proteome analysis of salt stress response in the cyanobacteriumsynechocystis sp. strain pcc 6803

RESEARCH ARTICLE

Proteome analysis of salt stress response in the

cyanobacterium Synechocystis sp. strain PCC 6803

Sabine Fulda 1*, Stefan Mikkat 2*, Fang Huang 3, Jana Huckauf 4, Kay Marin 4,Birgitta Norling 3 and Martin Hagemann 4

1 Universität Rostock, Institut Biowissenschaften, Pflanzengenetik, Rostock, Germany2 Universität Rostock, Medizinische Fakultät, Core Facility Proteomanalytik, Rostock, Germany3 Department of Biochemistry and Biophysics, Stockholm University,

Arrhenius Laboratories for Natural Sciences, Stockholm, Sweden4 Universität Rostock, Institut Biowissenschaften, Pflanzenphysiologie, Rostock, Germany

In the present study, changes in protein synthesis patterns after salt shock visualized by 35S-methionine labeling and the changed protein composition in salt-acclimated cells of the cyano-bacterium Synechocystis sp. strain PCC 6803 were analyzed by a combination of 2-DE for proteinseparation and PMF for protein identification. As a basis for the differential analysis, a proteomemap with 500 identified protein spots comprising 337 different protein species was established.Fifty-five proteins were found, which are induced by salt shock or accumulated after long-term saltacclimation. Some of the proteins are salt stress-specific, such as enzymes involved in the synthesisof the compatible solute glucosylglycerol, while most of them are involved in general stress accli-mation. Particularly, heat-shock proteins and proteins acting against lesions by reactive oxygenspecies were found. Moreover, changes in enzymes involved in basic carbohydrate metabolismwere detected. The dynamic of the proteome of salt-stressed Synechocystis cells was compared toprevious data concerning transcriptome analysis revealing that 89% of the proteins induced shortlyafter salt shock were also found to be induced at the RNA level. However, 42% of the stably up-regulated proteins in salt-acclimated cells were not detected previously using DNA microarrays.The comparison of transcriptomic and proteomic analyses shows the significance of post-tran-scriptional regulatory mechanisms in acclimation of Synechocystis to high salt concentrations.

Received: July 18, 2005Revised: November 22, 2005Accepted: December 1, 2005

Keywords:

2D-PAGE / Protein labeling / Transcriptome / Salt acclimation

Proteomics 2006, 6, 2733–2745 2733

1 Introduction

Changes in living cells after exposure to different environmen-tal stresses have been studied for many years using numerousprokaryotic and eukaryotic model organisms. Acclimation tohigh salt concentrations is of particular importance for basic as

well as applied research, since a high percentage of irrigatedland suffers from increasing levels of salts and cannot be furtherused to grow crop plants, which have generally a rather lowhalotolerance. The best characterized photoautotrophic micro-organism in relation to salt and osmotic stress response is themoderately halotolerant cyanobacterium Synechocystis sp.strain PCC 6803 (henceforth referred to as Synechocystis). Stud-ies using this cyanobacterium are strongly promoted by theknowledge of its complete genome sequence [1].

The basic response of Synechocystis cells to salt stress iswell understood [2]. As in most other living cells, it can bedistinguished into three phases. The first reaction after a sud-

Correspondence: Dr. Martin Hagemann, Universität Rostock,Institut Biowissenschaften/Pflanzenphysiologie, Albert EinsteinStreet 3a, D-18051 Rostock, GermanyE-mail: [email protected]: 149-381-4986112

Abbreviation: GG, glucosylglycerol * These authors contributed equally to this work.

DOI 10.1002/pmic.200500538

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

2734 S. Fulda et al. Proteomics 2006, 6, 2733–2745

den rise in concentration of NaCl includes a rapid influx ofNa1 and Cl2 within seconds into the cytoplasm after turgorcollapse. The second phase, which occurs within 60 min, ischaracterized by an exchange of Na1 by K1 leading to theelimination of toxic effects of high concentrations of Na1 onthe cell metabolism. In the third phase, which lasts for severalhours, the synthesis or uptake of compatible solutes takesplace in order to further stabilize the osmotic potential of thecytoplasm and to maintain structure of proteins and mem-branes. Synechocystis is able to acclimate to NaCl concentra-tions of up to 1.2 M by de novo synthesis of compatible solutes,mainly glucosylglycerol (GG) and small amounts of sucrose[3]. Additionally, salt stress activates an ABC-type transporterfor compatible solutes, such as GG, trehalose, and sucrose [4]and ion exchangers, such as Na1/H1-antiporters [5, 6] or a K1-uptake system [7]. Furthermore, this organism acclimates tosalt stress by tuning the main bioenergetic processes, namelyphotosynthesis and respiration, in enhancement of cyclicelectron-transport activity via photosystem I and the cyto-chrome oxidase activity [8], respectively.

A great impact on the global understanding of salt accli-mation had the identification of the complete set of salt- andosmo-regulated genes of Synechocystis cells using the DNA-microarray technique [9]. Among them, about 50% of the up-regulated genes encode so-called hypothetical proteins,which are not functionally characterized. Most of the earlygene inductions were found to be only transient, while inlong-term salt-acclimated cells only about 30 genes remainedstably up-regulated at RNA level [10]. The DNA-microarrayapproach was also successfully applied for identification ofregulatory proteins involved in salt- or osmotic stress-induced activation of gene expression. Among more than40 two-component systems, which are encoded in the Syne-chocystis genome, some have been characterized as sensorsinvolved in the acclimation to osmotic and salt stress [11, 12].However, these systems seem to sense not specifically salt orosmotic stress signals, since most of the regulated genesencode for more general stress proteins induced by differentenvironmental stimuli, such as cold or light stress [13, 14].

Transcriptome analyses display changes in gene expres-sion at RNA level, while biological function is mostly per-formed by proteins. Therefore, a more reliable picture ofphysiological mechanisms can be revealed by proteomicanalyses. Salt shock induces the synthesis of a set of stressproteins in Synechocystis cells [15], which have been dis-tinguished into proteins specific for salt stress and a largegroup of more general stress proteins [16]. However, in theseearlier studies proteins were mostly not identified. Thedevelopment of proteomic techniques in combination withknown genome sequences allows rapid protein identificationby PMF and peptide sequencing. In order to improve theprotein separation, proteomic studies on Synechocystis cellsused protein fractions such as soluble [17, 18] or periplasmicproteins [19] and membrane fractions, which were furtherseparated in cytoplasmic [20], outer [21], or thylakoid mem-branes [22, 23, 24]. All these studies used 2-DE and 1-DE

techniques and revealed the inventory of protein composi-tions of different compartments in Synechocystis cells. In arecent proteomic analysis of Synechocystis, different combi-nations of gel-depending and gel-free fractionation methodswere compared in combination with peptide identification byESI quadrupole TOF-MS/MS. By these different shotgunapproaches a total of 776 proteins were identified [25].

