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1 Department of Botany, Stockholm University, SE-106 91 Stockholm, Sweden
2 A. N. Belozersky Institute of Physico-Chemical Biology, M. V. Lomonosov State University, Moscow 119992, Russia
Correspondence
Olga A. Koksharova
OA-Koksharova{at}rambler.ru
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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, 2005; Lutkenhaus, 2002). The unicellular Synechococcus sp. strain PCC 7942 belongs to the ancient cyanobacterial group of photoautotrophic prokaryotes (Rippka et al., 1979), and has been used as a model organism for studying the genetic control of cyanobacterial cell division (Dolganov & Grossman, 1993; Koksharova & Wolk, 2002; Miyagishima et al., 2005) as well as plastid division in higher plants (Vitha et al., 2003). Recently, two new cell division genes were discovered in Synechococcus sp. strain PCC 7942 by transposon Tn5-692 mutagenesis, followed by mutant DNA cloning and sequencing (GenBank accession numbers AF421196 and AF421197) (Koksharova & Wolk, 2002). The genes/mutants were named ftn2/Ftn2 and ftn6/Ftn6 (ftn for filamentation), as the cells of these two mutants are highly elongated, more than 10 times longer than wild-type cells, due to the blocked regular cell division (Koksharova & Wolk, 2002). The Ftn2 protein contains a DnaJ domain, a single tetratricopeptide repeat (TPR) and a leucine zipper pattern, suggesting that Ftn2 may associate as a component in a protein complex and have a regulatory function. The Ftn6 protein was found to be specific for cyanobacteria and, as no detectable conserved domains have been found within the protein, knowledge about its precise function is still lacking (Koksharova & Wolk, 2002). Occasionally Ftn2 and Ftn6 mutant cells divide and septum formation takes place irregularly (Koksharova & Wolk, 2002) but only a diffuse localization of the key cell division protein FtsZ, a homologue of tubulin (Bi & Lutkenhaus, 1991), has been detected at these rare cell division constriction sites. However, only a small difference in FtsZ protein levels could be detected between the mutants and the wild-type strain using immunoblot analysis of total soluble protein extracts (Miyagishima et al., 2005). Therefore it has been suggested that these mutants are defective in recruitment of FtsZ to the division site or in subsequent assembly of the Z ring. Investigations of the Ftn2 protein orthologues in Arabidopsis thaliana (ARC6) and in the cyanobacterium Synechocystis sp. strain PCC 6803 (ZipN) have shown that a direct interaction between Ftn2 and FtsZ is most likely (Mazouni et al., 2004; Vitha et al., 2003). Ftn2 may function in a chaperone system, probably to stabilize FtsZ filaments. Whether the Ftn6 protein in cyanobacteria interacts directly or indirectly with FtsZ is not known, but a loss of the ftn6 gene results in aberrant cell division, similar to that seen in the Ftn2 mutant (Koksharova & Wolk, 2002; Mazouni et al., 2004; Miyagishima et al., 2005).
Cell division is a highly co-ordinated and fine-tuned process, and the precise regulation and positioning of the cell division apparatus requires a number of both structural and regulatory components, of which many are still unidentified. Mutagenic disruption of proper regulation in the cell division machinery often leads to the formation of elongated and/or mini cells. Such abnormal size changes may impose a strong internal stress, altering cell physiology. Metabolic pathways that are regulated by the cell cycle may also be affected. Hence, compensatory mechanisms to overcome the impaired cell division are expected. Although cell division is impaired in the Ftn2 and Ftn6 mutants, they have comparable growth rates (Koksharova & Wolk, 2002; Miyagishima et al., 2005).
2D PAGE is the pre-eminent tool for monitoring proteomic changes, for example during bacterial stress responses (for a review see Neidhardt & VanBogelen, 2000). However, proteomic studies of stress responses in cyanobacteria, including the potentially stressful condition that blocked cell division may impose, are so far limited. Proteome analysis has been successfully used for identifying periplasmic proteins of salt-stressed Synechocystis sp. strain PCC 6803 cells, and it resulted in the identification of proteins responding strongly to salt stress (Fulda et al., 2000, 2006; Huang et al., 2006). Proteomic analysis of the heat-shock response of wild-type and a mutant of the histidine kinase 34 gene has been performed in the cyanobacterium Synechocystis sp. strain PCC 6803 (Slabas et al., 2006). Moreover, 2D gel electrophoresis with in vivo [35S]methionine labelling has been used to investigate long-term chlorotic cells of Synechococcus (Sauer et al., 2001). In Synechococcus sp. strain PCC 7942, the first proteomic overview has been initiated (Koksharova et al., 2006). This became possible as the recently sequenced genome was released in publicly accessible databases (http://genome.jgi-psf.org/finished_microbes/synel/synel.home.html).
In this study, the pleiotropic responses induced in the strikingly affected cell morphologies of Ftn2 and Ftn6 mutants in Synechococcus sp. strain PCC 7942 were monitored. The proteomes of the two cell division mutants were compared to the wild-type in order to widen our knowledge about the cell division machinery using a new approach. Quantitative differences in the protein maps were detected and proteins with significant quantitative changes were identified. Hypothetical functions of these proteins are discussed to assess the impact of impaired cell division on cell physiology at the protein level.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) were grown in BG11 medium (Rippka et al., 1979) in 50 ml Erlenmeyer flasks at 25 °C in continuous light (18 µE m–2 s–1) on a rotary shaker for 3 days. Growth rates were monitored by measuring the optical density of the cultures at 710 nm.
Protein extraction and separation.
Three independent cultivations were made for each strain. Cultures of 25 ml were collected by centrifugation (3000 g) at 4 °C for 5 min, and frozen at –75 °C until use. The cyanobacterial pellets were dissolved in 150 µl extraction buffer [8 M urea, 0.5 % (v/v) IPG buffer (GE Healthcare) pH 4–7 or pH 3–10, 2 % (w/v) CHAPS, bromophenol blue (traces), protease inhibitors (Complete Mini protease inhibitor cocktail tablets, Roche Diagnostics Scandinavia), and 0.28 % (w/v) DTT]. The samples were then subjected to five cycles of freezing in liquid nitrogen and thawing followed by six cycles of sonication (BANDELIN Sonopuls HD 2070 MS72, DPC Scandinavia) for 2 s each, with cooling of samples for 40 s on ice between cycles to prevent carbamylation (McCarthy et al., 2003). Cell debris was pelleted by centrifugation using a bench-top centrifuge (15 800 g) for 10 min at room temperature. The supernatants with total proteins were retrieved and stored at –20 °C until use. Protein concentration was measured with the RC DC Protein Assay kit (Bio-Rad).
