©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The outB Gene of Bacillus subtilis Codes for NAD Synthetase (*)

(Received for publication, September 9, 1994; and in revised form, December 13, 1994)

Claudio Nessi (1)(§) Alessandra M. Albertini (1) Maria Luisa Speranza (2) Alessandro Galizzi (1)(¶)

From the  (1)Dipartimenti di Genetica e Microbiologia and (2)Biochimica and Centro Interuniversitario per lo Studio delle Macromolecole Informazionali, Università di Pavia, 27100 Pavia, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The outB gene of Bacillus subtilis is involved in spore germination and outgrowth and is essential for growth. The OutB protein was obtained by expression in Escherichia coli and purified to apparent homogeneity. Here we report experiments showing that OutB is a NH(3)-dependent NAD synthetase, the enzyme that catalyzes the final reaction in the biosynthesis of NAD. The enzyme is composed of two identical subunits of 30,240 Da and is NH(3)-dependent, whereas glutamine is inefficient as an amide donor. The NAD synthetase is highly resistant to heat, with a half-time of inactivation at 100 °C of 13 min. A mutant NAD synthetase was purified from a B. subtilis strain temperature-sensitive during spore germination and outgrowth. The mutant enzyme was 200 times less active than the wild-type one, with a lower temperature optimum and a non-hyperbolic kinetic versus NH(4). The time course of synthesis of OutB showed that synthesis of the enzyme started during germination and outgrowth, and reached the highest level at the end of exponential growth. The enzyme could be recovered from dormant spores.


INTRODUCTION

The outB gene of Bacillus subtilis was originally identified through the isolation of a mutant, outB81, which is temperature-sensitive during spore germination(1) . Strains with the mutation had a pleiotropic phenotype, being affected in vegetative growth at permissive temperature on various nitrogen sources and impaired in derepression of glutamine synthetase(2) . The deduced amino acid sequence of OutB showed high level identity to the product of the Escherichia coli essential gene efg, which was recently shown to code for NH(3)-dependent NAD synthetase(3) .

NAD plays a central role in cellular metabolism, as it functions as a cofactor in oxidation reduction reactions and as a substrate in others, such as DNA ligation and protein ADP-ribosylation. NAD can be synthesized de novo or through a pyridine salvage pathway. Both biosynthetic pathways have been characterized extensively in E. coli and Salmonella, and most of the genes involved have been described(4) . The nadB, nadA, and nadC genes code for L-aspartate oxidase, quinolinate synthetase, and quinolinic acid phosphoribosyltransferase, respectively, and are responsible for the first three metabolic steps of the de novo biosynthesis. The salvage pathway depends on the products of gene pncA (nicotinamide deamidase) and pncB (nicotinic acid phosphoribosyltransferase). The alternative pathways merge at the level of nicotinic acid mononucleotide, and the two final steps are in common; first nicotinic acid mononucleotide is adenylated by nicotinic acid adenine dinucleotide (NaAD) (^1)pyrophosphorylase, coded by nadD, and finally NaAD is converted to NAD by the following reaction catalyzed by NAD synthetase.

The gene encoding NAD synthetase was designed nadE(5) and has been cloned and sequenced(6) .

Less is known about the NAD biosynthetic pathways operating in B. subtilis, and they are generally considered to be similar to those reported for enterobacteria, even though the regulation of some steps appears to be different(7) . A number of nicotinic acid-requiring mutants of B. subtilis have been reported, and all mutations (nic) have been shown to map in the same region, located at approximately 240° on the genetic map(8) . Cloning, sequencing, and insertional mutagenesis of a segment of DNA derived from the nic locus showed the presence of three open reading frames, the inactivation of which resulted in a Nic-dependent phenotype(9) . One of the open reading frames corresponds to the gene encoding L-aspartate oxidase (nadB), whereas the function of the other two is still unknown.

In this report we describe the purification of OutB and show that it is a NH(3)-dependent NAD synthetase. We also report the purification and characterization of the enzyme from the outB81 mutant.


