A phosphate-stimulated NAD(P)+-dependent glyceraldehyde-3-phosphate dehydrogenase in Bacillus cereus

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A phosphate-stimulated NAD(P) ^þ -dependent

glyceraldehyde-3-phosphate dehydrogenase in Bacillus cereus

Abdelghani Iddar

^a

, Aurelio Serrano

^b;

, Abdelaziz Soukri

^a;

a Laboratoire de Biochimie, De¤partement de Biologie, Faculte¤s des Sciences A|«n-Chock, Universite¤ Hassan-II, Km 8 route d’El Jadida, B.P. 5366 Ma“arif, Casablanca, Morocco

b Instituto de Bioqu|¤mica Vegetal y Fotos|¤ntesis (CSIC-Universidad de Sevilla), Centro de Investigaciones Cient|¤¢cas Isla de la Cartuja, Ame¤rico Vespucio s/n, 41092 Sevilla, Spain

Received 11 January 2002 ; received in revised form 20 March 2002 ; accepted 25 March 2002 First published online 6 May 2002

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme of central carbon metabolism, was studied in aBacillus cereus strain isolated from the phosphate layer from Morocco. Enzymatic assays with cell extracts demonstrated that when grown on Luria^

Bertani (LB) medium,B. cereuscontains a major NAD^þ-dependent GAPDH activity and only traces of NADP^þ-dependent activity, but in cells grown on Pi-supplemented LB medium a strong increase of the NADP^þ-dependent activity, that became predominant, occurs concurrently with a GAPDH protein increase. Our results show thatB. cereus possesses two GAPDH activities, namely NAD^þ- and NADP^þ-dependent, catalyzed by two enzymes with distinct coenzyme specificity and different phosphate regulation patterns. The finding of a phosphate-stimulated NADP^þ-dependent GAPDH in B. cereus indicates that this bacterium can modulate its primary carbon metabolism according to phosphate availability. 8 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords : Glyceraldehyde-3-phosphate dehydrogenase ; NAD^þ; NADP^þ; Phosphate ; Soil bacterium;Bacillus cereus

1. Introduction

Phosphorus, like nitrogen, is a mineral nutrient required by plants and microorganisms, its major physiological role being energy storage and release during cellular metabolism [1]. In microorganisms, many proteins allow the man- agement of phosphate (P) in the cells. The synthesis of these proteins is either repressed or induced according to the orthophosphate (Pi) concentration [2]. Basically, the assimilation of any P compounds involves two early steps.

First, inorganic phosphate (mainly Pi) or an alternative P compound must be taken up, and second, Pi or the P in the alternative compound must be incorporated into ATP, the primary phosphoryl donor in metabolism [3^5].

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme involved in central pathways of carbon metabolism [6]. Three distinct GAPDH proteins with di¡erent subcellular localization and performing various roles have been found. (i) The typical NAD^þ-dependent glyco- lytic enzyme (EC 1.2.1.12) found in all organisms so far studied and located in the cytoplasm, that plays a pivotal role in the Embden^Meyerho¡ pathway not only in glycolysis but also in glyconeogenesis [6]. (ii) The NADP^þ- dependent GAPDH (EC 1.2.1.13), a key component of the reductive pentose-phosphate (RPP) cycle, which is located in the chloroplast stroma and the cyanobacterial cytoplasm and is involved in photosynthetic CO2 assimilation [7,8]. These two enzymes catalyze the oxidation of glyceraldehyde 3-phosphate (G3P) into diphosphoglyceric acid using the orthophosphate anion (PO³³₄ ) as a cofactor in their catalytic reactions. (iii) The cytosolic non-phosphorylating NADP^þ-dependent GAPDH (GAPDHN) (EC 1.2.1.9), also named Pi-independent G3P :NADP^þ oxido- reductase, that catalyzes an irreversible oxidation of G3P to 3-phosphoglycerate [7,9]. The gene gapN encoding the plant non-phosphorylating enzyme has been cloned and

* Corresponding author. Tel. : +34 (4) 48 95 24 ; Fax : +34 (4) 46 00 65.

** Also corresponding author. Tel. : +212 (2) 223 06 80/84 ; Fax : +212 (2) 223 06 74.

E-mail addresses :aurelio@cica.es (A. Serrano), a_soukri@hotmail.com (A. Soukri).

www.fems-microbiology.org

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shown to be a member of the aldehyde dehydrogenase superfamily [10].