2D gel electrophoresis.
Immobiline gel strips (GE Healthcare), 18 cm, pH range 4–7 and 3–10 NL, were rehydrated in the corresponding extraction buffer (see above) overnight at room temperature. Prior to the isoelectric focusing, extracts containing 600 µg protein for the gel strips pH 4–7 and 300 µg protein for the gel strips pH 3–10 NL were diluted into 100 µl with extraction buffer and loaded onto the gels by using application cups positioned at the acidic end of the gel strip. The first-dimensional electrophoresis was performed on an IPGphore (GE Healthcare) with voltage settings of 500 V for 1 h, 1000 V for 1 h and 8000 V for 10 h. The focusing time was adjusted to a total of 80 000 V h. Before SDS-PAGE the IPG strips were first equilibrated for 15 min in 50 mM Tris/HCl (pH 8.8), 6 M urea, 30 % (v/v) glycerol, 2 % (w/v) SDS, 1 % (w/v) DTT and then for 15 min in the same buffer with 2.5 % (w/v) iodoacetamide instead of DTT. After equilibration, the strip was placed on top of a 12 % polyacrylamide gel and embedded in 0.5 % heated low-melting agarose in SDS electrophoresis running buffer [25 mM Tris, 192 mM glycine, 0.1 % (w/v) SDS]. SDS-PAGE was performed in a PROTEAN II xi cell gel electrophoresis unit (Bio-Rad). Precision Plus Protein Standards (Bio-Rad) were used as protein molecular mass standards.
The separation was performed with constant current (5 mA per gel) overnight at 4 °C until the dye front reached the bottom edge of the gel. After the separation, the gel was incubated in fix solution [10 % (v/v) methanol and 7 % (v/v) acetic acid] for 30 min and subsequently stained for proteins with SYPRO Ruby protein gel stain (Molecular Probes) and by using the PlusOne silver staining kit (GE Healthcare). The silver staining method was modified to be compatible with mass spectrometry (MS) by omitting glutaraldehyde in the sensitization step and formaldehyde in the silver impregnation step (Yan et al., 2000).
Image analysis of 2D gels.
SYPRO Ruby-stained gels were digitized using a Typhoon 8600 laser scanner (GE Healthcare) with the following settings: emission filter Rox 610 BP30, PMTV 550V, Laser Green 532. The 2D gel imaging and analysis software PDQuest (Bio-Rad) version 7.2.0 was used for gel-to-gel matching and identifying differences between wild-type and mutant samples. Each of the six sets of samples (wild-type, Ftn2 and Ftn6; two different pH ranges) was represented by three independent biological replicates of 2D gels, giving a total of 18 analysed gels. The gel images were normalized in the PDQuest software, to even out differences in staining intensities between gels. Each matched protein spot was assigned a unique SSP (sample spot protein) number in the PDQuest software. For gel comparison, a statistical approach was applied when determining differentially expressed proteins using the PDQuest software. Student's t-test was performed with 95 % significance level to determine which proteins were differentially expressed between the wild-type and mutants. A minimum of 1.3-fold change was considered for the upregulated proteins and 0.7-fold for downregulated proteins.
In-gel digestion and peptide extraction.
In-gel digestion was performed on silver-stained protein spots according to Shevchenko et al. (2000), except for the reduction and alkylation steps, which were omitted since cysteines were already carbamidomethylated during 2D electrophoresis. Sequence-grade modified trypsin (Promega) was added (12.5 ng µl–1) in a digestion buffer containing 50 mM ammonium bicarbonate and 5 mM CaCl2 to the dehydrated gel pieces and allowed to absorb for 45 min on ice. Excess trypsin was removed and replaced with 10 µl 50 mM ammonium bicarbonate. In-gel digestion of protein was performed overnight at 37 °C, followed by three extractions of peptides with 50 % (v/v) acetonitrile/5 % (v/v) trifluoroacetic acid (TFA). Combined supernatants were dried in a vacuum centrifuge (DNA plus, Heto Lab Equipment) for 2–4 h. Dry pellets were resuspended in a small volume (3–5 µl) of 0.1 % (v/v) TFA in 50 % (v/v) acetonitrile and stored at –20 °C until analysis.
MALDI-TOF MS analysis and protein identification.
Mass spectra were recorded in positive reflection mode by using MALDI-TOF MS (Voyager-DE STR mass spectrometer, Applied Biosystems), equipped with a delayed ion extraction technology. The matrix used for facilitating the ionization of the tryptic peptides was 
-cyano-4-hydroxycinnamic acid (CHCA). The time of flight was measured using the following parameters: 20 kV accelerating voltage, 65 % grid voltage, 0 % guide wire voltage, 200 ns delay, low mass gate of 700 Da and acquisition mass range 800–4000 Da. The peptide mass profiles obtained were internally calibrated using MoverZ from http://www.genomicsolutionscanada.com, using known autolysis peaks from porcine trypsin. After subtraction of known background peaks derived from the matrix, trypsin and traces of keratin, the lists of peptide masses were compared to databases using the Mascot software (http://www.matrixscience.com). The following search parameters were applied for the Mascot searches: ‘NCBInr’ was used as the protein sequence database; taxonomy was set to ‘All entries’; a mass tolerance of 30 p.p.m was applied with one mis-cleavage allowed; possible fixed modification was considered to be alkylation of cysteine by carbamidomethylation, and oxidation of methionine. The program FindMod (http://www.expasy.org/tools/findmod/) was used for finding modifications and analysing unmatched peptide masses. For most peptide mass fingerprints, a single significant (P<0.05) hit with probability-based Mowse score >76 was obtained. In some cases the Mowse score was <76, which indicates that the protein was not identified with reliability above the level of significance. These protein spots was excluded from the results unless the top hit was from Synechococcus sp. strain PCC 7942, well separated from hits to other proteins, and unmatched peptides could be explained by FindMod program. If these criteria were fulfilled, the protein identification was considered as reliable and is presented.
