Carbohydrate metabolism in Bifidobacterium bifidum

BIOCHIMICA ET BIOPHYSICA ACTA

415

BBA 25748 CARBOHYDRATE METABOLISM IN BIFIDOBACTERIUM B I F I D U M WYTSKE DE ¥RIES, Sj. J. GERBRANDY AND A. H. STOUTHAMER

Microbiology Department, Laboratory for Botany, Free University, de Boelelaan Io87, Amsterdam (The Netherlands) (Received August 22nd, 1966)

SUMMARY

I. Washed suspensions of Bifidobacterium bifidum ferment glucose to approx. 1.5 moles of acetate and I mole of lactate. Xylose yields I mole of acetate and I mole of lactate. 2. Aldolase (EC 4.1.2.7) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49 ) are absent from cell-free extracts of B. bifidum. 3- An enzyme catalysing the conversion of fructose 6-phosphate into acetylphosphate and erythrose 4-phosphate is shown to be present in cell-free extracts: D-fructose-6-phosphate D-erythrose-4-phosphate-lyase (phosphate-acetylating). The trivial name fructose-6-phosphate phosphoketolase may be given. 4. By the action of transaldolase (EC 2.2.1.2) and transketolase (EC 2.2.1.1) pentose phosphates are formed from fructose 6-phosphate and erythrose 4-phosphate. 5. Pentose phosphates are split by xylulose-5-phosphate phosphoketolase (EC 4.1.2.9) into acetylphosphate and glyceraldehyde 3-phosphate. 6. Lactate dehydrogenase (EC 1.1.1.27) is shown to have an absolute requirement for fructose 1,6-diphosphate. This explains the presence of low amounts of phosphofructokinase (EC 2.7.1.11 ) in cell-free extracts. 7. The theoretical fermentation balances of glucose and xylose agree with those found experimentally. 8. The results strengthen the previous suggestions that the classification of B. bifidum in the genus Lactobacillus is not justified.

INTRODUCTION

Bifidobacterium bifidum, a strictly anaerobic bacterium found in the human intestinal flora, ferments glucose to acetate and lactate without evolution or uptake of CO s (NORRIS et al. 1, KUHN AND TIEDEMANN~, DEHNERT3, SEBALD, GASSER AND WERNER 4, BEERENS, GERARD AND QUILLAUMEg). KUHN AND TIEDEMANN2, only, gave some information on the pathway of degradation of glucose in B. bifidum. They concluded that both lactate and acetate are formed via pyruvate. No further details about carbohydrate metabolism in B. bifidum are known. According to Bergey's Manual of Determinative Bacteriology 6, B. bifidum belongs Abbreviations: P, phosphate ester group; Ery, erythrose; Rib, ribose; Xyl, xylulose; Glc, glucose; Fru, fructose; Sed, sedoheptulose.

Biochim. Biophys. Acta, 136 (I967) 415-425

416

W. DE VRIES, S. J. GERBRAND¥, A. H. STOUTHAMER

to the genus Lactobacillus. The classification of B. bifidum to the genus Lactobacillus would imply that fermentation is taking place either via the glycolysis or via the hexose monophosphate shunt. The glycolysis is the pathway of degradation of glucose in the homofermentative lactic acid bacteria. The hexose monophosphate shunt is characteristic of the heterofermentative lactic acid bacteria 7,8. However, formation of acetate not attended with evolution of CO 2, is difficult to be explained b y these pathways. Therefore it seemed worthwhile to investigate which fermentation routes are present in B. bifidum. The present paper reports the absence of the glycolytic route and the hexose monophosphate shunt from B. bifidum and describes an alternative route of degradation of glucose. MATERIALS AND METHODS

Maintenance and growth of the organism A pentose-fermenting strain (type V) z of B. bifidum, obtained from the Laboratory for Microbiology of the State University of Utrecht, was used for all experiments. B. bifidum was maintained as stab-cultures in tomato-agar (Oxoid) containing 2 % glucose and 0.2 % cysteine. They were subcultured weekly. Liquid cultures were grown at 37 ° in erlenmeyer flasks. A Mclntosh anaerobic jar was used to obtain anaerobic conditions. The gasphase was N2-CO 2 (95:5, v/v). The culture medium had the following composition (per 1 of water): IO g pepton (Oxoid), IO g beef extract (Oxoid), 5 g yeast extract (Oxoid), I ml Tween-8o, 2 g KzHPO4, 5 g sodium acetate.3 H,O, 2 g diammonium citrate, 0.2 g MgSO4"7 H20, o.o5 g MnSO4"4 H20, 20 g glucose, p H 6.8.

