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Evidence for the nitrate assimilationdependent nitrite excretion in cyanobacterium Nostoc MAC B.B. Singh, P.K. Pandey, S. Singh and P.S. Bisen” Nitrate uptake and nitrite efflux patterns in No&c MAC showed a rapid phase followed by their saturation. Nitrite efflux was maximum in nitrate medium whereas the cells incubated in N, and NH,+ media exhibited a decreased nitrite efflux activity. The simultaneous presence of NH,+ and nitrate significantly decreased nitrite efflux. L-Methionine-DL-sulphoximine (MSX) prevented inhibition of nitrite efflux by NH,+. In the dark there was negligible nitrite efflwr, whereas illumination increased the rate of nitrite efflwr significantly. The nitrite efflux system was maximally operative at pH 8.0,3O”C and a photon fluence rate of 50 pmol m-‘. s-‘. These results confirm that (i) the nitrite efflux system in Nostoc MAC is dependent upon nitrate uptake and assimilation and is repressible by NH,+; (ii) NH,+ itself is not the actual repressor of nitrite efflux; a product of NH,+ assimilation via glutamine synthetase (GS) is required for repression to occur: (iii) the catalytic function of GS does not appear to be involved in nitrate assimilation-dependent nitrite efflux, and (iv) the optimum pH, temperature and illumination for maximum nitrite efflux were found to be S.O,3O”C and 50 pmol rnm2. s respectively. Keyulords: Nitrate assimilation,

nitrite efflux, Nosfoc MAC.

Cyanobacteria are the only prokaryotes which can reduce nitrate to NH,+ b y u t’l’1 lzmg water as the primary electron donor and sunlight as the sole energy source. Nitrate assimilation in cyanobacteria comprises at least two consecutive steps which are transport of nitrate into the cells and its subsequent reduction to nitrite and NH,+ by ferredoxin-dependent nitrate and nitrite reductases, respectively (Manzano et al. 1976; Losada & Guerrero 1979; Guerrero & Lara 1987). NH, + , the end product of the nitrate assimilation pathway, is an effective antagonist of nitrate assimilation (Herrero & Guerrero 1986; Palod ef al. 1990; Singh 1992). Whereas nitrate metabolism in cyanobacteria has been extensively studied (Flores et al. 1983, 1987; Martin-Nieto et al. 1990; Bisen & Shanthy 1991; Singh 1992; Singh & Bisen 1994), so far there is no report concerning the nitrate assimilation-dependent nitrite efflux in cyanobacteria. Using Nostoc sp. MAC, we have provided

evidence that nitrite efflux is dependent on nitrate uptake and assimilation and is regulated at the level of NH,+ assimilation and not by NH, + uptake. Furthermore, the role of various environmental factors on nitrite efflux has been investigated.

Materials

,Organism and Culture Conditions Nostoc sp. MAC PCC 8009 (Het- Fox-) (obtained from Prof. J.C. Meeks, Department of Bacteriology, University of Davis, California, USA) was grown axenically in a four-fold dilution of medium containing KNO, as the sole nitrogen source (Allen & Amon 1955). The cultures were incubated at 24 f 1°C and illuminated with day-light fluorescent tubes having a photon fluence rate of 50 pm01 m-’ .s. The medium was buffered to pH 7.5 with 10 mM Hepes/NaOH. Nitrate

8.8. Singh. P.K. Pandey and P.S. Risen are with the Department of Microbiology, Barkatullah University, Bhopal 462026, India. SSingh is with the Department of Microbiology, School of Life Sciences, North Maharashtra University, Jalgaon. India; fax: 91-751-341450. ’ Corresponding author @ 1996 Rapid

Science

and Methods

Uptake

Assay

Nitrate uptake was assayed by measuring the depletion of nitrate from the external medium. Mid-log phase (6 d old) cultures of Nostoc sp. MAC were harvested by centrifugation (5000 xg, 5 min), washed thoroughly with sterile medium and resuspended in N,-medium to a final density of 400 pg protein ml-‘. The cells

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B.B. Singh, l?K. Pam&y, S. Singh and P.S. Bisen Analytical

Methods

Cellular protein was estimated by the method of Lowry using bovine serum albumin as the standard. Nitrate was estimated according to Avissar (1985), nitrite was assayed according to the diazo-coupling method of Snell & Snell (1949).

Results

0

8 12 Incubation time(h)

16

4

Figure 1. Kinetics of nitrate uptake (0) and nitrateassimilation-dependent nitrite efflux (e) in Nostoc sp. MAC.

