6.4.1.1) 127 721 5.41 A2 MS C167 PykA bg12661 Pyruvate kinase (EC 2.7.1.40) 61 999 4.88 A3, n MS168 PyrAA bg10715 Carbamoyl-phosphate synthetase

(glutaminase subunit) (EC 6.3.5.5)39 961 5.82 B1 MS

169 PyrAB bg10716 Carbamoyl-phosphate synthetase(catalytic subunit) (EC 6.3.5.5)

117 440 4.78 A3, n MS

170 PyrC bg10714 Dihydroorotase (EC 3.5.2.3) 46 445 5.43 B2 MS171 PyrR bg10712 Attenuation (antitermination) of the pyrimidine

operon (pyrPBCADFE) in the presence ofUMP, transcriptional attenuator and uracilphosphoribosyltransferase activity (minor)

20 119 4.99 C3 MS

172 RbsA bg10879 Ribose ABC transporter (ATP-binding protein) 54 363 6.18 A1 MS173 RbsK bg10877 Ribokinase (EC 2.7.1.15) 30 986 4.61 B3 MS; N174 Rho bg10416 Transcriptional terminator Rho 48 513 6.86 a MS175 RluB bg10530 Pseudouridine synthase B (EC 4.2.1.70)176 RncS bg11537 Ribonuclease III (EC 3.1.26.3) 28 276 8.08 a MS177 RocA bg10622 Pyrroline-5 carboxylate dehydrogenase

(EC 1.5.1.12)56 151 5.58 A2 MS

178 RocD bg10722 Ornithine aminotransferase (EC 2.6.1.13) 43 601 4.89 B3 MS

Proteinname

accordingto

SubtiLista)

Accessionnumber

inSu

btiList

TheoreticalM

r

Theoreticalpl

Gridsquare

b)

4.0–

7.0c

)

4.0–

12.0

c)

4.5–

5.5c

)

Charge-/mass–

modificationd

)




179 RocF bg10932 Arginase (EC 3.5.3.1) 32 001 4.93 B3 MS180 RplA bg10164 Ribosomal protein L1 (BL1) 24 776 9.73 a MS181 RplB bg11217 Ribosomal protein L2 (BL2) 30 181 11.0 a MS182 RplC bg11218 Ribosomal protein L3 (BL3) 22 537 10.3 a MS183 RplD bg11219 Ribosomal protein L4 22 245 10.5 a MS184 RplE bg10760 Ribosomal protein L5 (BL6) 20 003 9.95 a MS185 RplF bg11408 Ribosomal protein L6 (BL8) 19 366 9.92 a MS186 Rpll bg10009 Ribosomal protein L9 16 210 9.96 a MS187 RplJ bg11220 Ribosomal protein L10 (BL5) 17 885 5.49 C2, n MS188 RplK bg10163 Ribosomal protein L11 (BL11) 14 776 9.72 a MS189 RplL bg10726 Ribosomal protein L7/L12 (BL9) 12 611 4.36 D4 MS; N C190 RplM bg11970 Ribosomal protein L13 16 150 10.2 a MS191 RplP bg10755 Ribosomal protein L16 16 048 10.9 a MS192 RplU bg10333 Ribosomal protein L21 (BL20) 11 137 10.2 a MS193 RpoB bg0728 RNA polymerase (beta subunit) (EC 2.7.7.6) 13 3376 4.73 A3 MS194 RpsB bg19004 Ribosomal protein S2 27 817 6.26 B1, C3, n MS CM195 RpsC bg19005 Ribosomal protein S3 (BS3) 24 186 10.1 a MS196 RpsD bg10372 Ribosomal protein S4 (BS4) 22 690 10.2 a MS197 RpsE bg10442 Ribosomal protein S5 17 480 10.4 a MS198 RpsF bg10049 Ribosomal protein S6 (BS9) 10 986 4.99 D3 MS199 RpsG bg19006 Ribosomal protein S7 (BS7) 17 750 10.5 a MS200 RpsJ bg19008 Ribosomal protein S10 (BS13); syn TetA 11 527 10.2 a MS201 RsbV bg10733 Anti-anti sigma factor of �B 11 800 4.70 D3, D4 MS C202 RsbW bg10734 Anti sigma factor of �B 17 850 4.29 C4 MS203 SdhA bg10352 Succinate dehydrogenase (flavoprotein subunit)

