Wild-type Synechocystis Sp Research Articles

Ultrafast Primary Processes in PS I from Synechocystis sp. PCC 6803: Roles of P700 and A 0

The excitation transport and trapping kinetics of core antenna-reaction center complexes from photosystem I of wild-type Synechocystis sp. PCC 6803 were investigated under annihilation-free conditions in complexes with open and closed reaction centers. For closed reaction centers, the long-component decay-associated spectrum (DAS) from global analysis of absorption difference spectra excited at 660 nm is essentially flat (maximum amplitude <10 −5 absorbance units). For open reaction centers, the long-time spectrum (which exhibits photobleaching maxima at ∼680 and 700 nm, and an absorbance feature near 690 nm) resembles one previously attributed to (P700 + - P700). For photosystem I complexes excited at 660 nm with open reaction centers, the equilibration between the bulk antenna and far-red chlorophylls absorbing at wavelengths >700 nm is well described by a single DAS component with lifetime 2.3 ps. For closed reaction centers, two DAS components (2.0 and 6.5 ps) are required to fit the kinetics. The overall trapping time at P700 (∼24 ps) is very nearly the same in either case. Our results support a scenario in which the time constant for the P700 → A 0 electron transfer is 9–10 ps, whereas the kinetics of the subsequent A 0 → A 1 electron transfer are still unknown.

Type IV pilus biogenesis and motility in the cyanobacterium Synechocystis sp. PCC6803.

We have recently shown that phototactic movement in the unicellular cyanobacterium Synechocystis sp. PCC6803 requires type IV pilins. To elucidate further type IV pilus-dependent motility, we inactivated key genes implicated in pilus biogenesis and function. Wild-type Synechocystis sp. PCC6803 cells have two morphologically distinct pilus types (thick and thin pili). Of these, the thick pilus morphotype, absent in a mutant disrupted for the pilin-encoding pilA1 gene, appears to be required for motility. The thin pilus morphotype does not appear to be altered in the pilA1 mutant, raising the possibility that thin pili have a function distinct from that of motility. Mutants disrupted for pilA2, which encodes a second pilin-like protein, are still motile and exhibit no difference in morphology or density of cell-surface pili. In contrast, inactivation of pilD (encoding the leader peptidase) or pilC (encoding a protein required for pilus assembly) abolishes cell motility, and both pilus morphotypes are absent. Thus, the PilA1 polypeptide is required for the biogenesis of the thick pilus morphotype which, in turn, is necessary for motility (hence we refer to them as type IV pili). Furthermore, PilA2 is critical neither for motility nor for pilus biogenesis. Two genes encoding proteins with similarity to PilT, which is considered to be a component of the motor essential for type IV pilus-dependent movement, were also inactivated. A pilT1 mutant is (i) non-motile, (ii) hyperpiliated and (iii) accumulates higher than normal levels of the pilA1 transcript. In contrast, pilT2 mutants are motile, but are negatively phototactic under conditions in which wild-type cells are positively phototactic.

Dual-Mode EPR Detects the Initial Intermediate in Photoassembly of the Photosystem II Mn Cluster: The Influence of Amino Acid Residue 170 of the D1 Polypeptide on Mn Coordination

We report the first parallel polarization EPR signal from the Mn(III) ion formed by photooxidation of Mn(II) bound at the high affinity Mn-binding site of photosystem II (PSII). This species corresponds to the first photoactivation intermediate formed on the pathway to assembly of the water-splitting Mn cluster. The parallel mode EPR spectrum of the photooxidation product of 1.2/1 stoichiometry Mn(II)/Mn-depleted wild-type Synechocystis sp. PCC 6803 PSII particles consists of six well-resolved transitions split by a relatively small 55Mn hyperfine coupling (44 G). This spectral signature is absent in photooxidized Mn apoPSII complexes prepared from D1-Asp170Glu and D1-Asp170His mutants, providing direct spectral evidence for a role for this specific D1-Asp170 residue in the initial photoactivation chemistry. Temperature-dependence measurements and spectral simulations performed on the Mn(III) parallel mode EPR signal of the wild-type sample give an axial zero-field splitting value of D ≈ −2.5 cm-1 and a r...

Inactivation of the Open Reading Frame slr0399 in Synechocystis sp. PCC 6803 Functionally Complements Mutations near the QA Niche of Photosystem II: A POSSIBLE ROLE OF Slr0399 AS A CHAPERONE FOR QUINONE BINDING

The Synechocystis sp. PCC 6803 triple mutant D2R8 with V247M/A249T/M329I mutations in the D2 subunit of the photosystem II is impaired in Q(A) function, has an apparently mobile Q(A), and is unable to grow photoautotrophically. Several photoautotrophic pseudorevertants of this mutant have been isolated, each of which retained the original psbDI mutations of D2R8. Using a newly developed mapping technique, the site of the secondary mutations has been located in the open reading frame slr0399. Two different nucleotide substitutions and a deletion of about 60% of slr0399 were each shown to restore photoautotrophy in different pseudorevertants of the mutant D2R8, suggesting that inactivation of Slr0399 leads to photoautotrophic growth in D2R8. Indeed, a targeted deletion of slr0399 restores photoautotrophy in D2R8 and in other psbDI mutants impaired in Q(A) function. Slr0399 is similar to the hypothetical protein Ycf39, which is encoded in the cyanelle genome of Cyanophora paradoxa; in the chloroplast genomes of diatoms, dinoflagellates, and red algae; and in the nuclear genome of Arabidopsis thaliana. Slr0399 and Ycf39 have a NAD(P)H binding motif near their N terminus and have some similarity to isoflavone reductase-like proteins and to a subunit of the eukaryotic NADH dehydrogenase complex I. Deletion of slr0399 in wild type Synechocystis sp. PCC 6803 has no significant phenotypic effects other than a decrease in thermotolerance under both photoautotrophic and photomixotrophic conditions. We suggest that Slr0399 is a chaperone-like protein that aids in, but is not essential for, quinone insertion and protein folding around Q(A) in photosystem II. Moreover, as the effects of Slr0399 are not limited to photosystem II, this protein may also be involved in assembly of quinones in other photosynthetic and respiratory complexes.

