Identification of OmpR-family response regulators interacting with thioredoxin in the Cyanobacterium Synechocystis sp. PCC 6803

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 (Slr0947) and ManR (Slr1837), were identified as putative Trx targets [corrected].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.

Fig 1. Screening system to detect Trx-TF interaction in E. coli cells.

(A) Schematic representation of the E. coli co-expression screening system. The S-tagged mutant TrxM, whose active site C35 was substituted with serine and a His-tagged TF, are co-expressed in E. coli (Origami2 strain) cells. The Trx-TF complex can be detected by immunoblot analysis using either S-protein or a His-tag antibody. (B) Expression levels of PedR and Trx in the control Origami2 strain (Control), the strain expressing only TrxM_C35S (Trx), the strain expressing only PedR (PedR) and the strain expressing both TrxM_C35S and PedR (Trx-PedR). The whole cell extract (C), the soluble fraction (S) and the insoluble pellet fraction (P) were separated by 15% SDS-PAGE and stained with Coomassie Brilliant Blue (CBB). (C) Detection of the interaction between PedR and Trx. After the E. coli soluble protein fraction was separated by non-reducing SDS-PAGE, PedR and Trx were detected by immunoblot analysis using a His-tag antibody (left) and S-protein (right), respectively. ± indicates with or without 100 mM DTT treatment. Black arrow, white arrow and arrow head indicate the PedR monomer, the Trx monomer, and the Trx-PedR complex, respectively.

Fig 2. Examination of the interaction of RpaA, RpaB and ManR with Trx in E. coli cells.

Expression levels of (A) RpaA, (C) RpaB, (E) ManR and Trx in the control E. coli Origami2 strain (Control), the strain expressing only TrxM_C35S (Trx), the strain expressing only a TF and the strain expressing both TrxM_C35S and a TF were examined. The whole cell extract (C), the soluble fraction (S) and the insoluble pellet fraction (P) were separated by 15% SDS-PAGE and stained with CBB. The interaction of (B) RpaA, (D) RpaB and (F) ManR with Trx was examined by non-reducing 12% SDS-PAGE and immunoblot analysis of soluble proteins from each strain. TFs and Trx were detected using a His-tag antibody and S-protein, respectively. ± indicates with or without 100 mM DTT treatment. Black arrow, white arrow and arrow head indicate the TF monomer, the Trx monomer, and the Trx-TF complex, respectively.

Fig 3. Examination of the interaction of Rre3, CopR, NrsR and CcaR with Trx in E. coli cells.

Soluble proteins from the E. coli Origami2 strain (Control), the strain expressing only TrxM_C35S (Trx), the strain expressing only a TF and the strain expressing both TrxM_C35S and a TF were separated by non-reducing 12% SDS-PAGE and immunoblot analysis was performed. (A) Rre3, (B) CopR, (C) NrsR and (D) CcaR were detected using a His-tag antibody (left panel) and Trx was detected with S-protein (right panel). ± indicates with or without 100 mM DTT treatment. Black and white arrows indicate the TF monomer and the Trx monomer, respectively.

Fig 4. Examination of the interaction of Rre28, Rre37 and SphR with Trx in E. coli cells.

Soluble proteins from the E. coli Origami2 strain (Control), the strain expressing only TrxM_C35S (Trx), the strain expressing only a TF and the strain expressing both TrxM_C35S and a TF were separated by non-reducing 12% SDS-PAGE and immunoblot analysis was performed. (A) Rre28, (B) Rre37 and (C) SphR were detected using a His-tag antibody (left panel) and Trx was detected using S-protein (right panel). ± indicates with or without 100 mM DTT treatment. Black and white arrows indicate the TF monomer and the Trx monomer, respectively.

Fig 5. Effects of TrxM on the redox state of RpaA, RpaB and ManR.

Purified TFs were treated with oxidizing or reducing agents, modified with NEM or PEG-maleimide, fractionated by non-reducing 15% (for NEM) or 12% (for PEG maleimide) SDS-PAGE and stained with CBB. RpaA modified with NEM (A) or PEG-maleimide (B), RpaB modified with NEM (C) or PEG-maleimide (D), ManR modified with NEM (E) or PEG-maleimide (F). TFs (5 μM) were incubated with 100 mM DTT, H₂O₂ (1 or 10 mM) or aldrithiol (100 μM). Oxidized TFs were reduced by 100 mM DTT or TrxM (0.5 or 5 μM) with indicated concentration of DTT. After precipitation with 10% (w/v) trichloroacetic acid, TFs were subjected to the thiol modification using NEM or PEG-maleimide. Ox and Red indicate the oxidized and reduced forms of a TF, respectively.