Biologie VIII: Zellphysiologie1 and Biologie 12: Morphologie der Pflanzen und Feinbau der Zelle, Universität Bielefeld, D-33501 Bielefeld, Germany
Lehrstuhl für Biochemie der Pflanzen, Ruhr-Universität, D-44780 Bochum, Germany2
Author for correspondence: Elfriede K. Pistorius. Tel: +49 521 1065601. Fax: +49 521 1066410. e-mail: e.pistorius{at}biologie.uni-bielefeld.de
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ABSTRACT |
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Keywords: Synechocystis PCC 6803, iron deficiency, Slr1295, photosystem I and II
Abbreviations: ABC, ATP-binding cassette; Chl, chlorophyll; DCPIP, 2,6-dichlorophenol-indophenol; Dig, digoxigenin; MV, methylviologen; PBQ, phenyl-p-benzoquinone; PCV, packed cell volume; PS I and PS II, photosystem I and II
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INTRODUCTION |
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The association of IdiA with the thylakoid membranes of Synechococcus PCC 7942/PCC 6301 and the protective function on PS II came somewhat as a surprise, because IdiA has high similarity to the FbpA (also called SfuA) protein of pathogenic bacteria, such as Serratia marcescens (Angerer et al., 1990 ), Neisseria gonorrhoeae (Berish et al., 1990a
; Forng et al., 1997
), Neisseria meningitidis (Berish et al., 1990b
) and Pasteurella haemolytica (Kirby et al., 1998
) for example. FbpA belongs to the cluster 1 group of extracellular solute-binding proteins with specificity for ferric iron (Tam & Saier, 1993
). The FbpA protein of pathogenic bacteria is a component of a typical ATP-binding cassette (ABC) transporter for ferric iron across the inner membrane when iron is delivered as Fe3+-transferrin and/or Fe3+-lactoferrin. In the Fbp transport system FbpA is the periplasm-located component, FbpB is a cytoplasmic membrane protein and FbpC is a membrane-bound protein carrying a nucleotide-binding motif (Mietzner & Morse, 1994
; Adhikari et al., 1996
). A dendrogram was compiled for the pathogenic bacterial FbpA proteins, including the IdiA protein from Synechococcus PCC 6301, showing that IdiA has the highest similarity to FbpA from Pasteurella haemolytica (Kirby et al., 1998
).
Recently, a protein immunologically related to IdiA from Synechococcus PCC 6301/PCC 7942 was detected in three marine cyanobacterial species: Synechococcus sp., Crocosphaera sp. and Trichodesmium sp. (Webb et al., 2001). In the cyanobacterium Synechocystis PCC 6803, whose genome has been completely sequenced (Kaneko et al., 1996 ), there are two genes encoding proteins Slr0513 and Slr1295 which both have high similarity to IdiA from Synechococcus PCC 6301 and PCC 7942 (Slr1295, 53% identity and 69% similarity; Slr0513, 51% identity and 66% similarity). In addition, a third gene encoding a protein with lower similarity is present: Sll0237, with 26% identity and 42% similarity to IdiA. In a search for genes/proteins involved in iron acquisition in Synechocystis PCC 6803, Katoh et al. (2000
, 2001a
, b
) recently investigated a number of genes encoding polypeptides with similarity to proteins of ABC-type ferric iron transporters. These genes are slr1295, slr0513, slr0327 and sll1878, which were designated futA1, futA2, futB and futC, respectively. These genes are not located in an operon as is the case for the bacterial iron transporters (for a comparative listing, see Webb et al., 2001
). Inactivation of futB or futC or of both futA1 and futA2 greatly reduces the activity of ferric iron uptake and also growth. This group (Katoh et al., 2001a) concludes that the Fut system in Synechocystis PCC 6803 is related to the Sfu/Fbp family of bacterial iron transporters (Adhikari et al., 1996
). In the Synechocystis PCC 6803 transport system, proteins FutA1 (Slr1295) and FutA2 (Slr0513) are suggested to be the periplasm-located components which are assumed to play a role in iron binding. This is supported by the recent finding that the GST-tagged recombinant FutA1 was shown to bind ferric ion with high affinity (Katoh et al., 2001b). Since FutA1 and FutA2 are suggested to have redundant or overlapping substrate-binding functions, the phenotype with significantly reduced ferric iron uptake and consequently reduced growth is only obtained in the Synechocystis PCC 6803 double mutant lacking both proteins (Katoh et al., 2001a
, b
). In agreement with the assumed function of Slr0513 (FutA2) are the data of Fulda et al. (2000)
who have shown that Slr0513 is indeed present in the soluble protein fraction of the periplasm in Synechocystis PCC 6803. On the other hand, the localization of Slr1295 (FutA1) has remained uncertain, since it was not among the group of periplasm-located soluble proteins identified by this group. To obtain more information about Slr1295, we investigated the localization of Slr1295 together with the localization of Slr0513 and have performed a detailed study of Synechocystis PCC 6803 WT and an Slr1295-free mutant grown under iron-sufficient and iron-deficient conditions.
