Sequence-dependent cleavage site selection by RNase Z from the cyanobacterium Synechocystis sp. PCC 6803

Biosynthesis of transfer RNA requires processing from longer precursors at the 5'- and 3'-ends. In eukaryotes, in archaea, and in those bacteria where the 3'-terminal CCA sequence is not encoded, 3' processing is carried out by the endonuclease RNase Z, which cleaves after the discriminator nucleotide to generate a mature 3'-end ready for the addition of the CCA sequence. We have identified and cloned the gene coding for RNase Z in the cyanobacterium Synechocystis sp. PCC 6803. The gene has been expressed in Escherichia coli, and the recombinant protein was purified. The enzymatic activity of RNase Z from Synechocystis has been studied in vitro with a variety of substrates. The presence of C or CC after the discriminator nucleotide modifies the cleavage site of RNase Z so that it is displaced by one and two nucleotides to the 3'-side, respectively. The presence of the complete 3'-terminal CCA sequence in the precursor of the tRNA completely inhibits RNase Z activity. The inactive CCA-containing precursor binds to Synechocystis RNase Z with similar affinity than the mature tRNA. The properties of the enzyme described here could be related with the mechanism by which CCA is added in this organism, with the participation of two separate nucleotidyl transferases, one specific for the addition of C and another for the addition of A. This work is the first characterization of RNase Z from a cyanobacterium, and the first from an organism with two separate nucleotidyl transferases.     

Effect of increased PHA synthase activity on polyhydroxyalkanoates biosynthesis in Synechocystis sp. PCC6803

Polyhydroxyalkanoate (PHA) synthase activity in Synechocystis sp. PCC6803 was increased two-fold by introducing the PHA biosynthetic genes of Ralstonia eutropha. The resulting recombinant Synechocystis sp. PCC6803 strain was subjected to conditions that favor PHA accumulation and the effects of various carbon sources were studied. In addition, the fine structure of both wild-type and recombinant Synechocystis sp. PCC6803 was examined using freeze-fracture electron microscopy technique. The PHA granules in the recombinant Synechocystis sp. PCC6803 were localised near the thylakoid membranes. Maximum amount of PHA accumulation was obtained in the presence of acetate, where the number of granules in the recombinant cells ranged from 4 to 6 and their sizes were in the range of 70-240 nm. In comparison to wild-type Synechocystis sp. PCC6803, recombinant cells with increased PHA synthase activity showed only a marginal increase in PHA content suggesting that PHA synthase is not the rate limiting enzyme of PHA biosynthesis in Synechocystis sp. PCC6803.     

Swapping the domains of exoribonucleases RNase II and RNase R: conferring upon RNase II the ability to degrade ds RNA

RNase II and RNase R are the two E. coli exoribonucleases that belong to the RNase II super family of enzymes. They degrade RNA hydrolytically in the 3' to 5' direction in a processive and sequence independent manner. However, while RNase R is capable of degrading structured RNAs, the RNase II activity is impaired by dsRNAs. The final end-product of these two enzymes is also different, being 4 nt for RNase II and 2 nt for RNase R. RNase II and RNase R share structural properties, including 60% of amino acid sequence similarity and have a similar modular domain organization: two N-terminal cold shock domains (CSD1 and CSD2), one central RNB catalytic domain, and one C-terminal S1 domain. We have constructed hybrid proteins by swapping the domains between RNase II and RNase R to determine which are the responsible for the differences observed between RNase R and RNase II. The results obtained show that the S1 and RNB domains from RNase R in an RNase II context allow the degradation of double-stranded substrates and the appearance of the 2 nt long end-product. Moreover, the degradation of structured RNAs becomes tail-independent when the RNB domain from RNase R is no longer associated with the RNA binding domains (CSD and S1) of the genuine protein. Finally, we show that the RNase R C-terminal Lysine-rich region is involved in the degradation of double-stranded substrates in an RNase II context, probably by unwinding the substrate before it enters into the catalytic cavity.     

