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Journal of Bacteriology, October 2000, p. 5454-5461, Vol. 182, No. 19
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
An Operon for a Putative ATP-Binding Cassette
Transport System Involved in Acetoin Utilization of
Bacillus subtilis
Ken-Ichi
Yoshida,1,*
Yasutaro
Fujita,1 and
S. Dusko
Ehrlich2
Department of Biotechnology, Fukuyama
University, Fukuyama, Hiroshima 729-0292, Japan,1 and Génétique
Microbienne, Institut National de la Recherche Agronomique, Domaine de
Vilvert, 78352 Jouy-en-Josas Cedex, France2
Received 16 May 2000/Accepted 9 July 2000
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ABSTRACT |
The ytrABCDEF operon of Bacillus subtilis
was deduced to encode a putative ATP-binding cassette (ABC) transport
system. YtrB and YtrE could be the ABC subunits, and YtrC and YtrD are
highly hydrophobic and could form a channel through the cell membrane, while YtrF could be a periplasmic lipoprotein for substrate binding. Expression of the operon was examined in cells grown in a minimal medium. The results indicate that the expression was induced only early
in the stationary phase. The six ytr genes form a single operon, transcribed from a putative 
A-dependent promoter
present upstream of ytrA. YtrA, which possesses a
helix-turn-helix motif of the GntR family, acts probably as a repressor
and regulates its own transcription. Inactivation of the operon led to
a decrease in maximum cell yield and less-efficient sporulation,
suggesting its involvement in the growth in stationary phase and
sporulation. It is known that B. subtilis produces acetoin as an external carbon storage compound and then reuses it later during
stationary phase and sporulation. When either the entire ytr operon or its last gene, ytrF, was
inactivated, the production of acetoin was not affected, but the reuse
of acetoin became less efficient. We suggest that the Ytr transport
system plays a role in acetoin utilization during stationary phase and sporulation.
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INTRODUCTION |
Genome sequencing of Bacillus
subtilis revealed some 4,100 protein-coding genes, a quarter of
which corresponded to a number of gene families (8). The
largest family, of at least 78 members, encodes the ATP-binding
cassette (ABC) proteins (16), suggesting that the ABC
transport systems play an important role in this organism. Bacterial
ABC transport systems, functionally classified as the ABC importers and
extruders, are known as multisubunit machinery comprising
membrane-associated protein subunits (2). A typical ABC
transport system consists of two hydrophobic membrane-spanning domains
(MSDs) and two hydrophilic ABC domains localized at the cytoplasmic
face of the cell membrane. The ABC proteins are also called traffic
ATPases (1) and contain the ABC domains that supply the
energy for the active transport by hydrolyzing ATP. In the bacterial
ABC importers, the ABC domains and the MSDs are commonly present on
separate polypeptides, and in several systems, the ABC domains have
been shown to associate tightly with two hydrophobic proteins
containing MSDs (7). In addition, all the bacterial ABC
import systems include a periplasmic substrate-binding protein that
interacts with a coming substrate, binds to it, and presents it to the
import complex (2). It is also known that in gram-positive
species, all of such substrate-binding proteins are periplasmic
lipoproteins that are anchored to the membrane via N-terminal
hydrophobic lipid extension (16).
