The Presence of ADP-Ribosylated Fe Protein of Nitrogenase in Rhodobacter capsulatus Is Correlated with Cellular Nitrogen Status

Abstract

The photosynthetic bacterium Rhodobacter capsulatus has been shown to regulate its nitrogenase by covalent modification via the reversible ADP-ribosylation of Fe protein in response to darkness or the addition of external NH₄⁺. Here we demonstrate the presence of ADP-ribosylated Fe protein under a variety of steady-state growth conditions. We examined the modification of Fe protein and nitrogenase activity under three different growth conditions that establish different levels of cellular nitrogen: batch growth with limiting NH₄⁺, where the nitrogen status is externally controlled; batch growth on relatively poor nitrogen sources, where the nitrogen status is internally controlled by assimilatory processes; and continuous culture. When cultures were grown to stationary phase with different limiting concentrations of NH₄⁺, the ADP-ribosylation state of Fe protein was found to correlate with cellular nitrogen status. Additionally, actively growing cultures (grown with N₂ or glutamate), which had an intermediate cellular nitrogen status, contained a portion of their Fe protein in the modified state. The correlation between cellular nitrogen status and ADP-ribosylation state was corroborated with continuous cultures grown under various degrees of nitrogen limitation. These results show that in R. capsulatus the modification system that ADP-ribosylates nitrogenase in the short term in response to abrupt changes in the environment is also capable of modifying nitrogenase in accordance with long-term cellular conditions.

The reduction of dinitrogen to ammonia is catalyzed by the nitrogenase complex, which is composed of two electron-transferring proteins: an iron protein (Fe protein, dinitrogenase reductase) and a molybdenum-iron protein (MoFe protein, dinitrogenase). This reaction is an energy-demanding process which consumes 20 to 30 ATP molecules for each N₂ reduced, and N₂-fixing microorganisms have evolved efficient mechanisms to control both nitrogenase synthesis and its activity. Several nitrogen-fixing bacteria have been shown to regulate nitrogenase in the short term by posttranslational covalent modification via reversible ADP-ribosylation of the Fe protein in response to different environmental stimuli: ammonium addition, darkness, and the absence of oxygen (21, 22). This process is catalyzed by two non-nif-specific enzymes: dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase (19, 28) and has been particularly well characterized for the photosynthetic bacterium Rhodospirillum rubrum (4, 18, 20). In Rhodospirillum rubrum dinitrogenase reductase ADP-ribosyltransferase catalyzes the transfer of ADP-ribose from NAD to the Arg 101 residue of one subunit of the Fe protein homodimer in response to ammonium addition, darkness, or the presence of O₂ (22). This modification is accompanied by the loss of nitrogenase activity. After effector exhaustion or removal, dinitrogenase reductase-activating glycohydrolase removes ADP-ribose from modified Fe protein, restoring nitrogenase activity (22). Thus ADP-ribosylation of Fe protein provides one molecular basis for the fast and reversible inhibition of nitrogenase activity (nitrogenase switch-off) seen in vivo upon the addition of NH₄⁺.

Previously, this ADP-ribosylation process has been viewed as a transitory response to abrupt changes in culture conditions (22). The possible presence of ADP-ribosylated Fe protein in different cultures has received little or no attention. For example, numerous studies have shown that crude extracts of glutamate- or N₂-grown cells of Rhodospirillum rubrum, Rhodobacter capsulatus, and Rhodopseudomonas palustris contain modified (ADP-ribosylated) Fe protein (3, 10, 36, 37, 39). This was surprising, since glutamate-grown cultures exhibit high nitrogenase activity and are fully capable of regulating this activity in response to added NH₄⁺ (i.e., nitrogenase switch-off) (1, 10). It has been assumed that the observed modification is due to darkness, shown to induce rapid Fe protein ADP-ribosylation, during postculture manipulations (12). During a recent study of the regulation of nitrogenase activity in R. capsulatus (34), where we used a fast and sensitive immunochemical method for the analysis of the modification state of Fe protein, which avoids artifactual changes in modification state, we noticed that, depending upon the growth conditions, some culture types contained a proportion of the Fe protein as the ADP-ribosylated form without having been subjected to experimental manipulation. These observations led us to hypothesize that cells may naturally contain ADP-ribosylated Fe protein whose relative proportion depends upon cellular nitrogen status and to systematically examine the effects of culture conditions, particularly nitrogen status, on the cellular content of ADP-ribosylated Fe protein.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

