Effect of inhibitors on nitrogenase regulation in the cyanobacterium Nostoc linckia

Biochem. Physiol. Pflanzen 179, 623-629 (1984)

Effect of Inhibitors on Nitrogenase Regulation in the Cyanobacterium N ostoc linckia D. KUMAR and H. D. KUMAR Centre of Advanced Study in Botany, Banaras Hindu University, India Key Term Index: nitrogenase, heterocyst, glutamine synthetase; Nostoc linckia

Summary The effects of ammonium ion and certain inhibitors of protein synthesis on nitrogenase activity in N ostoc linckia were compared during heterocyst induction and exponential phases. Early stages of heterocyst differentiation and nitrogenase induction are prone to ammonium inhibition. Nitrogenase activity continued to increase even in presence of ammonium in 36 h old cultures. Addition of ammonium chloride in exponential, nitrogen-dependent cultures had little effect on nitrogenase activity up to 16 h whereafter the activity fell. The heterocyst frequency was higher in rifampicin and lower in chloramphenicol, as compared to control cultures.

Introduction

Recent progress in our understading of the genetics and regulation of nitrogen fixation genes (nit genes) has emanated largely from the finding that Klebsiella pneumoniae nit genes are linked and could be manipulated in Escherichia coli (MACNEIL et al. 1978). Similar genetic studies of nit gene in other nitrogen fixing species, especially in cyanobacteria, have been restricted by limitations in our ability to do the necessary genetic manipulations. While much is known about the biochemistry of initial stages of nitrogen fixation and assimilation, there has been little work on the regulation of nit genes in cyanobacteria. The mechanism of repression by NH4+ has been studied in some detail (STREICHER et al. 1974; HOUMARD and BOGUSZ 1981). It seems that glutamine is the metabolite most likely controlling nitrogenase biosynthesis. Short-term in vivo acetylene reduction assays indicate that, as in eubacterial systems, nitrogenase activity in cyanobacteria is not allosterically regulated by NH4 + (ROWELL et al. 1977; SINGH et al. 1983). However, these studies did not distinguish between the processes of nitrogen fixation and heterocyst differentiation. We have done in vivo experiments which suggest that induction becomes insensitive to NH4+-effected repression after the heterocyst differentiation process has progressed beyond the proheterocyst stage. The regulation of nitrogenase activity by certain other compounds or metabolic inhibitors has also been studied. Abbreviations: GS, glutamine synthetase; MSO, L-methionine-DL-sulfoximine; NH 4 +, ammonium chloride; Tris, Tris(hydoxymethyJ) methylamine/HCl

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D. KUMAR and H. D. KUMAR

Material and Methods Nos/oc linckia was grown axenically in the culture medium of ALLEN and ARNON (1955) adjusted to pH 7.5 after autoclaving and either lacking combined nitrogen or supplemented with 5 mmol of KN0 3 • The medium was buffered with 4 mmol of Tris to avoid any marked changes in pH. Cultures were maintained on 14 h light: 10 h dark cycle at an average temperature of 26 'f 2°C, and were illuminated with cool white fluorescent lights at about 2,000 lux. They were shaken by hand thrice daily. Heterocyst frequency was estimated by counting them in at least 12 filaments. Nitrogenase activity was estimated by the acetylene-ethylene assay (STEW ART et a!. 1968). Assay was done in calibrated triplicate serum bottles of 8 ml capacity. The partial pressure of acetylene was kept at 0.1 atm., and 2 ml cell suspensions were routinely injected in each bottle. Reactions were run for 30 min at 28°C, 3.000 lux. They were terminated by injecting 0.2 ml of HC!. Reaction mixtures were analyzed in a CIS Gas Chromatograph (Baroda) fitted with a Porapak R column and hydrogen flame ionization detector. Nitrogen was used as a carrier gas. Transferase activity of GS was determined at pH 7.0 by measuring the amount of y.glutamyl hydroxamate formed, as per the method of SHAPIRO and STADTMAN (1970). Whole cell GS activity was determined after treatment with toluene; into 0.5 ml cell suspension was added 0.25 ml of toluene and the mixture incubated for 10 min at 4°C. Following centrifugation, the toluene layer was removed and the cell pellet was suspended in 0.5 ml of 100 mmol imidazole buffer (pH 7). GS activity was expressed in terms of transferase activity (,1 OD 540 nm m!. sample- l 30 min-l). Protein was estimated as per the method of LOWRY et al. (1951). Chloramphenicol, rifampicin, glutamine, azaserine, MSO were purchased from Sigma Chemical Co., St. Louis. Ethylene was from Matheson Gas Co., Lyndhurst, USA; other gases were from Indian Oxygen Limited, Bombay.

