NITRATE REDUCTASE ACTIVITY OF SULPHATE-REDUCING BACTERIA Desulfomicrobium sp. CrR3 AT DIFFERENT CONDITIONS OF THE CULTIVATION

97 Sulphate-reducing bacteria are unique group of microorganisms that are able to use various inorganic compounds including sulphate and nitrate as terminal electron acceptors during the obtaining energy. This exact feature is of considerable interest in studying mechanisms of nitrate reduction activity of sulphate-reducing bacteria. Nitrate reduction is widely spread among prokaryotes and described in few sulphatereducing bacteria, particularly in the genera of Desulfovibrio [1, 2], Desulfomonas [1] and others. High concentrations of nitrates inhibit sulfate reduction [2, 3]. Nitrate reductase (EC 1.6.6.2; NR) is a key enzyme of nitrate reduction with molibdopterynguaninedinucleotide that is a part of the active site [4]. It belongs to dymethylsulphoxydreductase and is found both in eukaryotic and in prokaryotic organisms and plays an important role in converting nitrogen compounds [1]. Depending on its location and function in cells of microorganisms cytoplasmic assimilatory nitrate reductase (NAS), periplasmic dissimilatory nitrate reductase (NAP) and dissimilatory membrane nitrate reductase (NAR) are described [4]. For the first time the presence of nitrate reductase in sulphate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774 was shown by Bursakov and others. [5]. Some features of reduction of sulphates into the representatives of the genus of Desulfomicrobium at the presence of nitrates are described in the works of Sholyak and Dorosh [2, 3]. The aim of this work was to investigate nitrate reductase activity of sulphate-reducing bacteria Desulfomicrobium sp. CrR3 under the different culture conditions.

Sulphate-reducing bacteria are unique group of microorganisms that are able to use various inorganic compounds including sulphate and nitrate as terminal electron acceptors during the obtaining energy. This exact feature is of considerable interest in studying mechanisms of nitrate reduction activity of sulphate-reducing bacteria.
Nitrate reduction is widely spread among prokaryotes and described in few sulphatereducing bacteria, particularly in the genera of Desulfovibrio [1,2], Desulfomonas [1] and others.
Nitrate reductase (EC 1.6.6.2; NR) is a key enzyme of nitrate reduction with molibdopterynguaninedinucleotide that is a part of the active site [4]. It belongs to dymethylsulphoxydreductase and is found both in eukaryotic and in prokaryotic organisms and plays an important role in converting nitrogen compounds [1].
Depending on its location and function in cells of microorganisms cytoplasmic assimilatory nitrate reductase (NAS), periplasmic dissimilatory nitrate reductase (NAP) and dissimilatory membrane nitrate reductase (NAR) are described [4].
For the first time the presence of nitrate reductase in sulphate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774 was shown by Bursakov and others. [5].
Some features of reduction of sulphates into the representatives of the genus of Desulfomicrobium at the presence of nitrates are described in the works of Sholyak and Dorosh [2,3].
The aim of this work was to investigate nitrate reductase activity of sulphate-reducing bacteria Desulfomicrobium sp. CrR3 under the different culture conditions.

