Methylamine Regulation of Heterocyst and Nitrogenase in a Methylamine Resistant Blue-green Alga Anabaena doliolum

Methylamine Regulation of Heterocyst and Nitrogenase in a Methylamine Resistant Blue-green Alga Anabaena doHolum

J. PAPA RAo and H. N. SINGH School of Life Sciences, University of Hyderabad, Hyderabad-SOO 134, India Received August 28,1982 . Accepted November 8,1982

Summary The ammonium analog «methylamine" inhibits the growth, nitrogenase activity, photosynthetic ATP pool and glutamine synthetase activity of the parental Anabaena doliolum. MAcaused inhibition of growth, nitrogenase activity and photosynthetic ATP pool, all become resistant to MA inhibition in the methylamine resistant mutant of A. doliolum. The N2 growth and nitrogenase activity of the MAR strain is approximately half of that shown by the parent strain. The growing heterocystous N 2-fixing cultures of the MAR strain in N2 medium become nonheterocystous and nonfixer during growth in MA containing N2 medium, thus suggestin~ metabolic utilization of MA like a fixed nitrogen source as a mechanism of resistance of MA strain against inhibitory action of MA on growth, acetylene reducing activity and photosynthetic ATP pool. The MA grown culture of MAR strain shows higher ratio of glutamine synthetase biosynthetic to transferase activity than its MA free N2 grown cultures, apparently suggesting the involvement of GS in the metabolism of the ammonium analog like a fixed nitrogen source.

Key words: Anabaena doliolum, blue-green alga, glutamine synthetase, heterocyst, methylamine, nitrogenase.

Introduction Methylamine is an ammonium analog and is found to strongly inhibit in vitro photophosphorylation in chloroplasts (Good, 1960; Ortega et ai., 1977) and in bluegreen algae Nostoc muscorum and Plectonema boryanum (Padan and Schuldiner, 1978). It is found to inhibit photophosphorylation by causing collapse of photosynthetically generated chemical gradient (~pH) across their thylakoid membranes (Padan and Schuldiner, 1978). The fungus Aspergillus nidulans fails to grow in the fixed inorganic nitrogen media containing methylamine and its mutants resistant to methylamine are found to have become derepressed for ammonium repressible enzyme activities (Arts and Cove, 1969). The ammonium analog functions as an energy source in methylotrophs (Smith and Hoare, 1977) and as a nitrogen source in Pseudomonas strain MA (Bellion and Samkin, 1978) and is unable to influence either growth or N 2-fixation of Azotobacter vinelandii (Gordon and Brill, 1972). Methylamine in vivo inhibition of Abbreviations: MA: Methylamine; MAR: Methylamine resistant; GS: Glutamine synthetase (EC 6.3.1.2); CFU: Colony forming units; aD: Optical density.

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photophosphorylation or growth or nitrogen metabolism has not been investigated in any bluegreen alga except for the report of Singh et a1. (1979) and Singh and Singh (1981) which suggested utilization of methylamine as a nitrogen source by the methylamine resistant mutants of Nastac muscarum (Anabaena 7120). Anabaena dalialum is an obligate photoautotrophic heterocystous aerobic N2-fixer (Singh and Singh, 1964; Singh and Srivastava, 1968) showing strong sensitivity to growth inhibition by methylamine. We have isolated a large number of methylamine resistant mutants of this blue-green alga and have made a comparative study of the effect of methylamine on (a) photosynthetic ATP pool, (b) acetylene reducing activity, (c) heterocyst differentiation and frequency, and (d) glutamine synthetase (GS) biosynthetic and -y-glutamyl transferase activities in the parental strain and in one of its methylamine resistant strains. We report here results which suggest methylamine (a) to be inhibitory to photophosphorylation and nitrogenase activity in parental but not in methylamine resistant strain and (b) to be metabolized like a fixed nitrogen source in the methylamine resistant strain apparently via glutamine synthetase pathway.

