It has been suggested t h a t d u r i n g N2 fixation h y d r o x y l a m i n e is in-corporated into a m i n o acids via oximes, a n d this is discussed i n Chapter 5 b y V i r t a n e n a n d Miettinen. T h e condensation of h y d r o x y l a m i n e w i t h oxalacetic acid a n d reduction of t h e oxime to aspartic acid w h i c h is t h e n t r a n s a m i n a t e d to form other a m i n o acids h a s been suggested as a possible m e c h a n i s m (307, 3 0 8 - 3 1 1 ) . T h i s scheme, however, is not n o w generally accepted.
T h e incorporation of h y d r o x y l a m i n e into a m i n o acids via oximes i n Neurospora proposed b y Silver a n d M c E l r o y (269) has been mentioned previously. Silver a n d M c E l r o y found t h a t pyridoxinerequiring m u -tants of Neurospora a c c u m u l a t e d nitrite w h e n t h e v i t a m i n w a s de-ficient, a result suggesting t h a t pyridoxine is r e q u i r e d for n i t r i t e reduc-tion. T h e y suggest t h a t p y r i d o x i n e m a y act b y condensing w i t h h y d r o x y l a m i n e to form pyridoxal oxime, w h i c h could t h e n be reduced to a n a m i n e w i t h subsequent formation of a m i n o acids b y t r a n s a m i n a -tion reac-tions. Extracts of wild-type Neurospora however did not utilize pyridoxal phosphate oxime ( 2 1 1 , 212, 2 1 4 ) , a n d in a n o t h e r pyridoxine-r e q u i pyridoxine-r i n g m u t a n t t h e pyridoxine-r e w a s n o incpyridoxine-reased pyridoxine-r e q u i pyridoxine-r e m e n t fopyridoxine-r pypyridoxine-ridoxine w h e n it w a s g r o w n i n media containing n i t r a t e ( 2 1 4 ) . I t h a s been suggested b y Nicholas (212) t h a t one of t h e functions of oximes i n p l a n t s is to detoxicate h y d r o x y l a m i n e . I t is still a n open question w h e t h e r oximes a r e utilized b y microorganisms since care m u s t be taken i n interpreting data obtained w h e n large a m o u n t s of exogenous substrates a r e added to a n extract of a microorganism. I t is clear t h a t oximes (should t h e y be utilized) w o u l d occur i n v e r y small a m o u n t s i n
: Η 3 Ν202^ Ν20 N
t
2(8)
3. I N O R G A N I C N U T R I E N T N U T R I T I O N O F M I C R O O R G A N I S M S 395
association w i t h t h e e n z y m e s t h a t m i g h t convert t h e m to a m i n o acids.
T h e endogenous substrates or intermediates are also m a d e reactive in association w i t h t h e appropriate e n z y m e s in cell metabolism.
Little (152) claimed t h a t Neurospora crassa m i g h t utilize n i t r a t e via nitroaliphatic compounds such as n i t r o e t h a n e since t h e n i t r o e t h a n e oxidizing system, w h i c h produced nitrite, w a s induced b y n i t r a t e in t h e m e d i u m . T h e r e w a s n o definite correlation, however, between t h e ability to utilize n i t r a t e a n d t h e presence of nitroethane reductase in extracts of t h e fungal felts. T h e oxidation of organonitro compounds has been shown to occur in Pseudomonas a n d i n Nocardia species (46, It h a s been suggested that, w h e n nitrates are utilized, the n i t r a t e so formed combines w i t h a n organic compound w h i c h is t h e n reduced to a n a m i n e . T h u s Neurospora m u t a n t s w e r e u n a b l e to g r o w in a m i n i m a l m e d i u m containing a m m o n i a b u t utilized n i t r a t e a n d n i t r i t e ( 2 6 9 ) . Silver a n d M c E l r o y claim t h a t n i t r a t e or nitrite reduction involve a coupling to organic compounds w h i c h a r e t h e n reduced to a m i n o acids.
A n o t h e r interpretation is t h a t a m m o n i u m ions are toxic to these m u -tants a n d t h a t t h e m u t a n t s a r e also adversely affected b y t h e low p H of t h e a m m o n i u m cultures.
