Evidence for the role of the oxygen-evolving manganese complex in photoinhibition of Photosystem II

AbstractPhotoinhibition of PSII occurs at the same quantum efficiency from very low to very high light, which raises a question about how important is the rate of photosynthetic electron transfer in photoinhibition. We modulated electron transfer rate and light intensity independently of each other in lincomycin-treated pea leaves and in isolated thylakoids, in order to elucidate the specific effects of light and PSII electron transport on photoinhibition. Major changes in the rate of electron transport caused only small changes in the rate of photoinhibition, suggesting the existence of a significant photoinhibitory pathway that contains an electron-transfer-independent phase. We compared the action spectrum of photoinhibition with absorption spectra of PSII components that could function as photoreceptors of the electron-transfer-independent phase of photoinhibition and found that the absorption spectra of Mn(III) and Mn(IV) compounds resemble the action spectrum of photoinhibition, showing a steep decrease from UV-C to blue light and a low visible-light tail. Our results show that the release of a Mn ion to the thylakoid lumen is the earliest detectable step of both UV- and visible-light-induced photoinhibition. After Mn release from the oxygen-evolving complex, oxidative damage to the PSII reaction center occurs because the Mn-depleted oxygen-evolving complex cannot reduce P680+ normally

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

Singlet oxygen (O-1(2)) is a harmful species that functions also as a signaling molecule. In chloroplasts, O-1(2) is produced via charge recombination reactions in photosystem II, but which recombination pathway(s) produce triplet Chl and O-1(2) remains open. Furthermore, the role of O-1(2) in photoinhibition is not clear. We compared temperature dependences of O-1(2) production, photoinhibition, and recombination pathways. O-1(2) production by pumpkin thylakoids increased from -2 to +35 degrees C, ruling out recombination of the primary charge pair as a main contributor. S(2)Q(A)(-) or S(2)Q(B)(-) recombination pathways, in turn, had too steep temperature dependences. Instead, the temperature dependence of O-1(2) production matched that of misses (failures of the oxygen (O-2) evolving complex to advance an S-state). Photoinhibition in vitro and in vivo (also in Synechocystis), and in the presence or absence of O-2, had the same temperature dependence, but ultraviolet (UV)-radiation-caused photoinhibition showed a weaker temperature response. We suggest that the miss-associated recombination of P(680)(+)Q(A)(-) is the main producer of O-1(2). 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 O-1(2) or long-lived P-680(+) as damaging agents, dominate in red light

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 Delta sigBCE and Delta sigCDE strains show superior growth in chemically induced oxidative stress. All cyanobacterial strains, including a stress-sensitive strain Delta sigBCDE, grew well in KA2 for four days, indicating that KA2 did not cause immediate oxidative stress. Completely arrested growth and bleaching of Delta sigBCDE 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 Delta sigBCD was arrested early in un-buffered and Hepes buffered (pH 7.5) KA2. In Delta sigBCD, all phosphate transporter genes are upregulated in standard conditions, and Delta sigBCD cells showed growth defects in low-phosphate BG-11 medium. Delta sigBCD cells removed phosphate slower from KA2 than the control strain, but phosphate supplementation of KA2 did not improve growth of Delta sigBCD. The Delta sigBCE 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 Delta sigBCE 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

Group 2 Sigma Factors Are Central Regulators of Oxidative Stress Acclimation in Cyanobacteria

Regulatory σ factors of the RNA polymerase (RNAP) adjust gene expression according to environmental cues when the cyanobacterium Synechocystis sp. PCC 6803 acclimates to suboptimal conditions. Here we show central roles of the non-essential group 2 σ factors in oxidative stress responses. Cells missing all group 2 σ factors fail to acclimate to chemically induced singlet oxygen, superoxide or H2O2 stresses and lose pigments in high light. The SigB and SigD are the major σ factors in oxidative stress whereas SigC and SigE play only minor roles. The SigD factor is upregulated in high light, singlet oxygen and H2O2 stresses, and overproduction of the SigD factor in the ΔsigBCE strain leads to superior growth of ΔsigBCE cells in those stress conditions. Superoxide does not induce the production of the SigD factor but instead SigB and SigC factors are moderately induced. The SigB factor alone in ΔsigCDE can support almost as fast growth in superoxide stress as full complement of σ factors in the control strain but an overdose of the stationary-phase-related SigC factor causes growth arrest of ΔsigBDE in superoxide stress. Drastic decrease of the functional RNA polymerase limits the transcription capacity of the cells in H2O2 stress, which explains why cyanobacteria are sensitive to H2O2. Formation of RNAP-SigB and RNAP-SigD holoenzymes is highly enhanced in H2O2 stress and cells containing only SigB (ΔsigCDE) or SigD (ΔsigBCE) show superior growth in H2O2 stress

Inactivation of group 2 sigma 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 sigma factor SigA increases in the Delta sigBCDE strain of the cyanobacterium Synechocystis sp. PCC 6803 lacking all group 2 s 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, Delta sigBCDE contain more RNAPs and ribosomal subunits than the control strain. Extra RNAPs are fully active, and the RNA content of Delta sigBCDE cells is almost tripled compared to that in the control strain. Although Delta sigBCDE cells produce extra rRNAs and ribosomal proteins, functional extra ribosomes are not formed, and translation activity and protein content remained similar in Delta sigBCDE 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

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 sigma factor subunits of RNA polymerase (RNAP). The SigB and SigD sigma 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), Delta sigB, Delta sigD, Delta sigBCE (containing SigD as the only functional group 2 sigma factor), and Delta sigCDE (SigB as the only functional group 2 sigma 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 sigma factors does not lead to superior acclimation to high light or high temperature, indicating that all group 2 sigma 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.This work was supported by the Czech Science Foundation (project 19-29225X) and the Institutional Research Concept (RVO: 61388971). TT was supported by the Academy of Finland (265807).Peer ReviewedPostprint (published version

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 Delta pilD mutant and, by sequencing their genomes, identified secondary mutations in the SigF sigma factor, the gamma 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.</p