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 brasileizse DraT and DraG, respectively, were used to identify the corresponding genes of Rhodobacter capsulatus. Sequence analysis of a 1904bp 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 AzospirUum lipoferum, the R. capsulatus draTG genes were not located upstream of the structural genes of nitrogenase nzmDK but close to the dctP gene at a distance of about lo00 kb from the nzjHDK genes. Deletion mutations in the draTG gene region were constructed and introduced into R. capsulatus wild-type and a nijHDK 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 I1 of both the molybdenum and the alternative nitrogenase were detected in a ,draTG wild-type background by immunoblot analysis, whereas only the nonmodified 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 posttranslational regulation of both the molybdenum and the heterometal-free nitrogenase in R. capsulatus.

Comparison studies of dinitrogenase reductase ADP-ribosyl transferase/dinitrogenase reductase activating glycohydrolase regulatory systems in Rhodospirillum rubrum and Azospirillum brasilense

“…draT and draG have been cloned and sequenced from R. rubrum, R. capsulatus, and A. brasilense (5,17,30). The sequence comparison of draTG from these strains showed an extensive similarity, with some conserved regions (17).…”

“…However, this regulation has been well characterized only in Rhodospirillum rubrum, Azospirillum brasilense, Azospirillum lipoferum, and Rhodobacter capsulatus, in which it involves reversible mono-ADP ribosylation of dinitrogenase reductase, catalyzed by the dinitrogenase reductase ADP-ribosyl transferase (DRAT)/dinitrogenase reductase activating glycohydrolase (DRAG) regulatory system (5,6,17,30). DRAT (the gene product of draT) catalyzes the transfer of the ADP-ribose from NAD to the Arg-101 residue of one subunit of the dinitrogenase reductase dimer and thereby inactivates the enzyme.…”

“…The sequence comparison of draTG from these strains showed an extensive similarity, with some conserved regions (17). draTG mutants also were constructed and physiologically characterized in these organisms (15,17,30).In the cases of A. brasilense and R. rubrum, the activities of DRAG and DRAT themselves are subject to some form of posttranslational regulation by an unknown mechanism. Under derepressing (nitrogen-fixing) conditions, DRAG is active and DRAT is inactive (7,15,28,30).…”

“…The sequence comparison of draTG from these strains showed an extensive similarity, with some conserved regions (17). draTG mutants also were constructed and physiologically characterized in these organisms (15,17,30).…”

Altered residues in key proteins influence the expression and activity of the nitrogenase complex in an adaptive CO2 fixation-deficient mutant strain of Rhodobacter sphaeroides

“…Glutamine and a-ketoglutarate are then converted by glutamate synthase catalysis to form glutamate. Glutamine and a-ketoglutarate, the intermediates of ammonium assimilation, signal the nitrogen status of the cell (Dixon & Kahn, 2004;Jones & Monty, 1979;Leigh & Dodsworth, 2007;Masepohl et al, 1993;Oelze & Klein, 1996;Wall & Gest, 1979). When the intracellular concentration of glutamine is low and a-ketoglutarate is high, a signal is relayed throughout the cell, resulting in the activation of NifA.…”