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Nickel is essential for active hydrogenase in free-living Frankia isolated from Casuarina
FEMS MicrobiologyLetters70 (1990) 137-140 Published bY Elsevier
137
FEMSLE04057
Nickel is essential for active hydrogenase in free-living Frankia isolated from Casuarina Anita Sellstedt and Geoffrey D. Smith De,~artmentof Biochemistry, Facultyof Science, Australian National Unwersi,y.Canberra, Australia Received 12 March 1990 Accepted 19 March !990 Key words: Casuarina; Frankia; Hydrogenase, Nickel; Nitrogen fi×ation
1. SUMMARY Five free-living Frankia strains isolated from Casuarina were investigated for occurrence of hydrogenase activity. Nitrogenase activity (acetylene reduction) and hydrogen evolution were also evaluated, Acetylene redaction was recorded in all Frankia strains. None of the Frankia strains had any hydrogenase activity when grown on nickeldepleted medium and they released hydrogen in atmospheric air. After addition of nickel to the medium, the Frankia strains were shown to possess an active hydrogenase, which resulted in hydrogen uptake but no hydrogen evolution. The hydrogenase activity in Frankia strain KB5 increased from zero to 3.86 /tmol H 2 (mg protein) -1 h -t after addition of up to 1.0 ,aM Ni. It is likely that the hydrogenas~ activity could be enhanced even more as a response on further addition of Ni. It is indicated in this study that absence of hydrogenase activity in free-living Frankia isolated from Casuarina spp. is due to nickel deficiency. Frankia
Correspondence 1.9: Dr. Anita Sellstedt, Departmentof Plant Physiology,Universityof Umeh,S-90I 87 Ume/t,Sweden. * Permanent address: Departmentof Plant Physiology,University of UrneL S-901 87 Ume~ Sweden.
living in symbiosis with Casuarina spp. show hydrogenase acti,,ity. Therefore, the results also indicate that the hyaiogenase to some extent is regulated by the host plant and/or that the host plant supplies the symbiotic microorganism with nickel. Moreover, the result shows that this Frankia is somewhat different from Frankia isolated from Alnus incana and Comptonia peregrina., i.e. Frankia isolated from A. incana and C. peregrina showed a small hydrogen uptake activity even without addition of nickel.
2. INTRODUCTION All nitrogen-fixing organisms evolve hydrogen concomitantly with the reduction of atmospheric nitrogen. Some organisms have the ability to utilize this evolved hydrogen. They have an enzyme, hydrogenase, which catalyzes oxidation of hydrogen. Organisms that have this enzyme could regain some energy, and also scavenge H 2 and 02 from the sensitive nitrogenase [1]. In some bacteria Ni 2÷ is essential for synthesis of an active hydrogenase, including Alcaligenes eutrophus [2], Desulfovibrio gigas [3], Vil~rio succinogenes [4], Rhodobacter eapsulatus [51, Rhizobium japonicum [6] and Anabaena cylindrica
0378-1097/90/$03.50 © 1990 Federation of EuropeanMicrobiologicalSocieties
138 [7]. Frankia in symbiosis with Alnus and Comptonia have a great capacity ~or consumption of hydrogen gas [8,9]. Also, Frankia in symbiosis with Casuarina have a hydrogenase [10]. Hydrogenase has also been found in free-living actinomycetous Frankia, although it sometimes can be inactive [10,11]. No reports have been published, however, on free-living Frankia isolated from Casuarina. This is to our knowledge the first report on the absence of hydrogenase activity in the free-living form of a microorganism, which has been shown to have activity in the symbiotic state.
with a microtip (lOS), 15 s, output 7, duty cycle 50~) and NaOH was added to a final concentration of 1 M. Then the samples were heated (90 o C, 10 + :~ min), distilled H 2 0 was added and the samples were centrifuged (13000 rpm, 10 rain). Proteins were determined spectrophotometrieally at 562 nm using a bicinchoninic acid (BCA) and a 4% c~pper sulfate solution (BCA protein assay reagent kit, Pierce Chemical Co., Rockford, IL, USA). Bovine-v-globulin (Sigma) was used to standa-dize the assay procedure. Two determinations were made for each sample.
