Characterization of a naturally occurring trans-splicing intein from Synechocystis sp. PCC6803

A naturally occurring trans-splicing intein from the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) was used to characterize the intein-catalyzed splicing reaction. Trans-splicing/cleavage reactions were initiated by combining the N-terminal splicing domain of the Ssp DnaE intein containing five native N-extein residues and maltose binding protein as the N-extein with the C-terminal Ssp DnaE intein splicing domain (E(C)) with or without thioredoxin fused in-frame to its carboxy terminus. Observed rate constants (k(obs)) for dithiothreitol-induced N-terminal cleavage, C-terminal cleavage, and trans-splicing were (1.0 +/- 0.5) x 10(-3), (1.9 +/- 0.9) x 10(-4), and (6.6 +/- 1.3) x 10(-5) s(-1), respectively. Preincubation of the intein fragments showed no change in k(obs), indicating association of the two splicing domains is rapid relative to the subsequent steps. Interestingly, when E(C) concentrations were substoichiometric with respect to the N-terminal splicing domain, the levels of N-terminal cleavage were equivalent to the amount of E(C), even over a 24 h period. Activation energies for N-terminal cleavage and trans-splicing were determined by Arrhenius plots to be 12.5 and 8.9 kcal/mol, respectively. Trans-splicing occurred maximally at pH 7.0, while a slight increase in the extent of N-terminal cleavage was observed at higher pH values. This work describes an in-depth kinetic analysis of the splicing and cleavage activity of an intein, and provides insight for the use of the split intein as an affinity domain.     

Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme

Protein trans-splicing by the naturally split intein of the gene dnaE from Nostoc punctiforme (Npu DnaE) was demonstrated here with non-native exteins in Escherichia coli. Npu DnaE possesses robust trans-splicing activity with an efficiency of > 98%, which is superior to that of the DnaE intein from Synechocystis sp. strain PCC6803 (Ssp DnaE). Both the N- and C-terminal parts of the split Npu DnaE intein can be substituted with the corresponding fragment of Ssp DnaE without loss of trans-splicing activity. Protein splicing with the Npu DnaEN is also more tolerant of amino acid substitutions in the C-terminal extein sequence.     

Crystal structures of an intein from the split dnaE gene of Synechocystis sp. PCC6803 reveal the catalytic model without the penultimate histidine and the mechanism of zinc ion inhibition of protein splicing

The first naturally occurring split intein was found in the dnaE gene of Synechocystis sp. PCC6803 and belongs to a subclass of inteins without a penultimate histidine residue. We describe two high-resolution crystal structures, one derived from an excised Ssp DnaE intein and the second from a splicing-deficient precursor protein. The X-ray structures indicate that His147 in the conserved block F activates the side-chain N(delta) atom of the intein C-terminal Asn159, leading to a nucleophilic attack on the peptide bond carbonyl carbon atom at the C-terminal splice site. In this process, Arg73 appears to stabilize the transition state by interacting with the carbonyl oxygen atom of the scissile bond. Arg73 also seems to substitute for the conserved penultimate histidine residue in the formation of an oxyanion hole, as previously identified in other inteins. The finding that the precursor structure contains a zinc ion chelating the highly conserved Cys160 and Asp140 reveals the structural basis of Zn2+-mediated inhibition of protein splicing. Furthermore, it is of interest to observe that the carbonyl carbon atom of Asn159 and N(eta) of Arg73 are 2.6 angstroms apart in the free intein structure and 10.6 angstroms apart in the precursor structure. The orientation change of the aromatic ring of Tyr-1 following the initial acyl shift may be a key switching event contributing to the alignment of Arg73 and the C-terminal scissile bond, and may explain the sequential reaction property of the Ssp DnaE intein.     

