Engineering RNA polymerase to construct biotechnological host strains of cyanobacteria.

Application of cyanobacteria for bioproduction, bioremediation and biotransformation is being increasingly explored. Photoautotrophs are carbon-negative by default, offering a direct pathway to reducing emissions in production systems. More robust and versatile host strains are needed for constructing production strains that would function as efficient and carbon-neutral cyanofactories. We have tested if the engineering of sigma factors, regulatory units of the bacterial RNA polymerase, could be used to generate better host strains of the model cyanobacterium Synechocystis sp. PCC 6803. Overexpressing the stress-responsive sigB gene under the strong psbA2 promoter (SigB-oe) led to improved tolerance against heat, oxidative stress and toxic end-products. By targeting transcription initiation in the SigB-oe strain, we could simultaneously activate a wide spectrum of cellular protective mechanisms, including carotenoids, the HspA heat shock protein, and highly activated non-photochemical quenching. Yellow fluorescent protein was used to test the capacity of the SigB-oe strain to produce heterologous proteins. In standard conditions, the SigB-oe strain reached a similar production as the control strain, but when cultures were challenged with oxidative stress, the production capacity of SigB-oe surpassed the control strain. We also tested the production of growth-rate-controlled host strains via manipulation of RNA polymerase, but post-transcriptional regulation prevented excessive overexpression of the primary sigma factor SigA, and overproduction of the growth-restricting SigC factor was lethal. Thus, more research is needed before cyanobacteria growth can be manipulated by engineering RNA polymerase.

The genome sequence of Synechocystis sp. PCC 6803 substrain GT-T and its implications for the evolution of PCC 6803 substrains.

Synechocystis sp. PCC 6803 is a model cyanobacterium, glucose-tolerant substrains of which are commonly used as laboratory strains. In recent years, it has become evident that 'wild-type' strains used in different laboratories show some differences in their phenotypes. We report here the chromosome sequence of our Synechocystis sp. PCC 6803 substrain, named substrain GT-T. The chromosome sequence of GT-T was compared to those of two other commonly used laboratory substrains, GT-S and PCC-M. We identified 11 specific mutations in the GT-T substrain, whose physiological consequences are discussed. We also provide an update on evolutionary relationships between different Synechocystis sp. PCC 6803 substrains.

Singlet oxygen production by photosystem II is caused by misses of the oxygen evolving complex.

Singlet oxygen (1 O2 ) is a harmful species that functions also as a signaling molecule. In chloroplasts, 1 O2 is produced via charge recombination reactions in photosystem II, but which recombination pathway(s) produce triplet Chl and 1 O2 remains open. Furthermore, the role of 1 O2 in photoinhibition is not clear. We compared temperature dependences of 1 O2 production, photoinhibition, and recombination pathways. 1 O2 production by pumpkin thylakoids increased from -2 to +35°C, ruling out recombination of the primary charge pair as a main contributor. S2 QA - or S2 QB - recombination pathways, in turn, had too steep temperature dependences. Instead, the temperature dependence of 1 O2 production matched that of misses (failures of the oxygen (O2 ) evolving complex to advance an S-state). Photoinhibition in vitro and in vivo (also in Synechocystis), and in the presence or absence of O2 , had the same temperature dependence, but ultraviolet (UV)-radiation-caused photoinhibition showed a weaker temperature response. We suggest that the miss-associated recombination of P680 + QA - is the main producer of 1 O2 . Our results indicate three parallel photoinhibition mechanisms. The manganese mechanism dominates in UV radiation but also functions in white light. Mechanisms that depend on light absorption by Chls, having 1 O2 or long-lived P680 + as damaging agents, dominate in red light.

Roles of Close Homologues SigB and SigD in Heat and High Light Acclimation of the Cyanobacterium Synechocystis sp. PCC 6803.

