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(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
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 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) (
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
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
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 With glutamine as amide donor, the apparent K
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
Figure 4:
Kinetic response of the mutant NAD
synthetase to NH
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 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. ( 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
Volume 270,
Number 11,
Issue of March 17, 1995 pp. 6181-6185
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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.
-dependent NAD synthetase(3) .
)pyrophosphorylase, coded by nadD, and finally NaAD is converted to NAD by the following
reaction catalyzed by NAD synthetase.
-dependent NAD synthetase. We also report the
purification and characterization of the enzyme from the outB81 mutant.
Bacterial Strains, Plasmids, and Cultivation
Conditions
E. coli DH5
(F
, Y80d, lacZ 
M15 (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 DH5 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) . 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
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 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.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 NHCl, 10 mM MgCl
, 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.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.
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 DH5 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 DH5 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.
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.
= 67,000, lane 3) and chicken
egg albumin (M = 43,000, lane 4)
from Pharmacia.
The OutB Protein Is a NH
While this work was in progress, we were informed by
J. C. Willison that the E. coli protein homolog of OutB is the
NH-dependent NAD
Synthetase-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.
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. 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).
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. 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.
. The assay conditions are
described under ``Experimental
Procedures.''
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
), 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.
value is 10-fold higher than that reported for E. coli.)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., 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.
)
-D-galactopyranoside.)
Mass spectral data were provided by F. Corana of
Centro Grandi strumenti, Università di Pavia, who
was supported by a fellowship from Bracco SpA.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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