Françoise Joset

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.

Salt shock-inducible Photosystem I cyclic electron transfer in Synechocystis PCC6803 relies on binding of ferredoxin:NADP+ reductase to the thylakoid membranes via its CpcD phycobilisome-linker homologous N-terminal domain

Relative to ferredoxin:NADP(+) reductase (FNR) from chloroplasts, the comparable enzyme in cyanobacteria contains an additional 9 kDa domain at its amino-terminus. The domain is homologous to the phycocyanin associated linker polypeptide CpcD of the light harvesting phycobilisome antennae. The phenotypic consequences of the genetic removal of this domain from the petH gene, which encodes FNR, have been studied in Synechocystis PCC 6803. The in frame deletion of 75 residues at the amino-terminus, rendered chloroplast length FNR enzyme with normal functionality in linear photosynthetic electron transfer. Salt shock correlated with increased abundance of petH mRNA in the wild-type and mutant alike. The truncation stopped salt stress-inducible increase of Photosystem I-dependent cyclic electron flow. Both photoacoustic determination of the storage of energy from Photosystem I specific far-red light, and the re-reduction kinetics of P700(+), suggest lack of function of the truncated FNR in the plastoquinone-cytochrome b(6)f complex reductase step of the PS I-dependent cyclic electron transfer chain. Independent gold-immunodecoration studies and analysis of FNR distribution through activity staining after native polyacrylamide gelelectrophoresis showed that association of FNR with the thylakoid membranes of Synechocystis PCC 6803 requires the presence of the extended amino-terminal domain of the enzyme. The truncated DeltapetH gene was also transformed into a NAD(P)H dehydrogenase (NDH1) deficient mutant of Synechocystis PCC 6803 (strain M55) (T. Ogawa, Proc. Natl. Acad. Sci. USA 88 (1991) 4275-4279). Phenotypic characterisation of the double mutant supported our conclusion that both the NAD(P)H dehydrogenase complex and FNR contribute independently to the quinone cytochrome b(6)f reductase step in PS I-dependent cyclic electron transfer. The distribution, binding properties and function of FNR in the model cyanobacterium Synechocystis PCC 6803 will be discussed.

Molecular and genetical analysis of the fructose‐glucose transport system in the cyanobacterium Synechocystis PCC6803

Complementation for glucose transport capacity of deficient mutants from Synechocystis PCC6803 allowed the cloning of the corresponding gene, glcP. The protein predicted from one open reading frame (ORF) in the DNA sequence was 468 residues long. It showed 46–60% amino acid sequence homology and similarity in size and predicted structure (including twelve probable membrane‐spanning regions) with a group of non‐phosphorylating sugar transporters from mammals, yeasts and Escherichia coli. A second ORF, 64 base pairs downstream from glcP, was detected. Its function, dispensable under auto‐ and heterotrophic conditions, could not be determined. Genetic analysis of mutants confirmed that the resistance to fructose, acquired simultaneously with the deficiency in glucose transport, resulted from mutations in the glcP gene, whose approximate location could be determined.

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.

Hypothesis: versatile function of ferredoxin-NADP+ reductase in cyanobacteria provides regulation for transient photosystem I-driven cyclic electron flow

Pseudo-reversion of the high-CO2 requiring phenotype of the NADH dehydrogenase type 1-impaired mutant of Synechocystis PCC6803, strain M55, by salt stress coincides with partial restoration of PSI-driven cyclic electron transfer. In M55, the complete family of D proteins (D1-D6) that are needed for electron transfer through the complex is lacking. Adaptation to salt stress requires de novo synthesis of full-length 47-kDa ferredoxin-NADP+ reductase (FNR). A mutant created in the M55 background, which only expresses truncated chloroplast 37-kDa FNR cannot adapt to salt stress and refrains from growth in low CO2. A special feature of FNR in cyanobacteria is the relatively high molecular mass of 44-48 kDa. A positively charged extended N-terminal domain of the cyanobacterial enzyme defines the extra mass. The extension likely plays a key role in the salt-stress inducible enhancement of PSI-driven cyclic electron transfer, and in the pseudo-reversion of the high-CO2 requiring phenotype of M55. Data acquired with several other cyanobacteria and the oxychlorobacterium Prochlorothrix hollandica contributed to the present hypothesis. It proposes that FNR is involved in regulation of inducible and transient PSI cyclic electron transfer in cyanobacteria via a thylakoid surface charge and conditional-proteolysis steered mechanism.

