Physiological and Biochemical Characterization of. Chlorate-Resistant Mutants of ... algae [21, 29, 35], and higher plants [10, 11, 22,. 25]. In all these studies, ...
CURRENT MICROBIOLOGY Vol. 25 (1992), pp. 353-357
Current Microbiology 9 Springer-Verlag New York Inc. 1992
Physiological and Biochemical Characterization of Chlorate-Resistant Mutants of Anabaena doliolum P.S. Bisen and S. Shanthy Department of Microbiology, Barkatullah University, Bhopal, India
Abstract. Chlorate-resistant mutants of the filamentous cyanobacterium, Anabaena doliolum, were isolated by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) 1 mutagenesis. Three classes of mutants were obtained that were altered either in the nitrate uptake activity or nitrate reductase enzyme activity or both. These results suggest that the genetic determinant of the uptake system was distinct from that of the reductase system. Uptake studies of nitrite and ammonium and rate of nitrite reductase activity in the mutants revealed that the nitrite and ammonium metabolisms were not affected by this mutation. Both nitrate and chlorate acted like a pair of antagonists, with nitrate protecting the growth against chlorate with increase in its concentration; similarly, increasing chlorate concentrations counteracted the growth-protective action of nitrate.
Cyanobacteria reduce nitrate to ammonia by the joint action of the', photosynthetic, membranebound, ferredoxin-dependent nitrate reductase and nitrite reductase [14, 26]. The rate of nitrate reduction could be hampered by either changes in the uptake of nitrate or the reductase system, both of which are, in turn, focal points of ammonium controls [4, 6, 12-14, 17, 19, 26, 28]. Nontoxic chlorate, the structural analog of nitrate, is reduced to toxic chlorite by nitrate reductase [24, 36]. This enzyme does not discriminate between its natural substrate, NO3, and an unnatural one, C103, and can mediate the reduction of either. This results in the formatJ~on of CIO~, an anion so toxic that wild-type cells are killed while the enzymedeficient or uptake-defective mutants survive [1, 16, 18, 23-25]. Chlorate toxicity and mutations conferring resistance have been studied in bacteria [31, 37, 38], cyanobacteria [3, 30, 32, 33], fungi [5, 8, 9, 15, 23], algae [21, 29, 35], and higher plants [10, 11, 22, 25]. In all these studies, a loss of nitrate reductase activity, altered activity, or altered uptake were reported as a result of resistance to chlorate. i Abbreviations: HEPES, 4-(hydroxyethyl)-l-piperazine ethanesulfonic acid; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; MOPS, 4-morpholinepropanesulfonic acid; Tricine, N-tris(hydroxymethyl)methylglycine; TRIS, tris (hydroxymethyl)aminomethane.
This paper deals with the isolation of chlorateresistant mutants and the analyses of growth, heretocyst frequency, uptake and reductase systems of both the wild type and mutants. Materials and Methods Organism and culture conditions. Cultures ofAnabaena doliolum were grown in eight times diluted Allen and Arnon medium [2] with combined nitrogen (5 mM KNO3) and buffered with 50 mM HEPES-NaOH buffer, pH 7.5, at 28~ under illumination (6000 lux) with cool, white fluorescent light. The cultures were made chlorate tolerant by gradually subjecting them to increasing dosage of chlorate from 5 mM up to 30 raM. Growth and heterocyst frequency measurements. Growth was recorded by measuring the absorbance of the cultures at 650 nm with a spectrocolorimeter [6]. Heterocyst frequency was determined as a percentage of total vegetative cells per filament. Mutagenesis and isolation of chlorate-resistant mutants. The wild type and chlorate-tolerant cultures of Anabaena doliolum in the exponential phase of growth (106-107 cells - ml I in 50-ml volumes) were fragmented by sonication to reduce the filament size [39]. The fragmented cultures were washed twice with basal medium supplemented with 2.5 mM NHaC1 and buffered with 5 mM MOPS, pH 7.5. The cultures at a concentration of 10 6 to 2 x 10 7 cells/ml were suspended in 20 ml volume of basal medium for exposure to the mutagen, MNNG (100/zg/ml final concentration) and shaken gently for half an hour in dark. The cells were washed with basal medium after exposure to the mutagen. The treated cultures were screened on solid media for resistance to 30 mM chlorate after growth for 6 days in buffered liquid ammonium basal medium. The plates were divided into three set3: one set
Address reprint requests to: Dr. P.S. Bisen, Department of Microbiology, Barkatullah University, Bhopal 462 026 (M.P.), India.
