From the Department of Biochemistry and Biophysics, Arrhenius Laboratories of Natural Sciences, Stockholm University, SE-10691 Stockholm, Sweden; ¶ Department of Medical Nutrition and Biosciences, Karolinska Institute, Novum, SE-14186 Huddinge, Sweden; and || Department of Biochemistry, Umeå University, SE-90187 Umeå, Sweden
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ABSTRACT |
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The Synechocystis sp. PCC 6803 (henceforth referred to as Synechocystis) is a free-living freshwater cyanobacterium that is an attractive model organism to study oxygenic photosynthesis as well as other metabolic processes (4). Its genome information (8) on CyanoBase (www.kazusa.or.jp/cyano/Synechocystis/index.html) provides easy access to established studies with a broad scope. Proteomic studies have updated our knowledge considerably of this cyanobacterium, especially with respect to proteins present in the plasma and thylakoid membranes (5, 912). Many questions, however, currently remain open. There are fewer experimental data available for the cyanobacterial outer membranes compared with the other Gram-negative bacteria such as Escherichia coli (13).
In this work, we present a newly developed method for the isolation of pure outer membranes from Synechocystis. The purity of the membrane fractions was verified by immunoblot analysis using antibodies against membrane-specific marker proteins. As a first application, we have examined the protein composition of the outer membrane by two-dimensional (2D)1 gel electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis and identification via database search. We have identified 49 proteins corresponding to 29 different gene products (open reading frames, or ORFs). Surprisingly, a number of the proteins in the outer membrane preparation were also found as part of the plasma membrane proteome (5). The reason for this is discussed in the context of cyanobacterial cell envelope organization.
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EXPERIMENTAL PROCEDURES |
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Outer Membrane Isolation
Outer membranes of Synechocystis were purified by sucrose density centrifugation as the first step followed by aqueous two-phase partitioning as the second step. Cells were broken with glass beads, and the total membrane separation by sucrose density centrifugation was performed as described previously (5, 15). The pellet from the sucrose gradient was fractionated by two-phase extraction essentially as Norling et al. (15) with modifications as follows. The membrane suspension from the pellet (3.75 g) was applied to a 6.25-g polymer mixture yielding a two-phase system of 6.2% (w/w) Dextran T-500, 6.2% (w/w) polyethylene glycol 3350, 0.25 M sucrose, and 5 mM potassium phosphate (pH 7.8). A transfer system (20 g) with the same final concentrations (but without membrane sample) was prepared. In addition, a second repartitioning system (20 g) with 6.6% of both polymers in the same buffer and sucrose medium was prepared. The partition steps were performed by inverting the tube 35 times at 3 °C. Phase settling was facilitated by centrifugation for 5 min at 1000 x g, and the upper (T1) phase was transferred and repartitioned with lower phase from the first (6.2%) repartitioning system, yielding the T2 fraction. The T2 fraction was added to a 6.2% lower phase (in total 10 g) and supplemented with 0.4 g of the Dextran T-500 (20%) stock solution and 0.2 g of the polyethylene glycol 3350 (40%) stock solution, resulting in a two-phase system with 6.6% of each polymer (T3). To further purify outer membranes from minor cross-contamination with plasma membrane (data not shown), two more partitions in the 6.6% repartitioning system were performed. Completely pure outer membranes resulting in the final top phase (T5) were collected and washed by a centrifugation at 125,000 x g for 1 h (4 °C). The pellet was washed and resuspended in 5 mM potassium phosphate (pH 7.8) supplemented with 0.25 M sucrose. In order to prevent protein degradation, a protease inhibitor mixture (Sigma, St. Louis, MO) was added prior to cell breakage and during the membrane preparations.
Outer membranes were extracted twice with 0.1 M sodium carbonate (16) followed by two more washes with 40 mM Tris to remove excess sodium carbonate. The final pellet of outer membranes was resuspended in the 20 mM potassium phosphate buffer (pH 7.8) containing 0.25 M sucrose. Protein concentration was estimated according to Peterson (17), and the membranes were kept at 20 °C for further analyses.
SDS-PAGE and Immunoblot Detection
Membrane proteins were separated by SDS-PAGE (12.5% polyacrylamide) using a system prepared according to Laemmli (18). After electrophoresis, the proteins were electroblotted onto nitrocellulose, immunodetected with antibodies, and the signals were visualized by using enhanced chemiluminescence reagent (Amersham Bioscience, Piscataway, NJ). Antibody directed to Toc75 was a kind gift from U. C. Vothknecht (Christian-Albrecht-Universität, Kiel, Germany).
