Arch Microbiol (1982) 131:91-94

Archives of

Micrebiole!lY �9 Springer-Verlag 1982

Production of Ammonium Dependent on Basic L-Amino Acids by Anacystis nidulans

Enrique Flores, Antonia Herrero, and Miguel G. Guerrero

Departamento de Bioqulmica, Facultad de Biologia y C.S.I.C., Universidad de Sevilla, Spain

Abstract. The incubation of the cyanobacterium Anacystis nidulans with L-Arg, L-Lys or c-Orn, but neither with the corresponding D-isomers nor with other twenty L-amino acids, resulted in the production of large amounts of am- monium which accumulated in the outer medium. Relevant properties of this in vivo ammonium production activity have been studied in cell suspensions treated with the glutamine synthetase inactivator c-methionine-D,L-sulfoximine (MSX) to prevent assimilation by the cells of the resulting am- monium. In addition to its specificity for the basic L-amino acids, the system exhibited a set of properties (K,, value for substrates, requirement of oxygen which is taken up stoi- chiometrically with the production of ammonium, inhibition by o-phenanthroline and divalent cations) all of which are shared by a peculiar L-amino acid oxidase recently isolated from A. nidulans. The data strongly suggest the participation of this enzyme in the production of ammonium from basic amino acids by A. nidulans, an activity that could account for the ability of this cyanobacterium to use arginine as a nitrogen source.

Key words: Anacystis nidulans - Blue-green algae - Cyanobacteria - Ammonium production - Amino acid catabolism - Amino acid oxidase - Basic L-amino acids

As a part of the studies aimed to establish the nature of the ammonium assimilation product(s) that could be involved in the regulation by ammonium of nitrate assimilation in cyanobacteria (Flores et al. 1980; Herrero et al. 1981 a), we have tested the effect of different amino acids on the utilization of nitrate by Anacystis nidulans cells. For these experiments cell suspensions which had been treated with the glutamine synthetase inactivator MSX, to avoid the oper- ation of the main route for ammonium assimilation, were used. In the course of these experiments, the observation was made that a significant production of ammonium took place in the presence of certain amino acids, especially of basic L-amino acids.

Since the information presently available regarding the catabolism of amino acids in cyanobacteria is very scarce (Stanier and Cohen-Bazire 1977; Weathers et al. 1978), it seemed of interest to investigate the nature of the process(es) involved in the generation of ammonium from amino acids by

Offprint requests to M. G. Guerrero Abbreviations. DCCD, N,N'-dicyclohexylcarbodiimide; FCCP, car- bonyl cyanide p-trifluoromethoxy-phenylhydrazone; MSX, c-methi- onine-D,C-sulfoximine

A. nidulans. The present study concerns with the characteri- zation of this metabolic activity in MSX-treated Anacystis cells and provides evidence supporting the involvement of an L-amino acid oxidase specific for basic amino acids, enzyme that has been isolated and characterized recently (Pistorius et al. 1979; Pistorius and Voss 1980).

A preliminary account of this research has been presented at the II Meeting of the Federation of Spanisch Societies of Experimental Biology (Herrero et al. 1981b).

Materials and Methods

Organism and Culture Conditions. Anacystis nidulans (strain L1402-1 from the G6ttingen University's Algal Culture Collection) was grown photoautotrophically at 40~ as previously described (Herrero et al. 1981 a). All of the results shown below were obtained with ammonium-grown cells ; but the production of ammonium from c-Arg, c-Lys or L-Orn also took place with cells grown on nitrate as the nitrogen source, however.

Treatment of Anacystis Cells with MSX. The cells were collected by filtration on a Millipore HA 0.45 ~tm pore size filter, washed with 25 mM Tricine-NaOH buffer, pH 8.3, and suspended in the same buffer to a density of 2 ~tl cells per ml. The cell suspensions were incubated in the presence of i mM MSX with continuous shaking in air-opened conical flasks, at 40 ~ C, in the light (120 W. m-2, white light). After 15 rain, the cells were harvested by filtration, washed with 25 mM Tricine- NaOH buffer, pH 8.3, and finally suspended in the same buffer; these are the MSX-treated cell suspensions used in the ammonium accumulation assays.

Assays of Ammonium Release from Amino Acids. Unless otherwise indicated, the assays were carried out by incubating the corresponding 5 ml-reaction mixture under similar con- ditions to those described for the MSX treatment, except that illumination was ommited. The assay was initiated by the addition of an aliquot of the MSX-treated cell suspension, to give a final concentration of i gl cells per ml, to a flask containing a solution of amino acid in 25 mM Tricine-NaOH buffer, pH8.3. After a 15min incubation, the ammonium present in the outer medium was estimated in aliquots of the cell suspension, after rapid removal of the cells by filtration. Values were corrected for the amount of ammonium present at zero time.