354 overlayered with 5 mM KNO 3containing agar medium; the second set overlayered with 5 mM NaNO z containing agar medium; and the third set served as a control. The colonies growing on the plates were picked up separately and transferred to liquid basal medium. Uptake of inorganic nitrogen sources. The experiments on uptake of nitrate, nitrite, and ammonium were conducted in media supplemented with 100 txM KNO3, NaNO2, or NH4CI and buffered with 25 mN Tricine-NaOH buffer, pH 7.5. Nitrate uptake was estimated as in [19], nitrite uptake as in [34], and ammonium uptake as in [7]. Determination of nitrate reductase and nitrite reductase enzyme activities. The nitrate reductase activity was determined in a final volume of 1 ml in a reaction mixture containing 100 mM glycine/ KOH buffer (pH 10.5), 20 mM KNO3, 4 mM methyl viologen, 10 mM Na2S204 (0.1 ml Na2S204 stock solution prepared in 0.23 M NaHCO3) and cell-free extract. The supernatant was then used for measuring the enzyme activity colorimetrically by following the appearance of nitrite [6, 28]. Nitrite reductase activity was estimated from the consumption of nitrite [4]. The assay mixture contained 200 /xmoles tris buffer (pH 7.6), 3.0 /xmoles methyl viologen, 30 ~moles NazS204, 30 /zmoles NaHCO3 and appropriate amounts of cell free extract [20]. Analytical methods. Protein contents in the samples were measured as given in [27], with bovine serum albumin as the standard protein. Chlorate was dissolved in distilled water to obtain stock solution. Stock solution was filter sterilized (0.45 ~N millipore) before being added to the cultures. Chemicals. All chemicals at their analytical grades were procured from Sigma Chemical Co. (USA), British Drug House, and Sisco Research Laboratories (India).
Results Characterization of the chlorate-resistant (Clr) mutants o f Anabaena doliolum. The chlorate-resistant mutants were isolated f r o m the chlorate-containing nitrogen-free plates o v e r l a y e r e d with nitrate medium; chlorate-containing nitrogen-free plates overlayered with nitrite medium; and chloratecontaining nitrogen-free plates. T h e y were conveniently designated as CIr-N~, Clr-Nz, and Clr-N3 strains, respectively. The n u m b e r of chlorate-resistant mutant strains isolated include 32 colonies f r o m chlorate-containing nitrogen-free medium; 25 colonies from chlorate-containing nitrate medium; and 50 colonies f r o m chlorate-containing nitrite medium. The CIr-N 1 mutant showed no change in growth w h e n c o m p a r e d with the parent, in media containing nitrate, nitrite, a m m o n i u m , and under N2-fixing con-
CURRENT MICROBIOLOGY Vol. 25 (1992)
ditions (Table 1). This mutant f o r m e d h e t e r o c y s t s of the order of 7 - 8 % in nitrate-containing m e d i u m , in contrast to inhibition of h e t e r o c y s t formation in the parent (Table 1). The Clr-N2 strain exhibited no change in growth like the parent, but formed h e t e r o c y s t s (5-6%) in nitrate-containing m e d i u m (Table 1). The Clr-N3 mutant growing under N2-fixing conditions showed no alteration of growth, but differed from the parent in the formation o f h e t e r o c y s t s (6-7%) in nitrate m e d i u m (Table 1). Response of parent and chlorate-resistant mutants to uptake of inorganic nitrogen sources. The time course of nitrate, nitrite, and a m m o n i u m uptake by the parent and the