Oxidation of Translation Factor EF-Tu Inhibits the Repair of Photosystem II.

The repair of photosystem II (PSII) is particularly sensitive to oxidative stress and the inhibition of repair is associated with oxidative damage to the translational elongation system in the cyanobacterium Synechocystis sp. PCC 6803. However, the molecular mechanisms underlying this inhibition are unknown. We previously demonstrated in vitro that EF-Tu, a translation factor that delivers aminoacyl-tRNA to the ribosome, is inactivated by reactive oxygen species via oxidation of the Cys residue Cys-82. In this study, we examined the physiological role of the oxidation of EF-Tu in Synechocystis Under strong light, EF-Tu was rapidly oxidized to yield oxidized monomers in vivo. We generated a Synechocystis transformant that expressed mutated EF-Tu in which Cys-82 had been replaced with a Ser residue. Under strong light, the de novo synthesis of proteins that are required for PSII repair, such as D1, was enhanced in the transformant and photoinhibition of PSII was alleviated. However, photodamage to PSII, measured in the presence of lincomycin, was similar between the transformant and wild-type cells, suggesting that expression of mutated EF-Tu might enhance the repair of PSII. Alleviating photoinhibition through mutation of EF-Tu did not alter cell growth under strong light, perhaps due to the enhanced production of reactive oxygen species. These observations suggest that the oxidation of EF-Tu under strong light inhibits PSII repair, resulting in the stimulation of photoinhibition.

Oxidation of a Cysteine Residue in Elongation Factor EF-Tu Reversibly Inhibits Translation in the Cyanobacterium Synechocystis sp. PCC 6803.

Translational elongation is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803, and elongation factor G has been identified as a target of oxidation by ROS. In the present study we examined the sensitivity to oxidation by ROS of another elongation factor, EF-Tu. The structure of EF-Tu changes dramatically depending on the bound nucleotide. Therefore, we investigated the sensitivity to oxidation in vitro of GTP- and GDP-bound EF-Tu as well as that of nucleotide-free EF-Tu. Assays of translational activity with a reconstituted translation system from Escherichia coli revealed that GTP-bound and nucleotide-free EF-Tu were sensitive to oxidation by H2O2, whereas GDP-bound EF-Tu was resistant to H2O2. The inactivation of EF-Tu was the result of oxidation of Cys-82, a single cysteine residue, and subsequent formation of both an intermolecular disulfide bond and sulfenic acid. Replacement of Cys-82 with serine rendered EF-Tu resistant to inactivation by H2O2, confirming that Cys-82 was a target of oxidation. Furthermore, oxidized EF-Tu was reduced and reactivated by thioredoxin. Gel-filtration chromatography revealed that some of the oxidized nucleotide-free EF-Tu formed large complexes of >30 molecules. Atomic force microscopy revealed that such large complexes dissociated into several smaller aggregates upon the addition of dithiothreitol. Immunological analysis of the redox state of EF-Tu in vivo showed that levels of oxidized EF-Tu increased under strong light. Thus, resembling elongation factor G, EF-Tu appears to be sensitive to ROS via oxidation of a cysteine residue, and its inactivation might be reversed in a redox-dependent manner.

Identification of regulatory regions and regulatory protein complexes of the Spirulina desD gene under temperature stress conditions: role of thioredoxin as an inactivator of a transcriptional repressor GntR under low-temperature stress.

In the present study, electrophoretic mobility shift assays were used to identify temperature responsive elements in the 5' upstream region (5' UTR) of the Spirulina desD gene. Overlapping, synthetic oligonucleotides of both sense and anti-sense strands that spanned the entire 5' UTR of the gene were analyzed. The responsive DNA-binding protein complexes were identified using liquid chromatography-tandem mass spectrometry. The results indicated that the cold-responsive elements were located at -453 to -247, -197 to -151, -105 to -76, and -50 to -1, whereas the low-temperature specific regulatory regions were located at -372 to -352. Moreover, the heat-responsive elements were located at -347 to -243, -197 to -151, and -124 to -1, whereas the high-temperature specific elements were located between -130 to -101 and -30 to -1. In terms of regulatory protein complexes under the two stress conditions, Trx was only detected in the low-temperature responsive protein complex, and divalent cations were essential for the binding of the protein complex to the regulatory elements. Furthermore, Trx was shown to play a critical role as a reducing agent that inactivates the Spirulina desD repressor, GntR. Consequently, the desD gene expression is induced under the low-temperature condition.

Hik28-dependent and Hik28-independent ABC transporters were revealed by proteome-wide analysis of ΔHik28 under combined stress.