Recently, Quentin and coworkers reported a computerized prediction,
aiming to categorize the 78 ABC proteins of B. subtilis into
systems in combination with their MSD and substrate-binding protein
partners (16). They were able to classify the 78 systems into 11 subfamilies, further split into 6 subfamilies for importers and
5 for extruders. However, over two-thirds of the members of ABC
proteins of B. subtilis have not been functionally
characterized. Here we report characterization of one of those
functionally unknown ABC transport systems, the ytrABCDEF
gene cluster, found at 3118.3 kb on the B. subtilis
chromosome (8). YtrB and YtrE were predicted to encode ABC
proteins with a nucleotide-binding domain (8, 16). YtrB
exhibited weak similarity to PotG of Escherichia coli, involved in putrescine import (14), while YtrE showed
homology with BacH of Enterococcus faecalis, required for
bacteriocin export (23). YtrC and YtrD, which are
paralogous, with 47% identity in 333 amino acid residues, are highly
hydrophobic and are plausible MSD proteins, since they were predicted
to possess nine and eight transmembrane segments, respectively
(16). YtrF could be an MSD protein, because of its putative
four transmembrane segments (16), but was also predicted to
be a periplasmic lipoprotein (22). It was proposed that the
above five gene products could be split into two independent export
systems, YtrBCD and YtrEF (16). However, organization of the
six ytr genes suggests an alternative possibility, that they
might form a single operon for an integrated ABC transport system. The
first gene product of the putative operon, YtrA, was proposed to be a
transcriptional regulator, since it shares significant similarity with
N-terminal regions of many bacterial regulators that contain a
helix-turn-helix motif and belong to the GntR family (5,
26), such as FarR of E. coli (15), HutC of
Klebsiella aerogenes (20), and GntR of B. subtilis (10). It could possibly regulate this operon.
Acetoin is a product of fermentative metabolism in many prokaryotic and
eukaryotic microorganisms including Bacillus spp. (12). Strains of B. subtilis grown in media which
contain enough fermentative carbon sources, such as glucose or
fructose, produce acetoin as an external carbon storage compound and
then reuse it as a carbon and energy source later, during stationary
phase and sporulation (9). In B. subtilis, the
genes encoding the enzymes for acetoin production have been reported to
form a single operon, alsSD (17). In addition, it
has been proposed that there are at least two systems for acetoin
catabolism: one encoded by the acuABC genes and the other by
the acoABCLR genes (4, 6). However, the mechanism
for the production and utilization of acetoin by B. subtilis
is not fully understood, and nothing is known about transport systems
specific for acetoin. We report here that the ytrABCDEF
genes form a single operon, probably encoding an ABC transport system
involved in the reuse of acetoin.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and media.
Bacterial strains
used in this study are listed in Table 1.
Plasmid pMUTIN2mcs (lacZ lacI amp erm) is an integration
vector for the targeted gene inactivation in B. subtilis
(24). It replicates in E. coli but not in
B. subtilis and carries an erythromycin resistance gene that
is active in B. subtilis when the plasmid DNA is integrated
into the chromosome. In addition, it carries a promoterless
lacZ reporter derived from E. coli and the
spac promoter regulated by the lac repressor
encoded by lacI (25), which allows the expression
of the genes downstream of the integration site. E. coli
cells harboring plasmids were grown on Luria broth (LB) (18)
containing ampicillin (50 µg/ml). B. subtilis cells were
grown on the following media containing erythromycin (0.3 µg/ml) when
needed: tryptose blood agar base (Difco) supplemented with 0.18%
glucose (referred to as TBABG), a sporulation medium (DSM)
(19), a minimal medium containing 0.4% glucose as the carbon source (MM) (29), and S1 medium (21).
Construction of B. subtilis mutant strains.
B.
subtilis strains BFS45, BFS47, and FU349 were constructed as
follows. DNA fragments corresponding to part of the ytrA and ytrF genes (Fig. 1) were
amplified by PCR using specific primer pairs and chromosomal DNA of
B. subtilis 168 as a template. The specific primer pairs
used for the constructions were as follows (restriction sites are
underlined): for strain BFS45, ytrAE primer (5'-CCGGAATTCAAAGGTTCAGATCGTATAG-3') and ytrAB
(5'-CGCGGATCCATATATAGGTCCCTCTGC-3'); for BFS47,
ytrFH (5'-CCCAAGCTTTTTTAGGCTGTGTGATTG-3') and
ytrFB (5'-CGCGGATCCCGTTCGCATTATAATTCTC-3'); for
FU349, ytrFdH (5'-CCCAAGCTTGGCTTCGCGTCTTTATGACG-3') and ytrFdB
(5'-CGCGGATCCGTACGATTGCCTAAAGTAGC-3'). The PCR
product for the BFS45 construction was trimmed with
EcoRI and BamHI and ligated with pMUTIN2mcs
that had been digested with EcoRI and BamHI. For
construction of strains BFS47 and FU349, HindIII and BamHI were used instead of EcoRI and
BamHI. The ligated DNAs were used to transform E. coli C600 to ampicillin resistance on LB plates. Cloning of the
right PCR products into pMUTIN2mcs was confirmed by DNA sequencing. The
resulting plasmids, pytrA3B, pytrF1, and pytrFd, were used to transform
B. subtilis 168 to erythromycin resistance on TBABG,
yielding B. subtilis strains BFS45, BFS47, and FU349,
respectively. Correct integration of a single copy of the plasmids into
the respective positions by a single-crossover event was confirmed by
Southern blot analysis.
