Abstract
The redox state of the photosynthetic electron transport chain is known to act as a signal to regulate the transcription of key genes involved in the acclimation responses to environmental changes. We hypothesized that the protein thioredoxin (Trx) acts as a mediator connecting the redox state of the photosynthetic electron transport chain and transcriptional regulation, and established a screening system to identify transcription factors (TFs) that interact with Trx. His-tagged TFs and S-tagged mutated form of Trx, TrxMC35S, whose active site cysteine 35 was substituted with serine to trap the target interacting protein, were co-expressed in E. coli cells and Trx-TF complexes were detected by immuno-blotting analysis. We examined the interaction between Trx and ten OmpR family TFs encoded in the chromosome of the cyanobacterium Synechocystis sp. PCC 6803 (S.6803). Although there is a highly conserved cysteine residue in the receiver domain of all OmpR family TFs, only three, RpaA (Slr0115), RpaB (Slr0946) and ManR (Slr1837), were identified as putative Trx targets. The recombinant forms of wild-type TrxM, RpaA, RpaB and ManR proteins from S.6803 were purified following over-expression in E. coli and their interaction was further assessed by monitoring changes in the number of cysteine residues with free thiol groups. An increase in the number of free thiols was observed after incubation of the oxidized TFs with Trx, indicating the reduction of cysteine residues as a consequence of interaction with Trx. Our results suggest, for the first time, the possible regulation of OmpR family TFs through the supply of reducing equivalents from Trx, as well as through the phospho-transfer from its cognate sensor histidine kinase.
Highlights
Photosynthetic organisms rearrange their cellular components to balance the supply and consumption of photosynthetically derived energy in response to changes in environmental factors, PLOS ONE | DOI:10.1371/journal.pone.0119107 March 16, 2015Screening of OmpR-Type Regulators Interacting with Thioredoxin
A His-tagged transcription factors (TFs) and S-tagged TrxMC35S are co-expressed in E. coli Origami2 (DE3) cells in which the formation of disulfide bonds is promoted due to the lack of Trx reductase and glutathione reductase in that strain
The 32 kDa band might correspond to the Trx-PedR complex, but this complex was not detected using S-protein, probably because the S-tag was located inside the complex, and less accessible to the S-protein. These results indicated that this screening system provides an effective approach to detect the interaction between Trx and TFs
Summary
Photosynthetic organisms rearrange their cellular components to balance the supply and consumption of photosynthetically derived energy in response to changes in environmental factors, PLOS ONE | DOI:10.1371/journal.pone.0119107 March 16, 2015. It has been reported that in Synechococcus elongatus PCC 7942 (S.7942), the induction of psbAII/III gene expression under high-light (HL) conditions could be mimicked by adding the thiol-specific reductant dithiothreitol (DTT) under low-light (LL) conditions, and the opposite effects were observed by adding a thiol-specific oxidant, such as diamide, under high-light (HL) conditions [7] These data suggest that the expression of the psbAII/III genes was regulated by, and dependent on, the redox state of the thiols. We examined interactions between TrxM and ten OmpR-type response regulators that are encoded in the S.6803 genome and identified RpaA (Slr0115), RpaB (Slr0947) and ManR (Slr1837) as new candidate TrxM interacting partners We further confirmed their interaction with TrxM by the experiments using the corresponding recombinant proteins
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