Kieler Meeresforsch., Sonderh. 8, 369-375. Kiel 1991
Determination and significance of nitrate reductase in marine fungi
R. Rau* and H. P. Molitoris
Botanisches Institut an der Universitat Regensburg Postfach 397, D-8400 Regensburg, Germany
Abstract
A previous investigation has shown that many t e r r e s t r i a l fungi do not produce n i t r a t e reductase ( N a - R ) . R e c e n t l y , an e x a m i n a t i o n of marine fungi r e v e a l e d that a l l strains tested produce N a - R . Other authors, however, showed absence of this enzyme in a few marine strains. Our reinvestigation of an even broader spectrum of over 80 strains of marine fungi showed the presence of N a - R w i t h only a few exceptions. Since the test was based on determination of n i t r i t e , the f i r s t m e t a b o l i t e of N a - R a c t i v i t y , it cannot be excluded that the apparent a b - sence of N a - R in these few cases was due only to a rapid turnover of n i t r i t e by a very a c t i v e n i t r i t e reductase ( N i - R ) . T h e r e f o r e , in a l l cases in which the p r e - sence of N a - R in this test was u n c e r t a i n , the experiments were repeated by a method inhibiting N i - R by fluoride and therefore a c c u m u l a t i n g n i t r i t e which could then be d e t e c t e d . Using this method, it could be shown that in f a c t a l l marine fungi tested produce N a - R . N i t r o g e n often constitutes a l i m i t i n g f a c t o r for growth of fungi. N a t u r a l seawater contains considerable amounts of nitrogen in the f o r m of n i t r a t e (up to 600 u.g/1). If, however, marine fungi possess N a - R , they could use nitrate as a nitrogen source. Possession of N a - R therefore would constitute an important s e l e c t i v e advantage explaining our results that a l l m a - rine fungi investigated so far c o n t a i n this e n z y m e .
Introduction
In 1961 J O H N S O N and S P A R R O W pointed out in their comprehensive r e v i e w ,
" F u n g i in oceans and e s t u a r i e s " , that the investigation of the physiology of m a - rine fungi is a prerequisite to the understanding of their role in the marine e c o - s y s t e m . Since, however, only r e l a t i v e l i t t l e physiological work has been done w i t h marine fungi, most of this work has been c a r r i e d out on individual enzymes or fungi, and since the methods were often not c o m p a r a b l e , we set up a screening program to investigate c h a r a c t e r i s t i c enzymes of representative spe- cies of all s y s t e m a t i c and e c o l o g i c a l groups of marine and t e r r e s t r i a l fungi for comparison ( M O L I T O R I S and S C H A U M A N N 1986). More than 20 enzymes from s e v e r a l important m e t a b o l i c pathways (redox-, f a t - , c a r b o h y d r a t e - , n i t r o g e n - metabolism) were i n v e s t i g a t e d .
* P a r t of D i p l o m a Thesis of R. R a u
One of the objectives of our screening program was also to find c o m m o n proper- ties of marine fungi c o n t r a s t i n g w i t h other groups of fungi. Presence or absence of n i t r a t e reductase ( N a - R ) turned out to be such a d i f f e r e n t i a t i n g p r o p e r t y . A previous investigation by B R E S I N S K Y and S C H N E I D E R 1975, had shown that many t e r r e s t r i a l f u n g i , in p a r t i c u l a r B a s i d i o m y c e t e s , do not produce N a - R . R e - c e n t l y , our investigation of obligate marine fungi of d i f f e r e n t s y s t e m a t i c a l and e c o l o g i c a l groups ( M O L I T O R I S and S C H A U M A N N 1986) r e v e a l e d that a l l marine fungal strains tested are able to produce N a - R . O t h e r authors using the same t e s t , e.g. S C H A U M A N N et a l . 1986, however, showed absence of this e n z y m e for a few marine s t r a i n s , such as the marine A s c o m y c e t e Lulworthia. The o b j e c t i v e of the present paper is to c l a r i f y this discrepancy by an r e i n v e s t i g a t i o n using a s t i l l wider s e l e c t i o n of strains and employing additional methods.
Material and methods
B i o l o g i c a l m a t e r i a l : F u n g a l s t r a i n s , s y s t e m a t i c position and origins are given in Table 1.
