Complete genome structure of the unicellular cyanobacterium Synechocystis sp. PCC6803

Cyanobacteria are photoautotrophic organisms capable of oxygen-producing photosynthesis similar to that in eukaryotic algae and plants, and because of this, they have been used as model organisms for the study of the mechanism and regulation of oxygen-producing photosynthesis. To understand the entire genetic system in cyanobacteria, the nucleotide sequence of the entire genome of the unicellular cyanobacterium Synechocystis sp. PCC6803 has been determined. The total length of the circular genome is 3,573,470 bp, with a GC content of 47.7%. A total of 3,168 potential protein coding genes were assigned. Of these, 145 (4.6%) were identical to reported genes, and 1,259 (39.6%) and 342 (10.8%) showed similarity to reported and hypothetical genes, respectively. The remaining 1,422 (45.0%) showed no apparent similarity to any genes registered in the databases. Classification of the genes by their biological function and comparison of the gene complement with those of other organisms have revealed a variety of features of the genetic information characteristic of a photoautotrophic organism. The sequence data, as well as other information on the Synechocystis genome, is presented in CyanoBase on WWW [http:/(/)www.kazusa.or.jp/cyano/].     

Circadian rhythm of the cyanobacterium Synechocystis sp. strain PCC 6803 in the dark

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.     

Topography of the photosystem I core proteins of the cyanobacterium Synechocystis sp. PCC 6803

PsaA and PsaB are homologous integral membrane proteins that form the heterodimeric core of photosystem I. Domain-specific antibodies were generated to examine the topography of PsaA and PsaB. The purified photosystem I complexes from the wild type strain of Synechocystis sp. PCC 6803 were treated with eight proteases to study the accessibility of cleavage sites in PsaA and PsaB. Proteolytic fragments were identified using the information from N-terminal amino acid sequencing, reactivity to antibodies, apparent mass, and specificity of proteases. The extramembrane loops of PsaA and PsaB differed in their accessibility to proteases, which indicated the folded structure of the loops or their shielding by the small subunits of photosystem I. NaI-treated and mutant photosystem I complexes were used to identify the extramembrane loops that were exposed in the absence of specific small subunits. The absence of PsaD exposed additional proteolytic sites in PsaB, whereas the absence of PsaE exposed sites in PsaA. These studies distinguish PsaA and PsaB in the structural model for photosystem I that has been proposed on the basis of x-ray diffraction studies (Krauss, N., Schubert, W.-D., Klukas, O., Fromme, P., Witt, H. T., and Saenger, W. (1996) Nat. Struct. Biol. 3, 965-973). Using osmotically shocked cells for protease treatments, the N terminus of PsaA was determined to be on the n side of the photosynthetic membranes. Based on these data and available published information, we propose a topological model for PsaA and PsaB.     

Directed inactivation of the psbI gene does not affect photosystem II in the cyanobacterium Synechocystis sp. PCC 6803

PsbI is a small, integral membrane protein component of photosystem II (PSII), a pigment-protein complex in cyanobacteria, algae and higher plants. To understand the function of this protein, we have isolated the psbI gene from the unicellular cyanobacterium Synechocystis sp. PCC 6803 and determined its nucleotide sequence. Using an antibiotic-resistance cartridge to disrupt and replace the psbI gene, we have created mutants of Synechocystis 6803 that lack the PsbI protein. Analysis of these mutants revealed that absence of the PsbI protein results in a 25-30% loss of PSII activity. However, other PSII polypeptides are present in near wild-type amounts, indicating that no significant destabilization of the PSII complex has occurred. These results contrast with recently reported data indicating that PsbI-deficient mutants of the eukaryotic alga Chlamydomonas reinhardtii are highly light-sensitive and have a significantly lower (80-90%) titer of the PSII complex. In Synechocystis 6803, PsbI-deficient cells appear to be slightly more photosensitive than wild-type cells, suggesting that this protein, while not essential for PSII biogenesis or function, plays a role in the optimization of PSII activity.     

