The cyanobacterium Synechococcus sp. PCC 7942 possesses a close homologue to the chloroplast ClpC protein of higher plants

The Clp family consists of large, ubiquitous proteins that function as molecular chaperones and/or regulators of ATP-dependent proteolysis. A single copy gene coding for one of these proteins, ClpC, was cloned from the unicellular cyanobacterium Synechococcus sp. PCC 7942. The predicted polypeptide is most similar (ca. 88%) to the chloroplast-localized ClpC protein from higher plants. Using degenerate PCR primers specific for the two distinct ATP-binding domains characteristic of all ClpA-C proteins, partial sequences homologous to clpC from Synechococcus were also identified in five other cyanobacterial strains. The Synechococcus clpC gene is transcribed under standard growth conditions as a monocistronic message of around 2.7 kb. The level of this message, however, decreases slightly after a shift from 37 to 47.5°C for 2 h, similar to expression previously observed for clpC mRNA from heat-shocked higher plants. At the protein level, the amount of ClpC remains relatively unchanged during the high temperature shift, while that of the known heat shock protein GroEL rises considerably. In contrast, the constitutive level of ClpC in Synechococcus increases considerably under conditions of rapid growth, both with increasing light intensities or CO₂ concentrations. This, and the fact that attempts to inactivate clpC expression fail to produce a viable phenotype, suggest that ClpC activity is essential for growth in this obligate photoautotrophic cyanobacterium.     

Iron stress responses in the cyanobacterium Synechococcus sp. PCC7942

In the present study, we describe the sequential events by which the cyanobacterium Synechococcus sp. PCC 7942 adapts to iron deficiency. In doing so, we have tried to elucidate both short and long-term acclimation to low iron stress in order to understand how the photosynthetic apparatus adjusts to low iron conditions. Our results show that after an initial step, where CP43' is induced and where ferredoxin is partly replaced by flavodoxin, the photosynthetic unit starts to undergo major rearrangements. All measured components of Photosystem I (PSI), PSII and cytochrome (Cyt) ƒ decrease relative to chlorophyll (Chl) a . The photochemical efficiencies of the two photosystems also decline during this phase of acclimation. The well-known drop in phycobilisome content measured as phycocyanin (PC)/Chl was not due to an increased degradation, but rather to a decreased rate of synthesis. The largest effects of iron deficiency were observed on PSI, the most iron-rich structure of the photosynthetic apparatus. In the light of the recent discovery of an iron deficiency induced CP43' ring around PSI a possible dual function of this protein as both an antenna and a quencher is discussed. We also describe the time course of a blue shift in the low temperature Chl emission peak around 715 nm, which originates in PSI. The shift might reflect the disassembly and/or degradation of PSI during iron deficiency and, as a consequence, PSI might under these conditions be found predominantly in a monomeric form. We suggest that the observed functional and compositional alterations represent cellular acclimation enabling growth and development under iron deficiency, and that growth ceases when the acclimation capacity is exhausted. However, the cells remain viable even after growth has ceased, since they resumed growth once iron was added back to the culture.     

Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942.

A gene encoding the enzyme lycopene cyclase in the cyanobacterium Synechococcus sp strain PCC7942 was mapped by genetic complementation, cloned, and sequenced. This gene, which we have named crtL, was expressed in strains of Escherichia coli that were genetically engineered to accumulate the carotenoid precursors lycopene, neurosporene, and zeta-carotene. The crtL gene product converts the acyclic hydrocarbon lycopene into the bicyclic beta-carotene, an essential component of the photosynthetic apparatus in oxygen-evolving organisms and a source of vitamin A in human and animal nutrition. The enzyme also converts neurosporene to the monocyclic beta-zeacarotene but does not cyclize zeta-carotene, indicating that desaturation of the 7-8 or 7'-8' carbon-carbon bond is required for cyclization. The bleaching herbicide 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA) effectively inhibits both cyclization reactions. A mutation that confers resistance to MPTA in Synechococcus sp PCC7942 was identified as a point mutation in the promoter region of crtL. The deduced amino acid sequence of lycopene cyclase specifies a polypeptide of 411 amino acids with a molecular weight of 46,125 and a pI of 6.0. An amino acid sequence motif indicative of FAD utilization is located at the N terminus of the polypeptide. DNA gel blot hybridization analysis indicated a single copy of crtL in Synechococcus sp PCC7942. Other than the FAD binding motif, the predicted amino acid sequence of the cyanobacterial lycopene cyclase bears little resemblance to the two known lycopene cyclase enzymes from nonphotosynthetic bacteria. Preliminary results from DNA gel blot hybridization experiments suggest that, like two earlier genes in the pathway, the Synechococcus gene encoding lycopene cyclase is homologous to plant and algal genes encoding this enzyme.     

