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Co-denitrification by the denitrifying system of the fungus Fusarium oxysporum
FEMS MicrobiologyLetters 93 (1992) 177-1811 © 1992 Federation of European MicrobiologicalSocieties0378o11|97/92/$05.110 Published by Elsevier
177
FEMSLE 04897
Co-denitrification by the denitrifying system of the fungus Fusarium oxysporum T a t s u o T a n i m o t o ~, Ken-ichi H a t a n o % D u - h y u n Kim ", H i r o o Uchiyarna b and H i r o f u m i S h o u n " "htstitate of Applied Biochemistry. Unicersity of Tsukuba. and t, National tnstitatc for Em'ironmental Research. Tsukuba. Jalnm
Received 111 March 1902 Accepted 18 March 19o2 Key words: Co-denitrification; Nitrosation; Denitrification; Fusarium oxysponm~ 1. S U M M A R Y Nitlogen compounds such as azide, salicylhydroxamic acid, and possibly ammonium ions were converted to nitrous oxide (N_,O) or dinitrogen (N 2) by Fusarium oxysporum under denitrifying conditions. Nitrogen atoms in these compounds were combined with another nitrogen atom from nitrite to form a hybrid N.,O species. The fungus exhibited much higher converting activities as compared with similar reactions catalyzed by bacterial denitrifiers. We thus propose the phenomenon be called co-denitrification, which means that such nitrogen compounds are denitrifled by the system induced by nitrite (or nitrate) but are incapable by themselves of inducing the denitrifying system. 2. I N T R O D U C T I O N We recently found that the fungus Fusarium oxysporum exhibits a potent denitrifying activity Correspondence to: H. Shoun, Institute of Applied Biochem-
istry. Universityof Tsukuba, Tsukuba, Ibaraki 305, Japan.
[1]. Nitrate or nitrite was stoichiometricaily converted to nitrous oxide (N20) under anaerobic conditions. This was the first demonstration of a distinct denitrifying activity due to fungi. We further showed that a cytochrome P-450 is involved in the process. Results on effects of respiratory inhibitors, such as antimycin, suggested that the denitrification is associated with a respiratory chain. During studies on inhibitors, we noticed that much higher amounts of nitrogen were finaUy recovered in the denitrification products as compared with added nitrite, when nitrogen-containing inhibitors were used. In this paper we describe evidence for fungal denitrification that can utilize nitrogen compounds other than nit r a t e / nitrite as substrates.
3. M E T H O D S F. oxyspontm MT-811, previously shown to be capable of converting nitrate or nitrite dissimilatively to N~O [1], was used throughout this work. The fungus was incubated with azide (4 mM), ammonium chloride (4 mM), a n d / o r salicylhydroxamic acid (SHAM) (4 mM) under the denitri-
178 fying conditions [1] in the presence of 10 mM nitrite, in a 500-ml Erlcnmcycr flask with side arms. The flask contained 150 ml (final) of the medium that consisted of 1% glucose, 0.2% peptone, 10 mM sodium nitrite, and other inorganic salts [1]; the gas phase was replaced with helium after inoculation. The upper-space gas of incubation flasks was analyzed by gas chromatography (GC) and gas chromatograph-mass spectrometry (GC-MS) as previously reported [1]. [L~N]-nitrite (99 atom %) was obtained from Cambridge |sotope Laboratories. [15N]-azide (95 atom %) was obtainded from Berlin Chemie.
A
.~-~)1ir"
¢.,~ ~x" !~'~
~ . 1000
~
500
50
100
CULTIVATION TIME (h)
4. RESULTS Azide or SHAM (an inhibitor of cyanide-insensitive respiration, refs. [2,3]) repressed N:Oevolution at the initial stage of the anaerobic incubation of F. o x y s p o r u m with nitrite [1]. After a lag period for adaptation, however, a rapid evolution was observed as shown in Fig. !. The final level of evolved NzO far exceeded the stoichiometry against the amount of added nitrite, but was almost comparable to the sum of nitrogen atoms in nitrite and SHAM when both nitrogen compounds were added to the medium. When azide was further added, the evolution of dinitrogen (N 2) in addition to N_,O was observed. Here the recovery of nitrogen atoms in both gases was almost equal to the sum of nitrogen atoms in nitrite, azide and SHAM. The amount of N 2 (in mol) was equal to that of added azide, suggesting that N, was evolved from azide. Neither N , O nor N 2 were evolved when nitrite was omitted from the medium, i.e. when the fungus was inbubated only with azide a n d / o r SHAM. These results were highly indicative that the nitrogen compounds (azide and S H A M ) were 'co-denitrified' with nitrite by the fungal denitrification system. Above observations were further confirmed by GC-MS analyses employing heavy isotope (lSN) substrates as shown in Fig. 2. W h e n [l~N]-azide, [laN]-nitrite, and [14]-SHAM were used, ISN 2 ( m / z = 30) dinitrogen was recovered, indicating that both nitrogen atoms in dinitrogen were derived from azide. N 2 0 was the mixture of
600
B
~,~.~.-~;.;~li" .:
-~o
~ " .e" j ' 6
/ /
40o
,oo
2 _-.