In the present study the dynamics of the soluble proteinfraction of Synechocystis was analyzed during acclimation tosalt stress. We identified 500 protein spots visualized on2-DE gels corresponding to 337 different protein species.The proteomic comparison of control and salt-stressed cellsresulted in the identification of 55 proteins, which areinduced in salt-shocked cells and/or accumulated after long-term salt acclimation. The proteome variation was comparedto transcriptome data of salt-stressed Synechocystis cells [10],revealing that 38% of the proteomic changes were notdetected previously at the RNA level.

2 Materials and methods

2.1 Strain and culture conditions

Axenic cells of Synechocystis sp. strain PCC 6803 were grownat 307C aerated with CO2-enriched air (5% v/v) under con-tinuous illumination at 170 mM photons/m26s in Allen’smedium, which contains under control conditions 2 mMNaCl [26]. Salt-acclimated cells were obtained after cultiva-tion for 5 days in medium supplemented with 684 mMNaCl. Cells were transferred every second day into fresh me-dium at the same OD (A750 = 0.8). Short-term salt shockswere realized by adding solid NaCl directly to the culturevessel to obtain the desired salt concentration. Cells wereharvested by centrifugation at 35006g and 47C. Cell pelletswere immediately frozen and stored at 2807C.

2.2 Labeling of proteins and isolation of soluble

protein fraction

For labeling, 15 mL of cyanobacterial suspension (A750 = 0.8)was taken from exponential growing cultures (control andsalt-stressed) and immediately pulse-labeled for 30 min with8.3 MBq L-[35S]-methionine (specific activity .37 TBq/mmol, Amersham Biosciences, Freiburg, Germany). Pulselabeling was performed in polypropylene tubes withoutbubbling at 307C and illumination by the same light intensityas during the cultivation. Cells were collected by centrifuga-tion (50006g, 10 min, 47C), and the pellets were stored at2807C. To isolate the soluble protein fraction of unlabeled aswell as labeled cells, the cell pellets were suspended in ice-cold 10 mM HEPES/NaOH buffer (pH 7.0) supplementedwith the protease inhibitor PMSF. Homogenization wasachieved by treatment in a cell mill (E. Bühler, Bodels-hausen, Germany) cooled to 47C. The crude extracts werecentrifuged stepwise, first at 35006g to remove glass beads


Proteomics 2006, 6, 2733–2745 Microbiology 2735

and unbroken cells, and afterwards at 100 000g to separatemembrane and soluble protein fraction. Protein concentra-tions were estimated using the BioRad protein assay(BioRad, Munich, Germany).

2.3 Electrophoresis and Western-blot analysis

2-DE of the soluble protein fraction was carried out asdescribed in [19]. Lyophilized protein extracts (for stainedgels 200–400 mg protein) were dissolved in rehydration solu-tion (8 M urea, 2 M thiourea, 19.4 mM DTT, 1% CHAPS,0.5% carrier ampholytes (Pharmalyte, pH 3–10, AmershamBiosciences) and traces of BPB) and were separated in thefirst dimension on Immobilized Dry Strip Gels, pH 4–7/180 mm (Amersham Biosciences). The second dimensionwas carried out on SDS-containing polyacrylamide gels(12.5% acrylamide, 230623060.75 mm). A molecular massmarker (broad range, BioRad) was separated in the same gel.For Coomassie staining, gels were treated for 30 min instaining solution (50% methanol, 10% acetic acid,0.1% CBB R250) and destained with 20% methanol/10% acetic acid. Gels were washed with double distilledwater for 5–10 min and stored in closed plastic bags at 47C.Imaging was done using an UMAX Powerlook 1000 scannerat a resolution of 300 dpi (UMAX Systems GmbH, Willich,Germany). SYPRO Ruby protein gel stain (Molecular Probes,Eugene, OR, USA) was used according to the manufacturerprotocol. Stained proteins were visualized using a FLA-3000laser scanner at a resolution of 508 dpi (Fuji Photo Film,Tokyo, Japan) with 473 nm excitation and 580 nm long-passemission filters. To enable spot excision in visible light,SYPRO Ruby-stained gels were restained with colloidalCBB G-250 [27]. After separation of 35S-methionine-labeledproteins (500 000 cpm were loaded per gel, specific activity7000–20 000 cpm/mg protein), gels were dried and spotswere visualized on X-ray films. Using the transmitted lightadapter of a flatbed scanner, X-ray films were digitized at aresolution of 300 dpi (UMAX Powerlook 1000). 2-DE gelimage analysis software Delta2D, version 3.2 (Decodon,Greifswald, Germany) as well as Phoretix 2D Advanced Ver-sion 6.01 (Nonlinear Dynamics, Newcastle upon Tyne, UK)were used for gel to gel matching and spot quantification.The data were obtained from three independent experimentsincluding cultivation of cells (control and long-term salt-treated cells) and 2-DE. Each sample was separated threetimes to ensure good-quality resolution. Gels with the bestresolution were digitized and compared using Delta2D-soft-ware. The labeling experiments were repeated twice. Repre-sentative data are shown, while the induction factors aremean values from three experiments.

For immuno-blotting 10 mg protein was separated by 1-DE (12% acrylamide). Proteins were transferred onto NCmembranes (Hybond ECL, Amersham Biosciences) by elec-tro-blotting. The membranes were incubated with the anti-bodies at the following dilutions: GgpS and IsiB antibody5000-fold, StpA and Slr0924 antibody 1000-fold, GroEL anti-

body 20 000-fold. The binding of the antibodies was visual-ized on the basis of chemiluminescence using peroxidase-linked secondary antibodies specific for rabbit IgG (ECL kit,Amersham Biosciences).

2.4 Sample preparation for MS

Protein spots were excised from the Coomassie-stained gelsmanually using pipette tips that were cut to form an orifice ofapprox. 1.5 mm inner diameter. For identification of 35S-labeled, salt-induced proteins, they were visualized on X-rayfilm and corresponding gel pieces were excised from thedried gels after decay of radioactivity using a stainless steelpicking tool. The gel plugs were transferred into 96-wellplates that possess two small holes at the bottom of each well(pink microtiter plate-pierced, Genomic Solutions, AnnArbor, MI, USA). These pierced well plates were placed intodeep-well plates. During the digestion procedure, buffer andreagent solutions were added using electronic 12-channelpipettes and the liquids were removed by centrifugationthrough the pierced well bottom.

A low-salt digestion procedure adapted from the meth-ods described in [28, 29] was used. The gel plugs werewashed twice with 30% ACN in 25 mM ammonium bicar-bonate and 50% ACN in 10 mM ammonium bicarbonate,respectively, shrunk with ACN, and dried in a speedvacevaporator. The dried gel plugs were reswollen with 5 mL ofsequencing-grade trypsin solution (10 ng/mL in 3 mM Tris-HCl, pH 8.5, Promega, Madison, WI, USA) and incubatedfor 5–8 h at 377C. Thereafter, 5 mL of extraction solution(0.3% TFA, 50% ACN, 5 mM n-octyl-b-D-glucopyranoside)were added and the samples were agitated at room tempera-ture for 30–60 min before the peptide extracts were trans-ferred by centrifugation into the 96-well collection plates.