Synechocystis histidine kinase, Sll0474: Hik28, a signal protein in a two-component signal transduction system, plays a critical role in responding to a decrease in growth temperature and is also involved in nitrogen metabolism. In the present study, under combined stress from non-optimal growth temperature and nitrogen depletion, a comparative proteomic analysis of the wild type (WT) and a deletion mutant (MT) of Synechocystis histidine kinase, Sll0474: Hik28, in a two-component signal transduction system identified the specific groups of ABC transporters that were Hik28-dependent, e.g., the iron transporter, and Hik28-independent, e.g., the phosphate transporter. The iron transporter, AfuA, was found to be upregulated only in the WT strain grown under the combined stress of high temperature and nitrogen depletion. Whereas, the expression level of the phosphate transporter, PstS, was increased in both the WT and MT strains. Moreover, the location in the genome of the genes encoding Hik28 and ABC transporters in Synechocystis sp. PCC6803 were analyzed in parallel with the comparative proteomic data. The results suggested the regulation of the ABC transporters by the gene in a two-component system located in an adjacent location in the genome.

Comparative analysis of the Spirulina platensis subcellular proteome in response to low- and high-temperature stresses: uncovering cross-talk of signaling components.

The present study focused on comparative proteome analyses of low- and high-temperature stresses and potential protein-protein interaction networks, constructed by using a bioinformatics approach, in response to both stress conditions.The data revealed two important points: first, the results indicate that low-temperature stress is tightly linked with oxidative stress as well as photosynthesis; however, no specific mechanism is revealed in the case of the high-temperature stress response. Second, temperature stress was revealed to be linked with nitrogen and ammonia assimilation. Moreover, the data also highlighted the cross-talk of signaling pathways. Some of the detected signaling proteins, e.g., Hik14, Hik26 and Hik28, have potential interactions with differentially expressed proteins identified in both temperature stress conditions. Some differentially expressed proteins found in the Spirulina protein-protein interaction network were also examined for their physical interactions by a yeast two hybrid system (Y2H). The Y2H results obtained in this study suggests that the potential PPI network gives quite reliable potential interactions for Spirulina. Therefore, the bioinformatics approach employed in this study helps in the analysis of phenomena where proteome analyses of knockout mutants have not been carried out to directly examine for specificity or cross-talk of signaling components.

Subcellular proteomic characterization of the high-temperature stress response of the cyanobacterium Spirulina platensis.

The present study examined the changes in protein expression in Spirulina platensis upon exposure to high temperature, with the changes in expression analyzed at the subcellular level. In addition, the transcriptional expression level of some differentially expressed proteins, the expression pattern clustering, and the protein-protein interaction network were analyzed. The results obtained from differential expression analysis revealed up-regulation of proteins involved in two-component response systems, DNA damage and repair systems, molecular chaperones, known stress-related proteins, and proteins involved in other biological processes, such as capsule formation and unsaturated fatty acid biosynthesis. The clustering of all differentially expressed proteins in the three cellular compartments showed: (i) the majority of the proteins in all fractions were sustained tolerance proteins, suggesting the roles of these proteins in the tolerance to high temperature stress, (ii) the level of resistance proteins in the photosynthetic membrane was 2-fold higher than the level in two other fractions, correlating with the rapid inactivation of the photosynthetic system in response to high temperature. Subcellular communication among the three cellular compartments via protein-protein interactions was clearly shown by the PPI network analysis. Furthermore, this analysis also showed a connection between temperature stress and nitrogen and ammonia assimilation.

Oxidation of translation factor EF-G transiently retards the translational elongation cycle in Escherichia coli.

In Escherichia coli, elongation factor G (EF-G), a key protein in translational elongation, is particularly susceptible to oxidation. We demonstrated previously that EF-G is inactivated upon formation of an intramolecular disulphide bond. However, the details of the mechanism by which the oxidation of EF-G inhibits the function of EF-G on the ribosome remain to be elucidated. When we oxidized EF-G with hydrogen peroxide, neither the insertion of EF-G into the ribosome nor single-cycle translocation activity in vitro was affected. However, the GTPase activity and the dissociation of EF-G from the ribosome were suppressed when EF-G was oxidized. The synthesis of longer peptides was suppressed to a greater extent than that of a shorter peptide when EF-G was oxidized. Thus, the formation of the disulphide bond in EF-G might interfere with the hydrolysis of GTP that is coupled with dissociation of EF-G from the ribosome and might thereby retard the turnover of EF-G within the translational machinery. When we added thioredoxin to the suppressed translation system that included oxidized EF-G, translational activity was almost immediately restored. We propose that oxidation of EF-G might provide a regulatory mechanism for transient and reversible suppression of translation in E. coli under oxidative stress.