4. RES LILTS A N D DISCUSSION 3. MATERIALS AND METHODS 3.1. Media, growth conditions and cell preparation Cultures of Frankia strains ORS 020607 [12], HFP Cc13 [13], JCT 287 [14], KB 5 and BML [15] were grown in either P-medium [16] or in Smedium [17] containing nitrogen. Volumes of 100 ml media and cells were kept in 300ml flasks shaking at 27°C. Cells were collected by centrifugation (13000 x g, 10 rain), washed in N-free medium twice and then transferred to the N-free medium. Vesicles were induced after growth for 4 days on a N-free medium. In the first experiment, cells were either grown with or without 0.68 tLM NiSO4. In the other experiment, the cells were grown with different concentrations of NiSO4: (i) no addition, (ii) e.25 /tM nickel, (iii) 0.5 /tM nickel, (iv) 0.75/tM nickel a,-d (v) 1.0/~M nickel. Cells were bubb~'d w~:. ~f: for 30 s, 16 h prior to measurements and .~re transferred to carbonfree media 5-9 h prior to measurements. 3.2. Assays Nitrogenase activity was measured by acetylene reduction assay using a gas chromatograph as described earlier [18,19]. Hydrogen evolution and hydrogenase uptake activity was measured with a gas chromatograph [18,19]. Hydrogenase activity was also measured with an Oz-eleetrode converted for H 2 assays [8]. 3. 3. Protein determinations Samples were collected by centrifugation (13000 rpm, 10 rain), sonieated (Bransons B15
All Frankia strains actively fixed nitrogen as measured by acetylene reduction, irrespective of whether Ni was present or not (6.52 -t- 1.27/tmol C2H4 (mg protein) -1 h - l ) . H 2 evolution in air was also shown in all Frankia strains grown on Ni-deficient media (1.80 :t= 0.14 ttmol H 2 (mg protein) -1 h-I). These data show that the strains actively fixed nitrogen. In Frankia strains isolated from Alnus and Comptonia, hydrogenase was induced by bubbling through with H2 and by C- and N-starvation [11]. Therefore, in order to induce hydrogenase in Frankia isolated from Casuarina H 2 was bubbled in the saml~les prior to measurements. Substrate induction of hydrogenase activity and protein has been shown before for two Frankia strains and for AIcaligenes latus [10,11,21]. Hydrogenase activity could not, however, be detected in Frankia strains isolated from Casuarina spp. grown on nickel-depleted medium (Table 1). Not even after H2-induction, C*starvation a n d / o r withdrawal of N (induction of vesicles) could hydrogenase activity be detected. However, whc~ 0.~9 ~tM NiSO4 was added hydrogenase activities r o s e - t r o ~ : ' , _ / ) . 7 0 #real H2 (mg protein)- 1h - 1 in Frankia strain I~B~ hi,~. 2,~ 0.080 in Frankia strain HFP Cc13 (Table 2). Hvdrogenase activity even showed additional increase when 1.0 I~M nickel was added, reaching a value of 3.86/~mol H 2 (m$ protein)-lh - l (Fig. 1). Ni 2+ ions were essential for H 2 consumption in Frankia isolated from Casuarina. In this respect
139 Table 1 Hydroganaso activity in free-living Frankia strains under diffeeent treatments (~, n = 3) Treatment
Frankia strains" Hydroganaso activity btmol H 2oxidized (rag protein)- ]h- ]
C O
HFP KB5 BML ORS020607 JCT287 Cc13 +N, +C, - N i +N, -C, - N t - N . *C, - N i -N,-C,-Ni
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 *
i
[:] 0
Frankia strains f r o m Casuarina are different from other Frankia strains. F o r example in U G L 010010 isolated f r o m Alnus incana a n d C p l l isolated from Comptonia peregrina, Ni was not essential for hydrogenase activity [10,11,20]. However, Frankia isolated from A. incana and C. peregrina showed m u c h lower activities. It might be ,'hat Frankia f r o m Casuarina have a N i - d e p e n d e n t u p t a k e hydrogcnas¢ while the other Frankic strains have Fe-dependent uptake hydrogenases, i.e. these F,a~kia strains have different hydrogenases. It could also I~e t,'-,a* uptake bydrogenase activity in Frankia strains isolated i l , ~ the other host plants could have been increased with a d a i t i , ~ of nickel. Symbiotic Frankia f r o m Casuarina have I ~ n shown to possess hydrogenase activity ([8]; Sellstedt, unpublished). The results therefore show interesting differences between symbiotic a n d free-riving Frankia isolated from Casuarina spp. It is also indicated that the host plant might have
Table 2 Hydrogenase activity in two free-living Frankia sttalhS gr~~ in meCium containing Ni(X ± SE, n = 3) Treatment
+N, +N, -N, -N,
+C, -C, +C, -C,
Hydrogenase activity #mol H 2oxidized (mg protein)- ~h- i Frankia strain Franki, ~train H F P Ccl3 KB5 +Ni +Ni +Ni +Ni
0 0 0 0.08+0.01
0 0 0 0.70:t:0.01
0.0
0,2
OA
0.6
0.8
1,0
1,2
Concentration - ~ M NI Fig. 1. Activity of uptake hydrosenase in Frankia strain KB5 isolated from Casuarina equisetifolia.; ~, n = 4, SE is less than 5~. some m e c h a n i s m for regulating hydrogenase activity in Frankia. The results m a y offer means of studying interrelationships between the microorganism a n d its host plant.