The role of ApcD and ApcF in energy transfer from phycobilisomes to PS I and PS II in a cyanobacterium

The role of the phycobilisome core components, ApcD and ApcF, in transferring energy from the phycobilisome to PS I and PS II in the cyanobacterium Synechocystis sp. PCC6803 has been investigated. The genes encoding these proteins have been disrupted in the genomes of wild type Synechocystis sp. PCC6803 and a PS II deficient mutant, PsbD1CD2-, by inserting antibiotic resistance genes into their coding regions. Data from fluorescence emission spectra and pigment content analysis for these inactivation mutants is presented. These data suggest that both ApcD and ApcF are involved in the energy transfer route to PS II and PS I. In both cases, the energy transfer may to the reaction centres may be via the chromophore of ApcE (the L cm) or anchor polypeptide). The major route of energy transfer to both kinds of reaction centre appears to involve ApcF rather than ApcD. When both ApcF and ApcD are absent, the phycobilisomes are unable to transfer energy to either reaction centre. We suggest a model for the pathways of energy transfer from the phycobilisomes to PS I and PS II.

Yz. reduction kinetics in the absence of the manganese-stabilizing protein of photosystem II.

Decay of Signal IIvf of photosystem II (PSII), under repetitive flash conditions, was examined in whole cells of wild-type Synechocystis sp. PCC6803 and in cells of an engineered strain, delta psbO, which lacks the extrinsic 33 kDa manganese-stabilizing protein (MSP). Previous polarographic analysis had shown that O2 release during the S3-->[S4]-->S0 transition of the catalytic cycle is significantly retarded in the delta psbO strain relative to the wild-type [Burnap et al. (1992) Biochemistry 31, 7404-7410]. The present experiments provide evidence that a parallel retardation in the rate of reduction of photooxidized Yz by the H2O oxidation complex is due to the absence of MSP. The half-time of the Signal IIvf component, corresponding to Yz. reduction during the S3-->[S4]-->S0 transition, was estimated to be 1.2 and 6.0 ms in the wild-type and delta psbO cells, respectively.

Structure-function studies on the interaction of PsaC with the Photosystem 1 heterodimer

The interaction of PsaC with the core heterodimer of Photosystem 1 was studied in wild type Synechocystis sp. PCC 6803 and the site-directed mutant R561E of PsaB. The mutant reaction center was much less stable in urea and the functional reconstitution of the mutant core using PsaC was impaired. However, the extent of reconstitution increased in the presence of divalent cations whereas that of the wild type was inhibited. We conclude that the reaction center in the mutant is unstable, most likely due to the introduction of an unfavorable electrostatic interaction between surface-exposed residues on PsaC and the binding site for the subunit on the core heterodimer in support of the model proposed previously (Rodday et al. (1993) Photosynth Res 36: 1-9).

Regulation of Anabaena sp. strain PCC 7120 glutamine synthetase activity in a Synechocystis sp. strain PCC 6803 derivative strain bearing the Anabaena glnA gene and a mutated host glnA gene.

The glnA gene from Synechocystis sp. strain PCC 6803 was cloned by hybridization with the glnA gene from Anabaena sp. strain PCC 7120, and a deletion-insertion mutation of the Synechocystis gene was generated in vitro. A strain derived from Synechocystis sp. strain PCC 6803 which contained integrated into the chromosome, in addition to its own glnA gene, the Anabaena glnA gene was constructed. From that strain, a Synechocystis sp. glnA mutant could be obtained by transformation with the inactivated Synechocystis glnA gene; this mutant grew by using Anabaena glutamine synthetase and was not a glutamine auxotroph. A Synechocystis sp. glnA mutant could not be obtained, however, from the wild-type Synechocystis sp. The Anabaena glutamine synthetase enzyme was subject to ammonium-promoted inactivation when expressed in the Synechocystis strain but not in the Anabaena strain itself.

Sequence conservation among the glucose transporter from the cyanobacterium Synechocystis sp. PCC 6803 and mammalian glucose transporters

Synechocystis sp. PCC 6803 is capable of facultative photoheterotrophy with glucose as the sole carbon source. Eight mutants that were unable to take up glucose were transformed with plasmids from pooled gene banks of wild-type Synechocystis DNA prepared in an Escherichia coli vector that does not replicate in Synechocystis. One mutant (EG216) could be complemented with all gene banks to restore ability for photoheterotrophic growth. One of the gene banks was fractionated into single clones and plasmid DNA from each clone used to complement EG216. This yielded a 1.5 kb DNA fragment that was sequenced. It contained one complete open reading frame (gtr) whose putative gene product displayed high sequence conservation with the xylose transporter of E. coli and the mammalian glucose transporters. Further, the isolated gtr gene interrupted in vitro by a kanamycin resistance cassette could be used to construct mutants from wild-type Synechocystis sp. PCC 6803 that lacked a functional glucose transporter, thus confirming the identity of the gtr gene with the glucose transporter gene. This is the first prokaryotic glucose transporter known to share a sequence relationship with mammalian glucose transporters and the first sugar transporter from a cyanobacterium characterized at the sequence level.