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RESULTS AND DISCUSSION |
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and 2. The fluorescent chromophore-staining dye (SYPRO Ruby) is very sensitive (Berggren et al., 2000) and allows a digital image of the gel to be obtained that can be analysed by using PDQuest software. Normalization to circumvent slight variations in staining intensity between the nine gels for each pH range was achieved by using the PDQuest software. More than 800 protein spots on each gel were visualized, among which 76 protein spots in total were changed in quantity between the wild-type and the mutants as resolved by using the PDQuest software. These protein spots were subjected to MALDI-TOF MS, resulting in the identification of 53 protein spots representing 44 unique proteins, which were grouped into seven main functional categories (Table 1). Fifteen proteins were upshifted or induced in both cell division mutants, and 13 proteins were downshifted or repressed (Table 1). The changed proteins included a general increased level of proteins involved in cell cycle and morphogenesis as well as protein synthesis, post-translational processing and modification.
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). These results indicate that the Ftn2 protein may have a negative effect and the Ftn6 protein may have a positive effect on the level of some proteins in Synechococcus sp. PCC 7942, either directly or indirectly.
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). The 44 identified proteins affected in the mutants were organized into seven functional groups based on their sequence homology to proteins of known function (Table 1). These groups are described below.
Cell cycle/cell morphogenesis (group 1)
Several proteins involved in cell cycle control were affected in the Ftn2 and Ftn6 mutants, which could potentially explain the filamentous mutant phenotype. The β subunit (DnaN) of the multi-chain enzyme DNA polymerase III, a key enzyme in the replicative synthesis of bacteria, was upshifted twofold in the Ftn2 mutant (SSP 3608: Table 1, Fig. 2c). DnaN is required for the initiation of DNA replication and regulates the chromosomal replication cycle in Escherichia coli (Katayama et al., 1998). In E. coli, the function of DnaA is negatively regulated by DnaN (Katayama et al., 1998), and an interaction between the replication initiator DnaA and DnaN is required to regulate the chromosomal replication cycle (Katayama et al., 1998; Kawakami et al., 2001). Chromosome replication and cell division are highly co-ordinated processes, and the early stages of DNA replication play a key role in the precise positioning of the Z ring at the mid-cell and between replicating daughter chromosomes in Bacillus subtilis (Harry et al., 1999). Rhythmic expression of the dnaN gene in Synechococcus sp. strain PCC 7942 suggests that DNA replication could be under circadian control in this organism (Liu & Tsinoremas, 1996). It is likely that the increased amount of DnaN in cells of the Ftn2 mutant may affect DNA replication and consequently disturb cell division.
A protein identified as a chromosome segregation ATPase (SSP 0701, Fig. 2b) was downshifted twofold in the Ftn6 mutant (Table 1). Chromosome segregation has been well studied in the heterotrophic bacteria E. coli, B. subtilis and Caulobacter crescentus (Kruse et al., 2003; Sherratt, 2003), where proteins such as the Min system (Åkerlund et al., 2002), ParA and ParB (Easter & Gober, 2002), DivIVA (Thomaides et al., 2001), SMC proteins (Graumann, 2001) and SpoIIIE (Bath et al., 2000) have been proposed to be involved. It has been hypothesized that nucleoid occlusion does not regulate Z ring formation in cyanobacteria (Miyagishima et al., 2005). Since MreB (SSP 4401, see below) was also affected in the mutants, the processes of chromosome segregation and cell septation may be co-regulated at some level. However, how the chromosome segregation ATPase (SSP 0701) contributes to the process of cyanobacterial chromosome segregation and how it can be connected functionally with Ftn6 protein are presently unknown.
Bacteria possess a number of cytoskeletal elements (Mayer, 2003), including FtsZ, a bacterial tubulin homologue (Carballido-López & Errington, 2003; Michie & Löwe, 2006; Klint et al., 2007), MreB coiled structures similar to F-actin, the physiological polymer of eukaryotic actin (van den Ent et al., 2001), MinCDE coiled arrays (Shih et al., 2003) and intracellular protofilaments containing bacterial elongation factor Tu (EF-Tu) (Löwe et al., 2004). All of them may form cytoskeletal webs, which are important for the organization of intracellular structures and cell function (Mayer, 2003). Two of these proteins, MreB (SSP 4401; Fig. 1c) and EF-Tu (SSP 3603; group 2; Fig. 1d), were notably upshifted in the cell division mutants Ftn2 and Ftn6 (Table 1). It is likely that filamentous cells of the mutants may require an extended cytoskeletal web.
MreB, an actin homologue, is involved in shape determination in rod-shaped prokaryotic cells (Wachi et al., 1987; Jones et al., 2001; Figge et al., 2004; Gitai et al., 2004) and may or may not be involved in DNA replication in some bacterial species (Kruse et al., 2003, 2006; Gitai et al., 2005; Hu et al., 2007). Very little is known about MreB function in cyanobacterial cells. Recently it has been suggested (Hu et al., 2007) that in Anabaena sp. PCC 7120 this protein is involved in shape determination, but not in DNA segregation. A previous study revealed that cell division in E. coli is under negative control of the mreB gene (Wachi & Matsuhashi, 1989). While overexpression of wild-type MreB has been shown to inhibit cell division but not perturb chromosome segregation, overexpression of mutant forms of MreB causes, in addition to the inhibition of cell division, abnormal MreB filament morphology and induces severe localization defects of the nucleoid in E. coli (Kruse et al., 2003). This fact, together with enhanced expression of the cell division gene ftsI in mreB mutant E. coli cells (Wachi & Matsuhashi, 1989), may indicate a function of the mreB gene as a regulator for determining progression to cell division or elongation in E. coli. At the same time, in Synechococcus sp. strain PCC 7942, the upshift of the MreB protein in the two cell division mutants could reflect a direct or indirect negative regulation of mreB by the ftn2 and ftn6 genes and explain the filamentous phenotype of the mutant cells.
A molecular chaperone, heat-shock protein Hsp70 (SSP 0903; Fig. 1c) was upregulated in the Ftn2 mutant (Table 1). This 634 amino acid protein has an N-terminal MreB region (amino acids 1–371), and the protein shows 94 % sequence identity with the chaperone protein K2 (heat-shock protein 70-2) of Synechococcus sp. strain PCC 7942 (gi|1706478|sp|P50021|DNK2_SYNP7). Overproduction of DnaK2 has resulted in defects in cell septation and formation of cell filaments (Nimura et al., 2001), suggesting an interaction with key cell septation protein(s).
