J. Basic Microbiol. 34 (1994) 6, 401 -404

(Barkatullah University, Bhopal - 462026, India)

Inhibition of nitrite reductase and urease by arginine and proline in the cyanobacterium Anabaena cycadeae SURENDRA SINGH and P. S. BISEN

(Received 20 October 1993lAccepted 28 January 1994)

The arginine and proline inhibition of nitrite reductase and urease activities have been studied in the cyanobacterium Anabaena cycadeae and its mutant strain lacking glutamine synthetase (GS)') activity. Arginine and proline inhibited the nitrite reductase and urease activities in the parent strain, however, they could not do so in the mutant strain. The level of arginine- and proline-dependent NH: production in the outer medium was several fold higher in the mutant strain as compared to its parent strain. These results suggest that (1) nitrite reductase and urease activities are arginine- and proline-repressible; (2) the catalytic function of GS is necessarily required for the arginine and proline inhibition of nitrite reductase and urease systems; and ( 3 ) the NH: resulting from the catabolism of arginine and proline should be metabolized via GS for repression of nitrite reductase and urease to occur.

A clear understanding of inorganic and organic nitrogen metabolism in diazotrophic cyanobacteria is necessarily required for enhancing their potential in biotechnology. Arginine and proline which accumulate as a reserve material and as an osmoregulant in cyanobacteria, respectively (SIMON 1971, SPENCE and STEWART 1986) can be taken up and metabolized as sole nitrogen source by some diazotrophic cyanobacteria (VAIASHAMPAYAN 1982, SPENCE and STEWART 1986, HERRERO and FLORES 1990, SINGH 1993 a). Recently the effect of arginine and proline on heterocyst and nitrogenase formation and nitrate reductase have been reported in diazotrophic cyanobacteria (RAWSON 1985, SPENCE and STEWART 1986, HERRERO and FLORES 1990, SINGH 1993b, SINGH and BISEN 1994) but till date nothing is known regarding the arginine and proline regulation of nitrite reductase and urease in any N,-fixing cyanobacteria.

Studies with cyanobacterial mutant strain lacking GS activity would be helpful in clarifying the regulatory role of GS, arginine, proline and/or NH,f in arginine and proline inhibition of nitrite reductase and urease. Using the glutamine auxotrophic mutant of the diazotrophic cyanobacterium Anabaena cycadeae we have studied here for the first time the arginine and proline inhibition of nitrite reductase and urease.

Materials and methods

Organism and culture conditions: Anabaena cycndeae, an endophyte of Cycus coralloid root and its glutamine auxotrophic mutant were axenically grown in BG-I10 medium (RIPPKA et al. 1979) as described by SINGH (1993a). When needed KNO,, 5 mM; NH,CI, 1 mM; urea, 1 mM; and arginine and proline, 1 mM each were added and the medium was buffered to pH 7.5 with 10 mM Hepes/NaOH. N,-fixing cultures of the mutant strain were obtained by incubating the glutamine-grown cultures in fresh N,-medium for 48 h under normal growth conditions.

I ) Abbreviations: GOGAT, glutamate synthase; GS, glutamine synthetase; Hepes, 4-(2-hydroxyethyl)- 1 -piperazineethane sulphonic acid; MTA, mixed alkyltrimethyl ammonium bromide; MOPS, 3-(N-morpholine) propane sulphonic acid

402 S. SINGH and P. S. BISEN

Nitrite reductase activity: Nitrite reductase activity was measured in cells made permeable with MTA as described by HERRERO and GL:ERRERO (1986). For this assay cell suspension containing 400-500 pg protein ml- ' was added to the reaction mixture containing the following in a final volume of 1 ml: MTA, 100 pg; MOPS'NaOH buffer, pH 7.2, 25 pmol: KNO,; 0.5 pnol; methyl viologen, 5 pmol and Na,S,O,, 20 pmol in 0.1 ml of 0.3 M NaHCO,. The reaction mixture was incubated at 30 "C for 10 min and NO; was estimated in the corresponding buffers freed of cells. Blanks in which the reaction was stopped at zero time were also prepared. Activity was expressed in terms of nmol NO; removed pg-' protein min- ' .