In many eukaryotic organisms, more than one GAPDH has been identi¢ed, i.e. the three enzyme isoforms named TDH1^TDH3 of Saccharomyces cerevisiae [11]. Among bacteria, photosynthetic cyanobacteria possess two or three highly divergent GAPDH genes [12]. In one of these cyanobacterial species, Synechocystis sp. PCC 6803, two distinctgapgenes namedgap1andgap2encoding di¡erent GAPDHs have been characterized. The NAD^þ-dependent enzyme GAPDH1 was reported to be essential for glyco- lytic glucose breakdown whereas the NAD(P)^þ-dependent GAPDH2, which exhibits dual coenzyme speci¢city [13,14], was shown to be operative in the photosynthetic Calvin cycle and in heterotrophic gluconeogenesis [13].

Recently, two highly divergent gap genes encoding GAPDHs with opposite physiological roles have also been found in Bacillus subtilis [15,16] as well as in the genomes of the related strainsBacillus haloduransandBa- cillus anthracis ([17] ; www.ncbi.nih.gov :/Microb_blast/un-

¢nishedgenome.html).

The aim of this paper was to study the e¡ect of the elevated Pi concentrations on primary metabolism of a Bacillus cereus strain isolated from the phosphatic soil (P layer) from the Khouribga region (Morocco), and in particular the possible occurrence in this bacterium of a phosphate control on GAPDH, a key enzyme of central metabolism and bioenergetics that uses Pi as a cofactor.

This bacterial strain may serve as a model to study the physiological and biochemical relationships between primary carbon metabolism and phosphate utilization.

2. Materials and methods

2.1. Organisms and growth conditions

TheB. cereusstrain used in this work was isolated from soil rich in P (phosphate layer containing more than 65%

(w/w) P) sites in the Khouribga region (Morocco). A bacillus licheniformis strain was isolated from sol samples less rich in Pi from the same region. Biochemical analysis according to the standardized micromethod API 20 E and 20 NE (Biomereux) allowed bacterial identi¢cation. Bac- terial strains were cultured at 37‡C for 24 h in Luria^

Bertani (LB) medium [18], and in LB medium supplemented with NaH2PO4 (equivalent to 2 g l³¹ of PO³³₄ anion) to study the e¡ect of the Pi excess on the GAPDH enzyme.

2.2. Enzyme puri¢cation

The B. cereus GAPDH was puri¢ed from cultures on LB medium and LB medium supplemented with Pi by a procedure previously described for other GAPDHs [19].

Cells from 1 l of culture were harvested by centrifugation

at 8000Ug for 15 min, suspended in 20 mM Tris^HCl bu¡er (pH 7.5) containing 2 mM EDTA, 10 mM 2-mercaptoethanol and 2 mM PMSF and disrupted in the cold with a Branson model B-12 soni¢er (120 s at 70 W). Crude cell-free extracts (soluble protein fractions) were obtained by centrifugation at 20 000Ug for 40 min. The GAPDH was puri¢ed by ammonium sulfate precipitation and dye- a⁄nity chromatography (NAD^þ elution from a Blue- Sepharose CL-6B column).

2.3. Determination of enzyme activity

The enzymatic reaction was started by adding enzyme to the assay mixture containing 45 mM sodium pyrophos- phate bu¡er (pH 8.5), 3 mM 2-mercaptoethanol, 10 mM sodium arsenate, 1 mM NAD^þ or NADP^þ, and 1 mM

D-glyceraldehyde 3-phosphate at 25‡C. The change in ab- sorbance at 340 nm was followed. Kinetic constants were calculated from initial rates. For the determination of kinetic parameters, the concentrations of the respective ¢xed substrates for the oxidative phosphorylation reaction were 1 mM NAD^þ or NADP^þ and 0.2 mM G3P.Km andkcat

were determined from Lineweaver^Burk double-reciprocal plots, mean values P S.E. of three independent determinations being calculated. To determine optimal pH, enzymatic activity was measured over a wide range of pH (from 5 to 10) with di¡erent bu¡er solutions (acetate, imi- dazole, Tris and carbonate/bicarbonate) adjusted to the same ionic strength as the standard reaction mixture. Es- terase activity was measured using 150WM p-nitrophenyl acetate (pNPA) as substrate, in 50 mM Tricine^NaOH bu¡er, pH 7.0. Production of p-nitrophenolate ion was monitored at 400 nm [20].

