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Effect of Anilazine on Growth, Respiration and Enzyme Activity of Escherichia coli
Zbl. Bakt. II. Abt. 136 (1981),505-508
[Dept. of Microbiology and Dept. of Plant Pathology, Haryana Agricultural University, Hissar, India]
Effect of Anilazine on Growth, Respiration and Enzyme Activity of Escherichia coli F. C. GARG, P. TAURO, M. M. MrSHRA and R. K. GROVER With one Figure
Summary The effect of anilazine on growth, respiration and enzyme activity of E. coli has been studied. Anilazine delays the growth of E. coli by prolonging the lag period. It inhibits glucose oxidation by 60 % and succinate oxidation by 100 %. It also inhibits in vitro succinic dehydrogenase activity. It seems that the inhibition of E. coli by anilazine is because of inhibition of respiratory enzymes.
Zusammenfassung Anilazin unterdriickte das Wachstum von Escherichia coli durch Verlangerung der lag-Phase. Es inhibierte die Glukose-Oxydation zu 60 % und die Oxydation der Bernsteinsaure 100%ig. Desgleichen erfolgte eine Hemmung der Bernsteinsauredehydrogenase-Aktivitat. Die Wachstumshemmung von E. coli dureh Anilazin ist daher auf eine Hemmung der Atmung zuruckzufuhren,
The fungicides are primarily used for the control of pathogenic fungi, but other soil micro-organisms cannot escape from contact with these chemicals. One of such soil micro-organism, Rhizobium, is known to be sensitive to several fungicides (SUD and GUPTA 1972, ODEYAMI and ALEXANDER 1977). Benzimidazole is mutagenic to Salmonella (SEILER 1972). Thus these fungicides are required to be screened for their effect on non-target organisms to devise protective measures. Anilazine is a protective fungicide and has been found to inhibit the gr-owth of Rhizobium sp. (GARG et al.). We have examined the effect of anilazine on the growth, respiration and enzyme activity of a non-target micro-organism, i.e., E. coli. Though this organism is not exposed to this chemical but it was selected because it has been extensively used for biochemical and genetic studies and is thus a useful organism for understanding the mechanism of action of fungicides in bacteria.
Materials and Methods Cultures Escherichia coli K-10 used in this study was from the culture collection of the Department of Microbiology, H.A.U., Hissar. It was maintained on nutrient agar slant by regular transfer. A 24 h-old slant culture was used to inoculate the fresh liquid medium for various studies.
Fungicides Anilazine (2,4-Dich10ro-5-(0-chloro-anilino)-S-triazine) was technical grade 92 % W.P. from Nippon Soda Co. Ltd., Japan.
506
F. C.
GARG
et al,
Growth measurement For growth studies 50 ml of mineral salt base medium-56 (MS) (MONOD, COHEN-BAZTltE and COHN 1951) containing 1 % glucose were inoculated with 2 X 108 cells in the presence and absence of fungicide and incubated on rotary shaker at 30°C. Samples of 3 ml were withdrawn at 2 h interval up to 48 h and growth measured by determining optical density at 560 nm using Spectronic-20 spectrophotometer (Bausch & Lomb, U.S.A.).
Oxidation studies The capacity of intact cells to oxidise either glucose or succinate in the presence or absence of the fungicide was measured by polarographie method using Biologieal Oxygen Monitor (Yellow Spring Co., U.S.A.). Logarithmically growing cells were harvested by centrifugation at 10,000 rpm for 10 min., washed free of medium components by repeated washings (3 X) with 0.01 M phosphate buffer (pH 7) and finally suspended in the same buffer. Three ml reaction mixture contained phosphate buffer, 1 X 10 7 cells, 20 {'moles of either glucose or succinate, anilazine (50 ('g/ml) or no a.nilazine. The temperature was maintained at 30°C. The results are expressed as {'I of O 2 consumed per hour and mg dry weight of cells. Endogenous oxygen uptake was reeorded separately and deducted.
Dry weight The dry weight of cells was determined by drying 3 ml of cell suspension at 90°C to constant weight.
