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The metabolism of acetate and mevalonic acid by lactobacilli I. The effect of acetate and mevalonic acid on growth
BIOCHIMICA ET BIOPHYSICA ACTA
273
THE METABOLISM OF ACETATE AND MEVALONIC ACID BY LACTOBACILLI I. T H E E F F E C T OF ACETATE AND MEVALONIC ACID ON G R O W T H KAREEN
J. I. T H O R N E *
AND E. K O D I C E K
Dunn Nutritional Laboratory, University of Cambridge and Medical Research Council, Cambridge (Great Britain) (Received O c t o b e r 2nd, 1961 )
SUMMARY
The requirement of three species of lactobacillus for acetate and its replacement by mevalonic acid was investigated. It was found that, in the presence of 1 % glucose, Lactobacillus acidophilus had a requirement for sodium acetate which could be replaced by 1/8000 times its concentration of mevalonic acid, but when the glucose concentration was 2 %, acetate was no longer required. Lactobacillus arabinosus also required acetate, but mevalonic acid replaced only partially the acetate requirement. Acetate was not required, under the present experimental conditions for Lactobacillus casei. The acetate requirement of L. acidophilus could also be replaced by fl-hydroxyfl-methylglutaric acid and by 2,2'-dimethylacrylic acid, but not by farnesol or geraniol. INTRODUCTION
Acetate was found to be one of the necessary growth factors for lactobacilli 1, and it was shown that its activity was not related to its buffering effect 2. The same workers tested various aqueous extracts for their ability to replace acetate in the growth of lactobacilli a and were able to purify some material from yeast which had 440 times the activity of sodium acetate for the growth of Lactobacillus casei, but had little activity for Lactobacillus arabinosus. Ten years later, during the purification of lipoic acid from concentrates of dried distillers' solubles an acetate-replacing factor, mevalonic acid, was found for Lactobacillus acidophilus which had 16oo times the activity of sodium acetate 4. In liver and yeast systems mevalonic acid-acts as a precursor of ,°terols 5,6, however, it must have some other function in lactobacilli since bacteria do not appear to contain sterols 7. The present studies were undertaken to elucidate the function of mevalonic acid in lactobacilli. In the first paper of this series an account is given of a comparison of the growth-promoting effects of mevalonic acid and acetate for three different species of lactobacilli. A b b r e v i a t i o n s : MVA, m e v a l o n i c acid; HGM, f l - h y d r o x y - f l - m e t h y l g l u t a r i c acid; DMA, 2 , 2 ' - d i m e t h y l a c r y l i c acid. * P r e s e n t a d d r e s s : G r a d u a t e D e p a r t m e n t of B i o c h e m i s t r y , B r a n d e i s U n i v e r s i t y , W a l t h a m 54, Mass. (U.S.A.).
Biochim. Biophys. Acta, 59 (1962) 273-279
274
K.J.I.
THORNE, E. KODICEK
MATERIALS AND METHODS
Bacterial strains Lactobacillus acidophilus (ATCC 4963) and Lactobacillus arabinosus (17-5) were obtained from the D.S.I.R. National Collection of Industrial Bacteria. Lactobacillus casei (ATCC 7469) was a stock culture, originally obtained from Dr. E. E. SNELL. L. acidophilus was maintained in skim milk supplemented with 1% Bactotryptose* (Difco); L. arabinosus and L. casei were maintained on glucose-agar slopes s. Chemicals DL-MVA, as its N,N'-dibenzylethylenediamine salt was supplied by Merck Sharp & Dohme. The free acid was recovered b y the standard procedure of dissolving in water, taking to p H lO.5-11. 5 with 5 N alkali and removing the precipitated amine b y three extractions with diethyl ettler. The last traces of ether were removed by in vacuo evaporation. Acid-hydrolysed casein (vitamin-free) was obtained from Oxo; trypsin-digested casein was obtained from Difco. Growth media The semi-synthetic, basal medium for L. acidophilus was the same as that used for the original microbiological assay of MVA 4 but, as ~11 be described, it was found necessary to reduce the glucose concentration to I °/o (Table I). The medium of GUIRARD, S~ELL AND WILLIAMS* was used for L. arabinosus and L. casei (Table II). Concentrations of up to I mg sodium acetate/ml and up to o.5/~g MVA/ml were added to the basal media. Tubes containing IO ml medium were used experimentally. TABLE I COMPOSITION OF SEMI-SYNTHETIC MEDIUM FOR L. acidophilus (DOUBLE STRENGTH) The m i x t u r e was adjusted to p H 6.6 and sterilised by autoclaving at 134 °, 15 pounds] inL for 15 m i n .
