Abstract: Summary We pursued the characterization of the divisome of the spherical-celled cyanobacterium Synechocystis PCC6803, through deletion, site-directed mutagenesis, GFP tagging, two-hybrid and coimmunoprecipitation assays. We presently report that the DivIVA-like protein Cdv3 is essential to both cell growth and division, whereas the AmiC, AmpH, FtsE, FtsN, SpoIID, YlmD, YlmE and YlmG proteins are dispensable. With the exception of the self-interacting protein YlmD, none of these dispensable factors appeared to interact with ZipN, the crucial cytokinetic factor we previously characterized. By contrast, we found that ZipN interacts with itself and the self-interacting protein Cdv3, as well as with all other crucial cytokinetic factors we previously characterized, namely: FtsZ, FtsI, FtsQ, SepF and ZipS. We also identified ZipN aminoacids selectively involved in ZipN interaction with one of its following partners, Cdv3, FtsQ or SepF. Finally, we found no direct interaction between Cdv3, SepF and ZipS. Collectively, these results indicate that ZipN is a central player of divisome assembly in cyanobacteria, similarly to the FtsA protein of E. coli that is absent in cyanobacteria and chloroplast.
Abstract: Glutaredoxins (GRX) are redox proteins which use glutathione as a cofactor and are divided into two classes, monothiol and dithiol. In each class, several GRX have been shown to form [Fe2S2] cluster coordinating homodimers. The dithiol GRX homodimer is proposed to serve as a sequestration form and its iron-sulfur cluster as an oxidative stress sensor. In contrast, the monothiol GRX homodimer has been suggested to act as a scaffold for [Fe2S2] cluster delivery. We present here the structure of a monothiol GRX homodimer (Escherichia coli GRX4) coordinating a [Fe2S2] cluster that reveals the structural basis of intact iron-sulfur cluster delivery.
Abstract: BACKGROUND: DNA replication and cell cycle as well as their relationship have been extensively studied in the two model organisms E. coli and B. subtilis. By contrast, little is known about these processes in cyanobacteria, even though they are crucial to the biosphere, in utilizing solar energy to renew the oxygenic atmosphere and in producing the biomass for the food chain. Recent studies have allowed the identification of several cell division factors that are specifics to cyanobacteria. Among them, Ftn6 has been proposed to function in the recruitment of the crucial FtsZ proteins to the septum or the subsequent Z-ring assembly and possibly in chromosome segregation. RESULTS: In this study, we identified an as yet undescribed domain located in the conserved N-terminal region of Ftn6. This 77 amino-acids-long domain, designated here as FND (Ftn6 N-Terminal Domain), exhibits striking sequence and structural similarities with the DNA-interacting module, listed in the PFAM database as the DnaD-like domain (pfam04271). We took advantage of the sequence similarities between FND and the DnaD-like domains to construct a homology 3D-model of the Ftn6 FND domain from the model cyanobacterium Synechocystis PCC6803. Mapping of the conserved residues exposed onto the FND surface allowed us to identify a highly conserved area that could be engaged in Ftn6-specific interactions. CONCLUSION: Overall, similarities between FND and DnaD-like domains as well as previously reported observations on Ftn6 suggest that FND may function as a DNA-interacting module thereby providing an as yet missing link between DNA replication and cell division in cyanobacteria. Consistently, we also showed that Ftn6 is involved in tolerance to DNA damages generated by UV rays.
Abstract: Assembly of the tubulin-like cytoskeletal protein FtsZ into a ring structure at midcell establishes the location of the nascent division sites in prokaryotes. However, it is not yet known how the assembly and contraction of the Z ring are regulated, especially in cyanobacteria, the environmentally crucial organisms for which only one FtsZ partner protein, ZipN, has been described so far. Here, we characterized SepF and Ftn6, two novel septal proteins, in the spherical-celled strain Synechocystis PCC 6803. Both proteins were found to be indispensable to Synechocystis sp. strain PCC 6803. The depletion of both SepF and Ftn6 resulted in delayed cytokinesis and the generation of giant cells but did not prevent FtsZ polymerization, as shown by the visualization of green fluorescent protein (GFP)-tagged FtsZ polymers. These GFP-tagged Z-ring-like structures often appeared to be abnormal, because these reporter cells respond to the depletion of either SepF or Ftn6 with an increased abundance of total, natural, and GFP-tagged FtsZ proteins. In agreement with their septal localization, we found that both SepF and Ftn6 interact physically with FtsZ. Finally, we showed that SepF, but not Ftn6, stimulates the formation and/or stability of FtsZ polymers in vitro.