2.4. Protein techniques

Protein concentration was determined by the method of Bradford [21] using ovalbumin as a standard. Polyacryl- amide gel electrophoresis was carried out by the Laemmli method in 12% (w/v) acrylamide slab gels in the presence of sodium dodecyl sulfate (SDS^PAGE), using a Mini- Protean II apparatus (Bio-Rad, USA) and the Sigma MW-SDS protein markers.

The molecular mass of the native B. cereus GAPDH was estimated by the method of Hedrick [22] using native PAGE slab gels of di¡erent acrylamide concentrations in the absence of SDS. The protein markers used were ferri- tine (440 kDa), catalase (232 kDa), aldolase (154 kDa) and ovalbumin (43 kDa).

Isoelectric focusing was performed with the same elec- trophoretic system as described above in polyacrylamide slab gels (5% (w/v) acrylamide) holding ampholyte- generated pH gradients (pH range 3.5^10.0 ; Pharmalyte 3.5^10 ; Pharmacia Biotech, Uppsala, Sweden). 25 mM NaOH and 20 mM CH3COOH were used as cathode and anode solutions, respectively. The pIprotein markers

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kit used was the Sigma 3.6^9.3 IEF-Mix for isoelectric focusing.

The zymogram technique under non-denaturing conditions was carried out in 5% (w/v) acrylamide gel. The gel was stained for in situ location of NAD^þ- or NADP^þ- linked GAPDH activity bands by incubation for 30 min in the standard reaction mixture for the oxidative reaction, with either NAD^þor NADP^þ, supplemented with 20WM phenazine methosulfonate and 1 mMp-nitro blue tetrazolium chloride. The enzyme activity band was located by the appearance of a deep-purple band of the resulting in- soluble formazan salt.

A two-dimensional gel electrophoresis was carried out with an isoelectric focusing polyacrylamide gel (¢rst dimension), ¢tted over a SDS^PAGE polyacrylamide gel (second dimension) as described in [23].

2.5. Western blotting

The GAPDH protein was detected immunologically, in either cell-free extracts or puri¢ed preparations from bacteria, after SDS^PAGE (12% (w/v) acrylamide) and sub- sequent transfer to nitrocellulose. Samples were exposed to 1/800 dilution of a monospeci¢c polyclonal antibody raised in rabbit against the GAPDH1 ^ the product of thegap1gene ^ puri¢ed fromEscherichia colicells. Detec- tion of the GAPDH protein was performed with a 1/1000 dilution of a goat anti-rabbit IgG antibody^horseradish peroxidase conjugate (Sigma Chemical Co., St. Louis, MO, USA).

3. Results and discussion

3.1. The e¡ect of Pi addition to B. cereus cultures on GAPDH activity and protein levels

As shown in Fig. 1, only the NAD^þ-dependent GAPDH activity was clearly detected in cell-free extracts of B. cereus grown on normal LB medium. However, marked changes in GAPDH activity were observed in cells

grown on Pi-supplemented LB medium: whereas the NAD^þ-dependent GAPDH was only somewhat higher (ca. 20%), a dramatic stimulation (more than 100-fold the level found in normal LB medium cells) was observed for the NADP^þ-dependent GAPDH activity (see Fig. 1).

In contrast, aBacillus licheniformis strain isolated from a soil less rich in phosphate exhibited a slight decrease of both NAD^þ- and NADP^þ-dependent GAPDH activities after growth in the presence of supplementary Pi (Fig. 1).

The Pi-mediated stimulation of the NADP^þ-GAPDH activity inB.cereuscorrelated with an enlargement (about 8-fold) of a major protein band of 35 kDa, the expected molecular mass of the GAPDH subunit, found in SDS^

PAGE analyses of crude extracts (soluble protein fraction) of these cells (Fig. 2A). A polyclonal antibody raised against the puri¢ed GAPDH1 ^ the product of the gap1 gene ^ of E. coli was used for Western blot analysis of di¡erent crude protein preparations. This antibody clearly recognized a 35-kDa protein corresponding to the B. cereus GAPDH subunit in crude extracts (see Fig. 2B). The observed cross-reaction suggests some common epitopes between the E. coliand B. cereusGAPDH proteins. It is

Fig. 1. E¡ect of the Pi addition to the culture media on the NAD^þ- and NADP^þ-dependent GAPDH activity levels (U mg³¹of protein) of crude extracts ofB. cereusandB. licheniformis. Cells were from cultures grown on normal LB or Pi-supplemented LB medium.