Table 1. Relative contribution nitrate assimilation-dependent in the presence and absence (MSX). Nitrogen source

(nmol

k NH&I KNO, KNO,

+ NH,CI

Data shown are means

of

of different nitrogen sources on nitrlte efflux in Nostoc sp. MAC of L-methionine-oksulphoximine

nitrite

Nitrite Efflux excreted mg protein-‘.

- MSX

+ MSX

1.6 1.5

1.7 1.6

6.1 1.6

6.6 6.6

h

triplicate experiments.

were then equilibriated for I h at 25°C and at a photon fluence rate of 50 pm01 rnb2 .s prior to adding KNO, at 5 mM. Samples were withdrawn at intervals and cells separated from their bathing medium by rapid centrifugation (10,000 xg, 1 min). The cell-free supematants were analysed for residual nitrate. Nitrite Efflux Assay Nitrite efflux was assayed by measuring the appearance of nitrite in the external medium. The concentration of nitrite in nitrateincubated cells was estimated in cell-free supematants collected at regular time intervals. When needed, KNO, (5 mM) and NH,CI (2 mM) were added to the medium. L-Methionine-ot.-sulphoximine (MSX) was added to the medium at 50 PM 2 h prior to adding nitrate and/or NH,+ and incubated under normal growth conditions to allow entry of the chemical into the cells. Role ofpH, Temperature and Illumination To determine the effect of pH, temperature and illumination on nitrate efflux, mid-log phase cells (6 d old) were harvested by centrifugation (5000 x g, 5 min), washed with sterile distilled water and resuspended in phosphate buffer (0.02 M). The experiment was performed in three sets; in the first the cells were incubated over the pH range 4 to 10. The second and third sets were used to study the effects of illumination and temperature.

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and Discussion

Nitrate uptake and nitrate-dependent nitrite efflux in Nostoc sp. MAC cells were assayed simultaneously (Figure 1). Nitrate uptake showed a rapid phase up to 5 h followed by nutrient saturation with a total accumulation of 133 nmol NO,- mg protein-’ at 8 h. Whereas nitrate uptake started immediately, the nitrite efflux process had a lag of 5 h. This lag was consistent for the time taken for nitrate to be reduced to nitrite suggesting that nitrite efflux is nitrate assimilation-dependent. Nitrite efflux activity showed similar saturation kinetics to the nitrate uptake system. These results suggest that nitrite efflux is dependent on nitrate assimilation and the former is regulated by the latter. A glucose-dependent nitrite efflux has been reported in the cyanobacterium Synechocystis (Reyes et al. 1993) in which more than 60% nitrate is excreted in the form of nitrite. It has been suggested that this nitrite excretion is due to the different catalytic rates of the reductases. In the presence of glucose, the reduction of the ferredoxin pool by NADPH is not sufficient to maintain nitrite reduction and as a consequence nitrite is excreted. In order to further investigate the relative contribution of different nitrogen sources on nitrite efflux, the Nostoc sp. MAC cells were incubated in the presence or absence of nitrate and NH,+, with and without MSX. The amount of nitrite efflux was maximum in nitrate medium (Table I), however its amount was rather similar to that in cells incubated with either N, or NH,+ (Table 1). The simultaneous presence of NH, ’ in the nitrate medium always led to a reduction in nitrite efflux, indicating that the nitrite efflux system is NH,+ -repressible. The addition of MSX, a glutamate analogue and an irreversible inhibitor of GS, alleviated NH,+ inhibition of nitrite efflux, suggesting that the catalytic function of GS is required for the NH,’ inhibition of nitrite efflux, i.e. NH, + itself is not the repressor of nitrite efflux and a product of NH, + assimilation may be involved. The addition of MSX could not affect the nitrate efflux system, suggesting that the catalytic function of GS is not absolutely required for the nitrate assimilation-dependent nitrite efflux system. The effect of pH, temperature and illumination on nitrite efflux is shown in Figure 2. Nitrite efflux concentration increased with increased pH and was maximal at pH 8.0 (Figure 2). In contrast, cells assayed at pH 5.0 and pH 10.0 had a decreased rate of nitrite efflux compared to those assayed at pH 8.0. Nitrite efflux was maximal at 30°C and

to the University Grants Commission, providing financial assistance.

New

Delhi

for

c 7:

40

References

5 k E” 30 d E 5

20

X

3 c w !o” z

10

0

0

50

100 150 Light (,umol rii2d I I I I I 5 6 7 8 9 PH I I I 20 30 40 Temperature t°C 1

I 4 I 10 Figure 2. The (0) on the Nostoc incubated

sp.