(EC 1.3.99.1)65 174 5.71 A2 MS

204 SdhB bg10353 Succinate dehydrogenase (iron-sulfur protein)(EC 1.3.99.1)

28 267 7.99 a MS

205 SecA bg10741 Preprotein translocase subunit (ATPase) 95 334 5.34 A2 MS C206 SerA bg10509 Phosphoglycerate dehydrogenase

(EC 1.1.1.95)56 946 5.62 A2 MS

207 SerC bg12673 Phosphoserine aminotransferase(EC 2.6.1.52)

39 977 5.52 B2 MS

208 SerS bg10077 Seryl-tRNA synthetase (EC 6.1.1.11) 48 678 5.19 A2, n MS; N209 SigB bg10735 Sigma factor oB 29 995 5.42 B2 MS210 SodA bg11676 Superoxide dismutase (EC 1.15.1.1) 25 143 4.96 C3, n MS; N C211 SpeE bg12460 Spermidine synthease; syn YwhF 31 184 5.07 B3 MS212 Spo0A bg10765 Central role in the initiation of sporulation 29 540 5.99 C1 MS213 Spo0J bg10054 Stage 0 sporulation protein J 32 059 8.42 a MS214 Spo0M bg12229 Disruption or overproduction has

negative effects on sporulation; syn Ygal29 557 4.28 B3/4 MS

215 SpolVA bg10275 Required for proper spore cortex formationand coat assembly; syn SpoVP

55 007 4.55 A3, n MS

216 SpoVG bg10112 Required for spore cortex synthesis 10 755 5.11 D2 MS217 SrfAD bg10171 Surfactin synthetase; syn ComL 27 472 5.22 B2 MS218 Ssb bg10048 Single-strand DNA-binding protein 18 599 4.82 C3, n MS MS

Proteinname

accordingto

SubtiLista)

Accessionnumber

inSu

btiList

TheoreticalM

r

Theoreticalpl

Gridsquare

b)

4.0–

7.0c

)

4.0–

12.0

c)

4.5–

5.5c

)

Charge-/mass–

modificationd

)




219 SucC bg12680 Succinyl-CoA synthetase (beta subunit)(EC 6.2.1.5)

41 212 4.85 B3, n MS

220 SucD bg12681 Succinyl-CoA synthetase (alpha subunit)(EC 6.2.1.5)

31 229 5.59 B2 MS

221 TasA bg11697 Spore coat-associated protein; syn YqhF, CotN 28 154 5.44 B3, n MS MS222 ThiA bg11246 Biosynthesis of the pyrimidine moiety 65 740 5.26 A2 MS223 ThrB bg10462 Homoserine kinase (EC 2.7.1.39) 33 171 4.73 B3, n MS MS224 ThrC bg10461 Threonine synthase (EC 4.2.99.2) 37 307 5.19 B2, n MS MS225 ThrS bg10362 Threonyl-tRNA synthetase (EC 6.1.1.3) 73 335 5.21 A2, n MS226 ThrZ bg10421 Threonyl-tRNA synthetase (EC 6.1.1.3);

syn ThrS273 195 5.75 A1/2 MS

227 Tig bg19023 Trigger factor (prolyl isomerase) 47 324 4.22 A4, n MS; N C228 Tkt bg11247 Transketolase (EC 2.2.1.1) 72 163 4.80 A3, n MS229 Tpi bg10897 Triose phosphate isomerase (EC 5.3.1.1) 26 880 4.79 B3, n MS230 TrxA bg10348 Thioredoxin 11 254 4.31 D4 MS; N231 TrxB bg12398 Thioredoxin reductase (EC 1.6.4.5) 34 364 4.99 B3 MS232 TuaD bg12691 UDP-glucose 6-dehydrogenase,

biosynthesis of teichuronic acid; syn YvhD49 651 6.11 A1 MS C

233 TyrS bg10371 Tyrosyl-tRNA synthease (EC 6.1.1.1) 47 574 5.21 B2, n MS MS234 YaaD bg10075 Similar to proteins of unknown function

(member of UPF0019 family)31 457 5.09 B3, n MS; N MS

235 YaaQ bg10093 Similar ro proteins of unknown function 11 828 6.09 D1 MS236 YabJ bg10111 Similar ro proteins of unknown function

