Induction and repression of enzymes involved in exogenous purine compound utilization in Bacillus cereus

460

Biochimica et Biophysica Acta, 678 (1981) 460-466

Elsevier/North-Holland Biomedical Press BBA 29821

INDUCTION AND REPRESSION OF ENZYMES INVOLVED IN EXOGENOUS PURINE COMPOUND UTILIZATION IN B A C I L L US C E R E U S M.G. TOZZI, F. SGARRELLA and P.L. IPATA Laboratorio di Biochimica, Facolt~ di Scienze, Universitd di Pisa, Via A. Volta 4, 56100 Pisa (Italy)

(Received May 26th, 1981)

Key words: Purine utilization; Enzyme induction; Enzyme repression; (B. cereus)

5'-Nucleotidase, adenosine phosphorylase, adenosine deaminase and purine nucleoside phosphorylase, four enzymes involved in the utilization of exogenous purine compounds in Bacillus cereus, were measured in extracts of this organism grown in different conditions. It was found that adenosine deaminase is inducible by addition of adenine derivatives to the growth medium, and purine nucleoside phosphorylase by metabolizable purine and pyrimidine ribonucleosides. Adenosine deaminase is repressed by inosine, while both enzymes are repressed by glucose. Evidence is presented that during growth of B. cereus in the presence of AMP, the concerted action of 5'-nucleotidase and adenosine phosphorylase, two constitutive enzymes, leads to formation of adenine, and thereby to induction of adenosine deaminase. The ionsine formed would then cause induction of the purine nucleoside phosphorylase and repression of the deaminase. Taken together with our previous findings showing that purine nucleoside phosphorylase of B. cereus acts as a translocase of the ribose moiety of inosine inside the cell (Mum, U., Sgarrella, F. and Ipata, P.L. (1978) J. Biol Chem. 253, 7905-7909), our results provide a clear picture of the molecular events leading to the utilization of the sugar moiety of exogenous AMP, adenosine and inosine as an energy source.

Introduction In a previous paper [1], we have shown that AMP, added to intact vegetative cells of Bacillus cereus is quantitatively converted to hypoxanthine, via the successive action of 5'-nucleotidase, adenosine deaminase and purine nucleoside phosphorylase. The purine ring remains external, while the ribose moiety is translocated inside the cell as ribose 1-phosphate, by the action of a purine nucleoside phosphorylase, thus providing a system whereby the ribose moiety of exogenous AMP can be utilized as an energy source. In addition, we have found that in the same organism, adenosine is acted upon by a specific phosphorylase, an enzyme distinct from the purine nucleoside phosphorylase [2] ; however, the role of this enzyme has not been elucidated so far. In this paper, we have

studied the effect of a series of metabolites, added to a standard growth medium, on the levels of the tbur enzymes involved in the utilization of exogenous AMP in B. cereus. The results suggest that the concerted action of 5'-nucleotidase and adenosine phosphorylase, two constitutive enzymes, leads to production of adenine, which in turn acts as an inducer of adenosine deaminase. The inosine produced then acts as an inducer of purine nucleoside phosphorylase and as a repressor of the deaminase. Induction of purine nucleoside phosphorylase in B. cereus [3], as well as of other enzymes involved in purine compounds utilization in other bacterial species, have been observed before [ 4 - 9 ] . We now provide a clear picture of the sequential events responsible for exogenous AMP utilization in B. cereus.

