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Mutations affecting the glyconeogenesis of Escherichia coli
140
SHORT COMMUNICATIONS
nation for the difference in the effects of chlortetracycline on protein biosynthesis in the intact animal and in vitro may lie in the inability of the drug to reach the site of protein biosynthesis in liver cells in vivo rather than its metabolic degradation since KELLY AND BUYSKE9 reported that tetracycline is excreted largely unchanged in the rat. The absence of an inhibitory effect of chlortetracycline administration on liver protein biosynthesis in vivo would have been expected from the known low toxicity of the drug 1°. Puromycin, in contrast, is markedly toxic to animals at dose levels higher than IOO mg/kg (see ref. II). I am grateful to A. GODFREY,B. PEARSON AND A. GREIG for technical assistance.
Imperial Chemical Industries Limited, Pharmaceuticals Division, Alderley Park, MaccIesiield, Cheshire (Great Britain)
T . J . FRANKLIN
I T. J. FRANKLIN, Biochem. J., 87 (1963) 449T. K. NIKOLOV AND A. T. ILKOV, Proc. Intern. Congr. Biochem. 5th, Moscow, 1961, p. 44. 3 p. FEIGELSON AND O. GREENGARD, J. Biol. Chem., 237 (1962) 3714 • i W. E. KNox, Brit. J. Exptl. Pathol., 32 (1951 ) 462. 5 p. •. CAMPBELL AND O. G-REENGARD, Biochem. J., 71 (1959) 149. e lZ. J. MANS AND G. D. NOVELLI, Arch. Biochem. Biophys., 47 (1961) 497. IV[. B. YARMOLINSKY AND G'. L. DE LA HABA, Proc. Natl. Acad. Sci. U.S., 45 (1959) 1721. 8 A. M. 1NEMETH AND G'. L. DE LA HABA, J. Biol. Chem., 237 (1962) 119o. 9 ~:{.. Cr. KELLY AND D. A. BUYSKE, J. Pharmacol. Exptl. Therap., 13o (196o) 144. 10 D. A. BUYSKE, H. J. EISNER AND tZ. Cs. KELLY, J. Pharmacol. Exptl. Therap., 13o (196o) 15o. 11 j. F. SHERMAN, D. J. TAYLOR AND H. Vq'. BOND, Antibiot. Ann., (1954-1955) p. 757.
Received June 4th, 1963 Biochim. Biophys. Acta, 76 (1963) 138-14o
SC 7094 Mutations affecting the glyconeogenesis of Escherichia coli The term glyconeogenesis has been applied to the biosynthetic pathway involved in the formation of carbohydrates from non-carbohydrate precursors 1. GOTTO AND POGELL~ have recently suggested that the acid phosphatase(s) of Escherichia coli play a role in the glyconeogenesis of this organism. With this hypothesis as a background we have tried to isolate mutants of E. coli which require carbohydrates for growth in the hope that solne of these might lack one or several of the acid phosphatase components present in the cell-free extracts of normal bacteria 3. No strains with abnormally low acid phosphatase activity have so far been obtained, but the procedure employed for isolating the mutants appears to be suitable for detecting a variety of mutants with defects in their glyconeogenesis. Mutants were isolated from a KI2 strain of E. coli (a strain designed E I 5, which is alkaline phosphatase negative, was kindly provided by Dr. A. GAREN, University of Pennsylvania). Cells from aerobic cultures in the exponential state of growth on o.I o~ glucose in medium 63 (see ref. 4) were collected by centrifugation and mutagenized with ethyl methanesulfonate (Eastman) according to the procedure of LIN et al. 5. I h treatment of 2 m] cells (lO9 cells/m]) with 0.03 ml ethyl methanesu]Biochim. Biophys. Acta, 76 (1963) 14o-142
141
SHORT COMMUNICATIONS
fonate at 37 ° resulted in approx. 99.9 % decrease in the viable count. After two subcultures (treated cells diluted I : IO) in the original medium, cultures were started on 0.5 % succinate as the sole source of carbon. 40o units of penicillin were then added per ml culture, when this was in the exponential state of growth. Fig. I shows a
~o
~7
o ~6
I
0
I
2
J
I
4
t
I
6 Time (h)
t
8
ig. I. Lysis of E. coli growing on succinate to which penicillin was added at the time indicated b y the arrow. The g r o w t h was followed in an E p p e n d o r f p h o t o m e t e r at 546 m#.