Acclimation of cyanobacterium Synechocystis sp. PCC6803 to suboptimal conditions is largely dependent on adjustments of gene expression, which is highly controlled by the σ factor subunits of RNA polymerase (RNAP). The SigB and SigD σ factors are close homologues. Here we show that the sigB and sigD genes are both induced in high light and heat stresses. Comparison of transcriptomes of the control strain (CS), ΔsigB, ΔsigD, ΔsigBCE (containing SigD as the only functional group 2 σ factor), and ΔsigCDE (SigB as the only functional group 2 σ factor) strains in standard, high light, and high temperature conditions revealed that the SigB and SigD factors regulate different sets of genes and SigB and SigD regulons are highly dependent on stress conditions. The SigB regulon is bigger than the SigD regulon at high temperature, whereas, in high light, the SigD regulon is bigger than the SigB regulon. Furthermore, our results show that favoring the SigB or SigD factor by deleting other group 2 σ factors does not lead to superior acclimation to high light or high temperature, indicating that all group 2 σ factors play roles in the acclimation processes.

Mutations Suppressing the Lack of Prepilin Peptidase Provide Insights Into the Maturation of the Major Pilin Protein in Cyanobacteria.

Type IV pili are bacterial surface-exposed filaments that are built up by small monomers called pilin proteins. Pilins are synthesized as longer precursors (prepilins), the N-terminal signal peptide of which must be removed by the processing protease PilD. A mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking the PilD protease is not capable of photoautotrophic growth because of the impaired function of Sec translocons. Here, we isolated phototrophic suppressor strains of the original ΔpilD mutant and, by sequencing their genomes, identified secondary mutations in the SigF sigma factor, the γ subunit of RNA polymerase, the signal peptide of major pilin PilA1, and in the pilA1-pilA2 intergenic region. Characterization of suppressor strains suggests that, rather than the total prepilin level in the cell, the presence of non-glycosylated PilA1 prepilin is specifically harmful. We propose that the restricted lateral mobility of the non-glycosylated PilA1 prepilin causes its accumulation in the translocon-rich membrane domains, which attenuates the synthesis of membrane proteins.

Revealing secrets of the enigmatic omega subunit of bacterial RNA polymerase.

The conserved omega (ω) subunit of RNA polymerase (RNAP) is the only nonessential subunit of bacterial RNAP core. The small ω subunit (7 kDa-11.5 kDa) contains three conserved α helices, and helices α2 and α3 contain five fully conserved amino acids of ω. Four conserved amino acids stabilize the correct folding of the ω subunit and one is located in the vicinity of the ß' subunit of RNAP. Otherwise ω shows high variation between bacterial taxa, and although the main interaction partner of ω is always ß', many interactions are taxon-specific. ω-less strains show pleiotropic phenotypes, and based on in vivo and in vitro results, a few roles for the ω subunits have been described. Interactions of the ω subunit with the ß' subunit are important for the RNAP core assembly and integrity. In addition, the ω subunit plays a role in promoter selection, as ω-less RNAP cores recruit fewer primary σ factors and more alternative σ factors than intact RNAP cores in many species. Furthermore, the promoter selection of an ω-less RNAP holoenzyme bearing the primary σ factor seems to differ from that of an intact RNAP holoenzyme.

Action spectrum of the redox state of the plastoquinone pool defines its function in plant acclimation.

The plastoquinone (PQ) pool mediates electron flow and regulates photoacclimation in plants. Here we report the action spectrum of the redox state of the PQ pool in Arabidopsis thaliana, showing that 470-500, 560 or 650-660 nm light favors Photosystem II (PSII) and reduces the PQ pool, whereas 420-440, 520 or 690 nm light favors Photosystem I (PSI) and oxidizes PQ. These data were used to construct a model predicting the redox state of PQ from the spectrum of any polychromatic light source. Moderate reduction of the PQ pool induced transition to light state 2, whereas state 1 required highly oxidized PQ. In low-intensity PSI light, PQ was more oxidized than in darkness and became gradually reduced with light intensity, while weak PSII light strongly reduced PQ. Natural sunlight was found to favor PSI, which enables plants to use the redox state of the PQ pool as a measure of light intensity.