0304-4165/81/0000-0000/$02.50 © 1981 Elsevier/North-Holland Biomedical Press

461 Materials and Methods Chemicals. Nucleotides, nucleosides, bases, free and phosphorylated sugars, glycerol, citrate, pyruvate, acetate and a-ketoglutarate were from Sigma Chem. Co. or from Boehringer. All other chemicals were of reagent grade. Cellulose acetate filters were obtained from Sartorius GmbH. Bacteria and culture conditions. Bacillus cereus, strain NCIB 8122, was grown at 37°C with vigorous shaking in a medium of the following composition: 1.32 g K2HPO 4 • 3 H20/5 g (NH4)2SO4/0.1 g MnSO 4 • H20/0.8 g MgSO4/0.01 g ZnSO4/0.01 g CuSO4 " 5 H20/0.01 g CaC12/0.001 g FeSO4 • 7 H20/ 2 g yeast extract/distilled water up to 1 1. Bacterial growth was monitored by measuring the absorbance at 600 nm. 1 ml of a 4-h culture was used as standard inoculum. In the experiments described in this paper, this standard medium was supplemented with a number of compounds, as described later; cells were grown in 100-ml flasks, each containing 20 ml growth medium, and harvested by filtration through 1.2/lm pore size filters. 2-ml aliquots of the filtrate were used to determine purine nucleosides and bases (see below), while cells were washed with 10 ml 0.1 M Tris-HC1 buffer, pH 7.4, containing 50 mM KC1, and resuspended in the same buffer to an absorbance at 600 nm of about 10. Cells were then broken by ultrasonic treatment, and crude homogenates were assayed for 5'-nucleotidase, adenosine deaminase, adenosine phosphorylase and purine nucleoside phosphorylase activities. The specific activities of the inoculum, expressed as units/ml/A 6oo n m were: 5'-nucleotidase, 7.9 • 10-3; adenosine deaminase, 4.5 - 10-3; adenosine phosphorylase, 13.2 • 10 -3, and purine nucleoside phosphorylase, 14.1 • 10 -3. One enzyme unit is the amount of enzyme catalyzing the transformation of 1/lmol substrate/min. Enzymes. Xanthine oxidase (EC 1.2.3.2) (1 mg/ ml) and adenosine deaminase (EC 3.5.4.4) (5 mg/ml) were from Boehringer. Adenosine deaminase was diluted with water to a concentration of 20 #g]ml before use. Adenosine phosphorylase was partially purified from B. cereus as previously described [2]. Purine nucleoside phosphorylase was partially purified from B. cereus as follows. B. cereus was grown in

the presence of 20 mM inosine in order to repress adenosine deaminase (see Results). Stationary-phase cells were harvested by centrifugation at 15 000 × g, suspended in 0.1 M Tris-HC1 buffer, pH 7.4, and broken by ultrasonic treatment. The homogenate was subjected to ammonium sulfate fractionation between 30 and 70% saturation. The precipitate was then dissolved in a minimal volume of 0.05 M TrisHC1 buffer, pH 7.4, and chromatographed on a Sephadex G-100 column (70 × 5.5 cm) equilibrated with the same buffer. The flow rate was approx. 40 ml/h and 6-ml fractions were collected. Active fractions were pooled and concentrated with poly(ethylene glycol). The final enzyme preparation had a specific activity of 1.2 U/mg protein and was devoid of any adenosine deaminase activity. Enzyme assays. All enzyme activities were determined spectrophotometrically. 5'-Nucleotidase was assayed according to Ipata [ 10] ; adenosine deaminase and purine nucleoside phosphorylase were assayed according to Kalckar [I1,12]; adenosine phosphorylase was assayed according to Miech et al. [13]. Incubation of&tact cells with adenosine. Late logphase B. cereus cells were harvested by filtration through 1.2 /lm pore size filters, washed extensively with 0.1 M Tris-HC1 buffer, pH 7.4, at room temperature and resuspended in a small volume of the same buffer. The incubation mixture contained, in a final volume of 20 ml, 0.1 mM adenosine/0.1 M Tris-HC1 buffer, pH 7.4, and cells to a final absorbance at 600 nm of 0.34. The incubation was carried out at 37°C. 3-ml aliquots were rapidly filtered and the filtrate was heated for 2 min at 100°C. Adenosine, inosine, adenine and hypoxanthine were then determined in the filtrate. Determination o f purine nucleosides and bases. Adenosine, inosine, hypoxanthine and adenine were determined spectrophotometrically as described previously [14,15]. To determine inosine in the presence of adenosine, the partially purified purine nucleoside phosphorylase of B. cereus was used throughout instead of the commercial enzyme, which is significantly contaminated with adenosine deaminase. Results

Table I summarizes the effect of various metabolites added to the standard growth medium, on the

462 TABLE I EFFECT OF ADDITION OF VARIOUS COMPOUNDS TO THE STANDARD GROWTH MEDIUM ON THE LEVELS OF ENZYMES OF PURINE UTILIZATION IN B. CEREUS The cells were harvested at the 4th h, and enzymes were assayed. Enzyme levels are given as a multiple of basal enzyme level as determined in uninduced cells. Figures in parentheses indicate basal enzyme levels, expressed in units per absorbance unit. n.d., not determined. Compound

