257
Biochimica et Biophysica Acta, 428 (1976) 257--259 O Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA Report BBA 21422
DECREASED RIBOFLAVIN FORMATION IN MUTANTS OF
AEROBACTER (ENTEROBACTER) AEROGENES DEFICIENT IN THE BUTANEDIOL PATHWAY
K L A U S B R Y N and F R E D R I K C. ST~bRMER Department of Biochemistry, Universityof Oslo, and National Instituteof Public Health, Oslo (Norway) (Received January 8th, 1976)
Summary Riboflavin concentration increased linearly for more than 60 h in wild type cultures, whereas in three mutants deficient in the formation of acetoin and 2,3-butanediol the production ceased at the end of exponential growth.
During a physiological and biochemical survey of the butanediol pathway in Aerobacter (Enterobacter) aerogenes [1] we observed thatstationary cultures of the wild type turned yellow, in contrast to those of three mutants deficient in this pathway i.e. in the formation of acetolactate, acetoin, and 2,3-butanediol. Riboflavin has now been identified as the compound responsible for this color. Among several pathways leading to acetoin in microorganisms, the only one which has been well characterized is the butanediol pathway of A. aerogenes: 2 pyruvate -* acetolactate -* acetoin ~-) 2,3-butanediol. The three enzymes involved have been purified and characterized [2]. The formation of diacetyl, which can be irreversibly reduced to acetoin by the last enzyme in the sequence, still is unknown. Oxidation of acetoin to diacetyl occurs in some organisms [3]. In A. aerogenes two different enzymes can form acetolactate from pyruvate [4]. One is the pH 6 acetolactate-forming enzyme, catalyzing the first reaction in the butanediol pathway. The other is acetohydroxyacid synthetase involved in the biosynthesis of valine,: leucine, and isoleucine. Extensive studies have not elucidated all steps of riboflavin biosynthesis. The primary precursor is guanine, or a guanine compound [5] and in one of the latter steps a four carbon moiety is incorporated (Fig. 1). The nature of this moiety remains unknown, partly due to conflicting reports of incorporation experiments [6]. Acetoin or diacetyl has been proposed as the
258
O
HsC'~N~N/H Ou.n,o.
Ribitol Fig.1. B i o s y n t h e s i s o f r i b o f l a v i n [ 5 ] . T h e f o u r c a r b o n m o i e t y (C 4 ) i n c o r p o r a t e d as a p a r t o f 6,7-dim e t h y l - 8 - r i b i t y l l u m a z i n e is i n d i c a t e d .
origin of the four carbon part of riboflavin in Eremothecium ashbyii and Candida spp. [7,8], but this has not been proved. Investigators of riboflavin biosynthesis have utilized nonflavinogenic mutants of riboflavin overproducing yeasts, filamentous fungi, and bacteria, e.g.A, aerogenes [ 5,6]. It appears that nobody has utilized mutants defective in the formation of acetolactate (defect pH 6 acetolactate-forming enzyme), acetoin, or diacetyl. We have used A. aerogenes mutants III-45, IV-2, and P 14-43, which all form less than 1% of the wild type level of these metabolites [1]. The mutants have normal level of acetohydroxyacid synthetase, but I
I
I
24
36
1,0
z ~" O.5 J C~ ~Jo
12
48
HOURS
F i g . 2 . E x t r a c e l l u l a r r i b o f l a v i n in b a t c h c u l t u r e s o f A e r o b a c t e r aerogenes w i l d t y p e a n d t h r e e m u t a n t s w i t h d e f i c i e n c i e s i n t h e b u t a n e d i o l p a t h w a y . T h e o r g a n i s m s u s e d w e r e t h e w i l d t y p e s t r a i n 1 0 3 3 (e), a n d m u t a n t s I I I - 4 5 Gg, IV-2(~), a n d P 1 4 - 4 3 (*). M u t a n t I I I - 4 5 l a c k s all t h r e e e n z y m e s o f t h e b u t a n e d i o l p a t h w a y , a n d I V - 2 h a s less t h a n five p e r c e n t o f t h e f i r s t a n d l a s t e n z y m e [ 1 2 ] . T h e b a c t e r i a w