ACKNOWLEDGEMENTS W e are grateful to Drs P. Reddell for providing us with Frankia strains KB5 a n d BML, Diem for O R S 020607, Torrey for H F P . C c I 3 a n d W. Shipton for J C T 287. This w o r k was financially supported by grants f r o m Swedish Natural Science Council a n d Australian National University.
EFERENCES [1] Dixon, R.O.D. (1972) Arch. Microbiol. 85, 193-201. [2] Friedrich, C.G., Schneider. K. and Friedrich, B. (1982) J. Bact. 152, 42-48. [3] I.~Oall, J., Ljungdahl, P.O., Moura, l., Peck, Jr., H.D., Xavier, A.V., Moura, J.J.G., Texiera, M., Huynh, B.H. and DerVattanian, D.V. (1982) Biochem. Biophys. Res. Commnn. 106, 610-616. [4] Unden, G., B6cher. R.. Knecht, J., and Krt~ger,A. (1982) FEBS LeU. 145, 230-234.
140 [5] Takakuwa, S. and Wall, J.D. (1981) FEMS Microbiol Lett. 12, 359-363. [6] Klucas, R.V., Hanus, F.J., Russell, S.A. and Evans, HJ. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2253-2257. [7] Daday, A., platz, R.A. and Smith, G.D. (1977) Appl. Environ. Microbiol. 34, 478-483. [8] Sellstedt, A. and Winship, LJ. (1987) Physiol. Plant. 70, [9[ Sellstcdt, A. (1989) Physiol. Plant. 75, 304-308. [10] Liodblad, P. and Sellstedt. A. (1990) FEMS :,:~,icrobl~l. Lett. In press. [11] S¢llstedt, A. ~ d Lindblad, P. (1990) Plant Physiol. In press. [12] Diem, H.G. and Dommersues, Y.R. (1983) Can. J. Bot. GI, 2815-2821. [13] Zhan8, Z., Lope~ M.F. and Torrey, J.G. (1984) Plant Soil "/8, 79-90.
[14] Sh/pton, W.A. and Bursgranf, A.JA" (1983) Can. J. BoL 61, 2783-2792. [15] Rosbrook, P.A., Burggraaf, A.J.P. and Reddell, (1990) Plant and Soil, In press. [16] Burggraaf. A.J.P. and Shipton. W.A. (1983) Can. J. Bot. 61, 2774-2782. [17] Noridge, B.A, and Iknson. D.R. (1986) J. Bacteriol. 166, 301-305. [18] Lambert, G.R. and Smi:h. G.D. (1980) Arch. Biochem. Biophys. 211, 36-50. [19] Sellstedt, A., Huss-Danen, K. and Ahlqvist, A.-S. (1986) Physiol. Plant. 66, 99-107. [20] Murray, M. and Lopez, M.F. (1988) 7th Int©rnational Meeting on Actinorhizal plant~ and Frankia, Aa 8 1988, Univ. Connecticut, Storrs, CT 06268. U.S.A. [21] Doyle, C.M, and Arp. D.J. (1987) J. Bacterlul. ~,69, 4,;634468.