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METHODS |
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For growth of Synechocystis under iron limitation, BG11 medium without Fe(III) citrate was used. Prior to transfer of cells into iron-free medium, cultures were grown for several cycles of 2 days under regular conditions, then harvested by centrifugation and once washed with 20 mM sodium phosphate buffer, pH 7·0. In general, Synechocystis PCC 6803 [inoculation, 0·4 µl packed cell volume (PCV) ml-1] was grown for 48 h under the given conditions. Growth was determined by measuring the OD750 of Synechocystis PCC 6803 cultures [OD750 of 0·1 (up to a total absorbance of <1) corresponds to about 0·1 µl PCV ml-1] (see also Flores et al., 1982 ).
Preparation of cell suspensions and cell-free extracts.
For preparation of cell suspensions, Synechocystis cells were harvested by centrifugation for 30 min at 2200 g and then resuspended in MCMG buffer (20 mM MES/NaOH, pH 6·35, 5 mM CaCl2, 5 mM MgCl2, 25% glycerol) to give a cell density of 100 µl PCV ml-1.
When cell-free extracts of Synechocystis were used, the above cell suspension (100 µl PCV ml-1) was mixed with an equal volume of glass beads (0·170·18 mm) and treated in a Bead Beater (Biospec Products; chamber vol. 15 ml) 10 to 15 times for 25 s with cooling intervals of 4 min. After this treatment the cell extract was decanted from the beads and the beads were washed once with an equal volume of MCMG buffer. Subsequently, the two supernatants were combined. Thus, the obtained cell extract corresponded to a cell suspension of 50 µl PCV ml-1. The cell extract was centrifuged for 10 min at 2000 g to remove the residual glass beads and unbroken cells.
Determination of photosynthetic activities.
Photosynthetic O2 evolution/uptake with intact cells or cell-free extracts of Synechocystis PCC 6803 was measured in a Clark-type electrode (Rank Brothers) at a polarization voltage of 600 mV. The reaction temperature was 30 °C for cell suspensions and 20 °C for cell-free extracts. Continuous red light was provided by a halogen lamp (24 V, 250 W; Spindler and Hoyer). The light was filtered through a glass cuvette containing 2% (w/v) CuSO4 and a red plexiglass filter (RG1610; Schott). Light intensity on the water surface corresponded to 1995 µE m-2 s-1 (400740 nm).
O2 evolution of whole cells was determined in a reaction mixture of 3 ml containing 54 mM HEPES/NaOH, pH 7·0, 15 mM NaHCO3 in BG11 medium and cell suspension corresponding to 520 µg chlorophyll (Chl). Alternatively, a reaction mixture containing 54 mM HEPES/NaOH, pH 7·0, 1 mM phenyl-p-benzoquinone (PBQ), 50 mM CaCl2 and cell suspension was used.
PS I activity was determined with cell-free extracts. The reaction mixture contained in a total volume of 3 ml: 54 mM HEPES/NaOH, pH 7·0, 0·08 mM 2,6-dichlorophenol-indophenol (DCPIP), 3·33 mM sodium ascorbate, 0·17 mM methylviologen (MV), 0·4 mM KCN, 0·01 mM 3-(3,4-dichlorophenyl)-1,1-dimethylurea and cell-free extract corresponding to 520 µg Chl.
PS II activity was determined in a reaction mixture of 3 ml containing 54 mM HEPES/NaOH, pH 7·0, 50 mM CaCl2, 400 mM sucrose, 1·7 mM potassium ferricyanide and cell-free extract corresponding to 1020 µg Chl.
Since iron-deficient growth leads to Chl reduction, photosynthetic activity values are always given on Chl basis (per mg Chl) and in addition on the basis of PCV (per 100 µl PCV). PCV was calculated from the OD750 (see above).