Experiments were performed with R. capsulatus SB1003 (35) and strains JP23 and JP28, which are derivatives of strain RcM1 (ΔnifHDK) containing the conjugative plasmids pJP23 and pJP28, respectively. These plasmids bear the genes that encode wild-type MoFe protein and Fe protein with amino acid replacements of Arg 102 by Tyr (JP23) or by Lys (JP28) (27). Strains W107I and W107II are draTG deletion derivatives of B10S containing the gentamicin resistance interposon in different orientations (23). Cultures were grown essentially as previously described (34) in liquid RCV medium (32) containing 36 mM lactate and twice the normal concentration of K-phosphate (19.1 mM) to avoid a pH shift observed with concentrated cultures. Cultures from late exponential growth phase grown on RCV medium with NH₄⁺ excess (30 mM) were used as inocula (5% vol/vol). Batch cultures were grown in long tubes (1.6 by 20.5 cm) filled with 20 ml of RCV medium, which were sealed with rubber stoppers (Suba seal) with needles inserted for gas sparging (argon or N₂; 5 to 10 ml/min). Seven millimolar glutamate or N₂ or limiting amounts of NH₄⁺ (concentrations as indicated in the figures) were used as nitrogen sources. During growth, culture aliquots were withdrawn for the determination of cell density (A₆₆₀), in vivo nitrogenase activity was determined, and immunoblot analysis of nitrogenase proteins was carried out.

Continuous cultivation of R. capsulatus was performed with a photobioreactor specially designed for the continuous cultivation of photosynthetic microorganisms (30). Cultures were grown under photoheterotrophic conditions on the medium of Ormerod et al. (25) with 45 mM lactate at pH 7.0 and at 30°C with a light intensity of 81.4 W/m². (NH₄)₂SO₄ was used as the nitrogen source. Concentrations of 10 and 2 mM were used for turbidostat conditions and chemostat conditions, respectively. Cultures were continuously sparged with 98% argon plus 2% CO₂ (50 ml/min). All assays were performed under steady-state conditions after at least five culture doublings.

Determination of nitrogenase activity and Fe protein modification status.

Analysis of in vivo nitrogenase activity was performed by the acetylene reduction method (7) with 1- or 2-ml culture samples. Immunoblotting with chemiluminescence detection (33, 34) was used to monitor the modification state of Fe protein in R. capsulatus cells under different conditions. Samples (25 to 50 μl) were removed at the times indicated in the figures, brought to a 1× sodium dodecyl sulfate (SDS) sample buffer concentration (16), and immediately incubated in a boiling-water bath for 2 min. This method of sample preparation and subsequent SDS-polyacrylamide gel electrophoresis and immunoblot analysis present an accurate picture of the degree of in vivo nitrogenase modification since (i) samples that showed no treatment-induced ADP-ribosylation could be prepared by this method and (ii) increasing the time in the boiling-water bath did not change the amount of ADP-ribosylated subunit (as determined quantitatively by densitometric scanning). In our hands the SDS extraction method produced results equivalent to those of the previously described trichloroacetic acid precipitation procedure (38), but the latter method is more time-consuming and laborious.