A new type of asymmetrically acting beta-carotene ketolase is required for the synthesis of echinenone in the cyanobacterium Synechocystis sp. PCC 6803

We have isolated, based on the knowledge of the complete genomic sequence of the cyanobacterium Synechocystis sp. PCC 6803, an open reading frame (slr0088) similar to known bacterial carotene desaturases and have analyzed the function of the encoded protein. Surprisingly, this protein has no detectable desaturase activity with phytoene, hydroxyneurosporene, or zeta-carotene as substrates, but is rather a beta-carotene ketolase that acts asymmetrically introducing a keto group on only one of the two beta-ionone rings of beta-carotene to generate echinenone. This is in contrast to the so far characterized beta-carotene ketolases that act symmetrically, producing the di-keto carotenoid canthaxanthin from beta-carotene without significant accumulation of echinenone. We have designated this new gene crtO. The function of the crtO gene product has been demonstrated by 1) the biosynthesis of echinenone when the crtO gene is expressed in an Escherichia coli strain able to accumulate beta-carotene, 2) the in vitro biosynthesis of echinenone from beta-carotene with cell free extracts from E. coli cells that express the crtO gene, and 3) the absence of echinenone in a Synechocystis strain in which the crtO gene has been insertionally inactivated. The primary structure of the Synechocystis asymmetric ketolase bears no similarity with the known beta-carotene ketolases. crtO is not required for normal growth under standard or high light conditions, neither is the photosynthetic activity of the crtO-deficient strain affected.     

Light-dependent chlorophyll a biosynthesis upon chlL deletion in wild-type and photosystem I-less strains of the cyanobacterium Synechocystis sp. PCC 6803

Part of the chlL gene encoding a component involved in light-independent protochlorophyllide reduction was deleted in wild type and in a photosystem I-less strain of Synechocystis sp. PCC 6803. In resulting mutants, chlorophyll biosynthesis was fully light-dependent. When these mutants were propagated under light-activated heterotrophic growth conditions (in darkness except for 15 min of weak light a day) for several weeks, essentially no chlorophyll was detectable but protochlorophyllide accumulated. Upon return of the chlL- mutant cultures to continuous light, within the first 6 h chlorophyll was synthesized at the expense of protochlorophyllide at a rate independent of the presence of photosystem I. Chlorophyll biosynthesized during this time gave rise to a 685 nm fluorescence emission peak at 77 K in intact cells. This peak most likely originates from a component different from those known to be directly associated with photosystems II and I. Development of 695 and 725 nm peaks (indicative of intact photosystem II and photosystem I, respectively) required longer exposures to light. After 6 h of greening, the rate of chlorophyll synthesis slowed as protochlorophyllide was depleted. In the chlL- strain, greening occurred at the same rate at two different light intensities (5 and 50 microE m-2 s-1), indicating that also at low light intensity the amount of light is not rate-limiting for protochlorophyllide reduction. Thus, in this system the rate of chlorophyll biosynthesis is limited neither by biosynthesis of photosystems nor by the light-dependent protochlorophyllide reduction. We suggest the presence of a chlorophyll-binding 'chelator' protein (with 77 K fluorescence emission at 685 nm) that binds newly synthesized chlorophyll and that provides chlorophyll for newly synthesized photosynthetic reaction centers and antennae.     

Mutagenesis of the b'-subunit of Synechocystis sp. PCC 6803 ATP-synthase

We investigated the F0F1 ATP synthase of the cyanobacterium, Synechocystis sp. PCC 6803. The gene for the F0-subunit b', a peptide probably located at the interface between F0 and F1, has been partially or completely evicted from the bacterial genome. We found that the complete deletion of the subunit was lethal to the cells. However, the subunit could be truncated down to its hydrophobic N-terminal stretch without much harm. Since the gene for b' probably shares a common ancestor with the gene for subunit b and emerged by gene duplication, we propose that b' gathered a new role during evolution, perhaps in the regulation of photophosphorylation.     

