Kinematic analysis of patients with spinal muscular atrophy during spontaneous breathing and mechanical ventilation

Dinoseb is a herbicide known to inhibit photosystem II electron transfer like DCMU, triazine and phenolic-type herbicides. The mutant Din7 of the cyanobacterium Synechocystis sp. PCC 6803, selected for resistance to dinoseb, and the mutant Ins2, constructed by the insertion of the kanamycin resistance cassette into the drgA gene, were cross-resistant to other nitrophenolic herbicides (DNOC, 2,4-dinitrophenol) and to the cell inhibitor metronidazole but not to the photosystem II inhibitors DCMU or ioxynil. The Din7 mutant had the same characteristics of photosystem II inhibition by dinoseb as the wild type. This result suggested the existence of another site for dinoseb inhibition. The wild type cells modified dinoseb to a non-toxic product that gave an absorption spectrum similar to that of dithionite treated dinoseb containing reduced nitro groups. In contrast, the Din7 mutant did not modify dinoseb. These phenomena were controlled by the drgA gene encoding a protein which showed similarity to several enzymes having nitroreductase activity. The addition of superoxide dismutase to the medium relieved the toxic effect of dinoseb in wild type cells but not in Din7. It is proposed that in wild type cells of Synechocystis sp. PCC 6803 the DrgA protein is involved in detoxification of dinoseb via the reduction of the nitro group(s) and this process is accompanied by the formation of toxic superoxide anions. Mutations blocking the activity of the DrgA protein lead to the development of resistance to nitrophenolic herbicides and metronidazole.     

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.     

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.     

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.     

In vitro ferredoxin-dependent desaturation of fatty acids in cyanobacterial thylakoid membranes

Thylakoid membranes isolated from the cyanobacterium Synechocystis sp. strain PCC6803 were capable of desaturating the acyl groups in monogalactosyl diacylglycerol. This desaturation reaction required the reduced form of ferredoxin.     

Analysis of the psbU gene encoding the 12-kDa extrinsic protein of photosystem II and studies on its role by deletion mutagenesis in Synechocystis sp. PCC 6803

The gene encoding the 12-kDa extrinsic protein of photosystem II from Synechocystis sp. PCC 6803 was cloned based on N-terminal sequence of the mature protein. This gene, named psbU, encodes a polypeptide of 131 residues, the first 36 residues of which were absent in the mature protein and thus served as a transit peptide required for its transport into the thylakoid lumen. A psbU gene deletion mutant grew photoautotrophically in normal BG11 medium at almost the same rate as that of the wild type strain. This mutant, however, grew apparently slower than the wild type did upon depletion of Ca2+ or Cl- from the growth medium. Photosystem II oxygen evolution decreased to 81% in the mutant as compared with that in the wild type, and the thermoluminescence B- and Q-bands shifted to higher temperatures accompanied by an increase in the Q-band intensity. These results indicate that the 12-kDa protein is not essential for oxygen evolution but may play a role in optimizing the ion (Ca2+ and Cl-) environment and maintaining a functional structure of the cyanobacterial oxygen-evolving complex. In addition, a double deletion mutant lacking cytochrome c-550 and the 12-kDa protein grew photoautotrophically with a phenotype identical to that of the single deletion mutant of cytochrome c-550. This supports our previous biochemical results that the 12-kDa protein cannot bind to photosystem II in the absence of cytochrome c-550 (Shen, J.-R., and Inoue, Y. (1993) Biochemistry 32, 1825-1832).     

Bacillus isolates from the spermosphere of peas and dwarf French beans with antifungal activity against Botrytis cinerea and Pythium species

ClpP functions as the proteolytic subunit of the ATP-dependent Clp protease in eubacteria, mammals and plant chloroplasts. We have cloned a clpP gene, designated clpP1, from the cyanobacterium Synechococcus sp. PCC 7942. The monocistronic 591 bp gene codes for a protein 80% similar to one of four putative ClpP proteins in another cyanobacterium, Synechocystis sp. PCC 6803. The constitutive ClpP1 content in Synechococcus cultures was not inducible by high temperatures, but it did rise fivefold with increasing growth light from 50 to 175 micromol photons m(-2) s(-1). A clpP1 inactivation strain (delta clpP1) exhibited slower growth rates, especially at the higher irradiances, and changes in the proportion of the photosynthetic pigments, chlorophyll a and phycocyanin. Many mutant cells (ca. 35%) were also severely elongated, up to 20 times longer than the wild type. The stress phenotype of delta clpP1 when grown at high light was confirmed by the induction of known stress proteins, such as the heat shock protein GroEL and the alternate form of PSII reaction center D1 protein, D1 form 2. ClpP1 content also rose significantly during short-term photoinhibition, but its loss in delta clpP1 did not exacerbate the extent of inactivation of photosynthesis, nor affect the inducible D1 exchange mechanism, indicating ClpP1 is not directly involved in D1 protein turnover.     