Materials and Methods Axenic clonal culture of heterocystous blue-green alga Anabaena doliolum was grown routinely and maintained in modified Chu No. 10 medium (Gerloff et al., 1950) lacking fixed source of nitrogen (N z medium) or containing 2 mM NfuCI (NH4 + medium) as the only nitrogen source at a temperature of 28°C ± 2 under continuous light intensity of 2,500 Ix. Growth was measured as optical density changes at 663 nm in a spectrocolorimeter as described previously (Singh and Singh, 1978). Methylamine was used as methylamine hydrochloride and biological effects of its increasing concentrations were measured in terms of in vivo quantitative changes in growth, heterocyst frequency, acetylene reducing activity, ATP pool and glutamine synthetase biosynthetic and transferase activities.

Nitrogenase activity measurement The aerobic test samples were incubated under growth conditions for 1 h under the aerobic atmosphere of gas phase which contained 10% (v/v) acetylene in 7.0ml capacity serum bottles and then assayed for ethylene according to the method of Stewart and Lex (1970). The incubated samples were kept continuously shaken in a well illuminated (2500 Ix) orbital incubator operating at 30°C and rotating at a speed of about 120 rpm. The nitrogenase activity was expressed as n moles ethylene formed per I'g Chi a per h.

Isolation ofMethylamine resistant strain The method followed was that of Singh and Singh (1978). Since the parent alga was unable to grow with 1 mM MA in N 2 1iquid or on N2 solid nutrient medium, the N2 nutrient plates containing 2 mM MA were seeded with approximately 106 colony forming units of Nz grown parental strain and incubated in the growth chamber for two weeks. The growing colonies were counted and one of such colonies was raised into stock culture by inoculating it into a fresh Nz medium containing 2 mM MA. The stability of MAR phenotype was tested by growing it first in MA free N z medium for twelve successive subcultures and then in 2 mM MA containing N z medium.

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Measurement ofA TP Pool Both the parent and MAR strains were always grown in photoautotrophic growth conditions where photophosphorylation is the only major source for ATP production and, therefore, MA-induced decrease in the blue-green algal ATP pool size could be presumed to be due to the inhibitory action of MA on its photophosphorylation. ATP was extracted from variously treated samples using the method of Larsson and Olsson (1979). Aliquots of algal suspension were injected rapidly into the ice cold 1.5 ml of 3 M perchloric acid and 1 ml of 0.1 M EDTA. The samples were mixed vigorously and allowed to extract at O°C for 20 min. The extracts were neutralized to pH 7.75 and were then diluted to a final volume of 10 ml with Tris-HCI buffer, pH 7.75 at 0 °C. All extracts were stored at 0 °C for assay on the same day. The assay for ATP was performed by luciferin-Iuciferase technique as detailed by Bottomley and Stewart (1976).

Glutamine synthetase (EC 6.3.1. 2) Cell-free extracts were prepared from variously treated cultures at 4°C after harvesting the algal samples by centrifugation, washing twice in 50 mM Tris-HCI buffer pH 7.5 (buffer A) and then resuspending them in buffer B (buffer A supplemented with 5 mM MgCh, 10 mM Sodium glutamate,S mM 2-mercaptoethanol and 1 mM EDTA). These suspensions were then disrupted in a MSE MK-2 Sonicator at 4°C at maximum amplitude. An interval of 30 s was given after every 1 min sonication. Debris was removed by centrifugation at 36,000 g for 30 min. The supernatant thus obtained was used for Mg2+ dependent biosynthetic and Mn 2+ dependent "1glutamyl transferase assay of glutamine synthetase according to the procedure of Sampaio et al. (1979). The spectrophotometric assays of biosynthetic and transferase activities were carried out in a Gilford-250 spectrophotometer at 30°C at 340 nm and 540 nm respectively.

Protein estimation Method of Lowry et al. (1951) was used, with bovine serum albumin as the standard.