o
50
lO0
CULTIVATION TIME th|
Fig. 1. NzO (A) and N2 (B) evolution by the denitrifying system of F. o.~)'sporum F. o.o'sponon was incubated with nitrite, azide and/or SHAM as described in METHODS, and the evolved N~O and N, were determined by GC. Total amounts per flask were: nitrite, 1.5 mmol: azide and SHAM, 0.6 mmoL o, nitrite only: zx, nitrite plus azide; • , nitrite plus SHAM: e, nitrite plus azide and SHAM: v, azide only: v, SHAM only: El, azide and SHAM: t3, nitrite plus azide and SHAM but fungal cells were omitted. Each plot was performed at least 2 times. A representative plot is presented.
I~NI4NO ( m / z = 44) and 15NI4NO ( m / z = 45), showing that the third nitrogen in azide was used to form N20. Results on the GC-MS analyses upon various combinations of heavy and light substrates are summarized in Table 1. T h e approximate ratios in the table were qualitative rather than quantita-
. . . .
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30
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40
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.
30
.
.
.
i
.
40
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.
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i
50
m/z Fig. 2. Mass spectra of N., and N.,O formed by the denitrifying system of F. oxysponem. [tSNl-azidc (95 atom c;,) was incubated with [J4Nl-nitrite and [I'~N]-SHAM in the fungal system, and evolved N_, (upper) and N,O 0ower) were analyzed by GC-MS.
tive. t~N.~O ( m / z = 46) and laN,O ( m / z = 44) were formed, respectively, only when [ L~N]-nitrite and [laN]-nitrite were used. This means that at least one atom of nitrogen in N , O was derived
Table I Molecular species of N., and N,O evolved upon various combinations of tight and heavy substrates Substrate (nitrogen) [J4N] [15N] NO;
Product (m/z)/ approximateratio N: N,O
- " NO., NO~ +SHAM + N~ 31) N~ + NO~ 28 NO~ + Nj 30 SHAM + NO.~NH~ + NO.;NO,_ + NH.;,N~ +SHAM + NO_~ 28 SHAM + NO;- +N~- 3[)
44 46 44:45/I :1).5 46:45/I :0.1 46 46:45/1:0.2 46:45/I :0.4 46 46:45/1:11.5 46:45/1:0.2
Nitrogen compounds were incubated with F. o.~3"sporum as in Fig. | for 7 days, and evolved gases were analyzed by GC-MS. ~' Not detected.
from nitrite, i.e. that N , O could not be formed only from the nitrogen compounds (azidc, SHAM, and ammonium). The hybrid product, ~SNI~NO formed wht, ll [~~N]-nitrite was incubated with [14N]-azide, [ 14N]-ammonium, o r / a n d SHAM, or when [14N]-nitrite was incubated with [l~N]-azide and SHAM. The results show that nitrogen atoms in the compounds other than nitrite were utilized as a counterpart of nitrogen atoms in N_,O. Although the heavy isotope of S H A M was not a~'ailable, various combinations of heavy and light substrates indicated that nitrogen atoms in SHAM were utilized as the counterpart of nitrogen in N,O. Results with ammonium ions were not conclusive, however, suggesting that nitrogen atoms of ammonium were incorporated into N , O upon incubation with nitrite. It seems therefore that the fungus can utilize various nitrogen compounds as denitrification substrates.