The assays under anaerobic conditions were performed in 10ml glass tubes fitted with rubber stopper (Becton-

0302-8933/82/0131/0091/$01.00

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Dickinson Vacutainer) after three cycles of evacuation and flushing with argon.

Analytical Methods. Ammonium was determined with gluta- mate dehydrogenase (Bergmeyer 1974). Oxygen uptake measurements were carried out manometrically. The packed cell volume was estimated from the absorbance values at 750 nm of the cell suspensions, after a close correspondance between these two parameters had been established. One gl cells contains 150-175 gg protein and 5 - 8 ~tg chlorophyll a (Flores et al. 1980).

Chemicals. Tricine, ADP, MSX, L-canavanine, and L-glu- tamic dehydrogenase (Type II, from bovine liver) were pur- chased from Sigma Chemical Co., St. Louis. NADPH was from Boehringer, Mannheim. Other chemicals were products of Merck, Darmstadt, or of Sigma Chemical Co., St. Louis.

Results and Discussion

The main route for ammonium incorporation into nitro- genous organic compounds in the cyanobacterium Anacystis niduIans is the glutamine synthetase-glutamate synthase pathway (Ramos et al. 1980). MSX is an irreversible inhibitor of Nutamine synthetase (Prusiner and Stadtman 1973) that effectively impedes ammonium assimilation in a variety of organisms, including the strain of Anacystis used in this work (Flores et al. 1980).

Small amounts of ammonium were found to accumulate in the medium when Anacystis cell suspensions treated with MSX were incubated in the absence of any added nitrogen source under dark conditions. This ammonium is most probably derived from the catabolism of cellular nitrogenous compounds. The addition of certain amino acids to the MSX- treated cell suspensions resulted in ammonium accumulation values significatively higher than those of the controls without added amino acids. The extra ammonium release was repetitively recorded in the presence of L-Gln, L-Arg, L-Lys or L-Orn, being remarkably high for the case of the three latter amino acids, all of which have basic character (Table 1). Occasionally low amounts of ammonium were found to accumulate in the presence of L-Cys and L-His. No amino acid-dependent ammonium production could otherwise be detected in the presence of L-Ala, L-Asn, L-Asp, L- canavanine, L-citrulline, L-GIu, Gly, L-Ile, L-Leu, L-Met, L- Phe, L-Pro, L-Ser, L-Thr, L-Trp, L-Tyr or L-Val, when assayed up to 20 mM final concentration (data not shown). Results in Table 1 also show that the generation of ammonium from Arg, Lys or Orn was highly specific for the L-isomers of these basic amino acids, the corresponding D-isomers being in- effective as substrates.

Although the ammonium generation from L-Arg, L-Lys or L-Orn was also evident with untreated Anacystis (not shown), MSX-treated cells were routinously used in the assays to prevent the further assimilation of the ammonium produced. The ammonium release dependent on the basic amino acids was proportional to the incubation time for at least 30 min and to the amount of cells present in the assay (not shown), and it also proceeded effectively in the light at rates similar to those recorded under dark conditions.

The rate of ammonium release as a function of the concentration of the corresponding amino acid substrate has been studied for L-Arg, L-Lys and L-Orn. The obtained results

Table 1. Production of ammonium from some amino acids by Anacystis nidulans

Addition Ammonium produced (nmol - gl cells- 1)

None 20 L-GIn 63 L-Arg 631 L-Lys 662 L-Orn 264

o-Arg 19 D-Lys 12 D-Orn 15

Final concentration of the indicated amino acids was 25 mM. Incubation time, 30 min

E: 15

E c

b- < rv

LU 5

(D

IE

Z O IE

o

/ i I i

20 40

~AMINO AC[O l ( rnN)

Fig. 1. Effect of substrate concentration on the rate of ammonium production from L-Arg (O), L-Lys (A) and L-Orn (D) by A. nidulans

are shown in Fig. 1, where it can be seen that hyperbolic saturation curves were found for both L-Arg and L-Lys, whereas a more complex kinetic behavior was apparent for the case of L-Orn. The / ~ values, calculated from Hanes- Woolf plots of the results obtained for substrate con- centrations in the range 0 - 20 mM, were 4 mM for L-Arg and 8 mM for L-Lys. The maximal rates obtained with L-Arg and L-Lys were similar, in the range of 15 - 20 nmol per gl cells per min, for 50 mM of the substrate. At the same concentration of L-Orn, the rate of ammonium generation was about half of those obtained with L-Arg or L-Lys.

We have tried to determine whether the ammonium release from L-Arg, L-Lys and L-Orn are independent pro- cesses or, on the contrary, they share some common element. For this purpose, the ammonium production from mixtures of two of the basic amino acids was evaluated and compared with the values obtained with each amino acid, L-Arg, L-Lys or L-Orn, alone. Data for two different amino acid con- centrations are included in Table 2. It can be seen that the rates of the ammonium generation from mixtures of two of the amino acids were always lower than the sum of the rates obtained with each of the two substrates tested individually.