集胞藻PCC6803膜脂循环关键基因酶学性质和生理功能

集胞藻PCC6803野生型和其脂酰ACP合酶敲除突变株的自由脂肪酸含量和组成表明膜脂的重构和降解是细胞内自由脂肪酸的来源之一。在这一过程中脂肪酶起到关键性作用。通过基因组数据库检索,发现集胞藻PCC6803基因组中只有一个脂肪酶编码基因sll1969,但是还没有其功能相关的生化证据。为了确定该基因的功能及其在脂肪酸代谢途径中的作用,加深对集胞藻PCC6803脂肪酸代谢途径的了解,文中将sll1969基因在大肠杆菌中过表达和体外纯化,得到重组蛋白Sll1969,并对其酶学性质进行初步分析。在30℃条件下,测得Sll1969以对硝基苯丁酸酯作为底物时的Km和kcat值分别为(1.16±0.01)mmol/L和(332.8±10.0)/min;该脂肪酶的最适反应温度为55℃。通过比较分析sll1969突变株中脂肪酸含量和组成变化,发现sll1969的表达量与细胞自由脂肪酸的产量呈正相关,但Sll1969不是细胞中唯一的脂肪酶。     

细胞周期蛋白依赖性激酶抑制剂Roscovitine对3种蓝藻生长和活性的影响

为了明确蓝藻中丝氨酸/苏氨酸激酶的功能是否与调控细胞的生长分裂相关,以丝状鱼腥藻7120、单细胞集胞藻6803和聚球藻7002为对象,利用OD750光吸收测定和MTT方法研究了不同浓度丝氨酸苏氨酸激酶抑制剂roscovitine对其生长和脱氢酶活性的影响。结果表明:4 h roscovitine处理后对鱼腥藻7120和集胞藻6803生长量影响不大,对聚球藻7002的生长有促进作用。4 h roscovitine的处理对鱼腥藻7120有浓度依赖的显著抑制活性,对集胞藻6803的活性无影响,但是却促进聚球藻7002的活性。药物作用4 d后,7120的生长和活性均显著降低,并有浓度效应;6803的生长量较对照减少,但活性变化不明显;聚球藻7002的生长和活性均未受影响。显微观察结果显示,roscovitine对3种细胞形态没有影响,但药物作用4 d后的7120藻丝体较短。结果表明丝氨酸/苏氨酸抑制剂roscovitine影响丝状藻7120的生长和活性。     

Interrelation between cyanophycin synthesis, L-arginine catabolism and photosynthesis in the cyanobacterium Synechocystis sp. strain PCC 6803

Ultrastructural and immunocytochemical investigations gave evidence that cyanophycin (multi-L-arginyl-poly-L-aspartate) granules accumulate in the cyanobacterium Synechocystis sp. strain PCC 6803 under nutrient deficient growth conditions, especially under phosphate limitation. Besides nutrient deficiency, growth of Synechocystis PCC 6803 on L-arginine or L-asparagine as sole N-source also led to high increase of cyanophycin synthesis, while growth on the combination of L-arginine or L-asparagine with nitrate only caused minor cyanophycin accumulation. Growth of Synechocystis PCC 6803 on L-arginine as sole N-source caused substantial morphological and physiological changes, such as severe thylakoid membrane degradation with partial loss of pigments and photosynthetic activity leading to a phenotype almost like that seen under nutrient deficiency. In contrast to the wild type, the PsbO-free Synechocystis PCC 6803 mutant could grow on L-arginine as sole N-source with only minor morphological and physiological changes. Due to its fairly balanced growth, the mutant accumulated only few cyanophycin granules. L-arginine degrading activity (measured as ornithine and ammonium formation) was high in the PsbO-free mutant but not in the wild type when cells were grown on L-arginine as sole N-source. In both cells types the L-arginine degrading activity was high (although in the PsbO-free mutant about twice as high as in wild type), when cells were grown on L-arginine in combination with nitrate, and as expected very low when cells were grown on nitrate as sole N-source. Thus, net cyanophycin accumulation in Synechocystis PCC 6803 is regulated by the relative concentration of L-arginine to the total nitrogen pool, and the intracellular L-arginine concentration is greatly influenced by the activity of the L-arginine degrading enzyme system which in part is regulated by the activity status of photosystem II. These results suggest a complex interrelation between cyanophycin synthesis, L-arginine catabolism, and in addition photosynthesis in Synechocystis PCC 6803.     

Acclimation to the growth temperature and the high-temperature effects on photosystem II and plasma membranes in a mesophilic cyanobacterium, Synechocystis sp. PCC6803.