Testing the Potential of Regulatory Sigma Factor Mutants for Wastewater Purification or Bioreactor Run in High Light.

It is shown that a freshly inoculated culture of the model cyanobacterium Synechocystis sp. PCC 6803 consumed almost all phosphate and 50% of nitrate within 6 days from the nutrient-rich BG-11 growth medium, indicating potential of cyanobacteria to purify wastewaters. Synechocystis sp. PCC 6803 control strain also collected nutrients efficiently from a landfill leachate wastewater KA2 (5.9-6.9 mM ammonium and 0.073-0.077 mM phosphate). Wastewaters might induce oxidative stress to microalgae, which prompted us to test growth of sigma factor inactivation strains, as ΔsigBCE and ΔsigCDE strains show superior growth in chemically induced oxidative stress. All cyanobacterial strains, including a stress-sensitive strain ΔsigBCDE, grew well in KA2 for four days, indicating that KA2 did not cause immediate oxidative stress. Completely arrested growth and bleaching of ΔsigBCDE cells after one week in KA2 wastewater point to the importance of group 2 sigma factor-mediated changes in gene expression during wastewater treatment. The growth of ΔsigBCD was arrested early in un-buffered and Hepes buffered (pH 7.5) KA2. In ΔsigBCD, all phosphate transporter genes are upregulated in standard conditions, and ΔsigBCD cells showed growth defects in low-phosphate BG-11 medium. ΔsigBCD cells removed phosphate slower from KA2 than the control strain, but phosphate supplementation of KA2 did not improve growth of ΔsigBCD. The ΔsigBCE strain showed superior growth in a laboratory-scale bioreactor in bright light and removed phosphate even slightly more efficiently than the control strain if KA2 was Hepes buffered although ΔsigBCE grew slowly in un-buffered KA2 and in low-phosphate BG-11 medium. The results indicate that engineering expression of regulatory group 2 sigma factor(s) might be useful for practical applications.

Measurement of the redox state of the plastoquinone pool in cyanobacteria.

Here, we developed a method for measuring the in vivo redox state of the plastoquinone (PQ) pool in the cyanobacteria Synechocystis sp. PCC 6803. Cells were illuminated on a glass fiber filter, PQ was extracted with ethyl acetate and determined with HPLC. Control samples with fully oxidized and reduced photoactive PQ pool were prepared by far-red and high light treatments, respectively, or by blocking the photosynthetic electron transfer chemically before or after PQ in moderate light. The photoactive pool comprised 50% of total PQ. We find that the PQ pool of cyanobacteria behaves under light treatments qualitatively similarly as in plant chloroplasts, is less reduced during growth under high than under ambient CO2 and remains partly reduced in darkness.

Inactivation of group 2 σ factors upregulates production of transcription and translation machineries in the cyanobacterium Synechocystis sp. PCC 6803.

We show that the formation of the RNAP holoenzyme with the primary σ factor SigA increases in the ΔsigBCDE strain of the cyanobacterium Synechocystis sp. PCC 6803 lacking all group 2 σ factors. The high RNAP-SigA holoenzyme content directly induces transcription of a particular set of housekeeping genes, including ones encoding transcription and translation machineries. In accordance with upregulated transcripts, ΔsigBCDE contain more RNAPs and ribosomal subunits than the control strain. Extra RNAPs are fully active, and the RNA content of ΔsigBCDE cells is almost tripled compared to that in the control strain. Although ΔsigBCDE cells produce extra rRNAs and ribosomal proteins, functional extra ribosomes are not formed, and translation activity and protein content remained similar in ΔsigBCDE as in the control strain. The arrangement of the RNA polymerase core genes together with the ribosomal protein genes might play a role in the co-regulation of transcription and translation machineries. Sequence logos were constructed to compare promoters of those housekeeping genes that directly react to the RNAP-SigA holoenzyme content and those ones that do not. Cyanobacterial strains with engineered transcription and translation machineries might provide solutions for construction of highly efficient production platforms for biotechnical applications in the future.