Table 2. Ammonium generation by A. nidulans from mixtures containing two of the amino acids substrates

Amino acid Ammonium produced (nmol. gl cells- 1)

5 mM of each 50 mM amino acid

L-Arg 181 256 L-Lys 177 261 L-Orn 58 145

L-Arg, L-Lys 227 256 L-Lys, L-Orn 183 248 L-Orn, L-Arg 204 238

Incubation time, 15 rain

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Table3. Oxygen uptake associated with the production of ammonium from L-Arg, L-Lys and L-Orn by A. nidulans

Amino acid Oxygen taken up Ammonium O2/NH,/ (50 raM) (gmol) appeared ratio

(~tmol)

L-Arg 5.80 9.98 0.58 L-Lys 6.16 9.60 0.64 L-Orn 2.86 4.61 0.62

The experiments were carried out in Warburg vessels containing 11 ~tl cells in 2.75 ml Tricine buffer in the main compartment, and 0.25 ml of 20 % KOH in the center well. Oxygen uptake in the presence of the amino acids was corrected for endogenous respiration. Ammonimn appearance values were corrected for the amount of ammonium produced by the cells in the absence of added amino acids. Incubation time, 60rain

Addit ivi ty should have indicated the par t ic ipat ion of inde- pendent processes. The limited capacity of the cells to release ammonium from L-Arg, L-Lys and L-Orn rather suggests the par t ic ipat ion of a common factor in the catabolism of these basic amino adds .

An L-amino acid oxidase (deaminating), EC 1.4.3.2, spe- cific for basic amino acids has recently been characterized in A. nidulans by Pistorius et al. (1979) and Pistorius and Voss (1980). The best substrates for this enzyme are L-Arg and L- Lys, while L-Orn is oxidized at a rate approximately half of that for L-Arg. L-His is also a substrate for the purified enzyme, but much poorer than the other basic amino acids. The activity of ammonium accumulation by MSX-treated Anacystis cells with L-His is also low, only 1 - -2 % of that observed with similar concentrat ions of L-Arg. The analogies with regard to the amino acid specificity and to the K,, values for L-Arg exhibited by both the system we are dealing with here (4 mM) and the L-amino acid oxidase (5 mM, Pistorius and Voss 1980) point to a relationship between them.

If this L-amino acid oxidase is in fact responsible for the ammonium product ion from amino acids by whole A. nidulans cells, the in vivo process should proceed at lower or negligible rates under anaerobic conditions, and it should be associated to a concomitant oxygen uptake (Pistorius and Voss 1980). The ammonium release from L-Arg, L-Lys or L- Orn by A. nidulans was indeed greatly diminished under anaerobic conditions, with ammonium accumulat ion values between 4 and 10 ~ of those found in the presence of oxygen. On the other hand, we have also observed the occurrence of an oxygen uptake associated to the ammonium release from basic L-amino acids by A. nidulans cells under aerobic conditions, and have determined stoichiometry values of about 0 . 6 - 0 . 7 m o l oxygen per mol ammonium (Table3). These stoichiometries were only slightly affected by the presence of 1 - 5 mM KCN, conditions which inhibit the catalase-dependent oxygen evolution from H202 (not shown).

The in vivo ammonium product ion from L-Arg by Anacystis cells was inhibited by o-phenanthrol ine and by the divalent cations Mg 2+ and Ca 2§ (Table4). Both, divalent cations and o-phenanthrol ine are unusual inhibitors of amino acid oxidases, but they effectively inhibit the activity of the basic L-amino acid oxidase of Anacystis (Pistorius and Voss 1980). On the other hand, the rate of the ammonium generation from L-Arg by MSX-treated A. nidulans cells was not at all affected by the metabolic inhibitors ECCP (20 gM)

Table 4. Inhibition by divalent cations and o-phenanthroline of am- monium production from L-Arg by A. nidulans

Addition Concentration Inhibition (mM) (%)

MgCI z 0.1 43 MgC1 z 1 97

CaC1 z 0.1 7 CaC12 1 75

o-Phenanthroline 1 2 o-Phenanthroline I0 69

L-Arg at 25mM final concentration was used as the substrate. The ammonium production value of the control without added inhibit ors was 242 nmol. gl cells- 1 in 15 rain

or D C C D (30 ~tM). Both, FCCP and D C C D at the indicated concentrations effectively inhibit energy-requiring processes, such as the l ight-dependent CO2-fixation, in Anacystis (not shown). Cyanide at 5 mM inhibited by 90 ~ the endogenous dark respiration of Anacystis but it only caused a 10 inhibition of the ammonium generation from L-Arg, however (data not shown). As the L-amino acid oxidase o fA. nidulans appears to be intracellularly located (Pistorius et al. 1979; Pistorius and Voss 1980), the lack of effect of FCCP, D C C D and cyanide suggests that the entrance of basic amino acids into the cell should take place in a passive way, by diffusion, either facilitated or not. A passive type of influx of the basic amino acids into the cell would account for the fact that the / ~ values for L-Arg est imated for the isolated enzyme (Pistorius and Voss 1980) and for the in vivo process studied by us are very similar.

