Stefan Nordlund

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

In our efforts to identify the components participating in electron transport to nitrogenase in Rhodospirillum rubrum, we used mini-Tn5 mutagenesis followed by metronidazole selection. One of the mutants isolated, SNT-1, exhibited a decreased growth rate and about 25% of the in vivo nitrogenase activity compared to the wild-type values. The in vitro nitrogenase activity was essentially wild type, indicating that the mutation affects electron transport to nitrogenase. Sequencing showed that the Tn5 insertion is located in a region with a high level of similarity to fixC, and extended sequencing revealed additional putative fix genes, in the order fixABCX. Complementation of SNT-1 with the whole fix gene cluster in trans restored wild-type nitrogenase activity and growth. Using Western blotting, we demonstrated that expression of fixA and fixB occurs only under conditions under which nitrogenase also is expressed. SNT-1 was further shown to produce larger amounts of both ribulose 1,5-bisphosphate carboxylase/oxygenase and polyhydroxy alkanoates than the wild type, indicating that the redox status is affected in this mutant. Using Western blotting, we found that FixA and FixB are soluble proteins, whereas FixC most likely is a transmembrane protein. We propose that the fixABCX genes encode a membrane protein complex that plays a central role in electron transfer to nitrogenase in R. rubrum. Furthermore, we suggest that FixC is the link between nitrogen fixation and the proton motive force generated in the photosynthetic reactions.

Necessity of a membrane component for nitrogenase activity in Rhodospirillum rubrum

Acetylene reduction catalyzed by nitrogenase from Rhodospirillum rubrum has low activity and exhibits a lag phase. The activity can be increased by the addition of a chromatophore membrane component and the lag eliminated by preincubation with this component, which can be solubilized from chromatophores by treatment with NaCl. It is both trypsin- and oxygen-sensitive. Titration of the membrane component with nitrogenase and vice versa shows a saturation point. The membrane component interacts specifically with the Fe protein of nitrogenase, the interaction being ATP- and Mg2+-dependent.

Evidence for Conformational Protection of Nitrogenase against Oxygen in Gluconacetobacter diazotrophicus by a Putative FeSII Protein

The mechanisms protecting nitrogenase in Gluconacetobacter diazotrophicus from damage by oxygen were studied. Evidence is provided suggesting that in G. diazotrophicus these mechanisms include respiratory protection as well as conformational protection in which a putative FeSII Shethna protein is involved.

Transcription of the glnB and glnA genes in the photosynthetic bacterium Rhodospirillum rubrum

The PII protein, encoded by glnB, has a central role in the control of nitrogen metabolism in nitrogen-fixing prokaryotes. The glnB gene of Rhodospirillum rubrum was isolated and sequenced. The deduced amino acid sequence had very high sequence identity to other PII proteins. The glnA gene, encoding glutamine synthetase, was located 135 bp downstream of glnB and was partially sequenced. glnB is cotranscribed with glnA from a promoter with high similarity to the sigma 54-dependent promoter consensus sequence. A putative sigma 70 promoter was also identified further upstream of glnB. Northern blotting analyses showed that in addition glnA is either transcribed from an unidentified promoter or, more likely, that the glnBA transcript is processed to give the glnA mRNA. The total level of the two transcripts was much higher in nitrogen-fixing cells than in ammonia-grown cells.

The role of glutamine synthetase in the regulation of nitrogenase activity (“switch off” effect) in Rhodospirillum rubrum

We have studied the changes in the activities of both nitrogenase (“switch off”) and glutamine synthetase in Rhodospirillum rubrum upon addition of ammonium ions or glutamine to nitrogen fixing cultures. Both activities decrease drastically and return in a parallel manner when added ammonia is metabolized. The decrease in glutamine synthetase activity does not seem to be primarily due to adenylylation of the enzyme. Addition of glutamine to cells starved for nitrogen results in inactivation of glutamine synthetase but nitrogenase is only partially switched off.

Stabilization and partial characterization of the activating enzyme for dinitrogenase reductase (Fe protein) from rhodospirillum rubrum

Nitrogenase from Rhodospirillum rubrum grown on dinitrogen or glutamate as nitrogen source requires an activating factor in vitro. This factor can be solubilized from chromatophores of this organism by 0.5 M NaCl [ 1,2]. The activating factor acts on the Fe protein (i.e., dinitrogenase reductase) [ 1,2] by cleaving from it a covalently bound modifying group, thereby converting the inactive protein to its active form [3,4]. Preliminary evidence for the existence of this kind of activation of nitrogenase in other photosynthetic bacteria was provided in [5]. The activating factor has proven to be extremely labile and although a few sug gestions were made for its purification [2,3] a reproducible method was missing. We now have found that the activating factor can be stabilized in solutions containing 0.5 mM MnClz. Here, we provide evidence for its protein aceous nature and henceforth refer to it as the activating enzyme (AE). The enzyme is extremely oxygen-sensitive with a half-life in air of -2 min. Its Mr as estimated by gel filtration was 20 500 f 2000.