growth curve, where the resulting lysis is illustrated. Samples from the lysed culture were diluted and spread on agar containing medium 63 with o.I % glucose. After overnight incubation at 37 °, plates with a suitable number of colonies were replicated on agar containing succinate. Colonies which appeared only on the glucose plates were subsequently isolated and tested for growth in liquid cultures as well a~ on indicator agar. Table I summarizes the results of growth experiments with three mutant strains, each of which was isolated in a separate experiment. The mutant E 15/2 appears to require a carbohydrate for growth. However, when it was grown on limiting amounts of glucose plus increasing amounts of succinate an increase in the growth yield was obtained as illustrated in Fig. 2. This shows that although succinate cannot be used for the glyconeogenesis, it can still be metabolized by the cells. Similar results were obtained when glycerol or lactate were added to media containing limiting amounts of glucose or maltose. The strains E 15/1 and E 15/3 appear to be relatively similar in their physiological behaviour. No increase in growth yield was obtained when succinate was added to cultures of these strains grown on glucose. In contrast, the addition of 0.2 % succinate to a culture of E 15/1 growing on o.I % glucose caused a severe inhibition of growth. A similar but less pronounced inhibition by succinate was obtained also with strain E 15/3. These results indicate that the cells are permeable to succinate, and that this compound or a metabolic product of it is growth inhibitory. Biochim. Biophys. Acta, 76 (1963) 14o-142
142
SHORT COMMUNICATIONS
TABLE I AEROBIC GROWTH OF DIFFERENT STRAINS OF E. coli K I 2 IN THE MINERAL-SALTS MEDIUM 63 WITH VARIOUS SOURCES OF CARBON
E a c h c a r b o n s o u r c e w a s a d d e d a t a c o n c e n t r a t i o n of o.I }~o. + , g r o w t h w i t h i n 24 h; --, no g r o w l h within two days. Strain designation
Carbon source EI 5
D-Glucose D-Galactose D-Xylose Lactose Glycerol Lactate Acetate Malate Fumarate Succinate Acid-hydrolysed casein
EI5[I
EI5/2
EI513
+ + + + + + + + +
+ + + + + + + + +
~ + + + -----
+ + + + + + + + +
+
+
--
+
0.4 x
~0.3 ~D
,q~ 0.2 0.1 I
t
0.05 Glucose
o.10 (°/o)
Fig. 2. G r o w t h y i e l d of t h e m u t a n t E 15/2 o n g l u c o s e o n l y ( 0 - 0 ) , glucose + o.i % s u c c i n a t e ( × - × ) a n d g l u c o s e + 0.2 o/ /o s u c c i n a t e ( Q - O ) . T h e a m o u n t of g r o w t h "was d e t e r m i n e d p h o t o metrically. A l l the isolated mutants were found to have similar cell morphology and diagnostic properties as the parent strain, and they could also grow anaerobically on glucose. Further biochemical work is required to establish the metabolic block in each of these strains. The ease with which it appears to be possible to isolate mutants which are affected in their glyconeogenesis should make them suitable for studies of the enzyme reactions involved in this pathway. Presumably such strains may accumulate carbohydrate precursors if they are incubated in media containing other sources of carbon than carbohydrates.
Institule o/ Biochemistry,
BENGT V.
HOFSTEN
University o~ Uppsala, Uppsala (Sweden) H. A. KREBS, Bull. Johns Hopkins Hosp., 95 (1954) 19. 2 A. 3i. GOTTO AND B. M. POGELL, Biochem. Biophys. Res. Commun., 9 (1962) 381. 3 t3. v. HOFSTEN AND J. PORATH, Biochim. Biophys. Acta, 64 (1962) I. 4 A. B. PARDEE, F. JACOB AND J. ~-~IONOD, J . Mol. Biol., I (I959) 165. E. C. C. LIX, S. A. LERNER AND S. E. JORGENSEN, Biochim. Biophys. Acta, 60 (1962) 422.