Estimation ofchlorophyll a Chlorophyll a was extracted in 100 % methanol and its amount in the extract was determined by the method of Mackinney (1941). Chemicals of the growth medium were of BDH grade and rest including methylamine hydrochloride were of Sigma Grade.

Results

The parent A. dalialurn grew well in the N2 medium. However, it failed to grow in N2 medium containing 1 mM MA and its mutation to MAR phenotype occurred with a frequency of approximately 1.5 x 10-5 • One of the MAR clones on further testing by twelve successive subcultures in the N2 medium lacking MA, was found to retain the MAR phenotype, thus suggesting stable mutational origin of this phenotype in the blue-green alga. We used this MAR clonal culture and the parental clonal culture in the present investigation to study the biological basis of growth inhibition and resistance toMA. As expected the parental culture did not grow in the presence of MA in the N2 growth medium while its MAR mutant strain grew well under similar growth conditions {Fig.i}. The growth of MAR strain with and without MA remained almost

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similar. Its other most significant difference from the parental strain is seen in that it recorded progressively lower growth rate than the parental strain while assimilating N2 as the sole nitrogen source. In fact the parent recorded an OD value in its 8 d old N2 growing cultures which is nearly double of that recorded by the MAR strain under the similar growth conditions (Fig. 1). These results clearly suggest that mutation to MAR phenotype is simultaneously accompanied by a significant reduction in the mutant growth at the expense of N 2 • Microscopic examination of the N2 growing cultures of the parent and mutant strains revealed them to contain heterocystous filaments with a heterocyst frequency of nearly 5 % and 4 %, respectively. However, the N2 + MA growing 8 d old cultures

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of the MAR strain showed filaments completely devoid of heterocysts. Clearly MA, while not inhibiting mutant growth, does appear to completely inhibit its heterocyst differentiation process. Next we examined the short term effect of MA on acetylene reducing activitiy of the parent and the MAR strain grown with N2 as a nitrogen source and the results obtained are shown in Fig. 2. The acetylene reducing activity of the control parent culture showed slight increase with the passage of its growth incubation upto 16 h in N2 medium, thereafter it got stabilized. But when its culture was similarly incubated in N2 + MA medium, it showed rapid decrease in its acetylene reducing activity which approached almost zero value by the end of incubation period of 24 h. Apparently nitrogenase activity of the parental strain is extremely sensitive to the inhibitory action of MA. At any given time of incubation acetylene reducing activity of the N2 grown control culture of MAR strain is almost half of that shown by similar control culture of the parental strain. This observation is highly significant and relevant to the observed growth difference of the two strains in N2 medium (Fig. 1). The N2 + MA incubated culture of MAR strain under growth conditions showed much slower decrease in its acetylene reducing activity in comparison to the parental strain similarly treated and incubated, and the observed MA mediated decrease of its acetylene reducing activity at the end of 24 h of incubation period is less than half of

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Fig. 2: Time course pattern of the acetylene reducing activity of the parent and MAR strain cultures in N2 medium containing or lacking 2 mM methylamine hydrochloride. Methylamine hydrochloride was added to the culture at zero time. Parent (0); MAR strain (e); without methylamine hydrochloride (--); with methylamine hydrochloride (- -).

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its control value, whereas the parental culture by this time of incubation reaches a value of acetylene reduction approaching zero (Fig. 2). Apparently nitrogenase activity of the M/tR strain appears to suffer MA-mediated decrease at a considerably much slower rate than that of the parental strain. The MA-caused fast decrease in nitrogenase activity of the parental strain and MA-caused very slow decrease in nitrogenase activity of the M/tR strain coupled with MA inhibition of its heterocyst differentiation but not of its growth appear to have significant bearing on the differential growth response of the two strains to MA in N2 medium.

