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Proteomic analyses of the photoauto- and diazotrophically grown cyanobacterium Nostoc sp. PCC 73102

Fang Huang

Department of Botany, Stockholm University, SE-106 91 Stockholm, Sweden

Correspondence
Birgitta Bergman
bergmanb{at}botan.su.se

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
The filamentous cyanobacteria of the genus Nostoc are globally distributed, phenotypically complex organisms, capable of cellular differentiation and of forming symbiotic associations with a wide range of plants. To further our understanding of these processes and functions, the proteome of photoautotrophically and diazotrophically grown Nostoc sp. PCC 73102 (N. punctiforme) cells was examined. Extracted proteins were separated into membrane and soluble protein fractions and analysed using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). The analysis led to the identification of 82 proteins that could be divided into 12 functional categories. Significantly, 65 of these proteins have not been previously documented in the Nostoc proteome. Many of the proteins identified were readily recognized as housekeeping proteins involved in carbon, nitrogen and energy metabolism, but a number of proteins related to stress, motility, secretion and post-translational modifications were also identified. Ten unclassified proteins were also detected, representing potential novel functions. These proteins were highly expressed, suggesting that they play key roles during photoautotrophic and diazotrophic growth. Nineteen of the proteins expressed under the growth conditions examined contained putative thioredoxin (Trx) targets, a motif that functions in redox regulation via redox equivalent mediators and is known to be significant in a wide range of biological processes. These observations contribute to our understanding of the complex Nostoc life cycle.

The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are listed in the tables.

A supplementary table and figure are available with the online version of this paper.

Present address: Research Center of Photosynthesis, Institute of Botany, Chinese Academy of Sciences, Beijing, China.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Cyanobacteria are photosynthetic prokaryotic microbes, which make a substantial contribution to global CO₂ assimilation, O₂ production and N₂ fixation, and were evolutionary precursors of plant plastids. Nostoc is a nitrogen-fixing (diazotrophic) filamentous genus (Rippka et al., 1979), with multiple physiological properties, occupying a wide range of terrestrial and limnic niches (Dodds et al., 1995).

To survive in such varying environments (from cold arctic to tropical areas), the photosynthetic vegetative cells of Nostoc species have evolved elaborate developmental alternatives. Firstly, they may divide their vegetative cells into new photosynthetically competent cells; secondly, they may terminally differentiate a proportion of the vegetative cells (5–10 %) into highly specialized non-photosynthetic, but diazotrophic, heterocysts as a response to lack of combined nitrogen; thirdly, the vegetative cells may develop into transient spore-like cells, termed akinetes, able to withstand harsh environmental conditions; and finally, whole filaments may differentiate into motile hormogonia, functioning in short-distance dispersal and as ‘infective units' in cyanobacteria–eukaryote symbioses (Adams & Duggan, 1999; Meeks & Elhai, 2002). The multiple differentiation strategies allow Nostoc species to use a photoautotrophic and diazotrophic mode of growth, making them uniquely independent of combined carbon and nitrogen. There has been significant research into the developmental cycles exhibited by this organism and many of the genes involved in N₂ fixation for example have been characterized (Adams & Duggan, 1999) but still many unknowns remain, such as the factors that govern the highly symbiosis-competent nature of members of this genus (Rai et al., 2000), the regulatory elements central to their complex life cycle, and the factors that contribute to their global success in terrestrial systems (Dodds et al., 1995).

Whole-genome sequencing of Nostoc punctiforme (ATCC 29133; http://www.jgi.doe.gov), the only symbiotically competent cyanobacterium sequenced so far, revealed an exceptionally large prokaryotic genome approaching 10 Mb with 7432 predicted ORFs (Meeks et al., 2001). The availability of genomic information is just beginning to pave the way for the use of proteomics as a powerful tool for large-scale comparison of Nostoc protein levels and for the identification of these proteins. To date, approximately 35 cytoplasmic proteins of N. punctiforme (ATCC 29133) have been identified (Hunsucker et al., 2004), and 10 proteins in N. punctiforme (PCC 73102) are known to be differentially expressed during hormogonium differentiation (Klint et al., 2006).

Here, proteins were analysed from the free-living, photoautotrophic and diazotrophic life stage of Nostoc sp. PCC 73102, which is the predominant stage of its complex life cycle and at which cyanobacterial–plant interactions are initiated. To increase the number of identified proteins, cellular and subcellular fractionation procedures were employed together with a quantitative strategy. Proteins were identified by MALDI-TOF MS and divided into 12 categories, based on predicted functions; their putative functions are discussed.