Succinic dehydrogenase The activity of succinic dehydrogenase in cell free extract in the presence or absence of anilazine was determined as described by KING (1967). Cells were grown in MS medium for 24 h in the absence of fungicide and collected by centrifugation at 10,000 rpm for 10 min at 4°C. The cells were washed with phosphate buffer (0.01 M, pH 7.8) by repeated centrifugation. The cell pellet thus obtained was ground with acid washed river sand (Bacterial pellet: Sand: Water = 10: 10: 20) for 30 min. at 4°C. The mixture was first centrifuged at 2,000 rpm for 20 min. and then the resultant supernatant was centrifuged at 12,000 rpm for 20 min. at 4 °C. The clear supernatant was used to determine the enzyme activity.
p-galactosidase The effect of anilazine on p-galactosidase activity was determined by using tolunized cells grown in the presence of lactose by the method described by LEDERBERG (1950).
Results and Discussion Growth studies: To determine the effect of anilazine on the growth of E. coli 50 ml of MS medium with and without fungicide was inoculated with 2 X lOS actively growing cells and the growth determined as described earlier. Anilazine (50 f'gfml) delayed the growth of E. coli by prolonging the lag period. However, after a long lag, the growth was resumed but there was no effect on either the growth rate or final cell yield of E. coli. Oxidation of glucose and succinate: Most of the fungicides inhibit the growth of fungi either by inhibiting respiration or macromolecular synthesis (M.A.THRE 1970, DEKKER 1976). Thus the effect of anilazine on the oxidation of glucose and succinate by resting cells of E. coli was determined. Glucose oxidation was inhibited by 60 % in the presence of anilazine (Table 1) whereas succinate oxidation was completely inhibited in the presence of 50 f'g of anilazine per ml. Effect of anilazine on enzyme Activity: To determine whether inhibition of respiration was a consequence of specific inhibition of enzymes involved in respiration or general inhibition of enzymes, the effect of the fungicide on the in vitro activity of two enzymes, namely succinic dehydrogenase, a key enzyme involved in respiration, and
Effect of An ila zine
507
(I.D. f60 nm
I
; 3.0
I
· ~zt~-~ J /l=+-t!I : .
0. 5 !
0'
I
f-j~---t-----'- ----j 0- - 0
Con/rol
x- x Amluzine
Fi g_ 1 0.03
0
o
8
15 21, 32 'to 1,8
l ime f h ) T able 1. Effect of a n ila zine on oxidat ion and enzyme act.ivity
a». coli
Effect on
QO/) Glucose Su ccinate Succin ic dehydrogena se act. ivit.y-) (units/m l) ,a-Galactosidase ac t ivit ys) (units)
W it hout Ani la zine
W ith Anilazine
360 349
147 Zero Zero
23. 7
460
(50 J1gJm l )
460
1) J11 O 2 consumed h - 1 • mg - 1 d ry weigh t of cells. 2) The enzym e un it is defined as t he J1moles of su ccinat e oxidi zed (K ING 1967). 3) The enzyme unit is defined as the amount of en zyme t hat will liberate o-n it ro phen ol at tho r ate of 10- 8 moles per min. (LEUERBERG 1950).
p -gal actosid ase, an inducible enzy me not inv olv ed in resp ira tion, wa s examined. The incub a t ion of cell fr ee reacti on mi xtur e with anilazine completely inhibit ed succ in ic dehydrogenase acti vit y while p-galactosida se activity of tolunized E. coli cells wa s not affec ted at all (Table 1), suggesting t hat anilaz ine specifically inhib its enzy mes invo lved in respi ra t ion. Most fungicides a re eit her resp irat or y po isons or inhibit ma cromolecul ar synthes is in fungi . Carboxin is kn own t o inhibit respiration in fungi by inhibit ing succiniccy t ochrome C-red ucta se specifica lly. In t his study the a nila zine inhibited oxidation of glu cose by 60 % and succina te oxidation by 100 %. It also inhibited succinic d ehydrogen ase ac tiv it y in vitro complet ely but did not affect the p-gala ct osidase activity. It seems t hat t his fungicid e is a spec ific inhibitor of enz ymes involved in r espi ra ti on. We ha ve rep or ted earl ier t hat anila zine inhibits growth of Rhizobium. sp. (GARG et al.) by inhibiting respira tion. In Rh izobium sp. also, t his fungi cide inhibit s glucos e and succ inate by 65 % a nd 95 %, respecti vely . Thus t he ac tion of anilazine see ms to be similar both in E . coli a nd Rh izobiu m sp . References DE KKER, J . : Acquired resistance to fung icides. Ann. Rev. Phyt opath. 14 (197 6),405 -428. GARG, F. C., TAURO, P., MISHRA, M. M., and GROVER, R. K.: Effect of fungi cides on growth and on DNA, R N A an d protein an a bolism of a Rh izobium sp. (Com m un icated ).