Table 1

Puri¢cation of GAPDH fromB. cereusgrown on normal LB medium (A) and Pi-supplemented LB medium (B)

Fraction Total protein

(mg)

Total act. (U) Speci¢c act.

(U mg³¹of protein)

Puri¢cation factor (fold)^a

Yield^a(%)

Coenzyme in assays NAD^þ NADP^þ NAD^þ NADP^þ NAD^þ NAD^þ

A

Crude extract 905 181 4.53 0.20 0.005 1 100

Amm. sulfate (66^88%) 27.6 80.6 1.38 2.92 0.05 14.6 45

Blue Sepharose CL-6B 5.5 37.5 0.61 6.81 0.11 34.1 21

B

Crude extract 1076.9 283 657 0.26 0.61 1 100

Amm. sulfate (66^88%) 46.3 159 388.9 3.44 8.4 13.2 57

Blue Sepharose CL-6B 12.0 110 224.4 9.21 18.7 35.4 39

aPuri¢cation factor and yield were determined for the NAD^þ-dependent activity.

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interesting to note that the GAPDH proteins from some others Bacillusspecies (i.e. B. subtilis,Bacillus stearother- mophilus andBacillus megaterium) are not speci¢cally related to E. coli GAPDH1 [24]. Western blot analysis showed a ca. 8-fold increase of the single immunostained 35-kDa GAPDH subunit inB. cereus crude preparations from Pi-supplemented cultures (Fig. 2B). This result is in accordance with the SDS^PAGE analysis. As shown by the SDS^PAGE gels, the other protein bands remained substantially unchanged in these crude extracts (see Fig.

2A). It should be noted that the GAPDH proteins puri¢ed from both normal and Pi-supplementedB. cereuscultures (see below) were readily immunodetected by the polyclonal anti-GAPDH antibody used in this work (data not shown). Our results indicate a speci¢c increase of the amount of GAPDH protein in the soluble protein fraction fromB. cereusgrown on Pi-supplemented medium, which suggests a programmed response of this strain to the Pi surplus. It is interesting to note in this respect that phosphate has been reported to stimulate in many bacteria the expression of genes involved in macromolecular biosynthesis and of housekeeping genes required for growth and e⁄cient operation of intermediary metabolism [25]. Ac- cording to this, the Pi-supplemented B. cereus cultures exhibit higher growth rates and biomass production (ca.

1.5-fold) than normal cultures (data not shown).

3.2. Puri¢cation of GAPDH from normal and Pi-supplemented B. cereus cultures

Additional information was obtained after GAPDH pu-

ri¢cation from B. cereus grown on normal LB and Pi- supplemented LB media using a procedure involving con- ventional dye-ligand a⁄nity chromatography. As previously reported for other NAD^þ-dependent GAPDHs [26], chromatography on Blue-Sepharose is a very e¡ective and straightforward puri¢cation step forBacillusGADPH and, therefore, no additional puri¢cation steps were required to obtain homogeneous preparations of the enzyme (Table 1). Through the puri¢cation processes from B. cereus grown on normal and Pi-supplemented LB media both GAPDHs eluted from the dye-a⁄nity column as a single symmetrical activity peaks (with perfect overlapping of the NAD^þ- and NADP^þ-dependent activities), and the corresponding puri¢ed preparations exhibited under SDS^

PAGE a single protein band of 35 kDa, the expected molecular mass of the GAPDH subunit (data not shown). On the other hand, the ratio between NAD^þ-dependent and NADP^þ-dependent activities did not signi¢cantly change through the puri¢cation processes. All these results suggest the presence of a single GAPDH protein in each of the two puri¢ed preparations. In agreement with crude extract determinations, the GAPDH puri¢ed from B. cereus grown on normal LB medium shows a major NAD^þ-dependent activity and only traces of NADP^þ-dependent activity (Table 1A and Table 2) whereas the protein from Pi-supplemented cultures clearly exhibits higher activity with NADP^þ (18.7 U mg³¹ with NADP^þ versus 9.2 U mg³¹ with NAD^þ) (Table 1B and Table 2). More- over, as summarized in Table 2, the GAPDH puri¢ed from Pi-supplemented cultures exhibited much higher speci¢c activity than the enzyme from normal LB cultures.