e r e g r o w n a t 3 7 ° C a n d 1 3 0 r e v . / m i n in a m i n i m a l m e d i u m [ 1 3 ] , w i t h t r a c e e l e m e n t s [ 1 4 ] , a n d 1% g l u c o s e ( D i f c o ) . T h e media of the mutants contained L-leucine (12.5 rag), DL-isoleucine (25 mg), DL-vaiine (25 mg) [9], a n d 1 0 m g of g u a n i n e / l i t e r w a s a d d e d t o P 1 4 - 4 3 [ 1 1 ] . T h e e x p e r i m e n t s w e r e p e r f o r m e d in d a r k n e s s o r d i m l i g h t . S a m p l e s o f 5 m l w e r e w i t h d r a w n , a n d cells w e r e r e m o v e d b y c e n t r i f u g a t i o n a t 2 0 0 0 0 × g f o r 2 0 m i n . S u p e r n a t a n t s , a c i d i f i e d w i t h a n e q u a l v o l u m e o f ice c o l d 1 0 % t r i c h l o r o a c e t i c a c i d , w e r e c h r o m a t o g r a p h e d o n Florisil [ 1 5 ] (6 × 2 0 r a m ; 6 0 - - 8 0 m e s h , F l u k a A G , G e r m a n y ) . A f t e ~ w a s h i n g t h e c o l u m n w i t h 4 m l p o r t i o n s o f a c e t i c a c i d (2%), w a t e r , a n d p y r i d i n e (0.5%), a single c o m p o u n d w i t h y e l l o w f l u o r e s c e n c e w a s e l u t e d w i t h 5% p y r i d i n e . R i b o f l a v i n w a s d e t e r m i n e d i n e l u a t e s b y f l u o r i m e t r y , w i t h a u t h e n t i c r i b o f l a v i n as i n t e r n a l s t a n d a r d , a n d s o d i u m d i t h i o n i t e as a b l e a c h i n g a g e n t [ 1 6 ] . T h e identity of riboflavin was verified by the fluorescence characteristics (exltation maximum at 370 nm, e m i s s i o n a t 5 3 0 n m ) o f e l u a t e s f r o m t h e Flortsil c o l u m n . P a p e r c h r o m a t o g r a p h y o f 1 0 #1 e l u a t e o n W h a t m a n N o . 1 r e v e a l e d n e g l i g i b l e y e l l o w f l u o r e s c e n c e b e s i d e s r i b o f l a v i n s p o t s in t h e t h r e e s y s t e m s , I, 5% Na~ HPO 4 ; II, n-butanol~ethanol~5% Na~ HPO 4 (7:3:1); III, n - b u t a n o l / a c e t i c a c i d / w a t e r (4:1: 5, upper phase). In a sample exposed to ultraviolet light [16] the riboflavin spot disappeared, and a new s p o t m i g r a t e d as p h o t o d e c o m p o s e d a u t h e n t i c r i b o f l a v i n .
259
require valine, leucine, and isoleucine due to deficiencies in some of the other enzymes leading to their formation [9]. The accumulation of riboflavin in cultures of A. aerogenes is shown in Fig. 2. In the wild type, riboflavin concentration increased with a nearly constant rate for several days (at least for 60 h, not shown in Fig. 2), whereas exponential growth lasted for 6 hours [1]. In mutant cultures negligible increase in riboflavin levels were observed after about 8 hours. In a separate experiment the wild t y p e was grown in the presence of valine, which may be inhibitory u p o n the formation of diacetyl [10]. No effect on growth or riboflavin formation was observed. The very low amounts of riboflavin in the culture of mutant P 14-43 may in part be related to the guanine requirement of this strain. The normal amounts of riboflavin formed b y mutants III-45 and IV-2 (Fig. 2) during the 6 h exponential growth period [1] is not surprising. Acetolactate may have been provided by acetohydroxyacid synthetase, which is active during exponential growth [11 ]. These mutants have normal level of acetohydroxyacid synthetase [9]. It has also been shown that the mutants behave identically to the wild type in this period, with respect to growth rate and pH [1]. From our experiments it cannot be concluded that acetolactate or acetoin participate directly in the biosynthesis of riboflavin. The low pH levels which are always obtained in the mutant cultures [1], may have had an inhibitory effect u p o n the formation of riboflavin. It thus remains an open question b y which means the butanediol pathway exerts its effect, either b y controlling the pH, or by the formation of metabolite(s) to be incorporated. Obviously, mutants with known defects in the pathway leading to the presumed precursors of riboflavin are well suited for such studies. References 1 Johansen, L., Bryn, K. and St~rmer, F.C. (1975) J. Bacteriol. 