- Email: [email protected]
Structure and Site of Action of an Algicide from a Cyanobacterium, Oscillatoria late-virens
]. Plant Physiol. Vol. 146. pp. 372-374 (1995)
Short Communication
Structure and Site of Action of an Algicide from a Cyanobacterium, Oscillatoria late-virens
s. N. BAGCHI Department of Biological Sciences, R. D. University, Jabalpur - 482 001 (M.P.), India
Received October 17, 1994 · Accepted December 5, 1994
Summary
An algicidal (herbicidal) metabolite from cyanobacterium Oscillatoria late-virens was purified and the mechanism of action determined. The algicide had a molecular weight of 444 Da, an emperical formula of C 29 H4s0 3 , and contained phenol, Czo alkane and an a, ,6-unsaturated carbonyl residue. With K3 Fe(CN)6 and benzoquinones, 50% inhibition of photosynthetic electron flow (Iso) was attained by 1.25-2.35 J.tM of algicide. Chlorophyll fluorescence data indicate that the algicide interacted at the acceptor site of photosystem (PS)II, although the mechanism may be different from that of another herbicide, 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea (DCMU). Structural resemblance with phenolic herbicides indicates their common mode of interaction.
Key words: Cyanobacterium, Oscillatoria late-virens, algicidal/herbicidal metabolite, photosystem II acceptor site.
Introduction
Several filamentous cyanobacteria, under natural (Keating, 1977) and laboratory (Mason et al., 1982; Flores and Wolk, 1986; Bagchi et al., 1990) conditions, produce algicidal byproducts. Consequently, the algicides/herbicides from Scytonema hofmanni UTEX 2349 (Pignatello et al., 1983), Nostoc linkia CALU 892 (Gromov et al., 1991), Oscillatoria latevirens (Chauhan et al., 1992) and Fischerella sp. (Bagchi and Marwah, 1994) have been characterized for chemical and biological properties. They share a common function, viz. inactivation of PSII-mediated electron transport (Bagchi et al., 1993; Gleason and Case, 1986). While the Oscillatoria product was suspected to bind to the donor site of PSII (Chauhan et al., 1992), the others apparently shared the site of action of DCMU and atrazine known to interact with the quinone-binding protein (QB) at the acceptor site (Mallipudi and Gleason, 1989). In this paper the partial structure and site of action of 0. late-virens algicide were determined. © 1995 by Gustav Fischer Verlag, Stuttgart
Materials and methods
Axenic cultures of Synechococcus (PCC 7942), Synechocystis (PCC 6803) and 0. late-virens (local isolate; Bagchi et a!., 1990) were grown as previously (Chauhan et a!., 1992) in KN0 3-based BG-11 medium (Rippka et a!., 1979). Algicide from 0. late·virens was ether-extracted (Bagchi eta!., 1990), activity detected against Synechococcus growth (initial absorbancy at 665 mm = 0.05) and represented in terms of dry weight after solvent evaporation. The dried ether extract was refluxed for 6-8 h at 85 °C with 50 mL 2.5% methanolic-KOH. The condensed fraction was hydrolyzed with 25 mL 0.2 N H 2S04 at 45 °C, partitioned in ether and repeatedly washed with water. Further purification was achieved by thin-layer chromatography according to Pignatello et a!. (1983) using carbon tetrachloride: ethyl acetate (95: 5) as the solvent system. Ultraviolet-fluorescence material was extracted in methanol and applied to high performance liquid chromatography using a ODS C 18 column (Alltech Versapack) and methanol. Fractions absorbing at 254 mu were tested for algicidal activity, and active fractions were pooled and re-chromatographed. Preparation of spinach chloroplasts, thylakoids and cynobacterial suspension in BG-11 medium and measurement of chlorophyll
Structure and function of Oscillatoria algicide 100
57
444
350
400
450
Molecular ion mass < da Itons )
Fig.1: EI-Mass spectrum of an algicide from 0. late-virens. content and of 0 2 evolution rates with ferricyanide/benzoquinones were done as previously described (Allen and Holmes, 1986; Gleason and Case, 1986). Chloroplasts (40-45~-tgmL _, chlorophyll) were incubated with 80 ~-tg mL -I trypsin for 15 min followed by 100 ~-tg mL -I soybean trypsin inhibitor. The preparation was centrifuged, washed and suspended in buffer. Induction of chlorophyll fluorescence was recorded with a fluorometer (Heinz Walz Effeltrich) at a light intensity of 45~-tmol m - 2 s- 1• Fluorescence was measured through a filter transmitting at 640nm (Corning 254). Reactions were initiated after a 2-min dark pre-incubation, during which time inhibitors/methanol (solvent) were added. Experiments were repeated three times and deviation from the mean did not exceed 10%. Data from typical experiments are presented. All chemicals were purchased from commercial sources.
