H2O2-inducible genes and their functions
Using DNA microarrays, we demonstrated that the genome-wide pattern of gene expression in Synechocystis is dramatically changed by exogenous H2O2. Our results suggest that the response to peroxidative stress is effectively regulated at the level of gene expression in this microorganism. Peroxidative stress as a result of 0.25 mm H2O2 remarkably enhanced the expression of 77 genes in wild-type cells, with induction factors higher than 4.0. The genes can be divided into six groups with respect to the functions of the encoded proteins.
The first group includes genes for the regulation of the intracellular redox state, such as the ahpC gene for an antioxidative enzyme and the pgr5 gene for the regulation of the cyclic electron transport of photosystem I. The ahpC gene, identified in E. coli, encodes a homolog of a peroxiredoxin in the thiol-specific antioxidant (TSA)/AhpC family, which can scavenge H2O2 and alkylhydroxyperoxide (Jeong et al., 2000). In Synechocystis, two enzymes catalyze the reduction of H2O2 to H2O, namely, catalase (Tichy and Vermaas, 1999) and thioredoxin peroxidase (Yamamoto et al., 1999), which are encoded by the katG and tpx genes, respectively. These proteins seem to be expressed constitutively in cells that are grown in the light under conditions that allow intracellular concentrations of H2O2 to remain at low and harmless levels (Tichy and Vermaas, 1999; Ushimaru et al., 2002; Yamamoto et al., 1999). However, the activity of scavenging enzymes might be insufficient to meet the challenge presented by exogenous H2O2 at 0.25 mm. Therefore, expression of the ahpC gene might be necessary for the scavenging of ROS generated by reactions with various metal ions, such as the Fenton reaction. The H2O2-inducible expression of the ahpC gene was controlled by PerR. The pgr5 gene encodes a protein with substantial similarities of amino acid sequence to a component of the antimycin A-sensitive ferredoxin:plastoquinone reductase (FQR) in Arabidopsis thaliana (Munekage et al., 2002). The pgr5 mutant of Synechocystis is sensitive to strong light, suggesting that FQR might be important in controlling the intracellular redox state under adverse conditions (Yaremenko et al., 2005). The H2O2-inducible expression of the pgr5 gene was controlled by Hik33. These results also suggest that the Hik33-dependent and PerR-dependent pathways are important for the control of the intracellular redox state.
The second group includes genes for proteins related to the response to iron deficiency, such as fecE, futA1, futA2, isiA and isiB, that are not under the control of any of the Hiks or PerR. FutA1 and FutA2 are components of an ABC-type transport system, which is a major transporter of ferric ions in Synechocystis (Katoh et al., 2001). The FecE protein is a homolog of a component of the ABC-type ferric dicitrate transporter in E. coli (Staudenmaier et al., 1989). The isiA and isiB genes encode the chlorophyll-binding protein CP43’ (Burnap et al., 1993) and flavodoxin, respectively (Straus, 1994). Expression of these genes is also induced under iron-deficient conditions in Synechocystis (Kobayashi et al., 2004; Vinnemeier et al., 1998). The metabolism of iron and several iron-containing proteins might be closely related to cellular responses to oxidative stress (Hantke, 2001; Touati, 2000). Regulation of the expression of genes in this group might be important for acclimation to peroxidative stress.
The third group includes genes for the regulation of transcription, such as sigB and sigD for sigma factors, which were not under the control of any of Hiks and PerR (Table 1). The H2O2-inducible expression of these genes was also observed by Li et al. (2004). These findings may suggest that regulation of the transcription of certain genes is important for acclimation to peroxidative stress.