EXPERIMENTAL PROCEDURES

Bacterial Strains, Plasmids, and Cultivation Conditions

E. coli DH5alpha (F, Y80d, lacZ DeltaM15 Delta(lac ZYA - argF)U169, recA1, end A1, hrdR17 (rk, mk), supE44, , thi-1, gyrA, relA1) was used as a host for the expression plasmids. The 1-kilobase EcoRI-HindIII fragment of p999 (10) containing the outB gene was ligated with EcoRI-HindIII-cut pKQV4(11) , and this plasmid was named pKQ99. The outB81 mutant gene was cloned with the same procedure starting from plasmid p981(12) . The resultant plasmid was named pKQ81. Plasmid constructions were confirmed by restriction analysis and DNA sequence determination. Bacteria were grown on LB medium supplemented with ampicillin (100 µg/ml).

DNA Techniques

E. coli DH5alpha cells were made competent and transformed according to Hanahan(13) . Plasmid DNA from E. coli was isolated using standard procedures according to Sambrook et al.(14) .

Restriction enzymes and T4 DNA ligase were obtained from commercial suppliers and used according to their recommendations.

Purification of the OutB Protein

Two liters of LB medium were inoculated with E. coli containing pKQ99 and grown overnight. Cells were harvested by centrifugation, washed once with 50 mM Tris, pH 7.5, and resuspended in 8 ml of ice-cold buffer A (50 mM Tris, pH 7.5, 2 mM dithiothreitol, 400 µg/ml phenylmethylsulfonyl fluoride, 5% glycerol). The cells were disrupted by sonication and the crude extract recovered by centrifugation at 16,000 rpm for 20 min in a Sorvall centrifuge, using adapters for Eppendorf tubes. The supernatant was loaded onto a 100 times 3-cm Sephadex G-100 column equilibrated with 50 mM Tris, pH 7.5, 100 mM NaCl. The column was eluted with the same buffer. The proteins in the fractions were visualized by SDS-PAGE(14) . Fractions containing OutB were pooled and dialyzed overnight against 40 volumes of 1% glycine. The dialyzed fraction was brought to 95 ml with water, to which 2.5 ml of Ampholine (pH range 4-6) and 4 g of Ultrodex (both from Pharmacia Biotech Inc.) were added. The gel was poured in a 12.5 times 26-cm tray and kept at 7 °C for 8-9 h, until excess water had evaporated. Electrophoresis was run at 7 °C for 14 h at 8 watts. To locate the OutB protein, a sheet of Whatman No. 3 M paper was briefly applied to the gel, dried at 80 °C, washed three times for 15 min with 10% trichloroacetic acid, stained with Coomassie (0.2%, w/v, solution in methanol:water:acetic acid, 45:45:10) and destained with methanol:water:acetic acid. The portion of the gel corresponding to the OutB protein was spooned out and proteins eluted with 10 ml of 50 mM Tris, pH 7.5. After overnight dialysis against the same buffer, the fraction was reloaded onto a FPLC Mono-Q (HR 5/5 Pharmacia) column and eluted using a linear gradient (0-500 mM NaCl in 50 mM Tris pH 7.5). The OutB protein eluted at approximately 180 mM NaCl. This fraction was concentrated using Centricon-10 concentrators (Amicon) and stored at -20 °C in 25 mM Tris, pH 7.5, 90 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 50% glycerol. Protein concentrations were determined using the Bio-Rad protein assay with bovine serum albumin as standard. Amino acid analysis was performed in the laboratory of P. Iadarola (Dipartimento di Biochimica) using a Kontron Chromakon 500 automatic analyzer. Mass spectral data were obtained with a Finnigan Matt TSQ 700, equipped with an electrospray ionization source.

The protein coded by the mutant gene outB81 was purified with the same procedure.