Molecular characterization of a novel gene from Synechocystis sp. PCC 6803

A novel gene slr2049 was identified in Synechococcus sp. PCC7002 by homologous alignment. The features and possible functions of slr2049 gene were predicted by bioinformatics analysis. The function of slr2049 was analyzed in vitro with a heterologous Escherichia coli system with plasmids conferring biosynthesis of phycocyanobilin (PCB) and of the acceptor proteins, β-phycocyanin (CpcB). The resulting products were evaluated with SDS-PAGE and absorption spectra. The function of slr2049 was further analyzed via site-directed mutations. Two mutants, slr2049 (W14L) and slr2049 (Y132S) were generated. The results showed that Slr2049 could catalyze the chromophorylation of CpcB. Compared to wild type, mutant Slr2049 (W14L) had red-shifted absorbance maxima and was not highly fluorescent as the wild-type. However, mutant Slr2049 (Y132S) was almost the same as the wild-type. In conclusion, our study suggests that we have cloned a novel gene and this gene may play an important role in attachment of the chromophores to the apo-proteins.     

DNA-uptake in the naturally competent cyanobacterium, Synechocystis sp. PCC 6803

Abstract Eight species of halophilic Archaea were tested for the presence of the enzymes of the methylglyoxal bypass. Methylglyoxal synthase was found in extracts of all species tested, with the exception of Halobacterium salinarium and Halobacterium cutirubrum . The enzyme of Haloferax volcanii was most active at pH 7 in the absence of salt, and in the presence of 3 M NaCl or KCl activity was half of that without salt, and was inhibited by phosphate. Glyoxalase I was detected in all species tested. Optimal activity of H. volcanii glyoxalase I was found at pH 7 and 3 M KCl; in the absence of salt, activity was strongly reduced. Glutathione could be replaced by γ-glutamylcysteine as the acceptor of the D-lactoyl group. The work shows that the methylglyoxal bypass may be operative in representatives of the archaeal kingdom.     

In vivo protein cyclization promoted by a circularly permuted Synechocystis sp. PCC6803 DnaB mini-intein.

A synthetic Synechocystis sp. PCC6803 DnaB split mini-intein gene was constructed for the in vivo cyclization of recombinant proteins expressed in Escherichia coli. The system was used to cyclize the NH(2)-terminal domain of E. coli DnaB, the structure of which had been determined previously by NMR spectroscopy. Cyclization was found to proceed efficiently, with little accumulation of precursor, and the product was purified in high yield. The solution structure of cyclic DnaB-N is not significantly different from that of linear DnaB-N and it unfolds reversibly at temperatures approximately 14 degrees C higher. Improved hydrogen bonding was observed in the first and last helices, and the length of the last helix was increased, while the 9-amino acid linker used to join the NH(2) and COOH termini was found to be highly mobile. The measured thermodynamic stabilization of the structure (Delta Delta G approximately 2 kcal/mol) agrees well with the value estimated from the reduced conformational entropy in the unfolded form. Simple polymer theory can be used to predict likely free energy changes resulting from protein cyclization and how the stabilization depends on the size of the protein and the length of the linker used to connect the termini.     

Characterisation of acyltransferases from Synechocystis sp. PCC6803

As phylogenetic ancestors of plant chloroplasts cyanobacteria resemble plastids with respect to lipid and fatty acid composition. These membrane lipids show the typical prokaryotic fatty acid pattern in which the sn-2 position is exclusively esterified by C(16) acyl groups. In the course of de novo glycerolipid biosynthesis this prokaryotic fatty acid pattern is established by the sequential acylation of glycerol-3-phosphate with acyl-ACPs by the activity of different acyltransferases. In silico approaches allowed the identification of putative Synechocystis acyltransferases involved in glycerolipid metabolism. Functional expression studies in Escherichia coli showed that sll1848 codes for a lysophosphatidic acid acyltransferase with a high specificity for 16:0-ACP, whereas slr2060 encodes a lysophospholipid acyltransferase, with a broad acyl-ACP specificity but a strong preference for lysophosphatidyglycerol especially its sn-2 acyl isomer as acyl-acceptor. The generation and analysis of the corresponding Synechocystis knockout mutants revealed that lysophosphatidic acid acyltransferase unlike the lysophospholipid acyltransferase is essential for the vital functions of the cells.     