M e d i a , incubation and g r o w t h : The strains were grown in test tubes at 22 °C w i t h a p e r i o d i c i t y of t w e l v e hours light and twelve hours dark. The G P Y - m e d i u m consisted of glucose (1 g/l), peptone (0.5 g/1), yeast e x t r a c t (0.1 g/l) and agar (16 g/l), prepared w i t h s y n t h e t i c seawater ( R I L A products, T e a n e c k , N . J . , U S A ) , adjusted to p H 6.0 and a u t o c l a v e d for 20 min at 121 ° C .
N i t r a t e reductase ( N a - R - F ) , normal test: Tests for the presence of N a - R were p e r f o r m e d in the test tubes a f t e r 3 weeks of incubation by addition of the Griess-Ilosvaye reagents A and B to both, the test medium containing 15 g / l N a - n i t r a t e , and a c o n t r o l without n i t r a t e following the method of B R E S I N S K Y and S C H N E I D E R 1975. The development of a red colour indicating the presence of n i t r i t e was observed a f t e r 60 m i n . The a c t i v i t y is given in a r b i t r a r y units f r o m 0 (no a c t i v i t y ) to 4 (very a c t i v e ) as the d i f f e r e n c e between the readings w i t h and without n i t r a t e (control).
N i t r a t e reductase test w i t h inhibition of n i t r i t e reductase by the addition of f l u o r i d e (Na-R+F) f o l l o w i n g in p r i n c i p l e M O R T O N 1956: The test was conducted in the same way as above w i t h the f o l l o w i n g changes: The medium was made up w i t h deionized w a t e r and c o n t a i n e d 8.4 g/l N a F , 1.0 mg/1 N a2M o O i * (as in R I L A ) but no other R I L A salts because their C a+ + ions would p r e c i p i t a t e the f l u o r i d e . Since fluoride would inhibit the d e t e c t i o n of n i t r i t e by the G r i e s s - I l o s v a y e r e - agents, we used a liquid medium without agar, which allowed the r e m o v a l of f l u o r i d e by p r e c i p i t a t i o n w i t h 20 % aqueous C a C l2.
The c o m p a r a b i l i t y of the n i t r a t e reductase tests without and w i t h addition of f l u o r i d e was established by c o n t r o l experiments in which the medium did not c o n t a i n f l u o r i d e , seawater salts ( R I L A ) or molybdenum salts.
Results and discussion
M o r e than 80 strains of marine fungi comprising a l l major s y s t e m a t i c groups w i t h altogether 54 species (1 P h y c o m y c e t e , 15 A s c o m y c e t e s , 1 B a s i d i o m y c e t e , 21 D e u t e r o m y c e t e s , 6 ascosporous yeasts, 8 basidiosporous yeasts and 3 asporo- genous yeasts) were tested for the presence of N a - R by n i t r i t e d e t e c t i o n using the Griess-Ilosvaye reagent (Table 1 and F i g . 1).
Principle of the test a) b) c) d) Nitrate NO, Nitrate- Reductase (Na-R) • Nitrite N02 + Griess •red Ilosvaye colour Reagent Nitrite- Reductase (Ni-R) • Nitric NO oxide N03 no N02
NO, Na-R MjM inhib. no N02
NO, ^ Na-R N02 Ni-R NO
N03 Na-R • N02 ^ Ni-R inhibited 1 by fluoride 1 i NO Colour: colourless colourless colourless red colour Na-R activity present: none yes yes yes Na-R activity measured: none none none yes Explanation: no Na-R present Na-R present but inhibited
Na-R present, N02 not detectable because metabolized by very active Ni-R Na-R present and detectable because N02 degradation inhibited by fluoride Fig. 1. The Griess-Ilosvaye test for nitrate reductase. Principle and possible interpretations of results (a, b, c and d).
Table 1 (Part 1). M a r i n e fungal s t r a i n s , their origin and n i t r a t e reductase a c t i v i - ty without ( N a - R - F ) and w i t h inhibition of n i t r i t e reductase a c t i v i t y by addition
of fluoride ( N a - R + F ) .