Enzymatic Assays

The activity of the wild-type enzyme was routinely assayed in 0.5 ml of 60 mM HEPPS buffer, pH 8.5, containing 2 mM NaAD, 2 mM ATP, 10 mM NH(4)Cl, 10 mM MgCl(2), and 20 mM KCl. The reaction was started by the addition of 0.5 µg of enzyme. After 5 min of incubation at 37 °C, the reaction was stopped by addition of 0.5 ml of 0.1 M sodium pyrophosphate buffer, pH 8.9, containing 0.5% (w/v) semicarbazide hydrochloride. The NAD formed was measured spectrophotometrically at 340 nm, by the alcohol dehydrogenase method(16) . The activity of the mutant NAD synthetase was measured at 22 °C, using 5-10 µg of enzyme and extending the incubation to 30 min. The activity was expressed as nanomoles of NAD synthesized/min. One unit of enzyme was defined as that amount synthesizing 1 µmol of NAD/min.

To measure heat inactivation, the enzyme solutions in HEPPS buffer were put in a boiling water bath. At intervals aliquots were transferred into the incubation medium, at 37 °C or 22 °C, and the reaction was started by the addition of NaAD.

Polyacrylamide Gradient Gel Electrophoresis

Native proteins were electrophoresed on gels of increasing polyacrylamide concentrations from 5% to 20% in Tris borate-EDTA. After a pre-run at 4 °C overnight, protein samples (15 µg) were loaded and run at 4 °C for 5 h at 300 V. Proteins were visualized by Coomassie staining.

Protein Extraction and Western Immunoblotting

B. subtilis cells from Schaeffer sporulation medium (17) were harvested and frozen. The thawed pellet was resuspended in buffer B (100 mM HEPES buffer, pH 7.5, 2 mM phenylmethylsulfonyl fluoride), treated with lysozyme (0.5 mg/ml) for 10 min at 37 °C, and sonicated. Following centrifugation at 13,000 rpm for 20 min, the proteins in the supernatant were titrated, boiled in Laemmli buffer, and loaded onto SDS-PAGE gels(15) . After electrophoresis the proteins were electrotransferred to nitrocellulose and probed with anti-OutB antibodies. Dormant spores were purified by centrifugation in 70% (v/v) Urografin (Schering) according to Siccardi et al.(18) , lyophilized, and broken in a dental amalgamator(19) . The dry powder was extracted with 1 ml of buffer B, centrifuged at 13,000 rpm for 20 min. The supernatant was concentrated using Centricon-10 concentrators (Amicon), precipitated with acetone, and the pellet washed with ether and dissolved in Laemmli buffer. Protein extracts from germinating spores were prepared by sonication as for growing cells; the supernatant was concentrated and precipitated as described for dormant spores. For each time point the same aliquot (50 ml) of germinating spores was harvested. The low level of proteins made their titration unreliable; for this reason, the entire sample was loaded onto SDS-PAGE.


RESULTS

Expression of the OutB Protein in E. coli

To obtain sufficient OutB protein for its purification, we cloned the outB gene of B. subtilis in plasmid pKQV4, a vector suitable for expression of foreign genes in E. coli(11) . The complete coding sequence of outB and 140 upstream base pairs were cloned downstream of the IPTG-inducible promoter, to obtain pKQ99. The proper cloning and orientation in respect to the plasmid ptac promoter were confirmed by nucleotide sequence determination. E. coli DH5alpha cells containing pKQ99 produced large amount of a protein of about 36,000 daltons (Fig. 1). The protein band was absent from extract of E. coli DH5alpha cells containing the vector plasmid without added insert, and we tentatively considered the protein as the product of the outB gene. The size of the OutB protein, as predicted from the DNA sequence, is about 30,000 daltons (depending on the Met codon used to initiate translation and on the conservation or not of N-terminal methionine in the final product). The large discrepancy between the predicted mass and the value obtained from SDS-PAGE may depend on the low pI of the protein (4.8); acid proteins are known to migrate slower than expected in SDS-polyacrylamide gels. The same type of behavior was observed for the E. coli homolog of OutB (6) . Upon IPTG induction the level of the 36,000-dalton band did not increase further, suggesting transcription from an endogenous promoter. Previous experiments with plasmids bearing the outB promoter region, indicated high level of transcription from the outB P1 promoter in E. coli(10) . Thus the absence of any effect of IPTG induction on the level of expression of the 36,000-dalton protein band could be explained by the efficient use of the B. subtilis promoter.