Received June I7th , 1963 Biochim. Biophys. Acla, 76 (1963) 14o-142
SHORT COMMUNICATIONS
nation for the difference in the effects of chlortetracycline on protein biosynthesis in the intact animal and in vitro may lie in the inability of the drug to reach the site of protein biosynthesis in liver cells in vivo rather than its metabolic degradation since KELLY AND BUYSKE9 reported that tetracycline is excreted largely unchanged in the rat. The absence of an inhibitory effect of chlortetracycline administration on liver protein biosynthesis in vivo would have been expected from the known low toxicity of the drug 1°. Puromycin, in contrast, is markedly toxic to animals at dose levels higher than IOO mg/kg (see ref. II). I am grateful to A. GODFREY,B. PEARSON AND A. GREIG for technical assistance.
Imperial Chemical Industries Limited, Pharmaceuticals Division, Alderley Park, MaccIesiield, Cheshire (Great Britain)
T . J . FRANKLIN
I T. J. FRANKLIN, Biochem. J., 87 (1963) 449T. K. NIKOLOV AND A. T. ILKOV, Proc. Intern. Congr. Biochem. 5th, Moscow, 1961, p. 44. 3 p. FEIGELSON AND O. GREENGARD, J. Biol. Chem., 237 (1962) 3714 • i W. E. KNox, Brit. J. Exptl. Pathol., 32 (1951 ) 462. 5 p. •. CAMPBELL AND O. G-REENGARD, Biochem. J., 71 (1959) 149. e lZ. J. MANS AND G. D. NOVELLI, Arch. Biochem. Biophys., 47 (1961) 497. IV[. B. YARMOLINSKY AND G'. L. DE LA HABA, Proc. Natl. Acad. Sci. U.S., 45 (1959) 1721. 8 A. M. 1NEMETH AND G'. L. DE LA HABA, J. Biol. Chem., 237 (1962) 119o. 9 ~:{.. Cr. KELLY AND D. A. BUYSKE, J. Pharmacol. Exptl. Therap., 13o (196o) 144. 10 D. A. BUYSKE, H. J. EISNER AND tZ. Cs. KELLY, J. Pharmacol. Exptl. Therap., 13o (196o) 15o. 11 j. F. SHERMAN, D. J. TAYLOR AND H. Vq'. BOND, Antibiot. Ann., (1954-1955) p. 757.
Received June 4th, 1963 Biochim. Biophys. Acta, 76 (1963) 138-14o
SC 7094 Mutations affecting the glyconeogenesis of Escherichia coli The term glyconeogenesis has been applied to the biosynthetic pathway involved in the formation of carbohydrates from non-carbohydrate precursors 1. GOTTO AND POGELL~ have recently suggested that the acid phosphatase(s) of Escherichia coli play a role in the glyconeogenesis of this organism. With this hypothesis as a background we have tried to isolate mutants of E. coli which require carbohydrates for growth in the hope that solne of these might lack one or several of the acid phosphatase components present in the cell-free extracts of normal bacteria 3. No strains with abnormally low acid phosphatase activity have so far been obtained, but the procedure employed for isolating the mutants appears to be suitable for detecting a variety of mutants with defects in their glyconeogenesis. Mutants were isolated from a KI2 strain of E. coli (a strain designed E I 5, which is alkaline phosphatase negative, was kindly provided by Dr. A. GAREN, University of Pennsylvania). Cells from aerobic cultures in the exponential state of growth on o.I o~ glucose in medium 63 (see ref. 4) were collected by centrifugation and mutagenized with ethyl methanesulfonate (Eastman) according to the procedure of LIN et al. 5. I h treatment of 2 m] cells (lO9 cells/m]) with 0.03 ml ethyl methanesu]Biochim. Biophys. Acta, 76 (1963) 14o-142
141
SHORT COMMUNICATIONS
fonate at 37 ° resulted in approx. 99.9 % decrease in the viable count. After two subcultures (treated cells diluted I : IO) in the original medium, cultures were started on 0.5 % succinate as the sole source of carbon. 40o units of penicillin were then added per ml culture, when this was in the exponential state of growth. Fig. I shows a
~o
~7
o ~6
I
0
I
2
J
I
4
t
I
6 Time (h)
t
8
ig. I. Lysis of E. coli growing on succinate to which penicillin was added at the time indicated b y the arrow. The g r o w t h was followed in an E p p e n d o r f p h o t o m e t e r at 546 m#.