Abstract: MOTIVATION: Protein-protein interaction networks provide insights into the relationships between the proteins of an organism thereby contributing to a better understanding of cellular processes. Nevertheless, large-scale interaction networks are available for only a few model organisms. Thus, interologs are useful for a systematic transfer of protein interaction networks between organisms. However, no standard tool is available so far for that purpose. RESULTS: In this study, we present an automated prediction tool developed for all sequenced genomes available in Integr8. We also have developed a second method to predict protein-protein interactions in the widely used cyanobacterium Synechocystis. Using these methods, we have constructed a new network of 8783 inferred interactions for Synechocystis. AVAILABILITY: InteroPORC is open-source, downloadable and usable through a web interface at http://biodev.extra.cea.fr/interoporc/.
Abstract: BACKGROUND: Cadmium is a persistent pollutant that threatens most biological organisms, including cyanobacteria that support a large part of the biosphere. Using a multifaceted approach, we have investigated the global responses to Cd and other relevant stresses (H2O2 and Fe) in the model cyanobacterium Synechocystis PCC6803. RESULTS: We found that cells respond to the Cd stress in a two main temporal phases process. In the "early" phase cells mainly limit Cd entry through the negative and positive regulation of numerous genes operating in metal uptake and export, respectively. As time proceeds, the number of responsive genes increases. In this "massive" phase, Cd downregulates most genes operating in (i) photosynthesis (PS) that normally provides ATP and NADPH; (ii) assimilation of carbon, nitrogen and sulfur that requires ATP and NAD(P)H; and (iii) translation machinery, a major consumer of ATP and nutrients. Simultaneously, many genes are upregulated, such as those involved in Fe acquisition, stress tolerance, and protein degradation (crucial to nutrients recycling). The most striking common effect of Cd and H2O2 is the disturbance of both light tolerance and Fe homeostasis, which appeared to be interdependent. Our results indicate that cells challenged with H2O2 or Cd use different strategies for the same purpose of supplying Fe atoms to Fe-requiring metalloenzymes and the SUF machinery, which synthesizes or repairs Fe-S centers. Cd-stressed cells preferentially breakdown their Fe-rich PS machinery, whereas H2O2-challenged cells preferentially accelerate the intake of Fe atoms from the medium. CONCLUSION: We view the responses to Cd as an integrated "Yin Yang" reprogramming of the whole metabolism, we found to be controlled by the Slr1738 regulator. As the Yin process, the ATP- and nutrients-sparing downregulation of anabolism limits the poisoning incorporation of Cd into metalloenzymes. As the compensatory Yang process, the PS breakdown liberates nutrient assimilates for the synthesis of Cd-tolerance proteins, among which we found the Slr0946 arsenate reductase enzyme.
Abstract: When produced in Escherichia coli, the CGFS-type monothiol Grxs from this organism (EcGrx4p) and the model cyanobacterium Synechocystis (SyGrx3p) exist as a dimeric iron-sulfur containing holoprotein or as a monomeric apoprotein in solution. Spectroscopic and site-directed mutagenesis analyses show that the SyGrx3 holoprotein contains a subunit-bridging [2Fe-2S] cluster that is ligated by the catalytic cysteine located in the CGFS motif of each monomer and the cysteines of two molecules of glutathione. The biochemical characterization of several monothiol Grxs from the cyanobacteria Gloeobacter violaceus (GvGrx3p) and Thermosynechococcus elongatus (TeGrx3p), the yeast Saccharomyces cerevisiae (ScGrx3p, ScGrx4p, and ScGrx5p), the plant Arabidopsis thaliana (AtGrx5p), and human (HsGrx5p) indicate that the incorporation of a GSH-ligated [2Fe-2S] center is a common feature of prokaryotic and eukaryotic CGFS-active site monothiol Grxs. In light of these results, the involvement of these enzymes in the sensing of iron and/or the biogenesis and transfer of Fe-S cluster is discussed.
Abstract: The production of nanoparticles (NPs) is increasing rapidly for applications in electronics, chemistry, and biology. This interest is due to the very small size of NPs which provides them with many interesting properties such as rapid diffusion, high specific surface areas, reactivity in liquid or gas phase, and a size close to biomacromolecules. In turn, these extreme abilities might be a problem when considering a potentially uncontrolled exposure to the environment. For instance, nanoparticles might be highly mobile and rapidly transported in the environment or inside the body through a water or air pathway. Accordingly, the very fast development of these new synthetic nanomaterials raises questions about their impact on the environment and human health. We have studied the impact of a model water dispersion of nanoparticles (7 nm CeO2 oxide) on a Gram-negative bacteria (Escherichia coli). The nanoparticles are positively charged at neutral pH and thus display a strong electrostatic attraction toward bacterial outer membranes. The counting of colony forming units (CFU) after direct contact with CeO2 NPs allows for the defining of the conditions for which the contact is lethal to Escherichia coli. Furthermore, a set of experiments including sorption isotherms, TEM microscopy, and X-ray absorption spectroscopy (XAS) at cerium L3 edge is linked to propose a scenario for the observed toxic contact.