2D Electrophoresis
For preparative 2D gel analysis, about 4 mg of the outer membrane protein pooled from 12 preparations was precipitated according to a method described by Wessel and Flugge (19). The precipitate was solubilized in 250 µl of an electrofocusing solution containing 7 M urea, 2 M thiourea, 1% tetradecyanoylamido-propyl-dimethylammoniopropane-sulfonate (ASB-14) (w/v), 1% 3-(3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS) (w/v), 2 mM tributyl phosphine (TBP), and 0.5% v/v immobilized pH gradient (IPG) buffer, pH 310 (Amersham Bioscience). The mixture was incubated at room temperature for 1 h, then sonicated in the presence of protease inhibitor mixture (Sigma). After centrifugation at 9000 x g for 10 min at room temperature, the supernatant was incubated with a linear IPG strip, pH 47/13 cm. The strip was rehydrated for 12 h at 20 °C. The isoelectricfocusing was performed at the same temperature, and the running conditions were 300 V for 40 min, 500 V for 40 min, 1000 V for 1 h, and 8000 V until a total of 130 000 Vh was reached. The strip was equilibrated in a buffer described by Nouwens et al. (20) for 20 min then loaded on the top of SDS-PAGE (12.5% polyacrylamide) prepared according to Laemmli (18). The electrophoresis was carried out at 56 °C and 5 mA/gel for 1 h, then 20 mA/gel using a Hoefer S.E. 600 apparatus (Amersham Bioscience). Proteins were detected by Coomassie Brilliant Blue G-250 according to Nouwens et al. (20), then scanned using an image scanner and evaluated with the Image Master 2D Elite software version 4.01 (Amersham Bioscience).
MALDI-TOF Mass Spectrometry and Database Searching
MALDI-TOF analysis was performed on a Voyager-DE STR MALDI-TOF mass spectrometer from Applied Biosystems (Foster City, CA). Cutting of protein spots and sample preparation for MALDI-TOF analysis were done in the similar way as described by Fulda et al. (21), except that it was performed manually. External calibration was applied using Calibration Mixture 2 from the SequazymeTM Peptide Mass Standards Kit (PerSeptive Biosystems, Framingham, MA). Trypsin auto-digestion products (m/z values 842.5094 and 2211.1046, respectively) were used for internal calibration. The proteins were identified as the highest ranking result by searching in the NCBI database (NCBInr20021113) including all species using Mascot (www.matrixscience.com/cgi/index.pl?page=/search-form-select.html). The search parameters allowed for oxidation of methionine, carbamidomethylation of cysteine, one miscleavage of trypsin, and 30 ppm mass accuracy. At least 50% of the measured masses were required to match the theoretical masses deposited in the database. The identification was repeated at least once using spots from different gels. The presence of putative signal peptides and their cleavage sites were predicted using the SignalP program (www.cbs.dtu.dk/services/SignalP-2.0). The lipoproteins were predicted using PROSITE (us.expasy.org/prosite).
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RESULTS AND DISCUSSION |
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A number of proteins are resolved in multiple spots as shown in Fig. 2. This is most likely due to post-translational modifications. As in most cases only the pI is affected without changes in molecular mass, the modifications are mainly in the side chains of the amino acids rather than due to differential processing of the precursor proteins. However, Sll1307, a hypothetical protein, is found as two spots with a mass difference of about 1.5 kDa. One protein, denoted Sll1307b, has a molecular mass and pI quite similar to the theoretical values, whereas the other, Sll1307a, has a higher molecular mass and lower pI. This may be due to a post-translational modification that affects the migration of the protein, giving an anomalous molecular mass.
As precursors of outer membrane proteins should have an N-terminal signal peptide, each identified protein was analyzed by applying the SignalP program for Gram-negative bacteria (24). Indeed, all the identified proteins contain a putative signal peptide (Table I). For protease HtrA (Slr1204), we suggest that the methionine at position 34 is the actual start of the protein because a typical signal peptide can then be predicted at the N terminus (Table I). The prediction shows that 26 proteins contain typical Sec signal amino termini lacking sequence similarity (25, 26), while three proteins have the twin arginine signal peptide motif, Tat (27, 28), as summarized in Table II. Our results in this work and earlier proteome studies (5, 21) indicate that both Sec and Tat machineries contribute to the export of proteins across the plasma membrane of Synechocystis, although the Sec system seems to be the major route. By sequence alignment against Tat subunits in E. coli, we have previously identified the subunits of tatA, tatB, tatC, and tatD in the genome of Synechocystis (21). Most recently, it was observed by Spence et al. (29) that in a Synechocystis mutant, expressing a fusion protein comprising the Tat-specific signal of E. coli TorA linked to green fluorescence protein (GFP), GFP was almost exclusively located in the periplasm (29). Two components of the Sec machinery, SecY and SecA, are found in both the plasma and thylakoid membrane of Synechococcus PCC 7942 (30). In Synechocystis, a number of Sec proteins, but no Tat proteins, have been identified on the lumenal side of the thylakoid membrane (10). Thus, it is still an open question if the Tat machinery is present in the thylakoid membrane of cyanobacteria. Using the PROSITE program for the lipoprotein pattern, five of the identified proteins in the outer membrane are predicted to be lipoproteins (31), and two of these have a Tat motif (Table II).