Materials and Methods
The object of the study was sulphatereducing bacteria Desulfomicrobium sp. CrR3, resistant to the compounds of hexavalent chromium [6].
The bacteria were grown in the medium of Postgate C at 30 C in test tubes of 25 ml under anaerobic conditions. The test tubes were filled with the medium completely and closed with rubber plugs [7].
To determine nitrate reductase activity of bacteria they were cultivated in the modified medium of Postgate C of the following composition (g / l), potassium dehydrogen -0.5; calcium chloride hexahydrate -0.06; magnesium chloride hexahydrate -0,055; sodium lactate -6; yeast extract -1; sodium citrate dehydrate -0.3; pH -7.6. Nitrate was added in the form of aqueous solution of KNO 3 in the concentration of 10 mM.
Nitrate reductase activity of bacteria Desulfomicrobium sp. CrR3 was determined in cell-free extracts of bacteria grown in the modified medium of Postgate C under the argon atmosphere. For obtaining the cell-free extracts twice washed cells of Desulfomicrobium sp. CrR3 were resuspended in 0.05 M K + -phosphate buffer (pH 7.0) (10 -5 M of ethylendiamintetraacetate was added to it to bind heavy metal ions and 10 -5 M of phenylmethyl-sulphonylftoride was added for inhibition of proteases, which function at pH higher than 7.0) and destroyed by ultrasound (22 kHz) 5 times /30 s at 4 C with the help of the ultrasonic desintegrator UZDN-2T. The resulting disintegrate was centrifuged (4000 g, 30 min, 4 C), the precipitate was separated, and the supernatant was used for further research as cell-free extract [8].
Nitrate reductase activity was determined spectrophotometrically at a wavelength of 540 nm at a temperature of 30 C. The reaction mixture for the determination of nitrate reductase activity contained potassium phosphate buffer (25 mM, pH 7.3), 10 mM of K + -nitrate, 0.05 mM EDTA and freshly prepared 2 mM NADH. The reaction was started by adding cell-free extract. 5 min later the reaction was stopped by adding 58 mM solution of sulphanilamide 3 M HCl and 0.77 mM of NEDA reagent (N-(1-naphthyl)ethylendiamindehydrochloride) [9]. Protein content in each sample was determined by the Lowry method [10].
To investigate the ability of nitrate reductase NADH, NADPH and FADN as electron donors were used, being contributed to the reaction mixture at a concentration of 2 mM.
Kinetic parameters of nitrate reductase activity (K m , V max ) were determined in an incubation mixture under standard conditions described above. K m and V max were calculated by linearization data in the coordinates (1/V) and (1/S) [12]. From the resulting Lineweaver-Burke equation basic kinetic parameters of nitrate reductase of sulphate -reducing bacteria Desulfomicrobium sp. CrR3 were received.
Statistical analysis of the results was performed using Origin 6.1, and Microsoft Excel. The research results are presented as the average adjusted standard error (M ± m). Differences of averages are considered to be probable at a significance level of P  0,05 [13].