Overall, these results suggest that the two B. cereus GAPDHs puri¢ed in this work may be distinct proteins with di¡erent coenzyme speci¢city ^ the NAD(P)^þ-dependent being a Pi-stimulated protein ^ whose characteristics are yet to be fully described, and raised questions on their possible regulation and relationships with P utilization. It is relevant in this respect to emphasize that two GAPDHs

Table 2

Speci¢c activity and catalytic parameter values with nucleotide cofactors of GAPDHs puri¢ed fromB. cereusgrown on normal LB and Pi-supplemented LB media

Enzyme and culture conditions

NAD^þ NADP^þ NAD^þplus NADP^þ GAPDH from LB cultures

Speci¢c activity (U mg³¹) 6.52 0.12 6.49

k_cat(s³¹) 34.85 ^ NA

k_cat/K_m (M³¹s³¹) 1.493U10⁶ ^ NA

GAPDH from Pi-supplemented LB cultures

Speci¢c activity (U mg³¹) 7.85 17.03 17.25

k_cat(s³¹) 14.95 22.35 NA

k_cat/K_m (M³¹s³¹) 0.533U10⁶ 1.715U10⁶ NA All data are means of three di¡erent enzyme preparations with S.E. less than 10% of the presented values. NA, not applicable.

Fig. 2. A : Coomassie blue-stained SDS^PAGE electrophoretogram showing the protein patterns corresponding to cell-free preparations from B. cereus grown on normal LB medium (lane b) or Pi-supplemented LB medium (lane c); lane a, protein markers. B : Western blot analysis using a monospeci¢c antibody against the E. coliGAPDH of crude extracts fromB. cereuscells grown on normal (lane a) or Pi-supplemented (lane b) LB medium. Aliquots of cell extracts (about 50 Wg of protein per lane) were used. The 35-kDa protein band corresponding to the GAPDH subunit is indicated by the arrows.

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with di¡erent coenzyme speci¢city have been recently reported in B. subtilis [16].

3.3. Physicochemical and kinetic properties of two GAPDHs puri¢ed from B. cereus

A comparative study of several structural and biochemical properties of the two B. cereus GAPDHs puri¢ed in this work further supported that they should be indeed di¡erent proteins, although sharing certain characteristics that are common to most GAPDHs described to date.

Thus, both dehydrogenases exhibited the same molecular masses under denaturing (see Section 3.2) and non-denaturing PAGE, a homotetrameric structure with an estimated molecular mass of ca. 143 P 5 kDa being obtained for the native proteins, determined by Hedrick [22]. No signi¢cant di¡erences were found for optimal pH and tem- perature values (8.3 and 38‡C, respectively). However, a clear discrepancy was observed in the pI values determined by isoelectric focusing for the GAPDHs puri¢ed fromB. cereus cultures developed on normal LB medium (pI7.2, an almost neutral protein) or Pi-supplemented LB medium (pI4.8, an acidic protein) (Fig. 3A). These results were in agreement with the faster mobility observed in non-denaturing PAGE gels for the GAPDH puri¢ed from Pi-supplemented cultures (Fig. 3B). Since no strict NAD^þ-dependent protein band was found for this GAPDH preparation either in isoelectric focusing (Fig.

3A) or in two-dimensional PAGE (data not shown) it seems that only the acidic NAD(P)^þ-dependent GAPDH was produced by B. cereus under phosphate excess. It should be noted in this respect that a similar low pI value was reported for the NAD(P)^þ-dependent GAPDH of the cyanobacterium Synechocystissp. PCC 6803 [13].

Speci¢c activity levels were determined for the two puri¢ed B. cereus GAPDHs using NAD^þ, NADP^þ or both pyridine nucleotides in the assays (Table 2). No additivity was found between the NAD^þ-dependent and NADP^þ- dependent activities (no signi¢cant increase of enzymatic activity occurred in the presence of both coenzymes), strongly suggesting that only one enzyme was present in each puri¢ed preparation. Similar results were obtained with crude protein extracts (not shown). These data further support that B. cereus should possess two GAPDH isoforms, one being NAD^þ-dependent (present in normal cultures) and a second NAD(P)^þ-dependent (produced in Pi-supplemented cultures).

