The absence of deoxyriboaldolase activity in a thymineless mutant of Escherichia coli strain 15: A possible explanation for the low thymine requirement of some thymineless strains

PRELIMINARY NOTES

217

That the species differences m a y be due to contaminating RNA residues also seems unlikely, since differences in electrophoretic migration would have been observed as a function of per cent RNA contamination, and they were not. Furthermore, t h a t the contaminating RNA has very little effect on the electrophoretic pattern of ribosomal proteins has been shown by WALLER AND H A R R I S 6, who found no differences between the electrophoretic behaviour of isolated ribosomal proteins or entire ribosomes of Escherichia coli. It is possible that the residual RNA is detached from the proteins b y the effect of the ureaL In conclusion, our results show that ribosomal proteins of different species can be distinguished clearly in electrophoretograms, which exhibit species-specific patterning. No differences in ribosomal proteins of dilferent tissues or those at different developmental stages of the same embryo were detected with the same technique. In view of the importance of this finding, work is in progress to circumstantiate this evidence further. This investigation has been supported ill part by grants from the National Institutes of Health (TW-ooI99 to Dr. GIUBICE and RG-o26II to Dr. MONROY).

Laboratory o/ Comparative Anatomy o/ the University and Research Unit /or Molecular Embryology o~ the C.N.R., Palermo (Italy)

V. MUTOLO G. GIUDICE V. HOPPS G. DONATUTI

I S. A. YANKOFSRY AND S. SPIEGELMAN, Proc. Natl. Acad. Sci. U.S., 48 (1962) lO69. 2 A. I. ARONSON, Biochim. Biophys. Acta, 72 (1963) 176. 3 W'. S. ROBINSON, W. T. H s o , C. F. FOX AND S. ]3. WEISS, J. Biol. Chem., 239 (1964) 2944. 4 0 . H. LOWRY, N. J. ROSEBROUGH, A. L. I~'ARR AND R. J. RANDALL, J. Biol. Chem., 193 (1952) 265. 5 R. A. REISFELD, U. J. L E w I s AND D. E. WILLIAMS, Nature, 195 (1965) 281. J. V. WALLER AND J. I. HARRIS, Proc. Natl. Acad. Sci. U.S., 47 (1961) 18. 7 P. SPITNn~-ELSON, Biochim. Biophys. Acta, 55 (1962) 741. S G. GIVDICE AND V. MUTOLO, Biochim. Biophys. Acta, 138 (1967) 276.

Received December 5th, 1966

Biochim. Biophys. Acta, 138 (1967) 214-217

BBA 91157 The absence of deoxyriboaldolase activity in a thymineless mutant of Escherichia coli strain t5: A possible explanation for the low thymine requirement of some thymineless strains The transformation of a wild-type Escherichia coli to a strain requiring exogenous thymine at a concentration essentially equivalent to that utilized for DNA synthesis requires two mutations 1. The first mutation, the loss of thymidylate synthetase activity, results ill a strain having a thymine requirement approx. Io-fold greater than that utilized for DNA formation 2. The second mutation had not been identified.

Biochim. Biophys. Acta, 138 (1967) 217-22o

218

PRELIMINARY NOTES

However, previous results showed that of two thymineless mutants of E. coli strain 15, 'strain 7o'*, a one-step mutation from strain 15 (ref. 3) and 'strain 7oV3 '**, a one-step mutation from strain 7 o, only strain 7oV3 excreted deoxyribose during thymine starvation 1. This observation led to a study of the metabolism of deoxyribose phosphate in these two thymineless strains. The data presented in this paper show that strain 7oV3, when growing on dThd, catabolizes dThd at a lower rate than strains 15 and 7 ° and does not degrade the deoxyribose derived from dThd. Also shown is that strain 7oV3 has lost deoxyriboaldolase (2-deoxy-I~-ribose-5-phosphate acetaldehyde-lyase, EC 4.1.2.4) activity, accounting for its inability to degrade deoxyribose phosphates. Analysis of the supernatant fluid from cultures grown on I mM dThd show that the media from strain 7oV3 contained equimolar concentrations of thymine and deoxyribose while media from strains 15 and 7 ° contained only thymine (Table I). TABLE

I

METABOLISM

OF D E O X Y T H Y M I D I N E

BY GROWING CULTURES

OF E .

coli

E x p o n e n t i a l - p h a s e cells w e r e i n o c u l a t e d to a c o n c e n t r a t i o n of 4" lOT c e l l s / m l i n t o m i n i m a l m e d i a 4, c o n t a i n i n g 0.2 % glucose a n d i mM d T h d , a n d w e r e g r o w n to a c o n c e n t r a t i o n of 4" lOS c e l l s / m l a t 37 ° w i t h a e r a t i o n . T h e cells were r e m o v e d b y c e n t r i f u g a t i o n ; t h e s u p e r n a t a n t f r a c t i o n w a s a n a l y z e d for d e o x y r i b o s e b y BURTON'S 5 d i p h e n y l a m i n e p r o c e d u r e a n d for t h y m i n e b y t h e spect r o p h o t o m e t r i c m e t h o d of HOTCHKISS s. TO r e a c h a c o n c e n t r a t i o n of 4' 1°8 cells/ml, c u l t u r e s of s t r a i n s 15 a n d 7 ° r e q u i r e d 2 h, and, d u e to i n h i b i t i o n b y d T h d , c u l t u r e s of s t r a i n 7oV3 r e q u i r e d 3 h. Specific r a t e w a s c a l c u l a t e d a c c o r d i n g to KOCH ~.

