Monitoring photosynthesis in individual cells of Synechocystis sp. PCC 6803 on a picosecond timescale.

Picosecond fluorescence kinetics of wild-type (WT) and mutant cells of Synechocystis sp. PCC 6803, were studied at the ensemble level with a streak-camera and at the cell level using fluorescence-lifetime-imaging microscopy (FLIM). The FLIM measurements are in good agreement with the ensemble measurements, but they (can) unveil variations between and within cells. The BE mutant cells, devoid of photosystem II (PSII) and of the light-harvesting phycobilisomes, allowed the study of photosystem I (PSI) in vivo for the first time, and the observed 6-ps equilibration process and 25-ps trapping process are the same as found previously for isolated PSI. No major differences are detected between different cells. The PAL mutant cells, devoid of phycobilisomes, show four lifetimes: ∼20 ps (PSI and PSII), ∼80 ps, ∼440 ps, and 2.8 ns (all due to PSII), but not all cells are identical and variations in the kinetics are traced back to differences in the PSI/PSII ratio. Finally, FLIM measurements on WT cells reveal that in some cells or parts of cells, phycobilisomes are disconnected from PSI/PSII. It is argued that the FLIM setup used can become instrumental in unraveling photosynthetic regulation mechanisms in the future.

Synechocystis strain PCC 6803 cya2, a prokaryotic gene that encodes a guanylyl cyclase.

Synechocystis strain PCC 6803 exhibits similar levels of cyclic AMP (cAMP) and cyclic GMP (cGMP). A thorough analysis of its genome showed that Cya2 (Sll0646) has all the sequence determinants required in terms of activity and purine specificity for being a guanylyl cyclase. Insertional mutagenesis of cya2 caused a marked reduction in cGMP content without altering the cAMP content. Thus, Cya2 represents the first example of a prokaryotic guanylyl cyclase.

Deletion of the PB-loop in the L(CM) subunit does not affect phycobilisome assembly or energy transfer functions in the cyanobacterium Synechocystis sp. PCC6714.

In cyanobacteria, light energy is mainly harvested for photosynthesis by the phycobilisome (PBS): a large pigment-protein complex. This complex is composed of heterodimeric phycobiliproteins that are assembled with the aid of linker polypeptides in order to optimize light-energy absorbance and transfer to photosystem II. The core membrane linker subunit (L(CM)) is a fascinating multifunctional polypeptide that participates in the PBS structure, function and anchoring to the photosynthetic membrane. Sequence analysis has defined several domains within the L(CM) polypeptide. The C-terminal portion contains two to four repeated domains that are similar to the conserved domains of linker polypeptides and are believed to play the same role. The N-terminal portion is similar to phycobiliproteins (PB-domain) and carries, like phycobiliproteins, a covalently linked phycobilin chromophore. This domain is interrupted by a so-called PB-loop insertion. The PB-domain of the L(CM) is thus regarded as one of the core subunits, with its PB-loop protruding towards the photosynthetic membrane. The PB-loop was thought to be involved in the attachment of the PBS to the photosynthetic membrane. We generated an apcE gene (encoding L(CM)), in which we deleted the sequence encoding 54 amino acids of the PB-loop domain. The modified gene was expressed in a Synechocystis PCC6714 strain in which the apcE gene had been inactivated. The truncated polypeptide was functionally equivalent to the wild-type L(CM); PBSs were assembled and functioned as in the wild-type. The PB-loop of the L(CM) seems thus dispensable for the PBS biogenesis and function.

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.

P-type ATPase from the cyanobacterium Synechococcus 7942 related to the human Menkes and Wilson disease gene products.

DNA encoding a P-type ATPase was cloned from the cyanobacterium Synechococcus 7942. The cloned ctaA gene encodes a 790-amino acid polypeptide related to the CopA Cu(2+)-uptake ATPase of Enterococcus hirae, to other known P-type ATPases, and to the candidate gene products for the human diseases of copper metabolism, Menkes disease and Wilson disease. Disruption of the single chromosomal gene in Synechococcus 7942 by insertion of an antibiotic-resistance cassette results in a mutant cell line with increased tolerance to Cu2+ compared with the wild type.

Changes in the photosynthetic apparatus in the cyanobacterium Synechocystis sp. PCC 6714 following light-to-dark and dark-to-light transitions.