FutA2 is a ferric binding protein from Synechocystis PCC 6803

Synechocystis PCC 6803 has a high demand for iron (10 times greater than Escherichia coli) to sustain photosynthesis and is unusual in possessing at least two putative iron-binding proteins of a type normally associated with ATP-binding cassette-type importers. It has been suggested that one of these, FutA2, binds ferrous iron, but herein we clearly demonstrate that this protein avidly binds Fe(III), the oxidation state preference of periplasmic iron-binding proteins. Structures of apo-FutA2 and Fe-FutA2 have been determined at 1.7 and 2.7A, respectively. The metal ion is bound in a distorted trigonal bipyramidal arrangement with no exogenous anions as ligands. The metal-binding environment, including the second coordination sphere and charge properties, is consistent with a preference for Fe(III). Atypically, FutA2 has a Tat signal peptide, and its inability to coordinate divalent cations may be crucial to prevent metals from binding to the folded protein prior to export from the cytosol. A loop containing the His(43) ligand undergoes considerable movement in apo-versus Fe-FutA2 and may control metal release to the importer. Although these data are consistent with FutA2 being the periplasmic component involved in iron uptake, deletion of another putative ferric binding protein, FutA1, has a greater effect on the accumulation of iron and is more analogous to a DeltafutA1DeltafutA2 double mutant than DeltafutA2. Here, we also discover that there is a reduced level of ferric FutA2 in the periplasm of the DeltafutA1 mutant providing an explanation for its severe iron-uptake phenotype.     

Ferredoxin and flavodoxin from the cyanobacterium Synechocystis sp PCC 6803.

The unicellular cyanobacterium Synechocystis sp PCC 6803 is capable of synthesizing two different Photosystem-I electron acceptors, ferredoxin and flavodoxin. Under normal growth conditions a [2Fe-2S] ferredoxin was recovered and purified to homogeneity. The complete amino-acid sequence of this protein was established. The isoelectric point (pI = 3.48), midpoint redox potential (Em = -0.412 V) and stability under denaturing conditions were also determined. This ferredoxin exhibits an unusual electrophoretic behavior, resulting in a very low apparent molecular mass between 2 and 3.5 kDa, even in the presence of high concentrations of urea. However, a molecular mass of 10,232 Da (apo-ferredoxin) is calculated from the sequence. Free thiol assays indicate the presence of a disulfide bridge in this protein. A small amount of ferredoxin was also found in another fraction during the purification procedure. The amino-acid sequence and properties of this minor ferredoxin were similar to those of the major ferredoxin. However, its solubility in ammonium sulfate and its reactivity with antibodies directed against spinach ferredoxin were different. Traces of flavodoxin were also recovered from the same fraction. The amount of flavodoxin was dramatically increased under iron-deficient growth conditions. An acidic isoelectric point was measured (pI = 3.76), close to that of ferredoxin. The midpoint redox potentials of flavodoxin are Em1 = -0.433 V and Em2 = -0.238 V at pH 7.8. Sequence comparison based on the 42 N-terminal amino acids indicates that Synechocystis 6803 flavodoxin most likely belongs to the long-chain class, despite an apparent molecular mass of 15 kDa determined by SDS-PAGE.     

Functional analysis of the two homologous psbA gene copies in Synechocystis PCC 6714 and PCC 6803

The cyanobacteria Synechocystis 6803 and 6714 contain three genes (psbA) coding for the D₁ protein. This protein is an essential subunit of photosystem II (PSII) and is the target for herbicides. We have used herbicide-resistant mutants to study the role of the two homologous copies of the psbA genes in both strains (the third copy is not expressed). Several herbicide resistance mutations map within the psbAI gene in Synechocystis 6714 (G. Ajlani et al.), Plant Mol. Biol. 13 (1989): (469–479). We have looked for mutations in copy II. Results show that in Synechocystis 6714, only psbAI contains herbicide resistance mutations. Relative expression of psbAI and psbAII has been measured by analysing the proportions of resistant and sensitive D₁ in the thylakoid membranes of the mutants. In normal growth conditions, 95% resistant D₁ and 5% sensitive D₁ were found. In high light conditions, expression of psbAII was enhanced, producing 15% sensitive D₁. This enhancement is specifically due to high light and not to the decrease of D₁ concentration caused by photoinhibition. Copy I of Synechocystis 6714 corresponds to copy 2 of Synechocystis 6803 since it was always psbA2 which was recombined in Synechocystis 6803 transformants. PSII of the transformant strains was found to be 95% resistant to herbicides as in resistant mutants of Synechocystis 6714.     