Figure 1: Purification of OutB and SDS-PAGE electrophoresis. Molecular mass standards are shown in lane 1. Harvested cells were resuspended in buffer and lysed by sonication. After centrifugation, the lysate (lane 2) was loaded onto a Sephadex G-100 column. The fractions containing OutB were pooled (lane 3) and subjected to preparative isoelectric focusing. The proteins eluted from the isoelectric focusing gel (lane 4) were loaded onto a FPLC Mono-Q column. Purified OutB protein from this column is shown in lane 5.



Purification of the OutB Protein

The apparent lack of any E. coli protein co-migrating with OutB and the high level of expression of the protein in this host afforded a straightforward purification. In the early stages of this research, we did not have any enzymatic assay to follow the progress of purification; thus, we monitored it by SDS-PAGE fractionation. Later, when it become apparent that OutB is NAD synthetase, the same purification procedure was followed by both SDS-PAGE and enzymatic assay.

The OutB protein was purified according to the scheme described under ``Experimental Procedures,'' and the results obtained are illustrated in Fig. 1. After the last step, Mono-Q chromatography, the protein was estimated to be at least 95% pure, by SDS-PAGE and Coomassie staining. An aliquot of this preparation was used to determine the amino acid composition, which yielded values in good agreement with the ones deduced from the nucleotide sequence (Table 1). The mass of the purified protein was measured by mass spectrometry and gave a value of 30,240 Da, in very good agreement to the estimated mass assuming that translation initiates at the first ATG codon (20) and that the N-terminal methionine is removed from the mature protein.



Gel filtration experiments and nondenaturing polyacrylamide gels (Fig. 2) showed a molecular weight of about 60,000, indicating that the OutB protein is a dimer.


Figure 2: Molecular weight of OutB. Mobility of native wild-type OutB (lane 1) and mutant OutB81 (lane 2) proteins in non-denaturing polyacrylamide gradient (from 5% to 20%) gel. Standard proteins used were bovine serum albumin (M(r) = 67,000, lane 3) and chicken egg albumin (M(r) = 43,000, lane 4) from Pharmacia.



The OutB Protein Is a NH(3)-dependent NAD Synthetase

While this work was in progress, we were informed by J. C. Willison that the E. coli protein homolog of OutB is the NH(3)-dependent NAD synthetase(3) . We thus tested for NAD synthetase activity our purified protein; furthermore, we followed the purification by enzymatic assay. As shown in Table 2, NAD synthetase activity copurifies with the protein overproduced in E. coli containing plasmid pKQ99. The kinetic properties of the enzyme are summarized in Table 3.





Compared to the enzymes purified from E. coli(21) and Saccharomyces cerevisiae(22) , the NAD synthetase of B. subtilis shows a substantially higher specific activity. K(m) values for NaAD and ATP are almost identical to those reported for E. coli and S. cerevisiae, whereas the K(m) for NH(4) is 10 times higher than the E. coli one and 10 times lower than that of the yeast enzyme.

With glutamine as amide donor, the apparent K(m) was 9.1 times 10M; however, the glutamine sample contained up to 0.2% free ammonia, which fully accounted for the observed activity. The B. subtilis enzyme is remarkably resistant to heat inactivation, with 50% residual activity after 13 min (t) of incubation at 100 °C (Fig. 3).


Figure 3: Thermal stability of wild-type and mutant NAD synthetase. Enzyme solutions were incubated in a boiling water bath; at intervals aliquots were withdrawn and the residual activity measured at 37 °C (wild-type enzyme, open circles) or 22 °C (mutant enzyme, filled-in circles).



Enzyme stock solutions (2-20 mg/ml in HEPPS buffer) were stable up to 5 months, stored at -20 °C. Diluted (0.1 mg/ml) working solutions, submitted to frequent freezing and thawing, maintained about 80% of activity after 1 week.