Urease activity: Urease activity was estimated in cells made permeable by hexadecyltrimethyl ammonium bromide in the reaction mixture at a final concentration of 100 pg ml- ' as described by SINGH (1990). Activity was expressed in terms of nmol urea hydrolysed mg-' protein h- ' .

Analytical methods: Cellular protein content was estimated by the method of BRADFORD (1976) using bovine serum albumin as the standard. Urea was determined by using diacetylmonoxime (SIGMA Technical Bulletin No. 535 1980) whereas NO; was estimated following the method of SNELL and SNELL (1949). NH; in the medium was estimated following the method of SOLORZANO (1969).

Chemicals: Arginine, proline. Hepes. MOPS. MTA, hexadecyltrimethyl ammonium bromide, diacetyl- monoxime. coomassie brillient blue (3-250 and methyl viologen were purchased from SIGMA Chemical Co. St. Louis. Missouri (USA). All other chemicals were of analytical grade available from BDH, Poole. England.

Results and discussion

Table 1 presents the data on the role of GS activity in the arginine and proline inhibition of nitrite reductase and urease activities in the parent and glutamine auxotrophic mutant strains of Aiinhnrri~i q ~ ~ i t l r t i r . i n the parent strain, nitrite reductase activity was com- paratively more in NO;-medium than in N,-medium suggesting that NO; reduction process is NO,-inducible. In contrast, urease activity was similar in urea- as well as in N,-medium, suggesting that unlike nitrite reductase, urease activity system is constitutive. A similar trend was also observed having normal GS both nitrite reductase and urease activities were severely inhibited by arginine and proline, indicating that nitrite reductase and urease activities systems are under inhibitory control of arginine and proline. In contrast, arginine and proline did not inhibit the nitrite reductase and urease activities in the mutant

Table 1 Nitrite reductase and urease activity of the parent and mutant strain of Anahaenu c.yc.udpue

Nitrogen Nitrite reductase activity" Urease activityb source -

Parent Mutant Parent Mutant

Nz 10.3 f 0.20 60.9 k 0.40 485.5 3.15 978.3 k 4.65 NO; 37.5 0.45 251.4 f 3.70 489.3 & 3.50 982.2 k 6.15 NH; 0.6 k 0.10 61.1 k 0.45 39.8 f 0.25 938.7 k 5.85 Arginine 0.7 k 0.15 61.9 f 0.50 40.6 t- 0.35 965.8 f 4.45 Proline 0.9 f 0.20 68.3 F 0.65 48.1 k 0.45 975.2 k 4.35

a nmol NO; removed pg-' protein min-

The cells of A . cjrodeuc. and its mutant strain were incubated for 12 h with Nz, NO;, NHZ, arginine and proline. Samples were taken and the nitrite reductase and urease activities were assayed as described in Materials and methods. The concentration of nitrogen sources were as follows: KNO,, 5 mM; NH,CI, 1 mM; arginine and proline, 5 mp each. The data in each column are means +SE of three measurements from a single experiment done in triplicate.

~.

nmol urea hydrolyzed mg- protein h - '

Inhibition of nitrite reductase and urease in Anabnenn 403

strain, suggesting that catalytic function of GS is necessarily required for the arginine and proline inhibition of nitrite reductase and urease activities systems in A . cycadeae. Organic and inorganic nitrogen compounds can only be utilized as sole nitrogen source by cyanobacteria provided they are metabolized to generate glutamine via GS (SINGH 1993a). According to this view, arginine and proline should be catabolized to NH,f prior to their assimilation and thus it is logical to assume that the NH: resulting from the catabolism of arginine and proline should be metabolized to glutamine via GS for the repression of nitrite reductase and urease. Such NH; generating catabolic pathway of arginine and proline also explains why arginine and proline like NH; caused inhibition of nitrite reductase and urease in parent strain.

