Sylvie Bédu

Heterocyst differentiation and pattern formation in cyanobacteria: a chorus of signals

Heterocyst differentiation in filamentous cyanobacteria provides an excellent prokaryotic model for studying multicellular behaviour and pattern formation. In Anabaena sp. strain PCC 7120, for example, 5–10% of the cells along each filament are induced, when deprived of combined nitrogen, to differentiate into heterocysts. Heterocysts are specialized in the fixation of N2 under oxic conditions and are semi‐regularly spaced among vegetative cells. This developmental programme leads to spatial separation of oxygen‐sensitive nitrogen fixation (by heterocysts) and oxygen‐producing photosynthesis (by vegetative cells). The interdependence between these two cell types ensures filament growth under conditions of combined‐nitrogen limitation. Multiple signals have recently been identified as necessary for the initiation of heterocyst differentiation, the formation of the heterocyst pattern and pattern maintenance. The Krebs cycle metabolite 2‐oxoglutarate (2‐OG) serves as a signal of nitrogen deprivation. Accumulation of a non‐metabolizable analogue of 2‐OG triggers the complex developmental process of heterocyst differentiation. Once heterocyst development has been initiated, interactions among the various components involved in heterocyst differentiation determine the developmental fate of each cell. The free calcium concentration is crucial to heterocyst differentiation. Lateral diffusion of the PatS peptide or a derivative of it from a developing cell may inhibit the differentiation of neighbouring cells. HetR, a protease showing DNA‐binding activity, is crucial to heterocyst differentiation and appears to be the central processor of various early signals involved in the developmental process. How the various signalling pathways are integrated and used to control heterocyst differentiation processes is a challenging question that still remains to be elucidated.

Protein PII regulates both inorganic carbon and nitrate uptake and is modified by a redox signal in Synechocystis PCC 6803

In Synechocystis PCC 6803 as in other cyanobacteria, involvement of protein PII in the co‐regulation of inorganic carbon and nitrogen metabolism was established based on post‐translational modifications of the protein resulting from changes in the carbon/nitrogen regimes. Uptake of bicarbonate and nitrate in response to changes of the carbon and/or nitrogen regimes is altered in a PII‐null mutant, indicating that both processes are under control of PII. Modulation of electron flow by addition of methyl viologen with or without duroquinol, or in a NAD(P)H dehydrogenase‐deficient mutant, affects the phosphorylation level of PII. The redox state of the cells would thus act as a trigger for PII phosphorylation.

The signal transducer P(II) and bicarbonate acquisition in Prochlorococcus marinus PCC 9511, a marine cyanobacterium naturally deficient in nitrate and nitrite assimilation.

The amino acid sequence of the signal transducer P(II) (GlnB) of the oceanic photosynthetic prokaryote Prochlorococcus marinus strain PCC 9511 displays a typical cyanobacterial signature and is phylogenetically related to all known cyanobacterial glnB genes, but forms a distinct subclade with two other marine cyanobacteria. P(II) of P. marinus was not phosphorylated under the conditions tested, despite its highly conserved primary amino acid sequence, including the seryl residue at position 49, the site for the phosphorylation of the protein in the cyanobacterium Synechococcus PCC 7942. Moreover, P. marinus lacks nitrate and nitrite reductase activities and does not take up nitrate and nitrite. This strain, however, expresses a low- and a high-affinity transport system for inorganic carbon (C(i); K(m,app) 240 and 4 micro M, respectively), a result consistent with the unphosphorylated form of P(II) acting as a sensor for the control of C(i) acquisition, as proposed for the cyanobacterium Synechocystis PCC 6803. The present data are discussed in relation to the genetic information provided by the P. marinus MED4 genome sequence.

A protein involved in co-ordinated regulation of inorganic carbon and glucose metabolism in the facultative photoautotrophic cyanobacteriumSynechocystis PCC6803

The involvement of a gene ofSynechocystis PCC6803,icfG, in the co-ordinated regulation of inorganic carbon and glucose metabolism, was established. TheicfG gene codes for a 72 kDa protein, which shows no homology with those registered in data libraries. Expression oficfG required glucose, the actual inducer probably being glucose-6-phosphate, and was independent of light and of the external inorganic carbon concentration. Mutants carrying an inactivated copy oficfG were constructed. Their growth characteristics were identical to those of the wild type under all regimes except in limiting inorganic carbon with glucose being present either before or after the transfer to the limiting conditions. These conditions completely prevented growth, both in the light and in the dark. The inhibition could be relieved by several intermediates of the tricarboxylic acid cycle. Assays of various enzymic activities related to inorganic carbon uptake and to its assimilationvia either the Calvin cycle or phosphoenolpyruvate carboxylase did not reveal the level of action of IcfG. Possible models include a blockage of the assimilation of both carbon sources in the absence of IcfG, or the inhibition of Ci incorporation route(s) essential under limiting inorganic carbon conditions, even when glucose is present, and even in the dark.

Control of nitrogen and carbon metabolism in cyanobacteria

In the unicellular cyanobacteria that do not fix molecular nitrogen, interactions between N assimilation and C metabolism occur through the signal transducer PII and the global nitrogen regulator NtcA. Under high CO2 concentration, PII liganded to ATP and boundto 2-oxoglutarate becomes phosphorylated and negatively controls the high affinity transport for bicarbonate. In contrast, under low CO2, PII being only liganded to ATP becomes dephosphorylated and negatively controls the nitrate/nitrite active transport system. The redox state of the cells together with NtcA also modulate the phosphorylation state of PII. Moreover, the regulation of the expression of the gene encoding PII is at least in part NthA-dependent. This network of transcriptional and post-transcriptional regulations allows cells to rapidly acclimate by adjusting their carbon and nitrogen metabolism in response to changes in environmental coditions.

