Dinitrogenase Reductase-activating Glycohydrolase Research Articles

Posttranslational regulation of nitrogenase activity in Azospirillum brasilense ntrBC mutants: ammonium and anaerobic switch-off occurs through independent signal transduction pathways

Nitrogenase activity is regulated by reversible ADP-ribosylation in response to NH4+ and anaerobic conditions in Azospirillum brasilense. The effect of mutations in ntrBC on this regulation was examined. While NH4+ addition to ntrBC mutants caused a partial loss of nitrogenase activity, the effect was substantially smaller than that seen in ntr+ strains. In contrast, nitrogenase activity in these mutants was normally regulated in response to anaerobic conditions. The analysis of mutants lacking both the ntrBC gene products and dinitrogenase reductase activating glycohydrolase (DRAG) suggested that the primary effect of the ntrBC mutations was to alter the regulation of DRAG activity. Although nif expression in the ntr mutants appeared normal, as judged by activity, glutamine synthetase activity was significantly lower in ntrBC mutants than in the wild type. We hypothesize that this lower glutamine synthetase activity may delay the transduction of the NH4+ signal necessary for the inactivation of DRAG, resulting in a reduced response of nitrogenase activity to NH4+. Finally, data presented here suggest that different environmental stimuli use independent signal pathways to affect this reversible ADP-ribosylation system.

Cloning, sequencing, mutagenesis, and functional characterization of draT and draG genes from Azospirillum brasilense.

The Azospirillum brasilense draT gene, encoding dinitrogenase reductase ATP-ribosyltransferase, and draG gene, encoding dinitrogenase reductase activating glycohydrolase, were cloned and sequenced. Two genes were contiguous on the A. brasilense chromosome and showed extensive similarity to the same genes from Rhodospirillum rubrum. Analysis of mutations introduced into the dra region on the A. brasilense chromosome showed that mutants affected in draT were incapable of regulating nitrogenase activity in response to ammonium. In contrast, a mutant with an insertion in draG was still capable of ADP-ribosylating dinitrogenase reductase in response to ammonium but was no longer able to recover activity after ammonium depletion. Plasmid-borne draTG genes from A. brasilense were introduced into dra mutants of R. rubrum and restored these mutants to an apparently wild-type phenotype. It is particularly interesting that dra mutants of R. rubrum containing draTG of A. brasilense can respond to darkness and light, since A. brasilense is a nonphotosynthetic bacterium and its dra system does not normally possess that regulatory response. The nifH gene of A. brasilense, encoding dinitrogenase reductase (the substrate of dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase), is located 1.9 kb from the start of draT and is divergently transcribed. Two insertion mutations in the region between draT and nifH showed no significant effect on nitrogenase activity or its regulation.

Mutations affecting nitrogenase switch-off in Rhodobacter capsulatus

In vivo 'switch-off' and subsequent reactivation of nitrogenase activity in Rhodobacter capsulatus or Rhodospirillum rubrum in response to a variety of environmental stimuli, including the addition of fixed nitrogen, is thought to be due to the action of two nitrogenase Fe protein modifying activities; DRAT (dinitrogenase reductase ADP-ribosyl transferase) and DRAG (dinitrogenase reductase-activating glycohydrolase). Here it is demonstrated that strains, including one mutated in glnB, that constitutively express nif in the presence of fixed nitrogen are never-the-less capable of Fe protein modification. Thus the regulation of Fe protein modification is separate from that of its expression. The observations that Mn-deficient cultures are unable to fix nitrogen and that DRAG activity requires a divalent metal cation, most notably Mn2+, prompted the search for mutants (pseudo-prototrophs) capable of in vivo nitrogen fixation under Mn-deficient conditions. In the present study the isolation and partial characterization of several putative mutants is described. One, AF1, was shown to be altered in the in vivo regulation of N2ase activity in response to fixed nitrogen and to have an altered in vitro activity in glutamate grown cells. However, this strain was shown to possess in vitro DRAT activity and to have a modifiable Fe protein. Two-dimensional gel analysis indicates that this strain is altered in the synthesis of a 48 kDa protein of as yet unknown function. Thus, the mutation in this strain must affect, in an as yet undetermined manner, the response of the modifying system to fixed nitrogen.