N i t r o a r o m a t i c compounds a r e utilized b y m a n y microorganisms a n d are reduced to the corresponding a r y l a m i n e . Neurospora reduced m-dinitrobenzene ( I ) to m-dinitroaniline ( I V ) w i t h D P N H or T P N H as t h e h y d r o g e n donor ( 3 3 4 ) . T h e intermediates w e r e identified as m-nitrosonitrobenzene ( I I ) a n d m - n i t r o p h e n y l h y d r o x y l a m i n e ( I I I ) . 5 0 , 5 1 , 2 7 0 ) .
m-dinitrdbenzene m-nitroso-nitrobenzene
(I) (Π)
m-dinitroàniUne (IV)
m-nitrophenyl-hydroxylamine
(IH)
Escherichia coli reduced t h e nitro group i n chloramphenicol. T h e e n z y m e is a flavoprotein w h i c h requires m a n g a n e s e for its activity. It is of interest t h a t t h e differential response of nitroreductase to Aureo-m y c i n i n a resistant a n d nonresistant strain w a s related to t h e degree of binding of flavin a n d m a n g a n e s e to t h e e n z y m e . T h e flavin a n d m e t a l are less firmly b o u n d to t h e e n z y m e i n t h e nonresistant strain a n d t h e e n z y m e is therefore m o r e susceptible to t h e chelating action of A u r e o m y c i n ( 2 5 4 ) . A nitroreductase of a Nocardia species w a s purified 200-fold (305, 3 0 6 ) . T h e e n z y m e catalyzed t h e reduction of m- a n d p-dinitrobenzene w i t h D P N H as t h e h y d r o g e n donor. T h e purified e n z y m e w a s not fully characterized, b u t evidence w a s presented t h a t flavin a n d a n unidentified m e t a l m i g h t be involved i n electron transfer between D P N H a n d t h e nitro compound.
T h e physiological role of these e n z y m e s is less clear. T h e e n z y m e is induced i n Neurospora b y adding either m - or /?-dinitrobenzene only to t h e cultures w h e r e a s e n z y m e s i n t h e nitrate-reducing sequence to am-monia a r e induced b y nitrate. T h e r e is n o evidence t h a t a w i d e r a n g e of aromatic n i t r o compounds a r e assimilated b y extracts of Neurospora.
T h e enzymes, therefore, m a y only provide a detoxication m e c h a n i s m in t h e fungus a n d act as suitable redox acceptors only, since t h e r e is no evidence t h a t t h e y a r e incorporated into cell nitrogen ( 2 1 1 , 2 1 2 ) .
D . N I T R I F I C A T I O N
T h e oxidation of a m m o n i a to n i t r i t e a n d n i t r a t e i n soils is brought about p r i m a r i l y b y microorganisms a n d the process is u s u a l l y termed nitrification. T h e biochemical aspects of nitrification a r e discussed in detail i n Chapter 6. Schloesing a n d M ü n t z (257) a n d W a r i n g t o n
(317) showed t h a t chloroform vapor inhibited n i t r i t e formation in soil a n d demonstrated t h a t bacteria w e r e involved. W i n o g r a d s k y ( 3 2 6 ) , w h o h a d worked previously w i t h iron a n d sulfur bacteria, showed t h a t t h e bacteria w e r e autotrophs a n d concluded t h a t t h e y g r e w without organic m a t t e r . H e isolated Nitrobacter a n d Nitrosomonas a n d grew t h e m on silica gel plates. Since t h a t t i m e various forms of nitrifiers have been isolated from various habitats including soils a n d seas ( 1 4 4 - 1 5 1 ) . Cultures of Nitrosomonas a n d Nitrobacter can be m a i n t a i n e d in simple inorganic liquid m e d i u m containing a m m o n i a or nitrite, respec-tively, a n d trace metals, especially iron. Solid calcium carbonate was for m a n y years assumed to be essential for g r o w t h since it was thought t h a t t h e bacteria would grow only w h e n attached to particles (150, 180, 181). Engel a n d A l e x a n d e r (75, 76) a n d Skinner a n d W a l k e r ( 2 7 1 a ) , however, obtained good growth of Nitrosomonas i n particlefree m e -d i u m b y substituting potassium carbonate for calcium carbonate. U n -d e r
3. I N O R G A N I C N U T R I E N T N U T R I T I O N O F M I C R O O R G A N I S M S 397
these conditions 72.4 m g d r y w e i g h t yield w a s obtained w i t h 0.083 M nitrous acid produced. T h e e n e r g y derived from t h e oxidation of a m m o n i a to n i t r a t e is utilized for anabolic processes in t h e organism.
Kingman-Boltjes (132) showed t h a t no g r o w t h occurred in t h e absence of C 02, a n d recently C1 4-labeled carbonate was shown to be t h e sole source of cell carbon ( 1 5 0 ) . T h e s e results confirm t h a t Nitrosomonas is a n autotrophic organism, b u t t h e r e h a v e been suggestions t h a t certain growth factors m a y be required ( 1 0 1 a ) . A n u m b e r of workers, however, did not find a n y effect of adding a r a n g e of vitamins a n d a m i n o acids to the cultures b u t reported t h a t corn-steep liquor or a dialyzed extract of its ash reduced the lag phase of nitrite production ( 1 5 0 ) . Meiklejohn (180) found t h a t iron was r e q u i r e d at a relatively h i g h concentration, 6 m g per liter, b u t this is probably due to residual quinoline chelating the added iron in t h e m e d i u m .
