Sigma factor SigC is required for heat acclimation of the cyanobacterium Synechocystis sp. strain PCC 6803

The role of the primary-like sigma factor SigC was studied in Synechocystis. Under high temperature stress (48 degrees C) the DeltasigC inactivation strain showed a lower survival rate than the control strain. The DeltasigC strain grew poorly at 43 degrees C in liquid cultures under normal air. However, change to 3% CO(2) enhanced growth of DeltasigC at 43 degrees C. Differences in expression of many genes related to the carbon concentrating mechanisms between the control and the DeltasigC strain were recorded with a genome-wide DNA microarray. We suggest that low solubility of CO2 at high temperature is one of the factors contributing to the poor thermotolerance of the DeltasigC strain.     

Carbon status constrains light acclimation in the cyanobacterium Synechococcus elongatus

Acclimation to one environmental factor may constrain acclimation to another. Synechococcus elongatus (sp. PCC7942), growing under continuous light in high inorganic carbon (Ci; approximately 4 mm) and low-Ci (approximately 0.02 mm) media, achieve similar photosynthetic and growth rates under continuous low or high light. During acclimation from low to high light, however, high-Ci cells exploit the light increase by accelerating their growth rate, while low-Ci cells maintain the prelight shift growth rate for many hours, despite increased photosynthesis under the higher light. Under increased light, high-Ci cells reorganize their photosynthetic apparatus by shrinking the PSII pool and increasing Rubisco pool size, thus decreasing the photosynthetic source-to-sink ratio. Low-Ci cells also decrease their reductant source-to-sink ratio to a similar level as the high-Ci cells, but do so only by increasing their Rubisco pool. Low-Ci cells thus invest more photosynthetic reductant into maintaining their larger photosystem pool and increasing their Rubisco pool at the expense of population growth than do high-Ci cells. In nature, light varies widely over minutes to hours and is ultimately limited by daylength. Photosynthetic acclimation in S. elongatus occurs in both high and low Ci, but low-Ci cells require more time to achieve acclimation. Cells that can tolerate low Ci do so at the expense of slower photosynthetic acclimation. Such differences in rates of acclimation relative to rates of change in environmental parameters are important for predicting community productivity under variable environments.     

Essential role of glutathione in acclimation to environmental and redox perturbations in the cyanobacterium Synechocystis sp. PCC 6803

Glutathione, a nonribosomal thiol tripeptide, has been shown to be critical for many processes in plants. Much less is known about the roles of glutathione in cyanobacteria, oxygenic photosynthetic prokaryotes that are the evolutionary precursor of the chloroplast. An understanding of glutathione metabolism in cyanobacteria is expected to provide novel insight into the evolution of the elaborate and extensive pathways that utilize glutathione in photosynthetic organisms. To investigate the function of glutathione in cyanobacteria, we generated deletion mutants of glutamate-cysteine ligase (gshA) and glutathione synthetase (gshB) in Synechocystis sp. PCC 6803. Complete segregation of the ΔgshA mutation was not achieved, suggesting that GshA activity is essential for growth. In contrast, fully segregated ΔgshB mutants were isolated and characterized. The ΔgshB strain lacks reduced glutathione (GSH) but instead accumulates the precursor compound γ-glutamylcysteine (γ-EC). The ΔgshB strain grows slower than the wild-type strain under favorable conditions and exhibits extremely reduced growth or death when subjected to conditions promoting oxidative stress. Furthermore, we analyzed thiol contents in the wild type and the ΔgshB mutant after subjecting the strains to multiple environmental and redox perturbations. We found that conditions promoting growth stimulate glutathione biosynthesis. We also determined that cellular GSH and γ-EC content decline following exposure to dark and blue light and during photoheterotrophic growth. Moreover, a rapid depletion of GSH and γ-EC is observed in the wild type and the ΔgshB strain, respectively, when cells are starved for nitrate or sulfate.     

Designing and creating a modularized synthetic pathway in cyanobacterium Synechocystis enables production of acetone from carbon dioxide

Ketones are a class of important organic compounds. As the simplest ketone, acetone is widely used as solvents or precursors for industrial chemicals. Presently, million tonnes of acetone is produced worldwide annually, from petrochemical processes. Here we report a biotechnological process that can produce acetone from CO(2), by designing and creating a modularized synthetic pathway in engineered cyanobacterium Synechocystis sp. PCC 6803. The engineered Synechocystis cells are able to produce acetone (36.0 mgl(-1) culture medium) using CO(2) as the sole carbon source, thus opens the gateway for biosynthesis of ketones from CO(2).     