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Protease HtrA
The HtrA family of proteases represents one of the first well-studied protein quality control factors in cells (36). The HtrA family is a new class of oligomeric serine proteases that combines the chaperone and protease activities. Three members of HtrA family are found in the Synechocystis genome, i.e. HtrA, HhoA, and HhoB (37, 38). We found earlier that HhoA (Sll1679) is located in the periplasm of Synechocystis (21). Interestingly we found that HtrA (Slr1204) is present in the outer membrane of Synechocystis (Fig. 2), which supports the assigned physiological function for these proteases as a key factors dealing with misfolded protein degradation in the cell envelope (37). The higher plant HtrA homologue DegP2 has been shown to be involved in degradation of photosystem II reaction center protein D1 in in vitro studies (39). In Synechocystis, however, this function could not be assigned (38), which would be consistent with a localization far from the thylakoid membrane. Recently, a genetic study suggested that all three proteases of the HtrA family together are involved in protecting Synechocystis cells from light stress and in the repair of photosystem II (40). We have initiated proteomic studies of thylakoid membranes, which may provide further information about the localization of HtrA proteases in cyanobacteria.
GumB
Bacteria synthesize and secrete an array of complex carbohydrates including exopolysaccharides, capsular polysaccharides, lipopolysaccharides, and lipo-oligosaccharides, which provide protective functions to the cell (41). In Gram-negative bacteria outer membrane auxiliary proteins are required for export of exopolysaccharides and capsular polysaccharides. Based on computer analyses, GumB has been classified as one of the putative outer membrane auxiliary proteins in the bacterial plant pathogen Xanthomonas campestris (41). In X. campestris, it has also been demonstrated that the biosynthesis of the extracellular polysaccharide xanthan is directed by gum operon, an operon that is comprised of a cluster of 12 genes starting from gumB to gumM (42, 43). In the genome of Synechocystis, a gumB homologue has been identified, and our work shows that GumB (Sll1581) is expressed and localized in the outer membrane (Fig. 2). Searches in the Synechocystis genome revealed that in addition to the gumB gene, only gumD (slr0820, sll1535), gumH (slr1077, sll1231), and gumM (slr1118, slr1271) homologues are present. The Cyanobase annotations of these genes are putative glycosyltransferases, mannosyltransferase, putative UDP-N-acetyl-D-mannosaminuronic acid transferase. GumB in X. campestris and other putative outer membrane auxiliary proteins are predicted to be ß-barrel membrane proteins, and many of them also seem to be lipoproteins (41). The GumB homologue in Synechocystis was not found in the computer-based search for outer membrane auxiliary proteins (41), but it belongs to the polysaccharide export family. The Synechocystis GumB has a typical Sec signal sequence according to the SignalP program (Table I). However, the PROSITE prediction pattern for lipoproteins fits almost entirely for GumB. According to the PROSITE pattern, there should be a positive charge, a lysine or an arginine, between position 2 and 7 of the signal peptide. In GumB, the positive charge is at position 10. Although GumB in Synechocystis belongs to the polysaccharide export family, it is currently not known which carbohydrates are the substrates.
Pilus Proteins
The unicellular Synechocystis shows sporadic motility of twitching (44). It has been shown that in Pseudomonas aeruginosa the type IV pilus is responsible for the twitching motility (45). In Synechocystis, pil genes are composed of an operon of pilMNOQ (44, 45) and two other pil genes, pilA1 and -B1. All these genes are homologous to the type IV pilus genes in P. aeruginosa. In this work, we identified PilQ protein, Slr1277, in the outer membrane of Synechocystis (Fig. 2). We found previously that PilM, PilN, PilO, and PilA1 proteins are located in the plasma membranes (5, 15). Genetic studies demonstrate that mutations in any of the genes of the pil operon result in loss of both motility and competence for DNA uptake (44), whereas mutation of pilA1 only leads to loss of motility of Synechocystis (46).