Reaction mechanism of phosphoribulokinase from a cyanobacterium,Synechococcus PCC7942

The dependence of the activity of phosphoribulokinase isolated from a cyanobacterium, Synechococcus PCC7942, on Mg²⁺ showed that its real substrates were Mg-ATP and free D-ribulose 5-phosphate. On the basis of results of kinetic inhibition studies and previously reported result of affinity chromatography, an ordered bi bi mechanism in which Mg-ATP binds before ribulose 5-phosphate is proposed. The K_m values for ATP and D-ribulose 5-phosphate were 0.09 and 0.27 mM, respectively. K_i values of ADP and D-ribulose 1,5-bisphosphate were 0.32 and 10.0 mM, respectively. Inhibition constants K_i1 and K_i2 for 6-phosphogluconate were 9.3 and 0.49 mM. K_ia was 0.13 mM. New kinetics on PRK gave higher control coefficient than the kinetics on Spinach PRK did in the model with PRK activity from 175 to 1000 µmol min^–1 mg^–1 chl.     

Cloning of the phycobilisome rod linker genes from the cyanobacterium Synechococcus sp. PCC 6301 and their inactivation in Synechococcus sp. PCC 7942

Coexpression of pairs of nonhaemolytic H1yA mutants in the recombination-deficient (recA) strain Escherichia coli HB101 resulted in a partial reconstitution of haemolytic activity, indicating that the mutation in one H1yA molecule can be complemented by the corresponding wild-type sequence in the other mutant HlyA molecule and vice versa. This suggests that two or more HlyA molecules aggregate prior to pore formation. Partial reconstitution of the haemolytic activity was obtained by the combined expression of a nonhaemolytic HlyA derivative containing a deletion of five repeat units in the repeat domain and several nonhaemolytic HlyA mutants affected in the pore-forming hydrophobic region. The simultaneous expression of two inactive mutant HlyA proteins affected in the region at which HlyA is covalently modified by HlyC and the repeat domain, respectively, resulted in a haemolytic phenotype on blood agar plates comparable to that of wild-type haemolysin. However, complementation was not possible between pairs of HlyA molecules containing site-directed mutations in the hydrophobic region and the modification region, respectively. In addition, no complementation was observed between HlyA mutants with specific mutations at different sites of the same functional domain, i.e. within the hydrophobic region, the modification region or the repeat domain. The aggregation of the HlyA molecules appears to take place after secretion, since no extracellular haemolytic activity was detected when a truncated but active HlyA lacking the C-terminal secretion sequence was expressed together with a non-haemolytic but transport-competent HlyA mutant containing a deletion in the repeat domain.     

Characterization of a zwf mutant of Synechococcus sp. strain PCC 7942.

A mutant of the cyanobacterium Synechococcus sp. strain PCC 7942 carrying a disrupted gene encoding glucose-6-phosphate dehydrogenase (zwf) produced no detectable glucose-6-phosphate dehydrogenase as assessed by enzyme assay and Western blot (immunoblot) analysis. This mutant exhibited significantly impaired dark viability.     

Functional phycobilisome core structures in a phycocyanin-less mutant of cyanobacterium Synechococcus sp. PCC 7942

We have constructed a mutant Synechococcus sp. PCC 7942, termed R2HECAT, in which the entire phycobilisome rod operon has been deleted. In the whole cell absorption spectra of R2HECAT, the peak corresponding to phycocyanin (PC), max620 nm, could not be detected. However, a single pigment-protein fraction with max=654 nm could be isolated on sucrose gradients from R2HECAT. Analysis of this pigment-protein fraction by non-denaturing PAGE indicates an apparent molecular mass of about 1200–1300 kDa. On exposure to low temperature, the isolated pigment-protein complex dissociated to a protein complex with a molecular mass of about 560 kDa. When analysed by SDS-PAGE, the pigment-protein fraction was found to consist of the core polypeptides but lacked PC, 27, 33, 30, and the 9 kDa polypeptides which are a part of the rods. All the chromophore bearing polypeptides of the core were found to be chromophorylated. CD as well as absorption spectra showed the expected maxima around 652 and 675 nm from allophycocyanin (APC) and allophycocyanin B (APC-B) chromophores. Low temperature fluorescence and excitation spectra also showed that the core particles were fully functional with respect to the energy transfer between the APC chromophores. We conclude that PC and therefore the rods are dispensable for the survival of Synechococcus sp. PCC 7942. The results indicate that stable and functional core can assemble in absence of the rods. These rod-less phycobilisome core is able to transfer energy to Photosystem II.Abbreviations PS II Photosystem II - PC phycocyanin - APC allophycocyanin - APC-B allophycocyanin B - PAGE polyacrylamide gel electrophoresis - Cml chloramphenicol - kbp kilobase pairs     