An outer-membrane protein containing one transmembrane helix in the N-terminus was upregulated in both cell division mutants (SSP 1912; Table 1; Fig. 2c). The location of this protein in the outer membrane may suggest involvement in cell envelope biogenesis and/or secretion. Presumably, elongated mutant cells require an increased synthesis of cell membrane as well as an intensive intracellular traffic.
Protein synthesis and processing (group 2)
Three proteins (SSP 3603, SSP 6707, SSP 3305) involved in protein synthesis were upshifted in both mutants. The GTPase translation elongation factor (SSP 3603) has been mentioned above in the context of cytoskeletal webs, but this protein also has a role in protein translation (Rodnina et al., 2000). Polyribonucleotide nucleotidyltransferase (SSP 6707) was identified exclusively in the mutant cells (Fig. 2c). In addition, protein abundance was twofold higher in the Ftn2 mutant compared to the Ftn6 mutant (Table 1). Protein SSP 6707 possesses four 3' exoRNase family domains and three RNA-binding domains. The polynucleotide phosphorylase is a 3'-5' key exonuclease for mRNA decay and part of a multicomponent mRNA–protein complex (the ‘degradosome’) (Coburn & Mackie, 1999) that orchestrates mRNA decay in bacteria. Under stress conditions, polyribonucleotide nucleotidyltransferase has been shown to be upregulated in bacteria (Len et al., 2004), which may also be the situation in the Ftn2 and Ftn6 mutant cells.
The ribosomal protein S1 (SSP 3305; Fig. 1c), involved in ribosome binding to mRNA during translation, was upregulated in the mutants (Table 1). Moreover, the RNA-binding protein (RRM – RNA recognition motif – domain) (SSP 9001; Fig. 1b) was upshifted threefold in Ftn2 cells and downshifted almost sixfold in Ftn6 mutant (Table 1). There is no information available so far about the regulation of RNA-binding proteins during impaired cell division; however, it is known that RNA-binding proteins of cyanobacteria are under stress-responsive regulation, reacting, for example, to the temperature and nitrogen status (Maruyama et al., 1999; Mori et al., 2003).
In the cell division mutants, three proteins involved in post-translational protein processing and modifications (SSP 6913, SSP 0202 and SSP 8503) were upshifted (Table 1). SSP 6913 and SSP 0202 are chaperones, which may additionally reflect that the mutants are under a stressed condition; notably chaperonin GroEL (SSP 6913; Fig. 2c) was exclusively found in the mutant cells (Table 1). The GroEL/GroES system is a major chaperone system in all bacteria and its involvement in cyanobacterial stress responses has been extensively studied (Hihara et al., 2001; Kovacs et al., 2001; Mary et al., 2004). The twofold upshift of periplasmic protease (SSP 8503) was only obvious for the Ftn2 mutant (Table 1).
One protein (SSP 4203; Fig. 2b), identified as a TPR-containing protein, was distinctly upshifted in the Ftn6 mutant (Table 1). The tetratricopeptide repeat (TPR), a degenerate 34 amino acid sequence present in tandem arrays of 1–16 motifs and mediating protein–protein interactions, was found for the first time by Sikorski et al. (1990) in the cell division control protein Cdc23. TPR motifs are important for the function of chaperones, as well as cell-cycle, transcription, and protein-transport complexes (Blatch & Lässle, 1999). Interestingly, the TPR domain is also present in the cell division protein Ftn2 in Synechococcus sp. strain PCC 7942 (Koksharova & Wolk, 2002).
Two proteins, possibly involved in protein–protein interactions and protein processing/degradation, were, in contrast to the other proteins in this group, downshifted in both mutants. One, the leucyl aminopeptidase (SSP 2606; Fig. 2b; Table 1), was only detected in the wild-type. A second protein, containing the FHA (forkhead homology-associated) domain (SSP 2203; Fig. 2c), was absent in the Ftn6 mutant and downshifted in Ftn2 mutant cells (Table 1). FHA domains are implicated in many bacterial processes, including the regulation of cell shape, type III secretion, sporulation, pathogenic and symbiotic host–bacterium interactions, carbohydrate storage and transport, and signal transduction (Pallen et al., 2002).
Photosynthesis (group 3)
Four photosynthetis-related proteins were downregulated in one or both mutants. Protein PsaD, identified as two spots (SSP 7002 and SSP 9002) in two different pH range 2D gels [pH 3–10 (Fig. 1b) and pH 4–7 (Fig. 2b)], a small extrinsic polypeptide located on the stromal side of the photosystem I reaction centre complex, was strongly downshifted or undetectable in both cell division mutants (Table 1). Three hypothetical proteins (Selo03002341, Selo03000334 and Selo03000332) homologous to light-harvesting pigment-proteins were also downshifted in the mutant cells (Table 1). In contrast to these photosynthetic pigmented proteins, the Mn-stabilizing protein precursor (SSP 0209; Table 1) associated with the oxygen-evolving photosystem II was strongly upshifted in both mutants.
Oxidative stress response and redox control (group 4)
The upshift in the oxygen-evolving Mn-stabilizing protein precursor SSP 0209 may result in the production of reactive oxygen species and free radicals. Consequently, a protein involved in oxidative stress response, peroxiredoxin (SSP 0204; Table 1, Fig. 1b) (Wood et al., 2003) was identified as upregulated in both mutants. Additionally, an uncharacterized protein involved in ubiquitin biosynthesis (SSP 7203, SSP 8203) was upshifted in the Ftn2 mutant (Fig. 1c; Table 1). This protein possesses a region named coenzyme Q (ubiquinone) biosynthesis protein Coq4. Ubiquinones are essential redox components of the photosynthetic electron-transport chain in photoautotrophic organisms. They play vital roles in the management of oxidative stress and gene regulation (Soballe & Poole, 1999).
Thiol-disulfide isomerase (thioredoxin) was identified as spot SSP 0006 (Fig. 2b). This protein is downshifted in the Ftn6 mutant (Table 1). Recently, by using a proteomic approach, Kumar et al. (2004) identified 80 bacterial proteins associated with thioredoxin, implicating the involvement of thioredoxin in at least 26 distinct cellular processes including transcriptional regulation, cell division, energy transduction, and several biosynthetic pathways. Our data show that the protein level of thioredoxin may also have a direct or indirect connection with cell division in cyanobacteria.