2.5 MALDI-TOF-MS and protein identification

Extracted peptides were applied onto the MALDI target (384/600 mm AnchorChip, Bruker Daltonik, Bremen, Germany)according to [30]. The peptide sample (0.5–1.0 mL) was appliedonto the sample anchor and allowed to evaporate for a fewminutes. Prior to drying, 1.8 mL of CHCA solution (1.6 mMCHCA in ethanol/acetone, 67/33 v/v) was added and allowedto dry at room temperature. The sample was washed with 5 mLof 1% TFA that was removed after 30 s by means of filterpaper. After drying, 0.7 mL of recrystallization solution (etha-nol/acetone/1% TFA, 60/30/10 v/v/v) was added.

Peptide mixtures were analyzed by MALDI-TOF-MSusing a Reflex III mass spectrometer (Bruker Daltonik)equipped with the SCOUT source, delayed extraction, andoperated in positive ion reflector mode with an accelerationvoltage of 25 kV. Measurements were externally calibratedwith [M 1 H]1 ions of angiotensin II, angiotensin I, sub-stance P, bombesin, and adrenocorticotropic hormones(clip 1–17 and clip 18–39). Mass spectra were acquired andanalyzed automatically using Bruker software, but, if neces-



sary, peak picking and calibration were corrected manuallyusing XMASS/NT 5.1.5 (Bruker Daltonik). Database search-es were performed against a regularly updated in-houseSWALL sequence database (Swiss-Prot and TrEMBL) usingthe MASCOT 1.8 software via BioTools 2.1 software. A masstolerance of 80 ppm and one missing cleavage site wereallowed, oxidation of methionine residues was considered asvariable modification, and carboxyamidomethylation ofcysteines as fixed modification. The search was restricted toeubacterial proteins; thus, the number of sequences, whichwere used for the searches, increased from 538 140(June 2004) to 716 405 (May 2005) during our investigations.All results were examined carefully for reliability and occur-rence of multiple proteins in the same sample.

2.6 DNA-microarray data

The results of the proteomic analysis were compared to tran-scriptomic data. This data set was obtained with total RNApreparations from short-term as well as long-term salt-treatedSynechocystis cells, which were used in DNA-microarray anal-yses to estimate genome-wide changes in mRNA levels asdescribed in [10]. While in [10] only examples for up-regulatedgenes were given, which showed the highest induction values,for the present comparison the complete data set was newlyevaluated particularly for all proteins assumed to be changedin the proteome map. Genes showing inductions factorshigher than two were regarded as salt-induced.

3 Results and discussion

3.1 Proteome of soluble proteins

For the proteomic analysis of the soluble protein fraction weused the “classical” combination of 2-DE and PMF. The datawere obtained from three independent experiments includ-ing cultivation of cells and 2-DE. We were able to detectabout 1100 protein spots on SYPRO Ruby-stained gels(Fig. 1). The proteins from 500 spots were identified, whichrepresent 337 different protein species. In addition, fourunique proteins, which could not be assigned to visiblespots in the stained gels, were identified after pulse-labelingexperiments (see below); thus, a total of 341 different pro-teins were found. Therefore, about 10% of the total numberof proteins encoded by the Synechocystis genome [1] wasdisplayed, while 14% of potentially soluble proteins weredetected. This rather low coverage of the complete proteomecould be improved if the weak spots would be included inthe analysis. Furthermore, the lower left part of the gel wasdifficult to analyze. In this region proteins associated withphycobilisomes smear over a large part of the gel, whichmake the identification by PMF of low-abundance proteinsimpossible. The use of a phycobilisome-deficient mutantmay help to solve this problem. However, even with shot-gun proteome analyses it is at present not possible to ana-

lyze the whole set of soluble proteins of Synechocystis in oneapproach [25]. Recently, a comprehensive list of all Synecho-cystis proteins ever identified in proteome analyses has beenpublished (http://www.shef.ac.uk/wrightlab/synechocystis)[25]. According to this list, among the total of 341 proteinsidentified in the present study, 81 proteins were found forthe first time (indicated in the supplementary material andTable 1). Including proteins from previous proteomeapproaches with Synechocystis (see [25]), today 1039 proteinspecies were detected altogether, which corresponds to32.8% of the total proteins identified in the genome of thiscyanobacterium [1].

Our results were combined to establish a proteome mapof the soluble protein fraction of Synechocystis, in which500 identified protein spots are shown in pI range from 4.0to 7.0 and a MW range between 10 and 125 kDa (Fig. 1,identities are given in the Supplementary Material). Mixturesof two or three proteins were detected in 49 and 3 spots,respectively. Among the 337 identified proteins, 219 weredetected as a single spot, whereas 118 proteins were found in2 or more spots. Multiple spots of the same protein occurredin most cases with different pI values and similar molecularmasses. Proteins migrating to multiple spots of varying pIare known from 2-DE gels of many organisms, which mayreflect PTMs or may be generated during the proteinseparation [31, 32]. Generally, a good congruence betweentheoretical molecular mass and location in the 2-DE gels ofthe identified proteins was observed (Fig. 1 and Supplemen-tary Material). However, in a few cases multiple spots of thesame protein showed significant differences in their appar-ent molecular masses. From the sequence coverage of thepeptide maps this could be attributed to N-terminal trunca-tion (elongation factor Tu in spot 336 and 339 – full-lengthprotein in spots 191, 195, 196, 198; 60 kDa chaperonin 1,GroEL1 in spots 182 and 183 – full-length protein inspots 84, 88, 89, 90, 95) or C-terminal truncation (phycobili-some 32.1 kDa linker polypeptide, rod 1 in spot 430 – full-length protein in spots 357 and 365) of the respective pro-teins. The truncated protein variants of elongation factor Tuor GroEL1 were found for the first time and seem to berelated to the salt stress treatment (see below). Whether ornot these smaller proteins represent simple breakdownproducts or have special biological function, is not known yet.For the elongation factor Tu it was proposed that this domi-nant protein existing in different variants in a cell may bepart of the prokaryotic cytoskeleton [33]. Other deviations oftheoretical and apparent molecular masses were due to sig-nal peptide cleavage of periplasmic or lumenal proteins (e.g.,Slr2048 protein (PratA) in spot 324, photosystem II manga-nese-stabilizing polypeptide in spots 335, 348, and 350,Sll1306 protein in spot 351, Sll1762 protein in spots 361 and363, Slr0924 protein in spots 421 and 422) or intein splicing(DNA gyrase subunit B in spot 42). Moreover, some proteinsin the very acidic pI range showed a considerable higherapparent molecular mass in 2-DE gels as expected from theprediction, e.g., FtsY was identified in spot 22 among 80 kDa



Figure 1. Proteome of soluble fraction of Synechocystis sp. strain PCC 6803. Gel was obtained after separation of soluble proteins (200 mg)from control cells by 2-DE (first dimension: IEF pH range 4–7, second dimension: SDS-PAGE applying 12.5% acrylamide) and SYPRO Ruby-staining. Protein spots identified by PMF are numbered and their identities are given in the Supplementary Material.

proteins but has a predicted mass of only 53.9 kDa (furtherexamples in spots 94, 118, 127, and 151). We have, however,no explanation for this finding. Interestingly, DnaK1(Sll0058 in spot 40) was identified for the first time in thepresent work. The DnaK3 (Sll1932) was recently detected bypeptide sequencing as well [25]. Thus, all three dnaK genesare expressed in Synechocystis, whereby DnaK2 (Sll0170) is byfar the dominating DnaK protein also in our gels, but clearlynot the only expressed DnaK protein as believed before [34].