Strain

Thymine ([.*moles/ml supernatant [raction )

Deoxyribose (tzmoles /ml supernatant [raction

Speci[ic rate (ml~moles thymine [ormed × Io -8 bacteria × h -1)

15 7° 7oV3

0.89 0.85 0.29

0.04 0.04 o.29

200 200 52

Table I also shows that when all cultures had attained equal cell concentrations strains 15 and 7 ° had degraded a 3-fold greater amount of dThd than strain 7oV3. The specific rate of appearance of thymine in the media (Table I) is indicative of thymidine phosphorylase (EC 2.4.2.4) activity in vivo of cultures growing on dThd, a compound known to result in thymidine phosphorylase induction 8. The lower thymidine phosphorylase activity in vivo of strain 7oV3 as compared with strains 15 and 70 could have resulted from one or a combination of the following: (a) a lower induced level of thymidine phosphorylase; (b) a lower differential rate of synthesis of thymidine phosphorylase; and (c) an accumulation of deoxyribose phosphate. Results supporting each of these possibilities are shown below. (a) The data in Table I I show that sonic extracts of strains 15 and 7 ° grown on dThd have thymidine phosphorylase specific activities approx. 1.5-fold greater than strain 7oV3 . The specific activity of thymidine phosphorylase of strain 7oV3, given in Table II, is at a m a x i m u m since no further increase in specific activity occurs with growth on concentrations of dThd up to 2 raM. However, the thymidine phos* F u l l n a m e : E. coli s t r a i n 7o-462. *~ F u l l n a m e : E. eoli s t r a i n 7 ° V3-462.

Biochim. Biophys. Acta, 138 (1967) 217 220

219

PRELIMINARY NOTES TABLE II

THYMIDINE PHOSPHORYLASE AND DEOKYRIBOALDOLASE ACTIVITIES IN SONIC EXTRACTS FROM CULTURES OF E. coli GROWN ON THYMINE AND DEOXYTHYMIDINE Cells g r o w n on e i t h e r i mM d T h d or i mM t h y m i n e a n d i s o l a t e d as d e s c r i b e d in T a b l e I w e r e w a s h e d t w i c e in o.o 5 M Tris, p H 7. The cells were d i s r u p t e d b y s u s p e n d i n g 4' I°1° cells i n IO m l of o.05 M T ris ( c o n t a i n i n g O.Ol 4 M t h i o g l y c o l l i c acid a n d a d j u s t e d t o p H 7.1 w i t h HC1 a t 25 °) a n d s o n i c a l l y t r e a t i n g for o. 5 m i n w i t h a B r a n s o n S-75 Sonifier a t 8 A. D u r i n g sonic t r e a t m e n t , t h e s u s p e n s i o n w a s in a s t a i n l e s s s t e e l t u b e a r o u n d w h i c h c i r c u l a t e d c o o l a n t a t -- IO °. T h y m i d i n e p h o s p h o r y l a s e w a s a s s a y e d a c c o r d i n g to FRIEDKIN AND ROBERTS 9. D e o x y r i b o a l d o l a s e w a s a s s a y e d in a r e a c t i o n m i x t u r e b a s e d on PRICER AND HORECKER 1°. The r e a c t i o n m i x t u r e c o n t a i n e d 5/~mo les of p o t a s s i u m m a l e a t e buffer, p H 6.9; 20o m # m o l e s d R i b - 5 - P , a n d sonic e x t r a c t in a t o t a l v o l u m e of o . i ml. A f t e r i n c u b a t i o n a t 37 °, t h e r e a c t i o n w a s s t o p p e d b y t h e a d d i t i o n of o.9 ml of o.5 M p e r c h l o r i c acid. T h e m i x t u r e was a n a l y z e d for loss of d e o x y r i b o s e b y BURTON'S 5 d i p h e n y l a m i n e p r o c e d u r e . M e a s u r e m e n t of d R i b - 5 - P s y n t h e s i s a c c o r d i n g to PRICER AND HORECKER 1° g a v e c o m p a r a b l e a c t i v i t i e s . One u n i t of e n z y m e will d e g r a d e one m/zmole of s u b s t r a t e p e r rain.