Results and Discussion
From 100 g (fresh weight) of cells approximately 56 mg of purified algicide was recovered. The final minimum lethal concentration for algicidal activity from 20 ~tgmL _,in crude ether extract was reduced to 1.2 ~tgmL -I. The total activity recovered (units of minimum lethal concentration in total weight of the material) was 25 %. Thin layer and liquid chromatography showed only one ultraviolet-quenching fraction with aligicidal activity, even when some other organic solvents were used. The purified algicide contained 78 % C, and 11 % H and 0 each; it reacted positive with hexamine + hydrazine and
Fig. 2: Effect of graded concentrations of an algicide from 0. late-virens (A) on Hill activity (~-tmol 0 2 evolved h- 1 mg- 1 chlorophyll) in Synechococcus sp. (--0--; 100% = 125), Synechocystis sp. ( - - 0 - - ; 100% = 162) and spinach thylakoids ( - 6 - ; 100% = 355), of DCMU (B) and of algicide (C) on the activity in spinach chloroplasts ( - - 6 - - ; 100% = 275) and trypsin-treated chloroplasts ( - - 0 - - ; 100% = 325). Para benzoquinone, dichlorobenzoquinone and K3Fe(CN)6 were Hill acceptors for cyanobacteria, thylakoids and chloroplasts, respectively.
p-nitrophenyl hydrazine, indicating the presence of phenolic and a, {3-unsaturated carbonyl moieties (Anger and Oesper, 1966). The ultraviolet spectrum showed absorption at 210.5 mu and 270.5 mu. Upon 1H NMR a triplet at o0.8, a singlet at o1.2, two singlets at o5.25 and o5.6 and a doublet at o7.0 were detected, which represents the protons of -CHJ, -CH2-, H>C = C
The infrared spectrum was consistent with the proposed structure. The algicide inhibited 0 2 evolution in cyanobacterial cells, chloroplasts and thylakoids, lso' s being (~tM) 1.25, 2.35 and 2.0, respectively (Fig. 2). The permeability difference apparently accounted for variation in algicide-response in the two cyanobacteria. DCMU also inhibited the chloroplast reaction, albeit at lower concentrations (Fig. 2). Under non-saturating light the algicide treated thylakoids exhibited approximately 50% reduction in minimum (Fo)l maximum (Fm) fluorescence yields of untreated controls (Fig. 3). Electron flow between dichlorophenol indophenol (reduced) and methyl viologen (Mehler reaction) was not affected (data not shown), indicating that PSI was operative. The extent of sensitivity to the two herbicides was also examined in partial trypsin-digested chloroplasts (Fig. 2). DCMU-inhibition was reasonably alleviated probably due
A
>-
3 73
B
c
!::
> f= 75 u
l
w
g
~so
w u 25 0: w Q.
0 oL----IL---~2L---~3~--~0~--4~0~--~~--~\2~0~~8~0~0~--~~--~2~--~3~--~4
ALGICIDE cpM>
DCMU
ALGICIDE (pM>
374
s. N. BAGCHI
A
B
hanty, School of Life Sciences, Jawaharlal Nehru University, New Delhi, and Dr. A. P. Bhaduri, Central Drug Research Institute, Lucknow for helping in fluorescence and spectrometric analyses and the Department of Science and Technology, New Delhi for financial assistance (project SR/OY}B-09/90}.