The identified proteins (Supplementary Material) weresorted according to the functional categories defined by Cya-noBase (http://www.kazusa.or.jp/cyanobase/Synechocystis/

index.html) (Fig. 2). The main group (18%) of identified pro-teins comprises so-called hypothetical proteins. Despite simi-larities to proteins from other organisms, the function of suchproteins is mostly unknown. Their identification in 2-DE gelsoffers a possibility to study at least the regulation of such pro-teins under specific environmental conditions or in a specificmutant background. However, compared to their abundancein the total genome (54%) only a few of hypothetical proteinswere detected here indicating for their rather low quantitativelevels. In contrast, proteins involved in central intermediarymetabolism, energy metabolism, photosynthesis, respiration,and translation were identified in much higher ratios in the



Table 1. Salt shock proteins (2 h after adding 684 mM NaCl, short-term response) of Synechocystis sp. strain PCC 6803, which were iden-tified by PMF in the soluble protein fraction after labeling by 35S-methionine (see Fig. 4). Proteins in spots F, N, and S were identi-fied for the first time in the proteome of Synechocystis (shown in bold)

Spota) ORFb) Acc. Noc) Protein functionc) Gene inducedd)

A sll0170 P22358 Chaperone protein (DnaK2) SS 2 hB sll0416 P22034 60 kDa chaperonin 2 (GroEL2) SS 2 hC slr2076 Q05972 60 kDa chaperonin 1 (GroEL1) SS 2 hD slr1356 P73530 30S ribosomal protein S1 homolog A (Rps1) SS 0.5 hE slr1963 P74102 Hypothetical protein (Water-soluble carotenoid protein) SS 2 h, SAFe) slr1687 P74284 Hypothetical protein SS 2 h, SAG sll1621 P73728 Hypothetical protein (Ahp/TSA protein or peroxiredoxin 2 family) SS 2 hH slr1516 P77968 Superoxide dismutase (Fe, SodB) SS 2 hI slr1160 P74262 Hypothetical protein (periplasmic) NoJ sll1577 Q54714 C-phycocyanin beta chain (CpcB) NoK slr1894 P73321 Hypothetical protein (Probable DNA-binding stress protein, Dps family) SS 2 h, SAand sll1578 Q54715 C-phycocyanin alpha chain (CpcA) NoLe) sll1514 P72977 16.6 kDa small heat-shock protein (HspA) SS 2 h, SAM sll1746 P23349 50S ribosomal protein L7/L12 (Rpl12) SS 0.25 hNe) slr1674 P74262 Hypothetical protein SS 2 h, SAO ssr1480 P73557 RNA-binding protein (Rbp2) NoP sll1863 P74485 Hypothetical protein SS 2 h, SAQ slr2075 Q05971 10 kDa chaperonin (GroES) SS 2 hR sll0199 P21697 Plastocyanin precursor (PetE) SS 2 hSe) slr1915 P73113 Hypothetical protein SS 0.5 h

a) Spot-designations indicated in Fig. 4B.b) ORF designations according to CyanoBase (http://www.kazusa.or.jp/cyanobase/Synechocystis/index.html).c) Accession numbers and protein function according to Swiss-Prot and TrEMBL databases. In brackets gene names or, in the case of hy-

pothetical proteins, putative functions found in the new version of CyanoBase are given.d) Gene inductions according to transcriptome analyses in [10]; SS – gene induced in salt-shocked cells (time corresponds to peak

expression); SA – gene stably induced in salt-acclimated cells.e) No corresponding protein was identified in spots of the master gel (Fig. 1, Table 1 in Supplementary Material).

Figure 2. Distribution of all identified proteins (see Supplemen-tary Material) to the different functional categories, which weredefined for the Synechocystis proteins in CyanoBase (http://www.kazusa.or.jp/cyanobase/Synechocystis/index.html). Forcomparison the percentage of each functional group on total ge-nome basis is given in brackets.

proteome than in the genome, which corresponds to theiroccurrence as mass proteins in the normal and particularly instressed cells. Compared to the percentage in the whole ge-

nome, transport proteins and regulatory proteins are rareamong the identified proteins (Fig. 2). This is not surpris-ingly for hydrophobic membrane subunits of transporters,which are excluded from 2-DE. In contrast to typical gram-negative bacteria, such as Escherichia coli, whose substrate-binding subunits of ABC transporters are freely diffusible inthe periplasm, the cyanobacterial periplasmic binding pro-teins are mostly lipoproteins covalently attached to a lipidmoiety, and thus are also not found in the soluble fractionused in this work [22]. Regulatory proteins are mostly presentin rather low abundance and probably below the detectionlimit of the used staining procedures.

3.2 Proteome of short-term salt-shocked cells

The proteomic approach was further applied to comparecontrol and salt-stressed cells of Synechocystis. In order tooptimize the time scale for cell harvesting, main physiologi-cal parameters were traced after the salt shock (Fig. 3). Thetime courses for protein synthesis and photosynthesis showan immediate decrease in both activities after the addition ofsalt. While the protein synthesis was nearly completelyblocked, photosynthesis was reduced to about 60% of the



Figure 3. Changes of physiological parameters in cells of Syne-chocystis sp. PCC 6803 after addition of 684 mM NaCl to the me-dium at time point 0. Protein biosynthesis (triangles) was meas-ured as stable 35S-methionine incorporation into the cells (100%in control cells correspond to 110 cpm/min/mg protein). Photo-synthesis rate (squares) was estimated as oxygen evolution atsaturating light (100% in control cells correspond to 430 mmol O2/h/mg Chla). GG content (circles) after 24 h salt shock correspondto 133 mmol GG/mL/A750.

value in control cells. About 4 h after salt addition, new stablerates of protein synthesis and photosynthesis were observed.This first partial recovery is based on the accumulation of thecompatible solute GG (Fig. 3). Accordingly, we have chosentwo time points for investigation of the Synechocystis pro-teome. To investigate the short-term response cells shockedfor 2 h by 684 mM NaCl were characterized. Despite proteinsynthesis is still heavily affected at this time point, for mostof the transiently induced genes maximal mRNA levels wereobserved after 2 h of salt stress [10]. To reveal stable changesin the proteome (long-term response) in comparison to con-trol cells, cells grown for several days at high salinity(684 mM NaCl) were used.