Cotranscription of a GTPase gene from the cyanobacterium Synechocystis PCC 6803 and a P-type Ca(2+)-ATPase gene

Near-infrared spectroscopy is a technique used to monitor cerebral oxygenation. To validate the method, we measured regional oxygen saturation (rSo2) in the brains of 18 dead subjects (mean age, 74.4 +/- 14.6 years) 19.8 +/- 18.2 h (range, 1-73) after cessation of systemic circulation, and in 15 healthy probands (mean age, 34.2 +/- 8.7 years) with an INVOS 3100 cerebral oximeter. The mean (+/-SD) rSo2 in the dead subjects was 51.0 +/- 26.8% [range, 6-88%; left, 48.4 +/- 28.0% (n = 21); right, 54.4 +/- 25.7% (n = 16)]. The mean rSo2 in the control group was 68.4 +/- 5.2% (range, 60-76%; left, 68.1 +/- 5.0%; right, 68.7 +/- 5.6%). After removal of the brain at autopsy in five of the dead subjects, the rSo2 was 73.4 +/- 13.3% (15 measurements). Six of 18 of the dead subjects had values above the lowest values found in the healthy adults (> or = 60%). These findings raise concerns about the validity of cerebral rSo2 data in adults obtained by the INVOS 3100 system.     

The temperature-dependent expression of the desaturase gene desA in Synechocystis PCC6803

Oligonucleotide probes specific for 16S rRNA and capable of differentiating Streptococcus uberis and S. parauberis from each other and other esculin-hydrolyzing streptococci were developed. Use of a mini-RNA extraction technique for gram-positive cocci associated with bovine mastitis has allowed the probes to be used for identification of esculin-hydrolyzing streptococci from two dairy herds at the Institute for Animal Health, Compton, United Kingdom. One hundred seventy-nine of 206 isolates were identified as S. uberis, 3 were identified as S. parauberis, and 24 were not identified. Isolates not identified by the probes were tested biochemically and found to be mainly Enterococcus faecium, E. faecalis, or S. bovis.     

Site-specific mutations of the N-terminal threonines in the D1 protein affects photoautotrophic growth but not D1 protein stability in Synechocystis 6803

OBJECT: The goal of this study was to identify the neurological characteristics of patients with poststroke pain who show a favorable response to motor cortex (MC) stimulation used to control their pain. METHODS: The neurological characteristics of 31 patients treated by MC stimulation were analyzed. In 15 patients (48%), excellent or good pain control (pain reduction > 60%) was achieved for follow-up periods of more than 2 years by using MC stimulation at intensities below the threshold for muscle contraction. Satisfactory pain control was achieved in 13 (73%) of 18 patients in whom motor weakness in the painful area was virtually absent or mild, but in only two (15%) of the 13 patients who demonstrated moderate or severe weakness in the painful area (p < 0.01). Muscle contraction was inducible in the painful area in 20 patients when stimulated at a higher intensity. No such muscle response was inducible in the remaining 11 patients, no matter how extensively the authors attempted to determine appropriate stimulation sites. Satisfactory pain control was achieved in 14 (70%) of the 20 patients in whom muscle contraction was inducible, but in only one (9%) of the 11 patients in whom muscle contraction was not inducible (p < 0.01). No significant relationship was observed between pain control and various sensory symptoms, including the presence of hypesthesia, spontaneous dysesthesia, hyperpathia, and allodynia, or the disappearance of the N20 component of the median nerve-evoked somatosensory scalp potential. No significant relationship existed between the effect of MC stimulation on the pain and stimulation-induced phenomena, including paresthesia, improvement in motor performance, and attenuation of involuntary movements. CONCLUSION: These findings suggest that the pain control afforded by MC stimulation requires neuronal circuits that are maintained by the presence of intact corticospinal tract neurons originating from the MC. Preoperative evaluation of motor weakness of the painful area appears to be useful for predicting a favorable response to MC stimulation in the control of poststroke pain.     