123, 1124--1130 2 St#truer, F.C. (1975) M e t h o d s in E n z y m o l o g y (Colowick, S.P. and Kaplan, N.O., eds.), Vol. 41, part B, pp. 518--533, A c a d e m i c Press inc., New Y o r k 3 Sebek, O.K. and Randles, C.I. (1952) Arch. Biochem. Biophys. 40, 373--380 4 Halpern, Y.S. and Umbarger, H.E; (1959) J. Biol. Chem. 234, 3067--3071 5 Bacher, A. and Mail~'nder, B. (1973) J. Biol. Chem. 248, 6227--6231 6 Demain, A.L. (1972) Annu. Rev. Microbiol. 26~ 369--388 7 Goodwin, T.W. and Treble, D.H. (1958) Biochem. J. 70, 14 P 8 Schlee, D. and R e i n b o t h e , H. (1970) Z. AUg. Mikrobiol. 10, 77--80 9 Halpern, Y.S. and Even-Shosan, A. (1967) Biochim. Biophys. Acta 139, 502--504 10 Portno, A.D. (1966) J. Inst. Brew. 72, 193--196 11 Umbarger, H.E., Brown, B. and Eyring, E.J. (1960) J. Biol. Chem. 235, 1425--1432 12 St~rmer, F.C. (1968) FEBS Lett. 2, 36--38 13 Davis, B.D. and Mingioli, E.S. (1950) J. Bacteriol. 60, 17--28 14 Kogut, M. and Podoski, E.P. (1953) Biochem. J. 55, 800--808 15 Dimant, E., Sanadi, D.R. and H u e n n e k e n s , F.M. (1952) J. Am. Chem. Soc. 74, 5440--5444 16 Yagi, K. (1962) in M e t h o d s of B i o c h e m i c a l Analysis (Glick, D., ed.), Vol. 10, pp. 319--356s J o h n Wiley & Sons, N e w York
Biochimica et Biophysica Acta, 428 (1976) 257--259 O Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA Report BBA 21422
DECREASED RIBOFLAVIN FORMATION IN MUTANTS OF
AEROBACTER (ENTEROBACTER) AEROGENES DEFICIENT IN THE BUTANEDIOL PATHWAY
K L A U S B R Y N and F R E D R I K C. ST~bRMER Department of Biochemistry, Universityof Oslo, and National Instituteof Public Health, Oslo (Norway) (Received January 8th, 1976)
Summary Riboflavin concentration increased linearly for more than 60 h in wild type cultures, whereas in three mutants deficient in the formation of acetoin and 2,3-butanediol the production ceased at the end of exponential growth.
During a physiological and biochemical survey of the butanediol pathway in Aerobacter (Enterobacter) aerogenes [1] we observed thatstationary cultures of the wild type turned yellow, in contrast to those of three mutants deficient in this pathway i.e. in the formation of acetolactate, acetoin, and 2,3-butanediol. Riboflavin has now been identified as the compound responsible for this color. Among several pathways leading to acetoin in microorganisms, the only one which has been well characterized is the butanediol pathway of A. aerogenes: 2 pyruvate -* acetolactate -* acetoin ~-) 2,3-butanediol. The three enzymes involved have been purified and characterized [2]. The formation of diacetyl, which can be irreversibly reduced to acetoin by the last enzyme in the sequence, still is unknown. Oxidation of acetoin to diacetyl occurs in some organisms [3]. In A. aerogenes two different enzymes can form acetolactate from pyruvate [4]. One is the pH 6 acetolactate-forming enzyme, catalyzing the first reaction in the butanediol pathway. The other is acetohydroxyacid synthetase involved in the biosynthesis of valine,: leucine, and isoleucine. Extensive studies have not elucidated all steps of riboflavin biosynthesis. The primary precursor is guanine, or a guanine compound [5] and in one of the latter steps a four carbon moiety is incorporated (Fig. 1). The nature of this moiety remains unknown, partly due to conflicting reports of incorporation experiments [6]. Acetoin or diacetyl has been proposed as the
258
O
HsC'~N~N/H Ou.n,o.