Carbon dioxide fixation and concentrating mechanism (group 5)
A transcriptional regulator belonging to the LysR family (Henikoff et al., 1988) was upshifted slightly in Ftn2 and downshifted more than 2.5-fold in Ftn6 cells (SSP 8305; Table 1, Fig. 2b). The closest homologous proteins were found in Nostoc sp. strain PCC 7120 (gi|17133087|dbj|BAB75652.1| ORF_ID:all3953
transcriptional regulator, RbcR homologue, 83 % sequence identity). Hence, this protein is possibly involved in regulation of RuBisCO expression.
The protein identified as chain A of RuBisCO (SSP 7204) was not detected in protein extracts from the mutants (Table 1). The small subunit of RuBisCO was found on two different sets of gels: as spot SSP 5001 (Fig. 1b) and SSP 7004 (Fig. 2b). Protein spot SSP 5001 was downshifted in both mutants (Table 1; Fig. 1b), and protein spot SSP 7004 was absent in mutant Ftn2 (Table 1; Fig. 2b). The activity, synthesis and degradation of this enzyme are regulated by several mechanisms, one of them being by the redox potential (Marcus et al., 2003). Stress conditions, provoked by nitrate deprivation, also decrease RuBisCO content (Marcus et al., 2003). The interpretations for the low abundance of RuBisCO proteins (Table 1) in the cell division mutants Ftn2 and Ftn6 are speculative. Potentially, changes in the general cell redox status of the division mutants as well as possible deficiency of nitrogen may induce degradation of this protein.
A carbonic anhydrase/acetyltransferase (Badger & Price, 2003) was identified as a set of two spots (SSP 8801 and 8802; Fig. 1d). This protein was downshifted in the Ftn2 mutant and slightly upshifted in the Ftn6 mutant (Table 1).
Energy production and different biosynthetic processes (group 6)
Four proteins involved in purine biosynthesis were affected in the mutants (SSP 1909, SSP 2704, SSP 4301, and SSP 4504) (Table 1). Phosphoribosylformylglycinamidine (FGAM) synthase (PurL) (SSP 1909; Fig. 2b) is the fourth enzyme in the purine nucleotide biosynthesis pathway. The purL gene in Synechococcus sp. strain PCC 7942 is expressed under circadian cycle control in the same gene cluster as dnaN (see above) (Liu et al., 1996). The PurL protein was downregulated in the Ftn2 and Ftn6 cell division mutants. In contrast, two other purine biosynthesis enzymes were upregulated in both mutants (Table 1). The protein AICAR (aminoimidazole-4-carboxamide ribonucleotide) transformylase/IMP cyclohydrolase PurH (SSP 2704; Fig. 2c) catalyses the last two steps in de novo purine biosynthesis (Kappock et al., 2000), whereas IMP dehydrogenase/GMP reductase (guaB) (SSP 4504; Fig. 1c) catalyses the rate-limiting reaction of de novo GTP biosynthesis. The latter enzyme is another example of different regulation of Ftn2 and Ftn6 in Synechococcus: this enzyme was upregulated in Ftn2 mutant cells and downregulated in Ftn6 mutant cells (Table 1). Guanine nucleotides are important substrates for macromolecular synthesis and cell signalling, and have an evolutionarily conserved role during differentiation, proliferation and apoptosis (Yalowitz & Jayaram, 2000). An important observation is that these four nucleotide synthesis proteins and a component of DNA polymerase III (DnaN) involved in replicative synthesis (see above) were affected in the Ftn2 and Ftn6 mutants. This could be an indication that the cell cycle in the Ftn2 and Ftn6 mutants is affected in a stage prior to cell division due to the mutations, and thus expression of cell-cycle-dependent genes is modified. In addition, a chromosome segregation ATPase and MreB were affected (see above), also pointing in this direction.
A key enzyme of nitrogen metabolism, glutamine synthetase (GS), was strongly downshifted in both mutants (SSP 2703; Table 1; Fig. 2b). Inhibition of GS in Synechococcus sp. strain PCC 7942 leads to a rapid decrease of allophycocyanin mRNA and increase of nblA (the gene essential for degradation of the phycobilisome) levels, which is characteristic of nitrogen deprivation (Sauer et al., 1999). The low level of GS, phycobiliproteins (group 3) and RuBisCO proteins (group 5) are indications for nitrogen starvation and/or deficiency in amino acids in the mutants.
Three additional proteins involved in amino acid metabolism (SSP 1301, SSP 0207, SSP 3605; Table 1) were significantly changed in the cell division mutants, although different behaviour between the proteins was seen. Diaminopimelate epimerase (SSP 1301; Fig. 1d) is involved in peptidoglycan synthesis by catalysing the isomerization of LL- to DL-meso-diaminopimelate in the biosynthetic pathway leading from aspartate to lysine (Mirelman, 1979). N-Acetylglutamate synthase (N-acetylornithine aminotransferase) (SSP 0207; Fig. 2c) is a member of the ArgJ family involved in arginine biosynthesis (Caldovic & Tuchman, 2003). These two enzymes are upregulated in both cell division mutants (Table 1). A second enzyme involved in arginine biosynthesis, argininosuccinate synthase (SSP 3605; Fig. 1b), is downshifted in Ftn6 cells (Table 1).
Two proteins (SSP 4704 and SSP 7102; Table 1) involved in energy production and conversion were downshifted in both cell division mutants: pyruvate/2-oxoglutarate dehydrogenase complex, dihydrolipoamide dehydrogenase (E3) component (SSP 4704; Fig. 2b) and the 
subunit of F0F1-type ATP synthase (SSP 7102; Fig. 1b) (Table 1).
The first enzyme of the biosynthetic pathway to fatty acids, the β subunit of acetyl-CoA carboxylase, was also identified as downshifted in both cell division mutants (SSP 7306; Table 1, Fig. 1b). As fatty acids are primarily precursors of phospholipids, acetyl-CoA carboxylase activity can be correlated with cell growth and division, as well as with cell development (Gornicki et al., 1993).