Global Landscape of Native Protein Complexes in Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803(hereafter: Synechocystis) is a model organism for studying photosynthesis, energy metabolism, and environmental stress. Although known as the first fully sequenced phototrophic organism, Synechocystis still has almost half of its proteome without functional annotations. In this study, by using co-fractionation coupled with liquid chromatographytandem mass spectrometry(LC-MS/MS), we define 291 multi-protein complexes, encompassing24,092 protein±protein interactions(PPIs...     

Characterization of a sodium-regulated glutaminase from cyanobacterium Synechocystis sp. PCC 6803

Glutaminase is widely distributed among microorganisms and mammals with important functions. Little is known regarding the biochemical properties and functions of the deamidating enzyme glutaminase in cyanobacteria. In this study a putative glutaminase encoded by gene slr2079 in Synechocystis sp. PCC 6803 was investigated. The slr2079 was expressed as histidine-tagged fusion protein in Escherichia coli. The purified protein possessed glutaminase activity, validating the functional assignment of the genomic annotation. The apparent K _m value of the recombinant protein for glutamine was 26.6 ± 0.9 mmol/L, which was comparable to that for some of other microbial glutaminases. Analysis of the purified protein revealed a two-fold increase in catalytic activity in the presence of 1 mol/L Na⁺. Moreover, the K _m value was decreased to 12.2 ± 1.9 mmol/L in the presence of Na⁺. These data demonstrate that the recombinant protein Slr2079 is a glutaminase which is regulated by Na⁺ through increasing its affinity for substrate glutamine. The slr2079 gene was successfully disrupted in Synechocystis by targeted mutagenesis and the Δslr2079 mutant strain was analyzed. No differences in cell growth and oxygen evolution rate were observed between Δslr2079 and the wild type under standard growth conditions, demonstrating slr2079 is not essential in Synechocystis. Under high salt stress condition, however, Δslr2079 cells grew 1.25-fold faster than wild-type cells. Moreover, the photosynthetic oxygen evolution rate of Δslr2079 cells was higher than that of the wild-type. To further characterize this phenotype, a number of salt stress-related genes were analyzed by semi-quantitative RT-PCR. Expression of gdhB and prc was enhanced and expression of desD and guaA was repressed in Δslr2079 compared to the wild type. In addition, expression of two key enzymes of ammonium assimilation in cyanobacteria, glutamine synthetase (GS) and glutamate synthase (GOGAT) was examined by semi-quantitative RT-PCR. Expression of GOGAT was enhanced in Δslr2079 compared to the wild type while GS expression was unchanged. The results indicate that slr2079 functions in the salt stress response by regulating the expression of salt stress related genes and might not play a major role in glutamine breakdown in Synechocystis. Supported by the National Natural Sciences Foundation of China (Grant No. 30500108) and Hundred Talents Program of Chinese Academy of Sciences.     

Overexpression and reconstitution of a Rieske iron-sulfur protein from the cyanobacterium Synechocystis PCC 6803

Chloroplast cyt b6f complexes as well as mitochondrial and bacterial cyt bc1 complexes contain a high potential Rieske iron-sulfur protein which is essential for their function. To characterise the isolated Rieske protein from the mesophilic cyanobacterium Synechocystis PCC6803 we cloned the encoding gene into an expression vector and overexpressed the protein in E. coli. In cells overexpressing the protein no typical Rieske type EPR signal was detected neither in membranes nor in inclusion bodies where the majority of the protein was deposited. The inclusion bodies were isolated from the E. coli cells and denaturated with 8 M urea. With a single anion exchange chromatographic step a pure protein could be obtained which was used for further experiments. The NifS like protein IscS was recently reported to mediate the incorporation of iron-sulfur clusters into ferredoxin in vitro. We used the recombinant IscS protein for the incorporation of the cluster into the folded Rieske apoprotein. Spectroscopic characterisation of the resultant protein by CD and EPR spectroscopy showed the presence of a typical Rieske iron-sulfur centre.     

