(Received for publication, September 9, 1994; and in revised form, December 13, 1994)
From the
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-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
-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
. 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.
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-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) ()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-dependent NAD synthetase. We also report the
purification and characterization of the enzyme from the outB81 mutant.
Restriction enzymes and T4 DNA ligase were obtained from commercial suppliers and used according to their recommendations.
The protein coded by the mutant gene outB81 was purified with the same procedure.
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.
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.
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 = 67,000, lane 3) and chicken
egg albumin (M
= 43,000, lane 4)
from Pharmacia.
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 values for NaAD and ATP are almost identical to those reported
for E. coli and S. cerevisiae, whereas the K
for NH
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 was 9.1
10
M; 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.
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 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. 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.
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.
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 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. ()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, 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
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.