Lees a n d Quastel (143, 1 4 4 ) , using their ingenious soil percolation techniques (see Chapter 6 ) , studied i n detail nitrification in soil; t h e y concluded t h a t a m m o n i a is first adsorbed onto t h e surface of soil colloids before it is oxidized. T h i s m a y o n l y be a reflection of the site w h e r e the bacteria proliferate, w h e r e t h e r e is m a x i m u m aeration, a n d does not necessarily i m p l y a r e q u i r e m e n t per se for particles. Meyerhof (182) showed t h a t t h e p H o p t i m u m w a s between 8.5 a n d 8.8 for a m m o n i a concentration of 0.01 TV. T h e s e values for a m m o n i a a r e probably low since Engel a n d A l e x a n d e r h a v e shown t h a t 3 g m p e r liter is n o t toxic to Nitrosomonas (75, 7 6 ) . Lees (150) u s i n g washed suspensions of cells found t h a t a w i d e v a r i e t y of nitrogenous constituents w e r e not oxidized to n i t r a t e a n d t h a t chelating agents inhibit t h e oxidation of a m m o n i a . Hof m a n a n d Lees ( 1 1 1 , 112) showed t h a t a m m o n i a w a s quantitatively oxidized to nitrite a n d detected h y d r o x y l a m i n e as a n intermediate. A l k y l thiourea inhibited t h e oxidation of a m m o n i a non-competitively, b u t n o t t h a t of h y d r o x y l a m i n e . H y d r a z i n e inhibited the oxidation of h y d r o x y l a m i n e to nitrite. T h i s effect, also obtained i n cell-free extracts b y Nicholas a n d Jones ( 2 1 5 ) , w a s shown b y t h e m to be d u e to a competition for cytochrome c in t h e organism. T h e s e workers showed t h a t m e t h y l e n e blue, p h e n a z i n e methosulfate, a n d benzyl viologen w e r e suitable acceptors whereas ferricyanide, 2,3,6-trichloro-indophenol dye, D P N , T P N , or glutathione h a d little effect.
I n Nitrobacter, Lees a n d Simpson (151) reported absorption m a x i m a at λ 589, 557, 520, a n d 525 ταμ w h e n nitrite or dithionite w a s added to bacterial suspensions. A l e e m a n d N a s o n (4) suggested t h a t the oxida-tion of nitrite involved t h e transfer of electrons from n i t r i t e to molecular oxygen via cytochrome c a n d ατ (Eq. 9 ) .
N 02~ —• cytochrome c —» cytochrome αϊ —> 02 (9)
T h e y subsequently demonstrated t h a t t h e r e is a phosphorylation coupled to the oxidation of n i t r i t e although t h e P : 0 ratios w e r e n e v e r m o r e t h a n 0.2, a relatively inefficient system ( 5 ) . Malavolta et al. (168) h a v e also studied phosphorylation a n d t h e fixation of carbon dioxide i n extracts of Nitrobacter.
Heterotrophic fungi also oxidize a m m o n i a to nitrite a n d n i t r a t e . T h u s Aspergillus aureus, A. batatae produce n i t r a t e from n i t r i t e ( 2 5 1 ) . Aspergillus flavus w a s shown to oxidize a m m o n i a to n i t r i t e a n d n i t r a t e after it h a d completed its g r o w t h ( 2 5 8 ) . Nocardia corallina oxidizes p y r u v i c oxime a n d h y d r o x y l a m i n e to nitrite ( 1 2 6 ) . Streptomyces nitri-ficans w a s shown b y Isenberg a n d co-workers to form nitrite from u r e a or a m m o n i u m carbonate, b u t n i t r a t e w a s n o t formed ( 1 2 1 , 1 4 3 ) . A n u n u s u a l finding is t h a t preformed mycelia of Aspergillus niger will utilize t h e nitrogen from cyanide (122) a n d t h a t t h e latter compound stimulated g r o w t h i n Fusarium Uni ( 2 2 1 ) . T h e s e results a r e not u n -equivocal since t h e effect of c y a n i d e m i g h t be d u e to a metabolic disturbance diverting m o r e carbon into d r y m a t t e r ( 6 1 ) . A soil or-ganism, a n aerobic actinomycete, h a s been reported to u s e cyanide as t h e sole nitrogen a n d carbon source ( 5 8 ) .