Determination of fermentation balances 300 ml of a culture of B. bifidum grown either on glucose or on xylose was centrifuged in a MSE 18 centrifuge at 23000 x g for 20 rain. Cells were washed three times with 0.066 M phosphate buffer (pH 6.8) and resuspended in phosphate buffer to a concentration of approx. 7 mg of cells (dry wt.) per ml. Cell suspensions were allowed to ferment glucose or xylose respectively under an atmosphere of N2-CO z (95:5, v/v) at 37 °. Evolution or consumption of CO 2 was measured by the conventional Warburg technique 9. Control flasks were run in parallel with reaction flasks to permit correction for any endogenous formation of fermentation products. After 2 h (glucose) or 18 h (xylose), in which period the glucose and xylose were fermented completely, the incubation mixture was centrifuged. In the supernatant fluids glucose, xylose, lactate and acetate were determined. Glucose was determined by means of a Biochemica Test Combination (C. F. Boehringer und Soehne GmbH) containing glucose oxidase and peroxidase. Xylose was determined with the orcine reagent as described b y MEJBAUM 1°. Lactate was estimated by the procedure of BARKER AND SUMMERSON11. Acetate was determined by means of the enzymatic method of ROSE12 which is based on the colorimetric determination of acetyl phosphate according to LIPMANN AND TUTTLE 13.

_Preparation of cell-free extracts Cells obtained from 300 ml of a well-grown culture were washed three times with 0.85 % KC1 and resuspended in 20 ml 0.85 % KC1. Cell extracts were prepared

Biochim. Biophys. Acta, 136 (1967) 415-4'~5

CARBOHYDRATE METABOLISMIN B. bifidum

417

by submitting cell suspensions to ultrasonic oscillation in a MSE ultrasonic disintegrator (60 W at 20 kcycles/sec) for IO min. The temperature did not exceed io °. IR some cases cell extracts were prepared by means of a Sorvall Ribi cell fractionator at 26000 lb/inch 2. In this way the temperature did not exceed 2 °. The resultant homogenate was centrifuged at 120o0 × g for 15 min at 4 °. If necessary the crude cell-free extract was dialysed against 0.85 % KC1 during one night at 4 °. Cell-free extracts contained between 2 and 5 mg of protein per ml, as determined by the method of LowRY et al. ~4.

Spectro2bhotometric ])rocedures All spectrophotometric assays were performed at 25 ° in quartz cuvettes (I cm light-path) with a Unicam Sp 82o constant wavelength scanner. The decrease or increase in absorbance was followed at 340 m~ in the appropriate system. Colorimetric measurements were performed with a Unicam Sp 600 spectrophotometer. Spectra were drawn with a Unicam Sp 800 spectrophotometer. Enzyme assays Aldolase was determined colorimetrically by means of a Biochemica Test Combination (C. F. Boehringer und Soehne GmbH) and spectrophotometrically as described by BRUNS AND BERGMEYER15. Glucose-6-phosphate dehydrogenase, hexokinase (EC 2.7.i.i ), glucose-6-phosphate isomerase (EC 5.3.I.9), phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) were determined by the conventional spectrophotometric techniques16-Z°.The conversion of glycerate-3-phosphate into pyruvate was measured as described by DAWES et al. ~1. Lactate dehydrogenase was measured as described in the legend to Fig. 4. Fru-6-P phosphoketolase was demonstrated in three different ways: (a) spectrophotometrically, (b) by accumulation of Sed-7-P and (c) by measuring the formatiorL of acetyl phosphate. Details are mentioned in the legends to Figs. I and 2 and ir~ the heading of Table II. Transketolase and Xyl-5-P phosphoketolase were determined spectrophotometrically as described in the legend to Fig. 3. Xyl-5-P phosphoketolase was also determined by measuring the formation of acetyl phosphate from Rib-5-P at 3°o and at pH 6.0 according to the method described for Fru-6-P phosphoketolase. Transaldolase was assayed spectrophotometrically by measuring the rate of NADH oxidation in a reaction mixture (1.15 ml) containing in /zmoles: Tris-HCl buffer (pH 7.2), 25o; MgC12, 5; cysteine, io; Fru-6-P, 2.5; Ery-4-P, 2; NADH, 0.25; glycerol-3-phosphate dehydrogenase (EC I.I.I.8)/triose phosphate isomerase (EC 5.3.I.I), 0.7 I.U. ; cell-free extract, o.I mg of protein. Enzymes and chemicals Glycerol-3-P dehydrogenase/triose phosphate isomerase, aldolase, Glc-6-P dehydrogenase, NADH, NAD+, NADP+, ATP, Fru-6-P, Ery-4-P , Rib-5-P , and Glc-6-P were obtained from C.F. Boehringer und Soehne GmbH, Mannheim (Germany). Fru-I,6-P e and pyruvate were obtained from Fluka A.G. Other reagents were of analytical grade wherever possible.