Pigment content, protein content, SDS-PAGE and immunoblotting.
Chl content was estimated according to Grimme & Boardman (1972) , and phycocyanin and allophycocyanin content according to Tandeau de Marsac & Houmard (1988)
. Protein content was determined either according to Smith et al. (1985)
or Bradford (1976)
.
SDS-PAGE and immunoblotting were done as described previously (Exss-Sonne et al., 2000 ). Samples were denatured at 60 °C for 30 min. The antisera used were as follows: anti-IdiA (IdiA from Synechococcus PCC 6301; Michel & Pistorius, 1992
), anti-Slr0513 (Slr0513 was isolated from the soluble protein fraction of the periplasm from Synechocystis PCC 6803; a kind gift of Dr Sabine Fulda), anti-PsbA, anti-PsbB, anti-PsbO and anti-AtpA (proteins from oat or tobacco; Exss-Sonne et al., 2000), anti-PsaA/B (PsaA/B from Synechocystis PCC 6803) and anti-C-phycocyanin (from Aphanotheca halophytica; Sigma) and anti-allophycocyanin (from Anabaena variabilis; Boehringer).
77 K Chl a fluorescence measurements.
77 K Chl a fluorescence measurements were performed with a Perkin Elmer luminescence spectrometer LS50B. Excitation was at 435 nm and fluorescence emission was recorded between 650 and 800 nm. Cells suspensions cultivated under iron-sufficient or iron-deficient conditions were adjusted to equal Chl concentration by dilution with BG11 medium (±iron) to give a Chl concentration within the range of 210 µg Chl ml-1. Cells were immediately frozen in liquid nitrogen without any further pretreatment.
Isolation of Synechocystis PCC 6803 subcellular fractions.
Preparation of the soluble protein fraction from periplasm and of the spheroplast fraction was done according to Block & Grossman (1988) . The soluble protein fraction of the periplasm was concentrated (about eightfold) by dialysis against 20% carbowax (PEG 20000). The pellet containing the spheroplasts was resuspended in 5 mM Tris/HCl, pH 7·5, and broken by French pressure cell treatment at 138 MPa and 4 °C. Centrifugation for 1 h at 100000 g resulted in a soluble protein fraction and a pellet, which contained cytoplasmic and thylakoid membranes and which was resuspended in 5 mM Tris/HCl, pH 7·5.
The cytoplasmic and thylakoid membranes were isolated with slight modifications according to the procedure described by Omata & Murata (1984). Synechocystis WT cells (2·5 l cell suspension) were harvested by centrifugation, resuspended in 5 mM HEPES/NaOH, pH 7·0, containing 600 mM sucrose and 2 mM Na2EDTA, treated with lysozyme for 2 h at 28 °C and broken by passage through a prechilled French pressure cell at 40 MPa. The homogenate was centrifuged at 5000 g for 10 min to remove unbroken cells. The supernatant was subjected to density-gradient centrifugation as described by Omata & Murata (1984).
Isolation of PS I complexes from Synechocystis PCC 6803 was done as described by Wenk & Kruip (2000) , and isolation of PS II complexes was done as described by Burnap et al. (1989)
with slight modifications. The chromatography of the detergent-solubilized PS II complexes was done on a MonoQ column (Pharmacia) instead of a Toyopearl DEAE-M column and the NaCl gradient was extended to 500 mM NaCl instead of 200 mM NaCl.
Ultrastructural and immunocytochemical investigations.
Synechocystis PCC 6803 cells were grown and harvested as described above. A cell pellet obtained from 10 ml cell suspension was washed three times with EM buffer (50 mM KH2PO4/Na2HPO4, pH 7·0). The ultrastructural and immunocytochemical investigations were performed as described previously (Engels et al., 1997 ).
Construction of the Slr1295-free Synechocystis PCC 6803 mutant.
For construction of the Slr1295-free Synechocystis PCC 6803 mutant the primers P-5'-AACCTCCAGTTCCA-CTGACC-3' and P-5'-AGTCTGCCAACTGGTGACAA-3' were used to amplify a 1907 bp DNA fragment carrying the gene slr1295. The PCR-amplified fragment was cloned into SmaI-digested pK18mob (Schäfer et al., 1994 ). The insertion of a CmR cassette with flanking
terminators preventing transcriptional read-through activites, taken from plasmid pHP45
-Cm (Chang & Cohen, 1978
), into the AvaI-site leads to insertional inactivation of the slr1295 gene, leaving nearly equal flanking DNA regions on both sides of the fragment. Transformation of Synechocystis PCC 6803 with this 6987 bp construct (pK18mob1295CmR) was performed according to Laudenbach & Straus (1988)
.