TolC Efflux Pump
It has been suggested that in Gram-negative bacteria the TolC efflux pump is a complex protein machinery composed of three parts, i) the outer membrane TolC protein and ii) membrane fusion protein(s) connecting TolC to iii) specific plasma membrane channels (47). Due to this architecture, the TolC efflux pump can export small molecules up to large proteins directly from the cell interior, across the two membranes, to the extracellular environment without involving the periplasm. In this work, we verify that the TolC homologue Slr1270 is present in the outer membrane (Fig. 2) of Synechocystis. The crystal structure of TolC from E. coli has been resolved to 2.1 Å resolution (48). Three TolC monomers assemble to a continuous channel that is suggested to span both the outer membrane, as a ß-barrel, and the periplasmic space, as an -helical barrel, a novel substructure. We previously identified three membrane fusion proteins in the plasma membrane (5), which may be involved in this efflux system.
Cyanobacterial Porins, Outer Membrane Proteins with Tails
In the Synechocystis genome, six putative porin genes have been found, which are homologous to the genes somA and somB (Synechococcus outer membrane) (49). SomA and somB have been cloned and characterized as coding for two porins in Synechococcus PCC 6301. In the present work, we show that two (slr1841, slr1908) of these six putative porin genes in Synechocystis are expressed and localized as expected in the outer membrane. Bacterial porins are homotrimers of intimately associated subunits. Each subunit forms a completely antiparallel ß-barrel of 16 or 18 ß-strands (50). For most Gram-negative bacteria, the molecular mass of the monomer is between 30 and 40 kDa (51). The cyanobacterial porins characterized so far are larger and composed of monomers of about 5070 kDa. This is due to the presence of a 120-amino acid long N-terminal domain preceding the ß-barrel domain (49). The sequence of the N-terminal domain is furthermore shown to be homologous to the surface layer homology (SLH) domain, which is known to connect the cell wall or external layer proteins to the peptidoglycan layer (52). All six Synechocystis porins as well as SomA and SomB have extremely conserved SLH domains. The two expressed porins in Synechocystis (Slr1841, Slr1908) have 117 identical and one similar residue from a sequence of 118 amino acids each. The molecular masses of Slr1841 and Slr1908 are 65 kDa and 62 kDa, respectively and the ß-barrel has 18 and 16 predicted ß-strands, respectively (Rita Casadio, personal communication). Another protein identified in the outer membrane, Slr1272 (Fig. 2, Table I), is also annotated as a probable outer membrane protein in the Cyanobase. It also contains an SLH domain at the N-terminal, which is homologous to the SLH domains in the porins Slr1841 and Slr1908, but the molecular mass is about half, 25 kDa. The secondary structure has not been predicted for Slr1272.
Periplasmic and Outer Membrane Proteins
By comparing the proteome of the Synechocystis periplasm (21) and the present proteome of the outer membrane, it is found that eight proteins are present in both proteomes. These include three hypothetical proteins (Sll1307, Sll0319, and Slr1406) and three proteins with suggested functions, i.e. protease pqqE (Sll0915), oxalate decarboxylate (Sll1358), and virginiamycin b hydrolase (Sll0173). The presence of these proteins both in the outer membrane and the periplasm may indicate that these proteins in vivo are associated with outer membrane at the periplasmic side. The procedure for preparing the periplasmic fraction, involving cold osmotic shock, may also release loosely bound peripheral proteins. Furthermore, we found previously that virginiamycin b hydrolase (Sll0173) is enhanced, while oxalate decarboxylate (Sll1358) is reduced under salt stress conditions (21).
Proteins Present in the Plasma and Outer Membranes
Interestingly, we also found a number of proteins in the outer membrane of Synechocystis that we earlier identified in the plasma membrane (5). This overlap of proteins is however very specific and can hardly be explained by general cross-contamination of the two membrane preparations. The purity of the outer membrane fraction was demonstrated by immunoblot analysis using antibodies against i) NrtA, the nitrate/nitrite-binding lipoprotein in the plasma membrane, ii) CP47, the chlorophyll-binding protein of PSII in the thylakoid membrane, and iii) Toc75, the outer membrane protein (22). As shown in Fig. 1. neither NrtA nor CP47, only Toc75 was detectable in the outer membrane fraction. This indicates that the outer membrane preparation is not contaminated by plasma and/or thylakoid membranes. On the other hand, neither Toc75 nor CP47, but only NrtA was detected in the plasma membrane fraction. This also demonstrates that the plasma membrane preparation is not contaminated by outer or thylakoid membranes. Furthermore, there are a number of dominant protein spots present in one of the membranes, but absent in the other. Proteins, such as PilN (Slr1275) and PilO (Slr1276), could only be identified from the 2D gels of plasma membranes (5). On the other hand, Toc75 and a number of other proteins are pronounced in the outer membrane, but not present in the plasma membranes of Synechocystis. These include PilQ (Slr1277), GumB protein (Sll1581), and Hypo S-layer proteins (Slr1704, Slr1272) (Fig. 2). All these results provide strong evidence for the purity of the two distinct membranes.