137
FEMSLE04057
Nickel is essential for active hydrogenase in free-living Frankia isolated from Casuarina Anita Sellstedt and Geoffrey D. Smith De,~artmentof Biochemistry, Facultyof Science, Australian National Unwersi,y.Canberra, Australia Received 12 March 1990 Accepted 19 March !990 Key words: Casuarina; Frankia; Hydrogenase, Nickel; Nitrogen fi×ation
1. SUMMARY Five free-living Frankia strains isolated from Casuarina were investigated for occurrence of hydrogenase activity. Nitrogenase activity (acetylene reduction) and hydrogen evolution were also evaluated, Acetylene redaction was recorded in all Frankia strains. None of the Frankia strains had any hydrogenase activity when grown on nickeldepleted medium and they released hydrogen in atmospheric air. After addition of nickel to the medium, the Frankia strains were shown to possess an active hydrogenase, which resulted in hydrogen uptake but no hydrogen evolution. The hydrogenase activity in Frankia strain KB5 increased from zero to 3.86 /tmol H 2 (mg protein) -1 h -t after addition of up to 1.0 ,aM Ni. It is likely that the hydrogenas~ activity could be enhanced even more as a response on further addition of Ni. It is indicated in this study that absence of hydrogenase activity in free-living Frankia isolated from Casuarina spp. is due to nickel deficiency. Frankia
Correspondence 1.9: Dr. Anita Sellstedt, Departmentof Plant Physiology,Universityof Umeh,S-90I 87 Ume/t,Sweden. * Permanent address: Departmentof Plant Physiology,University of UrneL S-901 87 Ume~ Sweden.
living in symbiosis with Casuarina spp. show hydrogenase acti,,ity. Therefore, the results also indicate that the hyaiogenase to some extent is regulated by the host plant and/or that the host plant supplies the symbiotic microorganism with nickel. Moreover, the result shows that this Frankia is somewhat different from Frankia isolated from Alnus incana and Comptonia peregrina., i.e. Frankia isolated from A. incana and C. peregrina showed a small hydrogen uptake activity even without addition of nickel.
2. INTRODUCTION All nitrogen-fixing organisms evolve hydrogen concomitantly with the reduction of atmospheric nitrogen. Some organisms have the ability to utilize this evolved hydrogen. They have an enzyme, hydrogenase, which catalyzes oxidation of hydrogen. Organisms that have this enzyme could regain some energy, and also scavenge H 2 and 02 from the sensitive nitrogenase [1]. In some bacteria Ni 2÷ is essential for synthesis of an active hydrogenase, including Alcaligenes eutrophus [2], Desulfovibrio gigas [3], Vil~rio succinogenes [4], Rhodobacter eapsulatus [51, Rhizobium japonicum [6] and Anabaena cylindrica
0378-1097/90/$03.50 © 1990 Federation of EuropeanMicrobiologicalSocieties
138 [7]. Frankia in symbiosis with Alnus and Comptonia have a great capacity ~or consumption of hydrogen gas [8,9]. Also, Frankia in symbiosis with Casuarina have a hydrogenase [10]. Hydrogenase has also been found in free-living actinomycetous Frankia, although it sometimes can be inactive [10,11]. No reports have been published, however, on free-living Frankia isolated from Casuarina. This is to our knowledge the first report on the absence of hydrogenase activity in the free-living form of a microorganism, which has been shown to have activity in the symbiotic state.
with a microtip (lOS), 15 s, output 7, duty cycle 50~) and NaOH was added to a final concentration of 1 M. Then the samples were heated (90 o C, 10 + :~ min), distilled H 2 0 was added and the samples were centrifuged (13000 rpm, 10 rain). Proteins were determined spectrophotometrieally at 562 nm using a bicinchoninic acid (BCA) and a 4% c~pper sulfate solution (BCA protein assay reagent kit, Pierce Chemical Co., Rockford, IL, USA). Bovine-v-globulin (Sigma) was used to standa-dize the assay procedure. Two determinations were made for each sample.