	METHODS

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Cyanobacterial strains and culture conditions.
The cyanobacterium Nostoc sp. PCC 73102 (originally isolated from the coralloid roots of the cycad Macrozamia sp.; Rai et al., 2002) and equivalent to Nostoc ATCC 29133 and N. punctiforme, was grown in nitrogen-depleted (diazotrophic conditions) BG11₀ medium (Stanier et al., 1971) with constant light (18 µmol photons m^–2 s^–1) shaken at 63 r.p.m. at 25 °C.

Protein extraction and fractionation.
Two-week-old cyanobacterial cultures were harvested by centrifugation and the cell suspension was washed three times in fresh BG11₀ medium. The cell pellets were weighed and then ground in five times their volume of extraction buffer [40 mM Tris supplemented with a Complete Mini Protease Inhibitor Cocktail Tablet (Roche)] with a mortar and pestle in liquid nitrogen, followed by sonication (Bandelin Sonoplus, DPC Scandinavia) 10 times (70 % intensity) for 20 s each in an ice bath, with 40 s cooling breaks. The homogenates were centrifuged for 1 min at 13 600 g to remove unbroken cells and cell debris. Supernatants were pooled and centrifuged at 160 000 g for 40 min at 4 °C. These supernatants were designated the soluble protein fraction. The pellets were washed once in the same buffer, harvested by ultracentrifugation and designated the membrane fraction. The protein concentrations of the soluble and membrane protein fractions were determined using the DC Protein Assay kit (Bio-Rad). The proteins were solubilized in two-dimensional gel electrophoresis rehydration buffers: the soluble fraction proteins in 7 M urea, 2 M thiourea, 4 % (w/v) CHAPS, 30 mM DTT and 1 % (v/v) IPG buffer (GE Healthcare); and the membrane fraction proteins in 7 M urea, 2 M thiourea, 1 % (v/v) ASB-14, 4 % (w/v) CHAPS, 30 mM DTT and 2 % (v/v) IPG buffer, to a protein concentration of 2 mg ml^–1, and then stored at –80 °C until used.

Two-dimensional (2D) gel electrophoresis.
Protein extracts containing 200 µg protein were loaded onto rehydrated 18 cm immobiline gel strips (pH 4–7; GE Healthcare) by cup loading. The isoelectric focusing of the proteins was performed on an IPGphore (GE Healthcare) and the focusing time was adjusted to a total of 80 000 Vh. The strips were equilibrated in the equilibration buffer described by Nouwens et al. (2000) for 30 min then positioned on top of an SDS–polyacrylamide gel (10 % polyacrylamide) and sealed with 0.5 % (w/v) agarose. The second dimension was carried out in a Protean II xi 2-D cell (Bio-Rad) at 20 mA for 20 min and 40 mA for 5.5 h. The gels were stained with the fluorescent dye SYPRO Ruby (Molecular Probes) as described by the manufacturer. A laser-scanning instrument (Typhoon 8600, GE Healthcare) was used for digitizing protein spot maps of the gels.

Gel analysis, in-gel digestion and MALDI-TOF analysis.
The scanned gel images were further analysed, using the PDQuest software (Bio-Rad) for visualization of protein spot distribution and annotation. Reproducible spots (clearly detected at least three times) were selected for identification. In-gel digestion was performed manually according to Fulda et al. (2000), followed by analyses in a MALDI-TOF MS instrument (Voyager-DE STR mass spectrometer, Applied Biosystems). The peptide mass fingerprints obtained were internally calibrated in MoverZ software (http://www.genomicsolutionscanada.com) using known autolytic trypsin peaks.

Database search.
The proteins were identified by comparing peptide mass fingerprints to the NCBInr database using the Mascot search engine (http://www.matrixscience.com). The search parameters allowed for oxidation of methionines, carbamidomethylation of cysteines, one mis-cleavage of trypsin, and 30 p.p.m. mass accuracy. The proteins were successfully identified, based on the first-ranking result and Mascot scores >74, which indicates that the hits were significant. Some hits with Mascot scores <74, for which more than half of the peptides used in the search matched, and hits with the correct M_r and pI, were also accepted in this study.