Mutations in the draT and draG genes of Rhodospirillum rubrum result in loss of regulation of nitrogenase by reversible ADP-ribosylation

Reversible ADP-ribosylation of dinitrogenase reductase forms the basis of posttranslational regulation of nitrogenase activity in Rhodospirillum rubrum. This report describes the physiological effects of mutations in the genes encoding the enzymes that add and remove the ADP-ribosyl moiety. Mutants lacking a functional draT gene had no dinitrogenase reductase ADP-ribosyltransferase (DRAT, the draT gene product) activity in vitro and were incapable of modifying dinitrogenase reductase with ADP-ribose in vivo. Mutants lacking a functional draG gene had no dinitrogenase reductase-activating glycohydrolase (DRAG, the draG gene product) activity in vitro and were unable to remove ADP-ribose from the modified dinitrogenase reductase in vivo. Strains containing polar mutations in draT had no detectable DRAG activity in vitro, suggesting likely cotranscription of draT and draG. In strains containing draT and lacking a functional draG, dinitrogenase reductase accumulated in the active form under derepressing conditions but was rapidly ADP-ribosylated in response to conditions that cause inactivation. Detection of DRAT in these cells in vitro demonstrated that DRAT is itself subject to posttranslational regulation in vivo. Mutants affected in an open reading frame immediately downstream of draTG showed regulation of dinitrogenase reductase by ADP-ribosylation, although differences in the rates of ADP-ribosylation were apparent.

Cloning and expression of draTG genes from Azospirillum lipoferum

A genomic library of Azospirillum lipoferum was constructed with phage λEMBL4 as vector. From this library, the genes encoding dinitrogenase reductase ADP-ribosyltransferase (DRAT), draT, and dinitrogenase reductase-activating glycohydrolase (DRAG), draG, were cloned by hybridization with the heterologous probes of Rhodospirillum rubrum. As in R. rubrum, draT is located between draG and nifH, the gene encoding dinitrogenase reductase (a substrate for the DRAG/DRAT system). In the crude extract of Escherichia coli harboring the expression vector for this region, DRAT and DRAG enzyme activities were detected, confirming the identity of the cloned genes. Southern hybridization with genomic DNA from different Azospirillum spp., demonstrated a correlation between observable draTG hybridization and the biochemical demonstration of this covalent modification system.

Posttranslational regulatory system for nitrogenase activity in Azospirillum spp

The mechanism for "NH4+ switch-off/on" of nitrogenase activity in Azospirillum brasilense and A. lipoferum was investigated. A correlation was established between the in vivo regulation of nitrogenase activity by NH4Cl or glutamine and the reversible covalent modification of dinitrogenase reductase. Dinitrogenase reductase ADP-ribosyltransferase (DRAT) activity was detected in extracts of A. brasilense with NAD as the donor molecule. Dinitrogenase reductase-activating glycohydrolase (DRAG) activity was present in extracts of both A. brasilense and A. lipoferum. The DRAG activity in A. lipoferum was membrane associated, and it catalyzed the activation of inactive nitrogenase (by covalent modification of dinitrogenase reductase) from both A. lipoferum and Rhodospirillum rubrum. A region homologous to R. rubrum draT and draG was identified in the genomic DNA of A. brasilense as a 12-kilobase EcoRI fragment and in A. lipoferum as a 7-kilobase EcoRI fragment. It is concluded that a posttranslational regulatory system for nitrogenase activity is present in A. brasilense and A. lipoferum and that it operates via ADP-ribosylation of dinitrogenase reductase as it does in R. rubrum.

Regulation of Nitrogenase Activity by Reversible ADP Ribosylation

Publisher Summary This chapter discusses the molecular bases for Gest and Kamen's observations and attempts to put these observations into perspective with respect to the regulation of the nitrogenase enzyme complex by the reversible ADP ribosylation of dinitrogenase reductase. Although it is often difficult to demarcate where to begin a literature review, in this case the relevant literature begins precisely with the publication of consecutive papers by Gest and Kamen in Science in 1949. In these two brief papers, Gest and Kamen described their observations that the photosynthetic bacterium Rhodospirillum rubrum fixes nitrogen, that this activity is light-dependent, and that the activity is inhibited by ammonia. Thereafter, the discovery of dinitrogenase reductase-activating glycohydrolase (DRAG) allowed the purification and characterization of the dinitrogenase reductase from R. rubrum in its inactive form. The purification of the protein could be monitored by assaying the enzyme activity after activation. DRAG was purified based on its ability to activate ADP-ribosylated dinitrogenase reductase, even though it was not known at the time that ADP-ribose was the modifying group. The enzyme was found to be associated with the membrane fraction of extracts. Consequently, it was observed that Mono-ADP ribosylation exhibited a mechanism of action of the diphtheria toxin. This suggested that ADP ribosylation might play a role in normal cell metabolism as well by regulating the activity of key enzymes. The R. rubrum nitrogenase system provides an excellent experimental system for the investigation of such regulation.