three mutants were studied (Table 1). The rate of uptake of the nitrogen sources b y the CIr-N1 strain did not vary m u c h f r o m that of the parent. But the Clr-N2 and CIr-N3 strains s h o w e d quite low uptake rate of NO;-, i.e., 2.52 and 1.3 /xmoles NO3 taken up p e r / z g protein/rain c o m p a r e d with the parent, which showed a rate of 16.64/xmoles of NO3 taken up p e r / x g protein/rain. Nitrate reductase and nitrite reductase activities of the parent and chlorate-resistant mutants. The rate of nitrate reductase activity of the CIr-N~ type mutant was very low, i.e., 1.6/xmoles NO~ f o r m e d per /xg protein/h c o m p a r e d with the high rate of 38.2 /xmoles NO2 formed per/~g protein/h (Table 1). The rate of activity of this e n z y m e was c o m p a r a b l e to that of the parent in the Clr-N2 mutant, but no nitrate reductase activity was o b s e r v e d in the CIr-N 3 mutant. The rates of nitrite reductase activity of all three mutants were similar to those of the parent (Table 1). The rates of the e n z y m e activity of the parent, Clr-N 1, Clr-N 2, and Clr-N3 were 33, 32.3, 28.6, and 30.4/xmoles of N O 2 taken up p e r / x g protein/h, respectively. Growth response of the parent and the chlorate-resistant mutants o f Anabaena doliolum to graded concentrations of chlorate. The parent strain and the three chlorate-resistant mutants were e x p o s e d to graded concentrations of chlorate and the r e s p o n s e to growth was o b s e r v e d (Fig. I). The parent showed only 10% growth at 5 m g concentration of chlorate, whereas the CIr-N~, Clr-N2, and Clr-N3 mutant strains showed 92%, 90%, and 85% growth at that concentration. A chlorate concentration of m o r e than 5 mM inhibited the growth of the parent c o m -
355
P.S. Bisen and S. Shanthy: Chlorate-Resistant Mutants of Anabaena doliolum
Table 1. Growth, heterocyst frequency, nitrate, nitrite, ammonium uptake, nitrate and nitrite reductase activities of the parent and the chlorate-.resistant mutant strains (Clr-N) of Anabaena doliolum Heterocyst frequency b
Strains
N2
NO3
NO2
NH~
N 2
NO 3
NO 2
NH~
NO~
NO~
NH~
Nitrate reductase activity d
Parent Clr-N 1 Clr-N z Clr-N3
0.56 0.54 0.50 0.51
0.60 0.55 0.56 0.50
0.62 0.59 0.54 0.49
0.22 0.21 0.26 0.20
7-8 7-8 6-7 7-8
0.0 7-8 5-6 6-7
0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0
16.64 17.40 2.52 1.30
18.80 18.20 16.10 16.30
24.80 23.20 21.60 24.10
38.20 1.60 34.80 0.0
Growth ~
Uptake ~
Nitrite reductase activity e 33.0 32.30 28.60 30.40
The inoculum for various nitrogen media was NO3 grown cultures. The various culture characteristics were studied in cultures grown in the respective media fi~r 6 days. a Growth was recorded as OD at 663 nm. b Heterocyst frequency as percentage of total vegetative cells per filament. Uptake as tzmoles of the respective nitrogen source taken up/txg protein/rain. d Nitrate reductase activity as/xmoles NO 2 formed/p~g protein/h. e Nitrite reductase activity as/xmoles NOr taken up/tzg protein/h. The data are the mean of three independent experiments.
E3
[] = Parent
9
9
zx
A = CLr_Nz
9
=
C[r-N1
0.9-
9 = Ctr-N 3
-
-
-
-
- -
o A
= 5raM K N 0 3 l~.dium = 5mM K N O a + 5 m M K C i 0 3
9 o 9
=10raM K N O 3 + S m M KC[O3 Medium = 5mM KNO3+10mM KC[O 3 Medium = 5ram KCtO 3 Medium
/
0-8-
100
R~o 0.7-
90
Medium
o
//o
I I--
80
1.9
n-
70 60
w
50
O
0.6E c
~o 0 5 -
[]
>-
z~ 0.4-
Z iii
uIZ
30
a.
