Kouji Kojima

Light-driven Hydrogen Production by a Hybrid Complex of a [NiFe]-Hydrogenase and the Cyanobacterial Photosystem I

Abstract In order to generate renewable and clean fuels, increasing efforts are focused on the exploitation of photosynthetic microorganisms for the production of molecular hydrogen from water and light. In this study we engineered a ‘hard-wired’ protein complex consisting of a hydrogenase and photosystem I (hydrogenase–PSI complex) as a direct light-to-hydrogen conversion system. The key component was an artificial fusion protein composed of the membrane-bound [NiFe] hydrogenase from the β-proteobacterium Ralstonia eutropha H16 and the peripheral PSI subunit PsaE of the cyanobacterium Thermosynechococcus elongatus. The resulting hydrogenase-PsaE fusion protein associated with PsaE-free PSI spontaneously, thereby forming a hydrogenase–PSI complex as confirmed by sucrose-gradient ultracentrifuge and immunoblot analysis. The hydrogenase–PSI complex displayed light-driven hydrogen production at a rate of 0.58 μmol H2·mg chlorophyll−1·h−1. The complex maintained its accessibility to the native electron acceptor ferredoxin. This study provides the first example of a light-driven enzymatic reaction by an artificial complex between a redox enzyme and photosystem I and represents an important step on the way to design a photosynthetic organism that efficiently converts solar energy and water into hydrogen.

Oxidation of elongation factor G inhibits the synthesis of the D1 protein of photosystem II

Oxidative stress inhibits the repair of photodamaged photosystem II (PSII). This inhibition is due initially to the suppression, by reactive oxygen species (ROS), of the synthesis de novo of proteins that are required for the repair of PSII, such as the D1 protein, at the level of translational elongation. To investigate in vitro the mechanisms whereby ROS inhibit translational elongation, we developed a translation system in vitro from the cyanobacterium Synechocystis sp. PCC 6803. The synthesis of the D1 protein in vitro was inhibited by exogenous H2O2. However, the addition of reduced forms of elongation factor G (EF‐G), which is known to be particularly sensitive to oxidation, was able to reverse the inhibition of translation. By contrast, the oxidized forms of EF‐G failed to restore translational activity. Furthermore, the overexpression of EF‐G of Synechocystis in another cyanobacterium Synechococcus sp. PCC 7942 increased the tolerance of cells to H2O2 in terms of protein synthesis. These observations suggest that EF‐G might be the primary target, within the translational machinery, of inhibition by ROS.

Targeted inactivation of the hrcA repressor gene in cyanobacteria.

To determine if the CIRCE/HrcA system operates in cyanobacteria, we have inactivated the hrcA repressor gene in Synechocystis sp. PCC 6803 by gene targeting. In the hrcA mutant, the groESL1 operon and the groEL2 gene, both of which have the CIRCE operator in their upstream regions, were derepressed at 30°C without affecting expression of other major heat‐shock genes. However, expression of these groE genes in the mutant was not fully derepressed. Their transcription increased further upon heat shock, and was initiated from the same sites as those used under normal conditions. This suggests that their expression is regulated by at least two different mechanisms, a negative one controlled by HrcA and an unknown positive one. The heat‐induced expression of clpB1 and htpG was greatly repressed by the absence of HrcA. The hrcA mutant which constitutively overexpressed GroEL displayed improved cellular thermotolerance and also reduced photobleaching of phycocyanin under heat stress conditions.

Regulation of Translation by the Redox State of Elongation Factor G in the Cyanobacterium Synechocystis sp. PCC 6803