SDS samples prepared as described above were analyzed by Laemmli SDS-polyacrylamide gel electrophoresis (12.5% of total acrylamide) with low cross-linker gels (acrylamide–N,N′-methylenebis(acrylamide), 30:0.2) and an increased duration of electrophoresis to obtain increased resolution of Fe protein subunits (12). Equal amounts of total protein (1 mg/well) were loaded onto all gels. In this system, R. capsulatus Fe protein showed apparent molecular masses of 34.9 kDa for the unmodified subunit and 38.7 kDa for the ADP-ribosylated subunit. The slower-migrating band was assumed to be ADP-ribosylated Fe protein based on its recognition by anti-Fe-protein antibody, its appearance in response to the appropriate stimuli, its apparent molecular mass (38.7 kDa), and its absence in draT mutants as well as in strains containing an unmodifiable Fe protein (23, 27). Since only one of the two Fe protein subunits of the Fe protein dimer was modified, 100% modification of Fe protein dimers corresponds to two equal-intensity bands. The occasional occurrence of additional immunoreactive bands of Fe protein is due to the transient formation of incompletely denatured intermediates (results not shown). All bands identified and quantitated as either modified or unmodified were shown by control experiments (with, for example, culture extracts made from strains containing unmodifiable Fe protein) to be assignable to the appropriate category. For Fe protein quantitation, the X-ray films were scanned with a Molecular Dynamics personal densitometer and the percentages of unmodified and modified Fe protein dimers were calculated as described previously (20). The protein concentrations of culture samples were determined after 2 min of sonication by the Bradford method (2) with bovine serum albumin as the standard. The NH₄⁺ concentrations in culture supernatants were measured by the phenol-hypochlorite method (29).

Determination of intracellular glutamine concentrations.

Glutamine levels were measured by high-performance liquid chromatography analysis of o-phthalaldehyde-derivatized amino acid pools extracted from bacterial cultures by modifications of a previously described method (8). Samples were obtained with a no-harvest protocol with methanol extraction, which prevents metabolic depletion of the glutamine pool (8). Typically, 0.2 to 0.5 ml of culture was directly pipetted into ice-cold methanol to give a final concentration of 80% and the mixture was dried with a vacuum concentrator and stored at −70°C. Immediately prior to the assay, samples were dissolved in 100 μl of water and clarified by centrifuging them for 15 min at 4°C. Ten microliters of the o-phthalaldehyde-derivatized sample (8) was injected onto a C₁₈ Partisphere column (Whatman), which was developed with gradient elution (1 ml/min) with buffer A (90 mM sodium acetate [pH 7.2], 0.5% tetrahydrofuran) and buffer B (100% methanol [high-performance liquid chromatography grade]). The program was as follows: 0 to 5 min with 100% buffer A, 5 to 35 min with 0 to 30% buffer B, 35 to 45 min with 30 to 80% buffer B, and 45 to 50 min with 80 to 0% buffer B. A Shimadzu RF-551 fluorescence detector was used, with excitation at 340 nm and emission at 455 nm. Under these conditions, glutamate and glutamine eluted at 12.5 and 27.7 min, respectively.

RESULTS

We examined nitrogenase activity and the modification state of Fe protein using three types of culture conditions that differ in how nitrogen limitation is achieved. With ammonia-limited cultures, the nitrogen status is directly established by the experimentally set ratio of ammonia to carbon source (lactate). With either N₂- or glutamate-grown cultures, the nitrogen status is indirectly established by the assimilatory mechanisms of the cell. With continuous cultures, the nitrogen status of ammonia-limited cultures is controlled by the dilution rate (D).

Batch growth with limiting NH₄⁺ as the nitrogen source.

The possible relationship between the degree of cellular nitrogen limitation and Fe protein modification state was checked by varying the initial concentration of ammonium (0 to 25 mM NH₄⁺) in the growth media of batch cultures, producing cultures with different severities of nitrogen limitation (Fig. 1). In the initial NH₄⁺ concentration range of 0 to 11.4 mM, the final culture density was proportional to the initial NH₄⁺ concentration and the cells did not contain any ADP-ribosylated Fe protein. These cultures can be considered to be purely N limited. However, when the initial NH₄⁺ concentration in the medium was higher than 11.4 mM, the final culture density did not increase proportionally with an increase in ammonium. Under these conditions, the cultures appeared to be under dual limitation: they were still N limited up to 22.8 mM, since the nitrogenase proteins were still expressed, and, additionally, were light limited, since incubation at a higher light intensity resulted in a higher final cell density (e.g., growth of a 17.1 mM NH₄-limited culture at a higher light intensity gave a 10 to 15% increase in final optical density). A variety of genetic and molecular studies have described a complex regulatory machinery that ensures that nitrogenase is synthesized in R. capsulatus only under conditions of nitrogen limitation (15). Thus, we take the presence of nitrogenase proteins in cultures as indicative of nitrogen limitation. Dual limitation, by light and fixed nitrogen, of continuous cultures of R. capsulatus has been previously demonstrated (31). The final concentrations of extracellular ammonium were very low (10 to 20 μM) with 1.9 to 11.1 mM initial NH₄⁺, slightly higher (60 to 250 μM) with initial NH₄⁺ in the range 13.3 to 19.0 mM, and appreciably higher (≥0.7 mM) with initial NH₄⁺ exceeding 19 mM.