Harnessing transcription for bioproduction in cyanobacteria.

Sustainable production of biofuels and other valuable compounds is one of our future challenges. One tempting possibility is to use photosynthetic cyanobacteria as production factories. Currently, tools for genetic engineering of cyanobacteria are not good enough to exploit the full potential of cyanobacteria. A wide variety of expression systems will be required to adjust both the expression of heterologous enzyme(s) and metabolic routes to the best possible balance, allowing the optimal production of a particular substance. In bacteria, transcription, especially the initiation of transcription, has a central role in adjusting gene expression and thus also metabolic fluxes of cells according to environmental cues. Here we summarize the recent progress in developing tools for efficient cyanofactories, focusing especially on transcriptional regulation.

6S RNA plays a role in recovery from nitrogen depletion in Synechocystis sp. PCC 6803.

BACKGROUND: The 6S RNA is a global transcriptional riboregulator, which is exceptionally widespread among most bacterial phyla. While its role is well-characterized in some heterotrophic bacteria, we subjected a cyanobacterial homolog to functional analysis, thereby extending the scope of 6S RNA action to the special challenges of photoautotrophic lifestyles. RESULTS: Physiological characterization of a 6S RNA deletion strain (ΔssaA) demonstrates a delay in the recovery from nitrogen starvation. Significantly decelerated phycobilisome reassembly and glycogen degradation are accompanied with reduced photosynthetic activity compared to the wild type. Transcriptome profiling further revealed that predominantly genes encoding photosystem components, ATP synthase, phycobilisomes and ribosomal proteins were negatively affected in ΔssaA. In vivo pull-down studies of the RNA polymerase complex indicated that the presence of 6S RNA promotes the recruitment of the cyanobacterial housekeeping σ factor SigA, concurrently supporting dissociation of group 2 σ factors during recovery from nitrogen starvation. CONCLUSIONS: The combination of genetic, physiological and biochemical studies reveals the homologue of 6S RNA as an integral part of the cellular response of Synechocystis sp. PCC 6803 to changing nitrogen availability. According to these results, 6S RNA supports a rapid acclimation to changing nitrogen supply by accelerating the switch from group 2 σ factors SigB, SigC and SigE to SigA-dependent transcription. We therefore introduce the cyanobacterial 6S RNA as a novel candidate regulator of RNA polymerase sigma factor recruitment in Synechocystis sp. PCC 6803. Further studies on mechanistic features of the postulated interaction should shed additional light on the complexity of transcriptional regulation in cyanobacteria.

Acclimation to High CO2 Requires the ω Subunit of the RNA Polymerase in Synechocystis.

Inactivation of the nonessential ω-subunit of the RNA polymerase core in the ΔrpoZ strain of the model cyanobacterium Synechocystis sp. PCC 6803 leads to a unique high-CO2-sensitive phenotype. Supplementing air in the growth chamber with 30 mL L-1 (3%) CO2 accelerated the growth rate of the control strain (CS) 4-fold, whereas ΔrpoZ did not grow faster than under ambient air. The slow growth of ΔrpoZ during the first days in high CO2 was due to the inability of the mutant cells to adjust photosynthesis to high CO2 The light-saturated photosynthetic activity of ΔrpoZ in high CO2 was only half of that measured in CS, Rubisco content was one-third lower, and cells of ΔrpoZ were not able to increase light-harvesting phycobilisome antenna like CS upon high-CO2 treatment. In addition, altered structural and functional organization of photosystem I and photosystem II were detected in the ΔrpoZ strain compared with CS when cells were grown in high CO2 but not in ambient air. Moreover, respiration of ΔrpoZ did not acclimate to high CO2 Unlike the photosynthetic complexes, the RNA polymerase complex and ribosomes were produced in high CO2 similarly as in CS Our results indicate that the deletion of the ω-subunit specifically affects photosynthesis and respiration, but transcription and translation remain active. Thus, the specific effect of the ω-subunit on photosynthesis but not on all household processes suggests that the ω-subunit might have a regulatory function in cyanobacteria.