(member of YER057C/YIL051C/YJGF family)13 514 5.13 D3 MS; N

237 YcdF bg12761 Similar to glucose-1 dehydrogenase 27 625 5.74 B2 MS238 YceC bg12767 Similar to tellurium resistance protein 21 678 5.31 C2 MS239 YceD bg12768 Similar to tellurium resistance protein; syn TerD 20 549 4.29 C4 MS240 YcgN bg12012 Similar to 1-pyrroline-5-carboxylate

dehydrogenase56 320 5.37 A2 MS

241 YclM bg12033 Similar to homoserine dehydrogenase 49 661 4.78 A3, n MS MS242 YdaD bg12052 Belongs to short-chain

dehydrogenases/reductases30 930 4.78 B3 MS; N

243 YdaE bg12053 Unknown 19 109 4.88 C3 MS; N244 YdaG bg19017 Similar to general stress protein 15 735 5.20 C2 MS245 YdaP bg12063 Similar to pyruvate oxidase 62 965 5.22 A2 MS246 YdaT bg12067 Unknown 16 422 5.97 D1 MS247 YdbD bg12071 Similar to manganese-containing catalase 30 106 4.86 B3 MS; N248 YdbR bg12085 ATP dependent RNA helicase homolog 57 108 9.89 a MS

249 YdiS bg12791 Weekly similar to mcrB of E. coli 39 876 8.7 a MS250 YdjL bg12803 Similar to L-iditol 2-dehydrogenase 37 186 4.80 B3, n MS MS251 YfhB bg12877 Similar to proteins of unknown function 31 976 5.19 B2 MS252 YfhM bg12888 Similar to epoxide hydrolase 32 606 6.06 B1 MS253 YfkM bg12929 Similar to intracellular proteases 18 720 4.72 C3 MS; N254 YflT bg19020 Unknown 12 972 4.81 D3 MS255 YhdN bg13020 Similar to aldo keto reductase 37 156 4.77 B3 MS

Proteinname

accordingto

SubtiLista)

Accessionnumber

inSu

btiList

TheoreticalM

r

Theoreticalpl

Gridsquare

b)

4.0–

7.0c

)

4.0–

12.0

c)

4.5–

5.5c

)

Charge-/mass–

modificationd

)




256 YhfJ bg13055 Similar to lipoate protein ligase 37 865 5.84 B1 MS; N257 YhjL bg13078 Similar to sensory transduction

pleiotropic regulatory protein49 977 5.97 B1 MS

258 YisB bg13089 Unknown 11 311 9.94 B2 MS259 YitJ bg13115 Similar to proteins of unknown function,

probably S-box regulon67 751 5.37 A2 MS C

260 YjbC bg13132 Unknown 22 956 5.14 C2 MS261 YjbT bg13149 Similar to thiamin biosynthesis, probably ThiB 26 873 4.72 B/C3, n MS262 YjbW bg13152 Similar to enoyl-acyl-carrier protein reductase 28 905 5.72 B2 MS263 YjbX bg13153 Glutamic acid-rich protein 25 445 6.38 B1 MS264 YjcG bg13160 Unknown 19 518 5.57 C2 MS265 YjcH bg13161 Unknown 27 768 5.99 C1 MS266 YjcI bg13162 Similar to cystathionine gamma synthase,

probably S-box regulon41 546 5.02 B3, n MS MS C

267 YjcJ bg13163 Similar to cystathionine beta-lyase 42 332 5.76 B2 MS268 YjgD bg13191 Similar to proteins of unknown function

from B. subtillis21 126 6.72 a MS

269 YjlD bg13203 Similar to NADH dehydrogenase 41 794 6.29 B1 MS270 YjoA bg13215 Unknown 17 650 5.81 D2 MS; N271 YkpB bg13271 Similar to thiamine biosynthesis 33 419 6.14 B1 MS272 YkrS bg13278 Probably part of the S box regulon,

similar to initiation factor elF-2B(alpha subunit)

38 703 5.00 B3, n MS MS C

273 YkrT bg13279 Probably part of the S box regulon,unknown

45 187 4.86 B3, n MS MS C

274 YkrY bg13284 Probably part of the S box regulon,similar to proteins of unknown function

23 342 5.69 C2 MS

275 YkrZ bg13285 Probably S-box reguion, similar to proteinsof unknown function

20 680 4.41 C4, n MS

276 YkuQ bg13301 Similar to tetrahydrodipicolinate succinylase 24 838 4.68 C3, n MS277 YkwC bg13328 Similar to 3-hydroxyisobutyrate dehydrogenase 30 558 5.11 B/C2 MS278 YkzA bg19021 Similar to OsmC of E. coli 14 459 4.74 D3 MS; N279 YlaG bg13344 Similar to GTP binding elongation factor