A Synechococcus sp. PCC 7942 mutant with a higher tolerance towards bentazone

In this article we describe the partial characterization of a Synechococcus sp. PCC 7942 mutant Mu1 with an enhanced resistance towards the herbicide bentazone (3-isopropyl-1H-2,1,3-benzothiadiazine-4(3H)-one 2,2-dioxide). The mutant was derived from a random mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine (NSG) and exhibited superior growth rates, pigment content and overall photosynthetic activities under regular growth conditions compared to wild type. Whereas Synechococcus PCC 7942 wild type showed significant photoinhibition, especially in the presence of lincomycin, Mu1 was much more robust. A comparative analysis of the content of several photosynthesis-associated proteins revealed that Mu1 had an increased expression of PsbO on mRNA and protein level and that PsbO is tightly bound to Photosystem II, relative to wild type. This result was substantiated by mass spectrometer measurements of photosynthetic water oxidation revealing a higher stability and integrity of the water oxidizing complex in Mu1 cells grown under regular or calcium deficient conditions. Therefore, our results give rise to the possibility that the overexpression of PsbO in mutant Mu1 confers resistance to reactive oxygen species (ROS) formed as a consequence of bentazone binding to the acceptor side of PS II. In addition, we observed a significantly higher tolerance towards bentazone in iron depleted wild type cells, conditions under which the IdiA protein becomes expressed in highly elevated amounts. As we have previously shown, IdiA preferentially protects the acceptor site of PS II against oxidative stress, especially under iron limitation. Thus, it is likely that IdiA due to its topology interferes with bentazone binding or protects PS II against ROS generated in the presence of bentazone. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molecular characterization and redox regulation of phosphoribulokinase from the cyanobacterium Synechococcus sp. PCC 7942

We isolated and characterized a gene encoding phosphoribulokinase (PRK) from Synechococcus sp. PCC 7942. The isolated sequence consisted of a 999 bp open reading frame encoding 333 amino acid residues of PRK. The PRK contained a pair of cysteinyl residues corresponding to Cys16 and Cys55 of spinach PRK regulated by a ferredoxin-thioredoxin system. However, there were seventeen amino acid residues lacking between the two cysteinyl residues compared with those of the chloroplastic enzyme in higher plants. The recombinant PRK of Synechococcus sp. PCC 7942 accounted for about 6-13% of the total soluble protein in the Escherichia coli. The specific activity of the enzyme was 230 micro mol min(-1) (mg protein)(-1). The enzyme activity was completely inactivated by treatment with 5,5'-dithiobis (2-nitrobenzoic acid) (cysteinyl residue-specific oxidant) or was decreased by treatment with H(2)O(2), but was more tolerant to oxidation than that of chloroplast. The oxidized PRK was fully activated by treatment with excessive dithiothreitol. Furthermore, incubation with 3 mM ATP protected the oxidation of the enzyme by either 5,5'-dithiobis (2-nitrobenzoic acid) or H(2)O(2). These results suggest Synechococcus sp. PCC 7942 PRK can be regulated by reversible oxidation/reduction in vitro, but might be resistant to oxidative inactivation in vivo.     

Identification and cloning of a regulatory gene for nitrogen assimilation in the cyanobacterium Synechococcus sp. strain PCC 7942.

Twenty-seven mutants that were unable to assimilate nitrate were isolated from Synechococcus sp. strain PCC 7942. In addition to mutants that lacked nitrate reductase or nitrite reductase, seven pleiotropic mutants impaired in both reductases, glutamine synthetase, and methylammonium transport were also isolated. One of the pleiotropic mutants was complemented by transformation with a cosmid gene bank from wild-type strain PCC 7942. Three complementing cosmids were isolated, and a 3.1-kilobase-pair DNA fragment that was still able to complement the mutant was identified. The regulatory gene that was cloned (ntcA) appeared to be required for full expression of proteins subject to ammonium repression in Synechococcus sp.