None ATP ADP AMP IMP GMP CMP UMP Adenosine Deoxyadenosine Inosine Guanosine Xanthosine Cytidine Uridine Deo xyu ridine Adenine Hypoxanthine Cytosine Uracil Glucose Ribose Ribose-1 -P Ribose-5 -P Glycerol Acetate Pyruvate Citrate a-Ketoglutarate Adenosine + glucose Adenine + glucose

Concn. in growth medium (mM)

Enzyme levels 5'-Nucleotidase

Adenosine deaminase

Nucleoside phosphorylase

Adenosine phosphorylase

10 2 10 3.4 20 20 20 20 10 20 2 3 12 20 l0 20 20 20 20 20 20 1.1 2.5 20 20 20 20 20 2.5 + 20 2.5 + 20

1.0(7.9) 0.6 0.6 0.6 0.6 0.6 1.0 1.2 1.2 0.9 1.0 1.4 0.5 1.5 0.9 1.2 1.2 0.7 0.7 0.7 1.0 1.0 0.4 1.0 1.0 0.7 0.9 0.6 1.0 1.2 0.9

1.0(4.5) 13.6 8.0 15.0 1.0 0.8 0.8 0.8 12.0 20.0 0.0 0.5 1.0 1.1 0.6 0.7 20.0 1.6 1.1 0.8 0.2 0.5 1.0 0.9 0.8 1.0 0.6 0.5 1.0 1.0 0.7

1.0(14.1) 4.0 3.0 6.8 6.9 6A 1.5 4.3 6.7 3.2 5.6 4.8 0.6 1.6 3.0 3~ 1.1 1.6 0.8 1.3 0.5 0.6 0.9 1.0 0.9 1.0 0.6 0.6 1.0 1.7 0.5

1.0(13.2) 1.7 1.4 1.8 1.9 n.d. 0.8 1.0 0.9 1.1 1.0 1.3 1.0 1.2 1.2 1.2 1.0 0.9 0.7 0.7 1.0 0.7 1.0 1.0 0.7 0.6 0.7 0~ 0.8 1.0 0.9

levels o f t h e four e n z y m e s involved in the u t i l i z a t i o n o f e x o g e n o u s p u r i n e c o m p o u n d s in B. cereus. T h e activity o f a d e n o s i n e d e a m i n a s e significantly increases in t h e p r e s e n c e o f all a d e n i n e derivatives t e s t e d , w h e r e a s it is m a r k e d l y r e d u c e d in t h e p r e s e n c e o f glucose. F u r t h e r m o r e , glucose r e m o v e s t h e effect o f both adenine and adenosine on adenosine deaminase a n d p u r i n e n u c l e o s i d e p h o s p h o r y l a s e activities. T h e

levels o f 5 ' - n u c l e o t i d a s e a n d a d e n o s i n e p h o s p h o r y l a s e r e m a i n s u b s t a n t i a l l y u n a f f e c t e d . T h e a c t i o n o f inosine results in b o t h i n d u c t i o n o f p u r i n e n u c l e o s i d e phosp h o r y l a s e a n d r e p r e s s i o n o f a d e n o s i n e d e a m i n a s e . The f o r m e r e n z y m e is i n d u c e d also b y a d d i t i o n o f o t h e r g u a n i n e a n d uracil n u c l e o s i d e s a n d nucleosides. We have also m e a s u r e d t h e levels o f e n z y m e activities d u r i n g t h e g r o w t h cycle o f B. cereus g r o w n in the

463 presence of adenine, inosine and adenosine, repsectively. Addition of adenine (Fig. 1) to the standard growth medium causes a rapid increase of the adenosine deaminase activity during the early log-phase, followed by an abrupt decrease. The levels of the other three enzymes remain substantially unchanged. Addition of inosine (Fig. 2), results in a progressive decrease of adenosine deaminase. This enzyme disappears at the 4th h, but becomes again detectable at the 15th h, when inosine is quantitatively converted to hypoxanthine (see inset of Fig. 2); on the contrary, purine nucleoside phosphorylase activity rapidly increases and remains relatively high also during the stationary phase. Addition of adenosine (Fig. 3) results in the successive induction of adenosine deaminase and purine nucleoside phosphorylase. As shown in the inset of Fig. 3, the latter enzyme is induced when inosine accumulates in the medium. The levels of both 5'-nucleotidase and adenosine phosphorylase remain substantially unchanged in the conditions of Figs. 2 and 3.