508
F. C. GARG et al., Effect of Anilazine
KING, T. E.: Preparation of succinic dehydrogenase and reconstitution of succinate oxidase. In: Methods in Enzymology (EsTABRooK, R. W., and PULLMAN, M. E., eds.). Vol. X, pp. 322-331Academic Press, New York 1967. LEDERBERG, .J.: The poD-galactosidase of E. coli K-10. J. Bact. 60 (1951), 381-392. MATHRE, D. E.: Mode of action of oxathiin systemic fungicides. 1. Effect of carboxin and oxycarboxin on the general metabolism of several basidiomycetes. Phytopathology 60 (1970), 671-676. MONOD, J., COHEN-BAZIRE, G., and COHN, M.: Sur is biosynthese de la p-galactosidase (lactase) che Escherichia coli 1a specific de l'induction. Biochim, Biophys. Acta 7 (1951), 585-599. ODEYAMI, 0., and ALEXANDER, M.: Use of fungicide resistant rhizobia for legume inoculation. Soil. BioI. Biochem. 9 (1977), 247-251. SEILER, J. P.: The mutagenicity of benzimidazole and benzimidazole derivatives. 1. Forward and reverse mutations caused by benzimidazole and one of its derivatives. Mut. Res. 15 (1972), 273-276. SUD, R. K., and GUPTA, K. G.: On the sensitivity of isolates of Rhizobium spp. and Azotobacter chroococcum to TMTD and its degradation product Na-DDC. Arch. Microbiol. 85 (1972),19-22. ULRICH, J. T., and MATHRE, D. E.: Mode of action of oxathiin systemic fungicides. V. Effect on electron transport system of Ustilago maydis and Saccharomyces cereoisiae. J. Bact. 110 (1972), 628-632. Authors' addresses: Dr. F. C. GARG, Dr. M. M. MISHRA and Prof. P. TAuRo, Department of Microbiology, and Prof. R. K. GROVER, Department of Plant Pathology, Haryana Agricultural University, Hissar (India).
[Dept. of Microbiology and Dept. of Plant Pathology, Haryana Agricultural University, Hissar, India]
Effect of Anilazine on Growth, Respiration and Enzyme Activity of Escherichia coli F. C. GARG, P. TAURO, M. M. MrSHRA and R. K. GROVER With one Figure
Summary The effect of anilazine on growth, respiration and enzyme activity of E. coli has been studied. Anilazine delays the growth of E. coli by prolonging the lag period. It inhibits glucose oxidation by 60 % and succinate oxidation by 100 %. It also inhibits in vitro succinic dehydrogenase activity. It seems that the inhibition of E. coli by anilazine is because of inhibition of respiratory enzymes.
Zusammenfassung Anilazin unterdriickte das Wachstum von Escherichia coli durch Verlangerung der lag-Phase. Es inhibierte die Glukose-Oxydation zu 60 % und die Oxydation der Bernsteinsaure 100%ig. Desgleichen erfolgte eine Hemmung der Bernsteinsauredehydrogenase-Aktivitat. Die Wachstumshemmung von E. coli dureh Anilazin ist daher auf eine Hemmung der Atmung zuruckzufuhren,
The fungicides are primarily used for the control of pathogenic fungi, but other soil micro-organisms cannot escape from contact with these chemicals. One of such soil micro-organism, Rhizobium, is known to be sensitive to several fungicides (SUD and GUPTA 1972, ODEYAMI and ALEXANDER 1977). Benzimidazole is mutagenic to Salmonella (SEILER 1972). Thus these fungicides are required to be screened for their effect on non-target organisms to devise protective measures. Anilazine is a protective fungicide and has been found to inhibit the gr-owth of Rhizobium sp. (GARG et al.). We have examined the effect of anilazine on the growth, respiration and enzyme activity of a non-target micro-organism, i.e., E. coli. Though this organism is not exposed to this chemical but it was selected because it has been extensively used for biochemical and genetic studies and is thus a useful organism for understanding the mechanism of action of fungicides in bacteria.