Per litre Acid-hydrolysed, vitamin-free casein (Oxo) B a c t o - t r y p t o n e (Difco) DL-Tryptophan L-Cystine" HC1 DL-Alanine D-Glucose Adenine, guanine, uracil, x a n t h i n e T h y m i n e , orotic acid Salt solution A* Salt solution B* * Tween 80 Thiamine" HC1, riboflavin, calcium d-pantothenate, nicotinic acid P t e r o y l g l u t a m i c acid, pyridoxal, p-aminobenzoic acid Pyridoxin. HC1 Biotin (free acid) V i t a m i n Blz
io 5 o. 4 o.2 i 20 IO 4° 20 io 2
g g g g g g m g each m g each ml ml ml
o.2
m g each
i o.4 o.oi o.o2
m g each mg mg mg
* Salt solution A: 25 g KH2PO4, 25 g K~HPO 4 in 250 ml HzO. ** Salt solution B: xo g MgSO4-7HzO , o. 5 g NaC1, 0. 5 g MnSO4.4H~O , o, 5 g FeSO4.7HzO in 250 ml water.
Biochim. Biophys. Acta, 59 (1962) 273-279
METABOLISM OF ACETATE AND
MVA
BY LACTOBACILLI. I
275
TABLE II COMPOSITION OF SEMI-SYNTHETIC MEDIUM FOR L . c a s e i A N D L .
arabinosus (DOUBLE STRENGTH)
The m i x t u r e w a s a d j u s t e d to p H 6.8 a n d s t e r i l i s e d b y a u t o c l a v i n g a t 134 °, 15 p o u n d s / i n s for i o mi n.
Per litre A c i d - h y d r o l y s e d , v i t a m i n - f r e e casein (Oxo) D-Glucose KHsPO4 S a l t s o l u t i o n A* S a l t s o l u t i o n B** L-Cystine" HC1 DL-Tryptophan L-Asparagine Adenine, g u a n i n e , u r a c i l T h i a m i n e . HC1, riboflavin, c a l c i u m d-pantothenate N i c o t i n i c acid, p - a m i n o b e n z o i c acid Inositol Pteroylglutamic acid P y r i d o x i n . HC1 B i o t i n (free acid)
io 20 5 io IO 2o0 50 200 20 o.2 o.2 o I o.o2 2 1.6
g g g ml ml mg mg mg m g each m g each m g each mg mg mg /~g
* S a l t s o l u t i o n A: see T a b l e I. ** Salt s o l u t i o n B : see T a b l e I.
Growth of bacterial cultures Inocula of L. acidophilus were prepared from a culture grown for 24 h in the milk medium. The milk culture was diluted 4500 times with sterile saline and one drop was used for each experimental tube. Inocula of L. casei and L. arabinosus were obtained from cultures grown for 24 h in a complex medium ~ which was enriched by the addition of 1% Hepamino (Table III). The cells were harvested, washed twice with saline and re-suspended in enough saline to give about I / z g dry wt. per drop for each experimental tube. The tubes were incubated for 24 h at 37 °. The bacterial TABLE III COMPOSITION OF COMPLEX MEDIUM (DOUBLE STRENGTH) C o n d i t i o n s a s in T a b l e I I . Per l@re
B a c t o - p e p t o n e (Difco) L-Cystine. HC1 S a l t s o l u t i o n A* S a l t s o l u t i o n E** D-Glucose Potassium acetate (hydrated) Calcium d-pantothenate N i c o t i n i c acid B a c t o y e a s t e x t r a c t (Difco) Riboflavin H e p a m i n o (Evans)
2o 2oo 4° io 6o 6o i o. i 2 0.2 20
g mg ml ml g g mg mg g mg g
* Salt s o l u t i o n A: see T a b l e I. ** S a l t s o l u t i o n E : 6o g MgSO 4. 7HsO, 0. 5 g NaC1, 1. 5 g M n S O 4 - 4 H s O , 0.004 g F e S O 4- 7HsO, o.oo 4 g C u S O , . 5 H s O , o.oo 4 g ZnSO 4. 7 H 2 0 in 25o m l HsO.