5. DISCUSSION The present results demonstrate that the fungal denitrifying system is active on nitrogen compounds other than n i t r a t e / n i t r i t e . Nitrogen atoms in such compounds ~vere utilized as a counterpart of nitrogen atoms in the product N20. The denitrification from such compounds did not occur at all in the absence of nitrite. A similar phenomenon has been observed to occur with several denitrifying bacteria [4]. Dissimilatory nitrite reductase is responsible for the reaction [5]. The enzyme catalyzes nitrosyl transfer (nitrosation) from nitrite to N-nucleophiles such as azide. A ferrous heme-nitrosyi compound ( E - F e u • NO *) has been proposed as the enzymatic nitrosyl donor. Although there is precedence in bacteria as noted above, the fungal system exhibited unique features. Azidc or SHAM did not inhibit the overall denitrification (NzO evolution) by the fungal system (Fig. 1), unlike the inhibition by Nnucleophiles of bacterial systems [4]. And much higher amounts (50-70 raM) of nucleophiles were used to detect the nitrosation product (NzO) for the bacterial systems [4.5], whereas addition of only 4 mM azide o r / a n d S H A M increased after a
lag in the rate of N 2 0 evolution by the fungal system, and nitrogen atoms in these compounds were recovered 100% in NzO and N 2 (Fig. 1). N 2 evolution from azide has not been examined with bacterial systems, In bacterial systems, the nitrosation rates were at most only a few per cent of the reduction (denitrification) rate. In conclusion, the fungal system has a much higher nitrosation activity than bacterial systems. We thus propose to call the unique p h e n o m e n o n 'co-denitrification'. It is of mechanistic interest to know why the fungal system exhibits a high activity for nitrosation. Further studies concerning the p h e n o m e n o n will contribute to an understanding of both fungal and bacterial denitrification mechanisms.
ACKNOWLEDGEMENT This work was supported by the University of Tsukuba Project Research (A). REFERENCES [IJ Shoun, H. and Tanimo[o, T. (1991) J. Biol. Chem. 266, 11078-11082.
~2] Henry, M.F. and Nyns, E.J. (1975) Subeell. Bioehem. 4, 1-65.
[3] Janes. H.W. and Wies[, S.C. (1982) Plan[ Physiol. 70, 853-857. [4] Garber, E.A.E. and Hollocher. T.C. 0982) J. Biol. Chem. 257, 8091-8097. [5] Kim. C.-H. and Holloeher, T.C. (1984) J. Biol. Chem. 259, 2092-2099.
177
FEMSLE 04897
Co-denitrification by the denitrifying system of the fungus Fusarium oxysporum T a t s u o T a n i m o t o ~, Ken-ichi H a t a n o % D u - h y u n Kim ", H i r o o Uchiyarna b and H i r o f u m i S h o u n " "htstitate of Applied Biochemistry. Unicersity of Tsukuba. and t, National tnstitatc for Em'ironmental Research. Tsukuba. Jalnm
Received 111 March 1902 Accepted 18 March 19o2 Key words: Co-denitrification; Nitrosation; Denitrification; Fusarium oxysponm~ 1. S U M M A R Y Nitlogen compounds such as azide, salicylhydroxamic acid, and possibly ammonium ions were converted to nitrous oxide (N_,O) or dinitrogen (N 2) by Fusarium oxysporum under denitrifying conditions. Nitrogen atoms in these compounds were combined with another nitrogen atom from nitrite to form a hybrid N.,O species. The fungus exhibited much higher converting activities as compared with similar reactions catalyzed by bacterial denitrifiers. We thus propose the phenomenon be called co-denitrification, which means that such nitrogen compounds are denitrifled by the system induced by nitrite (or nitrate) but are incapable by themselves of inducing the denitrifying system. 2. I N T R O D U C T I O N We recently found that the fungus Fusarium oxysporum exhibits a potent denitrifying activity Correspondence to: H. Shoun, Institute of Applied Biochem-
istry. Universityof Tsukuba, Tsukuba, Ibaraki 305, Japan.
[1]. Nitrate or nitrite was stoichiometricaily converted to nitrous oxide (N20) under anaerobic conditions. This was the first demonstration of a distinct denitrifying activity due to fungi. We further showed that a cytochrome P-450 is involved in the process. Results on effects of respiratory inhibitors, such as antimycin, suggested that the denitrification is associated with a respiratory chain. During studies on inhibitors, we noticed that much higher amounts of nitrogen were finaUy recovered in the denitrification products as compared with added nitrite, when nitrogen-containing inhibitors were used. In this paper we describe evidence for fungal denitrification that can utilize nitrogen compounds other than nit r a t e / nitrite as substrates.