The cyanobacterium Synechocystis sp. strain PCC 6803 expresses a DNA methyltransferase specific for the recognition sequence of the restriction endonuclease PvuI

By use of restriction endonucleases, the DNA of the cyanobacterium Synechocystis sp. strain PCC 6803 was analyzed for DNA-specific methylation. Three different recognition sites of methyltransferases, a dam-like site including N6-methyladenosine and two other sites with methylcytosine, were identified, whereas no activities of restriction endonucleases could be detected in this strain. slr0214, a Synechocystis gene encoding a putative methyltransferase that shows significant similarities to C5-methylcytosine-synthesizing enzymes, was amplified by PCR and cloned for further characterization. Mutations in slr0214 were generated by the insertion of an aphII gene cassette. Analyses of chromosomal DNAs of such mutants demonstrated that the methylation pattern was changed. The recognition sequence of the methyltransferase was identified as 5'-CGATCG-3', corresponding to the recognition sequence of PvuI. The specific methyltransferase activity was significantly reduced in protein extracts obtained from mutant cells. Mutation of slr0214 also led to changed growth characteristics of the cells compared to wild-type cells. These alterations led to the conclusion that the methyltransferase Slr0214 might play a regulatory role in Synechocystis. The Slr0214 protein was also overexpressed in Escherichia coli, and the purified protein demonstrated methyltransferase activity and specificity for PvuI recognition sequences in vitro. We propose the designation M.Ssp6803I [corrected] (Synechocystis methyltransferase I) for the slr0214-encoded enzyme.     

Construction and characterization of a phycobiliprotein-less mutant of Synechocystis sp. PCC 6803

A mutant strain of the cyanobacterium Synechocystis PCC 6803, called PAL, (PC-, delta apcAB, delta apcE), lacking phycocyanin, allophycocyanin and the core-membrane linker (Lcm), was constructed. The strain was characterized by absorption and fluorescence spectroscopy. The mutant compensates for the absence of the major PS II antenna by increasing its PS II/PS I ratio. It is stable and grows well albeit more slowly than wild type.     

Changes in the reaction mechanism of electron transfer from plastocyanin to photosystem I in the cyanobacterium Synechocystis sp. PCC 6803 as induced by site-directed mutagenesis of the copper protein

The kinetic mechanism of plastocyanin oxidation by photosystem I in the cyanobacterium Synechocystis sp. PCC 6803 is drastically changed by modifying the metalloprotein by site-directed mutagenesis. The mutations herein considered concern four specific residues, two in the east face and the other two in the hydrophobic patch of plastocyanin. The first set of mutants include D44A, D44K, D47A, and D47R, as well as the double mutants D44A/D47A and D44R/D47R; the second set consists of L12A and K33E. The kinetic efficiency of all these mutant plastocyanins has been analyzed by laser-flash absorption spectroscopy. The plastocyanin concentration dependence of the observed electron transfer rate constant (kobs) is linear with most mutant plastocyanins, as with wild-type plastocyanin, but exhibits a saturation plateau at high protein concentration with the double mutant D44R/D47R, which suggests the formation of a plastocyanin-PSI transient complex. The effect of ionic strength on kobs varies from the wild-type plastocyanin to some of the mutants, for instance D44K, for which the salt concentration dependence of kobs is just the reverse as compared to the wild-type protein. The ionic strength dependence of kobs with D44R/D47R exhibits a bell-shaped profile, which is similar to that of green algae and higher plants. These findings indicate that the double mutant D44R/D47R follows a reaction mechanism involving not only complex formation with PSI but also further reorientation to properly accommodate the redox centers prior to electron transfer, as is the case in most evolved species, whereas the wild-type copper protein reacts with PSI by following a simple collisional kinetic model.     

Effect of dimethoate on photosynthesis and pigment fluorescence of Synechocystis sp. PCC 6803

The phylogenetic relationships of gibbons are still open questions. We have sequenced a mitochondrial cytochrome b gene fragment from Hylobates hoolock, H. concolor, H. lar and H. syndactylus. Combined with the sequences from Garza and Woodruff (1992), we have constructed a comprehensive phylogenetic tree of the gibbons using the maximum-parsimony analysis. Our results suggested that the gibbons should be divided into four groups: (1) hoolock, (2) syndactylus, (3) agilis, lar, muelleri and klossi, and (4) concolor, which correspond to the four morphological subgenera. There are at least four distinct clades in the concolor population, which indicates that the concolor may be divided into at least four species. Therefore, those four clades should be managed separately with the same conservation effort.