Biochim. Biophys. Acta, 59 (1962) 273-279
276
K. J, I. T H O R N E ,
E. K O D I C E K
growth was measured turbidimetrically with a photoelectric colorimeter, using a neutral grey filter. A calibration curve was made for each species so that the results could be obtained in terms of dry weight per millilitre. EXPERIMENTAL
Effect of glucose concentration on the acetate requirement Using a medium containing 2 % glucose no requirement for acetate could be demonstrated with L. acidophilus. However, it was found that if the medium was autoclaved for 2 5 min instead of for 15 min, acetate was required for growth. Since it was possible that this result could be explained by the destruction of glucose on autoclaving, the effect of acetate on growth was compared in media containing 1% ~Jgdry wf4ml 400300
L'°cid°l:~ilus
,ugdr wt./ml 400 L.ocidophilus
/
~0
300 200 0 IO0
0
jo I
I 0.2
0
I 0.4
I I I o.e 0.8 1.0 mg .=~ocliumacetate/ml
0
400
L.ard~nosus
f
f
I 0.1
I Q2
! 013 0.4 ~5 jJg MVA/ml 0.3rag sodium acetate
L.arabinosus
O
~
o
0~
300
200
0.2mg sodiumacetate ~0
0 0~
o /o, 0 1500[
0.2
100 no sodium acetate
(~o 0
t 0.4
L .casei
' 8 .8 ' O,O 1.0 mg sodium acetate/ml ~0
•
o
0 i
o.o2
d.o4
0
doo
I
008
&o
jag MVAIml
500L o---------
10I
L
o
d2
0.4
016
L
,
0,8 ~.0 so.urn acet.01e Irnl
F i g . I. E f f e c t of acetate and m e v a l o n i c acid o n g r o w t h of L. acidophilus, L. arabinosus a n d L. casei. Bacterial cultures were grown for 24 h f r o m s m a l l inocula. R e s u l t s are g i v e n in /,g dry w t . / m L
Biochim. Biophys. Acta, 59 (I962) 2 7 3 - 2 7 9
METABOLISM OF ACETATE AND
MVA
BY L A C T O B A C I L L I . I
277
and 2 % glucose after autoclaving for 15 min and for 25 min. The results are shown in Table IV. If the glucose concentration was reduced to 1% it was found that it was no longer necessary to autoclave for 25 min to obtain a requirement for acetate. The medium used for L. arabinosus and L. casei contained only 1% glucose. TABLE
IV
EFFECT OF GLUCOSE CONCENTRATION AND TIME OF AUTOCLAVING ON GROWTH STIMULATION OF L. acidophilus BY ACETATE
Growthof L. acidopkilus
Glucose (%)
I5min
25min
Blank (,ug dry wt./ml)
+ Acetate(z mg[ml) (Itg dry wt./ml)
Zl 480
485 590
63 46
45 ° 34 °
autoclave I 2 autoclave x 2
Effect of acetate and mevalonic acid on growth Fig. I shows the effect of different concentrations of sodium acetate and of MVA on the growth of the three species of lactobacillus. Three experimental tubes were used at each concentration and the graphs represent averages of several experiments. The three curves on the left represent the effect of sodium acetate on growth and the two curves on the right show the effect of MVA. As was demonstrated in the original experiments with mevalonic acid 4 it was found that in L. acidophilus acetate could be replaced by MVA, at a lower concentration, about 0.05 ~g MVA being equivalent to 0. 4 mg sodium acetate. For L. arabinosus, in the absence of acetate, MVA had no effect on growth. However, in the presence of some acetate, MVA stimulated growth, o.I Fg MVA/ml replacing 0.4 mg /3-HydroxY-r~-rr~thylglutar~acid
1rng Sodiumace~,o~e 0 800
400
.~
300
/
2.2-Oimethyl acrylicacid
1rng Sod,urnacetcrte
~
000
/
o
400' ~.
~100
o 2001
0
/ 0 ~ 0
0
0
I
0
I
I
002 0.04 mg HMG/ml
~
.05
F i g . 2. E f f e c t of f l - h y d r o x y - f l - m e t h y l g l u t a r i c
I
0
,.
I
. 005 0.1 mg DMA/rnl
acid and 2,2'-dimethylacrylic
,
0.15 acid on growth
of
L. acidophilus. B a c t e r i a l c u l t u r e s w e r e g r o w n f o r z4 h f r o m s m a l l i n o c u l a . R e s u l t s a r e g i v e n in /*g d r y w t . / m l . T h e a r r o w s m a r k t h e e q u i v a l e n t g r o w t h o b t a i n e d w i t h x m g s o d i u m a c e t a t e / m l .
Btochim. Biophys. Acta, 5 9 (1962) 2 7 3 - 2 7 9
278
K . J . I . THORNE, E. KODICEK
sodium acetate/ml in the presence of 0.3 mg sodium acetate/ml. No requirement for acetate could be demonstrated with L. casei under the present conditions.