High-temperature effects on Photosystem II and plasma membranes, temperature dependence of growth, and acclimation to the growth temperature were studied in a mesophilic cyanobacterium, Synechocystis sp. PCC6803. The following results were obtained. (1) Small but distinct temperature acclimation of the PSII reaction center activity was shown for the first time when the activity was measured at inhibitory high temperatures. However, the reaction center activity showed no apparent acclimation when it was measured at growth temperatures after heat stress. (2) Oxygen-evolving activity and the permeability of plasma membranes showed higher resistance to heat when PCC6803 cells were grown at higher temperatures. (3) Acclimation of photosynthesis to the growth temperature seemed to occur so as to maintain photosynthesis activity not at a maximum level but in a certain range at the growth temperatures. (4) Neither sensitivity to high-temperature-induced dissociation of phycobilisomes from the PSII reaction center complexes nor degradation of phycocyanin were altered by changes in the environmental temperature. (5) A close relationship between the viability of cells and the structural changes of plasma membranes (but not the inactivation of photosynthesis) was observed. The denaturation process of PSII complexes and the relationship between the temperature dependence of the growth of Synechocystis PCC6803 cells and that of the photosynthetic activity are also discussed.     

The deg proteases protect Synechocystis sp. PCC 6803 during heat and light stresses but are not essential for removal of damaged D1 protein during the photosystem two repair cycle

Members of the DegP/HtrA (or Deg) family of proteases are found widely in nature and play an important role in the proteolysis of misfolded and damaged proteins. As yet, their physiological role in oxygenic photosynthetic organisms is unclear, although it has been widely speculated that they participate in the degradation of the photodamaged D1 subunit in the photosystem two complex (PSII) repair cycle, which is needed to maintain PSII activity in both cyanobacteria and chloroplasts. We have examined the role of the three Deg proteases found in the cyanobacterium Synechocystis sp. PCC 6803 through analysis of double and triple insertion mutants. We have discovered that these proteases show overlap in function and are involved in a number of key physiological responses ranging from protection against light and heat stresses to phototaxis. In previous work, we concluded that the Deg proteases played either a direct or an indirect role in PSII repair in a glucose-tolerant version of Synechocystis 6803 (Silva, P., Choi, Y. J., Hassan, H. A., and Nixon, P. J. (2002) Philos. Trans. R. Soc. Lond. B Biol. Sci. 357, 1461-1467). In this work, we have now been able to demonstrate unambiguously, using a triple deg mutant created in the wild type strain of Synechocystis 6803, that the Deg proteases are not obligatory for PSII repair and D1 degradation. We therefore conclude that although the Deg proteases are needed for photoprotection of Synechocystis sp. PCC 6803, they do not play an essential role in D1 turnover and PSII repair in vivo.     

Analysis of the gene encoding the RNA subunit of ribonuclease P from cyanobacteria.

The genes encoding the RNA subunit of ribonuclease P from the unicellular cyanobacterium Synechocystis sp. PCC 6803, and from the heterocyst-forming strains Anabaena sp. PCC 7120 and Calothrix sp. PCC 7601 were cloned using the homologous gene from Anacystis nidulans (Synechococcus sp. PCC 6301) as a probe. The genes and the flanking regions were sequenced. The genes from Anabaena and Calothrix are flanked at their 3'-ends by short tandemly repeated repetitive (STRR) sequences. In addition, two other sets of STRR sequences were detected within the transcribed regions of the Anabaena and Calothrix genes, increasing the length of a variable secondary structure element present in many RNA subunits of ribonuclease P from eubacteria. The ends of the mature RNAs were determined by primer extension and RNase protection. The predicted secondary structure of the three RNAs studied is similar to that of Anacystis and although some idiosyncrasies are observed, fits well with the eubacterial consensus.     

Purification and characterization of class-I and class-II fructose-1,6-bisphosphate aldolases from the cyanobacterium Synechocystis sp. PCC 6803

The whole genome sequence database for Synechocystis sp. PCC 6803 has revealed the presence of genes encoding class-I (CI) and class-II (CII) fructose-1,6-bisphosphate aldolases (FBAs) in this organism. Two types of FBA from Synechocystis sp. PCC 6803 were separated by chromatography on phenyl-Sepharose. The activity of the enzyme in the major peak was inhibited by the presence of 25 mM EDTA; however, the activity in the minor peak was not. Therefore, the FBA in the former fractions was designated as CII-FBA, and in the latter designated as CI-FBA. CI-FBA was functionally redundant in Synechocystis sp. PCC 6803, while no disruptant for the gene encoding CII-FBA was obtained under photoautotrophic conditions. The kinetic parameters of CI- and CII-FBAs purified from Synechocystis sp. PCC 6803 in the cleavage reaction of FBP were generally similar, except in their reactivity for SBP. The SBP/FBP activity ratio of the CII-FBA was two times higher than that of the CI-FBA.     