N u m b e r S t r a i n C I .1 O r i g .2 N i t r a t e reduct, N a - R - F N a - R +
M 039 Acremonium furcatum D K M P B 2.5 4.0
M 037 Accemonium potronii D K M P B 2.0 4.0
M 038 Acremonium sp. D K M P B 2.5 4.0
M 141 Amylocarpus encephaloides A PP 1.5
M 001 Asteromyces cruciatus D K M P B 2.5 1.0
M 048 Asteromyces cruciatus D K M P B 2.5 2.0
M 118 Candida guiiiiermondii YI S C u
*
0.5M 119 Candida guiiiiermondii Y I S C u
*
0.5M 120 Candida guiiiiermondii YI S C 0 # 0.5
M 121 Candida guiiiiermondii YI S C u
*
0.5M 122 Candida guiiiiermondii YI S C u
*
0.5M 123 Candida guiiiiermondii YI S C 0
*
0.5M 148 Candida guiiiiermondii YI S C 0.5 0.5
M 124 Candida tropicalis YI S C 1.25 0.5
M 040 Cephalosporium sclerotigenum D K M P B 2.5 2.0
M 139 Cirrenalia tropicalis D PP 3.0
M 056 Corollospora lacera A K M P B 2.5
M 058 Corollospora lacera A J K 1.0
M 015 Corollospora maritima A K M P B 2.5
M 059 Corollospora maritima A J K 2.5
M 150 Corollospora maritima A M 3.0
5R 13 Corollospora maritima A SR 2.0
M 087 Corollospora trifurcata A J K 2.0
M 103 Cryptococcus albidus YI J F 2.5
M 061 Cytospora rhizophorae D J K 1.5
M 111 Debaryomyces hansenii Y A S C u
*
0.5M 112 Debaryomyces hansenii Y A S C 0 0.5
M 113 Debaryomyces hansenii Y A S C u 0.5
M 114 Debaryomyces hansenii Y A S C 0
*
0.5M 115 Debaryomyces hansenii Y A S C 0
*
0.5M 116 Debaryomyces hansenii Y A S C 1.0
*
0.5M 003 Dendryphiella salina D K M P B 2.5 1.5
M 094 Digitatispora marina B J K 0.5 0.5
M 125 Digitatispora marina B PP 0.5 0.5
M 126 Digitatispora marina B PP 0.5 0.5
M 131 Digitatispora marina B PP 1.0 0.5
M 147 Digitatispora marina B PP 1.0
M 062 Drechslera halodes D J K 0.5 0.5
M 050 Doratomyces sp. D K M P B 1.5 2.0
M 045 Fusarium sambucinum D K M P B 1.0
*
1.0M 065 Leptosphaeria australiensis A J K 1.5
M 090 Leptosphaeria obiones A J K 3.5 0.5
M 066 Leptosphaeria oraemaris A J K 1.0
M 142 Lulworthia lignoarenaria A PP 2.0
M 068 Lulworthia sp. I A J K u
*
0.5M 017 Lulworthia sp. II A K M P B 0.75* 1.0
Table 1 (Part 2). M a r i n e fungal strains, their origin and n i t r a t e reductase a c t i v i - ty without ( N a - R - F ) and w i t h inhibition of n i t r i t e reductase a c t i v i t y by addition
of fluoride ( N a - R + F ) .