Photosynthetic acclimation of the filamentous cyanobacterium, Plectonema boryanum UTEX 485, to temperature and light

Photosynthetic acclimation to temperature and irradiance was studied in the filamentous, non-heterocystous cyanobacterium Plectonema boryanum UTEX 485. Growth rates of this cyanobacterium measured at ambient CO2 were primarily influenced by temperature with minimal effects of irradiance. Both growth temperature and irradiance affected linolenic (18:3) and linoleic acid (18:2) levels in the four major lipid classes in an independent but additive manner. In contrast, photosynthetic acclimation was not due to either growth temperature or irradiance per se, but rather, due to the interaction of these environmental factors. P. boryanum grown at low temperature and moderate irradiance mimicked cells grown at high light. Compared to cells grown at either 29 degrees C/150 micromol m(-2) s(-1) (29/150) or 15/10, P. boryanum grown at either 15/150 or 29/750 exhibited: (1) reduced cellular levels of Chl a and phycobilisomes (PBS), and concomitantly higher content of an orange-red carotenoid, myxoxanthophyll; (2) higher light saturated rates (Pmax) when expressed on a Chl a basis but lower apparent quantum yields of oxygen evolution and (3) enhanced resistance to high light stress. P. boryanum grown at 15/150 regained normal blue-green pigmentation within 16 h after a temperature shift to 29 degrees C at a constant irradiance of 150 micromol m(-2) s(-1). DBMIB and KCN but not DCMU and atrazine partially inhibited the change in myxoxanthophyll/Chl a ratio following the shift from 15 to 29 degrees C. We conclude that P. boryanum responds to either varying growth temperature or varying growth irradiance by adjusting the ability to absorb light through decreasing the cellular contents of Chl a and light-harvesting pigments and screening of excessive light by myxoxanthophyll predominantly localized in the cell wall/cell membrane to protect PSII from over-excitation. The possible role of redox sensing/signalling for photosynthetic acclimation of cyanobacteria to either temperature or irradiance is discussed.     

NADPH dehydrogenase-mediated respiratory electron transport in thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803 is inactive in the light

Non-photochemical redox changes of the plastoquinone pools in darkness were investigated in the cyanobacterium Synechocystis sp. PCC 6803 by monitoring changes in Chl fluorescence yield during light-to-dark transitions. The inhibitors rotenone and mercury with or without 1 mM succinate fully suppressed the post-illumination increase in Chl fluorescence in both NADPH dehydrogenase-defective (M55) and deltaCtaI cells. The latter cells lack subunit I of cytochrome aa3-type cytochrome c oxidase. These results strongly suggest that NADPH dehydrogenase plays the major role in electron donation in the non-photo-chemical reduction of plastoquinone. The rising phase of post-illumination Chl fluorescence in both wild type pretreated with KCN, and deltaCtaI cells, was significantly slowed by low light illumination. We detected comparable photochemical levels of both photosystem (PS) II and PSI during steady state illumination in wild type and deltaCtaI cells. From these results, we suggest that respiratory electron flow involved in the non-photochemical redox change of plastoquinone is not likely to occur in the light.     

Role of mrgA in peroxide and light stress in the cyanobacterium Synechocystis sp. PCC 6803

In the unicellular cyanobacterium Synechocystis sp. PCC 6803, the mrgA gene is part of the PerR regulon that is upregulated during peroxide stress. We determined that an Δ mrgA mutant was highly sensitive to low peroxide levels and that the mutant upregulated a gene cluster ( sll1722-26 ) that encoded enzymes involved with exopolymeric substance (EPS) production. We made mutants in this EPS cluster in both a wild type and Δ mrgA background and studied the responses to oxidative stress by measuring cell damage with LIVE/DEAD stain. We show that Synechocystis sp. PCC 6803 becomes highly sensitive to oxidative stress when either mrgA or the sll1722-26 EPS components are deleted. The results suggest that the deletion of the EPS cluster makes a cell highly susceptible to cell damage, under moderate oxidative stress conditions. Mutations in either mrgA or the EPS cluster also result in cells that are more light and peroxide sensitive, and produce significantly less EPS material than in wild type. In this study, we show that in the absence of MrgA, which is known to be involved in the storage or mobilization of iron, cells can be more easily damaged by exogenous oxidative and light stress.     

sll1961 is a novel regulator of phycobilisome degradation during nitrogen starvation in the cyanobacterium Synechocystis sp. PCC 6803

The sll1961 gene was reported to encode a regulatory factor of photosystem stoichiometry in the cyanobacterium Synechocystis sp. PCC 6803. We here show that the sll1961 gene is also essential for the phycobilisome degradation during nitrogen starvation. The defect in phycobilisome degradation was observed in the sll1961 mutant despite the increased expression of nblA, a gene involved in phycobilisome degradation during nitrogen starvation. Photosystem stoichiometry is not affected by nitrogen starvation in the sll1961 mutant nor in the wild-type. The results indicate the presence of a novel pathway for phycobilisome degradation control independent of nblA expression.     

