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

Abstract

Reversible ADP-ribosylation of dinitrogenase reductase, catalyzed by the dinitrogenase reductase ADPribosyl transferase-dinitrogenase reductase-activating glycohydrolase (DRAT-DRAG) regulatory system, has been characterized in Rhodospirillum rubrum and other nitrogen-fixing bacteria. To investigate the mechanisms for the regulation of DRAT and DRAG activities, we studied the heterologous expression of R. rubrum draTG in Klebsiella pneumoniae glnB and glnK mutants. In K. pneumoniae wild type, the regulation of both DRAT and DRAG activity appears to be comparable to that seen in R. rubrum. However, the regulation of both DRAT and DRAG activities is altered in a glnB background. Some DRAT escapes regulation and becomes active under N-limiting conditions. The regulation of DRAG activity is also altered in a glnB mutant, with DRAG being inactivated more slowly in response to NH 4 ؉ treatment than is seen in wild type, resulting in a high residual nitrogenase activity. In a glnK background, the regulation of DRAT activity is similar to that seen in wild type. However, the regulation of DRAG activity is completely abolished in the glnK mutant; DRAG remains active even after NH 4 ؉ addition, so there is no loss of nitrogenase activity. The results with this heterologous expression system have implications for DRAT-DRAG regulation in R. rubrum.Biological nitrogen fixation, the conversion of atmospheric nitrogen to ammonium, is catalyzed by the nitrogenase complex, which consists of two proteins: dinitrogenase (or MoFe protein) and dinitrogenase reductase (or Fe protein) (7). It is a very energy-demanding process and is thus tightly regulated at both transcriptional and posttranslational levels.Transcriptional regulation of the nif genes has been found in all studied nitrogen-fixing bacteria and is best characterized in Klebsiella pneumoniae, a free-living nitrogen-fixing bacterium, where it involves the general nitrogen regulation (ntr) system (36). Analysis of the ntr regulatory system in K. pneumoniae and Escherichia coli (36, 39) has shown that it controls the transcription of many genes involved in nitrogen fixation and assimilation, such as glnA (encoding glutamine synthetase [GS]) and nifA (encoding the transcriptional activator for the other nif genes). The ntr system involves a number of gene products, including those of glnD, ntrA, ntrB, ntrC, and glnB, glnD encodes a bifunctional, uridylyltransferase-uridylyl-removing enzyme (UTase-UR) that is believed to be the sensor of the intracellular concentration of glutamine in the cell. UTase-UR reversibly controls the activity of the P II protein (the gene product of glnB) by uridylylation or deuridylylation. P II is responsible for sensing ␣-ketoglutarate (␣-KG) in E. coli (24), and it controls NtrB (NRII) activity. NtrB and NtrC (the gene products of ntrB and ntrC) belong to the family of twocomponent regulators. NtrB is a histidine kinase that phosphorylates NtrC (NRI) under nitrogen-limiting conditions and also can act as a phosphatase to dephosphorylate NtrC unde...

Yeast Two-Hybrid Studies on Interaction of Proteins Involved in Regulation of Nitrogen Fixation in the Phototrophic BacteriumRhodobacter capsulatus

“…After ammonium exhaustion, DraG becomes active, whereupon the modifying group is removed and nitrogenase activity is recovered. Recently, an involvement of PII-like proteins in the mechanism for the regulation of DraT and DraG has been demonstrated (32,40,64,65,66).…”

AmtB Is Necessary for NH 4 + -Induced Nitrogenase Switch-Off and ADP-Ribosylation in Rhodobacter capsulatus

“…In this regard, the very recent report of a reversible association of GlnK with the membrane that is AmtB and ammonium dependent is intriguing (8). The GlnK of Klebsiella pneumoniae has been shown to be involved in the posttranslational regulation of Rhodospirillum rubrum DraT and DraG when they are expressed in K. pneumoniae (55). While subsequent studies with R. rubrum have shown that these results cannot be strictly extrapolated to R. rubrum, it is clear that several of the R. rubrum P II proteins, namely, GlnB and GlnJ, are somehow involved in the posttranslational regulation of DraT and DraG in that organism (56).…”

Role of GlnK in NifL-Mediated Regulation of NifA Activity inAzotobacter vinelandii

“…In these experiments, as in other two-hybrid screens, caution should be used before any conclusions can be made concerning the validity of these new targets, and each will have to be verified in vivo or in vitro. On a positive note, however, an interaction with DRAT in this organism might be predicted from genetic evidence in the related organism Rhodospirillum rubrum (48).…”