Results

Fig. 1 shows the events occurring upon transference of NH4 +-grown filaments into nitrogen-free medium. Within 15 h, morphological differences became apparent in certain cells (called proheterocysts) spaced at regular intervals along the filament. The proheterocyst frequency rose to almost 4.5 % in 24 h and then fell to approximately 0.5 % by 42 h. The rise in heterocyst frequency corresponding to the decline in proheterocysts indicates that maturation of proheterocysts into heterocysts occurred during this period. Nitrogenase activity appeared about 19 h after the transfer of the material from ammonium medium to nitrogen-free medium; it increased gradually with time. Ammonium chloride (1 mmol) was added to separate aliquots of culture suspension at several time points after transfer into nitrogen-free medium. This was done to determine the developmental stage at which heterocyst differentiation becomes irreversible. Except when added at 36 h, NH4 + prevented the appearance of mature heterocysts (Table 1). The heterocyst frequency was lower than in control, but since growth resumed with available nitrogen, total heterocyst numbers were not significantly different from those in controls. At 24 h when proheterocysts had reached maximum frequency and heterocysts had just appeared, NH4 + prevented further maturation of proheterocysts. The results of experiments on the effects of chloramphenicol, rifampicin, and ammonium chloride are shown in Table 1. Fig. 2 shows the effect of adding 1 mmol of

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NH4CI at several points during the induction of nitrogenase activity in differentiating cultures. Following the reintroduction of NH4+ 24 h after transfer, the rate of increase in nitrogenase activity declined. However, when added at 12 h or before 12 hr, nitrogenase activity did not appear. 36 h after transfer, NH4 + had no inhibitory effect, and the activity rose linearly for over 10 h at the same rate as in the controls. For better evaluation of the level at which NH4+ acts in regulating nitrogen fixation, the action of NH4+ on nitrogenase induction in 48 h old cultures was compared Table 1. Effects of ammonium chloride, chloramphenicol and rifampicin on heterocyst frequency. N. B. Ammonium chloride (1 mmol), chloramphenicol and rifampicin (25,ug ml- 1 each) were added at stated h after transfer of heterocyst-free filaments into nitrogen-free medium. Average heterocyst frequency was estimated at the time of addition and 96 h following the start of the experiment Time of addition (h)

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Fig. 3. Effect of ammonium chloride (1 mmol, open triangles), chloramphenicol (25 flY ml-I, open circles) and rifampicin (25 flY ml- l , solid triangles) on nitrogenase activity of 48 h old cultures Each was added at the time of inoculation.

Fig. 2. Effect of ammonium chloride (1 mmol) on nitrogenase activity. Solid circles, control; open circles, NH4Cl addition at 12 h; solid triangles, addition at 24 h; open triangles, addition at 36 h after inoculation.