Properties of a Mutant Enzyme

Gene outB of B.subtilis was originally identified through the isolation of a mutant (outB81) temperature sensitive during spore outgrowth(1) . The mutation is a GC to AT transition, changing glycine 157 to glutamate(12) . The mutant allele was cloned in the pKQV4 expression vector, to give pKQ81 and the protein purified according to the procedure set up for the parental enzyme. It should be noted that the E. coli strains harboring pKQ81 grew slowly, did not survive upon freezing, and had a tendency to eliminate the B. subtilis insert from the plasmid.

The mutant enzyme differed from the parental one under several respects. As reported in Table 3, NAD synthesis is 200 times less efficient for the mutant protein than for the wild-type enzyme. An apparent K(m) for ammonia could not be calculated for the mutant enzyme; the double-reciprocal plot is not linear and indicates that the enzyme hardly reaches saturation (Fig. 4). The temperature optimum is remarkably lower for the mutant enzyme, which at 45 °C, i.e. the temperature optimum for the wild-type NAD synthetase, has no detectable activity ( Fig. 5and Table 3). The mutant enzyme shows a pH optimum moderately higher than the wild-type enzyme (Table 3). Finally, the half-time of inactivation at 100 °C (8 min, Fig. 3and Table 3), although lower than that of the wild-type enzyme, indicates that the mutant protein is still rather resistant to heat inactivation.


Figure 4: Kinetic response of the mutant NAD synthetase to NH(4). The assay conditions are described under ``Experimental Procedures.''




Figure 5: Temperature/activity profiles of wild-type and mutant NAD synthetase. The assay conditions are described under ``Experimental Procedures.'' Filled-in circles and open circles indicate wild-type and mutant enzyme, respectively. Note the change in scale on the ordinates.



Time Course of Synthesis of OutB Protein

Gene outB is transcribed from two promoters, P1 and P2. Transcription from the main P1 promoter is turned off at the beginning of stationary phase (T(0)), and a low level of constitutive transcription is maintained from the secondary promoter P2(20) . We wanted to see whether this was reflected at the protein level. We therefore measured the levels of OutB by immunoblot analysis.

We used the purified OutB protein to raise polyclonal antiserum in rabbits. In Western blot analysis, the antiserum detected a protein band in lysates of B. subtilis that comigrated with purified OutB (Fig. 6). The OutB antiserum was used to monitor the level and accumulation of OutB during the cell cycle of B.subtilis. A strain was grown under conditions that allow sporulation to occur, and samples of proteins taken at intervals were analyzed by Western immunoblotting. The results (Fig. 6) showed that OutB was present in growing cells, reached a peak at the transition from vegetative to stationary phase, and was still present 3 h after entering sporulation. These results are consistent with previous data relating to the transcription of outB(20) . The presence of OutB after asymmetric septation raised the possibility of its presence in the mature spore. By immunoblotting we showed that OutB is indeed present in purified spores (Fig. 7). During germination and outgrowth the level of OutB increases (Fig. 7), again in accordance with the results of transcription as measured in RNase protection experiments (20) or expression of outB-lacZ translational fusion(10) .


Figure 6: Synthesis of OutB protein. A, growth curve of B. subtilis strain PB 1424 in Schaeffer sporulation medium. Arrows indicate the time points at which samples were collected. B, time course of OutB synthesis during growth and sporulation. Cell extracts were separated by SDS-PAGE and transferred to a nitrocellulose filter. The filter was probed with anti-OutB antibodies, followed by a secondary antibody conjugated with horseradish peroxidase. The peroxidase activity was visualized with an enhanced chemiluminescence kit (Amersham Corp.). Lanes 1-4, purified OutB: 100, 50, 20, and 5 ng, respectively. Lanes 5 and 6, cell extracted from vegetative cells from a separate experiment. Lanes 7-14, cell extracts (3 µg of total protein) from samples of the culture whose growth curve is reported in A.




Figure 7: Synthesis of OutB during spore germination and outgrowth. Spores of strains PB1424 were heat-activated at 70 °C for 15 min and inoculated into 200 ml of nutrient broth containing 0.5% glucose. The starting absorbance at 540 nm was 0.4. At each time point, a sample of 50 ml was collected and proteins extracted as described under ``Experimental Procedures.'' Each lane was loaded with the whole cell extract. Lane 1, dormant spores; lanes 2-5, samples from germinating spores collected after 15, 45, and 60 min of incubation, respectively.