Biochim. Biophys. Acta, x36 (1967) 415-42N

418

W. DE VRIES, S. J. GERBRANDY, A. H. STOUTHAMER

RESULTS

Fermentation balances of glucose and xylose From the Warburg experiments it appeared that the amount of CO 2 evolved was about 0.05 mole per mole of glucose fermented. This is less than 1 % of the carbon added. Evolution of CO 2 from xylose was also negligible. From the analysis of acetate and lactate the following fermentation balances could be drawn up: I glucose ~

1.53 ~ 0.04 (4) a c e t a t e + 0.86 ~ 0.03 (4) lactate

i xylose - - + 0.98 a c e t a t e + 0.95 lactate

The fermentation balance of xylose was determined with one cell suspension. The C-recovery is 95 % ( ~ 2 %) for glucose and 96 % for xylose. In both cases the oxidation-reduction balance is I.O.

Aldolase and Glc-6-P dehydrogenase "Fable I records the specific activities of aldolase and Glc-6-P dehydrogenase in cell-free extracts of B. bifidum, Lactobacillus casei and Lactobacillus fermenti, prepared b y means of ultrasonic oscillation. To exclude any failure of the methods used, cell-free extracts of L. casei and L. fermenti were run in parallel as positive controls. I t can ,be seen that Glc-6-P dehydrogenase was completely absent from cell-free extracts of t3, bifidum. The specific activity of aldolase in cell-free extracts of B. bifidum was 0.06. Compared to the specific activity of aldolase in L. casei (I9.6), that in B. bifidum seemed very small. TABLE

1

SPECIFIC ACTIVITIES OF

ALDOLASE

AND

GIc-6-P DEHYDROGENASE

IN C E L L - F R I ~ E E X T R A C T S

OF

B. bifidum, L. 6asei AND L. fermenti Aldolase was d e t e r m i n e d colorimetrically. Specific activities are e x p r e s s e d as /~moles s u b s t r a t e t r a n s f o r m e d per m g of protein per h. Bacteria

A ldolase

Glc-6-P dehydrogenase

B. bifidum L. casei L. fermenti

o.o6 I9.6 o

o 3.8 34.5

Fermentation route

glycolysis hexose m o n o p h o s p h a t e s h u n t

Aldolase and Glc-6-P dehydrogenase were also assayed in a cell-free extract of

B. bifidum prepared with the Sorvall Ribi cell fractionator in which apparatus a more intensive cooling is possible than in the MSE ultrasonic disintegrator. In this case aldolase was determined spectrophotometrically. No aldolase could be detected although addition of crystalline muscle aldolase permitted immediate N A D H oxidation. Gtc-6-P dehydrogenase was also completely absent from this cell-free extract.

Fru-6-P phosphoketolase Fig. I shows the determination of Fru-6-P phosphoketolase. It can be seen that without added substrate (Curve a) a slow oxidation of N A D H took place due to the Biochim. Biophys. Acta, 136 (1967) 415-425

CARBOHYDRATE METABOLISM IN B. bifidum

419

presence of NADH oxidases in the extract. Comparing Curves b and c it appears clearly that phosphate was required for the formation of glyeeraldehyde-3-P from Fru-6-P. This may show that the following reactions were taking place: Fru-6-P + Pt ~

acetyl phosphate + Ery-4-P

Fru-6-P + Ery-4-P--÷ Sed-7-P + glyceraldehyde-3-P

From the slope of Curve c, corrected for endogenous NADH oxidation (Curve a), the specific activity of Fru-6-P phosphoketolase, in combination with transaldolase, was calculated (Table III, first number). The difference in slope of Curves a and b may be caused by contamination of Fru-6-P by inorganic phosphate. 1.6

8E

fextract

2.01

1.4

1.8!