The GAPDHs puri¢ed fromB. cereusgrown on normal and Pi-rich LB media were also analyzed by PAGE in non-denaturing gels by in situ speci¢c staining for G3P dehydrogenase activity using a tetrazolium dye and either NAD^þ or NADP^þ as the nucleotide cofactor. Both puri-

¢ed preparations exhibited only one activity band (Fig.

3B), a result that again is in agreement with the presence of only one GAPDH enzyme in each preparation. More- over, a virtually negligible activity with NADP^þ was de-

tected for the strict NAD^þ-dependent GAPDH puri¢ed fromB. cereusnormal cultures (Fig. 3B, top) ; in contrast to that, the GAPDH puri¢ed from B. cereus Pi-rich cultures exhibited a single activity band with both NAD^þand NADP^þ but with much higher intensity with the last cofactor (Fig. 3B, bottom) thus con¢rming the results presented above on the occurrence in this preparation of a GAPDH with dual cosubstrate speci¢city.

An esterase activity (0.1 U mg³¹ of protein) was also found in B. cereus cell-free extracts. As expected if this activity were mainly due to GAPDH, it clearly increased in cell-free extracts from Pi-supplemented cultures (up to 0.9 U mg³¹ of protein). Speci¢c activity values of ca. 4 and 31 U mg³¹ of protein were found for the puri¢ed

Fig. 3. A : Isoelectric focusing in polyacrylamide slab gel (5% w/v acrylamide) holding an ampholyte-generated pH gradient (pH range, 3.5^10) of theB. cereusGAPDH puri¢ed from cells grown on normal LB medium (lane a) and Pi-supplemented LB medium (lane b). B: Localiza- tion, with either NAD^þ or NADP^þ, after PAGE under non-denaturing conditions of the G3P dehydrogenase activity bands of B. cereus GAPDHs puri¢ed from cells grown on normal LB (a) or Pi-supplemented LB (b) medium. Aliquots containing about 20 Wg of puri¢ed protein were applied per lane.

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NAD^þ-dependent and NAD(P)^þ-dependent GAPDHs of B. cereus, respectively. This esterase activity is higher than those previously observed for other GAPDHs [27]. The physiological signi¢cance of the esterase activity ofB. cereus GAPDHs still remains to be investigated. Several studies have shown other enzymatic activities ofB. cereus, such as phospholipase C and alkaline phosphatase, to be a¡ected by changes in the concentration of phosphate in culture medium [28].

A comparative study of the kinetic parameters obtained by systematic variation of substrates for the two puri¢ed B. cereus GAPDHs (from normal and Pi-supplemented LB cultures) was carried out. No signi¢cant di¡erences were observed in the Km values for NAD^þ and G3P for both puri¢ed preparations, values of 28 P 5 WM and 188 P 15 WM being obtained, respectively. The Km value for NADP^þ, obtained only for the GAPDH fromB. cereus Pi-rich cultures, was 13 P 4 WM. Thus, the B. cereus NAD(P)^þ-dependent GAPDH exhibits signi¢cantly higher a⁄nity for NADP^þ, its preferred nucleotidic cofactor. Pre- vious investigations showed that a B. subtilis GAPDH presented a dual cofactor signature with preference to NADP^þand functions in the reverse direction during gluconeogenesis [16]. Finally, theB. cereusNAD(P)^þ-dependent GAPDH exhibited a lowerKm value for PO³³₄ than its strict NAD^þ-dependent counterpart found in normal LB medium cultures (3.5 P 0.3 mM and 6.1 P 0.5 mM, respectively), indicating a higher a⁄nity of the Pi-stimulated enzyme for this substrate. Comparison of the catalytic e⁄ciency values (kcat/Km ratios) for the nucleotide cofactors of the twoB. cereusGAPDHs (Table 2) revealed that when the NAD(P)^þ-dependent enzyme uses NADP^þ as a cofactor it is more active than its NAD^þ-dependent counterpart.

