Regulation of nitrogen fixation in Azospirillum brasilense.

The regulation of nitrogen fixation in Azospirillum brasilense is very complicated, and it responds to exogenous fixed nitrogen or a change of oxygen concentration. This regulation occurs at both transcriptional and posttranslational levels. Unlike regulation seen in Klebsiella pneumoniae, transcription of nifA does not require NTRB/NTRC in A. brasilense and the expression of nifHDK is controlled by posttranslational regulation of NIFA activity. Addition of NH4+ or a shift from microaerobic to anaerobic conditions also causes a rapid loss of nitrogenase activity in A. brasilense. This posttranslational regulation of nitrogenase activity involves the DRAT/DRAG regulatory system, which is similar to that of Rhodospirillum rubrum. Both DRAT and DRAG activities are regulated in vivo, but the mechanisms for their regulation are unknown.

Presence of a second mechanism for the posttranslational regulation of nitrogenase activity in Azospirillum brasilense in response to ammonium.

Although ADP-ribosylation of dinitrogenase reductase plays a significant role in the regulation of nitrogenase activity in Azospirillum brasilense, it is not the only mechanism of that regulation. The replacement of an arginine residue at position 101 in the dinitrogenase reductase eliminated this ADP-ribosylation and revealed another regulatory system. While the constructed mutants had a low nitrogenase activity, NH4+ still partially inhibited their nitrogenase activity, independent of the dinitrogenase reductase ADP-ribosyltransferase/dinitrogenase reductase activating glycohydrolase (DRAT/DRAG) system. These mutated dinitrogenase reductases also were expressed in a Rhodospirillum rubrum strain that lacked its endogenous dinitrogenase reductase, and they supported high nitrogenase activity. These strains neither lost nitrogenase activity nor modified dinitrogenase reductase in response to darkness and NH4+, suggesting that the ADP-ribosylation of dinitrogenase reductase is probably the only mechanism for posttranslational regulation of nitrogenase activity in R. rubrum under these conditions.

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

Reversible ADP ribosylation of dinitrogenase reductase, catalyzed by the dinitrogenase reductase ADP-ribosyl transferase (DRAT)/dinitrogenase reductase activating glycohydrolase (DRAG) regulatory system, has been characterized in both Rhodospirillum rubrum and Azospirillum brasilense. Although the general functions of DRAT and DRAG are very similar in these two organisms, there are a number of interesting differences, e.g., in the timing and extent of the regulatory response to different stimuli. In this work, the basis of these differences has been studied by the heterologous expression of either draTG or nifH from A. brasilense in R. rubrum mutants that lack these genes, as well as the expression of draTG from R. rubrum in an A. brasilense draTG mutant. In general, these hybrid strains respond to stimuli in a manner similar to that of the wild-type parent of the recipient strain rather than the wild-type source of the introduced genes. These results suggest that the differences seen in the regulatory response in these organisms are not primarily a result of different properties of DRAT, DRAG, or dinitrogenase reductase. Instead, the differences are likely the result of different signal pathways that regulate DRAG and DRAT activities in these two organisms. Our results also suggest that draT and draG are cotranscribed in A. brasilense.

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.

Posttranslational regulation of nitrogenase activity by anaerobiosis and ammonium in Azospirillum brasilense.

In the microaerophilic diazotroph Azospirillum brasilense, the addition of fixed nitrogen or a shift to anaerobic conditions leads to a rapid loss of nitrogenase activity due to ADP-ribosylation of dinitrogenase reductase. The product of draT (DRAT) is shown to be necessary for this modification, and the product of draG (DRAG) is shown to be necessary for the removal of the modification upon removal of the stimulus. DRAG and DRAT are themselves subject to posttranslational regulation, and this report identifies features of that regulation. We demonstrate that the activation of DRAT in response to an anaerobic shift is transient but that the duration of DRAT activation in response to added NH4+ varies with the NH4+ concentration. In contrast, DRAG appears to be continuously active under conditions favoring nitrogen fixation. Thus, the activities of DRAG and DRAT are not always coordinately regulated. Finally, our experiments suggest the existence of a temporary period of futile cycling during which DRAT and DRAG are simultaneously adding and removing ADP-ribose from dinitrogenase reductase, immediately following the addition of a negative stimulus.

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.

Regulation of nitrogenase activity by oxygen in Azospirillum brasilense and Azospirillum lipoferum.

The nitrogenase activity of the microaerophilic bacteria Azospirillum brasilense and A. lipoferum was completely inhibited by 2.0 kPa of oxygen (approximately 0.02 atm of O2) in equilibrium with the solution. The activity could be partially recovered at optimal oxygen concentrations of 0.2 kPa. In contrast to the NH4+ switch off, no covalent modification of the nitrogenase reductase (Fe protein) was involved, as demonstrated by Western-blotting and 32P-labeling experiments. However, the inhibition of the nitrogenase activity under anaerobic conditions was correlated with covalent modification of the Fe protein. In contrast to the NH4+ switch off, no increase in the cellular glutamine pool and no modification of the glutamine synthetase occurred under anaerobic switch-off conditions. Therefore, a redox signal, independent of the nitrogen control of the cell, may trigger the covalent modification of the nitrogenase reductase of A. brasilense and A. lipoferum.