1.-

1.6

1.0

~o~

/",,

~ 1.0

o

~ Oa 0.6

0.6 0.4

0.2 ~ 0

o

do

/

\

'\\

,//'"~\

0.4

C

t6o

,

0.2 ~o Time (sec)

48o ~6o

r~o

350

460

4,50 500 550 Wavelength (mJJ)

600

Fig. i. Influence of inorganic phosphate on the formation of glyceraldehyde-3-P from Fru-6-P. The complete reaction mixture (2.8 ml) contained in #moles: Tris-HC1 buffer (pH 7.2), 5oo; it organic phosphate, 3o; MgClz, IO; cysteine, 20; Fru-6-P, 30; NADH, o.7; glycerol-3-P dehydrogenase/triose phosphate isomerase, 1.4 1.15.; cell-free extract, approx. I mg of protein. Extract was added at the point indicated by an arrow. Fig. 2. Formation of Sed-7~P from Fru-6-P. The assay mixture (2.8 ml) contained in ffmoles: Tris-HC1 buffer (pH 7.2), 5o0; Pl, 30; MgC12, IO; cysteine, 2o; Fru-6-P, 30; NADH, 8; glycerol-3-P dehydrogenase/triose phosphate isomerase, 2.8 I.U.; cell-free extract, approx. 2 mg of protein. Samples were taken immediately after addition of the cell-free extract and after incubating at 25 ° for I h. Protein was precipitated with HzSO 4. In the supernatants the cysteine-sulfuric acid reaction of DlSC~IE2~ was carried out and spectra were drawn. Fructose gives an absorption peak at 415 raft, sedoheptulose at 508 m/z. - - , spectrum before incubation. - - - -, spectrum after incubation. A second a r g u m e n t for the occurrence of these reactions in B. bifidum was t h e a c c u m u l a t i o n of Sed-7-P (Fig. 2). To p r e v e n t f o r m a t i o n of pentose phosphates from Sed-7-P an d g l y c e r a l d e h y d e - 3 - P a large excess of N A D H was a d d e d to t h e reaction m i x t u r e . So an y g l y c e r a l d e h y d e - 3 - P formed was r ed u ced to glycerol-3-P i m m e d i a t e l y and a c c u m u l a t i o n of Sed-7-P could t a k e place. T h e peak at 508 m/~ shows t h e accu m u lation of Sed-7-P. T h e peaks at 415 m/~ show th e disappearance of F r u - 6 - P . A t h i r d a r g u m e n t for t h e phosphoketolase cleavage of F r u - 6 - P was the form a t i o n of acetyl phosphate. T a b l e n shows the specific a c t i v i t y of F r u - 6 - P phosphoketolase expressed a s / z m o l e s acetyl p h o s p h a t e formed p er m g of p r o t ei n per h u n d e r t he conditions m e n t i o n e d . C o m p a r i n g E x p t s . I an d 2 it appears t h a t t h e specific

Biochim. Biophys. Aaa, 136 (x967) 415-425

420

w . DE VRIES, S. J. GERBRANDY, A. H. STOUTHAMER

TABLE II SPECIFIC ACTIVITY OF

Fru-6-P

P~-IOSPHOKETOLASE UNDER

DIFFERENT

CONDITIONS

Specific activity is expressed as pmoles acetyl phosphate formed per mg of protein per h. The cell-free extract was prepared with the Sorvall Ribi cell fractionator. The reaction mixture contained per o.75 ml : 45/~moles of histidine buffer (pH 6.o) or 125 of #moles Tris (pH 7.2), 18/,moles of PI, o.25 #mole of thiamine pyrophosphate, 15/,moles of Fru-6-P, cell-free extract (between o.I and o.25 mg of protein) and, when indicated, 5/~moles of cysteine, 24/,moles of (NHa)2SO4, 2/,moles of NADH and o. 7 I.U. glycerol-3-P dehydrogenase/triose-P isomerase. After 3o rain incubation acetyl phosphate was measured according to LIPMANNAND T U T T L E 13. Expt. .No.

Temp.