FIG. 1.

Growth (final culture density), extracellular NH₄⁺, and Western immunoblot analysis of nitrogenase Fe protein in R. capsulatus batch cultures grown with different initial concentrations of NH₄⁺. Cultures were grown photoheterotrophically on RCV medium containing various NH₄⁺ concentrations (as indicated). After 40 h of growth the final culture densities (A₆₆₀) and extracellular NH₄⁺ concentrations were measured and samples were prepared for immunoblot analysis of Fe protein as described in Materials and Methods. (A) Final culture densities (A₆₆₀), extracellular-NH₄⁺ concentrations (NH₄⁺), and the proportions of ADP-ribosylated Fe protein, calculated from the scan of the immunoblot presented in panel B. (B) Immunoblot analysis of nitrogenase Fe protein in samples from cultures grown at the indicated initial concentrations of NH₄⁺ (results for 0 to 9.5 mM NH₄⁺ are not shown). One hundred percent modification of Fe protein dimers corresponds to two equal-intensity bands.

Immunoblot analysis of these cultures showed that some ADP-ribosylated Fe protein was present in cells grown on media containing 13.3 mM and higher concentrations of initial ammonium (until complete nitrogenase repression occurred at 24.7 mM NH₄⁺). The amount of ADP-ribosylated Fe protein was directly proportional to the initial NH₄⁺ concentration, with there being almost 90% modification in cells grown with 22.8 mM NH₄⁺. While the light limitation experienced by the weakly nitrogen-limited (high-density) cultures may have had some effect on the overall modification status, it is more likely that light limitation had an indirect effect. Indeed, that the intracellular nitrogen pool plays a more direct role than light intensity in establishing the ADP-ribosylation status can be seen in experiments where the nitrogen status of NH₄⁺-limited cells was varied, under conditions of a fixed concentration of NH₄⁺ (and therefore with a fixed cell density) and a static intensity of light, by varying the initial lactate concentration (15 to 35 mM). Immunoblot analysis (Fig. 2) clearly demonstrated the presence of ADP-ribosylated Fe protein in cells grown with a reduced amount of lactate (reduced nitrogen limitation) at an NH₄⁺ concentration where growth was not light limited. That these cultures were not light limited can be seen by a comparison of the results with the results presented in Fig. 1. At the limiting NH₄⁺ concentration used here (9.5 mM), the culture in Fig. 1 had a final A₆₀₀ of 6.0 whereas the 20 mM lactate culture shown in Fig. 2 had a final A₆₀₀ of 3.8.

FIG. 2.

Nitrogenase Fe protein immunoblot analysis of NH₄⁺-limited R. capsulatus cells grown with various lactate concentrations. Batch cultures were grown photoheterotrophically on RCV medium containing 9.5 mM NH₄⁺ as the nitrogen source and the indicated concentrations of lactate. After 40 h of growth the modification state of Fe protein was determined as described in Materials and Methods.

Assimilation of nitrogen requires the synthesis of only two central intermediates, glutamine and glutamate, from which other compounds derive nitrogen by secondary transfers. In the enteric bacteria Salmonella typhimurium and Klebsiella pneumoniae, the size of the intracellular glutamine pool appears to reflect the cellular nitrogen status, with external nitrogen limitation causing a drop in this pool (8). Therefore, we determined the levels of intracellular glutamine and glutamate in R. capsulatus cells grown with different initial concentrations of NH₄⁺ in the medium. As can be seen from the data presented in Fig. 3, the pool of intracellular glutamine was low (1 to 2 nmol/mg of cell protein) in cultures grown with initial concentrations of NH₄⁺ of 0 to 11.4 mM (no Fe protein modification) and increased (4.5 to 20 nmol/mg of cell protein) in cells grown with higher concentrations of ammonium in the medium and containing modified Fe protein. Comparison of Fig. 1 and 3 suggests that Fe protein ADP-ribosylation is more closely correlated with intracellular glutamine (nitrogen status) than with intracellular glutamate or the external concentration of residual NH₄⁺ in the medium.