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We also examined and compared the short-term effect of MA on photosynthetic pool of ATP in the two strains (Fig. 3). ATP pool of parent culture did not show much fluctuation with growth incubation period in N2 medium. This was true also for M/tR strain. However, M/tR strain always showed lower level of ATP pool than the parental strain. As shown in Fig. 3, the addition of MA to N2 growing cultures of the parent caused increasing reduction in its ATP pool level and 72 h or 96 h old such cultures appear to have an A TP pool level nearly three times less of the similar conZ. Pjlanzenphysiol. Ed. 109. S. 69-79. 1983.

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trol cultures. In comparison, MA inhibition of ATP pool level is low in MAR strain. Evidently mutation to MAR phenotype appears to be concomitantly accompanied by acquisition of resistance to MA inhibition of ATP pool as well. However, the invariably lower level of A TP pool shown by the MAR strain in comparison to its parental strain might be another causative factor of its observed lower growth in N2 medium. The differential growth response of the two strains might as well result from the differential response of their ATP pool level to MA. Since parental strain is sensitive to MA inhibition of growth and since MA is an analog of NJ-4 +, we raised nonheterocystous cultures of the MA sensitive parental

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strain by growing it with 2 mM N~ + medium. The requirement for the nonheterocystous filaments of the parent arose for comparing the kinetics of differentiation of its heterocysts and acetylene reducing activity with that of MA grown nonheterocystous mutant strain. In both the strains, heterocyst and nitrogenase activity appeared after a lag period of about 12 h after which the level of both the processes continued rising with the incubation period in N2 growth medium. Level of both the processes got stabilized by 48 h of incubation period. Heterocyst frequency of the MAR strain was slightly lower but its acetylene reducing activity was almost half of that shown by the parental strain (Fig. 4). In both the strains heterocyst differentiation was paralleled by the appearance of acetylene reducing activity. Both showed similar duration of lag period in differentiation of heterocyst and nitrogenase activity and both also showed similarity in the rising pattern of the two processes. Thus mutation to MAR phenotype does not seem to have influenced the time course pattern of the appearance of heterocyst and nitrogenase activity. However, it does seem to have adversely influenced mainly the level of nitrogenase activity. The level of glutamine synthetase biosynthetic activity in N2 grown culture of the parent and its MAR strain culture of the mutant was almost same. This was also true for the transferase activity of the two strains (Table 1). Thus one can infer from these Table 1: Biosynthetic and 'Y-glutamyl transferase activities of glutamine synthetase in extracts from N2 and N2+methylamine (N2+MA) grown 48 h old parental and MAR strains. The source of inoculum was 6 day old Nz grown cultures.

Biosynthetic activity 'Y-glutamyl transferase activity Biosynthetic ratio

Parental strain N 2 grown Nz+MAgrown

MAR strain N 2 grown Nz+MAgrown

20.0 210.0

19.5 215.0

0.095

9.5 103.0 0.092

0.090

30.5 125.0 0.244

Activities are expressed as n moles product formed min- I mg protein-I.

results that both the mutant strain (MAR strain) and the parent strain possess similar GS biosynthetic and transferase activities while fixing and assimilating N2. GS biosynthetic and transferase activities of the MA treated parental culture were found to be reduced to approximately 50 % of the control cultures while under similar growth conditions, the MAR strain showed significantly increased level of its GS biosynthetic activity and correspondingly reduced level of its GS transferase activity. In other words, the ratio of GS biosynthetic to transferase activities underwent significant alteration following the growth of MAR strain in MA containing growth medium (Table 1). In contrast extracts of N2 grown and N2 + MA grown parent and of N2 grown mutant strain gave almost similar ratio of GS biosynthetic to transferase activity.