pH

I z 3 4

3°o 3°o 25 ° 25°

6.o 7.2 7.2 7.2

Additions

Specific activity

cysteine, (NH4),zSO4 cysteine, NADH, enzymes

29 6.5 8.4 5.2

activity was much higher at pH 6 than at pH 7.2. This finding was also reported by SCHRAMM, KLYBAS AND RACKER 23 for Fru-6-P phosphoketolase in A cetobacter xylinum. I t must be emphasized that in the Expts. I and 2 acetyl phosphate formed both by Fru-6-P phosphoketolase and by Xyl-5-P phosphoketolase, was measured. Namely, because of high specific activities of transaldolase and transketolase, pentose phosphates were rapidly formed from Fru-6-P. In Expt. 4 formation of pentose phosphates was prevented by reducing glyceraldehyde-3-P with glycerol-3-P dehydrogenase/ triose phosphate isomerase and NADH. Therefore, in this case acetyl phosphate was formed by Fru-6-P phosphoketolase exclusively. Expt. 4 took place at p H 7.2, the optimum pH of the coupling enzymes. In Expt. 3 no enzymes and NADH were added. Otherwise the conditions were the same as in Expt. 4- (NH4) 2S04 was added because the coupling enzymes (Expt. 4) were dissolved in 2.4 M (NH4)2SO 4, a compound which appeared to decrease the specific activity of Fru-6-P phosphoketolase. Comparing the specific activities in Expts. 3 and 4, it can be seen that acetyl phosphate production from pentose phosphates formed from Fru-6-P is rather high. No information can be given as to how far the specific activity measured at pH 6.0 is based on formation of acetyl phosphate from pentose phosphates. The mean specific activity at pH 6.0 of seven extracts prepared by ultrasonic oscillation was 18.1 ~ 1.4 (Table In, second number). Note the much higher specific activity, namely 29, in an extract prepared by the Sorvall Ribi cell fractionator

(Table II). Transketolase and X y l - 5 - P phos2hhoketolase The determination of these enzymes is shown in Fig. 3. When omitting phosphate from the reaction mixture it was assumed that the transketolase reaction was taking place only (Curve b):

Xyl-5-P + Rib-5-P ~ glyceraldehyde-3-P + Sed-7-P To remove traces of phosphate the cell-free extract was dialysed. From the slope of Curve b, corrected for endogenous NADH oxidation (Curve a), the specific activity Biochim. Biophys. Acta, 136 (1967) 415-425

CARBOHYDRATE METABOLISM IN B. bifidum

42]

of transketolase was calculated. In the presence of phosphate (Curve c) both the transketolase reaction (see above) and the phosphoketolase reaction were assumed to be taking place. The phosphoketolase reaction is as follows: Xyl-5-P + PI --+ acctyl phosphate + glyceraldehyde-3-P

From the difference in rate of NADH oxidation without added phosphate and in the presence of phosphate, the specific activity of Xyl-5-P phosphoketolase was calculated. 2"0f 1.8 /extract

1.6 1.4

~ 1.2 c

b o.s <

0.6 0.4 0.2

o

~

~:~o ~,o ,~bo ~bo 6bo 76o ~o Time (see)

Fig. 3' Assay of transketolase and Xyl-5-P phosphoketolase. The complete reaction mixture (2.8 ml) contained in #moles: Tris-HC1 buffer (pH 7.2), 500; Pj, 30; MgC12, IO; cysteine, 2o; thiamine pyrophosphate, 0. 5/zmole; Rib-5-P, 3o; NADH, 0.7; glycerol-3-P dehydrogenase/triose phosphate isomerase, 1. 4 I.U.; cell-free extract, o.2 mg of protein. Extract (dialysed) was added at the point indicated by an arrow.

Only Rib-5-P was used for this determination because Xyl-5-P was not available. This must be formed by ribose-5-phosphate isomerase (EC 5.3.1.6) and ribulose phosphate 3-epimerase (EC 5.1.3.1 ) present in the extract before transketolase and Xyl-5-P phosphoketolase could be operating. From the beginning of Curves b and c it can be seen that indeed some time was required before NADH oxidation took place. Therefore specific activities were calculated from the straight part of the curves. It must be emphasized that only a coarse estimation of the specific activities could be obtained in this way. Mean specific activities are mentioned in Table I n .

Transaldolase, hexokinase, Glc-6-P isomerase, phosphofructokinase, glyceraldehyde-3-P dehydrogenase and the enzymes catalyzing the conversion of glycerate-3-phosphate into pyruvate The specific activities of these enzymes are mentioned in Table n I . Hexokinase could not be detected in cell-free extracts prepared by ultrasonic oscillation. For this reason this enzyme was supposed to be not very stable. Hexokinase was demonstrated in a cell-free extract prepared with the Sorvall Ribi cell fractionator quite well. Glyceraldehyde-3-P dehydrogenase was shown to be NAD + specific.