FIG. 3.

Levels of intracellular glutamine and glutamate in R. capsulatus batch cultures grown with different initial concentrations of NH₄⁺. Cultures were grown (40 h) photoheterotrophically on RCV medium containing various NH₄⁺ concentrations as indicated, and samples were prepared and analyzed for intracellular glutamine and glutamate as described in Materials and Methods. Values averages of results from at least five independent determinations, with standard deviations indicated by error bars.

Growth with N₂ or glutamate.

With NH₄⁺-limited cultures, nitrogenase is expressed only when the externally supplied ammonia is nearly exhausted, which at moderately to highly limiting concentrations of NH₄⁺ occurs at the beginning of the stationary growth phase (results not shown). Therefore, to determine the possible presence of modified Fe protein in actively growing cultures, we monitored the nitrogenase activity and the ADP-ribosylation status of the Fe protein during batch growth of R. capsulatus with N₂ or glutamate as the nitrogen source. With N₂ as the sole nitrogen source, in vivo nitrogenase activity reached a maximum in the middle of the exponential phase of growth and subsequently rapidly declined to a very low level (Fig. 4). Similar results have previously been reported for N₂-grown cultures of Rhodospirillum rubrum (24). In contrast to these dramatic effects the effects of changes in the content of the nitrogenase proteins (MoFe protein and Fe protein) were much less marked (not shown). Immunoblot analysis of the modification state of the Fe protein in these cells revealed that the ADP-ribosylated form of this protein was present, at various levels, throughout cultivation (Fig. 4B). Scanning densitometry showed that the content of ADP-ribosylated Fe protein dimers increased simultaneously with the increase of nitrogenase activity, reaching a maximum at approximately the same time (Fig. 4C, right y axis). In fact, after 20 to 25 h of cultivation, an N₂-grown R. capsulatus culture exhibited the highest nitrogenase-specific activity at the same time that 80 to 90% of the Fe protein dimers were in the ADP-ribosylated form. Analysis of NH₄⁺ in culture supernatants showed that there were only very low levels of extracellular NH₄⁺ (9 to 15 μM) throughout the period of growth examined. While at first glance it may seem surprising that cultures actively synthesize Fe protein and then immediately modify it, this may be a consequence of different rates of response of the controls for transcription and modification with respect to intracellular nitrogen status. Thus, a culture which has already synthesized an appreciable amount of nitrogenase proteins will rapidly assimilate N₂, leading to modification of preformed and newly synthesized Fe protein before transcription and synthesis can be shut off.

FIG. 4.

In vivo nitrogenase activity and the content of nitrogenase proteins in R. capsulatus cells during growth with N₂ as the nitrogen source. RCV medium containing no added nitrogen was inoculated (5%, vol/vol) with R. capsulatus cells grown on medium containing an excess of NH₄⁺ (30 mM) to give an initial culture density of 0.2 (A₆₆₀). At the times indicated, aliquots were removed for the determination of culture density (A₆₆₀), whole-cell nitrogenase activity, nitrogenase protein content, and Fe protein modification state as described in Materials and Methods. (A) Growth (A₆₆₀) and specific in vivo nitrogenase (N₂ase) activity. (B) Immunoblot analysis of nitrogenase proteins. (C) Changes in the content of nitrogenase proteins (calculated from the scan of the immunoblot presented in panel B) and in in vivo nitrogenase activity (from the graph in panel A). One hundred percent corresponds to (read from the left y axis, hatched bars) in vivo N₂ase activity (61 nmol of C₂H₄ · min⁻¹ · mg of protein⁻¹), MoFe protein (29.1 μg/mg of cell protein), unmodified Fe protein (3.1 μg/mg of cell protein), and (read from the right y axis, cross-hatched bars) ADP-ribosylated Fe protein (percentage of total Fe protein dimers present at the indicated time points, where bar numbers correspond to lane numbers in panel B). Each bar represents an average of results from at least three replicate assays.