N u m b e r S t r a i n C I .1 O r i g .2 N i t r a t e r e d u c t .3 N a - R - F N a - R + F
M 018 Lulworthia sp. Ill A K M P B 0 * 0.5
M 092 Lulworthia sp. V A J K 1
M 069 Macrophoma sp. D J K 1.5
M 143 Marinospora longissima A PP 0.5 1.0
M 071 Microascus senegalensis A J K 0 . 7 5 * 0.5
M 072 Microascus senegalensis A J K 0.5 * 0.5
M 019 Microascus trigonosporus A K M P B 0.5 3.0
M 006 Monodictys pelagica D K M P B 0 * 2.0
M 070 Monodictys pelagica D J K 0 . 7 5 *
M 052 Monodictys sp. D K M P B 2.5
M 144 Nautosphaeria cristaminuta A PP 0.5 1.0
M 140 Orbimyces spectabilis D PP 1.5
M 053 Phoma sp. D K M P B 1.0 * 0.5
M 145 Remispora stellata A PP 0.5 . 0.5
M 099 Rhodosporidium bisporidiis Y B J F 1.5
M 100 Rhodosporidium capitatum Y B J F u * 0.5
M 102 Rhodosporidium dacryoidum Y B J F 2.5
M 101 Rhodosporidium malvinellum Y B J F 2.0
M 080 Rhodosporidium sphaerocarpum Y B J F 1.5
M 081 Rhodosporidium diobovatum Y B J F 2.0
M 108 Rhodosporidium toruloides Y B J F 3.0
M 082 Rhodotorula minuta Y A J F 1.25*
M 104 Rhodotorula aurantiaca YI J F 1.5
M 106 Rhodotorula glutinis YI J F 2.5
M 107 Rhodotorula graminis YI J F 2.5
M 083 Rhodotorula rubra Y A J F 2.5
M 073 Savoriella paucispora A J K 1.5
M 137 Sigmoidea marina D S N 0 0.5
M 085 Sporobolomyces salmonicolor Y B J F 1.5
M 079 Trichocladium achrasporum D J K 1.25*
M 034 Ulkenia visurgensis P K M P B 1.0 * 0.5
M 074 Varicosporina ramulosa D J K 4.0 0.5
M 054 Verticillium lecanii D K M P B u * 1.5
M 008 Zalerion maritimum D K M P B 0.5 * 0.5
M 009 Zalerion maritimum D K M P B 2.5
D e t e r m i n a t i o n of enzyme a c t i v i t y a f t e r 3 weeks of growth.
C I .1: P = P h y c o m y c e t e s , A = A s c o m y c e t e s , B = B a s i d i o m y c e t e s , D = D e u t e r o m y - c e t e s , Y A = a s c o m y c e t o u s yeast, Y B = basidiomycetous yeast, YI = i m p e r f e c t yeast.
O r i g .2: S C = S. C r o w , A t h e n s ; J F = J . F e l l , M i a m i ; pp = G a r e t h Jones, P o r t s - mouth; J K = J . K o h l m e y e r , Morehead C i t y ; M = C o l l e c t i o n of marine fungi, U n i v . Regensburg; B S N = S. N e w e l l , Sapeloisland; SR = S. R o h r m a n n , R e g e n s - burg; K M P B = K . Schaumann, K u l t u r e n s a m m l u n g M a r i n e r P i l z e , B r e m e r h a v e n . N a - R a c t i v i t y3: a c t i v i t y given in a r b i t r a r y units f r o m 0 (no a c t i v i t y ) to 4 (very
strong a c t i v i t y ) ; u = u n c e r t a i n a c t i v i t y (positive and negative results in repe- titions); * = a c t i v i t y given as average of repetitions.
M o s t of the fungi showed the presence of N a - R , only a f e w showed low N a - R a c t i v i t y ; in 9 strains the results were u n c e r t a i n and in 8 strains no a c t i v i t y was found even in repeated tests (Table 1). This was e s p e c i a l l y true for the group of marine yeasts. G e n e r a l l y , in those cases where s e v e r a l strains of a given species were i n v e s t i g a t e d , the N a - R a c t i v i t i e s observed were rather s i m i l a r (Table 1).
The n o r m a l n i t r a t e reductase test ( N a - R - F ) used is based on the d e t e c t i o n of n i t r i t e ( F i g . l a - d ) , the f i r s t (and toxic) m e t a b o l i t e of N a - R a c t i v i t y . H o w e v e r , it cannot be e x c l u d e d , that an observed absence of red c o l o u r , i n d i c a t i n g the a b - sence of N a - R is due only to a rapid turnover of n i t r i t e by a very a c t i v e n i t r i t e reductase ( N i - R ) ( F i g . l c ) .
F o r this reason, a l l the few cases in w h i c h N a - R a c t i v i t y was found in the p r e v i - ous test to be l a c k i n g , u n c e r t a i n or low and in addition some strains w i t h r e l a - t i v e l y high N a - R a c t i v i t y were r e i n v e s t i g a t e d using a d i f f e r e n t m e t h o d . In this subsequent method N i - R was inhibited by the addition of f l u o r i d e , a s p e c i f i c i n - h i b i t o r ( M O R T O N 1956, N A S O N and E V A N S 1953), by w h i c h n i t r i t e a c c u m u l a t e s w h i c h can then be d e t e c t e d by the G r i e s s - I l o s v a y e reagent as explained in F i g . I d .