Results and Discussion
Nitrate reductase activity was studied in the process of cultivation of bacteria Desulfomicrobium sp. CrR3 in the medium of sulphates and nitrates as electron acceptors only.
While growing bacteria Desulfomicrobium sp. CrR3 in the environment of nitrates (10 mM) as a terminal electron acceptor the maximum of nitrate reductase activity was observed during the first days of cultivation. It was 12 μmol nitrite/min·mg protein. After the first day of the cultivation nitrate reductase activity decreased, which is obvious due to the decrease in the concentration of substrate in the medium (Fig. 1). The activity of nitrate reductase was found in the medium of sulphates as the sole electron acceptors. This allows us to assume that the nitrate reductase of bacteria Desulfomicrobium sp. CrR3 is constitutive enzyme (Fig. 2). Tarasova and others established the presence of the constitutive nitrate reductase in sulphatereducing bacteria D. vulgaris1388 [14].
To determine probable localization of nitrate reductase in the cells of Desulfomicrobium sp. CrR3 the enzymatic activity was determined in the culture fluid, soluble fraction and debris of cells (Fig. 3). The highest nitrate reductase activity was found in the soluble fraction (12 μM nitrite/min·mg protein). The activity in cell debris fraction was about 2 times less (5 μmol nitrite/min·mg protein), and in the supernatant nitrate reductase activity wasn't detected (Fig. 3).
The obtained results show that nitrate reductase of bacteria Desulfo microbium sp. CrR3 is localized in the cytoplasm (nitrate assimilation) or periplasmic space, that is proved by other scientists' researches [1,4,5].
The dependence of nitrate reductase activity from temperature was measured in the range of 15-45 C (Fig. 4, A).
The maximum nitrate reductase activity was detected at the temperature range of 25-35 C, which is the optimum temperature for life of the mesophilic sulphate-reducing bacteria Desulfomicrobium sp. CrR3 (Fig. 4, A).
Nitrate reductase activity of cells of Desulfomicrobium sp. CrR3 depends on the pH (Fig. 4, B). The curve of the dependence Fig. 2. Nitrate reductase activity of bacteria Desulfomicrobium sp. CrR3 at the absence and presence of nitrates in the cultural medium Here in after: * P < 0.05; ** P < 0.01 relative to control activity.
Nitrate reductase activity at the presence of NO 3in the medium served as a control activity of enzyme activity on pH is bell-shaped, and demonstrates the presence of at least one acid (H + ) and one alkaline (OH -) piece in the active site of the enzyme [15]. Maximum activity of the enzyme was observed at pH of 7-8. Thus, the maximum activity of nitrate reductase Desulfomicrobium sp. CrR3 showed at the temperature of 25-35 C and pH 7.8 (P  0,05).
The study of nitrate reductase activity of sulphate-reducing bacteria under the various culture conditions allows modeling the practical application of these bacteria in cleaning environment contaminated with nitrates and various organic substances that can be used as electron donors.
The results of research of the dependence of the activity of the enzyme on the concentration of the substrate (nitrate) are shown in Fig. 5. Basic kinetic properties of nitrate reductase reaction of bacteria Desulfomicrobium sp. CrR3, namely Michaelis-Menten constant and V max were determined by linearization of data in coordinates of (1/V) and (1/S), where V is the activity of the enzyme, and S is the concentration of substrate (nitrate).
The growth of concentration of nitrate from 1 to 4 mM does not affect the nitrate reductase activity of bacteria Desulfomicrobium sp. CrR3 (phase "plateau"). Vmax for studied enzyme is 15.7 μM nitrite / min · mg protein.
Thus, we found out that nitrate reductase activity of bacteria Desulfomicrobium sp. CrR3 tends to saturate the enzyme with substrate.
The scores of nitrate reductase of microorganisms are described [16,17], which allows us to compare the obtained data with results of other studies that is shown in the table.
The electron donor for nitrate reductase can be both NADH and NADPH. It depends on the type of organism and the type of nitrate reductase. It was found that FADN can not be a donor of electrons for nitrate reductase in bacteria Desulfomicrobium sp. CrR3, but NADH and NADPH can (Fig. 6).
At the presence of NADPH in the reaction mixture the enzyme activity was 7 μM nitrite/ min·mg protein, whereas at the presence of NADH it was 12 μmol nitrite / min · mg protein (P  0,05). Nitrate reductase of bacteria Azotobacter indicum can also accept the electrons from both NADH and NADPH [18].
The obtained results concerning the properties of nitrate reductase of bacteria Desulfomicrobium sp. CrR3 allow better understand the reduction of nitrate by sulphate-reducing bacteria, and find the optimal conditions (temperature, pH, substrate concentration, etc.) for effective nitrate reduction of these bacteria.  It was found that the nitrate reductase of the bacteria Desulfomicrobium sp. CrR3 was located in the cytoplasm (nitrate assimilation) or periplasmatic space.
The optimal conditions for nitrate reductase activity of the bacteria Desulfomicrobium sp. CrR3 are the following: the temperature of 25-35 C and the pH of 7-7.5. Lowering or raising the temperature or the pH results in decreasing activity of the enzyme.
Electron donor for nitrate reductase activity of bacteria Desul fo microbium sp. CrR3 can be both NADH and NADPH.
The value of K m for nitrate for investigated enzyme of bacteria Desulfomicrobium sp.CrR3 is 1.2 mM, V max is 15.7 mM nitrite / min · mg protein. This demonstrates the high affinity of the enzyme with the substrate.

Features of nitrate reductase activity of bacteria Desulfomicrobium sp. CrR3 compared with those of other species of microorganisms
Note: "ns" -not studied.