growth curve, where the resulting lysis is illustrated. Samples from the lysed culture were diluted and spread on agar containing medium 63 with o.I % glucose. After overnight incubation at 37 °, plates with a suitable number of colonies were replicated on agar containing succinate. Colonies which appeared only on the glucose plates were subsequently isolated and tested for growth in liquid cultures as well a~ on indicator agar. Table I summarizes the results of growth experiments with three mutant strains, each of which was isolated in a separate experiment. The mutant E 15/2 appears to require a carbohydrate for growth. However, when it was grown on limiting amounts of glucose plus increasing amounts of succinate an increase in the growth yield was obtained as illustrated in Fig. 2. This shows that although succinate cannot be used for the glyconeogenesis, it can still be metabolized by the cells. Similar results were obtained when glycerol or lactate were added to media containing limiting amounts of glucose or maltose. The strains E 15/1 and E 15/3 appear to be relatively similar in their physiological behaviour. No increase in growth yield was obtained when succinate was added to cultures of these strains grown on glucose. In contrast, the addition of 0.2 % succinate to a culture of E 15/1 growing on o.I % glucose caused a severe inhibition of growth. A similar but less pronounced inhibition by succinate was obtained also with strain E 15/3. These results indicate that the cells are permeable to succinate, and that this compound or a metabolic product of it is growth inhibitory. Biochim. Biophys. Acta, 76 (1963) 14o-142
142
SHORT COMMUNICATIONS
TABLE I AEROBIC GROWTH OF DIFFERENT STRAINS OF E. coli K I 2 IN THE MINERAL-SALTS MEDIUM 63 WITH VARIOUS SOURCES OF CARBON
E a c h c a r b o n s o u r c e w a s a d d e d a t a c o n c e n t r a t i o n of o.I }~o. + , g r o w t h w i t h i n 24 h; --, no g r o w l h within two days. Strain designation
Carbon source EI 5
D-Glucose D-Galactose D-Xylose Lactose Glycerol Lactate Acetate Malate Fumarate Succinate Acid-hydrolysed casein
EI5[I
EI5/2
EI513
+ + + + + + + + +
+ + + + + + + + +
~ + + + -----
+ + + + + + + + +
+
+
--
+
0.4 x
~0.3 ~D
,q~ 0.2 0.1 I
t
0.05 Glucose
o.10 (°/o)
Fig. 2. G r o w t h y i e l d of t h e m u t a n t E 15/2 o n g l u c o s e o n l y ( 0 - 0 ) , glucose + o.i % s u c c i n a t e ( × - × ) a n d g l u c o s e + 0.2 o/ /o s u c c i n a t e ( Q - O ) . T h e a m o u n t of g r o w t h "was d e t e r m i n e d p h o t o metrically. A l l the isolated mutants were found to have similar cell morphology and diagnostic properties as the parent strain, and they could also grow anaerobically on glucose. Further biochemical work is required to establish the metabolic block in each of these strains. The ease with which it appears to be possible to isolate mutants which are affected in their glyconeogenesis should make them suitable for studies of the enzyme reactions involved in this pathway. Presumably such strains may accumulate carbohydrate precursors if they are incubated in media containing other sources of carbon than carbohydrates.
Institule o/ Biochemistry,
BENGT V.
HOFSTEN
University o~ Uppsala, Uppsala (Sweden) H. A. KREBS, Bull. Johns Hopkins Hosp., 95 (1954) 19. 2 A. 3i. GOTTO AND B. M. POGELL, Biochem. Biophys. Res. Commun., 9 (1962) 381. 3 t3. v. HOFSTEN AND J. PORATH, Biochim. Biophys. Acta, 64 (1962) I. 4 A. B. PARDEE, F. JACOB AND J. ~-~IONOD, J . Mol. Biol., I (I959) 165. E. C. C. LIX, S. A. LERNER AND S. E. JORGENSEN, Biochim. Biophys. Acta, 60 (1962) 422.
Received June I7th , 1963 Biochim. Biophys. Acla, 76 (1963) 14o-142