Abstract: Using a bacterial two-hybrid system and a combination of in vivo and in vitro assays that take advantage of the green fluorescent reporter protein (GFP), we have investigated the localization and the protein-protein interaction of several key components of the cytokinetic machinery of cyanobacteria (i.e. the progenitor of chloroplast). We demonstrate that (i) the ftsZ and zipN genes are essential for the viability of the model cyanobacterium Synechocystis sp. PCC 6803, whereas the minCDE cluster is dispensable for cell growth; (ii) the GTP-binding domain of FtsZ is crucial to FtsZ assembly into the septal ring at mid-cell; (iii) the Z-ring of deeply constricted daughter cells is oriented perpendicularly to the mother Z-ring, showing that Synechocystis divides in alternating perpendicular planes; (iv) the MinCDE system affects the morphology of the cell, as well as the position and the shape of FtsZ structures; and (v) MinD is targeted to cell membranes in a process involving its C-terminal amphipathic helix, but not its ATP-binding region. Finally, we have also characterized a novel Z-interacting protein, ZipN, the N-terminal DnaJ domain of which is critical to the decoration of the Z-ring, and we report that this process is independent of MinCDE.
Abstract: Summary The cyanobacterial genes lexA, recA and ruvB were analysed in Synechocystis PCC6803, which is shown here to be more radiation resistant than the other unicellular model strain Synechococcus PCC7942. We found that cyanobacteria do not have an Escherichia coli-type SOS regulon. The Synechocystis lexA and recA promoters were found to be strong and UV insensitive, unlike the ruvB promoter, which is weak and UV-C inducible. Yet, lexA and recA are regulated by UV-C, but the control is negative and occurs at the post-transcriptional level. Two novel conserved elements were characterized in the lexA promoter: (i) an unusually long crucial box 5'-TAAAATTTTGTATCTTTT-3' (-64, -47); and (ii) a negatively acting motif 5'-TATGAT-3' (-42, -37). These elements were not found in the recA promoter, which appeared to be unusually simple in harbouring only a single crucial element (i.e. the canonical -10 box). RuvB, operating in recombination-dependent cellular processes, was found to be dispensable to cell growth, whereas LexA and RecA appeared to be critical to cell viability. Using DNA microarrays, we have identified 57 genes with expression that is altered, at least twofold, in response to LexA depletion. None of these genes is predicted to operate in DNA metabolism, arguing against the involvement of LexA in the regulation of DNA repair. Instead, most of the LexA-responsive genes were known to be involved in carbon assimilation or controlled by carbon availability. Consistently, the growth of the LexA-depleted strain was found to be strongly dependent on the availability of inorganic carbon.
Abstract: The Synechocystis fedI gene (petF, ssl0020) was found to be strongly expressed under the negative control of H2O2 or heavy metals, and the positive control of light fluence (regulation dependent on active photosynthesis) or carbon availability [under the control of NdhR, the regulator of the ndh3 operon encoding NAD(P)H dehydrogenase subunits]. The basic and constitutive promoter (BP) of fedI extending from -62 to +25 (relative to the transcription start point) is weakly active, presumably because it harbours a long (30 bp) spacer between the two crucial motifs: the -10 box (5'-TAgtAT-3', -13 to -8) and the '-35' box (5'-TTGctA-3', -49 to -44). BP strength is strongly enhanced by the two upstream regions, -113 to -82 and -151 to -114, mediating the 30-fold constitutive stimulation and the fourfold light activation respectively. Three well-conserved transcriptional elements were characterized for the first time, namely the -19 box (5'-TTTT-3') that is essential to transcription, and the two twice repeated elements that are both critical to light induction: the TTGyCA-3' box (-35 to -30, and -125 to -120) and the 5'-ATTTyA-3' box (-55 to -50, and -134 to -129). That two of these light induction motifs (5'-TTGtCA-3', -35 to -30; 5'-ATTTcA-3', -55 to -50) occur in the constitutive BP promoter indicate that in the fedI gene light activation and transcription per se are closely interacting. Interestingly, the fedI gene from marine strains was found to lack the three transcriptional elements presently described, as well as the 5'-AGGA-3' Shine-Dalgarno sequence, which are all conserved among the fedI from non-marine strains.