Protein patterns from control and short-term salt-shocked cells were first compared after Coomassie staining.

However, both spot patterns were almost identical (notshown). Probably, the short time after the shock and therather low-protein synthesis rate were not sufficient for theaccumulation of stress-specific proteins up to concentrationsnoticeable in stained gels. Newly synthesized proteins can beclearly detected after 35S-methionine labeling [19]. Therefore,pulse-labeling of newly synthesized proteins was applied toestablish a sensitive and specific detection method for saltstress proteins. As expected, pronounced differences be-tween control and salt-shocked cells became visible on auto-radiograms from radioactive 2-DE gels (Fig. 4). While incontrol cells a high number of labeled proteins appeared, inshort-term salt-shocked cells only a few prominent proteinspots remained labeled. Obviously, synthesis of most pro-teins from control conditions is switched off after shock andonly a specific set of stress proteins is induced or remainedunder active translation.

Eighteen different stress proteins appearing in short-term salt-shocked cells were identified by PMF after match-ing the radio-labeled spots visualized on X-ray films to thecorresponding position in the dried gels after decay of radio-activity (Fig. 4, Table 1). This strategy allowed the identifica-tion of more than half of the highly labeled protein spotsvisible on the autoradiogram, while the remaining proteinswere probably below the detection limit of the used massspectrometric method. It is noteworthy that four of the iden-tified salt-shock proteins in spots F, L, N, and S were notfound among the identified proteins from the master gel(Fig. 1, see Supplementary Material). Obviously, these pro-teins occur only in low amounts and were not selected foridentification from stained gels. Most of the identified pro-teins belong to the well-known group of general stress pro-teins, which are induced after different stress treatmentssuch as high light and heat-shock treatments as well as saltstress. The heat-shock proteins DnaK2, GroEL1, GroEL2,HspA, and GroES (labeled with A, B, C, L, and Q in Fig. 4)

Figure 4. Autoradiography of a2-DE separation of 35S-methio-nine-labeled proteins from cellsof Synechocystis sp. strainPCC 6803 grown in controlmedium (A) or shocked for 2 hwith 684 mM NaCl (B). Identityof salt-stress proteins labeled inpanel B is given in Table 1.



were found, which are known to protect or repair proteinsunder stress conditions. The probable DNA-binding stressprotein (spots K) and the RNA-binding protein (spot O) maybe necessary to stabilize nucleic acid structures, which areknown to be influenced by changes in water potential andionic strength. Moreover, proteins involved in the defenseagainst reactive oxygen species such as the water-solublecarotenoid protein (spots E), superoxide dismutase(spots H), and a peroxiredoxin-like protein (spots G) wereidentified, which are also induced at mRNA level in highlight-stressed cells [14]. As shown in Fig. 3, translation rate isstrongly affected in salt-shocked cells. The 30S ribosomalprotein S1 (spot D) and the 50S ribosomal protein L12(spot M) may be particularly instable, which is compensatedby their enhanced synthesis in short-term stressed cells.Additionally, five hypothetical proteins were identified, forwhich no speculation about their function is possible. Besideplastocyanin (spot R), a lumenal carrier involved in photo-synthetic electron transfer, two phycocyanin proteins (CpcAand CpcB, spots K and J) were detected, which serve as light-absorbing subunits in phycobilisomes. However, it is knownthat phycocyanin content is reduced in salt-stressed cells andphycobilisomes are quickly decoupled from photosystem II[35]. Therefore, we assume that the phycocyanins do notrepresent the salt-induced proteins found in the labeled pro-tein pattern. It is very likely that their dominating amount,which is smearing over this part of the 2-DE gel, disturbs theidentification of other proteins in minor concentration at thesame position. This view is supported by the fact that inmany protein spots of this region mixed identifications withphycocyanin were observed.

A further indication that proteins other than CpcA orCpcB were labeled can be taken from the previous tran-scriptome analysis, since the mRNA amount of the two cor-responding genes was reduced after salt shock [10]. With theexception of these phycobiliosme chromophor proteins andtwo other proteins (Slr1160 protein and RNA-binding pro-tein), the genes for all other short-term salt-induced proteinswere found to be up-regulated in correspondingly treatedSynechocystis cells (Table 1). Interestingly, most of them belongto the group of genes that were only transiently increased insalt-stressed cells. For 13 of these genes the maximumexpression was found 2 h after salt shock, the time point forlabeling of stress proteins. Moreover, for six proteins theexpression remained enhanced in completely salt-acclimatedcells [10]. Therefore, a good correspondence between prote-omic and transcriptomic analyses exist for short-term salt-shocked Synechocystis cells, since 89% of the newly labeledstress proteins also showed a salt-induced up-regulation inDNA-microarray experiments [10]. However, during the first2 h after salt shock about 200 genes were clearly up-regulatedbut only a few up-regulated proteins were detected in thelabeled protein pattern. Even if one takes into considerationthat only about 50% of the labeled proteins could be identifiedfrom the dried gels and that the 2-DE technique excludes mostof membrane and alkaline proteins, there is a big discrepancy

between the number of gene inductions and protein labeling.On the other hand, for most of the induced proteins in short-term shocked cells the corresponding gene was also found tobe induced by transcriptomics (Table 1). Highly abundantmRNAs, which showed mostly only transiently enhancedlevels in salt-shocked Synechocystis cells [10], may competemore successfully for the remaining protein synthesis capaci-ty in salt-stressed cells with mRNAs of unchanged amount.For example, the spot for the hypothetical protein Sll1863 isabsent in 2-DE gels with proteins from control cells, while it isstrongly labeled in stressed cells corresponding with the mas-sive induction (more than 200-fold) on the mRNA level [10].

3.3 Proteome of long-term salt-acclimated cells

In order to identify proteins which are accumulated in salt-acclimated cells, the spot intensity in 2-DE gels after Coo-massie- and SYPRO Ruby-staining was estimated. The pro-tein patterns of control and long-term salt-acclimated cellswere well comparable; most of the major protein spots werevisible in about the same quantities (not shown). To achievereliable quantifications of protein spots, the experimentalerror of the method was estimated by comparing corre-sponding spots from two sets of independent control gels.The obtained scatter plot indicates (Fig. 5) that the deviationof spot intensities is within the range of 2–0.5, respectively.Therefore, only protein spots which were reproduciblystained with more than two-fold higher intensity in 2-DE gelsfrom salt-acclimated cells were regarded as up-regulated.

