Introduction: In the marine bacterium Dinoroseobacter shibae the photosynthetic gene cluster (PGC), encoding all components for aerobic anoxygenic photosynthesis, is regulated in a light-dependent way. In our current regulatory model, PpsR inhibits the transcription of the PGC under white & blue light conditions by binding to promoter sequences. These sequences partially overlap with the -10 and -35 promoter regions. This prevents RNA polymerase binding. Under light or blue light conditions LdaP is in its inactive form and unable to bind to PpsR. Transcriptional activation under dark conditions is mediated by the interaction of dark state LdaP with PpsR preventing repressor function. In addition, the antirepressor PpaA is able to interact with PpsR in the presence of cobalamin and act as a second antirepressor of PpsR (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.

Results: The SPOT peptide arrays showed an interaction of LdaP with PpsR at the DNA binding domain of PpsR, specifically under dark conditions. EMSA analyses revealed specific PpsR binding to the conserved binding motif 5´-TGT-N12-ACA-3". Addition of LdaP did not abolish PpsR binding. Simultaneous EMSA and Western blot analyses revealed that only PpsR is present in the detected shift complex.

Summary: LdaP mediates expression of the PGC presumably by acting as an antirepressor of PpsR. Specific binding regions within the LdaP and PpsR proteins were identified. A light-dependent binding of PpsR in the presence of LdaP was not detected by in vitro binding studies. Specific binding of PpsR to the bchF promoter was determined.