The photosynthetic apparatus of Synechocystis sp. PCC 6714 cells grown chemoheterotrophically (dark with glucose as a carbon source) and photoautotrophically (light in a mineral medium) were compared. Dark-grown cells show a decrease in phycocyanin content and an even greater decrease in chlorophyll content with respect to light-grown cells. Analysis of fluorescence emission spectra at 77 K and at 20 °C, of dark- and light-grown cells, and of phycobilisomes isolated from both types of cells, indicated that in darkness the phycobiliproteins were assembled in functional phycobilisomes (PBS). The dark synthesized PBS, however, were unable to transfer their excitation energy to PS II chlorophyll. Upon illumination of dark-grown cells, recovery of photosynthetic activity, pigment content and energy transfer between PBS and PS II was achieved in 24-48 h according to various steps. For O2 evolution the initial step was independent of protein synthesis, but the later steps needed de novo synthesis. Concerning recovery of PBS to PS II energy transfer, light seems to be necessary, but neither PS II functioning nor de novo protein synthesis were required. Similarly, light, rather than functional PS II, was important for the recovery of an efficient energy transfer in nitrate-starved cells upon readdition of nitrate. In addition, it has been shown that normal phycobilisomes could accumulate in a Synechocystis sp. PCC 6803 mutant deficient in Photosystem II activity.

A new ioxynil-resistant mutant in Synechocystis PCC 6714: hypothesis on the interaction of ioxynil with the D 1 protein.

A new Synechocystis 6714 mutant, IoxIIA, resistant to the phenol-type herbicide ioxynil was isolated and characterized. The mutation found in the psbA gene (encoding the D 1 photosystem II protein) is at the same codon 266 as for the first ioxynil-resistant mutant IoxIA previously selected [G. Ajlani, I. Meyer, C. Vernotte, and C. Astier, FEBS Lett. 246, 207-210 (1989)]. In IoxIIA, the change of Asn 266 to Asp gives a 3 x resistance, whereas in IoxIA, the change of the same amino acid to Thr gives a 10 x resistance. The effect of these different amino acid substitutions on the ioxynil resistance phenotype has allowed us to construct molecular models and calculate the hydrogen-bonding energies between the hydroxyl group of ioxynil and the respective amino acids at position 266.

Molecular analysis of psbA mutations responsible for various herbicide resistance phenotypes in Synechocystis 6714.

Mutations conferring herbicide resistance in 3 mutant strains of the cyanobacterium Synechocystis 6714 have been characterized by gene cloning and sequencing. The mutants display very different phenotypes: DCMU-IIA is DCMU-resistant and atrazine-resistant, DCMU-IIB is DCMU-resistant and atrazine-sensitive, and Az-V is DCMU-sensitive, atrazine-resistant and presents particular photoinhibition properties. These mutants were originally obtained either by one-step selection (DCMU-IIA) or by two-step selection (DCMU-IIB and Az-V). psbA copies carrying herbicide resistance have been identified by transformation experiments as psb AI in all cases. Sequences of the psb AI copy of each mutant have been compared to the wild-type sequence. In the single mutant DCMU-IIA, a point mutation at codon 264 (Ser----Ala) results in resistance to both DCMU and atrazine. In the double mutants DCMU-IIB and Az-V, two point mutations were found. DCMU-IIB was derived from DCMU-IIA and had acquired a second mutation at codon 255 (Phe----Leu) resulting in a slight increase in DCMU resistance and complete abolition of atrazine resistance. Az-V contains two changes at codons 211 (Phe----Ser) and 251 (Ala----Val) resulting in high atrazine resistance but only slight DCMU resistance.

Mutations responsible for high light sensitivity in an atrazine-resistant mutant of Synechocystis 6714.

The primary target of photoinhibition is the photosystem II reaction center. The process involves a reversible damage, followed by an irreversible inhibition of photosystem II activity. During cell exposition to high light intensity, the D1 protein is specially degraded. An atrazine-resistant mutant of Synechocystis 6714, AzV, reaches the irreversible step of photoinhibition faster than wild-type cells. Two point mutations present in the psbA gene of AzV (coding for D1) lead to the modification of Phe 211 to Ser and Ala 251 to Val in D1. Transformation of wild-type cells with the AzV psbA gene shows that these two mutations are sufficient to induce a faster photodamage of PSII. Other DCMU- and/or atrazine-resistant mutants do not differ from the wild type when photoinhibited. We conclude that the QB pocket is involved in PSII photodamage and we propose that the mutation of Ala 251 might be related to a lower rate of proteolysis of the D1 protein than in the wild type.

Mutation in phenol-type herbicide resistance maps within the psbA gene in Synechocystis 6714.

A Synechocytis 6714 mutant resistant to the phenol-type herbicide ioxynil was isolated and characterized. Sensitivity to DCMU and atrazine was tf measured in whole cells and isolated thylakoids. The mutant presents the same sensitivity to atrazine as the wild type and a slightly increased sensitivity to DCMU. A point mutation has been found at codon 266 in the psbAI coding locus (AAC to ACC) resulting in an amino acid change from asparagine to threonine in the D1 protein.