The changes in the nitrogenase activities of these cultures appeared not to be correlated with the ADP-ribosylation status of the Fe protein. These results were corroborated by examining changes in nitrogenase activity during growth on N₂ of R. capsulatus draT draG mutants (W107I and W107II) which are unable to modify Fe protein. Changes in the nitrogenase activities of cultures of these strains were very similar to changes observed with the wild type (results not shown), with a very low level of activity occurring in the stationary phase of growth (about 5% of the maximal activity obtained in the mid-exponential phase). Obviously, the low level of final activity could not be attributed to ADP-ribosylation of the Fe protein and could only partially be explained by a modest decrease in the relative contents of nitrogenase proteins (20 to 30%).

We also examined the nitrogenase activities and the ADP-ribosylation states of the Fe protein of cultures growing on the relatively poor nitrogen source glutamate. Glutamate-grown cultures of R. capsulatus demonstrated two to five times greater nitrogenase specific activity than N₂-fixing cells (Fig. 5). There was a significant rate of C₂H₂ reduction even in the stationary phase of growth. A broadly maximal nitrogenase activity was observed after 20 to 30 h of cultivation, and the decline in nitrogenase activity in the stationary phase was significantly greater than was warranted by the modest decrease in levels of nitrogenase proteins. Western immunoblot analysis of Fe protein in these glutamate-grown cultures again demonstrated the presence, at various levels, of ADP-ribosylated Fe protein throughout the period of cultivation (Fig. 5B). Scanning densitometry clearly showed that the proportion of this form varied over a twofold range, reaching a maximum shortly after maximal nitrogenase activity was reached, at which point it consisted of 40% of the total Fe protein dimers (Fig. 5C, right y axis). The presence of ADP-ribosylated Fe protein in actively growing glutamate cultures can be rationalized in a number of ways. As is evident from the results presented here, regulation of transcription and regulation of modification appear to respond differently to the intracellular nitrogen status. In addition, while regulation of transcription might control the overall amounts of nitrogenase proteins available at any given time, the modification system may provide a mechanism to fine-tune the amount of active nitrogenase. Again, the ADP-ribosylation state of Fe protein appeared to be correlated with the nitrogen status of the cells, since the analysis of N₂- and glutamate-grown cultures revealed the presence of increased levels of intracellular glutamine in these cells, with N₂-grown cells (greater Fe protein ADP-ribosylation) showing higher levels of glutamine (7.3 to 11.2 nmol/mg of cell protein) than those in glutamate-grown cells (3.5 to 6.1 nmol/mg of cell protein).

FIG. 5.

In vivo nitrogenase activity and the content of nitrogenase proteins in R. capsulatus cells during growth with glutamate as the nitrogen source. RCV medium containing 7 mM glutamate was inoculated as described in the legend to Fig. 4. At the times indicated, aliquots were removed for the determination of culture density (A₆₆₀), whole-cell nitrogenase activity, nitrogenase protein content, and Fe protein modification state as described in Materials and Methods. (A) Growth (A₆₆₀) and specific in vivo nitrogenase activity (N₂ase). (B) Immunoblot analysis of nitrogenase proteins. (C) Changes in the content of nitrogenase proteins (calculated from the immunoblot in panel B) and in in vivo nitrogenase activity (from the graph in panel A). One hundred percent corresponds to (read from the left y axis, hatched bars) in vivo N₂ase activity (280 nmol C₂H₄ · min⁻¹ · mg of protein⁻¹), MoFe protein (29.1 μg/mg of cell protein), unmodified Fe protein (14.9 μg/mg of cell protein), and (read from the right y axis, cross-hatched bars) ADP-ribosylated Fe protein (percentage of total Fe protein dimers present at the indicated time points, where bar numbers correspond to lane numbers in panel B). Each bar represents an average of results from at least three replicate assays.