Effects of overexpressing photosynthetic carbon flux control enzymes in the cyanobacterium Synechocystis PCC 6803

Synechocystis PCC 6803 is a model unicellular cyanobacterium used in e.g. photosynthesis and CO₂ assimilation research. In the present study we examined the effects of overexpressing Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), sedoheptulose 1,7-biphosphatase (SBPase), fructose-bisphosphate aldolase (FBA) and transketolase (TK), confirmed carbon flux control enzymes of the Calvin-Bassham-Benson (CBB) cycle in higher plants, in Synechocystis PCC 6803. Overexpressing RuBisCO, SBPase and FBA resulted in increased in vivo oxygen evolution (maximal 115%), growth rate and biomass accumulation (maximal 52%) under 100 μmol photons m⁻² s⁻¹ light condition. Cells overexpressing TK showed a chlorotic phenotype but increased biomass by approximately 42% under 100 μmol photons m⁻² s⁻¹ light condition. Under 15 μmol photons m⁻² s⁻¹ light condition, cells overexpressing TK showed enhanced in vivo oxygen evolution. This study demonstrates increased growth and biomass accumulation when overexpressing selected enzymes of the CBB cycle. RuBisCO, SBPase, FBA and TK are identified as four potential targets to improve growth and subsequently also yield of valuable products from Synechocystis PCC 6803.     

A gene required for the regulation of photosynthetic light harvesting in the cyanobacterium Synechocystis 6803

A gene required for the short-term regulation of photosynthetic light harvesting (the state transition) has been identified in the cyanobacterium Synechocystis sp. PCC6803. The open reading frame is designated sll1926 in the complete Synechocystis gene sequence. The deduced amino acid sequence has no homologues in current sequence databases and no recognizable sequence motifs. It encodes a putative integral membrane protein of 16 kDa, which we have designated RpaC (regulator of phycobilisome association C). Fluorescence measurements of an insertional inactivation mutant of rpaC (Deltasll1926) show that it is specifically unable to perform state transitions. Deltasll1926 has approximately wild-type levels of PS1, PS2 and phycobilisomes. Measurements of oxygen evolution and uptake show Deltasll1926 to have no deficiency in electron transport rates. In vitro [gamma-32P]-ATP labelling experiments suggest that RpaC is not the 15 kDa membrane phosphoprotein previously implicated in state transitions. Deltasll1926 grows more slowly than the wild type only at very low light intensities.     

Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803

The sll1703 gene, encoding an Arabidopsis homologue of the thylakoid membrane-associated SppA peptidase, was inactivated by interposon mutagenesis in Synechocystis sp. strain PCC 6803. Upon acclimation from a light intensity of 50 to 150 microE m(-2) s(-1), the mutant preserved most of its phycobilisome content, whereas the wild-type strain developed a bleaching phenotype due to the loss of about 40% of its phycobiliproteins. Using in vivo and in vitro experiments, we demonstrate that the DeltasppA1 strain does not undergo the cleavage of the L(R)(33) and L(CM)(99) linker proteins that develops in the wild type exposed to increasing light intensities. We conclude that a major contribution to light acclimation under a moderate light regime in cyanobacteria originates from an SppA1-mediated cleavage of phycobilisome linker proteins. Together with changes in gene expression of the major phycobiliproteins, it contributes an additional mechanism aimed at reducing the content in phycobilisome antennae upon acclimation to a higher light intensity.     

The ATP-dependent Clp protease is essential for acclimation to UV-B and low temperature in the cyanobacterium Synechococcus

ClpP is the proteolytic subunit of the ATP-dependent Clp protease in eubacteria, mammals and plant chloroplasts. Cyanobacterial ClpP protein is encoded by a multigene family, producing up to four distinct isozymes. We have examined the importance of the first ClpP protein (ClpP1) isolated from the cyanobacterium Synechococcus sp. PCC 7942 for acclimation to ecologically relevant UV-B and low-temperature regimens. When the growth light of 50 μmol photons m^?2 s^?1 was supplemented with 0.5 W m^?2 UV-B for 8 h, the constitutive level of ClpP1 rose eightfold after an initial lag of 1 h. Wild-type cells readily acclimated to this UV-B level, recovering after the initial stress to almost the same growth rate as that before UV-B exposure. Growth of a clpP1 null mutant (ΔclpP1), however, was severely inhibited by UV-B, being eight times slower than the wild type after 8 h. In comparison, ClpP1 content increased 15-fold in wild-type cultures shifted from 37°C to 25°C for 24 h. Wild-type cultures readily acclimated to 25°C after 24 h, whereas the ΔclpP1 strain did not and eventually lost viability with prolonged cold treatment. During acclimation to either UV-B or cold, photosynthesis in the wild type was initially inhibited upon the shift but then recovered. Photosynthesis in ΔclpP1 cultures, however, was more severely inhibited by the stress treatment and failed to recover. Acclimation was also monitored by examining the exchange of photosystem II reaction centre D1 proteins that occurs in wild-type Synechococcus during conditions of excitation stress. During both cold and UV-B shifts, wild-type cultures replaced the acclimative form of D1 (D1:1) with the alternative D1 form 2 (D1:2) within the first hours. Once acclimated to either 25°C or 0.5 W m^?2 UV-B, D1:2 was exchanged back for D1:1. In ΔclpP1 cultures, this second exchange between D1 forms did not occur, with D1:2 remaining the predominant D1 form. Our results demonstrate that the ATP-dependent Clp protease is an essential component of the cold and UV-B acclimation processes of Synechococcus.