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1.
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The genome DNA of the cyanobacterium Synechocystis sp. PCC 6803 carries a number of insertion sequences (Kaneko, T. et al. 1996, DNA Res., 3, 109-136). We analyzed one of the abundant ISs (ISY203 group of IS4 family) in the common three substrains of Synechocystis and found that the four ISs with identical nucleotide sequences were present only in the "Kazusa" strain, whose complete genome sequence had been determined, while absent in ancestral strains (the original strain from Pasteur Culture Collection and its glucose-tolerant derivative). Three of these ISs were found in the genomic sequence as transposase genes of sll1474, sll1780 and slr1635. The fourth was on the plasmid, pSYSM. On the other hand, all three strains had a novel IS (denoted ISY203x), of which the nucleotide sequence was totally identical to the four ISs found only in the Kazusa strain. Since the flanking regions of ISY203x did not match any part of the genome or of the known plasmids of Synechocystis, it is presumably located on a yet uncharacterized plasmid. These suggest that the four ISs in Kazusa strain were recently transposed from ISY203x. Apparently, the transposition inactivated four preexisting genes, of which modified forms are presented as putative genes (sll1473, sll1475, slr1862, slr1863, slr1635 and ssl2982) in the list of the complete genome (CyanoBase: http://www.kazusa.or.jp/cyano/cyano.html). The possible effects of transposition of ISs in Synechocystis are discussed in relation to phenotypic mutations and microevolution.  相似文献   

3.
N J Silman  N G Carr    N H Mann 《Journal of bacteriology》1995,177(12):3527-3533
Glutamine synthetase (GS) inactivation was observed in crude cell extracts and in the high-speed supernatant fraction from the cyanobacterium Synechocystis sp. strain PCC 6803 following the addition of ammonium ions, glutamine, or glutamate. Dialysis of the high-speed supernatant resulted in loss of inactivation activity, but this could be restored by the addition of NADH, NADPH, or NADP+ and, to a lesser extent, NAD+, suggesting that inactivation of GS involved ADP-ribosylation. This form of modification was confirmed both by labelling experiments using [32P]NAD+ and by chemical analysis of the hydrolyzed enzyme. Three different forms of GS, exhibiting no activity, biosynthetic activity only, or transferase activity only, could be resolved by chromatography, and the differences in activity were correlated with the extent of the modification. Both biosynthetic and transferase activities were restored to the completely inactive form of GS by treatment with phosphodiesterase.  相似文献   

4.
Salt-sensitive mutants of Synechocystis were obtained by random cartridge mutagenesis, and one mutant (mutant 4) was characterized in detail. The salt tolerance of mutant 4 was reduced to about 20% of that of the wild-type. This was caused by a defect in the biosynthetic pathway of the osmoprotective compound glucosylglycerol (GG). Salt-treated cells of mutant 4 accumulated the intermediate glucosylglycerol-phosphate (GG-P). Only low levels of phosphate-free GG were detected. The phosphorylated form of GG was not osmoprotective and seemed to be toxic. In vitro enzyme assays revealed that GG-P-phosphatase activity was completely absent in mutant 4, while GG-P-synthase remained unchanged. The integration site of the aphII cartridge in mutant 4 and the corresponding wild-type region was cloned and sequenced. Mutant 4 was complemented to salt resistance after transformation by the cloned wild-type region. The integration of the cartridge led to a deletion of about 1.1 kb of the chromosomal DNA. This affected two of the identified putative protein coding regions, orfII and stpA. The ORFII protein shows a high degree of similarity to the receiver domain of response regulator proteins. Related sequences were not found for StpA. We assume that in mutant 4, regulatory genes necessary for the process of salt adaptation in Synechocystis are impaired. Received: 12 January 1996 / Accepted: 28 May 1996  相似文献   

5.
Ma W  Deng Y  Ogawa T  Mi H 《Plant & cell physiology》2006,47(10):1432-1436
We identified eight bands by staining native gels for NADPH-nitrobluetetrazolium oxidoreductase activity after electrophoresis ofn-dodecyl-ß-D-maltoside-treated membranes of Synechocystissp. strain PCC 6803. Among them, bands A, C, D and E were attributedto the activity of NADPH dehydrogenase (NDH-1). Band A is ahighly active supercomplex of NDH-1 (about 1,000 kDa) that wasabsent in the  相似文献   