4. RES LILTS A N D DISCUSSION 3. MATERIALS AND METHODS 3.1. Media, growth conditions and cell preparation Cultures of Frankia strains ORS 020607 [12], HFP Cc13 [13], JCT 287 [14], KB 5 and BML [15] were grown in either P-medium [16] or in Smedium [17] containing nitrogen. Volumes of 100 ml media and cells were kept in 300ml flasks shaking at 27°C. Cells were collected by centrifugation (13000 x g, 10 rain), washed in N-free medium twice and then transferred to the N-free medium. Vesicles were induced after growth for 4 days on a N-free medium. In the first experiment, cells were either grown with or without 0.68 tLM NiSO4. In the other experiment, the cells were grown with different concentrations of NiSO4: (i) no addition, (ii) e.25 /tM nickel, (iii) 0.5 /tM nickel, (iv) 0.75/tM nickel a,-d (v) 1.0/~M nickel. Cells were bubb~'d w~:. ~f: for 30 s, 16 h prior to measurements and .~re transferred to carbonfree media 5-9 h prior to measurements. 3.2. Assays Nitrogenase activity was measured by acetylene reduction assay using a gas chromatograph as described earlier [18,19]. Hydrogen evolution and hydrogenase uptake activity was measured with a gas chromatograph [18,19]. Hydrogenase activity was also measured with an Oz-eleetrode converted for H 2 assays [8]. 3. 3. Protein determinations Samples were collected by centrifugation (13000 rpm, 10 rain), sonieated (Bransons B15
All Frankia strains actively fixed nitrogen as measured by acetylene reduction, irrespective of whether Ni was present or not (6.52 -t- 1.27/tmol C2H4 (mg protein) -1 h - l ) . H 2 evolution in air was also shown in all Frankia strains grown on Ni-deficient media (1.80 :t= 0.14 ttmol H 2 (mg protein) -1 h-I). These data show that the strains actively fixed nitrogen. In Frankia strains isolated from Alnus and Comptonia, hydrogenase was induced by bubbling through with H2 and by C- and N-starvation [11]. Therefore, in order to induce hydrogenase in Frankia isolated from Casuarina H 2 was bubbled in the saml~les prior to measurements. Substrate induction of hydrogenase activity and protein has been shown before for two Frankia strains and for AIcaligenes latus [10,11,21]. Hydrogenase activity could not, however, be detected in Frankia strains isolated from Casuarina spp. grown on nickel-depleted medium (Table 1). Not even after H2-induction, C*starvation a n d / o r withdrawal of N (induction of vesicles) could hydrogenase activity be detected. However, whc~ 0.~9 ~tM NiSO4 was added hydrogenase activities r o s e - t r o ~ : ' , _ / ) . 7 0 #real H2 (mg protein)- 1h - 1 in Frankia strain I~B~ hi,~. 2,~ 0.080 in Frankia strain HFP Cc13 (Table 2). Hvdrogenase activity even showed additional increase when 1.0 I~M nickel was added, reaching a value of 3.86/~mol H 2 (m$ protein)-lh - l (Fig. 1). Ni 2+ ions were essential for H 2 consumption in Frankia isolated from Casuarina. In this respect
139 Table 1 Hydroganaso activity in free-living Frankia strains under diffeeent treatments (~, n = 3) Treatment
Frankia strains" Hydroganaso activity btmol H 2oxidized (rag protein)- ]h- ]
C O
HFP KB5 BML ORS020607 JCT287 Cc13 +N, +C, - N i +N, -C, - N t - N . *C, - N i -N,-C,-Ni
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 *
i
[:] 0
Frankia strains f r o m Casuarina are different from other Frankia strains. F o r example in U G L 010010 isolated f r o m Alnus incana a n d C p l l isolated from Comptonia peregrina, Ni was not essential for hydrogenase activity [10,11,20]. However, Frankia isolated from A. incana and C. peregrina showed m u c h lower activities. It might be ,'hat Frankia f r o m Casuarina have a N i - d e p e n d e n t u p t a k e hydrogcnas¢ while the other Frankic strains have Fe-dependent uptake hydrogenases, i.e. these F,a~kia strains have different hydrogenases. It could also I~e t,'-,a* uptake bydrogenase activity in Frankia strains isolated i l , ~ the other host plants could have been increased with a d a i t i , ~ of nickel. Symbiotic Frankia f r o m Casuarina have I ~ n shown to possess hydrogenase activity ([8]; Sellstedt, unpublished). The results therefore show interesting differences between symbiotic a n d free-riving Frankia isolated from Casuarina spp. It is also indicated that the host plant might have
Table 2 Hydrogenase activity in two free-living Frankia sttalhS gr~~ in meCium containing Ni(X ± SE, n = 3) Treatment
+N, +N, -N, -N,
+C, -C, +C, -C,
Hydrogenase activity #mol H 2oxidized (mg protein)- ~h- i Frankia strain Franki, ~train H F P Ccl3 KB5 +Ni +Ni +Ni +Ni
0 0 0 0.08+0.01
0 0 0 0.70:t:0.01
0.0
0,2
OA
0.6
0.8
1,0
1,2
Concentration - ~ M NI Fig. 1. Activity of uptake hydrosenase in Frankia strain KB5 isolated from Casuarina equisetifolia.; ~, n = 4, SE is less than 5~. some m e c h a n i s m for regulating hydrogenase activity in Frankia. The results m a y offer means of studying interrelationships between the microorganism a n d its host plant.