The glycolytic enzyme fructose/tagatose bisphosphate aldolase (SSP 4503 and 4507; Table 1, Fig. 1c) was upshifted in both cell division mutants. The protein was identified in two different protein spots (SSP 4503 and 4507) differing in both pI and in molecular mass on the 2D gel (Fig. 1c), indicative of protein modification. Such upshift in a glycolytic enzyme(s) could possibly be a mechanism to compensate for deficit of energy in the mutant cells, caused by the downregulation of many proteins involved in general cell metabolism.
Unknown and hypothetical proteins (group 7)
A hypothetical protein (SSP 8408; Table 1; Fig. 2b) possessing three S-layer homology (SLH) domains was upshifted 4.5-fold in the Ftn2 mutant. Three proteins [SSP 0107 (Fig. 2b), SSP 1003 and SSP 6608 (Fig. 2c)] were upshifted in both cell division mutants (Table 1), but do not show any significant homology to proteins with known function. The unknown protein SSP 6608 was found only in the mutants (Table 1). Future targeted inactivation of the genes encoding unknown and hypothetical proteins identified in this study may elucidate roles of these proteins in relation to cyanobacterial cell division.
Concluding remarks
In conclusion, the results show that mutations in the cell division genes ftn2 and ftn6 affect the cellular quantity of a complex series of known and unknown proteins. The pleiotropic responses represent a range of different physiological processes, for example cell division, DNA and protein synthesis, and chaperone activities. These responses may be a reflection of (1) adaptation to the abnormal cell morphology, (2) direct or indirect gene regulatory properties of Ftn2 and Ftn6, (3) that the cell cycle may be affected in a stage prior to septum formation and cytokinesis, and/or (4) compensatory mechanisms for the cell to overcome the blocked cell division. Identification of such differentially expressed proteins provides new targets for future studies that will allow assessment of their physiological roles and significance in cyanobacterial cell division. Interestingly, mutation in ftn2 and ftn6 can have opposite effects on some proteins in cyanobacterial cells: the Ftn2 protein appears to have a preferentially negative effect, and the Ftn6 protein a positive effect, directly or indirectly (Table 2). We therefore propose that Ftn2 and Ftn6 may have regulatory functions in Synechococcus cells and/or be a part of a regulatory cascade(s).
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ACKNOWLEDGEMENTS |
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Edited by: D. J. Scanlan
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Badger, M. R. & Price, G. D. (2003). CO2 concentrating mechanisms in cyanobacteria: molecular components, their diversity and evolution. J Exp Bot 54, 609–622.
Bath, J., Wu, L. J., Errington, J. & Wang, J. C. (2000). Role of Bacillus subtilis SpoIIIE in DNA transport across the mother cell-prespore division septum. Science 290, 995–997.
Berggren, K., Chernokalskaya, E., Steinberg, T. H., Kemper, C., Lopez, M. F., Diwu, Z., Haugland, R. P. & Patton, W. F. (2000). Background-free, high sensitivity staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels using a luminescent ruthenium complex. Electrophoresis 21, 2509–2521.[CrossRef][Medline]
Bi, E. F. & Lutkenhaus, J. (1991). FtsZ ring structure associated with division in Escherichia coli. Nature 354, 161–164.[CrossRef][Medline]
Blatch, G. L. & Lässle, M. (1999). The tetratricopeptide repeat: a structural motif mediating protein-protein interactions. Bioessays 21, 932–939.[CrossRef][Medline]
Caldovic, L. & Tuchman, M. (2003). N-Acetylglutamate and its changing role through evolution. Biochem J 372, 279–290.[CrossRef][Medline]
Carballido-López, R. & Errington, J. (2003). A dynamic bacterial cytoskeleton. Trends Cell Biol 13, 577–583.[CrossRef][Medline]
Coburn, G. A. & Mackie, G. A. (1999). Degradation of mRNA in Escherichia coli: an old problem with some new twists. Prog Nucleic Acid Res Mol Biol 62, 55–108.[Medline]
Dolganov, N. & Grossman, A. R. (1993). Insertional inactivation of genes to isolate mutants of Synechococcus sp. strain PCC 7942: isolation of filamentous strains. J Bacteriol 175, 7644–7651.
Easter, J., Jr & Gober, J. W. (2002). ParB-stimulated nucleotide exchange regulates a switch in functionally distinct ParA activities. Mol Cell 10, 427–434.[CrossRef][Medline]
Figge, R. M., Divakaruni, A. V. & Gober, J. W. (2004). MreB, the cell shape-determining bacterial actin homologue, co-ordinates cell wall morphogenesis in Caulobacter crescentus. Mol Microbiol 51, 1321–1332.[CrossRef][Medline]
Fulda, S., Huang, F., Nilsson, F., Hagemann, M. & Norling, B. (2000). Proteomics of Synechocystis sp. strain PCC 6803. Identification of periplasmic proteins in cells grown at low and high salt concentrations. Eur J Biochem 267, 5900–5907.[Medline]
Fulda, S., Mikkat, S., Huang, F., Huckauf, J., Marin, K., Norling, B. & Hagemann, M. (2006). Proteome analysis of salt stress response in the cyanobacterium Synechocystis sp. strain PCC 6803. Proteomics 6, 2733–2745.[CrossRef][Medline]
Gitai, Z., Dye, N. A. & Shapiro, L. (2004). An actin-like gene can determine cell polarity in bacteria. Proc Natl Acad Sci U S A 101, 8643–8648.
Gitai, Z., Dye, N. A., Reisenauer, A., Wachi, M. & Shapiro, L. (2005). MreB actin-mediated segregation of a specific region of a bacterial chromosome. Cell 120, 329–341.[CrossRef][Medline]
Gornicki, P., Scappino, L. A. & Haselkorn, R. (1993). Genes for two subunits of acetyl coenzyme A carboxylase of Anabaena sp. strain PCC 7120: biotin carboxylase and biotin carboxyl carrier protein. J Bacteriol 175, 5268–5272.
Graumann, P. L. (2001). SMC proteins in bacteria: condensation motors for chromosome segregation?. Biochimie 83, 53–59.[Medline]
Harry, E. J., Rodwell, J. & Wake, R. G. (1999). Co-ordinating DNA replication with cell division in bacteria: a link between the early stages of a round of replication and mid-cell Z ring assembly. Mol Microbiol 33, 33–40.[CrossRef][Medline]
Henikoff, S., Haughn, G. W., Calvo, J. M. & Wallace, J. C. (1988). A large family of bacterial activator proteins. Proc Natl Acad Sci U S A 85, 6602–6606.