Abstract: The three Synechocystis PCC6803 genes homologous to proteobacterial Calvin cycle regulators (cbbR) have been analyzed. sll0998 appeared to be crucial to cell viability, whereas both sll0030 and sll1594 were found to be dispensable for cell growth. Unlike what was predicted from their sequence homology, Sll0030 and Sll1594 did not appear to regulate the transcription of Calvin cycle key genes. Further analysis of Sll1594 showed that this protein plays an important role in the adaptation to inorganic carbon starvation and osmotic stress. Sll1594 mediates the response to these stress conditions by regulating the transcription of a new regulon including the monocistronic genes sll1594 and slr1727 (encoding a presumptive Na+/H+ antiporter) as well as the ndh3 operon encoding the NAD(P)H-dehydrogenase subunits F3 and D3 and a protein of unknown function. The sll1594 gene and the ndh3 operon are negatively controlled by Sll1594, which also regulates the expression of the slr1727 gene. Sequence alignment of the diverse Sll1594 DNA binding sites led us to propose the TCAATG-(N10)-ATCAAT sequence as the consensus motif. To our knowledge, this is the first report on the characterization and analysis of a transcriptional regulator for ndh genes in a photoautotrophic organism.
Abstract: The physiological function of the type 1 NAD(P)H dehydrogenase (Ndh-1) of Synechocystis sp. PCC6803 has been investigated by inactivating the gene ndhH encoding a subunit of the complex. Molecular analysis of independent transformants revealed that all clones were heteroploid, containing both wild-type and mutant ndhH copies, whatever the metabolic conditions used during genome segregation, including high CO(2) concentration. By replacing the chromosomal copy of the ndhH gene by a plasmidial copy under the control of a temperature-controlled promoter, we induce a conditional phenotype, growth being only possible at high temperature. This clearly shows for the first time that an ndh gene is indispensable to the survival of xD;Synechocystis sp. PCC6803.
Abstract: The two glyceraldehyde-3-phosphate dehydrogenase-encoding genes (gap) of Synechocystis were shown to be expressed as monocistronic transcripts. Whereas gap1 expression is slow and weak, gap2 gene induction is rapid xD;and strong. Transcription of the gap2 gene was shown to depend on functional photosynthetic electron transport and xD;on active carbon metabolism. The basal promoter of gap2 (P, -45 to +34, relative to the transcription start site) is xD;controlled by three cis-acting elements designated A (-443 to -45), B (+34 to +50, in the untranslated leader region) xD;and C (+50 to +167, in the coding region) that, together, promote a 100-fold stimulation of P activity. Element B xD;was found to behave as a transcriptional enhancer, in that it was active regardless of its position, orientation and xD;distance relative to P. All three cis-acting stimulatory elements exhibit a common 5'-agaTYAACg-3' nucleotide motif xD;that appears to be conserved in cyanobacteria and may be the target for a transcriptional enhancer. We also report that gap2 transcription depends on a Gram-positive-like -16 promoter box (5'-TRTG-3') that was obviously conserved xD;throughout the evolution of chloroplasts. This is the first report on the occurrence of a -16 promoter element in xD;photoautotrophic organisms.
Abstract: The Synechocystis PCC6803 secA gene was found to be essential to cell viability, and to be transcriptionally controlled by the redox state of the cells. The basic promoter (BP, -71 to +47 relative to the transcription start site) is controlled by three cis-acting elements which, together, mediate the four-fold light-induction of BP activity. The positively acting element (PE1, -361 to -71) upstream of BP exerts a two-fold stimulation of BP; the negative element (NE, +47 to +104) downstream of BP decreases BP-strength about six-fold. The PE2-element (+104 to +175) lying in the coding sequence overcomes NE-dependent down regulation of BP. BP harbors E. coli s70-like promoter elements -35 (5'-TTGAat-3') and -10 (5-'TAagAT-3'). The -10 motif, which has the features of an "extended -10" box, is absolutely essential to promoter activity. The -35 hexamer is critical to the enhancement of promoter-strength above BP-level and to light-inducibility, both features involving regulatory elements flanking BP. Most interestingly, reducing the length of the 30 bp spacing between the -35 and -10 boxes down to 17 bp was found to increase promoter activity, and to confer light-inducibility to BP. This demonstrate that promoter element spacing controls basal expression and light inducibility of the secA gene.