Strain

Thymidine phosphorylase (units[rag protein)

Deoxyriboaldolase (units/rag protein)

Grown on thymine 15 7° 7oV3

35 4° 22

14 14 < i

493 520 34 °

217 255 <5

Grown on dThd 15 7° 7°V3

phorylase specific activity of strains 15 and 7 ° are approx. 12oo units/rag protein when grown on 2 mM dThd. (b) For cultures growing on I mM dThd, the differential rates of synthesis of thymidine phosphorylase are: 5.3 units/#g protein for strains 15 and 7 o, and 3.0 units/ /,g protein for strain 7oV3. (c) While the specific activities for thymidine phosphorylase in sonic extracts of strains grown on I mM dThd (Table II) and the differential rates of synthesis of thymidine phosphorylase are qualitatively in agreement with measurements of thymidine phosphorylase activity in vivo (Table I), all inhibitor of thymidine phosphorylase activity also had to be considered since strain 7oV3 did not degrade deoxyribose in vivo (Table I). The catabolism of the deoxyribose moiety of dThd is mediated by the following 3 enzymatic reactions: thymidine phosphorylase dThd + Pt < > thymine+dRib-i-P dRib-i-P

phosphodeoxyribomut ase ~ dRib-5-P

deoxyriboaldolase dRib-5-P < ~ acetaldehyde+glyceraldehyde 3-phosphate

(i) (2) (3)

As shown in Table II, strain 7oV3 has no deoxyriboaldolase activity, accounting for its inability to degrade deoxyribose in vivo (Table I). Because the enzymatic reactions leading to the production of dRib-5-P from dThd are reversible, all intracellular accumulation of dRib-5-P would lead to an increase in d R i b - i - P concenBiochim. Biophys. Acta, 138 (1967) 217-22o

220

PRELIMINARY NOTES

tration and a decrease in dThd catabolism. Therefore, the 4-fold lower thymidine phosphorylase catabolic activity in vivo of strain 7oV3 compared to strains 15 and 7 ° (Table I) appears to be a result of less thymidine phosphorylase (Table II) and inhibition of dThd phosphorolysis by intracellular deoxyribose phosphate. The high thymine concentrations required b y strain 7 ° for optimal growth m a y function like the accumulation of deoxyribose phosphate in strain 7oV3 in decreasing dThd phosphorolysis. In addition, thymine is a potent inhibitor of dThd phosphorolysis in vitrog, 11 and m a y also function in this capacity in vivo. Both the high thymine concentration required for optimal growth of strain 7 ° and the mutation in strain 7oV3 probably allow for a net rate of dThd synthesis commensurate with the requirement for DNA synthesis. As shown in Table II, cells grown on dThd had approx. I5-fold greater thymidine phosphorylase and deoxyriboaldolase activities than ceils grown on thymine. These data suggest that the two enzymatic activities m a y be associated with the same protein or that they are under coordinate control. In either case they should have the same inducer. The induction of deoxyriboaldolase was first described by PRICER AND HORECKER10 in Lactobacillus plantarum grown on media containing deoxyribose. RAZZELL AND CASSHYAP11 have eliminated a nucleoside as an inducer of thymidine phosphorylase and have suggested that d R i b - I - P induces this enzyme. Another inducer compound which would be consistent with their results, as well as ours, is dRib-5-P. The failure of RAZZELL AND CASSHYAP to observe induction of thymidine phosphorylase by two thymineless mutants of E. coli K I 2 could possibly be because these mutants lack phosphodeoxyribomutase and cannot synthesize dRib-5-P from dThd.

Laboratory o/ Physiology, National Cancer Institute, National Institutes o/ Health, Bethesda, Md. (U.S.A.) I 2 3 4 5 6 7 8 9 IO ii

T. R. BREITMAN R. M. BRADFORD

T. R. BREITMAN AND R. M. BRADFORD, Biochem. Biophys. Res. Commun., 17 (1964) 786. T. R. BREITMAN AND R. M. BRADFORD, J. Bacteriol., in t h e press. R. R. ROEPKE AND V. E. MERCER, J. Bacteriol., 54 (1947) 731. B. D. DAVIS AND E. S. MINGIOLI, J. Bacteriol., 6o (195 o) 17. K. BURTON, Biochem. J., 62 (1956 ) 315 . R. D. HOTCHKISS, J. Biol. Chem., 175 (1948) 315 . A. L. KOCH, J. Biol. Chem., 217 (1955) 931M. RACHMELER, J. GERHART AND J. ROSNER, Biochim. Biophys. Acta, 49 (1961) 222. M. FRIEDKIN AND D. ROBERTS, J. Biol. Chem., 207 (1954) 245. V¢. E. PRICER, Jr. AND B. L. HORECKER, J. Biol. Chem., 235 (196o) 1292. VV. E. RAZZELL AND P. CASSHYAP, J. Biol. Chem., 239 (1964) 1789.

Received J a n u a r y 2nd, 1967 Biochim. Biophys. Acta, 138 (1967) 217-22o