(translation)68 212 4.97 A3, n MS MS

280 YlnB bg13378 Probably part of the S box regulon,similar to sulfate adenylyltransferase

42 725 5.65 B2 MS

281 YlnC bg13379 Similar to adenylylsulfate kinase 22 399 5.08 C3, n MS MS282 YlyB bg11796 Similar to proteins of unknown function 30 185 6.05 B2 MS283 YmdB bg13421 Similar to proteins of unknown function 29 138 6.50 a MS284 YmfA bg13422 Similar to proteins of unknown function 56 673 9.18 a MS285 YneT bg11827 Similar to proteins of unknown function 14 848 5.28 D2 MS; N286 YoaD bg13475 Similar to phosphoglycerate dehydrogenase,

probably S-box regulon38 473 5.62 B2, n MS

287 YocJ bg13523 Similar to acyl-carrier proteinphosphodiesterase

22 830 4.99 C3, n MS MS

288 YocK bg13524 Similar to YzwB, YteA and DnaK supressorDksA of E. coli

23 998 4.70 C4 MS

Proteinname

accordingto

SubtiLista)

Accessionnumber

inSu

btiList

TheoreticalM

r

Theoreticalpl

Gridsquare

b)

4.0–

7.0c

)

4.0–

12.0

c)

4.5–

5.5c

)

Charge-/mass–

modificationd

)




289 YoxD bg11048 Similar to 3-oxoacyl-acyl-carrier proteinreductase

25 150 5.33 C2 MS

290 YpcP bg11606 Similar to DNA polymerase I 32 790 5.22 B2, n MS291 YpfD bg11005 Similar to ribosomal protein S1 homolog 42 244 4.57 B3, n MS292 YpsB bg11454 Unknown 11 454 5.63 D2 MS; N293 YqfY bg11671 Similar to peptidoglycan acetylation 40 424 6.80 a MS294 YqgG bg11375 Phosphate ABC transporter binding protein PstS 31 530 4.85 B3 MS295 YqgJ bg11378 Phosphate ABC transporter (ATP) PstB1 29 852 7.90 C1 MS296 YqgK bg11379 Phosphate ABC transporter (ATP) PstB2 29 048 7.77 C1 MS297 YqhI bg11509 Similar to aminomethyltransferase 39 622 5.73 B1 MS298 YqiB bg11712 Putative exodeoxyribonuclease large subunit 50 854 6.55 a MS299 YqiV bg11725 Probable branched-chain alpha-keto acid

dehydrogenase E3 subunit(dihydrolipoamide dehydrogenase); syn Bkd

48 510 4.89 A3 MS

300 YqiX bg11727 Similar to amino acid ABC transporter(binding protein)

28 162 5.09 C3 MS

301 YqjI bg11738 Similar to 6-phosphogluconate dehydrogenase(pentose phosphate)

44 764 4.96 B3, n MS MS

302 YqjJ bg11739 Similar to glucose-6-phosphate1-dehydrogenase; syn Zwf

55 492 5.28 A2 MS

303 YqkF bg11761 Hypothetical oxidoreductase 34 563 5.13 B2 MS; N304 YraA bg13776 Similar to proteins of unknown function 16 655 4.77 C3, n MS; N305 YrbA bg13781 Similar to spore coat protein 43 071 5.75 A2 MS306 YsdC bg12317 Similar to endo-1,4-beta glucanase 39 059 5.55 B2 MS; N307 YsiB bg12331 Similar to 3-hydroxbutyryl-CoA dehydratase 27 404 4.94 C3 MS308 YsnB bg12334 Similar to proteins of unknown function 19 011 5.32 C2 MS309 YsnF bg12337 Unknown; syn Gsp9 16 036 4.61 A4 MS310 YsxC bg10339 Similar to proteins of unknown function,

hypothetical GTP binding21 881 9.71 a MS

311 YtdI bg13837 Similar to POS5 protein from S. cerevisiae 30 102 6.49 a MS312 YtgB bg13852 Similar to ABC transporter (ATP binding protein) 27 733 9.41 a MS313 YtgI bg13855 Similar to thiol peroxidase 18 072 4.70 C3, D3, n MS C314 YtoQ bg13899 Unknown 16 649 5.77 D2 MS315 YtxH bg10975 Unknown; syn Gsp14 16 543 5.11 C3 MS316 YtxJ bg10373 Unknown 12 263 5.09 D3 MS; N317 YuaE bg13945 Unknown 18 967 6.2 C1 MS318 YugG bg12361 Similar to transcriptional regulater