4 >_ 3

4

_u

3

~2 2

o o

0

, 0

5

10 Time ( h )

15

20

Fig. 2. Relative levels of 5'-nucleotidase (~), adenosine deaminase (o), purine nucleoside phosphorylase (o) and adenosine phosphorylase (A) in B. c e r e u s grown in the standard medium supplemented with 17 mM inosine; (. . . . . . ) indicates the absorbance of the culture at 600 nm. The inset shows the time course of inosine (D) breakdown and hypoxanthine (.) formation in the growth medium. The specific activities of the inoculum were those from the text.

,o[

2O ->

//

~ ....

I

2

u

.................................1

15

o

~

lO

//" I

0

> D

75

//

M

0

5

~0

Time (h)

~ 5c

lO 25

i) i

>

i

i

0

5

t t

0

10

g

10

1;

2'0

Time (h)

i

1'5

Time ( h ) Fig. 1. Relative levels o f 5 '-nucleotidase (A), adenosine deami-

nase (o), purine nucleoside phosphorylase (o) and adenosine phosphorylase (A) in B. c e r e u s grown in the standard medium supplemented with 5 mM adenine; (. . . . . . ) indicates the absorbance of the culture at 600 nm. The inset shows the levels of adenine (m) in the medium. The specific activities of the inoculum (zero time) are those from the text.

Fig. 3. Relative levels of 5'-nucleotidase (~), adenosine deaminase (o), purine nueleoside phosphorylase (e) and adenosine phosphorylase (A) in B. c e r e u s cells grown in the standard medium supplemented with 20 mM adenosine; (. . . . . -) indicates the absorbance of the culture at 600 rim. The inset shows the time course of adenosine (u) breakdown and of inosine (A) and hypoxanthine (m) formation in the growth medium. The specific activities of the inoculum were those from the text.

464 TABLE II EFFECT OF INCREASING CONCENTRATIONS OF ADENINE, INOSINE AND ADENOSINE ADDED TO THE STANDARD GROWTH MEDIUM ON THE LEVELS OF B. C E R E U S ADENOSINE DEAMINASE AND PURINE NUCLEOSIDE PHOSPHORYLASE The cells were harvested after 3.5 h of growth, and enzymes were assayed. Specific activities are expressed as units per absorbancc unit. Compound added: adenine (mM)

-

Specific activity: adenosine deaminase

Compound added: inosine (mM)

4 ~

0.04 0.1 0.3 0.5 0.7 1 5 10

-

6.6 10.2 14.2 21.3 33 41.1 51.8 51.4

0.04 0.1 0.2 0.4 1 10 20

Specific activity Adenosine deaminase

Purine nucleoside phosphorylase

4.5 4.5 4.5 4.7 4.5 4.5 0.99 0.00

19.3 20.4 26.3 27.9 28.2 50.3 91.6 92

Table II shows that the concentrations of adenine required to induce adenosine deaminase, are significantly lower than those of adenosine. Furthermore, repression of adenosine deaminase and induction of purine nucleoside phosphorylase by inosine occur at the same range of concentration of the nucleoside (between 10 and 20 raM). The utilization of adenosine by intact cells of B.

Adenosine deaminase

Purine nucleoside phosphorylase

0.04 0.1 0.2 0.4 1 5 10 20

4.7 6.7 7.1 9.3 16.4 32.2 63.3 65.3 65.9

19.3 19.4 21.3 21.6 25.6 38.6 74.6 74.0 74.3

Specific activity

grown under different conditions is shown in Fig. 4. It is evident that in incubations carried out either with cells grown in the standard medium (Fig. 4c) or in the standard medium supplemented with inosine (Fig. 4b), adenine is the first product appearing the incubation medium, most likely owing to the relatively low levels of the uninduced adenosine deaminase. On the other hand, adenine was undetect-

cereus

C

B

A

0.1 0

Compound added: adenosine (mM)

Z~

E

005

.