Materials and Methods Cultures Escherichia coli K-10 used in this study was from the culture collection of the Department of Microbiology, H.A.U., Hissar. It was maintained on nutrient agar slant by regular transfer. A 24 h-old slant culture was used to inoculate the fresh liquid medium for various studies.
Fungicides Anilazine (2,4-Dich10ro-5-(0-chloro-anilino)-S-triazine) was technical grade 92 % W.P. from Nippon Soda Co. Ltd., Japan.
506
F. C.
GARG
et al,
Growth measurement For growth studies 50 ml of mineral salt base medium-56 (MS) (MONOD, COHEN-BAZTltE and COHN 1951) containing 1 % glucose were inoculated with 2 X 108 cells in the presence and absence of fungicide and incubated on rotary shaker at 30°C. Samples of 3 ml were withdrawn at 2 h interval up to 48 h and growth measured by determining optical density at 560 nm using Spectronic-20 spectrophotometer (Bausch & Lomb, U.S.A.).
Oxidation studies The capacity of intact cells to oxidise either glucose or succinate in the presence or absence of the fungicide was measured by polarographie method using Biologieal Oxygen Monitor (Yellow Spring Co., U.S.A.). Logarithmically growing cells were harvested by centrifugation at 10,000 rpm for 10 min., washed free of medium components by repeated washings (3 X) with 0.01 M phosphate buffer (pH 7) and finally suspended in the same buffer. Three ml reaction mixture contained phosphate buffer, 1 X 10 7 cells, 20 {'moles of either glucose or succinate, anilazine (50 ('g/ml) or no a.nilazine. The temperature was maintained at 30°C. The results are expressed as {'I of O 2 consumed per hour and mg dry weight of cells. Endogenous oxygen uptake was reeorded separately and deducted.
Dry weight The dry weight of cells was determined by drying 3 ml of cell suspension at 90°C to constant weight.
Succinic dehydrogenase The activity of succinic dehydrogenase in cell free extract in the presence or absence of anilazine was determined as described by KING (1967). Cells were grown in MS medium for 24 h in the absence of fungicide and collected by centrifugation at 10,000 rpm for 10 min at 4°C. The cells were washed with phosphate buffer (0.01 M, pH 7.8) by repeated centrifugation. The cell pellet thus obtained was ground with acid washed river sand (Bacterial pellet: Sand: Water = 10: 10: 20) for 30 min. at 4°C. The mixture was first centrifuged at 2,000 rpm for 20 min. and then the resultant supernatant was centrifuged at 12,000 rpm for 20 min. at 4 °C. The clear supernatant was used to determine the enzyme activity.
p-galactosidase The effect of anilazine on p-galactosidase activity was determined by using tolunized cells grown in the presence of lactose by the method described by LEDERBERG (1950).
Results and Discussion Growth studies: To determine the effect of anilazine on the growth of E. coli 50 ml of MS medium with and without fungicide was inoculated with 2 X lOS actively growing cells and the growth determined as described earlier. Anilazine (50 f'gfml) delayed the growth of E. coli by prolonging the lag period. However, after a long lag, the growth was resumed but there was no effect on either the growth rate or final cell yield of E. coli. Oxidation of glucose and succinate: Most of the fungicides inhibit the growth of fungi either by inhibiting respiration or macromolecular synthesis (M.A.THRE 1970, DEKKER 1976). Thus the effect of anilazine on the oxidation of glucose and succinate by resting cells of E. coli was determined. Glucose oxidation was inhibited by 60 % in the presence of anilazine (Table 1) whereas succinate oxidation was completely inhibited in the presence of 50 f'g of anilazine per ml. Effect of anilazine on enzyme Activity: To determine whether inhibition of respiration was a consequence of specific inhibition of enzymes involved in respiration or general inhibition of enzymes, the effect of the fungicide on the in vitro activity of two enzymes, namely succinic dehydrogenase, a key enzyme involved in respiration, and
Effect of An ila zine
507
(I.D. f60 nm
I
; 3.0
I
· ~zt~-~ J /l=+-t!I : .
0. 5 !