Southern blotting.
Southern blotting was performed as described in Sambrook et al. (1989) . Genomic DNA from Synechocystis PCC 6803 WT and the Slr1295-free mutant was isolated by the Sarkosyl method, purified by the phenol extraction procedure (Williams, 1988
) and then digested with SspI and ScaI or XmnI, or AlwNI and KpnI. The digested DNA was separated in a 1% agarose gel and transferred via capillary blotting onto a positively charged nylon membrane (Hybond-N+; Amersham). For hybridization a digoxigenin (Dig)-labelled pK18mob1295CmR probe was used, according to the manufacturers recommendation.
Northern blotting.
Northern blotting was done as previously described (Michel et al., 1999 ). The primers used to amplify the probes were 5'-AACCTCCAGTTCCACTGACC-3' and 5'-AGTCTGCCAACTGGTGACAA-3' for slr1295, 5'-GGCAAGGATGGACAGCAGTA-3' and 5'-GCATTACTGCCAGCCAACTT-3' for slr0513, and 5'-GGTGGACAGAGACGCTTTATT-3' and 5'-TGGCTAATGACTAGGTTTG-CA-3' for isiA.
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RESULTS AND DISCUSSION |
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Expression of Slr1295 and Slr0513 at the protein level was analysed with an anti-IdiA antiserum raised against IdiA isolated from Synechococcus PCC 6301 (Michel & Pistorius, 1992 ), and with an antiserum raised against the Slr0513 protein which was isolated from the soluble protein fraction of the periplasm from Synechocystis PCC 6803. The results of the immunoblot analysis are given in Fig. 2
. Two very weak protein bands were detectable after immunostaining with the anti-IdiA antiserum in WT cells cultivated under iron-sufficient conditions. The expression of both proteins was highly increased under iron limitation. As expected, in the Slr1295-free mutant only one protein with an increased expression under iron limitation was detected, corresponding to the lower band in WT cells. Thus, the upper band in WT cells represents Slr1295, which is missing in the mutant, and the lower band represents Slr0513, which is present in the mutant. The results in Fig. 2
also show that the anti-Slr0513 antiserum only recognizes the lower band in WT cells and thus only cross-reacts with Slr0513, the protein against which the antiserum was raised. This antiserum is slightly more sensitive in detecting small amounts of Slr0513 than anti-IdiA antiserum. Thus, a 10% SDS-polyacrylamide gel (Tris-glycine buffer system) can resolve the two proteins under investigation: Slr1295 migrates slightly slower (upper band, apparent molecular mass 35 kDa) than Slr0513 (lower band, apparent molecular mass 32 kDa). Both proteins are detected by anti-IdiA antiserum, while the anti-Slr0513 antiserum only cross-reacts with Slr0513.
Comparative analysis of growth, pigment content and photosynthetic activities
Growth, pigment content and photosynthetic activities of Synechocystis PCC 6803 WT and the Slr1295-free mutant were analysed under iron-sufficient and iron-deficient growth conditions. The results (Table 1) show that growth measured as OD750 was reduced after 4 days of iron-deficient conditions (-Fe: 4d) from 3·1 to 1·7 and from 2·9 to 1·1 for the WT and mutant, respectively, showing that the reduction was higher in the mutant than in the WT (45 vs 62%). A higher decrease was also observed in pigment content. The reduction in Chl, phycocyanin and allophycocyanin content in the WT corresponded to 42, 67 and 66%, respectively; the corresponding values in the Slr1295-free mutant were 71, 80 and 82%, respectively.