The fourth group includes genes for heat-shock proteins, such as hspA, dnaJ, dnaK2 and htpG. The enhanced expression of heat-shock genes suggests that chaperones might be important for acclimation of Synechocystis to peroxidative stress. We found previously that protein synthesis de novo in Synechocystis is strongly inhibited by exogenous H2O2 (Nishiyama et al., 2001). Enhanced expression of genes for chaperones might be necessary for unhindered translation. Apart from induction by H2O2 of the htpG gene, which was controlled by Hik34, the H2O2-induced expression of heat-shock proteins was regulated by an as-yet uncharacterized mechanism. The expression of these heat-shock genes was induced transiently by heat shock (Inaba et al., 2003: Suzuki et al., 2005), strong-light stress (Hihara et al., 2003), UV irradiation (Huang et al., 2003), hyperosmotic stress (Paithoonrangsarid et al., 2004) and salt stress (Shoumskaya et al., 2005). These findings indicate that the induction of the heat-shock proteins is very rapid and a general response to various kinds of stress.
The fifth group includes genes for proteases, such as ftsH, clpB1 and ctpA, and for proteins of phycobilisome degradation, such as nblA1 and nblA2. The expression of these genes is also induced by UV irradiation (Huang et al., 2003). In Synechocystis, H2O2 inhibits de novo synthesis of the D1 protein in photosystem II (Nishiyama et al., 2001), and thus decreases the rate of turnover of the D1 protein in photosystem II. The induction expression of genes for proteases, including FtsH, which are located in thylakoid membranes and are involved in the degradation and rapid turnover of the D1 protein (Itzhaki et al., 1998; Lindahl et al., 2000; Nixon et al., 2005), may be necessary for the rapid turnover of D1 under peroxidative stress. The expression of nblA1 and nblA2 genes was controlled by both Hik33 and PerR, whereas that of ftsH genes was controlled only by Hik33.
The sixth group includes 35 genes for hypothetical proteins of unknown function (Table 1). Among these 35 genes, four genes, namely, slr0967, slr0589, sll0157 and slr1259, the induction of which by H2O2 was regulated by Hik16 plus Hik41, PerR, Hik33 and an as-yet uncharacterized mechanism, respectively, are conserved in A. thaliana and in eight species of cyanobacteria, the genomes of which have been sequenced (Cyanobase; http://www.kazusa.or.jp/cyanobase/). These genes might be important for the acclimation of plants and cyanobacteria to peroxidative stress. By contrast, 27 of the 35 genes are conserved in other cyanobacteria but not in A. thaliana, and four genes, such as ssl2162, ssl1533, ssl2501 and slr0587, are found only in Synechocystis. These results suggest that a number of hypothetical proteins, the functions of which are unknown, might be involved in the acclimation of cyanobacterial cells to peroxidative stress.
Genes whose expression was decreased by incubation with H2O2 includes the genes for phycobilisome components, photosystem-I components and a number of hypothetical proteins (the whole set of data is available from http://www.genome.jp/kegg/expression/). The expression of these genes was also decreased by UV irradiation (Huang et al., 2003) and strong-light stress (Hihara et al., 2001). Rapid reduction of photosystem I and phycobilisome components may suggest that the regulation of the photosynthetic activity by decreasing the expression of these genes is one of the primary responses for the acclimation to many kinds of stress.
Peroxidative stress-inducible genes in other organisms
In E. coli, the expression of 140 genes (about 3% of the total number of genes) is induced by 1 mm H2O2 with induction factors of either 4.0 or higher (Zheng et al., 2001). In A. thaliana, the expression of 113 genes is induced by 20 mm H2O2 with induction factors of either 1.5 or higher (Desikan et al., 2001). Comparing the H2O2-inducible genes in E. coli with those in Synechocystis, we failed to identify any homologous genes whose expression was induced by H2O2 in both microorganisms. However, the expression of the hspA, htpG, dnaJ, slr0442 and sll1615 genes, which correspond to hsp17, hsp83, AA04366 (EST accession no.), N37850 (EST accession no.) and the ras gene for GTP-binding protein in A. thaliana, respectively, is induced by H2O2 in both Synechocystis and A. thaliana. As only a small number of genes are induced in common, it is possible that the responses to H2O2 differ among species.
