Effects of Perturbations of the Nitrogenase Electron Transfer Chain on Reversible ADP-Ribosylation of Nitrogenase Fe Protein in Klebsiella pneumoniae Strains Bearing the Rhodospirillum rubrum dra Operon

Abstract

The redox state of nitrogenase Fe protein is shown to affect regulation of ADP-ribosylation in Klebsiella pneumoniae strains transformed by plasmids carrying dra genes from Rhodospirillum rubrum. The dra operon encodes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase, enzymes responsible for the reversible inactivation, via ADP-ribosylation, of nitrogenase Fe protein in R. rubrum. In bacteria containing the dra operon in their chromosomes, inactivation occurs in response to energy limitation or nitrogen sufficiency. The dra gene products, expressed at a low level in K. pneumoniae, enable transformants to reversibly ADP-ribosylate nitrogenase Fe protein in response to the presence of fixed nitrogen. The activities of both regulatory enzymes are regulated in vivo as described in R. rubrum. Genetic perturbations of the nitrogenase electron transport chain were found to affect the rate of inactivation of Fe protein. Strains lacking the electron donors to Fe protein (NifF or NifJ) were found to inactivate Fe protein more quickly than a strain with wild-type background. Deletion of nifD, which encodes a subunit of nitrogenase MoFe protein, was found to result in a slower inactivation response. No variation was found in the reactivation responses of these strains. It is concluded that the redox state of the Fe protein contributes to the regulation of the ADP-ribosylation of Fe protein.Biological nitrogen fixation is a capability found in diverse genera of bacteria, including plant root-associated symbionts, free-living bacteria, and cyanobacteria. The conversion of N 2 to NH 3 is catalyzed by the nitrogenase enzyme complex, which comprises two separable components. Nitrogenase MoFe protein is an ␣ 2 ␤ 2 tetramer containing the cofactor FeMoco, believed to be the site of nitrogen reduction. Nitrogenase Fe protein is the obligate electron donor to MoFe protein. Fe protein is a homodimer, containing a [Fe 4 S 4 ] cluster bound by cysteinyl ligands at the dimer interface (reviewed in reference 9). Nitrogen fixation is extremely expensive in terms of the biological energy requirement. No fewer than 16 high-energy phosphoanhydride bond cleavages (ATP energy equivalents) are required for the reduction of a single dinitrogen molecule (21). In response to this requirement in the unstable microbial environment, some nitrogen-fixing bacteria have adopted a posttranslational regulatory system in which the activity of the electron carrier, Fe protein, is regulated. Inhibition of Fe protein activity is achieved by ADP-ribosylation of a specific arginine residue located on the surface of Fe protein that interacts with MoFe protein (6, 26).The ADP-ribosylation system has been best characterized in the purple nonsulfur bacterium Rhodospirillum rubrum. The NAD ϩ -dependent ADP-ribosylation of R101 of the R. rubrum Fe protein is catalyzed by dinitrogenase reductase ADP-ribosyltransferase (DRAT) in response to energy limitation or nitrogen sufficiency (16). Upon relief of these negative ...

The fixABCX Genes in Rhodospirillum rubrum Encode a Putative Membrane Complex Participating in Electron Transfer to Nitrogenase

“…Since the fixC mutant was severely affected in nitrogen fixation, the switch-off response of the mutant was studied. Halbleib and coworkers have previously shown that oxidized dinitrogenase reductase is the preferred substrate for dinitrogenase reductase ADP ribosyltransferase (DRAT), whereas the reduced form is the preferred substrate for dinitrogenase reductase-activating glycohydrolase (DRAG) (17,18). Glutamate-grown SNT-1 showed a normal switch-off response to ammonia and light, while the response of diazotrophically grown SNT-1 was variable; sometimes diazotrophically grown SNT-1 showed a normal switchoff response, while at other times little or no switch-off was observed.…”

Characterization of the DraT/DraG System for Posttranslational Regulation of Nitrogenase in the Endophytic Betaproteobacterium Azoarcus sp. Strain BH72

“…It was previously assumed for phototrophs (12,38) that the electron transport might play a role in the regulation of nitrogenase activity. In Azoarcus sp.…”

“…In addition, it has been demonstrated that the Fe protein redox status is important for the interaction with DraT or DraG, respectively (12). DraT exhibited higher activity with the Fe protein in its oxidized form, whereas DraG had no activity with oxidized Fe protein.…”

Effect of P II and Its Homolog GlnK on Reversible ADP-Ribosylation of Dinitrogenase Reductase by Heterologous Expression of the Rhodospirillum rubrum Dinitrogenase Reductase ADP-Ribosyl Transferase–Dinitrogenase Reductase-Activating Glycohydrolase Regulatory System in Klebsiella pneumoniae

“…A 15 to 25-ml culture was transferred to a 60-ml serum vial with a rubber stopper, degassed and flushed with argon, and then derepressed for nitrogenase for 4 to 5 h under anaerobic conditions at 30°C. Assay of whole-cell nitrogenase activity has been described previously (15). To maintain plasmids, appropriate antibiotics were added in all media.…”

“…A freeze-thaw protocol was used for the transformation of plasmids into K. pneumoniae (46). pCH7 (P tac -draTG) was constructed by digestion of pCH1 (P tac -draTGB) (15) with NcoI and religated, resulting in the deletion of draB.…”