Continuous (chemostat) cultivation under NH₄⁺ limitation.

In order to directly test the role of the cellular nitrogen status in the regulation of nitrogenase in R. capsulatus, we checked the effects of different dilution rates on nitrogenase activity and the Fe protein modification states of cells growing in NH₄⁺-limited chemostat cultures. Continuous cultivation makes it possible to vary the degree of culture limitation by nitrogen while keeping other parameters (carbon supply, light, pH, etc.) at constant levels. In an NH₄⁺-limited chemostat, an increase in D reduces the degree of nitrogen limitation and, conversely, a reduction in D increases the degree of nitrogen limitation. As presented in Fig. 6, R. capsulatus cells growing in an NH₄⁺-limited chemostat culture expressed nitrogenase activity within a wide range of dilution rates (0.01 to 0.21 h⁻¹), with a broad maximum at 0.04 to 0.08 h⁻¹. At higher rates of dilution nitrogenase activity was decreased due to a gradual suppression of nitrogenase synthesis and activity by an increased supply of nitrogen, and at lower rates of dilution it was decreased because of the effect of severe nitrogen limitation on cell metabolism and nitrogenase synthesis and activity. A similar effect of dilution rate on the nitrogenase activity of R. capsulatus cells grown in NH₄⁺-limited chemostat cultures has also been observed by other investigators (9).

FIG. 6.

In vivo nitrogenase activity and the content of ADP-ribosylated Fe protein dimers in R. capsulatus cells grown in an NH₄⁺-limited continuous (chemostat) culture at different flow rates (D). Inflowing medium contained 1 mM (NH₄)₂SO₄. After the culture had reached steady state at a given flow rate, aliquots were removed for the analysis of in vivo nitrogenase (N₂ase) activity and Fe protein modification state as described in Materials and Methods.

Immunoblot analysis demonstrated the absence of ADP-ribosylated Fe protein at low rates of dilution (0.01 to 0.02 h⁻¹; strong nitrogen limitation) and the presence of increasing amounts of this form at higher rates of dilution (decreased nitrogen limitation) (Fig. 6). The presence of ADP-ribosylated Fe protein at high rates of dilution is not due to nitrogenase switch-off by NH₄⁺ being added with the fresh medium since even at maximal rates of dilution (0.21 h⁻¹) the steady-state concentration of NH₄⁺ in the photobioreactor was less than 5 μM (as ascertained with an NH₄⁺ electrode), which is below the threshold necessary for the nitrogenase switch-off response. In the D range of 0.02 to 0.06 h⁻¹ the increase in the content of ADP-ribosylated Fe protein occurred simultaneously with an increase in nitrogenase activity. A possible role of light intensity in Fe protein modification in R. capsulatus cells was checked with an NH₄⁺-limited chemostat culture (D = 0.02 h⁻¹) grown under decreasing light intensities (from 226.5 to 2.7 W/m²). The culture became purely light limited at 6.6 W/m², as judged by our inability to detect nitrogenase component proteins (data not shown). Since no ADP-ribosylated Fe protein was observed in this experiment, it appears that the cellular nitrogen status (degree of nitrogen limitation) was the main factor controlling the modification state of the Fe protein, which therefore supports the conclusion reached from the experiments with batch cultures reported here.

DISCUSSION

The short-term regulation of nitrogenase, termed nitrogenase switch-off, in photosynthetic bacteria has been extensively investigated over the past 2 decades. With R. capsulatus two different, presumably independent, control mechanisms have been demonstrated. Like Rhodospirillum rubrum, R. capsulatus contains draT and draG (23) and carries out the reversible ADP-ribosylation of its Fe protein in response to the addition of NH₄⁺ or darkness (6). The enzymatic system that carries out this process appears to be highly similar in most respects to that of Rhodospirillum rubrum (6, 11). Nevertheless, much remains to be learned about the signal transduction processes for both organisms. Moreover, in addition to covalent modification, R. capsulatus has been shown to carry out an ADP-ribosylation-independent regulation of nitrogenase activity since strains carrying Fe protein mutant alleles that are unable to be ADP-ribosylated (26) or strains with mutations in draT or draG (34) are nonetheless capable of the switch-off of nitrogenase activity. The basis of this response is obscure, although it has been suggested that it results from the gating of electron flow to nitrogenase (26, 34).