Our results indicate thatB. cereuspossesses two distinct GAPDHs that may be encoded by di¡erent gap genes, thus resembling what has been found for B. subtilis and other non-photosynthetic bacterial species, i.e. Neisseria meningitidis,Neisseria gonorrhoeae, andHelicobacter pylo- ri [16]. Up to now and to our knowledge, no information on the B. cereus genome is present in the databases. Our results also indicate that phosphate may control the GAPDH system inB. cereus. In this connection it should be noted that many enzymes of the central pathways of bacterial primary metabolism are stimulated by phosphate, thus providing greater amounts of intermediates required for macromolecular biosynthesis during the increased growth that occurs in the presence of high Pi concentrations [25]. Since most anabolic reactions require NADPH, a highly active Pi-stimulated NADP^þ-dependent GAPDH can play a signi¢cant role in the intense gluconeogenesis and in the generation of the increasing amounts of substrates necessary for the intense anabolism that takes place in the bacterial cell in the presence of elevated Pi concentrations. We are currently investigating the molecular mechanisms involved in a possible Pi-dependent

induction of the NAD(P)^þ-dependent GAPDH in B. cereus as well as the possible role played by this enzyme in phosphate utilization.

Acknowledgements

This work was supported by grants from CNCPRST, project PARS (Morocco) ; PB 97-1135 (Spain) ; group PAI CVI-261 (Junta de Andaluc|¤a, Spain) ; and Convenio Co- laboracio¤n Univ. Marroqu|¤es (Junta de Andaluc|¤a, Spain).

We thank Prof. Manuel Losada for his generous encour- agement and help, Prof. Ru«diger Cer¡, the reviewer for his competent and constructive comments on the manuscript and Mr. N. Cha¢k for helpful corrections of the text.

References

[1] Alexander, M. (1977) Introduction to Soil Microbiology. Wiley, New York.

[2] Pourriot, R. and Meybeck, M. (1995) Limnologie Ge¤nerale, pp. 252^

253. Masson, Paris.

[3] Wanner, B.L. (1987) Phosphate regulation of gene expression. In : Escherichia coliandSalmonella typhimuriumCellular and Molecular Biology, Vol. 2 (Neidhardt, F.C., Ingraham, J., Low, K.B., Magasa- nik, B., Schaechter, M. and Umbarge, H.E., Eds.), pp. 1326^1333.

Am. Soc. Microbiol., Washington, DC.

[4] Wanner, B.L. (1990) Phosphorus assimilation and its control of gene expression inEscherichia coli. In : The Molecular Basis of Bacterial Metabolism (Hauska, G. and Thauer, R., Eds.), pp. 152^163. Spring- er-Verlag, Heidelberg.

[5] Wanner, B.L. (1992) Is cross regulation by phosphorylation of two- component response regulator proteins important in bacteria ? J. Bac- teriol. 174, 2053^2058.

[6] Forthergill-Gilmore, L.A. and Michels, P.A.M. (1993) Evolution of glycolysis. Prog. Biophys. Biol. 95, 105^135.

[7] Cer¡, R. (1982) Separation and puri¢cation of NAD^þ- and NADP^þ- linked glyceraldehyde-3-phosphate dehydrogenase from higher plants. In : Methods in Chloroplast Molecular Biology (Edekman, M., Hallik, R.B. and Chua, N.H., Eds.), pp. 683^694. Elsevier Bio- medical Press, Amsterdam.

[8] Brinkman, H., Cer¡, R., Salomon, M. and Soll, J. (1989) Cloning and sequence analysis of cDNAs encoding the cytosolic precursors of subunits GapA and GapB of chloroplast glyceraldehyde-3-phosphate dehydrogenase from pea and spinach. Plant Mol. Biol. 13, 81^

94.

[9] Serrano, A., Maetos, M.I. and Losada, M. (1993) ATP-driven trans- hydrogenation and ionization of water in a reconstituted glyceraldehyde-3-phosphate dehydrogenase (phosphorylating and non-phosphorylating) model system. Biochem. Biophys. Res. Commun. 197, 1348^1356.

[10] Habenicht, A., Hellman, U. and Cer¡, R. (1994) Non-phosphorylating GAPDH of higher plants is a member of the aldehyde dehydrogenase superfamily with no sequence homology to phosphorylating GAPDH. J. Mol. Biol. 237, 165^171.

[11] Delgado, M.L., O’Connor, J.E., Azorin, I.I., Renau-Piqueras, J., Gill, M.L. and Gozalbo, D. (2001) The glyceraldehyde-3-phosphate dehydrogenase polypeptides encoded by Saccharomyces cerevisiae TDH1, TDH2 and TDH3 genes are also cell wall proteins. Micro- biology 147, 411^417.