Lactate dehydrogenase Although it was clear that lactate dehydrogenase must be present in B. bifidum, great difficulties were met with the demonstration of this enzyme in cell-free extracts. Biochim. Biophys. ,data, x36 (x967) 4x5-425

W. DE VRIES, S. J. GERBRANDY, A. H. STOUTHAMER

422 TABLE III

S P E C I F I C A C T I V I T I E S OF T H E E N Z Y M E S I N V O L V E D IN T H E D E G R A D A T I O N

OF G L U C O S E BY

]~. bifidum

Unless otherwise m e n t i o n e d cell-free e x t r a c t s were p r e p a r e d b v ultrasonic oscillation. Specific activities were e x p r e s s e d as follows: hexokinase, Glc-6-P isomerase, g l y c e r a l d e h y d e - 3 - P d e h y d r o genase as /,moles N A D P + (NAD +) reduced p e r m g of p r o t e i n p2r h; F r u - 6 - P p h o s p h o k e t o l a s e , X y l - 5 - P p h o s p h o k e t o l a s e as # m o l e s N A D H oxidized p e r m g of protein p : r h (first n u m b e r ) a n d as ~ m o l e s acetyl p h o s p h a t e f o r m e d per m g of protein per h (second n u m b e r ) ; transaldolase, t r a n s ketolase, lactate d e h y d r o g e n a s e , p h o s p h o f r u c t o k i n a s e a s / * m o l e s N A D H oxidized par m g of protein per h; f o r m a t i o n of p y r u v a t e from g!ycerate-3-P as # m o l e s p y r u v a t e f o r m e d par m g of p r o t e i n per h. R e s u l t s are given with t h e s t a n d a r d error of t h e m e a n ; n u m b e r of e x t r a c t s tested in parentheses.

Enzyme

Specific activity

Fru-6-P phosphoketolase Transketolase Xyl-5-P phosphoketolase Transaldolase Hexokinase* Glc-6-P isomerase Phosphofructokinase* Glyceraldehyde-3-P dehydrogenase F o r m a t i o n of p y r u v a t e from g l y c e r a t e - 3 - P Lactate dehydrogenase*

*

4.I ± 0.4 (8); i8. I ~_ 1. 4 (7 ~ 1. 7 (IO) I3.8 ~ I. 4 (IO); 28. 7 ~ 2. 5 (7)

II. 5 4-

Io.2 ~_ i.i

(9)

6.1 45 I.I 4.7 8. 5 59

T h e e x t r a c t used was p r e p a r e d with Sorvall Ribi cell fractionator.

Pyruvate did not cause any oxidation of NADH when incubated with the cell-free extract. DL-Lactate did not reduce NAD + or NADP + in the presence of hydrazine. No reduction of 2,6-dichlorophenolindophenol by DL-lactate could be observed. However, a highly active lactate dehydrogenase could be detected in the presence of Fru-I,6-P 2 (Fig. 4)- Addition of Fru-I,6-P 2 to the assay mixture caused a rapid oxidation of NADH (Fig. 4a). Fig. 4 b shows that the oxidation of NADH was not caused by some conversion of Fru-I,6-P 2. The specific activity is mentioned in Table III. From Fig. 5 it can be seen that minute concentrations of Fru-I,6-P 2 were sufficient to activate lactate dehydrogenase. Fru-6-P had not any influence on lactate

1.6

extract FOP 1,6 -2 1.4

extract _ _ ~ F PRY

1.4

DP

1.2

1.2

8~.c

~ 1.0 u

c

E

0.8

# o.e b o.e

.8 o.e < 0.4

< 0.4 0.2

0.2

1do 25o 360 46o 56b (a)

Time (sec)

o'

1do 2bo ~ o 450 5'o0

(b)

Time (see)

Fig. 4. R e q u i r e m e n t of lactate d e h y d r o g e n a s e for F r u - I , 6 - P 2. T h e complete reaction m i x t u r e (2.75 ml) c o n t a i n e d in /~moles: Tris-HC1 buffer (pH 7-4), 500; N A D H , o.8; p y r u v a t e , 2o; F r u 1,6-Pz, 2; cell-free extract, o.I m g of protein. P y r u v a t e (PYR), F r u - I , 6 - P z (FDP) a n d e x t r a c t were a d d e d a t t h e p o i n t s indicated b y arrows.