Elongation factor G (EF-G), a key protein in translational elongation, was identified as a primary target of inactivation by reactive oxygen species within the translational machinery of the cyanobacterium Synechocystis sp. PCC 6803 (Kojima, K., Oshita, M., Nanjo, Y., Kasai, K., Tozawa, Y., Hayashi, H., and Nishiyama, Y. (2007) Mol. Microbiol. 65, 936–947). In the present study, we found that inactivation of EF-G (Slr1463) by H2O2 was attributable to the oxidation of two specific cysteine residues and formation of a disulfide bond. Substitution of these cysteine residues by serine residues protected EF-G from inactivation by H2O2 and allowed the EF-G to mediate translation in a translation system in vitro that had been prepared from Synechocystis. The disulfide bond in oxidized EF-G was reduced by thioredoxin, and the resultant reduced form of EF-G regained the activity to mediate translation in vitro. Western blotting analysis showed that levels of the oxidized form of EF-G increased under strong light in a mutant that lacked NADPH-thioredoxin reductase, indicating that EF-G is reduced by thioredoxin in vivo. These observations suggest that the translational machinery is regulated by the redox state of EF-G, which is oxidized by reactive oxygen species and reduced by thioredoxin, a transmitter of reducing signals generated by the photosynthetic transport of electrons.

A novel light- and heat-responsive regulation of the groE transcription in the absence of HrcA or CIRCE in cyanobacteria

The inactivation of the hrcA gene resulted in de‐repression of the two CIRCE‐containing groE genes in a cyanobacterium Synechocystis sp. strain PCC6803, indicating that the CIRCE operator/HrcA repressor system operates in the cyanobacterium. We found that the groE expression in the hrcA mutant is greatly induced by heat and/or light. Removal of a K‐box containing and an N‐box containing region upstream of the groESL1 promoter abolished light‐induced transcription of a luxAB reporter gene fused with the groESL1 promoter. Similar sequences to the K‐box, GTTCGG‐NNAN‐CCNNAC, were also found upstream of the dnaK2 genes. A specific binding of a protein(s) to the N‐box, GATCTA, was detected by a gel mobility shift assay with using cell extracts. We propose that the cyanobacterial groEL expression is regulated by a putative positive mechanism mediated by these novel elements in addition to the HrcA/CIRCE system. The groEL2 genes from Synechococcus sp. strain PCC 7942 and Thermosynechococcus elongatus, which lack CIRCE, K‐box, and N‐box naturally, were also induced by heat and/or light, indicating that the control mechanism of the unique light‐responsive groE expression is highly diversified in cyanobacteria.

Comparative analysis of the hspA mutant and wild-type Synechocystis sp. strain PCC 6803 under salt stress: evaluation of the role of hspA in salt-stress management.

DNA microarray analysis has previously revealed that hspA, which encodes a small heat-shock protein, is the second most highly expressed gene under salt stress in Synechocystis sp. strain PCC 6803. Consequently, an hspA deletion mutant was studied under various salt stresses in order to identify a potential role of HspA in salt stress management. The mutant had a growth disadvantage under moderate salt stress. It lost the ability to develop tolerance to a lethal salt treatment by a moderate salt pre-treatment when the tolerance was evaluated by cell survival and the level of major soluble proteins, phycocyanins, while the wild-type acquired tolerance. Under various salt stresses, the mutant failed to undergo the ultrastructural changes characteristic of wild-type cells. The mutant, which showed higher survival than the wild-type after a direct shift to lethal salt conditions, accumulated higher levels of groESL1 and groEL2 transcripts and the corresponding proteins, GroES, GroEL1, and GroEL2, suggesting a role for these heat-shock proteins in conferring basal salt tolerance. Under salt stress, heat-shock genes, such as hspA, groEL2, and dnaK2, were transcriptionally induced and greatly stabilized, indicating a transcriptional and post-transcriptional mechanism of acclimation to salt stress involving these heat-shock genes.