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Discussion The MA is observed to inhibit growth, phototrophic ATP pool, nitrogenase activity of GS activity in the parental strain. All these observed inhibitory actions of MA are found to be absent in the MAR strain. Since MA is a strong inhibitor of photophosphorylation in oxygenic photosynthetic organisms including blue-green algae (Padan and Schuldiner, 1978) and since ATP is a definite requirement in nitrogenase reaction and in glutamine synthetase mediated synthesis of glutamine from glutamate and ammonia, the only biosynthetically significant primary pathway of ammonium assimilation in heterocystous N2 fixing blue green algae (Rowell et ai., 1977), it is possible that all the observed inhibitory actions of MA result primarily from its inhibitory action on photophosphorylation in parental strain. This view is quite in agreement with observed strong inhibitory action of MA on the phototrophic pool of ATP in the parental strain. The absence of the various observed inhibitory actions of MA in MAR strain could be either due to the mutational acquisition of resistance against photophosphorylation inhibitory action of the ammonium analog or due to the mutational acquisition of enzymatic activity to utilize MA like a fixed nitrogen source. The fact that MA grown cultures of MAR strain lack both heterocyst and nitrogenase activity, both of which appear on subsequent transfer of such cultures to a fresh N2 medium, it can be safely inferred that MAR strain is capable of metabolizing MA to a product(s) inhibitory to both heterocyst formation and nitrogenase synthesis. As MA causes complete inhibition of both heterocyst formation and nitrogenase synthesis in MAR strain in N2 medium, in which it is found to grow a little better than its N2 control heterocystous and N2-fixing culture, the product(s) of MA metabolism while remaining inhibitory to heterocyst differentiation and nitrogenase synthesis, is apparently found slightly growth promotory thus suggesting its utilization like a fixed nitrogen source in MAR strain. The metabolic product of GS activity, but not ammonia by itself is suggested to be the actual coreppressor of heterocyst differentiation and nitrogenase synthesis (Stewart and Rowell, 1975; Rowell et ai., 1977). Since MA appears to influence the process of heterocyst and nitrogenase synthesis in MAR strain in a way ammonium does in this as well as in parental strain, the mutant strain may be producing a GS analogous to the GS of the parental strain but with greatly enhanced affinity for methylamine as a substrate. This view finds strong support in the observation that MA grown cultures of MAR strain are found to have significantly high ratio of GS biosynthetic to transferase activities in comparison to its MA free N2 grown cultures of the parental strain. The non-photosynthetic bacterium Pseudomonas MA strain capable of using MA as a nitrogen source has been shown to do so by synthesizing a methylamine inducible enzyme similar in activity to GS but having a higher specificity for MA (Kung and Wagner, 1969). Z. Pjlanzenphysiol. Ed. 109. S. 69-79. 1983.

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J. PAPA RAo and H. N. SINGH Acknowledgements

We thank the Dean, School of Life Sciences for the facilities, University of Hyderabad for providing M. Phil Fellowship to J. P. Rao and also thank Mr. P. Omprakash for typing the manuscript.

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SINGH, R. K. and H. N. SINGH: Isolation and preliminary characterization of mutants of the cyanobacterium Nostoc muscorum resistant to growth inhibition by methylamine. Mol. Gen. Genet. 184, 334-336 (1981). SINGH, R. N. and H. N. SINGH: Ultra-violet induced mutants of blue-green alga II. Anabaena doliolum Bharadwaja. Arch. Microbiol. 48,118-121 (1964). SMITH, A. J. and D. S. HOARE: Specialist phototrophs, lithotrophs and methylotrophs: a unit among a diversity of prokaryotes. Bacteriol. Rev. 41, 419--448 (1977). STEWART, W. D. P. and M. LEX: Nitrogenase activity in the blue-green alga Plectonema boryanum strain 594. Arch. Microbiol. 73,250-260 (1970). STEWART, W. D. P. and P. ROWELL: Effects of L-methionine-DL-sulfoximine on the assimilation of newly fixed NH 3 , acetylene reduction and heterocyst production in Anabaena cylindrica. Biochem. Biophys. Res. Commun. 65,846--856 (1975).

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