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Since a substantial amount of Slr1295 was associated with the thylakoid membranes, the question was asked how tightly this protein is associated and whether it interacts preferentially with PS I or PS II. Neither Slr1295 nor Slr0513 was detectable in isolated PS I complexes (not shown). For PS II isolation, the membrane fraction obtained after breaking the cells was washed first with a low concentration of dodecyl maltoside to remove phycobili proteins and only loosely associated proteins. In the wash supernatant a small amount of Slr0513 but no Slr1295 was detectable, implying that Slr1295 was relatively tightly associated with the thylakoid membranes, while Slr0513 was only loosely bound (Fig. 8, SPF 1). Subsequently, PS II complexes were solubilized with a combination of dodecyl maltoside and octyl glucopyranoside. In the PS II-containing fraction an enrichment of Slr1295 was clearly seen in parallel to the enrichment of the PsbA protein (Fig. 8
, SPF 2). The PS II complexes were further purified on an FPLC-MonoQ column. Subsequent immunoblot analysis gave evidence that the Slr1295 protein co-purified with the reaction centre of PS II (Fig. 9
). Only a very slight shift of Slr1295 relative to the D1 polypeptide was seen, most likely due to different subpopulations of PS II.
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A comparison of the presumed signal peptide sequence of Slr1295 and Slr0513 of Synechocystis PCC 6803 (not shown) provided evidence that both proteins have a characteristic sequence motif in the N-terminal region before the hydrophobic region [core sequence of (S/T)-R-R-x-F] that includes two consecutive and invariant arginine residues characteristic for the bacterial Tat (twin-arginine translocation) system (Berks 1996 ; Berks et al., 2000
). Bacterial Tat signal peptides are characterized by a high occurrence of a proline residue at position -6 to the signal peptidase cleavage site and of basic amino acids in the c-region. When analysing the N-terminal amino acid region in the two proteins, it became apparent that Slr0513 meets all these criteria, while in Slr1295 the corresponding proline is missing and there is an acidic amino acid in the c-region before the signal peptidase cleavage site (PSORT prediction; von Heijne, 1986
). Moreover, there is a possible additional cleavage site recognized by the program SignalP (Nielsen et al., 1999
). This might be the reason why only significant amounts of Slr0513, but not of Slr1295, are found in the soluble protein fraction isolated from the periplasm. It has been shown that the
pH-dependent protein import pathway of thylakoid membranes in plant chloroplasts and the bacterial Tat system are very closely related (Klösgen 1997
; Brink et al., 1998
; Wexler et al., 1998
; Halbig et al., 1999
). This might explain the high amounts of gold labels located intracellularly and suggests that Slr0513 is transported into the periplasm and also into the intrathylakoid space due to some misdirection. In contrast, Slr1295 does not seem to be exported in significant amounts into the periplasm (or lumen) at least not under mild iron limitation and when Slr0513 is present.
Concluding remarks
The intracellular localization and the co-purification of Slr1295 with PS II in Synechocystis PCC 6803 suggest a function for Slr1295 in protecting PS II under iron limitation. This is in agreement with the higher susceptibility of PS II for inactivation in the Slr1295-free mutant as compared to the WT. Thus, the function of Slr1295 in Synechocystis PCC 6803 is comparable to the suggested function of IdiA in Synechococcus PCC 6301/PCC 7942 (Michel et al., 1996 ; Exss-Sonne et al., 2000
) and to some extent also to the function of a plant PS II-associated 22 Ku heat-shock protein (Downs et al., 1999a
, b
). The cyanobacterial Slr1295 and IdiA, as well as the plant 22 Ku protein, represent proteins that are not integral proteins of PS II, but function in protecting PS II under specific stress conditions. In the case of Slr1295 (and also IdiA), we suggest that PS II becomes protected or more effectively supplied with iron on the D1/D2 reaction centre heterodimer (QA-Fe-QB). We think that it is highly likely that specific proteins exist to optimize PS II function under iron limitation since iron limitation is a commonly occurring nutrient limitation in natural habitats (see e.g. Geider & La Roche, 1994
; Behrenfeld & Kolber, 1999
). PS II has been proven to be the most labile reaction of the photosynthetic process, and the D1 (and also D2) polypeptide has an extraordinary high turnover under various environmental stresses (Aro et al., 1993
; Ke, 2001
), thus having an elevated iron requirement due to the PS II repair cycle. Based on our results, Slr1295 seems to be a good candidate for such an optimizing and protective function, since due to its assumed original function as a component of an iron transporter it is expressed in elevated amounts under iron limitation and can interact with iron. From an evolutionary point of view, Slr1295 seems to be a protein acquiring a different and/or additional function during evolution (for review see Murzin, 1993
), but retaining significant homology to the protein family of its original function.
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ACKNOWLEDGEMENTS |
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Received 4 March 2002;
revised 24 June 2002;
accepted 25 June 2002.