The ispB gene encoding octaprenyl diphosphate synthase is essential for growth of Escherichia coli

The Escherichia coli ispB gene encoding octaprenyl diphosphate synthase is responsible for the synthesis of the side chain of isoprenoid quinones. We tried to construct an E. coli ispB-disrupted mutant but could not isolate the chromosomal ispB disrupted mutant unless the ispB gene or its homolog was supplied on a plasmid. The chromosomal ispB disruptants that harbored plasmids carrying the ispB homologs from Haemophilus influenzae and Synechocystis sp. strain PCC6803 produced mainly ubiquinone 7 and ubiquinone 9, respectively. Our results indicate that the function of the ispB gene is essential for normal growth and that this function can be substituted for by homologs of the ispB gene from other organisms that produce distinct forms of ubiquinone.     

Ferredoxin reduction by photosystem I from Synechocystis sp. PCC 6803: toward an understanding of the respective roles of subunits PsaD and PsaE in ferredoxin binding

The process of ferredoxin reduction by photosystem I has been extensively investigated by flash-absorption spectroscopy in psaD and psaE deleted mutants from Synechocystis sp. PCC 6803. In both mutants, the dissociation constant for the photosystem I/ferredoxin complex at pH 8 is considerably increased as compared to the wild type: approximately 25- and 100-fold increases are found for PsaD-less and PsaE-less photosystem I, respectively. However, at high ferredoxin concentrations, submicrosecond and microsecond kinetics of electron transfer similar to that observed in the wild type are present in both mutants. The presence of these fast kinetic components indicates that the relative positions of ferredoxin and of the terminal photosystem I acceptor are not significantly disturbed by the absence of either PsaD or PsaE. The second-order rate constant of ferredoxin reduction is lowered 10- and 2-fold for PsaD-less and PsaE-less photosystem I, respectively. Assuming a simple binding equilibrium between photosystem I and ferredoxin, PsaD appears to be important for the guiding of ferredoxin to its binding site (main effect on the association rate) whereas PsaE seems to control the photosystem I/ferredoxin complex lifetime (main effect on the dissociation rate). The properties of electron transfer from photosystem I to ferredoxin were also studied at pH 5. 8. In the psaE deleted mutant as in the wild type, the change of pH from 8 to 5.8 induces a 10-fold increase in affinity of ferredoxin for photosystem I. In the absence of PsaD, this pH effect is not observed, in favor of this subunit being mostly responsible for the low pH increased affinity.     

Paternal care enhances male reproductive success in pine engraver beetles

His117 of the D2 protein of photosystem II (PS II) is a conserved residue in the second transmembrane region of the protein and has been suggested to bind chlorophyll. Nine site-directed mutations were introduced at residue 117, using both photosystem I (PS I)-containing and PS I-less background strains of the cyanobacterium Synechocystis sp. PCC 6803. Of these nine, four (H117C, H117M, H117N, and H117T) were photoautotrophic in the PS I-containing background. The other mutants (H117F, H117L, H117P, H117R, and H117Y) did not accumulate appreciable amounts of PS II in their thylakoids. The type of residues that can functionally replace His117 support the notion of His117 serving as a chlorophyll ligand. The properties of the H117N and H117T mutants were characterized in more detail. Whereas the properties of the H117N mutant were close to those of wild type, in the H117T mutant the 77-K fluorescence emission spectrum shows a much smaller amplitude at 695 nm than expected on the basis of the amount of PS II that is present. Moreover, in H117T, the amount of light needed to half-saturate O2-evolution rates was twofold higher than in the control strain, and the variable fluorescence yield was quenched. However, O2 evolution rates at saturating light intensity and electron-transport kinetics were normal in the mutant. Also, the radical accessory chlorophyll (Chlz+) formed by donation of an electron to the PS-II reaction center could be generated normally by illumination at low temperature in the H117T mutant. We conclude that the chlorophyll associated with residue 117 of the D2 protein is important for efficient excitation transfer between the proximal antenna and the PS II reaction center. A possible mechanism involving a chlorophyll cation to explain the quenching in the H117T mutant is discussed.     

Mutational analysis of the genes encoding the photosystem II proteins in Cyanobacterium synechocystis sp. 6803

Chlorophyll--binding protein CP43 and cytochrome b559, encoded by psbC and psbE/F genes, are the components of photosystem II (PS II). Three psbC- and four psbE/F- mutants were isolated from the collection of PS II-deficient mutants of the cyanobacterium Synechocystis sp. 6803. Restoration of photosynthetic activity was achieved by transformation of psbE/F- mutants with cloned psbE/F gene cluster from wild type cells and each of psbC- mutants--with specific part of wild type psbC gene. DNA fragments carrying the mutations were isolated from mutant cells and sequenced. The mutations which affect PS II activity were identified in psbC gene as "frameshift" mutation, stop-codon formation, or as deletion of three nucleotides resulting in loss of one of three Phe residues in position 422-424 of CP43. Sequence of mutant psbE/F genes revealed single mutations resulting in deletion of Phe-36 or substitution of Pro-63 for Leu in alpha-subunit and Val-29 for Phe in beta-subunit of cytochrome b559.