Identifizierung von Interaktionspartnern des response Regulators RegA aus Rhodobacter capsulatus : Die Rolle der Interaktion von RegA und NtrX bei der Regulation von Photosynthesegenen

Abstract

Rhodobacter capsulatus is a facultatively phototrophic purple bacterium. It is able to perform anoxygenic photosynthesis under anaerobic conditions in the presence of light. The formation of pigment protein complexes in facultatively photosynthetic bacteria of the genus Rhodobacter is only induced when the oxygen tension drops below a threshold value. In R. capsulatus the sensor kinase RegB autophosphorylates in a redox-dependent manner. It then transfers the phosphatidyl residue to the response regulator RegA, a DNA binding protein which activates transcription of a number of genes for pigment binding proteins and for pigment syntheses. Among the genes activated by RegA at low oxygen tension are the genes of the puf and puc operons. The puf operon comprises genes encoding pigment binding proteins of the light harvesting (LH) I complex and of the reaction center complex, and regulatory proteins. Genes required for the formation of the LH II complex are part of the puc operon. The RegB/RegA two component system is also involved in redox-dependent regulation of genes for nitrogen fixation, Calvin cycle enzymes, hydrogenase, and for enzymes of the respiratory chain. In order to test whether the coregulation of different redox-controlled regulons also involves a direct interaction of regulatory proteins the yeast two-hybrid system was applied to search for proteins interacting with the response regulator RegA. NtrX, the response regulator of the NtrY/NtrX two component system was identified as interaction partner of RegA. In Azorhizobium caulinodans the NtrY/NtrX two component system is involved in the regulation of nitrogen fixation genes. The interaction of RegA and NtrX was confirmed by GST pulldown experiments. To learn more about the role of NtrX in R. capsulatus, Rhodobacter strains were constructed, which harbor a knock-out of the chromosomal ntrX gene. These mutants showed a different composition of their photosynthetic components compared to the wild type. The change in composition was dependent on the amount of ammonium added to the medium. Additionally, all NtrX mutants showed more photosynthetic complexes than the wild type strain. When NtrX was overexpressed in R. capsulatus the cells contained lower amounts of photosynthetic complexes than wild type cells. NtrX seems to be a negative regulator of the photosynthetic complexes in R. capsualtus. Since NtrX has a typical helix-turn-helix motif for DNA-binding, gel retardation assays should show, whether NtrX can directly interact with the promoter regions of the photosynthesis operons puf and puc. An interaction of NtrX with the puf promoter region was only detectable, when a NtrX variant was used in the gel retardation assay. This NtrX variant harbored an amino acid exchange at the putuative phosphorylation site of the protein so that the conserved aspartate at position 52 was changed to glutamate. Theses results lead to the suggestion that the NtrX mediated negative regualtion of photosynthetic complexes in R. capsulatus is due to a direct interaction of NtrX with the puf promoter region.