Effect of other materials on growth Since, unlike L. casei and L. arabinosus, L. acidophilus had a specific requirement for acetate which could be replaced by MVA, this species was used to test the growthpromoting activities of some substances biochemically related to MVA. Two types ~f substances were tested: precursors of MVA and intermediates in the synthesis of cholesterol from MVA. Two MVA precursors were used: fl-hydroxy-fi-methylglutaric acid which, as its coenzyme A derivative, is an intermediate between acetate and MVA1°, and 2,2'-dimethylacrylic acid which is an intermediate in the conversion of leucine to HMG-CoA1~.The results are shown in Fig. 2. The equivalent growth obtained with I mg sodium acetate/ml was measured at the same time and the result is marked with an arrow on the two graphs. Levels of o.o6 mg HMG/ml and o.17 mg DMA/ml could replace I mg sodium acetate. The two substances geraniol and farnesol, which as their pyrophosphates are precursors of squalene and sterols ~2-~4, were also tested for the ability to replace acetate. Neither compound was active at concentrations of up to I mg/ml. DISCUSSION The replacement of acetate by MVA for L. acidophilus confirmed previous experiments 4, although the MVA was found te be somewhat more active (8000 times as active as acetate instead of I600 times). L. arabinosus however could only replace part of its acetate requirement by MVA. This is in agreement with the early experiments 3 with a substance resembling partially purified MVA in which it was found that the acetate requirement of L. casei but not that of L. arabinosus could be replaced. Since a significant portion of the acetate requirement of L. arabinosus can be replaced by MVA it appears that part of the acetate is required for conversion to MVA and thence to compounds derived from it and the remainder is required for a different purpose. In L. acidophilus when only MVA was present the second requirement would have to be met from another source, such as glucose. The reported requirement of L. casei for acetate 2 could not be demonstrated in the present experiments. It is possible that acetate is being formed from glucose via pyruvic acid by the dismutation reactionl~: 2CHa'CO'COOH + CoA~CHa'CH(OH)'COOH + CHa'CO'CoA -I CO~ This reaction is catalysed by lipoic acid and it is reported that acetate can be replaced by lipoic acid in L. casei. This could also explain why L. acid@hilus required acetate only when the glucose concentration was kept low. The replacement of acetate by somewhat lower concentrations of HMG suggests that in lactobacilli, as in yeast and liver, HMG is an intermediate in the conversion of acetate to MVA. Similarly the utilisation of DMA suggests that it can be converted to MVA in lactobacilli as well as in other systems, It must be noted that it is the coenzyme A derivatives of HMG and DMA which are the intermediates in these pathways. The negative results obtained with farnesol and geraniol could be due to impermeability. WRIGHT16 has shown that certain substances, including HMG and
13iochim, Biophys..4cta, 59 (t962) 273-279
METABOLISM OF ACETATE AND
MVA
BY LACTOBACILLI. I
279
DMA, are antimetabolites in that they inhibit the growth of L. acidophilus in the presence of MVA. It is difficult to reconcile this with their growth-promoting properties. However the present authors did find a similar "antimetabolite" effect with farnesol and geraniol in the presence of MVA ACKNOWLEDGEMENTS
One of us (K. J. I. THORNE, formerly K. J. I. CHALK)is indebted to the Medical Research Council for a studentship. We are indebted to Merck Sharp & Dohme, Inc. for a generous supply of DL-mevalonic acid. REFERENCES 1 E. E. SNELL, E. L. TATUM AND W. 1-[. PETERSON, J. Bacteriol., 33 (1937) 207. 2 B. M. GUIRARD, E. E. SNELL AND R. J. WILLIAMS, .4rch. Biochem., 9 (1946) 36I. s B. M. GUIRARD, E. E. SNELL AND R. J. WILLIAMS, Arch. Biochem., 9 (1946) 381. 4 H. R. SKEGGS, L. D. WRIGHT, E. L. CRESSON,G. D. E. MACRAE, C. H. HOFFMAN,n . E. WOLF AND K. FOLKERS, J. Bacteriol., 72 (1956) 519. 5 p. A. TAVORMINA, M. H. GIBBS ANP J. W. HUFF, J . . 4 m . Chem. Soc., 78 (1956) 4498. 6 B. H. AMDUR, H. RILLING AND K. BLOCH, J. Am. Chem. Soc., 79 (1957) 26467 A. FIERTEL AND H. P. KLEIN, J. Bacteriol., 78 (1959) 738. 8 K. M. CLEGG, E. KODlCEK AND S. P. MISTRY, Biochem. J., 5 ° (1952) 326. 9 E. KODICEK, l i n d International Conference on Biochemical Problems of Lipids, Ghent, Butterw o r t h s Scientific Publications, London, 1956, p. 4Ol. ill j . KNAPPE, E. RINGELMANN AND F. LYNEN, Biochem. Z., 332 (1959) 195. 11 M. J. CooN, W. G. ROBINSON AND B. K. BACHHAWAT, Symposium on Amino .4cid ,~letabolism, J o h n s H o p k i n s Press, Baltimore, 1955, p. 431. 12 F. LYNEN, B. W. AGRANOFF, H. EGGERER, U. HENNING AND E. M. MOSLEIN, .4ngew. Chem., 71 (1959) 657. 13 DEW. S. GOODMAN AND G. POPJAK, J. Lipid Research, 1 (196o) 286. 14 G. POPJAK, J. W. CORNFORTH, R. l-I. CORNFORTH AND DEW. S. GOODMAN, Biochem. Biophys. Research Communs., 4 (1961) 2o4. 16 j . S. FRUTON AND S. SIMMONDS, General Biochemistry, J o h n Wiley and Sons, Inc., New York, London, 1959, p. 481. 16 L. D. WRIGHT, Proc. Soc. Exptl. Biol. Med., 96 (1957) 364.