Ribitol Fig.1. B i o s y n t h e s i s o f r i b o f l a v i n [ 5 ] . T h e f o u r c a r b o n m o i e t y (C 4 ) i n c o r p o r a t e d as a p a r t o f 6,7-dim e t h y l - 8 - r i b i t y l l u m a z i n e is i n d i c a t e d .
origin of the four carbon part of riboflavin in Eremothecium ashbyii and Candida spp. [7,8], but this has not been proved. Investigators of riboflavin biosynthesis have utilized nonflavinogenic mutants of riboflavin overproducing yeasts, filamentous fungi, and bacteria, e.g.A, aerogenes [ 5,6]. It appears that nobody has utilized mutants defective in the formation of acetolactate (defect pH 6 acetolactate-forming enzyme), acetoin, or diacetyl. We have used A. aerogenes mutants III-45, IV-2, and P 14-43, which all form less than 1% of the wild type level of these metabolites [1]. The mutants have normal level of acetohydroxyacid synthetase, but I
I
I
24
36
1,0
z ~" O.5 J C~ ~Jo
12
48
HOURS
F i g . 2 . E x t r a c e l l u l a r r i b o f l a v i n in b a t c h c u l t u r e s o f A e r o b a c t e r aerogenes w i l d t y p e a n d t h r e e m u t a n t s w i t h d e f i c i e n c i e s i n t h e b u t a n e d i o l p a t h w a y . T h e o r g a n i s m s u s e d w e r e t h e w i l d t y p e s t r a i n 1 0 3 3 (e), a n d m u t a n t s I I I - 4 5 Gg, IV-2(~), a n d P 1 4 - 4 3 (*). M u t a n t I I I - 4 5 l a c k s all t h r e e e n z y m e s o f t h e b u t a n e d i o l p a t h w a y , a n d I V - 2 h a s less t h a n five p e r c e n t o f t h e f i r s t a n d l a s t e n z y m e [ 1 2 ] . T h e b a c t e r i a w e r e g r o w n a t 3 7 ° C a n d 1 3 0 r e v . / m i n in a m i n i m a l m e d i u m [ 1 3 ] , w i t h t r a c e e l e m e n t s [ 1 4 ] , a n d 1% g l u c o s e ( D i f c o ) . T h e media of the mutants contained L-leucine (12.5 rag), DL-isoleucine (25 mg), DL-vaiine (25 mg) [9], a n d 1 0 m g of g u a n i n e / l i t e r w a s a d d e d t o P 1 4 - 4 3 [ 1 1 ] . T h e e x p e r i m e n t s w e r e p e r f o r m e d in d a r k n e s s o r d i m l i g h t . S a m p l e s o f 5 m l w e r e w i t h d r a w n , a n d cells w e r e r e m o v e d b y c e n t r i f u g a t i o n a t 2 0 0 0 0 × g f o r 2 0 m i n . S u p e r n a t a n t s , a c i d i f i e d w i t h a n e q u a l v o l u m e o f ice c o l d 1 0 % t r i c h l o r o a c e t i c a c i d , w e r e c h r o m a t o g r a p h e d o n Florisil [ 1 5 ] (6 × 2 0 r a m ; 6 0 - - 8 0 m e s h , F l u k a A G , G e r m a n y ) . A f t e ~ w a s h i n g t h e c o l u m n w i t h 4 m l p o r t i o n s o f a c e t i c a c i d (2%), w a t e r , a n d p y r i d i n e (0.5%), a single c o m p o u n d w i t h y e l l o w f l u o r e s c e n c e w a s e l u t e d w i t h 5% p y r i d i n e . R i b o f l a v i n w a s d e t e r m i n e d i n e l u a t e s b y f l u o r i m e t r y , w i t h a u t h e n t i c r i b o f l a v i n as i n t e r n a l s t a n d a r d , a n d s o d i u m d i t h i o n i t e as a b l e a c h i n g a g e n t [ 1 6 ] . T h e identity of riboflavin was verified by the fluorescence characteristics (exltation maximum at 370 nm, e m i s s i o n a t 5 3 0 n m ) o f e l u a t e s f r o m t h e Flortsil c o l u m n . P a p e r c h r o m a t o g r a p h y o f 1 0 #1 e l u a t e o n W h a t m a n N o . 1 r e v e a l e d n e g l i g i b l e y e l l o w f l u o r e s c e n c e b e s i d e s r i b o f l a v i n s p o t s in t h e t h r e e s y s t e m s , I, 5% Na~ HPO 4 ; II, n-butanol~ethanol~5% Na~ HPO 4 (7:3:1); III, n - b u t a n o l / a c e t i c a c i d / w a t e r (4:1: 5, upper phase). In a sample exposed to ultraviolet light [16] the riboflavin spot disappeared, and a new s p o t m i g r a t e d as p h o t o d e c o m p o s e d a u t h e n t i c r i b o f l a v i n .