[12] Figge, R.M., Schubert, M., Brinkman, H. and Cer¡, R. (1999) Glyc- eraldehyde phosphate dehydrogenase gene diversity in eubacteria an

(7)

eukaryotes : evidence for intra- and inter-kingdom gene transfer. Mol.

Biol. Evol. 16, 429^440.

[13] Valverde, F., Losada, M. and Serrano, A. (1997) Functional comple- mentation of anEscherichia coli gapmutant supports an amphibolic role for NAD(P)^þ-dependent glyceraldehyde-3-phosphate dehydrogenase ofSynechocystissp. strain PCC 6803. J. Bacteriol. 179, 4513^

4522.

[14] Koksharova, O., Schubert, M., Shestakov, S. and Cer¡, R. (1998) Genetic and biochemical evidence for distinct key functions of two highly divergent GAPDH genes in catabolic and anabolic carbon

£ow of the cyanobacterium Synechocystis sp. PCC 6803. Plant Mol. Biol. 36, 183^194.

[15] Kunst, F., Ogasawara, N. and Moszer, I. et al. (1997) The complete genome sequence of the Gram-positive bacterium Bacillus subtilis.

Nature 390, 249^256.

[16] Fillinger, S., Boschi-Muller, S., Azza, S., Dervyn, E. and Branlant, G.

(2000) Two glyceraldehyde-3-phosphate dehydrogenases with opposite physiological roles in a nonphotosynthetic bacterium. J. Biol.

Chem. 275, 14031^14037.

[17] Takami, H., Nakasome, K., Takaki, Y., Maeno, G., Sasaki, Y., Masui, N., Fuji, F., Hirama, C., Nakamura, Y., Ogasawara, N., Kuhara, S. and Horikoshi, K. (2000) Complete genome sequence of the alkaliphilic bacteriumBacillus haloduransand genomic sequence comparison withBacillus subtilis. Nucleic Acids Res. 28, 4317^4331.

[18] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Clon- ing, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

[19] Soukri, A., Valverde, F., Ha¢d, N., Elkebbaj, M.S. and Serrano, A.

(1995) Characterization of muscle glyceraldehyde-3-phosphate dehy-

drogenase isoforms from euthermic and induced hibernatingJaculus orientalis. Biochim. Biophys. Acta 1243, 161^168.

[20] Darryl, P.A. and Regina, P. (1992) Modi¢cation of aldehyde dehydrogenase with dicyclohexylcarbodiimide : separation of dehydrogenase from esterase activity. J. Protein Chem. 11, 59^70.

[21] Bradford, M.M. (1976) A rapid and sensitive method for the quanti-

¢cation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248^254.

[22] Hedrick, J.L. and Smith, A.J. (1968) Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch. Biochem. Biophys. 162, 155^164.

[23] Phillips, T.A. (1988) Two-dimensional Polyacrylamide Gel Electro- phoresis of Proteins in DNA and Protein Engineering Techniques, Vol. 1, pp. 5^9. Alan R. Liss, New York.

[24] Figge, R.M. and Cer¡, R. (2001) GAPDH gene diversity in spiro- chetes: a paradigm for genetic promiscuity. Mol. Biol. Evol. 18, 2240^2248.

[25] Liras, P., Villanueva, J.R. and Martin, J.F. (1977) Sequential expression of macromolecule biosynthesis and candicidin formation in Streptomyces griseus. J. Gen. Microbiol. 102, 669^672.

[26] Thompson, S.T., Cass, K.H. and Stellwagen, E. (1975) Blue dextran- Sepharose: an a⁄nity column for the dinucleotide fold in proteins.

Proc. Natl. Acad. Sci. USA 72, 669^672.

[27] Behme, M.T.A. and Cordes, E.H. (1967) Kinetics of glyceraldehyde- 3-phosphate dehydrogenase catalysed hydrolysis of p-nitro-phenyl acetate. J. Biol. Chem. 242, 5500^5509.

[28] Vinter, V., Smid, F. and Smrckova, I. (1987) Factors in£uencing the activity of cellular alkaline phosphatase during growth and sporula- tion ofBacillus cereus. Folia Microbiol. 32, 89^95.