It has been widely assumed that the modification of Fe protein observed with N₂- or glutamate-grown cultures of photosynthetic bacteria is due to the darkness associated with cell concentration by centrifugation (12, 21), and the present model of nitrogenase regulation in photosynthetic bacteria assumes that only the addition or removal of external NH₄⁺ or darkness can induce modification or demodification of Fe protein (21, 22). However, here we have shown that the system that ADP-ribosylates nitrogenase in the short term in response to abrupt changes in the environment is also capable of modifying nitrogenase in accordance with long-term cellular conditions. We used a fast and sensitive immunochemical method for the analysis of the modification state of the Fe protein in R. capsulatus cells which avoids sample concentration by centrifugation. Our results clearly demonstrate that a significant proportion (30 to 90%) of nitrogenase Fe protein is present, in vivo, as the ADP-ribosylated form in R. capsulatus cells grown with different nitrogen sources. ADP-ribosylated Fe protein was found in cells in both exponential (N₂, glutamate) and stationary (NH₄⁺-limited) growth phases. Analysis of the glutamine contents of the various culture types indicated that the modification state of Fe protein is directly proportional to the level of intracellular glutamine and therefore is presumably a function of the cellular nitrogen status. Here we used glutamine levels as an indicator of cellular nitrogen status (8). The role for glutamine as a controller of the Fe protein ADP-ribosylation process is presently unclear. Previously it has been suggested, on the basis of short-term incubation experiments using another photosynthetic bacterium, Rhodospirillum rubrum, that the intracellular glutamine pool may serve as the signal for nitrogenase modification (17). However, other researchers have found that the glutamine concentration varied independently of Fe protein ADP-ribosylation (13). That the modification status of the Fe protein is correlated with cellular nitrogen status was directly demonstrated here with chemostat cultures of R. capsulatus where ADP-ribosylation was shown to be inversely proportional to the severity of nitrogen limitation.

In this study, under some conditions substantial nitrogenase activity was observed at the same time that a significant proportion of Fe protein was found to be ADP-ribosylated. In fact, all experimental conditions tested showed wide variations in levels of nitrogenase activity with small or no changes in the levels of both nitrogenase proteins (determined by immunoblotting). While some of this disparity might be due to differing ADP-ribosylation states of the Fe protein in cultures of the wild-type strain, this is clearly not the case with draT G mutant strains, and the contents of unmodified Fe protein dimers in both the wild type and the draT G mutant strains did not correspond to the level of nitrogenase activity. These results strongly suggest that in vivo nitrogenase activity is not limited by the concentration of catalytically active nitrogenase proteins. This conclusion was corroborated by determining the in vitro nitrogenase activities of extracts of cells grown under conditions where there was a wide variation in in vivo activity. For example, N₂-grown cultures showed a 100-fold variation in in vivo activity but only a 20% decline in in vitro activity (results not shown). A similar, although less drastic variation has been reported for Azotobacter vinelandii, which is incapable of Fe protein ADP-ribosylation, with a twofold increase in nitrogenase activity without an increase in the content of nitrogenase proteins (14). These observations suggest that at least under some conditions whole-cell nitrogenase activity may be determined mainly by electron flow to nitrogenase and/or ATP supply and not be the levels of nitrogenase proteins or the proportion of unmodified Fe protein. This regulation may be due to the ADP-ribosylation-independent response previously noted in studies of the short-term regulation of nitrogenase.

Thus, both regulatory systems may be operative under conditions where no changes in the external milieu are taking place. These two levels of control may thus mirror elements previously shown to be involved in the short-term switch-off of nitrogenase. Both systems may be necessary to fine-tune the nitrogenase system in response to internal cellular conditions. Thus, nitrogenase in R. capsulatus is subject to both a short-term regulation of activity in response to sudden environmental changes and a long-term adaptation of the activity of preformed enzyme to specific growth conditions.