MAC. To in phosphate

determination were suspended to ranges temperature 4 h and

effect of pH (0) temperature nitrate-assimilation-dependent determine buffer

nitrite

the role of pH (0.02 M), pH range

of the role of illumination in phosphate buffer (0.02

of illumination (IO-50°C) efflux

(10-200 respectively. was

measured

(A)

pmol

and nitrite

200 1 10

I 50 illumination efflux

in

the cells were 4 to 10. For the

and temperature, M, pH 8.0) and

cells exposed

mm2 s-’ photon flux) Cultures were incubated

and for

as described.

any further increase or decrease in the temperature resulted in a decreased nitrite efflux. Illumination at the photon fluence rate of SO pmol m-‘.s-’ supported maximal nitrite efflux in this cyanobacterium. Further increase in illumination resulted in pigment bleaching leading to reduced nitrite excretion. We conclude that (i) the nitrite efflux system in Nosfoc sp. MAC is dependent upon nitrate uptake and assimilation and is NH,+ -repressible; (ii) NH,+ itself is not the repressor of nitrite efflux but a product of NH,+ assimilation via glutamine synthetase (GS) is required for repression; (iii) GS is not necessarily involved in the nitrite efflux process; and (iv) pH, temperature and illumination play important roles in the regulation of the nitrite efflux system.

Allen, M.B. & Amon, D.I. 1955 Studies on nitrogen fixing bluegreen algae. I. Growth and nitrogen fixation by Anabaena cyhndrica Lemn. Plant Physiology 30, 366-372. Avissar, Y.J. 1985 Induction of nitrate assimilation in the cyanobacterium Anabaena uariabilis. Physiologia Planfarum 65, IO5108. Bisen, P.S. & Shanthy, S. 1991 Regulation of assimilatory nitrate reductase in the cyanobacterium Anabaena doliolum. Cwrent Microbiology 23, 239-244. Flores, E., Ramos, J.L., Herrero, A. & Guerrero, M.G. 1983 Nitrate assimilation by cyanobacteria. In: Photosynthetic Prokaryofes: Cell Differentiation and Function, eds Papageorgiou, G.C. & Packer, L. pp. 363-387. New York: Elsevier Science Publishers. Flares, E., Herrero, A. & Guerrero, M.G. 1987 Nitrate uptake and its regulation in the cyanobacterium Anacysfis nidulans. Biochimica et Biophysics Acfa 896, 103-108. Guerrero, M.G. & Lara, C. 1987 Assimilation of inorganic nitrogen. In: The cyanobacferia, eds Fay, P. & Van Baalen, C. pp. 163186. Amsterdam: Elsevier Science Publishers. Herrero, A. & Guerrero, M.G. 1986 Regulation of nitrite reductase in the cyanobacterium Anacysfis nidulans. Journal of General Microbiology 132, 2463-2468. Losada, M. 81 Guerrero, M.G. 1979 The photosynthetic reduction of nitrate and its regulation. In: Photosynthesis in Relation to Model Systems, ed Barber, J. pp. 365-408. Amsterdam: Elsevier Science Publishers. Manzano, C., Candau, P., Gomez-Moreno, C., Relimpio, A.M. & Losada, M. 1976 Ferredoxin dependent photosynthetic reduction of nitrate and nitrite by particles of Anacysfis nidulans. Molecular and Cellular Biochemistry 10, 161-169. Martin-Nieto, J., Flores, E. & Herrero, A. 1990 Mutants of Anabaena variabihs requiring high levels of molybdate for nitrate reductase and nitrogenase activity. FEMS Microbiology Letters 67, l-4. Palod, A., Chauhan, V.S. & Bagchi, S.N. 1990 Regulation of nitrate reduction in a cyanobacterium Phormidium uncinafwn: distinctive modes of ammonium repression of nitrate and nitrite reductases. FEMS Microbiology Letters 63, 285-288. Reyes, C., Chavez, S., Muro-Paster, MI., Candau, P. & Florencio, F.J. 1993 Effect of glucose utilization on nitrite excretion by the unicellular cyanobacterium Synechocystis sp. strain PCC 6803. Applied and Environmental Microbiology 59, 3161-3163. Singh, S. 1992 Regulation of nitrate uptake in cyanobacteria: effect of nitrogen starvation. Biomedical Letters 47, 229-233. Singh, S. & Bisen, P.S. 1994 Glutamine synthetase and arginine inhibition of nitrate reductase in Anabaena cycadeae. World lotrrnal of Microbiology and Biotechnology 10, 191-193. Snell, F.D. & Snell, CT. 1949 Calorimetric Methods of Analysis, volume 3. New York: Van Nostrand.

Acknowledgements The authors thank the Department of Biotechnology (Govt. of India), New Delhi for financial assistance. BBS is grateful

(Received

in revised

form

9 Januay

World]oumal

of Microbmlogy

1996;

accepted

73 Januay

7996)

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