Cell-type specific modification of PII is involved in the regulation of nitrogen metabolism in the cyanobacterium Anabaena PCC 7120

In the heterocystous cyanobacterium Anabaena PCC 7120, the modification state of the signalling PII protein is regulated according to the nitrogen regime of the cells, as already observed in some unicellular cyanobacteria. However, during the adaptation to diazotrophic growth conditions, PII is phosphorylated in vegetative cells while unphosphorylated in heterocysts. Isolation of mutants affected on PII modification state and analysis of their phenotypes allow us to show the implication of PII in the regulation of molecular nitrogen assimilation and more specifically, the requirement of unmodified state of PII in the formation of polar nodules of cyanophycin in heterocysts.

A noncanonical WD-repeat protein from the cyanobacterium Synechocystis PCC6803: structural and functional study.

Synechocystis PCC6803 possesses several open reading frames encoding putative WD‐repeat proteins. One, the Hat protein, is involved in the control of a high‐affinity transport system for inorganic carbon that is active when the cells are grown under a limiting concentration of this carbon substrate. The protein is composed of two major domains separated by a hydrophobic linker region of 20 amino acid residues. The N‐terminal domain of Hat has no homolog in standard databases and does not display any particular structural features. Eleven WD repeats have been identified in the C‐terminal moiety. The region encompassing the four terminal WD repeats is essential for growth under a limiting inorganic carbon regime. The region encompassing the two most terminal WD repeats is required for the activity of the high‐affinity transport system. However, because the Hat protein is located in the thylakoids, it should not be itself an element of the transport system. The structural organization of the WD‐containing domain of Hat was modeled from the crystal structure of the G protein β subunit (with seven WD repeats) and of hemopexin (a structural analog with four blades). Functional and structural data argue in favor of an organization of the Hat WD moiety in two subdomains of seven and four WD repeats. The C‐terminal 4‐mer subdomain might interact with another, yet unknown, protein/peptide. This interaction could be essential in modulating the stability of the 4‐mer structure and, thus, the accessibility of this subdomain, or at least of the region encompassing the last two WD repeats.

UPTAKE OF INORGANIC CARBON IN THE CYANOBACTERIUM SYNECHOCYSTIS PCC6803 : PHYSIOLOGICAL AND GENETIC EVIDENCE FOR A HIGH-AFFINITY UPTAKE SYSTEM

Synechocystis PCC6803 displays two inorganic carbon‐uptake processes, a low‐affinity one (apparent Km: 300–400 µM) functional in cells grown under standard or limiting inorganic carbon concentrations, and one with a higher affinity (60±12 µM), detected only in cells adapted to limiting inorganic carbon conditions. A mutational and screening procedure allowed the isolation of a mutant deficient in the high‐affinity system, but only slightly impaired in its growth capacities. The mutated genomic region revealed two open reading frames (ORFs), possibly belonging to an operonic structure. A clone in which the downstream ORF, hatR (high‐affinity transport), had been inactivated showed a phenotype close to that of the original mutant. Inactivation of the other ORF, hatA, yielded a clone unable to grow in limiting inorganic carbon conditions. The deduced HatA protein showed no homology with any registered protein. It possessed three hydrophobic domains, including a putative signal peptide. Several hypotheses are considered as to its role. The deduced HatR protein, which possessed the features characteristic of the response regulators of the two‐component regulatory systems ubiquitous in bacteria, might be a regulator controlling the activity of the high‐affinity transport process. It would belong to the subclass of these molecules lacking the DNA‐binding domain.

Inactivation of spkD, encoding a Ser/Thr kinase, affects the pool of the TCA cycle metabolites in Synechocystis sp. strain PCC 6803.

The inactivation of sll0776 (spkD), a gene encoding a protein Ser/Thr kinase in Synechocystis PCC 6803, led to a pleiotropic phenotype of the SpkD null mutant. This mutant is impaired in its growth ability under low concentration of inorganic carbon (C(i)), though its C(i)-uptake system is not affected. Addition of glucose, phosphoglyceraldehyde or pyruvate does not allow the mutant to grow under low-C(i) conditions. In contrast, this growth defect can be restored when the low-C(i) culture medium is supplemented with metabolites of the TCA cycle. Growth of the mutant is also inhibited when ammonium is provided as nitrogen source, whatever the carbon regime of the cells, due to the high demand for 2-oxoglutarate, which is the carbon skeleton for ammonium assimilation. When mutant cells are cultured under standard growth conditions, the intracellular concentration of 2-oxoglutarate is 20 % lower than is observed in the wild-type strain. However, this decrease of 2-oxoglutarate level only slightly affects the phosphorylation state of PII, a protein that regulates nitrogen and carbon metabolism according to the intracellular levels of 2-oxoglutarate. Properties of the SpkD mutant suggest that the Ser/Thr kinase SpkD could be involved in adjusting the pool of the TCA cycle metabolites according to C(i) supply in the culture medium.

Phosphate uptake in the yeast Candida tropicalis: purification of phosphate-binding protein and investigations about its role in phosphate uptake

The purification of a phosphate-binding protein (PiBP2) by immunoadsorption is described. The entire anti phosphate-binding protein 2 antibodies as well as the Fab fragments obtained from these antibodies inhibit Pi uptake by whole cells. The inhibition is a mixed type of inhibition (Vm and Km are affected). These results should be regarded as a possible involvement of phosphate-binding protein 2 in Pi uptake. The binding of 125I-labelled fragments prepared from anti phosphate-binding protein 2 antibodies to whole cells, to shocked cells and to protoplasts has been investigated. The results confirm the release of phosphate-binding protein by osmotic shock and during protoplast formation. From these findings, a cell-wall localisation, near the cell surface of the phosphate-binding protein should be proposed.