Roles of Group 2 Sigma Factors in Acclimation of the Cyanobacterium Synechocystis sp. PCC 6803 to Nitrogen Deficiency.

Acclimation of cyanobacteria to environmental conditions is mainly controlled at the transcriptional level, and σ factors of the RNA polymerase have a central role in this process. The model cyanobacterium Synechocystis sp. PCC 6803 has four non-essential group 2 σ factors (SigB, SigC, SigD and SigE) that regulate global metabolic responses to various adverse environmental conditions. Here we show that although none of the group 2 σ factors is essential for the major metabolic realignments induced by a short period of nitrogen starvation, the quadruple mutant without any group 2 σ factors and triple mutants missing both SigB and SigD grow slowly in BG-11 medium containing only 5% of the nitrate present in standard BG-11. These ΔsigBCDE, ΔsigBCD and ΔsigBDE strains lost PSII activity rapidly in low nitrogen and accumulated less glycogen than the control strain. An abnormally high glycogen content was detected in ΔsigBCE (SigD is active), while the carotenoid content became high in ΔsigCDE (SigB is active), indicating that SigB and SigD regulate the partitioning of carbon skeletons in low nitrogen. Long-term survival and recovery of the cells after nitrogen deficiency was strongly dependent on group 2 σ factors. The quadruple mutant and the ΔsigBDE strain (only SigC is active) recovered more slowly from nitrogen deficiency than the control strain, and ΔsigBCDE in particular lost viability during nitrogen starvation. Nitrogen deficiency-induced changes in the pigment content of the control strain recovered essentially in 1 d in nitrogen-replete medium, but little recovery occurred in ΔsigBCDE and ΔsigBDE.

Oxygen produced by cyanobacteria in simulated Archaean conditions partly oxidizes ferrous iron but mostly escapes-conclusions about early evolution.

The Earth has had a permanently oxic atmosphere only since the great oxygenation event (GOE) 2.3-2.4 billion years ago but recent geochemical research has revealed short periods of oxygen in the atmosphere up to a billion years earlier before the permanent oxygenation. If these "whiffs" of oxygen truly occurred, then oxygen-evolving (proto)cyanobacteria must have existed throughout the Archaean aeon. Trapping of oxygen by ferrous iron and other reduced substances present in Archaean oceans has often been suggested to explain why the oxygen content of the atmosphere remained negligible before the GOE although cyanobacteria produced oxygen. We tested this hypothesis by growing cyanobacteria in anaerobic high-CO2 atmosphere in a medium with a high concentration of ferrous iron. Microcystins are known to chelate iron, which prompted us also to test the effects of microcystins and nodularins on iron tolerance. The results show that all tested cyanobacteria, especially nitrogen-fixing species grown in the absence of nitrate, and irrespective of the ability to produce cyanotoxins, were iron sensitive in aerobic conditions but tolerated high concentrations of iron in anaerobicity. This result suggests that current cyanobacteria would have tolerated the high-iron content of Archaean oceans. However, only 1 % of the oxygen produced by the cyanobacterial culture was trapped by iron, suggesting that large-scale cyanobacterial photosynthesis would have oxygenated the atmosphere even if cyanobacteria grew in a reducing ocean. Recent genomic analysis suggesting that ability to colonize seawater is a secondary trait in cyanobacteria may offer a partial explanation for the sustained inefficiency of cyanobacterial photosynthesis during the Archaean aeon, as fresh water has always covered a very small fraction of the Earth's surface. If oxygenic photosynthesis originated in fresh water, then the GOE marks the adaptation of cyanobacteria to seawater, and the late-Proterozoic increase in oxygen concentration of the atmosphere is caused by full oxidation of the oceans.