ACKNOWLEDGEMENTS W e are grateful to Drs P. Reddell for providing us with Frankia strains KB5 a n d BML, Diem for O R S 020607, Torrey for H F P . C c I 3 a n d W. Shipton for J C T 287. This w o r k was financially supported by grants f r o m Swedish Natural Science Council a n d Australian National University.
EFERENCES [1] Dixon, R.O.D. (1972) Arch. Microbiol. 85, 193-201. [2] Friedrich, C.G., Schneider. K. and Friedrich, B. (1982) J. Bact. 152, 42-48. [3] I.~Oall, J., Ljungdahl, P.O., Moura, l., Peck, Jr., H.D., Xavier, A.V., Moura, J.J.G., Texiera, M., Huynh, B.H. and DerVattanian, D.V. (1982) Biochem. Biophys. Res. Commnn. 106, 610-616. [4] Unden, G., B6cher. R.. Knecht, J., and Krt~ger,A. (1982) FEBS LeU. 145, 230-234.
140 [5] Takakuwa, S. and Wall, J.D. (1981) FEMS Microbiol Lett. 12, 359-363. [6] Klucas, R.V., Hanus, F.J., Russell, S.A. and Evans, HJ. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2253-2257. [7] Daday, A., platz, R.A. and Smith, G.D. (1977) Appl. Environ. Microbiol. 34, 478-483. [8] Sellstedt, A. and Winship, LJ. (1987) Physiol. Plant. 70, [9[ Sellstcdt, A. (1989) Physiol. Plant. 75, 304-308. [10] Liodblad, P. and Sellstedt. A. (1990) FEMS :,:~,icrobl~l. Lett. In press. [11] S¢llstedt, A. ~ d Lindblad, P. (1990) Plant Physiol. In press. [12] Diem, H.G. and Dommersues, Y.R. (1983) Can. J. Bot. GI, 2815-2821. [13] Zhan8, Z., Lope~ M.F. and Torrey, J.G. (1984) Plant Soil "/8, 79-90.
[14] Sh/pton, W.A. and Bursgranf, A.JA" (1983) Can. J. BoL 61, 2783-2792. [15] Rosbrook, P.A., Burggraaf, A.J.P. and Reddell, (1990) Plant and Soil, In press. [16] Burggraaf. A.J.P. and Shipton. W.A. (1983) Can. J. Bot. 61, 2774-2782. [17] Noridge, B.A, and Iknson. D.R. (1986) J. Bacteriol. 166, 301-305. [18] Lambert, G.R. and Smi:h. G.D. (1980) Arch. Biochem. Biophys. 211, 36-50. [19] Sellstedt, A., Huss-Danen, K. and Ahlqvist, A.-S. (1986) Physiol. Plant. 66, 99-107. [20] Murray, M. and Lopez, M.F. (1988) 7th Int©rnational Meeting on Actinorhizal plant~ and Frankia, Aa 8 1988, Univ. Connecticut, Storrs, CT 06268. U.S.A. [21] Doyle, C.M, and Arp. D.J. (1987) J. Bacterlul. ~,69, 4,;634468.