F r o m the data presented above it is evident that the properties of the system responsible for the in vivo generation of ammonium from basic L-amino acids in A. nidulans parallel in many respects with those of the enzyme L-amino acid oxidase specific for basic amino acids isolated from the same organism (Pistorius et al. 1979; Pistorius and Voss 1980). It seems therefore logical to assume that this enzyme is re- sponsible for the process herein characterized for whole Anacvstis cells. The evaluation of the rates of ammonium generation from L-Arg or L-Lys by MSX-treated A. nidulans can be used as a good estimate of the cellular levels of the enzyme.

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An aspect worth to be underlined is the significant magnitude at which ammonium generation by A. nidulans cells takes place in the presence of added amino acids. Under our experimental conditions, the rates of ammonium pro- duct~on from L-Arg or L-Lys were in the range of 15 - 20 nmol per gl cells per rain. This compares quite favorably with the rates of assimilation of inorganic nitrogen (nitrate, nitrite or ammonium), which are 1 - 4 nmol per gl cells per min (Flores et al. 1980), and it is of about the same order of magnitude that the CO2-dependent photosynthetic oxygen evolution: about 45 nmol per ~1 cells per min. The ammonium gen- eration from L-Arg might account for the capability of Anacystis to grow on this amino acid as the sole nitrogen source. In fact, our estimates for the growth rate ofA. nidulans on L-Arg are quite close to those obtained with nitrate or ammonium (generation time, 4 h).

The physiological function of this metabolic activity under conditions of absence of added amino acids remains to be established, although possible functions have been suggest- ed for the L-amino acid oxidase specific for basic amino acids found in Anaeystis (Pistorius et al. 1979; Pistorius and Voss 1980). The stoichiometry values we have determined in whole A. nidulans cells for the L-Arg-dependent oxygen uptake as related to the concomitant ammonium production are lower than those determined for the isolated enzyme (Pistorius and Voss 1980). This might suggest that under in vivo conditions a part of the reducing power resulting from the oxidation of the amino acids would be channeled to acceptors other than molecular oxygen.

Acknowledgements. This work was supported by a grant from the Fundaci6n Ram6n Areces (Spain). We are deeply grateful to Prof.

Manuel Losada for his generous encouragement and help and to Dr. Elfriede K. Pistorius for helpful criticism and suggestions.

References

Bergmeyer HU (1974) Methoden der enzymatischen Analyse, 2nd ed. Verlag Chemie, Weinheim

Flores E, Guerrero MG, Losada M (1980) Short-term ammonium inhibition of nitrate utilization by Anacystis nidulans and other cyanobacteria. Arch Microbiol 128:137-144

Herrero A, Flores E, Guerrero MG (1981a) Regulation of nitrate reductase levels in the cyanobacteria Anaeystis nidulans, Anabaena sp. strain 7119, and Nostoc sp. strain 6719. J Bacterio1145 : 175 - 180

Herrero A, Flores E, Guerrero MG (1981 b) Generaci6n de amonio a partir de L-Arg, L-Lys y L-Orn por c61ulas de la cianobacteria Anacystis nidulans. Abstract 491, 2nd FESBE Congress, Madrid

Pistorius EK, Jetschman K, Voss H, Vennesland B (1979) The dark respiration of Anacystis nidulans. Production of HCN from histidine and oxidation of basic amino acids. Biochim Biophys Acta 585 : 630- 642

Pistorius EK, Voss H (1980) Some properties of a basic L-amino acid oxidase from Anaeystis nidulans. Biochim Biophys Acta 611:227- 240

Prusiner S, Stadtman ER (1973) The enzymes of glutamine metabolism. Academic Press, New York London

Ramos JL, Flores E, Guerrero MG (1980) Glutamine synthetase- glutamate synthase: the pathway of ammonium assimilation in Anacystis nidulans. Abstract 226 A, 2nd FESPP Congress, Santiago de Compostela

Stanier RY, Cohen-Bazire G (1977) Phototrophic prokaryotes: the cyanobacteria. Ann Rev Microbiol 31 : 225 - 274

Weathers PJ, Chee HL, Allen MM (1978) Arginine catabolism in Aphanocapsa 6308. Arch Microbiol 118 : 1 - 6

Received August 7, 1981/Accepted November 30, 1981