259
require valine, leucine, and isoleucine due to deficiencies in some of the other enzymes leading to their formation [9]. The accumulation of riboflavin in cultures of A. aerogenes is shown in Fig. 2. In the wild type, riboflavin concentration increased with a nearly constant rate for several days (at least for 60 h, not shown in Fig. 2), whereas exponential growth lasted for 6 hours [1]. In mutant cultures negligible increase in riboflavin levels were observed after about 8 hours. In a separate experiment the wild t y p e was grown in the presence of valine, which may be inhibitory u p o n the formation of diacetyl [10]. No effect on growth or riboflavin formation was observed. The very low amounts of riboflavin in the culture of mutant P 14-43 may in part be related to the guanine requirement of this strain. The normal amounts of riboflavin formed b y mutants III-45 and IV-2 (Fig. 2) during the 6 h exponential growth period [1] is not surprising. Acetolactate may have been provided by acetohydroxyacid synthetase, which is active during exponential growth [11 ]. These mutants have normal level of acetohydroxyacid synthetase [9]. It has also been shown that the mutants behave identically to the wild type in this period, with respect to growth rate and pH [1]. From our experiments it cannot be concluded that acetolactate or acetoin participate directly in the biosynthesis of riboflavin. The low pH levels which are always obtained in the mutant cultures [1], may have had an inhibitory effect u p o n the formation of riboflavin. It thus remains an open question b y which means the butanediol pathway exerts its effect, either b y controlling the pH, or by the formation of metabolite(s) to be incorporated. Obviously, mutants with known defects in the pathway leading to the presumed precursors of riboflavin are well suited for such studies. References 1 Johansen, L., Bryn, K. and St~rmer, F.C. (1975) J. Bacteriol. 123, 1124--1130 2 St#truer, F.C. (1975) M e t h o d s in E n z y m o l o g y (Colowick, S.P. and Kaplan, N.O., eds.), Vol. 41, part B, pp. 518--533, A c a d e m i c Press inc., New Y o r k 3 Sebek, O.K. and Randles, C.I. (1952) Arch. Biochem. Biophys. 40, 373--380 4 Halpern, Y.S. and Umbarger, H.E; (1959) J. Biol. Chem. 234, 3067--3071 5 Bacher, A. and Mail~'nder, B. (1973) J. Biol. Chem. 248, 6227--6231 6 Demain, A.L. (1972) Annu. Rev. Microbiol. 26~ 369--388 7 Goodwin, T.W. and Treble, D.H. (1958) Biochem. J. 70, 14 P 8 Schlee, D. and R e i n b o t h e , H. (1970) Z. AUg. Mikrobiol. 10, 77--80 9 Halpern, Y.S. and Even-Shosan, A. (1967) Biochim. Biophys. Acta 139, 502--504 10 Portno, A.D. (1966) J. Inst. Brew. 72, 193--196 11 Umbarger, H.E., Brown, B. and Eyring, E.J. (1960) J. Biol. Chem. 235, 1425--1432 12 St~rmer, F.C. (1968) FEBS Lett. 2, 36--38 13 Davis, B.D. and Mingioli, E.S. (1950) J. Bacteriol. 60, 17--28 14 Kogut, M. and Podoski, E.P. (1953) Biochem. J. 55, 800--808 15 Dimant, E., Sanadi, D.R. and H u e n n e k e n s , F.M. (1952) J. Am. Chem. Soc. 74, 5440--5444 16 Yagi, K. (1962) in M e t h o d s of B i o c h e m i c a l Analysis (Glick, D., ed.), Vol. 10, pp. 319--356s J o h n Wiley & Sons, N e w York