Biochim. Biophys. A aa, a 3 6 "(I967) 415-425

CARBOHYDRATE METABOLISM IN B. bifidum

423

dehydrogenase. These results are similar to those of WOLIN14 who found that lactate dehydrogenase of Streptococcus boris specifically requires Fru-i,6-P 2 for activity. 60

//f

50 4O >

~D 3 0

u 20

~

~o

I x

I

/ o.~

abs

o112 o'.16 o12o Q~4 o'.28

Fru -1,6 -P2 (jumol/mt assay mixture)

Fig. 5- I n f l u e n c e of the concentration of F r u - I , 6 - P 2 on the specific activity of lactate d e h y d r o genase. Specific a c t i v i t y is expressed as/~moles N A D H oxidized per mg of protein per h. DISCUSSION

Members of the genus Lactobacillus ferment glucose either vm the glycolytic system or via the hexose monophosphate shunttS. These pathways are not involved in the degradation of glucose by B. bi/~dum although this microorganism is classified by BERGEY6 as belonging to the genus Lactobacillus. Namely aldolase, an enzyme unique to glycolysis, could not be detected in cell-free extracts of B. bifidum. Phosphofrnctokinase, also characteristic for glycolysis, was demonstrated in only low amounts. The absence of Glc-6-P dehydrogenase from B. bil~dum rules out the operation of the hexose monophosphate shunt. GIc I ATP G[c-6-P

Rib - 5 -P

Xy!. -AT-P- ~" XyL - 5 - P

Acetyl - P

!

Ac tate

Gtyceratdehyde - 3 -P

See - 7 - P

Gtyceratdehyde - 3 - P

,

T

T

Fru - 6 -P

Ery-4 -P

AcetyL - P

Acetate

Lactate

F i g . 6. Degradation of glucose and xylose b y B. bi#dum. Interrupted arrows: more than one step

is involved.

An alternative route of glucose degradation is shown in Fig. 6. The key reaction of this scheme is a phosphoketolase cleavage of Fru-6-P, which was found before in A. xylinum by SCHRAMM, KLYBAS AND RACKER23. By this reaction Fru-6-P is converted into acetyl phosphate and Ery-4-P. Strong arguments for the occurrence of Biochim. Biophys. Acta, 136 (1967) 4 1 5 - 4 2 5

424

W. DE VRIES, S. J. GERBRAND¥, A. H. STOUTHAMER

this reaction are available. Ery-4-P reacts with Fru-6-P to form Rib-5-P and Xyl-5-P. This conversion is catalysed by transaldolase and transketolase. Pentose phosphates are converted into acetyl phosphate and glyceraldehyde-3-P by Xyl-5-P phosphoketolase. Via reactions common to glycolysis lactate is formed from glyceraldehyde3-P. The most important enzymes of this scheme were shown to be present in cell-free extracts of B. bifidum (Table III). No attempts were made to demonstrate acetate kinase (EC 2.7.2.1 ) and pentose-transforming enzymes. The scheme mentioned gives the following fermentation balances for glucose and xylose: I glucose ~ 1. 5 a c e t a t e + i.o l a c t a t e I x y l o s e ---+ I.O a c e t a t e + I.O l a c t a t e

These theoretical fermentation balances agree with the fermentation balances found experimentally rather well. The presence of phosphofructokinase does not fit into the reaction scheme described. However, phosphofructokinase is by no means superfluous for B. bifidum, for lactate dehydrogenase is shown to have an absolute requirement for Fru-I,6-P v the product of action of phosphofructokinase. Minute concentrations of Fru-I,6-P~ are sufficient to demonstrate a highly active lactate dehydrogenase. In this way the rapid degradation of glucose by B. bifidum is easily understood. For formation of lactate from xylose also an activator must be present. Small amounts of Fru-I,6-P 2 may be formed from Xyl-5-P by transketolase, transaldolase and phosphofructokinase or traces of aldolase may catalyse the conversion of glyceraldehyde-3-P into Fru-I,6-P v The fermentation route found in the present investigation does not agree with the findings of KUHN AND TIEDEMANN2. They mentioned the formation of [14C]acetate and [14CJlactate from [I-l~C]glucose, whereas according to the fermentation route described in the present paper lactate formed from [I-14CIglucose is not radioactive. Unlike reported in the present paper, they detected aldolase in cell-free extracts of the microorganism used. However, the specific activity was not determined. Lactate dehydrogenase demonstrated by them, had not a specific requirement for Fru-I,6-P 2 as reported in the present paper. No explanation for these discrepancies can be given. The pathway given in Fig. 7 was also found in a number of other strains of Bifidobacterium bifidum ~. As these strains belonged to several types of B. bifidum it may be concluded that this pathway is specific for all strains of this species. From the present investigation it can be concluded that B. bifidum ferments glucose via a pathway which is different from those found in members of the genus Lactobacillus. Therefore classification of B. bifidum to the genus Lactobacillus does not seem justified. SEBALD, GASSERAND WERNER¢ came to this conclusion on account of the difference in percentage GC of the DNA of B. bifidum and members of the genus Lactobacillus. The present paper confirms the conclusion of SEBALD, GASSER AND WERN~ER 4, that the name Lactobacillus bifidus is not correct, and must be replaced by the name Bifidobacterium bifidum. This name, already proposed by ORLA-JENSEN~5 in 1924 and used by several other investigators 4, is therefore used throughout this paper. NOTE ADDED IN PROOF (Received March 9th, 1967) In Ber. Wiss. Biol., 267 (1967) 191, the authors found an abstract of a paper of V. SCARDOVIoriginally published in Ann. Microbiol. Enzimol., 15 (1965) 19. In this Biochim. Biophys. Acta, 136 (1967) 415-425

