Pucelik Saskia, Becker Miriam, Heyber Steffi, Wöhlbrand Lars, Rabus Ralf, Jahn Dieter, Härtig Elisabeth
Institute of Microbiology, Technische Universität Braunschweig, Braunschweig, Germany.
Institute for Chemistry and Biology of the Marine Environment (ICBM), Carl von Ossietzky University of Oldenburg, Oldenburg, Germany.
Front Microbiol. 2024 Feb 7;15:1351297. doi: 10.3389/fmicb.2024.1351297. eCollection 2024.
In the marine α-proteobacterium more than 40 genes of the aerobic anoxygenic photosynthesis are regulated in a light-dependent manner. A genome-wide screen of 5,605 clones from a transposon library for loss of pigmentation and changes in bacteriochlorophyll absorbance identified 179 mutant clones. The gene encoding the LOV-domain containing protein Dshi_1135 was identified by its colorless phenotype. The mutant phenotype was complemented by the expression of a Dshi_1135-strep fusion protein in trans. The recombinantly produced and chromatographically purified Dshi_1135 protein was able to undergo a blue light-induced photocycle mediated by bound FMN. Transcriptome analyses revealed an essential role for Dshi_1135 in the light-dependent expression of the photosynthetic gene cluster. Interactomic studies identified the repressor protein PpsR as an interaction partner of Dshi_1135. The physical contact between PpsR and the Dshi_1135 protein was verified using the bacterial adenylate cyclase-based two-hybrid system. In addition, the antirepressor function of the Dshi_1135 protein was demonstrated testing of a reporter gene fusion in a heterologous -based host system. We therefore propose to rename the Dshi_1135 protein to LdaP (light-dependent antirepressor of PpsR). Using the bacterial two-hybrid system, it was also shown that cobalamin (B) is essential for the interaction of the antirepressor PpaA with PpsR. A regulatory model for the photosynthetic gene cluster in was derived, including the repressor PpsR, the light-dependent antirepressor LdaP and the B-dependent antirepressor PpaA.
在海洋α-变形杆菌中，超过40个需氧不产氧光合作用基因以光依赖方式受到调控。从转座子文库中对5605个克隆进行全基因组筛选，以寻找色素沉着丧失和细菌叶绿素吸光度变化的情况，共鉴定出179个突变克隆。编码含LOV结构域蛋白Dshi_1135的基因通过其无色表型得以鉴定。通过反式表达Dshi_1135-链霉亲和素融合蛋白对突变表型进行了互补。重组产生并经色谱纯化的Dshi_1135蛋白能够经历由结合的黄素单核苷酸介导的蓝光诱导光循环。转录组分析揭示了Dshi_1135在光合基因簇光依赖表达中的重要作用。相互作用组学研究确定阻遏蛋白PpsR是Dshi_1135的相互作用伙伴。使用基于细菌腺苷酸环化酶的双杂交系统验证了PpsR与Dshi_1135蛋白之间的物理接触。此外，通过在基于异源宿主系统中测试报告基因融合，证明了Dshi_1135蛋白的抗阻遏功能。因此，我们建议将Dshi_1135蛋白重命名为LdaP（PpsR的光依赖抗阻遏蛋白）。使用细菌双杂交系统还表明，钴胺素（B12）对于抗阻遏蛋白PpaA与PpsR的相互作用至关重要。由此得出了该菌光合基因簇的调控模型，包括阻遏蛋白PpsR、光依赖抗阻遏蛋白LdaP和B12依赖抗阻遏蛋白PpaA。