Bioinformatic analyses.
Signal peptides and their cleavage sites were predicted using the SignalP program (www.cbs.dtu.dk/services/SignalP-3.0). The prediction of transmembrane helices in identified proteins was performed using the TMHMM program (www.cbs.dtu.dk/services/TMHMM). Putative thioredoxin (Trx) targets were predicted based on homology (BLAST) to sequences of known Trx-linked proteins. To determine the number of conserved cysteines in cyanobacterial Trx targets, homologues (see Supplementary Table S1, available with the online version of this paper) from cyanobacteria, plants or green alga were compared and analysed by CLUSTALW (Lemaire et al., 2004; Lindahl & Florencio, 2003) (Supplementary Fig. S1). For each alignment, at least one of the selected proteins was previously identified as a Trx target.

	RESULTS

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Protein fractionation, 2D electrophoresis and protein identification
To achieve optimal cell protein coverage, total protein extracts from vegetative filaments (containing vegetative cells and heterocysts) of Nostoc sp. PCC 73102 were first fractionated into soluble and membrane proteins. After separation of the cyanobacterial proteins using 2D gel electrophoresis, gels were stained with SYPRO Ruby, which is more sensitive than Coomassie brilliant blue (Berggren et al., 2000). About 180 of the most abundant and reproducible (clearly detected on at least three gels) protein spots were selected for identification. As expected, a considerably higher number of protein spots, distributed over the entire gel, were detected in the soluble fraction as compared to the membrane fraction (composed of thylakoids, and inner and outer cell membranes) of the vegetative cells (Fig. 1a, b). The latter proteins dominated in the lower pH and higher-M_r region. Of the 180 protein spots, about 130 were successfully identified using peptide mass fingerprints (MALDI-TOF MS) and by searching public databases. The protein spots identified corresponded to 82 proteins, 60 soluble (Fig. 1a) and 22 membrane (Fig. 1b) fraction proteins from the whole-filament extractions (dominated by vegetative cells; typically about 92–95 % of the total cell population under N₂-fixing conditions). Of the proteins identified, 65 were detected in the Nostoc proteome for the first time. Some proteins, particularly in the membrane fraction, appeared as multiple spots, possibly due to post-translational modifications. However, the nature of the modifications was not discernible from the MALDI-TOF spectra, but in most cases resulted in a shift in pI rather than in M_r.

View larger version (61K): [in this window] [in a new window]   Fig. 1. 2D protein patterns of Nostoc sp. PCC 73102 from (a) the cellular soluble fraction and (b) the membrane fraction, of photoautotrophically and diazotrophically grown cells. The proteins were separated using a linear pH 4–7 immobilized gradient and 10 % SDS–PAGE. The gels were stained with SYPRO Ruby fluorescent dye.

View this table: [in this window] [in a new window]   Table 1. Proteins identified in the cellular soluble fraction (Sp) of Nostoc sp. PCC 73102, corresponding to the proteins marked in Fig. 1(a)

View larger version (32K): [in this window] [in a new window]   Fig. 2. Categories and relative proportion of the proteins identified in the proteome of Nostoc sp. PCC 73102. The 82 proteins identified from the two fractions (Sp and Mp) were divided into categories, based on their expected functions. The number of proteins identified in each category is given, and also expressed as percentage of the total number of identified proteins (in parentheses).