Careful examination of the gels by means of Delta2D-software and visual control led to the discovery of 63 proteinspots that showed a greater staining intensity (.2-fold) inextracts from salt-acclimated cells. Most of these spots, listedin Table 2, were at least identified twice in independent gelsand their increase in spot intensity was verified in three in-dependent experiments. Finally, 45 different proteins werefound to be accumulated in salt-acclimated cells of Synecho-

Figure 5. Scatter plot showing the deviation of spot quantifica-tion after comparison of about 100 corresponding spots fromstained control gels, which were quantified using the Delta2Dsoftware package. Deviation of single spots is plotted against itsrelative quantity.



Table 2. List of the 45 proteins accumulated more than two-fold in salt-acclimated cells (long-term response, growth for 5 days at 684 mMNaCl) of Synechocystis sp. strain PCC 6803, which were identified by PMF in the soluble protein fraction after separation by 2-DEand Coomassie- or SYPRO Ruby-staining (Fig. 6 for selected proteins)

Spot Noa) Ind/total spotsb) Ind.c) ORFd) Protein functione) Gene ind.f)

493 (P)g) 1/1 20.0 sll1863 Hypothetical protein SS, SA182, 183 2/7 11.6 slr2076 60 kDa chaperonin 1 (GroEL1), C-terminal fragment SS336 1/7 8.4 sll1099 Elongation factor Tu (EF-Tu), C-terminal fragment No209 1/7 7.2 sll1099 Elongation factor Tu (EF-Tu) No117, 120 2/2 5.9 sll1566 GG-phosphate synthase SS, SA438 1/1 5.7 slr0001 Hypothetical protein No83 1/1 5.6 sll0550 Diflavin flavoprotein 1 (Flv1) No421, 422 2/2 4.9 slr0924h) Hypothetical protein (periplasmic fraction, TIC22) No466, 470 (K) 2/2 4.9 slr1894 Hypothetical protein (DNA-binding protein, Dps) SS, SA289 1/2 4.7 sll0934 Carboxysome formation protein (CcmA) No291 1/1 4.4 slr0537 Hypothetical protein (putative sugar kinase) No221 1/1 4.0 slr0746 and

sll0373GG-phosphate phosphataseGamma-glutamyl phosphate reductase

SS, SANo

361, 363 2/2 4.0 sll1762 Hypothetical protein No14, 15 2/3 3.8 slr1367 Glycogen phosphorylase SA388 1/1 3.8 slr0315 Hypothetical protein (probable oxidoreductase) SS124 1/3 3.6 sll0726 Phosphoglucomutase No481 1/1 3.6 slr0623 Thioredoxin (TRX) No375, 381 2/2 3.4 sll0807 Ribulose-phosphate 3-epimerase SS254, 275 2/2 3.2 slr1793 Transaldolase No218 1/1 3.1 sll1536 Molybdopterin biosynthesis protein (MoeB) No322, 323 (E) 2/5 3.0 slr1963 Hypothetical protein (Water-soluble carotenoid protein) SS, SA316 1/1 3.0 sll0057 GrpE protein (HSP-70 cofactor) No465 1/1 3.0 slr1992 Glutathione peroxidase SS317, 318 2/2 2.9 slr0952 Fructose-1,6-bisphosphatase SS379 1/1 2.7 sll0596 Hypothetical protein SA239 1/1 2.5 slr1938 Probable methylthioribose-1-phosphate isomerase SS, SA99, 100 2/2 2.5 sll1676 4-alpha-glucanotransferase No498 (R) 1/1 2.5 sll0199 Plastocyanin precursor (PetE) SS162 1/2 2.5 slr1331 Processing protease SS172 1/3 2.5 sll0179 Glutamyl-tRNA synthetase No160 1/5 2.5 slr1176 Glucose-1-phosphate adenyltransferase No346 1/2 2.4 sll1058 Dihydrodipicolinate reductase SS314, 315 2/2 2.3 slr1485 Hypothetical protein SS, SA52, 53 (A) 2/4 2.3 sll0170 Chaperone protein DnaK2 SS157 1/1 2.3 slr0877 Glutamyl-tRNA(Gln) amidotransferase subunit A SS407, 414 2/2 2.2 sll1549 Hypothetical protein SA429 (H) 1/1 2.2 slr1516 Superoxide dismutase (Fe, SodB) SS380 1/1 2.2 slr2144 Hypothetical protein No150 1/1 2.2 slr1535 Hypothetical protein No125 1/3 2.1 slr0898 Ferredoxin-nitrite reductase SS54, 58, 59 3/5 2.1 sll1070 Transketolase No426 (G) 1/3 2.1 sll1621 Hypothetical protein (AhpC/TSA or peroxiredoxin 2) SS248, 249 2/3 2.1 slr0394 Phosphoglycerate kinase SS453 1/3 2.0 sll0248 Flavodoxin (IsiB) SA280 1/1 2.0 sll0408 Hypothetical protein (Peptidyl-prolyl cis-trans isomerase) No449 1/1 2.0 slr1171 and

slr0711Putative glutathione peroxidaseHypothetical protein

SS, SANo

a) Spot-numbering according to Fig. 1.b) The number of salt-induced spots is compared to the total number of spots, in which this protein has been identified.c) Induction factors were estimated by comparison of three-independent gels with the Delta2D-software package. For proteins with mul-

tiple spots mean values are shown.d) ORF designations according to CyanoBase (http://www.kazusa.or.jp/cyanobase/Synechocystis/index.html).e) Protein function according to Swiss-Prot and TrEMBL databases. In brackets gene names or, in the case of hypothetical proteins, puta-

tive functions found in the new version of CyanoBase are given.f) Gene inductions according to transcriptome analyses by [10]; SS – gene transiently induced in salt-shocked cells; SA – gene stably

induced in salt-acclimated cells.g) Designation of corresponding salt-induced proteins in 35S-labeled protein pattern (Fig. 4, Table 1).h) Previously identified as salt-induced periplasmic protein by [21].



Figure 6. Changes in the proteome of soluble proteins in Syne-chocystis sp. strain PCC 6803 after long-term salt acclimation(cells cultivated for 5 days at 684 mM NaCl). Proteins accumu-lated in salt-acclimated cells were detected after staining in 2-DEgels. Magnifications of regions showing the accumulations ofselected proteins in high-salt-grown cells are shown (GG-syn-thesizing enzymes: A – Sll1566, GgpS; B – Slr0746, StpA, chaper-onin: C – Slr2076, GroEL1 truncated, hypothetical proteins:D – Sll0924, E – Slr1963, F – Sll1863, carbohydrate metabolism:G – Slr1793, transaldolase, H – Sll0807, ribose phosphate 3-epi-merase, I – Sll0726, phosphoglucomutase). Complete list of pro-teins accumulated in salt-acclimated cells is given in Table 2.

cystis more than two-fold. 26 of them occur in two or morespots on the gels; however, for 16 of these proteins onlyselected spots were significantly increased, while the otherspots of the same protein are only slightly different or remain

Figure 7. Western blotting experiments to confirm the accumu-lation of selected proteins in salt-acclimated cells of Synecho-cystis sp. strain PCC 6803. Specific antibodies for the proteinswere used to monitor the protein concentration after separationof soluble protein extracts in 1-DE (12% acrylamide).

unchanged. However, in most cases the main spots contain-ing the majority of the corresponding protein showed theincrease in staining. Moreover, seven of the proteins havebeen already identified in the group of short-term salt stressproteins (compare Tables 1, 2). Correspondingly, for three ofthem a stable up-regulation of gene expression was reported[10]. For five of the up-regulated proteins we had specificantibodies, which allowed comparing their amounts in con-trol and salt-acclimated cells by Western blotting. In all casesthe increased amount of these proteins in long-term salt-treated Synechocystis cells was verified by this independenttechnique (Fig. 7).

