The draTG gene region of Rhodobacter capsulatus is required for post-translational regulation of both the molybdenum and the alternative nitrogenase

Abstract

Synthetic oligonucleotides, which were designed according to amino acid sequences conserved between Rhodospirillum rubrum and Azospirillum brasilense DraT and DraG, respectively, were used to identify the corresponding genes of Rhodobacter capsulatus. Sequence analysis of a 1904 bp DNA fragment proved the existence of R. capsulatus draT and draG. These two genes were separated by 11 bp only, suggesting that R. capsulatus draT and draG were part of one transcriptional unit. In contrast to R. rubrum, A. brasilense and Azospirillum lipoferum, the R. capsulatus draTG genes were not located upstream of the structural genes of nitrogenase nifHDK but close to the dctP gene at a distance of about 1000 kb from the nifHDK genes. Deletion mutations in the draTG gene region were constructed and introduced into R. capsulatus wild-type and a nifHDK deletion strain. The resulting mutant strains were examined for post-translational regulation of the molybdenum and the alternative nitrogenase in response to ammonia and darkness. Under 'switch-off' conditions the modified (ADP-ribosylated) and the non-modified forms of component II of both the molybdenum and the alternative nitrogenase were detected in a draTG wild-type background by immunoblot analysis, whereas only the non-modified forms were present in the draTG deletion strains. Nitrogenase activity in these strains was followed by the acetylene reduction assay. In contrast to the wild-type, draTG mutants were not affected in nitrogenase activity in response to ammonia or darkness. These results demonstrated that the draTG genes are required for post-translational regulation of both the molybdenum and the heterometal-free nitrogenase in R. capsulatus.