Interaction of the signal transduction protein GlnJ with the cellular targets AmtB1, GlnE and GlnD in Rhodospirillum rubrum: dependence on manganese, 2-oxoglutarate and the ADP/ATP ratio

The PII family of signal transduction proteins is widespread amongst the three domains of life, and its members have fundamental roles in the general control of nitrogen metabolism. These proteins exert their regulatory role by direct protein-protein interaction with a multitude of cellular targets. The interactions are dependent on the binding of metabolites such as ATP, ADP and 2-oxoglutarate (2-OG), and on whether or not the PII protein is modified. In the photosynthetic nitrogen-fixing bacterium Rhodospirillum rubrum three PII paralogues have been identified and termed GlnB, GlnJ and GlnK. In this report we analysed the interaction of GlnJ with known cellular targets such as the ammonium transporter AmtB1, the adenylyltransferase GlnE and the uridylyltransferase GlnD. Our results show that the interaction of GlnJ with cellular targets is regulated in vitro by the concentrations of manganese and 2-OG and the ADP : ATP ratio. Furthermore, we show here for the first time, to our knowledge, that in the interactions of GlnJ with the three different partners, the energy signal (ADP : ATP ratio) in fact overrides the carbon/nitrogen signal (2-OG). In addition, by generating specific amino acid substitutions in GlnJ we show that the interactions with different cellular targets are differentially affected, and the possible implications of these results are discussed. Our results are important to further the understanding of the regulatory role of PII proteins in R. rubrum, a photosynthetic bacterium in which the nitrogen fixation process and its intricate control mechanisms make the regulation of nitrogen metabolism even more complex than in other studied bacteria.

Dependence on divalent cations of the activation of inactive Fe-protein of nitrogenase from rhodospirillum rubrum

Abstract The activation of inactive Fe-protein from Rhodospirillum rubrum is dependent on the presence of ATP and a divalent cation. By preactivation of inactive Fe-protein in the presence of activating enzyme, ATP and different divalent cations, the efficiency of the metal ions used was studied. Mn2+, as the only divalent cation present, will support maximal rate of activation in vitro. Mn2+ is reqired both as free ion and as MnATP. MgATP will function as the meta(II)-nucleotide needed, but Mg2+ is a poor activator as free ion. Fe+ alone can support activation to the same extent as Mn2+. It was also found that Ba2+ is a competitive inhibitor versus Mn2+ in the activation but has little effect on acetylene reduction by nitrogenase when active Fe-protein is used.

Mechanism of ADP-ribosylation removal revealed by the structure and ligand complexes of the dimanganese mono-ADP-ribosylhydrolase DraG

ADP-ribosylation is a ubiquitous regulatory posttranslational modification involved in numerous key processes such as DNA repair, transcription, cell differentiation, apoptosis, and the pathogenic mechanism of certain bacterial toxins. Despite the importance of this reversible process, very little is known about the structure and mechanism of the hydrolases that catalyze removal of the ADP-ribose moiety. In the phototrophic bacterium Rhodospirillum rubrum, dinitrogenase reductase-activating glycohydrolase (DraG), a dimanganese enzyme that reversibly associates with the cell membrane, is a key player in the regulation of nitrogenase activity. DraG has long served as a model protein for ADP-ribosylhydrolases. Here, we present the crystal structure of DraG in the holo and ADP-ribose bound forms. We also present the structure of a reaction intermediate analogue and propose a detailed catalytic mechanism for protein de-ADP-ribosylation involving ring opening of the substrate ribose. In addition, the particular manganese coordination in DraG suggests a rationale for the enzymes preference for manganese over magnesium, although not requiring a redox active metal for the reaction.

Properties of the nitrogenase system from a photosynthetic bacterium, Rhodospirillum rubrum

Soluble nitrogenase from Rhodospirillum rubrum has been isolated and separated into its two components, the MoFe protein and the Fe protein. The MoFe protein has been purified to near homogeneity and has a molecular weight or 215 000. It contains two Mo, 25--30 Fe and 19--22 acid-labile sulphide and consists of four subunits, Mw 56 000. The Fe protein has a molecular weight 65 000. It contains approximately four Fe and four acid-labile sulphide and consists of two subunits, Mw 31 500. The highest specific activities for the purified components are 920 and 1260 nmol ethylene produced per min per mg protein, respectively. The purified components require the membrane component for activity (Nordlund, S., Eriksson, U. and Baltscheffsky, H. (1977) Biochim. Biophys. Acta 462, 187--195). Titration of the MoFe protein with the Fe protein shows saturation and excess MoFe protein over Fe protein is inhibitory. Addition of Fe2+ or Mn2+ to the reaction mixture increases the activity apparently through interaction with the membrane component.