Abstract: The genes encoding (2Fe-2S) plant-like ferredoxins were studied in the widely-used cyanobacterium Synechocystis PCC6803. The fedI gene (ssl0020) coding for the most abundant ferredoxin product was found to be strongly expressed as a light-induced monocistronic transcript, whereas the other fed genes appeared to be silent (slr1828), or moderately expressed as polycistronic transcripts regulated by either light fluence (slr0150, negative control) or glucose availability (sll1382). fedI was found to be critical to Synechocystis PCC6803 viability in spite of slr0150-, sll1382- or flavodoxin-induction, even after the addition of glucose that compensate for the loss of photosynthesis. Nevertheless, fedI could be deleted from all chromosome copies in cells propagating a fedI gene (even of heterologous origin) on a replicating plasmid. This strain was used as the host for the subsequent introduction of fedI mutant alleles propagated on a second vector. Analysis of the fedI mutant strains generated after plasmid exchange, showed that the C18-C85 disulfide bridge is central neither to the tight compaction of ferredoxin I nor to its reduction by photosystem I, and demonstrate that the length of the FedI carboxy-terminus is important for effective PSI/FedI interactions. The plasmid shuffling strategy presently described has general applicability for mutational analysis of essential genes in many organisms since it is based on promiscuous plasmids.
Abstract: The gdhA gene of Synechocystis PCC 6803, which encodes an NADP- dependent glutamate dehydrogenase (NADP-GDH), has been cloned by complementation of an Escherichia coli glutamate auxotroph. This gene was found to code for a polypeptide of 428 amino acid residues, whose sequence shows high identity with those of archaebacteria (42-47%), some Gram-positive bacteria (40-44%) and mammals (37%). The minimal fragment of Synechocystis DNA required for complementation (2kb) carries the gdhA gene preceded by an open reading frame (ORF2) encoding a polypeptide of 130 amino acids. ORF2 and gdhA are co-transcribed as a 1.9 kb mRNA, but shorter transcripts including only gdhA were also detected. Two promoter regions were identified upon transcriptional fusion to the cat reporter gene of a promoter probe plasmid. Transcription from the promoter upstream of ORF2 was found to be regulated depending on the growth phase of Synechocystis, in parallel to NADP-GDH activity. This promoter is expressed in Escherichia coli too, in contrast to the second promoter, located between ORF2 and gdhA, which was silent in E. coli and did not respond to the stage of growth in Synechocystis. Disruption of the cyanobacterial gdhA gene with a chloramphenicol resistance cassette yielded a mutant strain totally lacking NADP-GDH activity, demonstrating that this gene is not essential to Synechocystis 6803 under our laboratory conditions.
Abstract: The facultative heterotrophic cyanobacterium Synechocystis sp. strain PCC 6803 was transformed by HaeII Cmr fragments ligated at random to HaeII DNA fragments of the host genome. A similar transformation was done with an AvaII Kmr marker ligated to AvaII host DNA fragments. Integration of the resistance markers into the host genome led to a high frequency of stable Kmr and Cmr transformants. Physical analysis of individual transformants indicated that this result was due to homologous recombination by conversionlike events leading to insertion of the Cmr (or Kmr) gene between two HaeII (or AvaII) sites of the host genome, with precise deletion of the host DNA between these sites. In contrast, integrative crossover of circular DNA molecules with homology to the host DNA is very rare in this cyanobacterium. Strain PCC 6803 was shown to have about 12 genomic copies per cell in standard growth conditions, which complicates the detection of recessive mutations induced by chemical or UV mutagenesis. Random disruption of the host DNA by insertional transformation provides a convenient alternative to transposon mutagenesis in cyanobacteria and may help to overcome the difficulties encountered in generating recessive mutants by classical mutagenesis.
Abstract: Photosynthetic mutants of the cyanobacterium Synechocystis PCC 6803 were produced by a random cartridge mutagenesis method leading to gene inactivation. This procedure relies on random ligation of an Escherichia coli kanamycin resistance (Kmr) gene to restriction fragments of genomic DNA from the host. Then recombination occurring during transformation promotes integration of the marker gene into the genome of the recipient cells. Several mutants impaired in photosynthesis were obtained by this procedure. All are partially or totally defective in photosystem II activity and some of them also harbour a functionally modified photosystem I. Restriction and recombination data showed that one mutant (AK1) is best explained as an insertion of the Kmr gene into an AvaII restriction site of the gene psbD-1. All others harbour a deletion, ranging from at least 1.15 kb (AK3) to more than 50 kb (AK9), which partly or fully overlaps the genes psbB and/or psbD-1, depending on the mutant. A genetic-physical map of the more than 60 kb region of the cyanobacterial genome harbouring the genes psbB, psbC and psbD-1 was constructed by combining published sequence data on these genes with the results of recombination and restriction mapping.
Notes: Can produce large deletions. Silent gene is responsible for self transformation?