In vivo recruitment analysis and a mutant strain without any group 2 σ factor reveal roles of different σ factors in cyanobacteria.

In eubacteria, replacement of one σ factor in the RNA polymerase (RNAP) holoenzyme by another one changes the transcription pattern. Cyanobacteria are eubacteria characterized by oxygenic photosynthesis, and they typically encode numerous group 2 σ factors that closely resemble the essential primary σ factor. A mutant strain of the model cyanobacterium Synechocystis sp. PCC 6803 without functional group 2 σ factors (named as ΔsigBCDE) could not acclimate to heat, high salt or bright light stress, but in standard conditions ΔsigBCDE grew only 9% slower than the control strain. One-fifth of the genes in ΔsigBCDE was differently expressed compared with the control strain in standard growth conditions and several physiological changes in photosynthesis, and pigment and lipid compositions were detected. To directly analyze the σ factor content of RNAP holoenzyme in vivo, a His-tag was added to the γ subunit of RNAP in Synechocystis and RNAPs were collected. The results revealed that all group 2 σ factors were recruited by RNAP in standard conditions, but recruitment of SigB and SigC increased in heat stress, SigD in bright light, SigE in darkness and SigB, SigC and SigE in high salt, explaining the poor acclimation of ΔsigBCDE to these stress conditions.

The ω subunit of RNA polymerase is essential for thermal acclimation of the cyanobacterium Synechocystis sp. PCC 6803.

The rpoZ gene encodes the small ω subunit of RNA polymerase. A ΔrpoZ strain of the cyanobacterium Synechocystis sp. PCC 6803 grew well in standard conditions (constant illumination at 40 µmol photons m(-2) s(-1); 32°C; ambient CO2) but was heat sensitive and died at 40°C. In the control strain, 71 genes were at least two-fold up-regulated and 91 genes down-regulated after a 24-h treatment at 40°C, while in ΔrpoZ 394 genes responded to heat. Only 62 of these heat-responsive genes were similarly regulated in both strains, and 80% of heat-responsive genes were unique for ΔrpoZ. The RNA polymerase core and the primary σ factor SigA were down-regulated in the control strain at 40°C but not in ΔrpoZ. In accordance with reduced RNA polymerase content, the total RNA content of mild-heat-stress-treated cells was lower in the control strain than in ΔrpoZ. Light-saturated photosynthetic activity decreased more in ΔrpoZ than in the control strain upon mild heat stress. The amounts of photosystem II and rubisco decreased at 40°C in both strains while PSI and the phycobilisome antenna protein allophycocyanin remained at the same level as in standard conditions. The phycobilisome rod proteins, phycocyanins, diminished during the heat treatment in ΔrpoZ but not in the control strain, and the nblA1 and nblA2 genes (encode NblA proteins required for phycobilisome degradation) were up-regulated only in ΔrpoZ. Our results show that the ω subunit of RNAP is essential in heat stress because it is required for heat acclimation of diverse cellular processes.

The omega subunit of the RNA polymerase core directs transcription efficiency in cyanobacteria.