0

b

T

,

I0

15

0

20

40

0

20

40

Time ( m i n )

Fig. 4. Breakdown of adenosine by intact cells of B. cereus grown 4 h in the standard medium supplemented with 20 mM adenosine (A), 20 mM inosine (B) or without additions (C). (z~)Residual adenosine; (e) hypoxanthine; (A) adenine; (o) inosine.

465

able in incubations carried out with intact cells grown in the presence of adenosine (Fig. 4a). Discussion

Two out of the four enzymes involved in the utilization of exogenous AMP in B. cereus, adenosine deaminase and purine nucleoside phosphorylase, are subjected to genetic regulation. They are metabolically related, since they catalyze the successive deamination of adenosine and phosphorolysis of inosine. However, they do not appear to be coordinated with simultaneous induction, since: (a) inosine causes induction of purine nucleoside phosphorylase and repression of adenosine deaminase (Fig. 2); (b) adenine causes induction of the deaminase, without affecting the level of the phosphorylase (Fig. 1); (c) both enzymes respond to adenosine addition to the growth medium; however, induction of the phosphorylase follows that of the deaminase (Fig. 3), and apparently occurs when inosine accumulates in the medium at the concentration (approx. 5 mM) needed for enzyme induction (see also Table II); (d) the deaminase increases by addition of adenine compounds to the growth medium, whereas purine nucleoside phosphorylase activity increases also in the presence of guanine and uracil compounds (Fig. 1 and Table I). Induction of adenosine deaminase by adenine compounds might be a consequence of adenine formation through the action of adenosine phosphorylase, a constitutive enzyme. For instance, induction by AMP might occur through the sequential action of 5'-nucleotidase, the other constitutive enzyme, and adenosine phosphorylase: AMP 5'-nucleotidase adenosine adenosine phosphorylase adenine

5'-Nucleotidase exists in B. cereus as an extoenzyme [17]. After induction, the deaminase becomes responsible for the utilization of adenosine, together with the purine nucleoside phosphorylase, which in turn is induced by inosine. In these conditions, there is no appreciable formation of adenine: most likely, in accord with our previous results [1], deamination of

the nucleoside by intact cells is much faster than its phosphorolysis. This idea is strengthened by the results of Fig. 4b and c, showing that adenine accumulates in the incubation medium, when adenosine is added to washed intact cells of B. cereus, previously grown either in the standard medium, or in the presence of inosine, when the deaminase is at the lowest uninduced level. It must be added that C. cereus does not possess any adenase activity. Exogenous deoxyadenosine rapidly undergoes phosphorolysis to adenine, without appreciable deamination to deoxyinosine [16] which explains its action as an inducer of adenosine deaminase. The rapid fall of adenosine deaminase after induction by adenine (Fig. 1) contrasts with the slower decrease of the enzyme activity, induced in the presence of adenosine (Fig. 3), and suggests a process of enzyme inactivation occurring in the absence of metabolizable adenosine. Purine nucleoside phosphorylase is induced by compounds which are either substrates, or can be converted into substrates of purine and pyrimidine nucleoside phosphorylases. The lack of induction by xanthosine and cytidine is not surprising, since the former is not a substrate of B. cereus purine nucleoside phosphorylase [3], and the latter is not attacked by intact cells of this organism (Tozzi, M.G., unpublished results). The ribose moiety, or one of its metabolites, of exogenous nucleosides might be involved in the induction of purine nucleoside phosphorylase, in view of the action of this enzyme as a 'translocase' of ribose 1-phosphate inside B. cereus [ 1]. Moreover, free bases did not induce the enzyme (Table I). In conclusion, adenine formed by the action of the constitutive 5'-nucleotidase and adenosine phosphorylase, and the subsequent induction of adenosine deaminase and purine nucleoside phosphorylase, which catalizes the translocation of the sugar moiety into B. cereus, would lead to the utilization of the ribose moiety of exogenous AMP as an energy source. Repression of both adenosine deaminase and purine nucleoside phosphorylase by glucose is in line with this idea. Finally, the utilization of the purine ring remaining in the medium might be accomplished by the action of a phosphoribosyltransferase, which acts on exogenous hypoxanthine, and endogenous 5-phosphorybosyl-1 -pyrophosphate [18,19].

466 Acknowledgements This paper was supported by the Italian CNR and by the Italian Board of Education.

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