The salt-accumulated proteins belong to four differentfunctional groups: (i) salt-specific stress proteins, i.e.,enzymes involved in the synthesis of the compatible soluteGG; (ii) general stress proteins; (iii) enzymes of the basiccarbohydrate metabolism; and (iv) hypothetical proteins,which are not characterized regarding possible functions.The identification of increased amounts of the enzymesinvolved in GG biosynthesis – GG-phosphate synthase andGG-phosphate phosphatase (Fig. 6A, B) – verifies our meth-od, since it was known [2] that the activity and amount(Fig. 7) of both proteins increased in salt-acclimated cells aswell as their gene expression was stably up-regulated [10, 35].As most other salt-accumulated proteins, GG-phosphatephosphatase (spot 221) was identified from two independentgels. While in one sample only GG-phosphate phosphatasewas identified, the sample from the other gel also containeda second protein (gamma-glutamyl phosphate reductase).Although the identification of this protein was reliable, it wasbased on only a few low peptide ion signals, which repre-sented 12% of the total peptide ion intensity of the respective



mass spectrum. In contrast, all higher ion signals represent-ing 70% of the total peptide ion intensity were assigned toGG-phosphate phosphatase. This analysis indicated thatgamma-glutamyl phosphate reductase (that was also foundclose to spot 221 in spot 220) is only a minor component inspot 221 whereas the increased labeling of spot 221 arisesfrom GG-phosphate phosphatase. Furthermore, gamma-glutamyl phosphate reductase did not exhibit any salt-de-pendent regulation in DNA-microarray experiments. More-over, the accumulation of glucose-1-phosphate adenyl-transferase (ADP-glucose pyrophosphorylase, Table 2) wasdetected after 2-DE, while the corresponding mRNA levelwas not changed. This enzyme is responsible for the synthe-sis of ADP-glucose, one precursor for the biosynthesis of thecompatible solute GG.

The general stress proteins include again heat-shockproteins acting as chaperones, which play important roles inprotein folding and stabilization. Obviously, the decreasedwater content in salt-acclimated cells and the higher saltconcentration require higher amounts of such proteins tomaintain protein integrity. Accordingly, GroEL1, DnaK2, andGrpE were found among the accumulated proteins. How-ever, while in the short-term salt-stressed cells GroEL1 wasfound at the expected molecular mass in 2-DE gels (Fig. 4),only truncated forms of GroEL1 comprising its C-terminalpart (Fig. 6C) showed an increased amount in the 2-DE pat-terns of salt-acclimated cells. A similar situation was foundfor the elongation factor Tu. The two spots 336 and 209increasingly stained in salt-acclimated cells were found in alower molecular mass range in 2-DE gels compared to theprobable full-length protein (spots 191, 195, 196, 198 inFig. 1). Another group of general stress proteins, such asglutathione peroxidase, thioredoxin, and superoxide dis-mutase, is responsible for dealing with imbalances in redoxstate and reactive oxygen species. To this group may alsobelong the diflavin flavoprotein (Sll0550), which was shownto be involved in the so-called Mehler reaction, the photo-reduction of oxygen [36], and the water-soluble carotenoidprotein (Fig. 6E), which is involved in the process of statetransition balancing the relative excitation of photosystem Iand II (Kirilovsky, personal communication). Both thio-redoxin (Slr0623) and the water-soluble carotenoid proteinhave been shown to be associated with the thylakoid mem-brane [37]. As mentioned earlier, the changed cytoplasmiccomposition may lead to perturbations in electron transfer,which is also indicated by the increased accumulation of thesoluble electron carries around photosystem I, flavodoxin,and plastocyanin (Table 2). With the exception of thio-redoxin, diflavin flavoprotein, and GrpE, for all general stressproteins the corresponding genes were found to be stably orat least transiently up-regulated (Table 2).

The third group of salt-accumulated proteins compris-ing enzymes of basic carbon metabolism was not expected,since carbon fixation and growth is reduced in salt-stressedcells. Nevertheless, a clear accumulation of spots for trans-aldolase, ribulose-phosphate 3-epimerase, phosphogluco-

mutase (Fig. 6G–I), transketolase, glycogen phosphorylase,phosphoglycerate kinase, and fructose-1,6-bisphosphatase(Table 2) was reproducibly found. These proteins areinvolved in the main organic carbon pathways such asglycolysis, Calvin cycle (reductive pentose-phosphate cycle),and oxidative pentose-phosphate cycle. The latter two per-form most of the carbohydrate metabolism in cyano-bacteria, since the Krebs cycle is incomplete in cyano-bacteria. Interestingly, such enzymes are known to beassociated with thioredoxins probably regulating their ac-tivity depending on the cellular redox potential, i.e., light/dark activation/inactivation [38]. With the exception of gly-cogen phosphorylase for none of the accumulated enzymesof basic carbohydrate metabolism the corresponding genesare stably up-regulated in salt-acclimated cells. For three ofthese proteins, the corresponding mRNA levels were tran-siently increased (Table 2). Therefore, post-transcriptionalregulation may be responsible for these changes. Thisassumption is further supported by the finding that someof the carbohydrate-metabolizing enzymes were found inmultiple spots, which differ mainly in pI. Only one spot ofphosphoglucomutase appear up-regulated, while the twoothers are almost unchanged (spot 124 increased,spots 119, 121 unchanged). A similar situation was foundfor transketolase, while all spots identified for transaldolaseor fructose-1,6-bisphosphatase were increased (Table 2).Moreover, the glycogen phosphorylase was detected amongthe salt-regulated proteins. Since glycogen is accumulatedin salt-acclimated cyanobacteria [39], this enzyme could beinvolved in glycogen synthesis rather than breakdown. Themassive accumulation of the compatible solute GG in salt-acclimated cells could be a further reason that basic carbonmetabolism is changed.