(Lrp/AsnC family)18 617 4.88 C3, n MS

319 Yugl bg12363 Similar to RNA-binding motif of S1and PnpA; syn Gsp13

14 143 5.92 D1 MS; N

320 YugJ bg12364 Similar to NADH-dependent butanoldehydrogenase

42 572 5.22 B2, n MS; N MS

321 YugK bg12365 Similar to NADH-dependent butanoldehydrogenase

43 245 4.64 B3 MS

322 YugU bg12373 Similar to proteins of unknown function(member of UPF0047 family)

16 167 6.59 D2 MS; N

Proteinname

accordingto

SubtiLista)

Accessionnumber

inSu

btiList

TheoreticalM

r

Theoreticalpl

Gridsquare

b)

4.0–

7.0c

)

4.0–

12.0

c)

4.5–

5.5c

)

Charge-/mass–

modificationd

)




323 YumC bg12391 Similar to thioredoxin reductase 36 685 5.57 B3, n MS324 YurP bg14002 Similar to opine catabolism 36 720 5.44 B2 MS325 YurY bg14011 Similar to ABC transporter (ATP-binding protein) 28 879 4.62 C3, n MS326 YusC bg14015 Similar to ABC transporter (ATP-binding protein) 37 729 7.3 a MS327 YusJ bg14022 Similar to butyryl-CoA dehydrogenase 65 160 5.15 A2 MS328 YvaB bg14058 Similar to NAD(P)H dehydrogenase (quinone) 23 125 5.11 C3, n MS MS329 YvbY bg14091 Similar to proteins of unknown function 26 128 5.16 C2, n MS MS330 YvgN bg14096 Similar to plant-metabolite dehydrogenase 31 511 5.07 B3, n MS MS331 YvgQ bg14099 Similar to sulfite reductase 64 634 5.49 A2 MS332 YvgR bg14100 Similar to sulfite reductase 67 083 4.57 A3, n MS333 YvrE bg14145 Similar to RNA polymerase,

belongs to the SMP30/CGR1 family33 051 4.62 B3 MS

334 YvyD bg10740 Similar to o54 modulation factors 21 834 5.18 C2, n MS C335 YwaA bg10546 Similar to branched-chain amino acid

aminotransferase40 164 4.95 B3, n MS MS

336 YwbD bg10565 Similar to proteins of unknown function 44 283 9.18 a MS337 Ywfl bg10633 Similar to proteins of unknown function 29 335 4.97 C3 MS338 YwhD bg12458 Unknown 19 313 6.51 a MS339 YwjH bg10413 Similar to transaldolase 19 830 5.61 C2 MS C340 YxaB bg11104 Similar to proteins of unknown function 36 394 4.58 B3 MS341 YxiE bg11134 Similar to UspA of E. coli

(member of the UPF0022 (UspA) family)15 801 6.34 D1 MS; N

342 YxjG bg11156 Similar to proteins of unknown function fromB. subtilis, probably part of the S boxregulon

37 861 5.29 B3, n MS

343 YxjH bg11157 Similar to proteins of unknown function fromB. subtilis, probably part of the S boxregulon

38 142 5.08 B3, n MS MS

344 YyaF bg10050 Similar to proteins of unknown function 39 910 4.66 B3, n MS MS345 YybJ bg10021 Hypothetical ABC transporter

(ATP-binding protein)24 125 8.68 a MS

346 YybQ bg10014 Putative exopolyphosphatase 33 834 4.51 B3/4, n MS

a) In three cases there are different names of genes and their corresponding products (tufA and EF-Tu, fus and EF-G, tsfand EF-Ts). The reader should pay attention to this obstacle in case of database searches.

b) The grid section is given for proteins that are labelled on the reference map (Fig. 2). For multiple spots the localization ofall forms are specified. Occurrence on the maps of the narrow or the alkaline pI gradient is indicated by n and a, respec-tively.

c) The identification method (MS – peptide mass finger printing, N – Edman degradation) is indicated in the correspondingpI range of the gels that were used.

d) The existence of modifications and the type (C – in charge, M – in mass) is also recorded.

Proteinname

accordingto

SubtiLista)

Accessionnumber

inSu

btiList

TheoreticalM

r

Theoreticalpl

Gridsquare

b)

4.0–

7.0c

)

4.0–

12.0

c)

4.5–

5.5c

)

Charge-/mass–

modificationd

)


A comprehensive two-dimensional map of cytosolic proteins ofBacillus subtilis

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