Introduction: In the marine bacterium Dinoroseobacter shibae the photosynthetic gene cluster (PGC), encoding all components for aerobic anoxygenic photosynthesis, is specifically activated under dark growth conditions. The photosynthetic repressor PpsR inhibits PGC transcription under white & blue light conditions by binding to promoter sequences and preventing RNA polymerase binding. In our current regulatory model, the light-dependent, FMN containing LOV protein LdaP interacts with PpsR only in its dark state, while light state LdaP is inactive. Thereby LdaP acts as a PpsR antirepressor only under dark growth conditions. (Pucelik et al., 2024, Front. Microbiol. 15:1351297. doi: 10.3389/fmicb.2024.1351297).

Goals: Characterising the light-dependent interaction of the antirepressor LdaP with the PpsR repressor and its influence on DNA binding properties of PpsR.

Methods: Homologous produced & purified LdaP protein was incubated under blue light or dark conditions with SPOT peptide arrays representing the PpsR protein sequence and vice versa. Specific interactions were detected using LdaP or PpsR specific antisera. The DNA binding properties of PpsR were analysed by EMSA using a 100 bp bchF promoter DNA fragment and recombinantly produced PpsR. In addition, in vivo DNA binding studies of PpsR were performed using chromatin immunoprecipitation followed by qPCR. The oligomeric state of LdaP & PpsR was investigated using in vitro crosslinking.

Results: The SPOT peptide arrays showed specific interaction of LdaP with PpsR at the DNA binding domain of PpsR only under dark conditions. Single amino acid exchanges involved in the FMN coordination of LdaP abolished this interaction. EMSA analyses revealed specific PpsR binding to the conserved binding motif 5´-TGT-N12-ACA-3". In EMSA assays, LdaP did not abolish PpsR binding, but instead altered complex formation only under dark conditions. In vitro crosslinking experiments indicated the formation of PpsR tetramers and LdaP dimers as well as potential tetramers. This led to the suggestion that, under dark conditions, PpsR, LdaP and DNA form an altered complex that enables the transcription of the PGC.

Summary: LdaP mediates the activation of PGC expression, presumably by acting as an antirepressor of PpsR. Specific binding regions within the LdaP and PpsR proteins were identified. Light-dependent complex formation of PpsR, LdaP and DNA was demonstrated. An octameric protein complex of PpsR and LdaP is proposed.