CARBOHYDRATE METABOLISM IN B .

bifidum

425

p a p e r t h e p r e s e n c e of f r u c t o s e - 6 - p h o s p h a t e p h o s p h o k e t o l a s e i n cell-free e x t r a c t s of

B . bifidum w a s d e s c r i b e d : T h e p a t h w a y of c a r b o h y d r a t e m e t a b o l i s m w a s f u r t h e r c o n f i r m e d b y s t u d y i n g t h e b r e a k d o w n of [I-14C]glucose. ACKNOWLEDGEMENTS T h e a u t h o r s a r e g r a t e f u l t o Miss F . K . DE MEIJERE a n d Mr. F. FLENTGE f o r technical assistance.

REFERENCES i R. F. NORRIS, T. FLANDERS, R. M. TOMARELLI AND D. GY6RGY, J. Bacteriol., 60 (195 o) 681. 2 R. KUHN AND H. TIEDEMANN, Z. Naturforsch., 8 b (1953) 428.

3 J. DEHNERT, Zbl. Bakt. I. Abt. Ref., 163 (1957) 481. 4 M. SEBALD, F. GASSER AND H. WERNER, Ann. Inst. Pasteur, lO9 (1965) 251. 5 H. BEERENS, A. GERARD AND J. GUILLAUME, Ann. Inst. Pasteur, Lille, 9 (1957) 77. 6 Bergey's Manual of Determinative Bacteriology, Baltimore, 1957. 7 G. BUYZE, C. J. A. VAN DEN HAMER AND P. G. DE HAAN, Antonie van Leeuwenhoek J. Microbiol.

Serol., 23 (1957) 345. 8 C. J. A. VAN DEN HAMER, Thesis, University of Utrecht, 196o. 9 W. W. UMBREIT, R. H. BURRIS AND J. F. STAUFFER, Manometric Techniques, Burgess, Minne-

io II 12 13 14 15 16 17 18 19 20

21 22 23 24 25 26

sota, 1964 . W. MEJBAUM,Z. Physiol. Chem., 258 (1939) 117. S. B. BARKER AND W. H. SUMMERSON,J. Biol. Chem., 138 (1941) 535I. A. ROSE, M. GRUNBERG-MANAGO,S. R. KOREY AND S. OCHOA,J. Biol. Chem., 211 (1954) 737. F. LIPMANN AND L. C. TUTTLE, J. Biol. Chem., 159 (1945) 21. O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL,J. Biol. Chem., 193 (1951) 265 . F. H. BRUNS AND H. U. BERGMEYER, in H. U. BERGMEYER, Methoden der Enzymatischen Analyse, Verlag Chemie, Weinheim, 1962, p. 724 . A. KORNBERG AND B. L. HORECKER, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. I, Academic Press, New York, 1955, p. 323 • M. W. SLEIN, G. T. CORI AND C. F. CORI, J. Biol. Chem., 186 (195 o) 763 . M. W. SLEIN, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. I, Academic Press, New York, 1955, p. 299. t(. H. LING, W. L. BYRNE AND H. LARDY, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. I, Academic Press, New York, 1955, p. 3°6. S. F. VELICK, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. I, Academic Press, New York, 1955, p. 4 ol. E. A. DAWES, D. W. RIBBONS AND P. J. LARGE, Biochem. J., 98 (1966) 795. Z. DlSCHE AND E. BORENFREUND, J. Biol. Chem., 18o (1949) 1297. M. SCHRAMM,V. KLYBAS AND E. RACKER, J. Biol. Chem., 233 (1958) 1283. M. J. WOLIN, Science, 146 (1964) 775. S. ORLA-JENSEN, Lair, 4 (1924) 469 • W. DE VRIES AND A. H. STOUTHAMER,J. Bacteriol., in the press.

Biochim. Biophys. Acta, 136 (1967) 415-425