DISCUSSION

The identification of OutB as NAD synthetase was made possible by the high degree of homology to the characterized E. coli enzyme. The B. subtilis enzyme, like the E. coli one, requires ammonia as amide donor, but its apparent K(m) value is 10-fold higher than that reported for E. coli.

A peculiarity of the B.subtilis enzyme is its remarkable resistance to heat inactivation, with a half-life at 100 °C of 13 min. This aspect may reflect an adaptation to the life style of this Gram-positive spore-former, and in fact the enzyme was found in the dormant spore. Alternatively the heat resistance may be an accidental characteristic, acquired by adaptation to other enzymatic parameters. Dormant spores have significant levels of NAD, that is rapidly reduced to NADH during germination, as soon as metabolism is resumed(23) . De novo synthesis of NAD can be observed after 30 min from the beginning of germination(23) , and transcription of outB occurs early during germination, being detectable at 12 min(10, 20) . Thus it is not obvious why NAD synthetase is stored in mature spores. It cannot be excluded that the enzyme is passively trapped in the forespore during sporulation. It has been reported (24) that OutB becomes phosphorylated at the end of exponential growth. This raised the possibility of a programmed modification of NAD synthetase. We were unsuccessful in our attempts to show OutB phosphorylation, by immunoprecipitation with crude B.subtilis extracts or purified protein. (^2)Thus the physiological meaning of the presence of NAD synthetase in mature spores and its role, if any, during germination and outgrowth are still obscure.

The properties of the outB81 mutant enzyme explain a number of observations concerning the phenotype of the temperature-sensitive mutant. The optimal temperature is 22 °C versus 37 °C and above for the parental; in addition, at 45 °C and higher temperatures no activity could be detected with the mutant enzyme. Strains with the outB81 mutation are temperature-sensitive (at 46 °C) during early stages of spore germination and outgrowth, whereas their vegetative growth is not impaired at high temperature (1) . This observation can be explained by the assumption that the increase in the number of molecules per cell of NAD synthetase, which ensues during spore outgrowth, can statistically compensate for the lower activity and higher sensitivity to temperature of the mutant enzyme.

The low activity of the mutant enzyme compared to the wild-type enzyme may also explain the slow growth of strains with the outB81 mutation on poor nitrogen sources(2) . As for the relationship between structure and function, we can only make general considerations. The substitution of a glutamic acid for a glycine in the mutant enzyme puts a bulkier and charged residue in place of the small glycine hydrogen. The dramatic effect on enzyme activity, and the modification of the kinetic versus NH(4), suggest that the substitution might involve the active site and be related to the binding of the ammonium ion. The presence of a glutamyl residue near the NH(4) binding site could impair a proper and productive binding, mostly at low ammonium concentrations. It is also conceivable that an impaired binding could be adversely affected by a rise in temperature. Alternatively the substitution of glycine by glutamic acid might induce a ``loosening'' in the structure of the active site, which could be worsened by a rise in temperature.


FOOTNOTES

*
This research was supported in part by grants from Ministero Dell'Università E Della Ricerca Scientifica E Technologica (40%) and Progetto Finalizzato Ingegneria Genetica Consiglio Nazionale delle Ricerche. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a fellowship from Fonds National Suisse de la Recherche Scientifique.

To whom correspondence should be addressed: Dipartimento di Genetica e Microbiologia, Via Abbiategrasso 207, 27100 Pavia, Italy. Tel.: 39-382-505548; Fax: 39-382-528496.

(^1)
The abbreviations used are: NaAD, nicotinic acid adenine dinucleotide; PAGE, polyacrylamide gel electrophoresis; FPLC, fast protein liquid chromatography; HEPPS, 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid; IPTG, isopropyl-1-thio-beta-D-galactopyranoside.

(^2)
C. Nessi and A. Galizzi, unpublished data.


ACKNOWLEDGEMENTS

Mass spectral data were provided by F. Corana of Centro Grandi strumenti, Università di Pavia, who was supported by a fellowship from Bracco SpA.


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