Transformation of glutamate to delta-aminolevulinic acid by soluble extracts of Synechocystis sp. PCC 6803 and other oxygenic prokaryotes

delta-Aminolevulinic acid is the first committed precursor in the biosynthesis of hemes, phycobilins, and chlorophylls. Plants and algae synthesize delta-aminolevulinic acid from glutamate via an RNA-dependent 5-carbon pathway. Previous reports demonstrated that cyanobacteria form delta-aminolevulinic acid from glutamate in vivo. We now report the direct measurement of this activity in vitro. Three oxygenic prokaryotes were examined, the unicellular cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 (Agmenellum quadruplicatum PR-6) and the chlorophyll a- and b-containing filamentous prochlorophyte Prochlorothrix hollandica. delta-Aminolevulinic acid-forming activity was detected in soluble extracts of all three species. delta-Aminolevulinic acid formation by Synechocystis extracts was further characterized. Activity depended upon addition of reduced pyridine nucleotide, ATP, and Mg2+ to the incubation mixture. NADPH was a more effective pyridine nucleotide than NADH at low concentrations, but NADPH inhibited delta-amino-levulinic acid formation above 1 mM, whereas NADH did not. The pH optimum was about 7.6, and the ATP concentration optimum was 0.1 mM. Activity was stimulated by addition of RNA derived from Synechocystis or Chlorella, and abolished by preincubation with RNase A. After RNase inactivation, activity was restored by addition of RNasin to block further RNase action, followed by supplementation with Synechocystis RNA. Activity was inhibited by micromolar concentrations of hemin, as was previously found with plant and algal extracts. Complete dependence on added glutamate could not be achieved. Radioactivity was incorporated into delta-aminolevulinic acid when the incubation mixture contained 1-[14C]glutamate. Activity in the Synechocystis enzyme extract was stimulated by the addition of a partially purified enzyme fraction from Chlorella. It thus appears that prokaryotic oxygenic organisms share with chloroplasts the capacity for biosynthesis of photosynthetic pigments from glutamate via the RNA-dependent 5-carbon pathway.     

Targeted random mutagenesis to identify functionally important residues in the D2 protein of photosystem II in Synechocystis sp. strain PCC 6803

To identify important residues in the D2 protein of photosystem II (PSII) in the cyanobacterium Synechocystis sp. strain PCC 6803, we randomly mutagenized a region of psbDI (coding for a 96-residue-long C-terminal part of D2) with sodium bisulfite. Mutagenized plasmids were introduced into a Synechocystis sp. strain PCC 6803 mutant that lacks both psbD genes, and mutants with impaired PSII function were selected. Nine D2 residues were identified that are important for PSII stability and/or function, as their mutation led to impairment of photoautotrophic growth. Five of these residues are likely to be involved in the formation of the Q(A)-binding niche; these are Ala249, Ser254, Gly258, Ala260, and His268. Three others (Gly278, Ser283, and Gly288) are in transmembrane alpha-helix E, and their alteration leads to destabilization of PSII but not to major functional alterations of the remaining centers, indicating that they are unlikely to interact directly with cofactors. In the C-terminal lumenal tail of D2, only one residue (Arg294) was identified as functionally important for PSII. However, from the number of mutants generated it is likely that most or all of the 70 residues that are susceptible to bisulfite mutagenesis have been altered at least once. The fact that mutations in most of these residues have not been picked up by our screening method suggests that these mutations led to a normal photoautotrophic phenotype. A novel method of intragenic complementation in Synechocystis sp. strain PCC 6803 was developed to facilitate genetic analysis of psbDI mutants containing several amino acid changes in the targeted domain. Recombination between genome copies in the same cell appears to be much more prevalent in Synechocystis sp. strain PCC 6803 than was generally assumed.     