Biochim. Biophys. Acta, 59 (1962) 273-279
273
THE METABOLISM OF ACETATE AND MEVALONIC ACID BY LACTOBACILLI I. T H E E F F E C T OF ACETATE AND MEVALONIC ACID ON G R O W T H KAREEN
J. I. T H O R N E *
AND E. K O D I C E K
Dunn Nutritional Laboratory, University of Cambridge and Medical Research Council, Cambridge (Great Britain) (Received O c t o b e r 2nd, 1961 )
SUMMARY
The requirement of three species of lactobacillus for acetate and its replacement by mevalonic acid was investigated. It was found that, in the presence of 1 % glucose, Lactobacillus acidophilus had a requirement for sodium acetate which could be replaced by 1/8000 times its concentration of mevalonic acid, but when the glucose concentration was 2 %, acetate was no longer required. Lactobacillus arabinosus also required acetate, but mevalonic acid replaced only partially the acetate requirement. Acetate was not required, under the present experimental conditions for Lactobacillus casei. The acetate requirement of L. acidophilus could also be replaced by fl-hydroxyfl-methylglutaric acid and by 2,2'-dimethylacrylic acid, but not by farnesol or geraniol. INTRODUCTION
Acetate was found to be one of the necessary growth factors for lactobacilli 1, and it was shown that its activity was not related to its buffering effect 2. The same workers tested various aqueous extracts for their ability to replace acetate in the growth of lactobacilli a and were able to purify some material from yeast which had 440 times the activity of sodium acetate for the growth of Lactobacillus casei, but had little activity for Lactobacillus arabinosus. Ten years later, during the purification of lipoic acid from concentrates of dried distillers' solubles an acetate-replacing factor, mevalonic acid, was found for Lactobacillus acidophilus which had 16oo times the activity of sodium acetate 4. In liver and yeast systems mevalonic acid-acts as a precursor of ,°terols 5,6, however, it must have some other function in lactobacilli since bacteria do not appear to contain sterols 7. The present studies were undertaken to elucidate the function of mevalonic acid in lactobacilli. In the first paper of this series an account is given of a comparison of the growth-promoting effects of mevalonic acid and acetate for three different species of lactobacilli. A b b r e v i a t i o n s : MVA, m e v a l o n i c acid; HGM, f l - h y d r o x y - f l - m e t h y l g l u t a r i c acid; DMA, 2 , 2 ' - d i m e t h y l a c r y l i c acid. * P r e s e n t a d d r e s s : G r a d u a t e D e p a r t m e n t of B i o c h e m i s t r y , B r a n d e i s U n i v e r s i t y , W a l t h a m 54, Mass. (U.S.A.).
Biochim. Biophys. Acta, 59 (1962) 273-279
274
K.J.I.
THORNE, E. KODICEK
MATERIALS AND METHODS
Bacterial strains Lactobacillus acidophilus (ATCC 4963) and Lactobacillus arabinosus (17-5) were obtained from the D.S.I.R. National Collection of Industrial Bacteria. Lactobacillus casei (ATCC 7469) was a stock culture, originally obtained from Dr. E. E. SNELL. L. acidophilus was maintained in skim milk supplemented with 1% Bactotryptose* (Difco); L. arabinosus and L. casei were maintained on glucose-agar slopes s. Chemicals DL-MVA, as its N,N'-dibenzylethylenediamine salt was supplied by Merck Sharp & Dohme. The free acid was recovered b y the standard procedure of dissolving in water, taking to p H lO.5-11. 5 with 5 N alkali and removing the precipitated amine b y three extractions with diethyl ettler. The last traces of ether were removed by in vacuo evaporation. Acid-hydrolysed casein (vitamin-free) was obtained from Oxo; trypsin-digested casein was obtained from Difco. Growth media The semi-synthetic, basal medium for L. acidophilus was the same as that used for the original microbiological assay of MVA 4 but, as ~11 be described, it was found necessary to reduce the glucose concentration to I °/o (Table I). The medium of GUIRARD, S~ELL AND WILLIAMS* was used for L. arabinosus and L. casei (Table II). Concentrations of up to I mg sodium acetate/ml and up to o.5/~g MVA/ml were added to the basal media. Tubes containing IO ml medium were used experimentally. TABLE I COMPOSITION OF SEMI-SYNTHETIC MEDIUM FOR L. acidophilus (DOUBLE STRENGTH) The m i x t u r e was adjusted to p H 6.6 and sterilised by autoclaving at 134 °, 15 pounds] inL for 15 m i n .