Hihara, Y., Kamei, A., Kanehisa, M., Kaplan, A. & Ikeuchi, M. (2001). DNA microarray analysis of cyanobacterial gene expression during acclimation to high light. Plant Cell 13, 793–806.
Hu, B., Yang, G., Zhao, W., Zhang, Y. & Zhao, J. (2007). MreB is important for cell shape but not for chromosome segregation of the filamentous cyanobacterium Anabaena sp. PCC 7120. Mol Microbiol 63, 1640–1652.[CrossRef][Medline]
Huang, F., Fulda, S., Hagemann, M. & Norling, B. (2006). Proteomic screening of salt-stress-induced changes in plasma membranes of Synechocystis sp. strain PCC 6803. Proteomics 6, 910–920.[CrossRef][Medline]
Jones, L. J., Carballido-Lopez, R. & Errington, J. (2001). Control of cell shape in bacteria: helical, actin-like filaments in Bacillus subtilis. Cell 104, 913–922.[CrossRef][Medline]
Kappock, T. J., Ealick, S. E. & Stubbe, J. (2000). Modular evolution of the purine biosynthetic pathway. Curr Opin Chem Biol 4, 567–572.[CrossRef][Medline]
Katayama, T., Kubota, T., Kurokawa, K., Crooke, E. & Sekimizu, K. (1998). The initiator function of DnaA protein is negatively regulated by the sliding clamp of the E. coli chromosomal replicase. Cell 94, 61–71.[CrossRef][Medline]
Kawakami, H., Iwura, T., Takata, M., Sekimizu, K., Hiraga, S. & Katayama, T. (2001). Arrest of cell division and nucleoid partition by genetic alterations in the sliding clamp of the replicase and in DnaA. Mol Genet Genomics 266, 167–179.[CrossRef][Medline]
Klint, J., Rasmussen, U. & Bergman, B. (2007). FtsZ may have dual roles in the filamentous cyanobacterium Nostoc/Anabaena sp. strain PCC 7120. J Plant Physiol 164, 11–18.[CrossRef][Medline]
Koksharova, O. A. & Wolk, C. P. (2002). A novel gene that bears a DnaJ motif influences cyanobacterial cell division. J Bacteriol 184, 5524–5528.
Koksharova, O. A., Klint, J. & Rasmussen, U. (2006). The first protein map of Synechococcus sp. strain PCC 7942. Mikrobiologiia 75, 765–774.[Medline]
Kovacs, E., van der Vies, S. M., Glatz, A., Torok, Z., Varvasovszki, V., Horvath, I. & Vigh, L. (2001). The chaperonins of Synechocystis PCC 6803 differ in heat inducibility and chaperone activity. Biochem Biophys Res Commun 289, 908–915.[CrossRef][Medline]
Kruse, T., Möller-Jensen, J., Löbner-Olesen, A. & Gerdes, K. (2003). Dysfunctional MreB inhibits chromosome segregation in Escherichia coli. EMBO J 22, 5283–5292.[CrossRef][Medline]
Kruse, T., Blagoev, B., Løbner-Olesen, A., Wachi, M., Sasaki, K., Iwai, N., Mann, M. & Gerdes, K. (2006). Actin homolog MreB and RNA polymerase interact and are both required for chromosome segregation in Escherichia coli. Genes Dev 20, 113–124.
Kumar, J. K., Tabor, S. & Richardson, C. C. (2004). Proteomic analysis of thioredoxin-targeted proteins in Escherichia coli. Proc Natl Acad Sci U S A 101, 3759–3764.
Len, A. C., Harty, D. W. & Jacques, N. A. (2004). Stress-responsive proteins are upregulated in Streptococcus mutants during acid tolerance. Microbiology 150, 1339–1351.
Liu, Y. & Tsinoremas, N. F. (1996). An unusual gene arrangement for the putative chromosome replication origin and circadian expression of dnaN in Synechococcus sp. strain PCC 7942. Gene 172, 105–109.[CrossRef][Medline]
Liu, Y., Tsinoremas, N. F., Golden, S. S., Kondo, T. & Johnson, C. H. (1996). Circadian expression of genes involved in the purine biosynthetic pathway of the cyanobacterium Synechococcus sp. strain PCC 7942. Mol Microbiol 20, 1071–1081.[CrossRef][Medline]
Löwe, J., van den Ent, F. & Amos, L. A. (2004). Molecules of the bacterial cytoskeleton. Annu Rev Biophys Biomol Struct 33, 177–198.[CrossRef][Medline]
Lutkenhaus, J. (2002). Dynamic proteins in bacteria. Curr Opin Microbiol 5, 548–552.[CrossRef][Medline]
Marcus, Y., Altman-Gueta, H., Finkler, A. & Gurevitz, M. (2003). Dual role of cysteine 172 in redox regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase activity and degradation. J Bacteriol 185, 1509–1517.
Margolin, W. (2001). Spatial regulation of cytokinesis in bacteria. Curr Opin Microbiol 4, 647–652.[CrossRef][Medline]
Margolin, W. (2005). FtsZ and the division of prokaryotic cells and organelles. Nat Rev Mol Cell Biol 6, 862–871.[CrossRef][Medline]
Maruyama, K., Sato, N. & Ohta, N. (1999). Conservation of structure and cold-regulation of RNA-binding proteins in cyanobacteria: probable convergent evolution with eukaryotic glycine-rich RNA-binding proteins. Nucleic Acids Res 27, 2029–2036.
Mary, I., Tu, C.-J., Grossman, A. & Vaulot, D. (2004). Effects of high light on transcripts of stress-associated genes for the cyanobacteria Synechocystis sp. PCC 6803 and Prochlorococcus MED4 and MIT9313. Microbiology 150, 1271–1281.