References
I
0
4
2 Time
(S)
Fig. 3: Induction of chlorophyll fluorescence in spinach thylakoids incubated with (A} methanol and (B) an algicide from 0. late-virens (4.21J.M final concentration). to elimination of target site(s) on OB protein (Rensen, 1989), but the algicide-sensitivity was sustained. The data indicate that the algicide interacted with the acceptor rather than donor site of PSII as proposed earlier (Bagchi et al., 1993). Further, we suggest that the target site may be different from that of DCMU. Owing to structural resemblance with «phenol-type» herbicides, discussed by Trebst and Draber (1986), it is reasonable to assume that the algicide shares a common mechanism of action (also see Rensen, 1989). Acknowledgements
The author thanks the Head, Department of Biological Sciences, R. D. University, Jabalpur for laboratory facilities, Prof. P. Mo-
ALLEN, J. F. and N. G. HoLMES: In: HIPKINS, M. F. and N. R. BAKER (eds.}: Photosynthesis: energy transduction (IRL Press, Oxford and Washington DC}, pp. 103-141 (1986}. ANGER, V. and R. E. 0ESPER (eds.}: Spot tests in organic analysis. Elsevier Publishing Co., Amsterdam, London and New York (1966}. BAGCHI, S. N., V. S. CHAUHAN, and J. B. MARWAH: Curr. Microbiol. 26, 223-228 (1993}. BAGCHI, S. N. andJ. B. MARwAH: Microbios. 79, 187-193 (1994}. BAGCHI, S. N., A. PALOD, and V. S. CHAUHAN: J. Basic Microbiol. 30, 21-29 (1990}. CHAUHAN, V. S., J. B. MARwAH, and S. N. BAGCHI: New Phytol. 120, 251-257 (1992}. FLORES, E. and C. P. Wou: Arch. Microbiol. 145, 215-219 (1986}. GLEASON, F. K. and D. E. CASE: Plant Physiol. 80, 834-837 (1986}. GROMOV, B. v., A. A. VEPRITSKIY, N. N. TITOVA, K. A. MAMAKAYEVA, and 0. V. ALEXANDROVA: J. Appl. Phycol. 3, 55-59 (1991}. KEATING, K. 1.: Science 196, 885-887 (1977}. MALL!Pum, L. R. and F. K. GLEASON: Plant Sci. 60, 149 -154 (1989). MAsoN, C. P., K. R. EDwARDs, R. E. CARLSoN, J. PIGNATELLO, F. K. GLEASON, andJ. M. WooD: Science 215, 400-402 (1982). PIGNATELLO, J. J., J. PoRwou, R. E. CARLSoN, A. XAviER, F. K. GLEASON, andJ. M. WooD: J. Org. Chern. 48, 4035-4037 (1983}. RENSEN, J. J. S.: In: DoDGE, A. B. (ed.): Herbicides and Plant Metabolism, Soc. Expl. Biol. Seminar Series 38 (Cambridge University Press}, pp. 21-36 (1989}. RIPPKA, R., J. DERUELLES, J. WATERBURY, M. HERDMAN, and R. Y. STANIER: J. Gen. Microbiol. 111, 1-61 (1979). TREBsT, A. and W. DRABER: Photosynth. Res. 10, 381-392 (1986).
Short Communication
Structure and Site of Action of an Algicide from a Cyanobacterium, Oscillatoria late-virens
s. N. BAGCHI Department of Biological Sciences, R. D. University, Jabalpur - 482 001 (M.P.), India
Received October 17, 1994 · Accepted December 5, 1994
Summary
An algicidal (herbicidal) metabolite from cyanobacterium Oscillatoria late-virens was purified and the mechanism of action determined. The algicide had a molecular weight of 444 Da, an emperical formula of C 29 H4s0 3 , and contained phenol, Czo alkane and an a, ,6-unsaturated carbonyl residue. With K3 Fe(CN)6 and benzoquinones, 50% inhibition of photosynthetic electron flow (Iso) was attained by 1.25-2.35 J.tM of algicide. Chlorophyll fluorescence data indicate that the algicide interacted at the acceptor site of photosystem (PS)II, although the mechanism may be different from that of another herbicide, 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea (DCMU). Structural resemblance with phenolic herbicides indicates their common mode of interaction.
Key words: Cyanobacterium, Oscillatoria late-virens, algicidal/herbicidal metabolite, photosystem II acceptor site.