Per litre Acid-hydrolysed, vitamin-free casein (Oxo) B a c t o - t r y p t o n e (Difco) DL-Tryptophan L-Cystine" HC1 DL-Alanine D-Glucose Adenine, guanine, uracil, x a n t h i n e T h y m i n e , orotic acid Salt solution A* Salt solution B* * Tween 80 Thiamine" HC1, riboflavin, calcium d-pantothenate, nicotinic acid P t e r o y l g l u t a m i c acid, pyridoxal, p-aminobenzoic acid Pyridoxin. HC1 Biotin (free acid) V i t a m i n Blz
io 5 o. 4 o.2 i 20 IO 4° 20 io 2
g g g g g g m g each m g each ml ml ml
o.2
m g each
i o.4 o.oi o.o2
m g each mg mg mg
* Salt solution A: 25 g KH2PO4, 25 g K~HPO 4 in 250 ml HzO. ** Salt solution B: xo g MgSO4-7HzO , o. 5 g NaC1, 0. 5 g MnSO4.4H~O , o, 5 g FeSO4.7HzO in 250 ml water.
Biochim. Biophys. Acta, 59 (1962) 273-279
METABOLISM OF ACETATE AND
MVA
BY LACTOBACILLI. I
275
TABLE II COMPOSITION OF SEMI-SYNTHETIC MEDIUM FOR L . c a s e i A N D L .
arabinosus (DOUBLE STRENGTH)
The m i x t u r e w a s a d j u s t e d to p H 6.8 a n d s t e r i l i s e d b y a u t o c l a v i n g a t 134 °, 15 p o u n d s / i n s for i o mi n.
Per litre A c i d - h y d r o l y s e d , v i t a m i n - f r e e casein (Oxo) D-Glucose KHsPO4 S a l t s o l u t i o n A* S a l t s o l u t i o n B** L-Cystine" HC1 DL-Tryptophan L-Asparagine Adenine, g u a n i n e , u r a c i l T h i a m i n e . HC1, riboflavin, c a l c i u m d-pantothenate N i c o t i n i c acid, p - a m i n o b e n z o i c acid Inositol Pteroylglutamic acid P y r i d o x i n . HC1 B i o t i n (free acid)
io 20 5 io IO 2o0 50 200 20 o.2 o.2 o I o.o2 2 1.6
g g g ml ml mg mg mg m g each m g each m g each mg mg mg /~g
* S a l t s o l u t i o n A: see T a b l e I. ** Salt s o l u t i o n B : see T a b l e I.
Growth of bacterial cultures Inocula of L. acidophilus were prepared from a culture grown for 24 h in the milk medium. The milk culture was diluted 4500 times with sterile saline and one drop was used for each experimental tube. Inocula of L. casei and L. arabinosus were obtained from cultures grown for 24 h in a complex medium ~ which was enriched by the addition of 1% Hepamino (Table III). The cells were harvested, washed twice with saline and re-suspended in enough saline to give about I / z g dry wt. per drop for each experimental tube. The tubes were incubated for 24 h at 37 °. The bacterial TABLE III COMPOSITION OF COMPLEX MEDIUM (DOUBLE STRENGTH) C o n d i t i o n s a s in T a b l e I I . Per l@re
B a c t o - p e p t o n e (Difco) L-Cystine. HC1 S a l t s o l u t i o n A* S a l t s o l u t i o n E** D-Glucose Potassium acetate (hydrated) Calcium d-pantothenate N i c o t i n i c acid B a c t o y e a s t e x t r a c t (Difco) Riboflavin H e p a m i n o (Evans)
2o 2oo 4° io 6o 6o i o. i 2 0.2 20
g mg ml ml g g mg mg g mg g
* Salt s o l u t i o n A: see T a b l e I. ** S a l t s o l u t i o n E : 6o g MgSO 4. 7HsO, 0. 5 g NaC1, 1. 5 g M n S O 4 - 4 H s O , 0.004 g F e S O 4- 7HsO, o.oo 4 g C u S O , . 5 H s O , o.oo 4 g ZnSO 4. 7 H 2 0 in 25o m l HsO.