Mayer, F. (2003). Cytoskeletons in prokaryotes. Cell Biol Int 27, 429–438.[CrossRef][Medline]
Mazouni, K., Domain, F., Cassier-Chauvat, C. & Chauvat, F. (2004). Molecular analysis of the key cytokinetic components of cyanobacteria: FtsZ, ZipN and MinCDE. Mol Microbiol 52, 1145–1158.[CrossRef][Medline]
McCarthy, J., Hopwood, F., Oxley, D., Laver, M., Castagna, A., Righetti, P. G., Williams, K. & Herbert, B. (2003). Carbamylation of proteins in 2-D electrophoresis – myth or reality?. J Proteome Res 2, 239–242.[CrossRef][Medline]
Michie, K. A. & Löwe, J. (2006). Dynamic filaments of the bacterial cytoskeleton. Annu Rev Biochem 75, 467–492.[CrossRef][Medline]
Mirelman, D. (1979). Biosynthesis and assembly of cell wall peptidoglycan. In Bacterial Outer Membranes, pp. 115–166. Edited by M. Inouye. New York: Wiley.
Miyagishima, S. Y., Wolk, C. P. & Osteryoung, K. W. (2005). Identification of cyanobacterial cell division genes by comparative and mutational analyses. Mol Microbiol 56, 126–143.[CrossRef][Medline]
Mori, S., Castoreno, A., Mulligan, M. E. & Lammers, P. J. (2003). Nitrogen status modulates the expression of RNA-binding proteins in cyanobacteria. FEMS Microbiol Lett 227, 203–210.[CrossRef][Medline]
Neidhardt, F. C. & VanBogelen, R. A. (2000). Proteomic analysis of bacterial stress responses. In Bacterial Stress Responses, pp. 445–452. Edited by G. Storz & R. Hengge-Aronis. Washington, DC: ASM Press.
Nimura, K., Takahashi, H. & Yoshikawa, H. (2001). Characterization of the dnaK multigene family in the cyanobacterium Synechococcus sp. strain PCC7942. J Bacteriol 183, 1320–1328.
Pallen, M., Chaudhuri, R. & Khan, A. (2002). Bacterial FHA domains: neglected players in the phospho-threonine signalling game?. Trends Microbiol 10, 556–563.[CrossRef][Medline]
Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M. & Stanier, R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111, 1–61.
Rodnina, M. V., Stark, H., Savelsbergh, A., Wieden, H. J., Mohr, D., Matassova, N. B., Peske, F., Daviter, T., Gualerzi, C. O. & Wintermeyer, W. (2000). GTPases mechanisms and functions of translation factors on the ribosome. Biol Chem 381, 377–387.[CrossRef][Medline]
Sauer, J., Görl, M. & Forchhammer, K. (1999). Nitrogen starvation in Synechococcus PCC 7942: involvement of glutamine synthetase and NtcA in phycobiliprotein degradation and survival. Arch Microbiol 172, 247–255.[CrossRef][Medline]
Sauer, J., Schreiber, U., Schmid, R., Volker, U. & Forchhammer, K. (2001). Nitrogen starvation-induced chlorosis in Synechococcus PCC 7942. Low-level photosynthesis as a mechanism of long-term survival. Plant Physiol 126, 233–243.
Sherratt, D. J. (2003). Bacterial chromosome dynamics. Science 301, 780–785.
Shevchenko, A., Chernushevich, I., Wilm, M. & Mann, M. (2000). De novo peptide sequencing by nanoelectrospray tandem mass spectrometry using triple quadrupole and quadrupole/time-of-flight instruments. Methods Mol Biol 146, 1–16.[Medline]
Shih, Y. L., Le, T. & Rothfield, L. (2003). Division site selection in Escherichia coli involves dynamic redistribution of Min proteins within coiled structures that extend between the two cell poles. Proc Natl Acad Sci U S A 100, 7865–7870.
Sikorski, R. S., Boguski, M. S., Goebl, M. & Hieter, P. (1990). A repeating amino acid motif in CDC23 defines a family of proteins and a new relationship among genes required for mitosis and RNA synthesis. Cell 60, 307–317.[CrossRef][Medline]
Slabas, A. R., Suzuki, I., Murata, M., Simon, W. J. & Hall, J. J. (2006). Proteomic analysis of the heat shock response in Synechocystis PCC6803 and a thermally tolerant knockout strain lacking the histidine kinase34 gene. Proteomics 6, 845–864.[CrossRef][Medline]
Soballe, B. & Poole, R. K. (1999). Microbial ubiquinones: multiple roles in respiration, gene regulation and oxidative stress management. Microbiology 145, 1817–1830.
Thomaides, H. B., Freeman, M., El Karoui, M. & Errington, J. (2001). Division site selection protein DivIVA of Bacillus subtilis has a second distinct function in chromosome segregation during sporulation. Genes Dev 15, 1662–1673.
van den Ent, F., Amos, L. A. & Lowe, J. (2001). Prokaryotic origin of the actin cytoskeleton. Nature 413, 39–44.[CrossRef][Medline]
Vitha, S., Froehlich, J. E., Koksharova, O. A., Pyke, K. A., van Erp, H. & Osteryoug, K. W. (2003). ARC6 is a J-domain plastid division protein and an evolutionary descendant of the cyanobacterial cell division protein Ftn2. Plant Cell 15, 1918–1933.
Wachi, M. & Matsuhashi, M. (1989). Negative control of cell division by mreB, a gene that functions in determining the rod shape of Escherichia coli cells. J Bacteriol 171, 3123–3127.
Wachi, M., Doi, M., Tamaki, S., Park, W., Nakajima-Iijima, S. & Matsuhashi, M. (1987). Mutant isolation and molecular cloning of mre genes, which determine cell shape, sensitivity to mecillinam, and amount of penicillin-binding proteins in Escherichia coli. J Bacteriol 169, 4935–4940.
Wood, Z. A., Schroder, E., Robin Harris, J. & Poole, L. B. (2003). Structure, mechanism and regulation of peroxiredoxins. Trends Biochem Sci 28, 32–40.[CrossRef][Medline]
Yalowitz, J. A. & Jayaram, H. N. (2000). Molecular targets of guanine nucleotides in differentiation, proliferation and apoptosis. Anticancer Res 20, 2329–2338.[Medline]
Yan, J. X., Wait, R., Berkelman, T., Harry, R. A., Westbrook, J. A., Wheeler, C. H. & Dunn, M. J. (2000). A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 21, 3666–3672.[CrossRef][Medline]
Received 19 February 2007;
revised 18 April 2007;
accepted 23 April 2007.
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