0'
I
f-j~---t-----'- ----j 0- - 0
Con/rol
x- x Amluzine
Fi g_ 1 0.03
0
o
8
15 21, 32 'to 1,8
l ime f h ) T able 1. Effect of a n ila zine on oxidat ion and enzyme act.ivity
a». coli
Effect on
QO/) Glucose Su ccinate Succin ic dehydrogena se act. ivit.y-) (units/m l) ,a-Galactosidase ac t ivit ys) (units)
W it hout Ani la zine
W ith Anilazine
360 349
147 Zero Zero
23. 7
460
(50 J1gJm l )
460
1) J11 O 2 consumed h - 1 • mg - 1 d ry weigh t of cells. 2) The enzym e un it is defined as t he J1moles of su ccinat e oxidi zed (K ING 1967). 3) The enzyme unit is defined as the amount of en zyme t hat will liberate o-n it ro phen ol at tho r ate of 10- 8 moles per min. (LEUERBERG 1950).
p -gal actosid ase, an inducible enzy me not inv olv ed in resp ira tion, wa s examined. The incub a t ion of cell fr ee reacti on mi xtur e with anilazine completely inhibit ed succ in ic dehydrogenase acti vit y while p-galactosida se activity of tolunized E. coli cells wa s not affec ted at all (Table 1), suggesting t hat anilaz ine specifically inhib its enzy mes invo lved in respi ra t ion. Most fungicides a re eit her resp irat or y po isons or inhibit ma cromolecul ar synthes is in fungi . Carboxin is kn own t o inhibit respiration in fungi by inhibit ing succiniccy t ochrome C-red ucta se specifica lly. In t his study the a nila zine inhibited oxidation of glu cose by 60 % and succina te oxidation by 100 %. It also inhibited succinic d ehydrogen ase ac tiv it y in vitro complet ely but did not affect the p-gala ct osidase activity. It seems t hat t his fungicid e is a spec ific inhibitor of enz ymes involved in r espi ra ti on. We ha ve rep or ted earl ier t hat anila zine inhibits growth of Rhizobium. sp. (GARG et al.) by inhibiting respira tion. In Rh izobium sp. also, t his fungi cide inhibit s glucos e and succ inate by 65 % a nd 95 %, respecti vely . Thus t he ac tion of anilazine see ms to be similar both in E . coli a nd Rh izobiu m sp . References DE KKER, J . : Acquired resistance to fung icides. Ann. Rev. Phyt opath. 14 (197 6),405 -428. GARG, F. C., TAURO, P., MISHRA, M. M., and GROVER, R. K.: Effect of fungi cides on growth and on DNA, R N A an d protein an a bolism of a Rh izobium sp. (Com m un icated ).
508
F. C. GARG et al., Effect of Anilazine
KING, T. E.: Preparation of succinic dehydrogenase and reconstitution of succinate oxidase. In: Methods in Enzymology (EsTABRooK, R. W., and PULLMAN, M. E., eds.). Vol. X, pp. 322-331Academic Press, New York 1967. LEDERBERG, .J.: The poD-galactosidase of E. coli K-10. J. Bact. 60 (1951), 381-392. MATHRE, D. E.: Mode of action of oxathiin systemic fungicides. 1. Effect of carboxin and oxycarboxin on the general metabolism of several basidiomycetes. Phytopathology 60 (1970), 671-676. MONOD, J., COHEN-BAZIRE, G., and COHN, M.: Sur is biosynthese de la p-galactosidase (lactase) che Escherichia coli 1a specific de l'induction. Biochim, Biophys. Acta 7 (1951), 585-599. ODEYAMI, 0., and ALEXANDER, M.: Use of fungicide resistant rhizobia for legume inoculation. Soil. BioI. Biochem. 9 (1977), 247-251. SEILER, J. P.: The mutagenicity of benzimidazole and benzimidazole derivatives. 1. Forward and reverse mutations caused by benzimidazole and one of its derivatives. Mut. Res. 15 (1972), 273-276. SUD, R. K., and GUPTA, K. G.: On the sensitivity of isolates of Rhizobium spp. and Azotobacter chroococcum to TMTD and its degradation product Na-DDC. Arch. Microbiol. 85 (1972),19-22. ULRICH, J. T., and MATHRE, D. E.: Mode of action of oxathiin systemic fungicides. V. Effect on electron transport system of Ustilago maydis and Saccharomyces cereoisiae. J. Bact. 110 (1972), 628-632. Authors' addresses: Dr. F. C. GARG, Dr. M. M. MISHRA and Prof. P. TAuRo, Department of Microbiology, and Prof. R. K. GROVER, Department of Plant Pathology, Haryana Agricultural University, Hissar (India).