Biochim. Biophys. Acta, 59 (1962) 273-279
276
K. J, I. T H O R N E ,
E. K O D I C E K
growth was measured turbidimetrically with a photoelectric colorimeter, using a neutral grey filter. A calibration curve was made for each species so that the results could be obtained in terms of dry weight per millilitre. EXPERIMENTAL
Effect of glucose concentration on the acetate requirement Using a medium containing 2 % glucose no requirement for acetate could be demonstrated with L. acidophilus. However, it was found that if the medium was autoclaved for 2 5 min instead of for 15 min, acetate was required for growth. Since it was possible that this result could be explained by the destruction of glucose on autoclaving, the effect of acetate on growth was compared in media containing 1% ~Jgdry wf4ml 400300
L'°cid°l:~ilus
,ugdr wt./ml 400 L.ocidophilus
/
~0
300 200 0 IO0
0
jo I
I 0.2
0
I 0.4
I I I o.e 0.8 1.0 mg .=~ocliumacetate/ml
0
400
L.ard~nosus
f
f
I 0.1
I Q2
! 013 0.4 ~5 jJg MVA/ml 0.3rag sodium acetate
L.arabinosus
O
~
o
0~
300
200
0.2mg sodiumacetate ~0
0 0~
o /o, 0 1500[
0.2
100 no sodium acetate
(~o 0
t 0.4
L .casei
' 8 .8 ' O,O 1.0 mg sodium acetate/ml ~0
•
o
0 i
o.o2
d.o4
0
doo
I
008
&o
jag MVAIml
500L o---------
10I
L
o
d2
0.4
016
L
,
0,8 ~.0 so.urn acet.01e Irnl
F i g . I. E f f e c t of acetate and m e v a l o n i c acid o n g r o w t h of L. acidophilus, L. arabinosus a n d L. casei. Bacterial cultures were grown for 24 h f r o m s m a l l inocula. R e s u l t s are g i v e n in /,g dry w t . / m L
Biochim. Biophys. Acta, 59 (I962) 2 7 3 - 2 7 9
METABOLISM OF ACETATE AND
MVA
BY L A C T O B A C I L L I . I
277
and 2 % glucose after autoclaving for 15 min and for 25 min. The results are shown in Table IV. If the glucose concentration was reduced to 1% it was found that it was no longer necessary to autoclave for 25 min to obtain a requirement for acetate. The medium used for L. arabinosus and L. casei contained only 1% glucose. TABLE
IV
EFFECT OF GLUCOSE CONCENTRATION AND TIME OF AUTOCLAVING ON GROWTH STIMULATION OF L. acidophilus BY ACETATE
Growthof L. acidopkilus
Glucose (%)
I5min
25min
Blank (,ug dry wt./ml)
+ Acetate(z mg[ml) (Itg dry wt./ml)
Zl 480
485 590
63 46
45 ° 34 °
autoclave I 2 autoclave x 2
Effect of acetate and mevalonic acid on growth Fig. I shows the effect of different concentrations of sodium acetate and of MVA on the growth of the three species of lactobacillus. Three experimental tubes were used at each concentration and the graphs represent averages of several experiments. The three curves on the left represent the effect of sodium acetate on growth and the two curves on the right show the effect of MVA. As was demonstrated in the original experiments with mevalonic acid 4 it was found that in L. acidophilus acetate could be replaced by MVA, at a lower concentration, about 0.05 ~g MVA being equivalent to 0. 4 mg sodium acetate. For L. arabinosus, in the absence of acetate, MVA had no effect on growth. However, in the presence of some acetate, MVA stimulated growth, o.I Fg MVA/ml replacing 0.4 mg /3-HydroxY-r~-rr~thylglutar~acid
1rng Sodiumace~,o~e 0 800
400
.~
300
/
2.2-Oimethyl acrylicacid
1rng Sod,urnacetcrte
~
000
/
o
400' ~.
~100
o 2001
0
/ 0 ~ 0
0
0
I
0
I
I
002 0.04 mg HMG/ml
~
.05
F i g . 2. E f f e c t of f l - h y d r o x y - f l - m e t h y l g l u t a r i c
I
0
,.
I
. 005 0.1 mg DMA/rnl
acid and 2,2'-dimethylacrylic
,
0.15 acid on growth
of
L. acidophilus. B a c t e r i a l c u l t u r e s w e r e g r o w n f o r z4 h f r o m s m a l l i n o c u l a . R e s u l t s a r e g i v e n in /*g d r y w t . / m l . T h e a r r o w s m a r k t h e e q u i v a l e n t g r o w t h o b t a i n e d w i t h x m g s o d i u m a c e t a t e / m l .
Btochim. Biophys. Acta, 5 9 (1962) 2 7 3 - 2 7 9
278
K . J . I . THORNE, E. KODICEK
sodium acetate/ml in the presence of 0.3 mg sodium acetate/ml. No requirement for acetate could be demonstrated with L. casei under the present conditions.