20
hi
LJJ s
< 0'3-
10 [] ' ~ q 7 00
5
E o
n 10
1'5
0.2-
2'0 0.1-
CONCENTRATION (mM)
Fig. 1. Growth response of the parent strain and the chlorateresistant mutants Clr-N 1, Clr-N 2, and Clr-N3 ofAnabaena doliolum to graded concentrations of chlorate.
0.0
i
~
5 i ~ TIME (days)
~
~
Fig. 2. Growth interaction of nitrate and chlorate on the parent strain of Anabaena doliolum.
pletely, but the mutant strains Clr-N~, Clr-N2, and Clr-N 3 showed only 19%, 28%, and 23% inhibition even at 15 mM chlorate concentration. Effect of nitrate and chlorate interactions on the growth of the parent, Anabaena doliolum. The toxicity of 5 mM chlorate on the growth of the parent was decreased to a significant level by 5 mM nitrate
(Fig. 2). The protective action of nitrate against chlorate increased with increase in nitrate concentration. The inhibitory action of chlorate against nitrate increased with increase in chlorate concentration (Fig. 2), thus counteracting the growth-promoting actions of nitrate. The parent exhibited good growth in the
356 presence of only 5 mM nitrate, in contrast to 8% growth in the p r e s e n c e of 5 mM chlorate.
Discussion The toxic effect of chlorate, the structural analog of nitrate, on microbial growth results from the reduction of nontoxic chlorate by the nitrate reductase to the toxic chlorite ion [1, 3, 16, 18, 23-25, 36]. The growth and h e t e r o c y s t formation of the parent and the chlorate-resistant mutants indicated that the mutation conferring chlorate resistance also conferred a relief of h e t e r o c y s t formation from nitrate inhibition. The nature of this mutation seems to be pleiotrophic, suggesting a c o m m o n genetic basis for the chlorate resistance and derepression of heterocyst formation in nitrate medium. The chlorate-resistant mutants showed either a normal nitrate uptake and reductase system or a defective one, or completely lacked the e n z y m e system when c o m p a r e d with the normal assimilation of nitrate in the parent. F r o m these results, it is clear that there are different genetic determinants for the functional nitrate uptake and reductase systems. Chlorate resistance in these mutants could possibly be owing to two different mechanisms: in the Clr-N 1 mutant, chlorate was p r o b a b l y reduced at a lower rate b e c a u s e of the low level of NR; in the Clr-N2 type there was lower uptake of chlorate; and in the Clr-N3 type it m a y be a combination of both these mechanisms. Similar mutants with altered nitrate assimilation h a v e b e e n isolated in c y a n o b a c t e r i u m N o s t o c m u s c o r u m [3] and higher plants [10-11, 22]. It is clear f r o m the results that the uptake of NO~ and N H 2 ions remains unaffected, indicating that this mutation did not affect the genes of nitrite and a m m o n i u m metabolism. Chlorate-resistant mutants o f Aspergillus nidulans [9], Aspergillus oryzae [15], R h i z o b i u m sp. [34], and N o s t o c m u s c o r u m [30] that were able to utilize nitrite and a m m o n i u m have also been isolated. G r o w t h repression by chlorate in the parent could be explained by its mimicking nitrate catabolism with formation of the toxic product, chlorite. This is in accord with the theory first suggested by Aberg [1] on the basis of experiments with young wheat plants. Chlorate toxicity was also o b s e r v e d in c y a n o b a c t e r i a [33], fungi [24, 25], green algae [21, 29, 35] and higher plants [10, 11, 22]. The growth-protective action of nitrate against chlorate increased with increasing nitrate concentration. Similarly, increasing chlorate concentrations counteracted the growth-promoting action of ni-
CURRENT MICROBIOLOGYVo1.25 (1992)
trate. Thus, both substrates acted like a pair of antagonists in A n a b a e n a doliolum. A similar d e p e n d e n c e of chlorate toxicity on nitrate concentration was reported in A r a b i d o p s i s thaliana [22], Chlorella vulgaris [34], Aspergillus nidulans and Penicillium c h r y s o g e n u m [5, 9], and N o s t o c m u s c o r u m [33]. Since chlorate and nitrate can be reduced b y the same e n z y m e , the questions can be raised w h e t h e r the uptake of these ions is related as well. Thus, the isolation of mutants blocked in chlorate uptake would be very interesting.