6.
Posttranslational regulation of nitrate assimilation was studied in the cyanobacterium Synechocystis sp. strain PCC 6803. The ABC-type nitrate and nitrite bispecific transporter encoded by the nrtABCD genes was completely inhibited by ammonium as in Synechococcus elongatus strain PCC 7942. Nitrate reductase was insensitive to ammonium, while it is inhibited in the Synechococcus strain. Nitrite reductase was also insensitive to ammonium. The inhibition of nitrate and nitrite transport required the PII protein (glnB gene product) and the C-terminal domain of NrtC, one of the two ATP-binding subunits of the transporter, as in the Synechococcus strain. Mutants expressing the PII derivatives in which Ala or Glu is substituted for the conserved Ser49, which has been shown to be the phosphorylation site in the Synechococcus strain, showed ammonium-promoted inhibition of nitrate uptake like that of the wild-type strain. The S49A and S49E substitutions in GlnB did not affect the regulation of the nitrate and nitrite transporter in Synechococcus either. These results indicated that the presence or absence of negative electric charge at the 49th position does not affect the activity of the PII protein to regulate the cyanobacterial ABC-type nitrate and nitrite transporter according to the cellular nitrogen status. This finding suggested that the permanent inhibition of nitrate assimilation by an S49A derivative of PII, as was previously reported for Synechococcus elongatus strain PCC 7942, is likely to have resulted from inhibition of nitrate reductase rather than the nitrate and nitrite transporter.  相似文献   

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S Aoki  T Kondo  H Wada    M Ishiura 《Journal of bacteriology》1997,179(18):5751-5755
The cyanobacterium Synechocystis sp. strain PCC 6803 exhibited circadian rhythms in complete darkness. To monitor a circadian rhythm of the Synechocystis cells in darkness, we introduced a PdnaK1::luxAB gene fusion (S. Aoki, T. Kondo, and M. Ishiura, J. Bacteriol. 177:5606-5611, 1995), which was composed of a promoter region of the Synechocystis dnaK1 gene and a promoterless bacterial luciferase luxAB gene set, as a reporter into the chromosome of a dark-adapted Synechocystis strain. The resulting dnaK1-reporting strain showed bioluminescence rhythms with a period of 25 h (on agar medium supplemented with 5 mM glucose) for at least 7 days in darkness. The rhythms were reset by 12-h-light-12-h-dark cycles, and the period of the rhythms was temperature compensated for between 24 and 31 degrees C. These results indicate that light is not necessary for the oscillation of the circadian clock in Synechocystis.  相似文献   

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11.
Accumulation of poly-beta-hydroxybutyrate (PHB) by photoautotrophic microorganisms makes it possible to reduce the production cost of PHB. The Synechocystis sp. PCC6803 cells grown in BG11 medium under balanced, nitrogen-starved or phosphorus-starved conditions were observed by transmission electron microscope. Many electron-transparent granules in the nitrogen-starved cells had a diameter up to 0.8 micron. In contrast, the number of granules in the normally cultured cells decreased obviously and only zero to three much smaller granules were in each cell. These granules were similar to those in bacteria capable of synthesizing PHB. They were proved to be PHB by gas chromatography after subjecting the cells to methanolysis. Effects of glucose as carbon source and light intensity on PHB accumulation in Synechocystis sp. PCC6803 under nitrogen-starved cultivation were further studied. Glucose and illumination promoted cell growth but did not favor PHB synthesis. After 7 days of growth under nitrogen-starved photoautotrophic conditions, the intracellular level of PHB was up to 4.1% of cellular dry weight and the PHB concentration in the culture broth was 27 mg/l.  相似文献   

12.
Summary. Among prokaryotes, cyanobacteria are unique in having highly differentiated internal membrane systems. Like other Gram-negative bacteria, cyanobacteria such as Synechocystis sp. strain PCC 6803 have a cell envelope consisting of a plasma membrane, peptidoglycan layer, and outer membrane. In addition, these organisms have an internal system of thylakoid membranes where the electron transfer reactions of photosynthesis and respiration occur. A long-standing controversy concerning the cellular ultrastructures of these organisms has been whether the thylakoid membranes exist inside the cell as separate compartments, or if they have physical continuity with the plasma membrane. Advances in cellular preservation protocols as well as in image acquisition and manipulation techniques have facilitated a new examination of this topic. We have used a combination of electron microscopy techniques, including freeze-etched as well as freeze-substituted preparations, in conjunction with computer-aided image processing to generate highly detailed images of the membrane systems in Synechocystis cells. We show that the thylakoid membranes are in fact physically discontinuous from the plasma membrane in this cyanobacterium. Thylakoid membranes in Synechocystis sp. strain PCC 6803 thus represent bona fide intracellular organelles, the first example of such compartments in prokaryotic cells. Supplementary material to this paper is available in electronic form at Correspondence and reprints: Department of Biology, CB1137, Washington University, St. Louis, MO 63130, U.S.A.  相似文献   