Putative thioredoxin (Trx) targets
Thioredoxin (Trx) targets among the proteins found were identified by sequence similarity to known Trx targets detected in cyanobacteria, plants and algae that had been characterized previously using Trx affinity chromatography coupled to proteomic analysis. The numbers of cysteines and conserved cysteines in the cyanobacterial proteins were first determined by multiple sequence alignments (Table 3). Nineteen of the cyanobacterial proteins expressed under the growth conditions examined were predicted to be putative Trx targets (Table 3). These included one reductase (Sp51) and proteins involved in carbohydrate and energy metabolism (Sp7, 25, 28, 31, 36, 52, 20 and Mp5,); amino acid biosynthesis (Sp8, 29, 30, 43 and 46); translation (Sp23); protein folding and degradation (Sp6, 9 and 10); and fatty acid biosynthesis (Sp3). All of the predicted Trx target proteins contained at least one cysteine residue, and 16 of the proteins contained conserved cysteines as in reported Trx targets (Table 3).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Interestingly, our data indicated that growth under free-living conditions in light under combined N-depletion may be stressful. This is however not a surprising result, since two incompatible processes, aerobic photosynthesis and anaerobic nitrogen fixation, occur within single filaments. For instance, the known stress-related enzyme superoxide dismutase (Sp59) (Kliebenstein et al., 1998) was strongly expressed, together with three reductases (Sp42, 44 and 51), which also may have roles in stress defence and repair systems. Additionally, four molecular chaperones (Sp1, 6, 9 and 10) known to play roles in stabilizing and refolding proteins during exposure of cells to stress, for instance in the GroEL system (Bukau & Horwich, 1998; Fink, 1999), were highly expressed (Fig. 1a). The proteins represented by spots Sp49 and 60, representing peptidyl-prolyl cis-trans isomerases (PPIase), are additional protein-folding catalysts related to stress (Lodish et al., 1999). The genes encoding the Sp53 (an ATP-dependent Clp protease) and Sp32 (an uncharacterized protein containing a vWA domain) proteins were located in a conserved gene cluster. The Clp protease removes oxidatively damaged proteins in response to environmental stress (Adam & Clarke, 2002; Agrawal et al., 2002; Zheng et al., 2002), while the vWA domain is found in cell adhesion proteins and promotes protein–protein interactions via a highly conserved metal ion-dependent adhesion site (Whittaker & Hynes, 2002; Lee et al., 1995). These proteins may function in reducing or repairing misfolded proteins, facilitate the refolding of proteins, and prevent protein aggregation.

NifK (Sp11; one of the larger subunits of dinitrogenase) was detected in total extracts from vegetative filaments (Fig. 1a). This shows that the proteomic procedure allows detection of proteins present only in 5–10 % of the cells (heterocysts). The nitrogen fixed in heterocysts is efficiently scavenged into amino acids to avoid inhibition by the ammonium ions synthesized after fixation, and indeed, 24 proteins (Table 1) were identified as being involved in amino acid and protein biosynthesis or in protein modification. In fact, this was one of the largest categories based on the number of proteins identified under N₂-fixing conditions (Fig. 2).

Proteins involved in energy and carbohydrate metabolism together represented the largest fraction of the proteome. Both photosynthesis and diazotrophy are associated with high energy demands, the latter requiring cell differentiation, synthesis of the nitrogenase complex and 12–16 ATPs per N₂ molecule fixed. Many of the components of the Calvin cycle were identified (Sp2, 7, 12, 28, 30, 36 and 52) and most of them were highly abundant (Sp7 and 36; Fig. 1). The oxidative pentose phosphate (OPP) pathway enzyme 6-phosphogluconate dehydrogenase (6PGD, Sp25) was highly abundant, while glucose-6-phosphate dehydrogenase (G6PD) (Summers et al., 1995; Hagen & Meeks, 2001) was not identified. The latter finding corroborates earlier data showing that light inhibits the activity of G6PD in Anabaena sp. PCC 7120 (Gleason, 1996). In contrast, G6PD protein levels in Nostoc symbionts are up-shifted under the dark heterotrophic conditions offered in plant symbiosis (Ekman et al., 2006). This result is expected since the OPP pathway is a major route for providing reductants (NADPH) to nitrogenase in heterocysts (Winkenbach & Wolk, 1973; Bothe & Neuer 1988). This illustrates the metabolic flexibility of the genus and the findings are supported by our data.

Two proteins (Mp19 and Sp56), annotated as hypothetical proteins in the Nostoc sp. ATCC 29133 (N. punctiforme) genome, are listed in the energy metabolism category. Mp19 contains a conserved domain of MSP (manganese-stabilizing protein) and shows high similarity (95 %) to an MSP protein (P13907) in Nostoc sp. PCC 7120, a 33 kDa protein associated with photosytem II in plants and cyanobacteria (Borthakur & Haselkorn, 1989). We propose that Sp56 (Table 1) is equivalent to a phycobilisome (PBS) rod–core linker polypeptide due to the high homology to such proteins (67 % identity to NP440438 in Synechocystis sp. PCC 6803), and that it contains a PBS-linker domain as does protein Sp48, a ferredoxin : NADP⁺ reductase (FNR).