The PedR transcriptional regulator interacts with thioredoxin to connect photosynthesis with gene expression in cyanobacteria

The redox state of the photosynthetic electron transport chain acts as a critical sensing mechanism by regulating the transcription of key genes involved in the acclimation response to a change in the environment. In the present study we show that the small LuxR-type regulator PedR interacts with Trx (thioredoxin) to achieve photosynthetic electron-transport-dependent transcriptional regulation in the cyanobacterium Synechocystis sp. PCC 6803. TrxM, an isoform of Trx, was isolated as an interacting factor of PedR by pull-down assays. In vitro analysis revealed that the intermolecular disulfide bond formed between Cys80 residues of the PedR homodimer was reduced by both TrxM and TrxX. It has been shown previously that, although PedR is active under low-light conditions, it becomes transiently inactivated following a shift to high-light conditions, with a concomitant conformational change [Nakamura and Hihara (2006) J. Biol. Chem. 281, 36758-36766]. In the present study, we found that the conformational change of PedR and the change in the transcript level of its target gene were minimal when mutants of Synechocystis that lack ferredoxin-Trx reductase or NADPH-Trx reductase were exposed to high levels of light. These results indicate that the reduction of PedR by Trx causes transient inactivation of PedR upon the shift of cyanobacterial cells to high-light conditions.

A change in the sensitivity of elongation factor G to oxidation protects photosystem II from photoinhibition in Synechocystis sp. PCC 6803

The repair of photosystem II (PSII) after photodamage is particularly sensitive to oxidative stress and inhibition of such repair is associated with the oxidation of specific cysteine residues in elongation factor G (EF‐G), a key translation factor, in the cyanobacterium Synechocystis sp. PCC 6803. Expression of mutated EF‐G with a target cysteine residue replaced by serine in Synechocystis resulted in the protection of PSII from photoinhibition. This protection was attributable to the enhanced repair of PSII via acceleration of the synthesis of the D1 protein, which might have been due to reduced sensitivity of protein synthesis to oxidative stress.

Elongation Factor G Is a Critical Target during Oxidative Damage to the Translation System of Escherichia coli

Background: Elongation factor G of Escherichia coli is sensitive to oxidation. Results: Elongation factor G is inactivated via the formation of an intramolecular disulfide bond. Conclusion: Elongation factor G is a critical target during oxidative damage to the translation system. Significance: Oxidation of elongation factor G suggests a novel mechanism for the redox regulation of translation. Elongation factor G (EF-G), a key protein in translational elongation, is known to be particularly susceptible to oxidation in Escherichia coli. However, neither the mechanism of the oxidation of EF-G nor the influence of its oxidation on translation is fully understood. In the present study, we investigated the effects of oxidants on the chemical properties and function of EF-G using a translation system in vitro derived from E. coli. Treatment of EF-G with 0.5 mm H2O2 resulted in the complete loss of translational activity. The inactivation of EF-G by H2O2 was attributable to the oxidation of two specific cysteine residues, namely, Cys114 and Cys266, and subsequent formation of an intramolecular disulfide bond. Replacement of Cys114 by serine rendered EF-G insensitive to oxidation and inactivation by H2O2. Furthermore, generation of the translation system in vitro with the mutated EF-G protected the entire translation system from oxidation, suggesting that EF-G might be a primary target of oxidation within the translation system. Oxidized EF-G was reactivated via reduction of the disulfide bond by thioredoxin, a ubiquitous protein that mediates dithiol-disulfide exchange. Our observations indicate that the translational machinery in E. coli is regulated, in part, by the redox state of EF-G, which might depend on the balance between the supply of reducing power and the degree of oxidative stress.

Constitutive expression of small heat shock protein in an htpG disruptant of the cyanobacterium Synechococcus sp. PCC 7942

In cyanobacteria, a disruptant of hspA encoding a small heat shock protein homologue, shows decreased cell growth rates at moderately high temperatures, and loss of both basal and acquired thermo-tolerances, which resemble the phenotype of an htpG disruptant. In vitro studies have shown that both small heat shock protein and Hsp90 can bind and keep non-native proteins in a refolding-competent state under denaturing conditions. The aim of the present study is to elucidate whether constitutive expression of HspA can functionally replace HtpG, a prokaryotic homolog of Hsp90, in the cyanobacterium Synechococcus sp. PCC 7942. HspA did not improve the viability of the htpG disruptant at a lethal temperature, although it did that of the wild type. It did not improve an iron-starved phenotype of the mutant under normal growth conditions, a novel phenotype found in the present study. These results suggest that cellular function of HtpG may differ significantly from that of HspA.