As a fourth group 14 hypothetical proteins were foundamong the accumulated proteins after 2-DE separation ofproteins from salt-acclimated cells of Synechocystis (Fig. 6Dand F). For some of these proteins probable functions havebeen proposed during the last years (given in brackets inTable 2). Particularly interesting is the Sll1863 protein(Fig. 6F), which was found before to be synthesized atincreased rate in short-term salt-treated cells. The genesll1863 showed by far the highest induction factor in tran-scriptome analyses [10]. Obviously, it is cotranscribed withsll1862; however, the corresponding protein was notdetected in our 2-DE analyses. A mutant defective in thishighly salt-stimulated operon did not show any differencesregarding salt tolerance in comparison to wild-type cells[10]. The Sll1863 protein seems to be unique for Synecho-cystis, since none of the other fully sequenced cyano-bacteria as well as other organisms has a gene homologousto sll1863. A good correspondence between transcriptomeand proteome data was also found for some other hypo-thetical proteins such as Slr1894, Sll0596, and Slr1485,while other hypothetical proteins showed an accumulationin the proteome but no increase at the transcriptional levelwas observed (Table 2).



The comparison of stably up-regulated proteins (Table 2)and genes found to be stably up-regulated [10] revealed amuch lower overlap than found before for proteins in theshort-term response. Among the 337 proteins identified inthe stained gels, for 21 proteins a more than two-foldincrease in the corresponding mRNA level was found inlong-term salt-acclimated cells. However, only 11 of theseproteins were revealed as salt-accumulated protein spots in2-DE, while for the other proteins no significant increasedespite their gene induction was found. On the other hand,for 42% of the stably up-regulated proteins (Table 2) nochange in transcriptional activity was reported. Therefore,the data sets from proteome and trancriptome analyses differfor almost half of the proteins or genes in long-term salt-acclimated cells of Synechocystis.

Most of the findings regarding salt-regulated changes ofthe Synechocystis proteome are in line with reports fromother organisms. Many of the salt-induced and salt-accumu-lated proteins identified in Synechocystis were also found inproteome analyses of salt-stressed cells from other organ-isms. Cells of the halotolerant green alga Dunaliella salinaincreased the amount of about 100 proteins after a shift from0.5 to 3.0 M NaCl [40]. These proteins belonged to the func-tional groups of antioxidants, chaperones, carbon assim-ilatory proteins, proteins involved in energy production, andprotein synthesis. Similar proteins were also found in pro-teome studies with salt-treated higher plants such as rice andArabidopsis thaliana [41, 42]. As found in our study with salt-treated Synechocystis cells, in salt-shocked cells of Arabidopsisthe protein synthesis rate was clearly affected. Nevertheless,proteins such as glutathione transferase, peroxiredoxin,DnaK-type heat-shock proteins, elongation factor Tu, andribosomal proteins were identified among the salt-respon-sive spots. Moreover, many enzymes of basic carbohydratemetabolism such as fructose-bisphosphate aldolase werefound to be salt-induced [42], which indicates that similarfunctional groups of proteins are up-regulated in salt-treatedcells of photoautotrophic organisms such as Arabidopsis,Dunaliella, and Synechocystis. In the gram-positive, hetero-trophic model bacterium Bacillus subtilis the acclimation tohigh salt and other stresses is exceptional well studied. Using2-DE and PMF 16 salt-induced protein spots were detectedcontaining proteins involved in the biosynthesis and trans-port of compatible solutes, hypothetical proteins, and sur-prisingly some proteins known to be iron-starvation induced[43]. Salt stress is known to induce the SigB regulon inB. subtilis, which comprises about 100 genes encoding pro-teins of various functions to prepare the cell against futureunfavorable growth conditions. In Synechocystis, a structuralsimilar sigma factor, the SigF, has been found to be alsoinvolved in the induction of several salt stress proteins [44].DNA-microarray analysis showed that many more genes areunder control of SigB in B. subtilis than were found before in2-DE [45]. Many of them were also induced on RNA level bysalt stress. Comparable to the situation in Synechocystis, thespectra of salt-regulated genes were clearly different between

short-term and long-term salt-treated B. subtilis cells [46] andalso different from the proteome pattern. As found in ourstudies with Synechocystis, among the SigB-regulated genesin B. subtilis various proteins were found, which areobviously responsible to deal with oxidative stress, while incontrast to our findings and the work on Dunaliella and Ara-bidopsis [40, 42], no induction of typical heat-shock proteinssuch as DnaK and GroEL has been reported for salt-treatedB. subtilis cells.

4 Concluding remarks

During our analysis of the soluble protein fraction by 2-DEand PMF, we identified nearly half of the stained proteinspots. This led to the discovery of 341 protein species, whichis slightly more than 10% of the total number of proteinsencoded in the Synechocystis genome [1]. Among them,81 proteins were detected for the first time. For Synechocystisa whole genome DNA microarray is available, which allowsthe quantification of the mRNA levels of all genes on thechromosome. In contrast, a complete display of all proteinsof a cell is presently impossible and a higher number of pro-teins can be only seen by combining different approaches[25]. The analysis of the Synechocystis proteome using 2-DE iscomplicated by the occurrence of many proteins in multiplespots. Future investigations should clarify to what extentthese multiple spots represent post-translational modifiedprotein species or just artifacts of 2-DE. Multiple spots fur-ther amplify the general problem of spot overlapping [47],i.e., merging of two or more polypeptides in the envelope ofthe same spot.

A combination of 35S-methionine labeling and total pro-tein staining allowed the discovery of 55 short-term-inducedand/or stable accumulated proteins during acclimation tohigh salt concentrations. Beside a few salt-specific stressproteins, which are involved in biosynthesis of the compati-ble solute GG, most salt-regulated proteins are involved inthe general stress response as chaperones or enzymes deal-ing with reactive oxygen species as well as in basic carbonmetabolism. Such salt-stress induced proteins seem to becharacteristic for photosynthetic organisms, since similarprotein classes were identified in the salt-stress proteome ofDunaliella and Arabidopsis [40, 42]. This indicates that Syne-chocystis may be still a valuable model for plants in order tocharacterize acclimation to environmental stresses. For thevast majority of the 18 short-term-induced proteins, anincreased mRNA concentration was found after salt shock ina previous transcriptome analysis [10]. However, for 19 out of45 proteins that were accumulated in salt-acclimated Syne-chocystis cells, a gene induction was not observed in the DNA-microarray experiments, indicating the involvement of post-transcriptional regulation in salt acclimation. However, themechanisms leading to enhanced protein amounts, whichare obviously not directly based on increased transcriptionalrate, remain to be elucidated.



This work was supported by a grant from the Deutsche For-schungsgemeinschaft (to M. H.) and from Carl Trygger andMagnus Bergvall Foundation (to B. N.). We would like to thankProfessor N. Murata, NIBB Okazaki, Japan, for his generoushelp with DNA-microarray analyses of salt-stressed Synechocystiscells in his laboratory. The help of M. Kreutzer, Proteome CenterRostock, Germany, with Mascot server maintenance and datahandling is greatly acknowledged.

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proteome analysis of salt stress response in the cyanobacteriumsynechocystis sp. strain pcc 6803

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