Introduction
Several filamentous cyanobacteria, under natural (Keating, 1977) and laboratory (Mason et al., 1982; Flores and Wolk, 1986; Bagchi et al., 1990) conditions, produce algicidal byproducts. Consequently, the algicides/herbicides from Scytonema hofmanni UTEX 2349 (Pignatello et al., 1983), Nostoc linkia CALU 892 (Gromov et al., 1991), Oscillatoria latevirens (Chauhan et al., 1992) and Fischerella sp. (Bagchi and Marwah, 1994) have been characterized for chemical and biological properties. They share a common function, viz. inactivation of PSII-mediated electron transport (Bagchi et al., 1993; Gleason and Case, 1986). While the Oscillatoria product was suspected to bind to the donor site of PSII (Chauhan et al., 1992), the others apparently shared the site of action of DCMU and atrazine known to interact with the quinone-binding protein (QB) at the acceptor site (Mallipudi and Gleason, 1989). In this paper the partial structure and site of action of 0. late-virens algicide were determined. © 1995 by Gustav Fischer Verlag, Stuttgart
Materials and methods
Axenic cultures of Synechococcus (PCC 7942), Synechocystis (PCC 6803) and 0. late-virens (local isolate; Bagchi et a!., 1990) were grown as previously (Chauhan et a!., 1992) in KN0 3-based BG-11 medium (Rippka et a!., 1979). Algicide from 0. late·virens was ether-extracted (Bagchi eta!., 1990), activity detected against Synechococcus growth (initial absorbancy at 665 mm = 0.05) and represented in terms of dry weight after solvent evaporation. The dried ether extract was refluxed for 6-8 h at 85 °C with 50 mL 2.5% methanolic-KOH. The condensed fraction was hydrolyzed with 25 mL 0.2 N H 2S04 at 45 °C, partitioned in ether and repeatedly washed with water. Further purification was achieved by thin-layer chromatography according to Pignatello et a!. (1983) using carbon tetrachloride: ethyl acetate (95: 5) as the solvent system. Ultraviolet-fluorescence material was extracted in methanol and applied to high performance liquid chromatography using a ODS C 18 column (Alltech Versapack) and methanol. Fractions absorbing at 254 mu were tested for algicidal activity, and active fractions were pooled and re-chromatographed. Preparation of spinach chloroplasts, thylakoids and cynobacterial suspension in BG-11 medium and measurement of chlorophyll
Structure and function of Oscillatoria algicide 100
57
444
350
400
450
Molecular ion mass < da Itons )
Fig.1: EI-Mass spectrum of an algicide from 0. late-virens. content and of 0 2 evolution rates with ferricyanide/benzoquinones were done as previously described (Allen and Holmes, 1986; Gleason and Case, 1986). Chloroplasts (40-45~-tgmL _, chlorophyll) were incubated with 80 ~-tg mL -I trypsin for 15 min followed by 100 ~-tg mL -I soybean trypsin inhibitor. The preparation was centrifuged, washed and suspended in buffer. Induction of chlorophyll fluorescence was recorded with a fluorometer (Heinz Walz Effeltrich) at a light intensity of 45~-tmol m - 2 s- 1• Fluorescence was measured through a filter transmitting at 640nm (Corning 254). Reactions were initiated after a 2-min dark pre-incubation, during which time inhibitors/methanol (solvent) were added. Experiments were repeated three times and deviation from the mean did not exceed 10%. Data from typical experiments are presented. All chemicals were purchased from commercial sources.
Results and Discussion
From 100 g (fresh weight) of cells approximately 56 mg of purified algicide was recovered. The final minimum lethal concentration for algicidal activity from 20 ~tgmL _,in crude ether extract was reduced to 1.2 ~tgmL -I. The total activity recovered (units of minimum lethal concentration in total weight of the material) was 25 %. Thin layer and liquid chromatography showed only one ultraviolet-quenching fraction with aligicidal activity, even when some other organic solvents were used. The purified algicide contained 78 % C, and 11 % H and 0 each; it reacted positive with hexamine + hydrazine and
Fig. 2: Effect of graded concentrations of an algicide from 0. late-virens (A) on Hill activity (~-tmol 0 2 evolved h- 1 mg- 1 chlorophyll) in Synechococcus sp. (--0--; 100% = 125), Synechocystis sp. ( - - 0 - - ; 100% = 162) and spinach thylakoids ( - 6 - ; 100% = 355), of DCMU (B) and of algicide (C) on the activity in spinach chloroplasts ( - - 6 - - ; 100% = 275) and trypsin-treated chloroplasts ( - - 0 - - ; 100% = 325). Para benzoquinone, dichlorobenzoquinone and K3Fe(CN)6 were Hill acceptors for cyanobacteria, thylakoids and chloroplasts, respectively.
p-nitrophenyl hydrazine, indicating the presence of phenolic and a, {3-unsaturated carbonyl moieties (Anger and Oesper, 1966). The ultraviolet spectrum showed absorption at 210.5 mu and 270.5 mu. Upon 1H NMR a triplet at o0.8, a singlet at o1.2, two singlets at o5.25 and o5.6 and a doublet at o7.0 were detected, which represents the protons of -CHJ, -CH2-, H>C = C
The infrared spectrum was consistent with the proposed structure. The algicide inhibited 0 2 evolution in cyanobacterial cells, chloroplasts and thylakoids, lso' s being (~tM) 1.25, 2.35 and 2.0, respectively (Fig. 2). The permeability difference apparently accounted for variation in algicide-response in the two cyanobacteria. DCMU also inhibited the chloroplast reaction, albeit at lower concentrations (Fig. 2). Under non-saturating light the algicide treated thylakoids exhibited approximately 50% reduction in minimum (Fo)l maximum (Fm) fluorescence yields of untreated controls (Fig. 3). Electron flow between dichlorophenol indophenol (reduced) and methyl viologen (Mehler reaction) was not affected (data not shown), indicating that PSI was operative. The extent of sensitivity to the two herbicides was also examined in partial trypsin-digested chloroplasts (Fig. 2). DCMU-inhibition was reasonably alleviated probably due
A
>-
3 73
B
c
!::
> f= 75 u
l
w
g
~so
w u 25 0: w Q.