ACKNOWLEDGMENTS
We are grateful to the Council of Scientific and Industrial Research, New Delhi, for awarding a Senior Research Fellowship to S. Shanthy.
Literature Cited
1. Aberg B (1947) On the mechanism of the toxic action of chlorates and some related substances upon young wheat plants. Kungl Lantbrukshi6gskolans Ann 15:37-107 2. Allen MB, Arnon DI (1955) Studies on nitrogen fixing bluegreen algae. I. Growth and nitrogen fixingby Anabaena cylindrica. Lemm Plant Physiol 30:366-372 3. BagchiSN, SinghHN(1984)Geneticcontrolofnitratereduction in the cyanobacterium Nostoc muscorurn. Mol Gen Genet 193:82-84 4. Bagchi SN, Rai UN, Rai AN, Singh HN (1985) Nitrate metabolism in the cyanobacterium Anabaena cycadeae: regulation of nitrate uptake and reductase by ammonia. Physiol P1 63:322-326 5. Birkett JA, Rowlands RT (1981) Chlorate resistance and nitrate assimilation in industrial strains of Penicillium chrysogenum. J Gen Microbiol 123:281-285 6. Bisen PS, Shanthy S (1991) Regulation of assimilatory nitrate reductase in the cyanobacterium Anabaena doliolum. Curr Microbiol 23:239-244 7. Burris RH, Wilson PW (1957) Methods of nitrogen fixation. Methods Enzymol 4:355-367 8. Cove DJ (1976a)Chlorate toxicity inAspergillus nidulans : the selection and characterization of chlorate-resistant mutants. Heredity 36:191-203 9. Cove DJ (1976b) Chlorate toxicity in Aspergillus nidulans: studies of mutants altered in nitrate assimilation. Mol Gen Genet 146:147-159 10. Fietje J Oostindi6r-Braaksma (1970) A chlorate-resistant mutant of Arabidopsis thaliana. Arabidopsis Inform Serv 7:24 11. Fietje J Oostindi6r-Braaksma, Feenstra WJ (1972) Chlorateresistant mutants of Arabidopsis thaliana II. Arabidopsis Inform Serv 9:9-10 12. 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 13. Flores E, Guerrero MG, Losada M (1981) Photosynthetic nature of nitrate uptake and reduction in the cyanobacterium Anacystis nidulans. Biochim Biophys Acta 722:408-416 14. Flores E, Ramos JL, Herrero A, Guerrero MG (1983) Nitrate
P.S. Bisen and S. Shanthy: Chlorate-Resistant Mutants of Anabaena doliolum
15.
16.
17. 18.
19. 20.
21.
22.
23.
24. 25.
26.
27.