Further, to ascertain and to obtain more conclusive evidence regarding the arginine- and proline-dependent NHd generation prior to their assimilation, NH; generation in the outer medium was estimated in both parent and its mutant strain in the presence and absence of arginine and proline (Table 2). The NH; generation was only confined to mutant strain incubated in N,, NO;, urea, arginine and proline whereas no detectable or very low NH: was generated by the identical cultures of the parent strain, suggesting that GSiGOGAT pathway is the main NH: assimilating pathway in diazotrophic cyanobacteria. Furthermore, the mutant strain quantitatively released in the external medium NHZ resulting from the catabolism ofarginine or proline individually or in combination with NO; or urea (Table 2). NH,f generation can thus be used as a tool for determining the extent at which arginine and proline catabolism and NO; reduction and urea hydrolysis occur. These results also ascertain the relative contribution of arginine + NO;, arginine urea, proline + NO; and proline + urea in NH: generation when present together and also suggest that the NH,f generation and accumulation by the mutant strain in the presence of arginine + NO;, arginine + urea, proline + NO; and proline + urea is most probably derived from the

Table 2 N;, NO;-, urea-, arginine-, proline-, arginine + NO;-, arginine + urea-, proline + NO;- and proline + urea-dependent NH: generation by the parent and mutant strains of Annbnenn cycadeae

Addition or incubation condition

NH,' in the medium (nmol mg- protein h ~ I )

Parent Mutant

N2 NO; Urea Arginine Proline Arginine + NO; Proline + NO; Arginine + urea Proline + urea

ND ND ND 1.7 + 0.15 1.3 + 0.10 2.1 + 0.20 1.9 + 0.25 3.9 + 0.35 2.7 + 0.30

9.5 + 0.40 197.6 + 5.25

2044.6 + 9.75 210.7 + 6.35 203.9 + 6.85

383.5 + 6.40 2239.3 + 9.55 2215.7 + 9.35

397.8 + 5.45

The cells of A. cycadeae and its mutant strain were incubated for 1 h with N,, NO;, urea, arginine, arginine + NO;, arginine + urea, proline, proline + NO; and proline + urea, and NH,' in the outer medium was estimated as detailed in Materials and methods in aliquots of the cell suspension, after removal of the cells by rapid centrifugation. Values were corrected for the amount of NHf present at zero time. The concentration of N-sources were as follows: KN02, 5 inM, urea, 1 mM, arginine and proline, 5 mM each. The data in each column are means f SE of three measurements from a single experiment done in triplicate. ND, not detectable

404 S. SINGH and P. S. BISEN

catabolism of arginine and proline and also from the reduction and hydrolysis of NO; and urea, respectively. This NH,f generation from arginine and proline might account for the capability of the parent strain to utilize these amino acids as sole nitrogen source (data not shown). These results also affirm the contention of the earlier report (SINGH 1993a) that organic and inorganic nitrogenous compounds can not be utilized as sole nitrogen source unless they are metabolized to generate glutamine via GS.

Thus, overall it is concluded that (i) nitrite reductase activity in A . cycadeae is NO;-inducible as in Nostoc ANTH (SINGH 1992) whereas urease activity is constitutive as in Anabaena doliolum (SINGH 1990); (ii) nitrite reductase and urease activities systems are arginine- and proline-repressible; (iii) catalytic function of GS is necessarily required for the arginine and proline inhibition of nitrite reductase and urease; and (iv) the NH: resulting from the catabolism of arginine and proline is not the actual repressor signal but its meta- bolism via GS is being required for the repression of nitrite reductase and urease to occur.

Acknowledgements

We thank DSTP, CSIR, New Delhi (India) for financial assistance and Head, Department of Bio- chemistry, North-Eastern Hill University, Shillong (India) for providing necessary laboratory facilities.

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Mailing address: Prof. P. S. BISEN, Department of Microbiology, Barkatullah University, Bhopal - 462026, India