13.
The transport of putrescine into a moderately salt tolerant cyanobacterium Synechocystis sp. PCC 6803 was characterized by measuring the uptake of radioactively-labeled putrescine. Putrescine transport showed saturation kinetics with an apparent K(m) of 92 +/- 10 microM and V(max) of 0.33 +/- 0.05 nmol/min/mg protein. The transport of putrescine was pH-dependent with highest activity at pH 7.0. Strong inhibition of putrescine transport was caused by spermine and spermidine whereas only slight inhibition was observed by the addition of various amino acids. These results suggest that the transport system in Synechocystis sp. PCC 6803 is highly specific for polyamines. Putrescine transport is energy-dependent as evidenced by the inhibition by various metabolic inhibitors and ionophores. Slow growth was observed in cells grown under salt stress. Addition of low concentration of putrescine could restore growth almost to the level observed in the absence of salt stress. Upshift of the external osmolality generated by either NaCl or sorbitol caused an increased putrescine transport with an optimum 2-fold increase at 20 mosmol/kg. The stimulation of putrescine transport mediated by osmotic upshift was abolished in chloramphenicol-treated cells, suggesting possible involvement of an inducible transport system.  相似文献   

14.
Periplasmic proteins were obtained from control cells and salt-adapted cells of the cyanobacterium Synechocystis sp. PCC 6803 using the method of cold osmotic shock. Two of these proteins (PP 1, apparent mol. mass 27.6 kDa, and PP 3, apparent mol. mass 39.9 kDa) were accumulated in high amounts in the periplasm of salt-adapted cells, while the major periplasmic protein (PP 2, apparent mol. mass 36.0 kDa) was accumulated independently from salt. After isolation from gels and partial sequencing, the proteins could be assigned to proteins deduced from the complete genome sequence of Synechocystis. Neither salt-induced periplasmic proteins (PP 1, Slr0924 and PP 3, Slr1485) exhibited sequence similarity to proteins of known function from databases. The major protein (PP 2-Slr0513) showed significant sequence similarities to iron-binding proteins. All proteins included typical leader sequences at their N-terminus. Received: 21 September 1998 / Accepted: 17 December 1998  相似文献   

15.
In the cyanobacterium Synechocystis sp. PCC 6803 five open reading frames (scpAscpE) have been identified that code for single-helix proteins resembling helices I and III of chlorophyll a/b-binding (Cab) antenna proteins from higher plants. They have been named SCPs (small Cab-like proteins). Deletion of a single scp gene in a wild-type or in a photosystem I-less (PS I-less) strain has little effect. However, the effects of functional deletion of scpB or scpE were remarkable under conditions where chlorophyll availability was limited. When cells of a strain lacking PS I and chlL (coding for a polypeptide needed for light-independent protochlorophyllide reduction) were grown in darkness, the phycobilin and protochlorophyllide levels decreased upon deletion of scpB or scpE and the protoheme level was reduced in the strain lacking scpE. Addition of -aminolevulinic acid (ALA) in darkness drastically increased the level of Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester in the PS I-less/chlL /scpE strain, whereas PChlide accumulated in the PS I-less/chlL /scpB strain. In the PS I-less/chlL control strain ALA supplementation did not lead to large changes in the levels of tetrapyrrole biosynthesis intermediates. We propose that ScpE and ScpB regulate tetrapyrrole biosynthesis as a function of pigment availability. This regulation occurs primarily at an early step of tetrapyrrole biosynthesis, prior to ALA. In view of the conserved nature of chlorophyll-binding sites in these proteins, it seems likely that regulation by SCPs occurs as a function of chlorophyll availability, with SCPs activating chlorophyll biosynthesis steps when they do not have pigments bound.  相似文献   