So which proteins are found in both the plasma and the outer membrane? All five lipoproteins identified in the outer membrane (Table II) were also found in the plasma membrane (5). There are however additional lipoproteins only present in the plasma membrane (Ref. 5 and Huang et al., unpublished results). These are interesting results in view of what is known about lipoprotein sorting between plasma and outer membranes of E. coli. In E. coli, five proteins have been identified that appear to be involved in localization of lipoproteins to the outer membrane (53). LolCDE in the plasma membrane releases outer membrane-directed lipoproteins. A periplasmic chaperone, LolA, is then suggested to transport the lipoproteins to the outer membrane receptor LolB, which mediates the anchoring of the lipoproteins to the outer membrane. No lolABCDE sequence homologues are present in the sequenced cyanobacterial genomes of Synechocystis or Anabaena sp. PCC 7120 (54). Thus, if cyanobacteria have a similar mechanism for sorting lipoproteins to the plasma and outer membranes as does E. coli, the proteins involved in this process must be different at the sequence level.
Most surprising is the finding that the typical outer membrane ß-barrel porins Slr1841 and Slr1908, as well as the ß-barrel protein TolC, are found not only in the outer membrane but also in the plasma membrane. A possible explanation is that during the transport of proteins to the final destination in the outer membrane, these proteins can be found in the plasma membrane. But why only certain outer membrane proteins are detected in the plasma membrane is intriguing. One of the putative membrane fusion proteins (Sll0180), which is suggested (47, 55) to couple TolC to its inner membrane counterpart, is also found in both membranes (5), whereas two other membrane fusion proteins are only found in the plasma membrane (5). As discussed above, the overlap between the plasma and outer membranes cannot be explained by general cross-contamination of the two membranes. A possible explanation relates to putative contact points between the plasma and outer membrane that are involved in direct translocation of components through the plasma and outer membrane without the intervening periplasm. These contact points are built up from outer membrane proteins (probably porins), the membrane fusion proteins (often lipoproteins), which connect the two membranes, and the plasma membrane translocase proteins (47). Upon breakage of the cells, membrane fragments containing these contact points are formed and these fragments have the same density as outer membranes. Thus these contact point membranes are recovered in the outer membrane preparation. By performing counter current distribution of the outer membrane preparation in an aqueous polymer two-phase system, it may be possible to separate the contact point membranes from the general outer membrane vesicles (56, 57). Three hypothetical proteins (Slr1506, Slr0431, Sll1835) with unknown functions are also found in both the plasma and the outer membrane.
In summary, our present work offers a newly developed method for isolating the outer membrane from Synechocystis. We show that pure outer membranes can be separated from total cyanobacterial membranes in two steps using sucrose density centrifugation combined with aqueous polymer two-phase partitioning. The isolated outer membranes are used in a systematic investigation of this cell compartment in a 2D gel-based proteomic approach. As a primary investigation, 29 proteins have been identified, such as the TolC efflux pump, as well as ß-barrel porins, transporters, pilus proteins, proteases, and the Toc75 homologue, whose function remains puzzling in prokaryotes. The results also suggest the presence of possible contact points between outer and plasma membranes involved in transport processes. More insights can be expected using complimentary approaches that enable identification of more integral membrane proteins located in the different membranes of this cyanobacterium.
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FOOTNOTES |
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Published, MCP Papers in Press, February 26, 2004, DOI 10.1074/mcp.M300137-MCP200
1 The abbreviations used are: 2D, two-dimensional; ASB-14, tetradecyanoylamido-propyl-dimethylammoniopropane-sulfonate; CHAPS, 3-(3-cholamidopropyl)dimethylammonio)-1-propanesulfonate; IPG, immobilized pH gradient; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; TBP, tributyl phosphine; ORF, open reading frame; SLH, surface layer homology.
* This work was supported by funding from Carl Trygger Foundation (to B. N. and W. P. S.) and The Swedish Research Council (to C. F. and W. P. S.).
F. H. and E. H. contributed equally to this work.
** To whom correspondence should be addressed: Department of Biochemistry and Biophysics, Arrhenius Laboratories of Natural Sciences, Stockholm University, SE-10691 Stockholm, Sweden. Tel.: 46-8-162460; Fax: 46-8-153679; E-mail: birgitta{at}dbb.su.se
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REFERENCES |
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