Regulation by Hiks of the H2O2-inducible expression of genes
As noted above, mechanisms for the H2O2-induced regulation of the expression of genes seem to differ among species. In this study, we found that Hiks are involved in the expression of a large number of H2O2-inducible genes in Synechocystis. Figure 5 shows a hypothetical scheme for the perception of H2O2 stress signals and the subsequent regulation of gene expression. Localization of Hik molecules either in the cytoplasm or within the membrane was predicted by the smart program (http://smart.embl-heidelberg.de/) and the sosui program (http://bp.nuap.nagoya-u.ac.jp/sosui/) for individual Hiks. The amino acid sequence of Hik33 indicates that it has two transmembrane regions, a histadine kinases, adenylyl cyclases, methyl binding proteins and phosphatases (HAMP)-linker, a period circadian protein, Ah receptor nuclear translocator protein, single-minded protein (PAS) domain and a Hik domain (Mikami et al., 2002). Hik34 has a Hik domain (Marin et al., 2003) and a proline- and leucine-rich region at its amino terminus. Hik16 has seven membrane-spanning domains, a cGmp-specific phosphodiesterases (GAF) domain and a Hik domain (Marin et al., 2003). Hik41 has a receiver domain and a Hik domain at its amino and carboxyl termini, respectively (Marin et al., 2003). From the results in Table 1 and the domain structures of Hik16 and Hik41, we propose that Hik16 is located in the membrane, perceives the signal caused by H2O2 and transfers the signal, as a phosphate group, to the receiver domain of Hik41, which is located in the cytosol (Marin et al., 2003).
Figure 5. A hypothetical scheme for perception and transduction of hydrogen peroxide (H2O2) stress signals in Synechocystis sp. PCC 6803. Putative signal cascades are represented by bold arrows. Representative genes, the expression of which is selectively induced by H2O2, are listed in boxes. The numbers enclosed by squares indicate the total numbers of H2O2-inducible genes with induction factors higher than 4.0 and with RE values of less than 15%. The contribution of histidine kinase 33 (Hik33) and PerR in the expression of the ndhD2,nblA1 and nblA2 genes is suggested by the finding that a mutation of either of these components eliminated the H2O2-induced expression of these genes (see Table 1).
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It has been suggested that PAS and GAF domains might bind co-factors, such as heme and cGMP, respectively (Galperin et al., 2001). Although the PAS domain of Hik33 does not contain the cysteine residues that are necessary for binding to a heme molecule, these functional domains might be involved in the perception of signals caused by H2O2 by an as-yet unknown mechanism. It has been suggested that the integrity of the periplasmic region of Sln1, a Hik in S. cerevisiae, might be essential for sensing osmotic stress and oxidative stress (Reiser et al., 2003). As the structure of Sln1 is similar to that of Hik33 (Mikami et al., 2002), it is possible that the periplasmic region of Hik33 might also be related to the perception of peroxidative signals in Synechocystis.
Tu et al. (2004) and Hsiao et al. (2004) reported that Hik33 negatively regulates the expression of hli genes under control conditions. Their conclusion was based on the changes in the global expression of genes under normal conditions, and the complementation of ΔHik33 cells of Synechocystis sp. Pcc. 7942 with the nblS gene from Synechococcus with respect to the light-induced expression of the hli genes assayed by the northern blotting analysis. As the complementation test was not performed by the genome-wide expression of genes, it is likely that their ΔHik33 cells contain a mutation in addition to the mutation of the hik33 gene (Murata and Los, 2006).
Our results demonstrated that Hiks and PerR regulate the H2O2-induced expression of a number of genes. It is likely that as-yet unknown mechanisms are also involved in the H2O2-regulated expression of genes. The data presented here will form the basis for future research on the entire pathway of H2O2 signal transduction.