U s i n g this m o d i f i e d version of the N a - R test ( N a - R + F ) , it could be shown that in f a c t a l l marine fungi tested produce N a - R , even in those cases where the n o r m a l test had given negative or u n c e r t a i n results (Table 1). In a d d i t i o n , it was shown that generally those strains w h i c h display high a c t i v i t y in the n o r m a l N a - R t e s t , also exhibit high a c t i v i t y in the m o d i f i e d test (Table 1).
N i t r o g e n o f t e n c o n s t i t u t e s a g r o w t h - l i m i t i n g f a c t o r in fungi. N i t r o g e n reductase enables the organism producing this e n z y m e to use n i t r a t e as a nitrogen supply.
B R E S I N S K Y and S C H N E I D E R (1975) in their investigation of nearly 200 t e r r e s - t r i a l f u n g i , using the same test for N a - R as in this paper have shown that most of the D e u t e r o m y c e t e s t e s t e d produce this e n z y m e whereas most of the 135 B a - s i d i o m y c e t e s investigated lack N a - R .
M O L I T O R I S and S C H A U M A N N (1986) have shown the presence of N a - R in a l l obligate and f a c u l t a t i v e marine fungi they i n v e s t i g a t e d . S C H A U M A N N et a l . (1986), however, using the same test did not d e t e c t this e n z y m e in some species of Lulworthia. In this paper by far the majority of the 81 m a r i n e fungal strains investigated showed N a - R a c t i v i t y . Only a few strains gave u n c e r t a i n results or did not show this e n z y m e at a l l . This was found in p a r t i c u l a r for the marine yeasts, for a few D e u t e r o m y c e t e s and for a few A s c o m y c e t e s as in some of the Lulworthia strains i n v e s t i g a t e d . G e n e r a l l y , in those cases where s e v e r a l strains of a given species were i n v e s t i g a t e d , the N a - R a c t i v i t i e s observed were rather s i m i l a r .
Since the n i t r i t e produced f r o m n i t r a t e by N a - R can be m e t a b o l i z e d very q u i c k l y by fungi ( N I C H O L A S 1965) and might therefore evade d e t e c t i o n in the medium by our test, we inhibited N i - R by the addition of fluoride ( M O R T O N 1956, m o d i - fied) to prevent the m e t a b o l i z a t i o n of n i t r i t e and to obtain the a c c u m u l a t i o n of this m e t a b o l i t e ( F i g . Id).
U s i n g this m o d i f i e d test a l l strains including those w i t h low, u n c e r t a i n or no N a - R in the n o r m a l test now produced the red c o l o u r , i n d i c a t i n g the presence of n i t r i t e and t h e r e f o r e p r o d u c t i o n of N a - R .
O f a l l the enzymes i n v e s t i g a t e d so far using the same method ( M O L I T O R I S and S C H A U M A N N 1986) n i t r a t e reductase is the only one c o m m o n to a l l marine fungi investigated w i t h the same methodology. Since natural s e a w a t e r contains
considerable amounts of nitrogen in the f o r m of n i t r a t e (up to 600 ^ig/l), marine fungi producing this e n z y m e would be able to use n i t r a t e as a nitrogen source.
Possession of N a - R t h e r e f o r e would c o n s t i t u t e an important s e l e c t i v e advantage for these organisms, explaining our result that a l l marine fungi investigaterd so f a r contain this e n z y m e .
S i m i l a r l y , the lack of n i t r a t e reductase in many t e r r e s t r i a l fungi, could be e x - plained by the lack of an urgent need for nitrogen in this environment because of the a v a i l a b i l i t y of o f t e n high amounts of nitrogen-containing organic m a t e r i a l . The absence of N a - R observed in p a r t i c u l a r in many t e r r e s t r i a l B a s i d i o m y c e t e s can be easily understood since they comprise a high number of m y c o r r h i z a l strains which by their symbiosis w i t h higher plants f u l f i l their nitrogen r e q u i r e - ments by d i r e c t import of organic m a t e r i a l from these plants.
Acknowledgements
The authors are g r a t e f u l to S. C r o w , J . F e l l , E . B . G . Jones, J . K o h l m e y e r , S.
R o h r m a n n and K . Schaumann, for kindly providing fungal cultures and to R . O w e n , for c r i t i c a l l y reading the English t e x t .
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