16.
We have cloned and sequenced the dnaA region of Synechocystis sp. strain PCC6803, a bacterium with a light-dependent cell cycle. The dnaA gene product, DnaA, is the central factor for replication initiation in bacteria. The deduced amino acid sequence of the protein encoded by the cyanobacterial dnaA gene is 45% identical to DnaA of Bacillus subtilis and fits very well into the homology pattern of the known eubacterial DnaA proteins. The genetic environment of the Synechocystis sp. strain PCC6803 dnaA gene is completely different from the one in other eubacteria. An open reading frame of unknown function, orf134, was detected upstream of dnaA. The purT gene homolog encoding the glycinamide ribonucleotide transformylase T starts about 200 bp away from this open reading frame in the opposite direction. Downstream of the dnaA gene we detected the start of the psbDC operon, which codes for the photosystem II reaction center proteins D2 and CP43 that are involved in the positioning of chlorophyll a.  相似文献   

17.
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.  相似文献   

18.
The flavodi-iron proteins, also named FDPs, are an extensive family of enzymes able to reduce dioxygen to water and/or nitric oxide to nitrous oxide. These proteins are formed by a metallo-β-lactamase-like module with a di-iron catalytic site fused to a flavodoxin-like module bearing an FMN. However, in cyanobacteria, which are oxygenic photosynthetic organisms widespread in Nature, FDPs have an extra NAD(P)H:flavin reductase-like domain as a C-terminal extension. Interestingly, cyanobacteria contain more than one gene encoding FDP-like proteins, with the genome of Synechocystis sp. PCC6803 containing four genes coding for putative FDPs. However, the function of those proteins remains unclear. In the present study, we have analysed the expression profile of these genes under oxidative and nitrosative stress conditions. The results indicate that one of the flavodi-iron genes, the so-called flv1, is induced in cells exposed to nitrosative stress. By conducting a broad analysis on the primary sequences of FDPs, we have identified that the FDPs of cyanobacteria and oxygenic photosynthetic eukaryotes may be divided into multiple types (1-12), according to the amino acid residues of the di-iron catalytic site.  相似文献   

19.
S Aoki  T Kondo    M Ishiura 《Journal of bacteriology》1995,177(19):5606-5611
The expression of the dnaK gene in the cyanobacterium Synechocystis sp. strain PCC 6803 was continuously monitored as bioluminescence by an automated monitoring system, using the bacterial luciferase genes (luxAB) of Vibrio harveyi as a reporter of promoter activity. A dnaK-reporting bioluminescent Synechocystis strain was constructed by fusing a promoterless segment of the luxAB gene set downstream of the promoter region of the Synechocystis dnaK gene and introduction of this gene fusion into a BglII site downstream of the ndhB gene in the Synechocystis chromosome. Bioluminescence from this strain was continuously monitored and oscillated with a period of about 22 h for at least 5 days in continuous light. The phase of the rhythm was reset by the timing of the 12-h dark period administered prior to the continuous light. The period of the rhythm was temperature compensated between 25 and 35 degrees C. Thus, the bioluminescence rhythm satisfied the three criteria of circadian rhythms. Furthermore, the abundance of dnaK mRNA also oscillated with a period of about 1 day for at least 2 days in continuous light conditions, indicating circadian control of dnaK gene expression in Synechocystis sp. strain PCC 6803.  相似文献   

20.
To advance our knowledge of the model cyanobacterium Synechocystis sp. PCC 6803 we investigated the three-dimensional organization of the cytoplasm using standard transmission electron microscopy and electron tomography. Electron tomography allows a resolution of ~5 nm in all three dimensions, superior to the resolution of most traditional electron microscopy, which is often limited in part by the thickness of the section (70 nm). The thylakoid membrane pairs formed layered sheets that followed the periphery of the cell and converged at various sites near the cytoplasmic membrane. At some of these sites, the margins of thylakoid membranes associated closely along the external surface of rod-like structures termed thylakoid centers, which sometimes traversed nearly the entire periphery of the cell. The thylakoid membranes surrounded the central cytoplasm that contained inclusions such as ribosomes and carboxysomes. Lipid bodies were dispersed throughout the peripheral cytoplasm and often juxtaposed with cytoplasmic and thylakoid membranes suggesting involvement in thylakoid maintenance or biogenesis. Ribosomes were numerous and mainly located throughout the central cytoplasm with some associated with thylakoid and cytoplasmic membranes. Some ribosomes were attached along internal unit-membrane-like sheets located in the central cytoplasm and appeared to be continuous with existing thylakoid membranes. These results present a detailed analysis of the structure of Synechocystis sp. PCC 6803 using high-resolution bioimaging techniques and will allow future evaluation and comparison with gene-deletion mutants.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.  相似文献   

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