The eubacterial RNA polymerase core, a transcription machinery performing DNA-dependent RNA polymerization, consists of two α subunits and ß, ß' and ω subunits. An additional σ subunit is recruited for promoter recognition and transcription initiation. Cyanobacteria, a group of eubacteria characterized by oxygenic photosynthesis, have a unique composition of the RNA polymerase (RNAP) core due to splitting of the ß' subunit to N-terminal γ and C-terminal ß' subunits. The physiological roles of the small ω subunit of RNAP, encoded by the rpoZ gene, are not yet completely understood in any bacteria. We found that although ω is non-essential in cyanobacteria, it has a major impact on the overall gene expression pattern. In ΔrpoZ strain, recruitment of the primary σ factor into the RNAP holoenzyme is inefficient, which causes downregulation of highly expressed genes and upregulation of many low-expression genes. Especially, genes encoding proteins of photosynthetic carbon concentrating and carbon fixing complexes were down, and the ΔrpoZ mutant showed low light-saturated photosynthetic activity and accumulated photoprotective carotenoids and α-tocopherol. The results indicate that the ω subunit facilitates the association of the primary σ factor with the RNAP core, thereby allowing efficient transcription of highly expressed genes.

Oxidative stress and photoinhibition can be separated in the cyanobacterium Synechocystis sp. PCC 6803.

Roles of oxidative stress and photoinhibition in high light acclimation were studied using a regulatory mutant of the cyanobacterium Synechocystis sp. PCC 6803. The mutant strain ΔsigCDE contains the stress responsive SigB as the only functional group 2 σ factor. The ∆sigCDE strain grew more slowly than the control strain in methyl-viologen-induced oxidative stress. Furthermore, a fluorescence dye detecting H2O2, hydroxyl and peroxyl radicals and peroxynitrite, produced a stronger signal in ∆sigCDE than in the control strain, and immunological detection of carbonylated residues showed more protein oxidation in ∆sigCDE than in the control strain. These results indicate that ∆sigCDE suffers from oxidative stress in standard conditions. The oxidative stress may be explained by the findings that ∆sigCDE had a low content of glutathione and low amount of Flv3 protein functioning in the Mehler-like reaction. Although ∆sigCDE suffers from oxidative stress, up-regulation of photoprotective carotenoids and Flv4, Sll2018, Flv2 proteins protected PSII against light induced damage by quenching singlet oxygen more efficiently in ∆sigCDE than in the control strain in visible and in UV-A/B light. However, in UV-C light singlet oxygen is not produced and PSII damage occurred similarly in the ∆sigCDE and control strains. According to our results, resistance against the light-induced damage of PSII alone does not lead to high light tolerance of the cells, but in addition efficient protection against oxidative stress would be required.

Group 2 sigma factor mutant ΔsigCDE of the cyanobacterium Synechocystis sp. PCC 6803 reveals functionality of both carotenoids and flavodiiron proteins in photoprotection of photosystem II.

Adjustment of gene expression during acclimation to stress conditions, such as bright light, in the cyanobacterium Synechocystis sp. PCC 6803 depends on four group 2 σ factors (SigB, SigC, SigD, SigE). A ΔsigCDE strain containing the stress-responsive SigB as the only functional group 2 σ factor appears twice as resistant to photoinhibition of photosystem II (PSII) as the control strain. Microarray analyses of the ΔsigCDE strain indicated that 77 genes in standard conditions and 79 genes in high light were differently expressed compared with the control strain. Analysis of possible photoprotective mechanisms revealed that high carotenoid content and up-regulation of the photoprotective flavodiiron operon flv4-sll0218-flv2 protected PSII in ΔsigCDE, while up-regulation of pgr5-like, hlipB or isiA genes in the mutant strain did not offer particular protection against photoinhibition. Photoinhibition resistance was lost if ΔsigCDE was grown in high CO2, where carotenoid and Flv4, Sll0218, and Flv2 contents were low. Additionally, photoinhibition resistance of the ΔrpoZ strain (lacking the omega subunit of RNA polymerase), with high carotenoid but low Flv4-Sll0218-Flv2 content, supported the importance of carotenoids in PSII protection. Carotenoids likely protect mainly by quenching of singlet oxygen, but efficient nonphotochemical quenching in ΔsigCDE might offer some additional protection. Comparison of photoinhibition kinetics in control, ΔsigCDE, and ΔrpoZ strains showed that protection by the flavodiiron operon was most efficient during the first minutes of high-light illumination.