3. M E T H O D S F. oxyspontm MT-811, previously shown to be capable of converting nitrate or nitrite dissimilatively to N~O [1], was used throughout this work. The fungus was incubated with azide (4 mM), ammonium chloride (4 mM), a n d / o r salicylhydroxamic acid (SHAM) (4 mM) under the denitri-
178 fying conditions [1] in the presence of 10 mM nitrite, in a 500-ml Erlcnmcycr flask with side arms. The flask contained 150 ml (final) of the medium that consisted of 1% glucose, 0.2% peptone, 10 mM sodium nitrite, and other inorganic salts [1]; the gas phase was replaced with helium after inoculation. The upper-space gas of incubation flasks was analyzed by gas chromatography (GC) and gas chromatograph-mass spectrometry (GC-MS) as previously reported [1]. [L~N]-nitrite (99 atom %) was obtained from Cambridge |sotope Laboratories. [15N]-azide (95 atom %) was obtainded from Berlin Chemie.
A
.~-~)1ir"
¢.,~ ~x" !~'~
~ . 1000
~
500
50
100
CULTIVATION TIME (h)
4. RESULTS Azide or SHAM (an inhibitor of cyanide-insensitive respiration, refs. [2,3]) repressed N:Oevolution at the initial stage of the anaerobic incubation of F. o x y s p o r u m with nitrite [1]. After a lag period for adaptation, however, a rapid evolution was observed as shown in Fig. !. The final level of evolved NzO far exceeded the stoichiometry against the amount of added nitrite, but was almost comparable to the sum of nitrogen atoms in nitrite and SHAM when both nitrogen compounds were added to the medium. When azide was further added, the evolution of dinitrogen (N 2) in addition to N_,O was observed. Here the recovery of nitrogen atoms in both gases was almost equal to the sum of nitrogen atoms in nitrite, azide and SHAM. The amount of N 2 (in mol) was equal to that of added azide, suggesting that N, was evolved from azide. Neither N , O nor N 2 were evolved when nitrite was omitted from the medium, i.e. when the fungus was inbubated only with azide a n d / o r SHAM. These results were highly indicative that the nitrogen compounds (azide and S H A M ) were 'co-denitrified' with nitrite by the fungal denitrification system. Above observations were further confirmed by GC-MS analyses employing heavy isotope (lSN) substrates as shown in Fig. 2. W h e n [l~N]-azide, [laN]-nitrite, and [14]-SHAM were used, ISN 2 ( m / z = 30) dinitrogen was recovered, indicating that both nitrogen atoms in dinitrogen were derived from azide. N 2 0 was the mixture of
600
B
~,~.~.-~;.;~li" .:
-~o
~ " .e" j ' 6
/ /
40o
,oo
2 _-.
o
50
lO0
CULTIVATION TIME th|
Fig. 1. NzO (A) and N2 (B) evolution by the denitrifying system of F. o.~)'sporum F. o.o'sponon was incubated with nitrite, azide and/or SHAM as described in METHODS, and the evolved N~O and N, were determined by GC. Total amounts per flask were: nitrite, 1.5 mmol: azide and SHAM, 0.6 mmoL o, nitrite only: zx, nitrite plus azide; • , nitrite plus SHAM: e, nitrite plus azide and SHAM: v, azide only: v, SHAM only: El, azide and SHAM: t3, nitrite plus azide and SHAM but fungal cells were omitted. Each plot was performed at least 2 times. A representative plot is presented.
I~NI4NO ( m / z = 44) and 15NI4NO ( m / z = 45), showing that the third nitrogen in azide was used to form N20. Results on the GC-MS analyses upon various combinations of heavy and light substrates are summarized in Table 1. T h e approximate ratios in the table were qualitative rather than quantita-
. . . .
i
--|
30
|-
....
,--
40
,
50
m/z
.
.
.
.
i
.
.
.
.
,
.
30
.
.
.
i
.
40
.
.
.
.
.
.
i
.
.
.