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.

Identificaton of plastoquinone-cytochrome b6f reductase pathways in direct or indirect photosystem I driven cyclic electron flow in Synechocystis PCC 6803

In cyanobacteria and chloroplasts, concerted action of PS2 and PS1 in linear oxygenic photosynthetic electron transfer renders NADPH and ATP according to the Z-scheme. Maintenance of a stable phosphate potential is secured by PS1 cyclic photophosphorylation (1, 2). In regular photoautotrophic growth, the latter has a very limited capacity (2-4), and is probably used for finetuning of ATP generation capacity and regulation (5). More pronounced usage of PS1 cyclic photophosphorylation and demonstration of the involvement of other PQ reductase pathways which under standard conditions may remain repressed or be only marginally active, can be envisaged during Abbreviations: flvd, flavodoxin; FNR, ferredoxin-NADP+ oxidoreductase; M55N, mutant M55 in normal medium; M55S, ibid in medium plus 550 mM NaCl; NEM, N-ethyl maleimide; P700, the reaction center of PS1; PAS, photoacoustic spectrometry; PAM, pulse amplitude modulation fluorimetry; PS1, photosystem 1; PQ, plastoquinone; WTN/S, wildtype in normal/salt added medium.

Biosynthesis of the branched-chain amino acids in the cyanobacterium Synechocystis PCC6803: existence of compensatory pathways.

Complementation of an E. coli mutant auxotrophic for the branched-chain amino acids (BCAA)—valine, leucine, and isoleucine—by the ilvG gene (slr2088) of the cyanobacterium Synechocystis PCC6803 indicates that this gene encodes an active α-acetohydroxy acid synthase. Differences of response of the recombinants to the addition of the essential amino acids suggested a lower specificity for the initial reaction of the valine/leucine chain than for the isoleucine one. Inactivation of ilvG in Synechocystis led to a leaky phenotype, suggesting a capacity to compensate the auxotrophies by other processes. This observation is discussed in view of the general difficulty of obtaining auxotrophs in cyanobacteria.

A protein is involved in accessibility of the inhibitor acetazolamide to the carbonic anhydrase(s) in the cyanobacterium Synechocystis PCC 6803

A gene, zam (for resistance to acetazolamide), controlling resistance to the carbonic anhydrase inhibitor acetazolamide, is described. It has been cloned from a spontaneous mutant, AZAr-5b, isolated from the cyanobacterium Synechocystis PCC 6803, for its resistance to this drug (Bédu et al., Plant Physiol 93: 1312–1315, 1990). This mutant, besides its resistance to acetazolamide, displayed an absence of catalysed oxygen exchange activity on whole cells, suggestive of a deficiency in carbonic anhydrase activity. The gene was isolated by screening a genomic library of AZAr-5b, and selecting for the capacity to transfer the AZAr phenotype to wild-type cells. A system leading to forced homologous recombination in the host chromosome, using a platform vector, was devised in order to bypass direct selection difficulties. The putative encoded protein, 782 amino acids long, showed some homology with four eukaryotic and prokaryotic proteins involved in different cellular processes, one of them suppressing a phosphatase deficiency. The mutated allele of AZAr-5b showed an in-frame 12 nucleotide duplication, which should not interfere with translation, and might result from transposition of a mobile element. Integration into a wild-type genome of either the spontaneous mutated allele or one inactivated by insertional mutagenesis conferred the character of resistance, but not the deficiency in oxygen exchange, indicating that the two phenotypic aspects of AZAr-5b corresponded to two independent mutations. A working hypothesis explaining the phenotypes of the mutants is that the presence of the Zam protein would be necessary for the inhibitor to reach (one of) the two carbonic anhydrases present in this strain. This, however, would be a secondary action, the physiological role of the protein still being cryptic.