Cloning, expression, purification, and preliminary characterization of a putative hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803

The genome of the unicellular cyanobacterium Synechocystis sp. PCC 6803 contains a gene (slr2097, glbN) encoding a 123 amino-acid product with sequence similarity to globins. Related proteins from cyanobacteria, ciliates, and green algae bind oxygen and have a pronounced tendency to coordinate the heme iron with two protein ligands. To study the structural and functional properties of Synechocystis sp. PCC 6803 hemoglobin, slr2097 was cloned and overexpressed in Escherichia coli. Purification of the hemoglobin was performed after addition of hemin to the clarified cell lysate. Recombinant, heme-reconstituted ferric Synechocystis sp. PCC 6803 hemoglobin was found to be a stable helical protein, soluble to concentrations higher than 500 microM. At neutral pH, it yielded an electronic absorption spectrum typical of a low-spin ferric species, with maxima at 410 and 546 nm. The proton NMR spectrum revealed sharp lines spread over a chemical shift window narrower than 40 ppm, in support of low-spin hexacoordination of the heme iron. Nuclear Overhauser effects demonstrated that the heme is inserted in the protein matrix to produce one major equilibrium form. Addition of dithionite resulted in an absorption spectrum with maxima at 426, 528, and 560 nm. This reduced form appeared capable of carbon monoxide binding. Optical data also suggested that cyanide ions could bind to the heme in the ferric state. The spectral properties of the putative Synechocystis sp. PCC 6803 hemoglobin confirmed that it can be used for further studies of an ancient hemoprotein structure.     

Purification and properties of glutamine synthetases from the cyanobacteria Synechocystis sp. strain PCC 6803 and Calothrix sp. strain PCC 7601.

Glutamine synthetases (GSs) from two cyanobacteria, one unicellular (Synechocystis sp. strain PCC 6803) and the other filamentous (Calothrix sp. strain PCC 7601 [Fremyella diplosiphon]), were purified to homogeneity. The biosynthetic activities of both enzymes were strongly inhibited by ADP, indicating that the energy charge of the cell might regulate the GS activity. Both cyanobacteria exhibited an ammonium-mediated repression of GS synthesis. In addition, the Synechocystis sp. showed an inactivation of GS promoted by ammonium that had not been demonstrated previously in cyanobacteria.     

The serine protease HhoA from Synechocystis sp. strain PCC 6803: substrate specificity and formation of a hexameric complex are regulated by the PDZ domain

Enzymes of the ATP-independent Deg serine endopeptidase family are very flexible with regard to their substrate specificity. Some family members cleave only one substrate, while others act as general proteases on unfolded substrates. The proteolytic activity of Deg proteases is regulated by PDZ protein interaction domains. Here we characterized the HhoA protease from Synechocystis sp. strain PCC 6803 in vitro using several recombinant protein constructs. The proteolytic activity of HhoA was found to increase with temperature and basic pH and was stimulated by the addition of Mg(2+) or Ca(2+). We found that the single PDZ domain of HhoA played a critical role in regulating protease activity and in the assembly of a hexameric complex. Deletion of the PDZ domain strongly reduced proteolysis of a sterically challenging resorufin-labeled casein substrate, but unlabeled beta-casein was still degraded. Reconstitution of the purified HhoA with total membrane proteins isolated from Synechocystis sp. wild-type strain PCC 6803 and a DeltahhoA mutant resulted in specific degradation of selected proteins at elevated temperatures. We concluded that a single PDZ domain of HhoA plays a critical role in defining the protease activity and oligomerization state, combining the functions that are attributed to two PDZ domains in the homologous DegP protease from Escherichia coli. Based on this first enzymatic study of a Deg protease from cyanobacteria, we propose a general role for HhoA in the quality control of extracytoplasmic proteins, including membrane proteins, in Synechocystis sp. strain PCC 6803.     

From genome to enzyme: analysis of key glycolytic and oxidative pentose-phosphate pathway enzymes in the cyanobacterium Synechocystis sp. PCC 6803

Activities of glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucose isomerase, phosphofructokinase (PFK), enolase, pyruvate kinase (PK) and phosphoenolpyruvate (PEP) carboxylase were determined in extracts of photoautotrophic, mixotrophic, and heterotrophic cultures of Synechocystis sp. PCC 6803. Annotated genomes of Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 were analyzed for the respective predicted physical properties of each enzyme investigated here. Enzymatic activity was largely unaffected by nutritional mode, with the exception of glucokinase and PK whose activities were significantly elevated in heterotrophic cultures of Synechocystis sp. PCC 6803. PFK activity was insensitive to bacterial PFK-A (allosteric) effectors such as PEP, implying that Synechocystis PFK should be classified as a PFK-B (non-allosteric). Immunoblot and kinetic studies indicated that irrespective of nutritional mode, the Synechocystis PK corresponds to a PK-A (AMP activated) rather than PK-F (fructose-1,6-bisphosphate activated).