Effect of other materials on growth Since, unlike L. casei and L. arabinosus, L. acidophilus had a specific requirement for acetate which could be replaced by MVA, this species was used to test the growthpromoting activities of some substances biochemically related to MVA. Two types ~f substances were tested: precursors of MVA and intermediates in the synthesis of cholesterol from MVA. Two MVA precursors were used: fl-hydroxy-fi-methylglutaric acid which, as its coenzyme A derivative, is an intermediate between acetate and MVA1°, and 2,2'-dimethylacrylic acid which is an intermediate in the conversion of leucine to HMG-CoA1~.The results are shown in Fig. 2. The equivalent growth obtained with I mg sodium acetate/ml was measured at the same time and the result is marked with an arrow on the two graphs. Levels of o.o6 mg HMG/ml and o.17 mg DMA/ml could replace I mg sodium acetate. The two substances geraniol and farnesol, which as their pyrophosphates are precursors of squalene and sterols ~2-~4, were also tested for the ability to replace acetate. Neither compound was active at concentrations of up to I mg/ml. DISCUSSION The replacement of acetate by MVA for L. acidophilus confirmed previous experiments 4, although the MVA was found te be somewhat more active (8000 times as active as acetate instead of I600 times). L. arabinosus however could only replace part of its acetate requirement by MVA. This is in agreement with the early experiments 3 with a substance resembling partially purified MVA in which it was found that the acetate requirement of L. casei but not that of L. arabinosus could be replaced. Since a significant portion of the acetate requirement of L. arabinosus can be replaced by MVA it appears that part of the acetate is required for conversion to MVA and thence to compounds derived from it and the remainder is required for a different purpose. In L. acidophilus when only MVA was present the second requirement would have to be met from another source, such as glucose. The reported requirement of L. casei for acetate 2 could not be demonstrated in the present experiments. It is possible that acetate is being formed from glucose via pyruvic acid by the dismutation reactionl~: 2CHa'CO'COOH + CoA~CHa'CH(OH)'COOH + CHa'CO'CoA -I CO~ This reaction is catalysed by lipoic acid and it is reported that acetate can be replaced by lipoic acid in L. casei. This could also explain why L. acid@hilus required acetate only when the glucose concentration was kept low. The replacement of acetate by somewhat lower concentrations of HMG suggests that in lactobacilli, as in yeast and liver, HMG is an intermediate in the conversion of acetate to MVA. Similarly the utilisation of DMA suggests that it can be converted to MVA in lactobacilli as well as in other systems, It must be noted that it is the coenzyme A derivatives of HMG and DMA which are the intermediates in these pathways. The negative results obtained with farnesol and geraniol could be due to impermeability. WRIGHT16 has shown that certain substances, including HMG and
13iochim, Biophys..4cta, 59 (t962) 273-279
METABOLISM OF ACETATE AND
MVA
BY LACTOBACILLI. I
279
DMA, are antimetabolites in that they inhibit the growth of L. acidophilus in the presence of MVA. It is difficult to reconcile this with their growth-promoting properties. However the present authors did find a similar "antimetabolite" effect with farnesol and geraniol in the presence of MVA ACKNOWLEDGEMENTS
One of us (K. J. I. THORNE, formerly K. J. I. CHALK)is indebted to the Medical Research Council for a studentship. We are indebted to Merck Sharp & Dohme, Inc. for a generous supply of DL-mevalonic acid. REFERENCES 1 E. E. SNELL, E. L. TATUM AND W. 1-[. PETERSON, J. Bacteriol., 33 (1937) 207. 2 B. M. GUIRARD, E. E. SNELL AND R. J. WILLIAMS, .4rch. Biochem., 9 (1946) 36I. s B. M. GUIRARD, E. E. SNELL AND R. J. WILLIAMS, Arch. Biochem., 9 (1946) 381. 4 H. R. SKEGGS, L. D. WRIGHT, E. L. CRESSON,G. D. E. MACRAE, C. H. HOFFMAN,n . E. WOLF AND K. FOLKERS, J. Bacteriol., 72 (1956) 519. 5 p. A. TAVORMINA, M. H. GIBBS ANP J. W. HUFF, J . . 4 m . Chem. Soc., 78 (1956) 4498. 6 B. H. AMDUR, H. RILLING AND K. BLOCH, J. Am. Chem. Soc., 79 (1957) 26467 A. FIERTEL AND H. P. KLEIN, J. Bacteriol., 78 (1959) 738. 8 K. M. CLEGG, E. KODlCEK AND S. P. MISTRY, Biochem. J., 5 ° (1952) 326. 9 E. KODICEK, l i n d International Conference on Biochemical Problems of Lipids, Ghent, Butterw o r t h s Scientific Publications, London, 1956, p. 4Ol. ill j . KNAPPE, E. RINGELMANN AND F. LYNEN, Biochem. Z., 332 (1959) 195. 11 M. J. CooN, W. G. ROBINSON AND B. K. BACHHAWAT, Symposium on Amino .4cid ,~letabolism, J o h n s H o p k i n s Press, Baltimore, 1955, p. 431. 12 F. LYNEN, B. W. AGRANOFF, H. EGGERER, U. HENNING AND E. M. MOSLEIN, .4ngew. Chem., 71 (1959) 657. 13 DEW. S. GOODMAN AND G. POPJAK, J. Lipid Research, 1 (196o) 286. 14 G. POPJAK, J. W. CORNFORTH, R. l-I. CORNFORTH AND DEW. S. GOODMAN, Biochem. Biophys. Research Communs., 4 (1961) 2o4. 16 j . S. FRUTON AND S. SIMMONDS, General Biochemistry, J o h n Wiley and Sons, Inc., New York, London, 1959, p. 481. 16 L. D. WRIGHT, Proc. Soc. Exptl. Biol. Med., 96 (1957) 364.
Biochim. Biophys. Acta, 59 (1962) 273-279