Characterization of a sodium-regulated glutaminase from cyanobacterium Synechocystis sp. PCC 6803

Glutaminase is widely distributed among microorganisms and mammals with important functions. Lit-tle is known regarding the biochemical properties and functions of the deamidating enzyme glutami-nase in cyanobacteria. In this study a putative glutaminase encoded by gene slr2079 in Synechocystis sp. PCC 6803 was investigated. The slr2079 was expressed as histidine-tagged fusion protein in Es-cherichia coli. The purified protein possessed glutaminase activity, validating the functional assign-ment of the genomic annotation. The apparent Km value of the recombinant protein for glutamine was 26.6 ± 0.9 mmol/L, which was comparable to that for some of other microbial glutaminases. Analysis of the purified protein revealed a two-fold increase in catalytic activity in the presence of 1 mol/L Na . Moreover, the Km value was decreased to 12.2 ± 1.9 mmol/L in the presence of Na . These data demon-strate that the recombinant protein Slr2079 is a glutaminase which is regulated by Na through in-creasing its affinity for substrate glutamine. The slr2079 gene was successfully disrupted in Synecho-cystis by targeted mutagenesis and the △slr2079 mutant strain was analyzed. No differences in cell growth and oxygen evolution rate were observed between △slr2079 and the wild type under standard growth conditions, demonstrating slr2079 is not essential in Synechocystis. Under high salt stress condition, however, △slr2079 cells grew 1.25-fold faster than wild-type cells. Moreover, the photosyn-thetic oxygen evolution rate of △slr2079 cells was higher than that of the wild-type. To further charac-terize this phenotype, a number of salt stress-related genes were analyzed by semi-quantitative RT-PCR. Expression of gdhB and prc was enhanced and expression of desD and guaA was repressed in △slr2079 compared to the wild type. In addition, expression of two key enzymes of ammonium assimi-lation in cyanobacteria, glutamine synthetase (GS) and glutamate synthase (GOGAT) was examined by semi-quantitative RT-PCR. Expression of GOGAT was enhanced in △slr2079 compared to the wild type while GS expression was unchanged. The results indicate that slr2079 functions in the salt stress re-sponse by regulating the expression of salt stress related genes and might not play a major role in glutamine breakdown in Synechocystis.     

Red antenna states of photosystem I from Synechocystis PCC 6803

Single-molecule spectroscopy at low temperatures was used to elucidate spectral properties, heterogeneities, and dynamics of the red-shifted chlorophyll a (Chl a) molecules responsible for the fluorescence from photosystem I (PSI). Emission spectra of single PSI complexes from the cyanobacterium Synechocystis PCC 6803 show zero-phonon lines (ZPLs) as well as broad intensity distributions without ZPLs. ZPLs are found most frequently on the blue side of the broad intensity distributions. The abundance of ZPLs decreases almost linearly at longer wavelengths. The distribution of ZPLs indicates the existence of at least two pools with maxima at 699 and 710 nm. The pool with the maximum at 710 nm is assigned to chlorophylls absorbing around 706 nm (C706), whereas the pool with the maximum at 699 nm (F699) can be assigned to chlorophylls absorbing at 692, 695, or 699 nm. The broad distributions dominating the red side of the spectra are made up of a low number of emitters assigned to the red-most pool C714. The properties of F699 show close relation to those of F698 in Synechococcus PCC 7002 and C708 in Thermosynechococcus elongatus. Furthermore, a high similarity is found between the C714 pool in Synechocystis PCC 6803 and C708 in Synechococcus PCC 7002 as well as C719 in T. elongatus.