The unknown protein category was prominent. Some of its protein members were abundant, such as Mp6, Mp10, Sp33 and Sp40 (Fig. 1), suggesting that they may have vital functions. SLH (S-layer homology) domains are present in two of the abundant proteins (Mp6 and Mp10). The latter, Mp10, also shows high similarity (78 % identity) to a component in the phosphotransferase system of Anabaena ATCC 29413 (ZP_00158087). The gene downstream of Mp10 is predicted to encode a signal transduction histidine kinase and the gene upstream of Mp6 is predicted to encode a cAMP-binding protein. Therefore, these two proteins may be involved in signal transduction and/or adhesion, which makes them candidate components for intracellular communication in cyanobacteria. The high homology (92 % similarity) between the hypothetical protein Sp39 and the ALAase, an actin-like ATPase in Anabaena variabilis, suggests a role in morphogenesis in Nostoc PCC 73102.

The protein Sp33 was identified as an ‘uncharacterized protein conserved in bacteria’ showing similarity to the ‘akinete marker’ protein (AvaK) in A. variabilis, particularly in the conserved N-terminus (93 % similarity) (Zhou & Wolk, 2002). AvaK is proposed to be exclusive to akinetes (Argueta et al., 2004), but as Sp33 was strongly expressed in the actively growing vegetative cells in Nostoc, other functions related to this protein must be considered. The PRC domain of Sp33 is widespread and ancient, and has been associated with a variety of biological processes, ranging from RNA processing to photosynthesis (Anantharaman & Aravind, 2002).

Signal peptides were identified in several of the membrane category proteins, which confirms their membrane location. This category was primarily related to cell motility, secretion and cell envelope biogenesis (Table 2). Six proteins were identified as outer-membrane proteins, and Mp7 and Mp18 were predicted to contain two conserved domains: the OEP (outer-membrane efflux protein) domain and the TolC protein domain. The OEP family forms trimeric channels allowing export of a variety of substrates in bacteria (Johnson & Church, 1999), whereas TolC proteins are outer-membrane, multifunctional proteins producing peptide antibiotics (Delgado et al., 1999) and maintaining membrane integrity (Bernadac et al., 1998). Located upstream of Mp18 are two genes encoding protein components of the ABC-type antimicrobial peptide transport system, and we predict that Mp7 and Mp18 may function in outer-membrane secretion systems and antimicrobial signalling. As the outer-membrane proteins Mp3 and Mp4 both contain a bacteria surface antigen (Bac surface Ag ) domain, a family including protective surface antigens (Ruffolo & Adler, 1996), they are likely to serve a protective function in Nostoc sp. PCC 73102. Mp4 also contains a domain homologous to FhaC (homolysin activation/secretion protein) known to be a membrane transporter with a role in intracellular trafficking and secretion (Guedin et al., 2000). Due to the unique symbiotic competence of Nostoc sp. PCC 73102, we propose that Mp9, a polysaccharide biosynthesis/export protein, and Sp19 phosphomannomutase, are related to lipopolysaccharide biosynthesis (Maroda & Valvano, 1993), and possibly of symbiotic significance.

Trxs are small, multifunctional, widely distributed redox-active proteins operating through a reversible reduction of intra- or intermolecular disulfides (Balmer et al., 2004; Lindahl & Florencio, 2003). Recently, numerous putative Trx targets have been identified in plant chloroplasts (Balmer et al., 2003, 2004; Marchand et al., 2004), in green algae (Lemaire et al., 2004) and in cyanobacteria (Lindahl & Florencio, 2003). About 200 proteins appear to be linked to Trx in plants (Buchanan & Balmer, 2005). Since 19 of the proteins identified here in Nostoc were predicted Trx targets (Table 3), Trx-linked processes also appear to be essential in Nostoc. Trx targets were detected in fructose-1,6-bisphosphatase, translation elongation factors, the Rubisco large subunit, chaperones, ATPase and peroxiredoxins of both plants and cyanobacteria (Table 3). All putative Trx targets identified here contained at least one cysteine, and 16 of them contained cysteines in conserved positions (Table 3). The presence of single cysteines in some of the proteins (Sp23, 46, 9, 1 and Mp5) suggests a regulatory function, although biochemical evidence is required to verify the regulation.

In summary, the protein profiling and expression data presented here provide an expanded view of the proteins expressed and involved in growth under photoautotrophic and diazotrophic conditions and illustrate the complexity of the proteome and lifestyle of Nostoc sp. PCC 73102 (N. punctiforme).

	ACKNOWLEDGEMENTS

 
The Swedish Research Council and the Swedish Research Council for Environment, Agricultural Science and Spatial Planning are gratefully acknowledged for financial support.

Edited by: M. Hecker

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Received 7 June 2006; revised 4 October 2006; accepted 23 October 2006.

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