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with that of other metabolic inhibitors. Fig. 3 shows the effect of NH4 +, chloramphenicol, and rifampicin on nitrogenase activity in such cultures. Chloramphenicol and rifampicin significantly inhibited nitrogenase activity. Fig. 4 shows the time course of changes which occur upon adding NH4+ to 8 d old nitrogen-fixing cultures. Contrary to the results obtained with 48 h cultures, nitrogenase activity declined slowly up to 16 h and then declined rapidly; after 48 h there was only about 26% of the initial activity. GS activity declined some 50% from its initial level within this time. Fig. 5 shows the effect of glutamine (1 mmol), azaserine (4,umol) and MSO (l,umol) on nitrogenase activity. The activity fell by about 47% within 48 h after addition of glutamine. The addition of MSO, which causes NH3 excretion in Anabaena eylindriea (STEWART et al. 1975), had no effect on nitrogenase activity (Fig. 5), but inhibited GS activity; in 1,umol of MSO, this activity was 0.04 units as compared to 0.34 in control and in azaserine, and 0.27 in NH4+ (the concentrations of azaserine and NH4CI in these experiments were 4,umol and 5 mmol respectively). Azaserine inhibits glutamate synthase activity in eubacteria (LEA and MIFLIN 1975); we found that it has no effect on GS activity in N ostoe linekia.

Discussiou

The induction experiments indicate that both nitrogenase synthesis and heterocyst differentiation are inhibited when NH4+ is added at an early stage of differentiation. The extent of inhibition of both the processes is greater the sooner the NH4+ is introduced. However, nitrogenase biosynthesis continues unaltered (for more than 10 h) 41*

628

D.

KUMAR

and H. D.

KUMAR

when NH4+ is added 36 h after the start of the induction experiment. In free-living bacteria, nitrogenase synthesis is rapidly repressed by ammonia (BRILL 1980). KLEINER (1974) has shown that nitrogenase synthesis in Azotobacter vinelandii is repressed by ammonia if it is present in the medium in concentrations exceeding 25,umol. Ammonia at a concentration of 2-3 mmol is sufficient to completely repress nitrogenase synthesis in all common laboratory strains of heterocystous cyanobacteria, whereas repression by nitrate is often only partial (STEWART 1977). Nif expression is controlled at the level of transcription or at a site prior to transcription (BRILL 1980). Our results on comparisons of ammonium-affected nitrogenase repression and with inhibitors of protein synthesis and transcription indicate a difference in the kinetics of NH4+ from those of inhibitors of transcription and translation. Nitrogenase activity in 48 h old cultures was unaffected by the addition of ammonium chloride, and enzyme activity remained almost at the same level. However, NH4+ appears only to have inhibited further heterocyst differentiation. Although nitrogenase ~ctivity was apparently not inhibited by NH4 + in short term in vivo experiments, it probably plays some indirect role in regulating nitrogenase activity under some conditions. Nitrogenase activity was lost after prolonged NH4+ treatment of 8 d old cultures. On considering the possible ways in which GS might regulate the synthesis of nitrogenase in N. linckia, we have noted that in long term experiments, nitrogenase activity fell more rapidly as compared to GS activity. Our results are in accord with the findings of ROWELL et al. (1977) and TULI and THOMAS (1980) that MSO inhibits GS activity and causes secretion of ammonia into the medium but does not inhibit nitrogenase. However, the addition of azaserine did inhibit nitrogenase. Rather than NH4 +, glutamine (or its metabolite) may be involved in nitrogenase regulation. Perhaps the glutamine synthesized might be quickly lost from heterocysts, thus enabling the nitrogenase to remain derepressed. These results indicate that when ammonium-grown filaments are transferred into nitrogen-free medium, they remain unaffected by ammonium-mediated repression of nitrogenase synthesis at certain stages. Tough chemicals such as MSO, DL-7-azatryptophan, and rifampicin increase heterocyst frequency, they do not affect nitrogenase activity proportionally (BOTTOMLEY et al. 1980). On the other hand metronidazole inhibits nitrogenase without affecting heterocyst differentiation (TETLEY and BISHOP 1979). Under aerobic conditions, heterocyst differentiation is a pre-requisite for the expression of the oxygen-labile nitrogenase. Our results suggest that nitrogen fixation under aerobic conditions in filamentous cyanobacteria probably requires both ket and nif genes in a programmed sequence, i.e. nitrogenase expression follows heterocyst differentiation. Acknowledgements D. K. thanks the University Grants Commission for the award of a research fellowship. All experimental work was done by D. K. This work forms part of the Advanced Centre Programme in the Core Area of Biological Nitrogen Fixation.