assimilalLion in cyanobacteria. In: Papageorgiou GC, Packer L (eds) Photosynthetic prokaryotes: cell differentiation and function. Amsterdam: Elsevier Science Publishing Co, pp 363-385 GoksCyr J (1951) On the effect of chlorate upon the nitrate reduction of plants. I. Experiments with Aspergillus oryzae. Physiol P1 4:498-513 GoksCyr J (1952) On the effect of chlorate upon the nitrate reduction of plants II. The effect upon the nitrate-reducing system in Escherichia coli. Physiol PI 5:228-240 Guerrero MG, Ramos JL, Losada M (1982) Photosynthetic production of ammonia. Experientia 38:53-58 Hackenthal E, Hackenthal R (1965) Die Nitratreduktion durch ze.llfreie Extrakte aus Bacillus cereus. Biochim Biophys Acta 107:189-202 Herbert D, Phipps PJ, Strange RE (1971) Chemical analysis of microbial cells. Methods Microbiol 5B:209-344 Herrero A, Flores E, Gnerrero MG (1981) Regulation of nitrate reductase levels in the cyanobacteria Anacystis nidulans, Anabaena sp strain 7119 and Nostoc sp strain 6719. J Bacteriol 145:175-180 Huskey RJ, Semenkovich CF, Griffin BE, Cecil PO, Callahan AM, Chace KV, Kirk DL (1979) Mutants of Volvox carteri affecting nitrogen assimilation. Mol Gen Genet 169:157-161 Laan PH van der, Fietje J Oostindi~r-Braaksma, Feenstra WJ (1971) Chlorate-resistant mutants of Arabidopsis thaIiana. Arabidopsis Inform Serv 8:22 Lewin RA (1988) Making mutants and influencing genes--the genetic exploitation of algae and cyanobacterial. In: Rogers LJ, Gallon JR (eds) Biochemistry of the algae and cyanobacteria. Oxford: Clarendon Press, pp 351-360 Lewis CM, Fincham JRS (1970) Genetics ofnitrate reductase in Ustilago maydis. Genet Res 16:151-163 Liljestr6m S, Aberg B (1966) Studies on the mechanism of chlorate toxicity. Kungl Lantbruksh6gskolans Ann 32:93-107 Losada M, Guerrero MG, Vega JM (1981) The assimilatory reduction of nitrate. In: Bothe H, Trebst A (eds) Biology of inorganic: nitrogen and sulfur. Berlin: Springer Verlag, pp 30-63 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951)
28.
29.
30.
31.
32.
33.
34.
35. 36.
37.
38.
39.
357
Protein measurements by folin phenol reagent. J Biol Chem 193:266-275 Manzano C, Candau P, Gomez-Moreno C, Relimpio AM, Losada M (1976) Ferredoxin dependent photosynthetic reduction of nitrate and nitrite by particles of Anacystis nidulans. Mol Cell Biochem 10:161-169 Nicholas GL, Syrett PJ (1978) Nitrate reductase deficient mutants of Chlamydomonas reinhardii, isolation and genetics. J Gen Microbiol 108:71-77 Pandey RD, Singh PK (1984) Isolation and characterization of nitrate reductase mutants and regulation of nitrate reductase and nitrogen in the cyanobacterium Nostoc rnuscorum. Mol Gen Genet 195:180-185 Piechaud M, Puig J, Pichinoty F, Azoulay E, Le Minor L (1967) Mutation affectant la nitrate r6ductase A et d'autre enzymes bact6riennes d' oxydo-reduction, l~tude preliminaire. Annales de l'Institut Pasteur 112:24-37 Singh HN, Sonie KC (1977) Isolation and characterization of chlorate-resistant mutants of the blue green alga Nostoc rauscorum. Mutat Res 43:205-212 Singh HN, Sonie KC, Singh HR (1977) Nitrate regulation of heterocyst differentiation and nitrogen fixation in a chlorate resistant mutant of the blue green alga Nostoc muscorum. Mutat Res 42:447-452 Singh RK, Singh RN (1984) Diverse effects of formate on the assimilatory metabolism of nitrate and nitrite in Rhizobium. J Biosci 6:181-184 Snell FD, Snell CT (1949) Colorimetric methods of analysis. New York: D Van Nostrand Co, VoI. 3, pp 785-807 Solomonson LP, Vennesland B (1972) Nitrate reductase and chlorate toxicity in Chlorella vulgaris Beijerinck. P1 Physiol 50:421-424 SorgerGJ (1969) Regulation of nitrogen fixation inAzotobacter vinelandii OP; the role of nitrate reductase. J Bacteriol 98:56-61 Stouthamer AH (1967) Nitrate reduction in Aerobacter aerogenes I. Isolation and properties of mutant strains blocked in nitrate assimilation and resistant against chlorate. Arch Mikrobiol 56:68-75 Wolk CP, Wojcinch E (1973) Simple methods for plating single vegetative cells of and for replica plating filamentous blue green algae. Arch Mikrobiol 91:91-95
