Corinne Cassier-Chauvat

Function and regulation of the cyanobacterial genes lexA, recA and ruvB: LexA is critical to the survival of cells facing inorganic carbon starvation.

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′‐TAT GAT‐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.

Molecular analysis of the key cytokinetic components of cyanobacteria : FtsZ, ZipN and MinCDE

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.

Glutathionylation in the photosynthetic model organism Chlamydomonas reinhardtii: a proteomic survey

Protein glutathionylation is a redox post-translational modification occurring under oxidative stress conditions and playing a major role in cell regulation and signaling. This modification has been mainly studied in nonphotosynthetic organisms, whereas much less is known in photosynthetic organisms despite their important exposure to oxidative stress caused by changes in environmental conditions. We report a large scale proteomic analysis using biotinylated glutathione and streptavidin affinity chromatography that allowed identification of 225 glutathionylated proteins in the eukaryotic unicellular green alga Chlamydomonas reinhardtii. Moreover, 56 sites of glutathionylation were also identified after peptide affinity purification and tandem mass spectrometry. The targets identified belong to a wide range of biological processes and pathways, among which the Calvin-Benson cycle appears to be a major target. The glutathionylation of four enzymes of this cycle, phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, ribose-5-phosphate isomerase, and phosphoglycerate kinase was confirmed by Western blot and activity measurements. The results suggest that glutathionylation could constitute a major mechanism of regulation of the Calvin-Benson cycle under oxidative stress conditions.

Characterization and analysis of an NAD(P)H dehydrogenase transcriptional regulator critical for the survival of cyanobacteria facing inorganic carbon starvation and osmotic stress.

The three Synechocystis PCC6803 genes homologous to proteobacterial Calvin cycle regulators (cbbR) have been analysed. sll0998 appeared to be crucial to cell viability, whereas both sll0030 and sll1594 were found to be dispensable for cell growth. In spite of 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.

Direct and indirect CeO2 nanoparticles toxicity for Escherichia coli and Synechocystis

Abstract Physico-chemical interactions between nanoparticles and cell membranes play a crucial role in determining the cytotoxicity of nanoparticles, which may thereby vary depending on the nature of the target microorganisms. We investigated the responses of two different models of unicellular bacteria to cerium oxide (CeO2) nanoparticles. These organisms are: Synechocystis PCC6803 a representative of environmentally important cyanobacterial organisms (producer of biomass for aquatic food chains), and Escherichia coli a representative of intestine-colonizing bacteria. Coupling physico-chemical (adsorption isotherms and electrophoretic mobility), biological (survival tests), microscopical (SEM, TEM and EDS) and spectroscopic (XANES) methods, we enlightened two distinct mechanisms for the CeO2 nanoparticles toxicological impact: A ‘direct’ mechanism that requires a close contact between nanoparticles and cell membranes, and an ‘indirect’ influence elicited by the acidity of nanoparticles stabilizing agents. We showed that E. coli is sensitive to the ‘direct’ effects of nanoparticles, whereas Synechocystis being protected by extracellular polymeric substances preventing direct cellular contacts is sensitive only to the ‘indirect’ mechanism. Consequently, our findings demonstrate the importance of the ‘direct/indirect’ effects of nanoparticles on cell fitness, a phenomenon that should be systematically investigated with appropriate techniques and dose metrics to make meaningful environmental and/or health recommendations.

A conjugative plasmid vector for promoter analysis in several cyanobacteria of the genera Synechococcus and Synechocystis

A promoter-probe vector, pSB2A, based on the plasmid RSF1010 and the promoterless chloramphenicol acetyl transferase (cat) reporter gene, has been constructed. pSB2A appeared to be most efficiently transferred by conjugation to the widely used cyanobacteria Synechocystis strains PCC6803 (S.6803) and PCC6714 (S.6714) and Synechococcus strains PCC7942 (S.7942) and PCC6301 (S.6301), where it replicates stably even though it contains no cyanobacterial DNA. Using pSB2A we found that (1) a light-regulated promoter from S.6803 remains controlled by light intensity in S.7942 while it is silent in Escherichia coli, and (2) the E. coli tac promoter behaves as a strong and light-independent promoter in the four cyanobacterial hosts tested.

An Arabidopsis Homolog of the Bacterial Cell Division Inhibitor SulA Is Involved in Plastid Division

Plastids have evolved from an endosymbiosis between a cyanobacterial symbiont and a eukaryotic host cell. Their division is mediated both by proteins of the host cell and conserved bacterial division proteins. Here, we identified a new component of the plastid division machinery, Arabidopsis thaliana SulA. Disruption of its cyanobacterial homolog (SSulA) in Synechocystis and overexpression of an AtSulA-green fluorescent protein fusion in Arabidopsis demonstrate that these genes are involved in cell and plastid division, respectively. Overexpression of AtSulA inhibits plastid division in planta but rescues plastid division defects caused by overexpression of AtFtsZ1-1 and AtFtsZ2-1, demonstrating that its role in plastid division may involve an interaction with AtFtsZ1-1 and AtFtsZ2-1.

Targeted deletion and mutational analysis of the essential (2Fe-2S) plant-like ferredoxin in Synechocystis PCC6803 by plasmid shuffling.

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 expressed strongly 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 compensates 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 disulphide bridge is not central either to the tight compaction of ferredoxin I or to its reduction by photosystem I and demonstrated 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, as it is based on promiscuous plasmids.

Multidisciplinary evidences that Synechocystis PCC6803 exopolysaccharides operate in cell sedimentation and protection against salt and metal stresses.

Little is known about the production of exopolysaccharides (EPS) in cyanobacteria, and there are no genetic and physiological evidences that EPS are involved in cell protection against the frequently encountered environmental stresses caused by salt and metals. We studied four presumptive EPS production genes, sll0923, sll1581, slr1875 and sll5052, in the model cyanobacterium Synechocystis PCC6803, which produces copious amounts of EPS attached to cells (CPS) and released in the culture medium (RPS) as shown here. We show that sll0923, sll1581, slr1875 and sll5052 are all dispensable to the growth of all corresponding single and double deletion mutants in absence of stress. Furthermore, we report that sll0923, sll1581 and slr1875 unambiguously operate in the production of both CPS and RPS. Both sll1581 and slr1875 are more important than sll0923 for CPS production, whereas the contrary is true for RPS production. We show that the most EPS-depleted mutant, doubly deleted for sll1581 and slr1875, lacks the EPS mantle that surrounds WT cells and sorbs iron in their vicinity. Using this mutant, we demonstrate for the first time that cyanobacterial EPS directly operate in cell protection against NaCl, CoCl2, CdSO4 and Fe-starvation. We believe that our EPS-depleted mutants will be useful tools to investigate the role of EPS in cell-to-cell aggregation, biofilm formation, biomineralization and tolerance to environmental stresses. We also suggest using the fast sedimenting mutants as biotechnological cell factories to facilitate the otherwise expensive harvest of the producer cell biomass and/or its separation from products excreted in the growth media.

ZipN, an FtsA-like orchestrator of divisome assembly in the model cyanobacterium Synechocystis PCC6803

We pursued the characterization of the divisome of the spherical‐celled cyanobacterium Synechocystis PCC6803, through deletion, site‐directed mutagenesis, GFP tagging, two‐hybrid and co‐immunoprecipitation 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 amino acids 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.