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References ALLEN, M. B., and ARNON, D. I.: Studies on nitrogen-fixing blue-green algae. I. Growth and nitrogen fixation by Anabaena cylindrica. Plant Physiol. 30, 366-372 (1955). BOTTOMLEY, P. J., VAN BAALEN, C., and TABITA, F. R.: Heterocyst differentiation and tryptophan metabolism in the cyanobacterium Anabaena sp. CA. Arch. Biochem. Biophys. 203, 204-213 (1980). BRILL, W. J.: Biochemical genetics of nitrogen fixation. Microbiol. Rev. 44, 449-467 (1980). HOUMARD, J., and BOGusz, D.: Kinetic study of the expression of K. pneumoniae nitrogen fixation (nif) genes under conditions of inhibited transcription. Biochem. Biophys. Res. Commun. 100, 1237-1244 (1981). KLEINER, D.: Quantitative relations for the repression of nitrogenase synthesis in Azotobacter vinelandii by ammonia. Arch. Microbiol. 101, 153-159 (1974). LEA, P. J., and MIFLIN, B. J.: Glutamine (amide), 2-oxyglutarate aminotransferase in blue-green algae. Biochem. Soc. Trans. 3, 381-384(1975). LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J.: Protein measurement with the folin phenol reagent. J. BioI. Chern. 193, 263-275 (1951). MACNEIL, T., MACNEIL, D., ROBERTS, G. P., SUPIANO, M. A., and BRILL, W. J.: Fine structure mapping and complementation analysis of nit genes in Klebsiella pneumoniae. J. Bacteriol. 136, 253-266 (1978). ROWELL, P., ENTICOTT, S., and STEWART, W. D. P.: Glutamine synthetase and nitrogenase activity in the blue-green alga Anabaena cylindrica. New Phytol. 79, 41-54 (1977). SHAPIRO, B. M., STADTMAN, E. R.: Glutamine synthetase (Escherichia coli). Methods in Enzymol. 17 A, 910-922 (1970). SINGH, H. N., RAI, U. N., RAO, V. U., and BAGCHI, S. N.: Evidence for ammonia as an inhibitor of heterocyst and nitrogenase formation in the cyanobacterium Anabaena cycadeae. Biochem. Biophys. Res. Commun. 111, 180-187 (1983). STEWART, W. D. P.: Blue-green Algae. In: HARDY, R. W. F., and SILVER, W. S. (eds.) A Treatise on Dinitrogen Fixation. Wiley, New York, pp. 63-123 (1977). STEWART, W. D. P., FITZGERALD, G. P., and BURRIS, R. H.: Acetylene reduction by nitrogen-fixing blue-green algae. Areh. Microbiol. 62, 336-348 (1968). STEWART, W. D. P., ROWELL, P., and TEL-OR, E.: Nitrogen fixation and the heterocysts in bluegreen algae. Biochem. Soc. Trans. 3, 357-361 (1975). STREICHER, S. L., SHANMUGAM, K. T., AUSUBEL, F. M., and GOLDBERG, R.: RegUlation of nitrogen fixation in Klebsiella pneumoniae: evidence for a role of glutamine synthetase as a regulator of nitrogenase biosynthesis. J. Bacteriol. 120, 815-821 (1974). TETLEY, R. M., and BISHOP, N. 1.: The differential action of metronidazole on nitrogen fixation, H2 metabolism, photosynthesis and respiration in Anabaena and Scenedesmus. Biochim. Biophys. Acta 046, 43-53 (1979). TULI, R., and THOMAS, J.: Regulation of glutamine synthetase in the blue-green alga Anabaena L-31. Biochim. Biophys. Acta 613, 526-533 (1980). Received November 30, 1983; accepted January 25, 1984

Author's address: Professor Dr. H. D. KUMAR, Centre of Advanced Study in Botany, Banaras Hindu University, VARANASI - 221005, India.