0 oL----IL---~2L---~3~--~0~--4~0~--~~--~\2~0~~8~0~0~--~~--~2~--~3~--~4
ALGICIDE cpM>
DCMU
ALGICIDE (pM>
374
s. N. BAGCHI
A
B
hanty, School of Life Sciences, Jawaharlal Nehru University, New Delhi, and Dr. A. P. Bhaduri, Central Drug Research Institute, Lucknow for helping in fluorescence and spectrometric analyses and the Department of Science and Technology, New Delhi for financial assistance (project SR/OY}B-09/90}.
References
I
0
4
2 Time
(S)
Fig. 3: Induction of chlorophyll fluorescence in spinach thylakoids incubated with (A} methanol and (B) an algicide from 0. late-virens (4.21J.M final concentration). to elimination of target site(s) on OB protein (Rensen, 1989), but the algicide-sensitivity was sustained. The data indicate that the algicide interacted with the acceptor rather than donor site of PSII as proposed earlier (Bagchi et al., 1993). Further, we suggest that the target site may be different from that of DCMU. Owing to structural resemblance with «phenol-type» herbicides, discussed by Trebst and Draber (1986), it is reasonable to assume that the algicide shares a common mechanism of action (also see Rensen, 1989). Acknowledgements
The author thanks the Head, Department of Biological Sciences, R. D. University, Jabalpur for laboratory facilities, Prof. P. Mo-
ALLEN, J. F. and N. G. HoLMES: In: HIPKINS, M. F. and N. R. BAKER (eds.}: Photosynthesis: energy transduction (IRL Press, Oxford and Washington DC}, pp. 103-141 (1986}. ANGER, V. and R. E. 0ESPER (eds.}: Spot tests in organic analysis. Elsevier Publishing Co., Amsterdam, London and New York (1966}. BAGCHI, S. N., V. S. CHAUHAN, and J. B. MARWAH: Curr. Microbiol. 26, 223-228 (1993}. BAGCHI, S. N. andJ. B. MARwAH: Microbios. 79, 187-193 (1994}. BAGCHI, S. N., A. PALOD, and V. S. CHAUHAN: J. Basic Microbiol. 30, 21-29 (1990}. CHAUHAN, V. S., J. B. MARwAH, and S. N. BAGCHI: New Phytol. 120, 251-257 (1992}. FLORES, E. and C. P. Wou: Arch. Microbiol. 145, 215-219 (1986}. GLEASON, F. K. and D. E. CASE: Plant Physiol. 80, 834-837 (1986}. GROMOV, B. v., A. A. VEPRITSKIY, N. N. TITOVA, K. A. MAMAKAYEVA, and 0. V. ALEXANDROVA: J. Appl. Phycol. 3, 55-59 (1991}. KEATING, K. 1.: Science 196, 885-887 (1977}. MALL!Pum, L. R. and F. K. GLEASON: Plant Sci. 60, 149 -154 (1989). MAsoN, C. P., K. R. EDwARDs, R. E. CARLSoN, J. PIGNATELLO, F. K. GLEASON, andJ. M. WooD: Science 215, 400-402 (1982). PIGNATELLO, J. J., J. PoRwou, R. E. CARLSoN, A. XAviER, F. K. GLEASON, andJ. M. WooD: J. Org. Chern. 48, 4035-4037 (1983}. RENSEN, J. J. S.: In: DoDGE, A. B. (ed.): Herbicides and Plant Metabolism, Soc. Expl. Biol. Seminar Series 38 (Cambridge University Press}, pp. 21-36 (1989}. RIPPKA, R., J. DERUELLES, J. WATERBURY, M. HERDMAN, and R. Y. STANIER: J. Gen. Microbiol. 111, 1-61 (1979). TREBsT, A. and W. DRABER: Photosynth. Res. 10, 381-392 (1986).