.
i
50
m/z Fig. 2. Mass spectra of N., and N.,O formed by the denitrifying system of F. oxysponem. [tSNl-azidc (95 atom c;,) was incubated with [J4Nl-nitrite and [I'~N]-SHAM in the fungal system, and evolved N_, (upper) and N,O 0ower) were analyzed by GC-MS.
tive. t~N.~O ( m / z = 46) and laN,O ( m / z = 44) were formed, respectively, only when [ L~N]-nitrite and [laN]-nitrite were used. This means that at least one atom of nitrogen in N , O was derived
Table I Molecular species of N., and N,O evolved upon various combinations of tight and heavy substrates Substrate (nitrogen) [J4N] [15N] NO;
Product (m/z)/ approximateratio N: N,O
- " NO., NO~ +SHAM + N~ 31) N~ + NO~ 28 NO~ + Nj 30 SHAM + NO.~NH~ + NO.;NO,_ + NH.;,N~ +SHAM + NO_~ 28 SHAM + NO;- +N~- 3[)
44 46 44:45/I :1).5 46:45/I :0.1 46 46:45/1:0.2 46:45/I :0.4 46 46:45/1:11.5 46:45/1:0.2
Nitrogen compounds were incubated with F. o.~3"sporum as in Fig. | for 7 days, and evolved gases were analyzed by GC-MS. ~' Not detected.
from nitrite, i.e. that N , O could not be formed only from the nitrogen compounds (azidc, SHAM, and ammonium). The hybrid product, ~SNI~NO formed wht, ll [~~N]-nitrite was incubated with [14N]-azide, [ 14N]-ammonium, o r / a n d SHAM, or when [14N]-nitrite was incubated with [l~N]-azide and SHAM. The results show that nitrogen atoms in the compounds other than nitrite were utilized as a counterpart of nitrogen atoms in N_,O. Although the heavy isotope of S H A M was not a~'ailable, various combinations of heavy and light substrates indicated that nitrogen atoms in SHAM were utilized as the counterpart of nitrogen in N,O. Results with ammonium ions were not conclusive, however, suggesting that nitrogen atoms of ammonium were incorporated into N , O upon incubation with nitrite. It seems therefore that the fungus can utilize various nitrogen compounds as denitrification substrates.
5. DISCUSSION The present results demonstrate that the fungal denitrifying system is active on nitrogen compounds other than n i t r a t e / n i t r i t e . Nitrogen atoms in such compounds ~vere utilized as a counterpart of nitrogen atoms in the product N20. The denitrification from such compounds did not occur at all in the absence of nitrite. A similar phenomenon has been observed to occur with several denitrifying bacteria [4]. Dissimilatory nitrite reductase is responsible for the reaction [5]. The enzyme catalyzes nitrosyl transfer (nitrosation) from nitrite to N-nucleophiles such as azide. A ferrous heme-nitrosyi compound ( E - F e u • NO *) has been proposed as the enzymatic nitrosyl donor. Although there is precedence in bacteria as noted above, the fungal system exhibited unique features. Azidc or SHAM did not inhibit the overall denitrification (NzO evolution) by the fungal system (Fig. 1), unlike the inhibition by Nnucleophiles of bacterial systems [4]. And much higher amounts (50-70 raM) of nucleophiles were used to detect the nitrosation product (NzO) for the bacterial systems [4.5], whereas addition of only 4 mM azide o r / a n d S H A M increased after a
lag in the rate of N 2 0 evolution by the fungal system, and nitrogen atoms in these compounds were recovered 100% in NzO and N 2 (Fig. 1). N 2 evolution from azide has not been examined with bacterial systems, In bacterial systems, the nitrosation rates were at most only a few per cent of the reduction (denitrification) rate. In conclusion, the fungal system has a much higher nitrosation activity than bacterial systems. We thus propose to call the unique p h e n o m e n o n 'co-denitrification'. It is of mechanistic interest to know why the fungal system exhibits a high activity for nitrosation. Further studies concerning the p h e n o m e n o n will contribute to an understanding of both fungal and bacterial denitrification mechanisms.
ACKNOWLEDGEMENT This work was supported by the University of Tsukuba Project Research (A). REFERENCES [IJ Shoun, H. and Tanimo[o, T. (1991) J. Biol. Chem. 266, 11078-11082.
~2] Henry, M.F. and Nyns, E.J. (1975) Subeell. Bioehem. 4, 1-65.
[3] Janes. H.W. and Wies[, S.C. (1982) Plan[ Physiol. 70, 853-857. [4] Garber, E.A.E. and Hollocher. T.C. 0982) J. Biol. Chem. 257, 8091-8097. [5] Kim. C.-H. and Holloeher, T.C. (1984) J. Biol. Chem. 259, 2092-2099.