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FIG. 1.
Genetic organization of the ytr locus of each
of B. subtilis strains used in this study. The
ytr genes and the pMUTIN2mcs-borne genes are presented as
solid and open arrows, respectively. Beneath the genetic organization
of strain 168 (wild type), the DNA stretches cloned into pMUTIN2mcs
(open boxes) or replaced (thick line) are indicated with the name of
the respective mutants. Positions of a probe for the Northern analysis
(Fig. 2) and a primer used for extension analysis (Fig. 3) are
indicated with a closed box and a small arrowhead, respectively.
Positions of the ytr promoter (Pytr) and two
putative terminators (T) deduced from the DNA sequence are also
indicated. In the diagrams of the four mutant strains, dotted lines
indicate integration of the pMUTIN2mcs into the chromosome, and
truncated genes are shaded. The IPTG-inducible spac promoter
(Pspac), which is regulated by the lac repressor
encoded by lacI, drives expression of the downstream genes.
The lacZ reporter is expressed under the control of the
region upstream of the integration site.
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B. subtilis strain BFS53 with a deletion of
ytrA
was constructed as follows. Two DNA fragments (approximately 1.5 kb)
were
amplified by PCR using specific primer pairs and chromosomal DNA
of
B. subtilis 168 as a template; one fragment corresponded
to
a region upstream of the
ytrA gene, and the other
corresponded
to a downstream one. All the PCR was done using GeneAmp XL
PCR
kit (Perkin-Elmer). The specific primer pairs used were as follows
(restriction sites are underlined): for the upstream fragment,
ytrAU1
primer (5'-CATTTTTCGTGCATGCGG-3') and ytrAU2
(5'-CGC
GGATCCTTTCTATACGATCTGAACC-3');
and for
the downstream fragment, ytrAD1
(5'-CCC
AAGCTTAGGAAATCAGCGCTGATG-3')
and ytrAD2
(5'-ATAGAAAGCGGATTTGCC-3'). The upstream and downstream
fragments were trimmed with
BamHI and
HindIII, respectively, and
then ligated with
pMUTIN2mcs that had been digested with
BamHI
and
HindIII. Ligated DNA was directly used for
transformation
of
B. subtilis 168 to erythromycin resistance
on TBABG. In such
transformants,
ytrA gene was expected to
be replaced by pMUTIN2mcs
via a double-crossover event. The replacement
was confirmed by
PCR analysis using the primer pair of ytrAU1 and
ytrAD2 (Fig.
1).
RNA techniques.
Cells of B. subtilis strains were
inoculated into MM to an optical density at 600 nm (OD600)
of 0.05 and incubated at 37°C with shaking. The cells were harvested
5 h (T
1, 1 h before the beginning of
stationary phase), 6 h (T0, point defined
as the beginning of stationary phase), 7 h
(T1, 1 h after the beginning of stationary
phase), and 8 h (T2, 2 h after the
beginning of stationary phase) after inoculation, at OD600s
of approximately 1.5, 2.8, 4.0, and 4.1, respectively. Total RNA of the
cells was extracted by mixing the cells with glass beads, phenol, and
cetyltrimethylammonium bromide, and then purified as described
previously (3). Alternatively, the cells were inoculated
into DSM and grown for 1.5 h (exponential growth), 3 h
(transition between exponential growth and stationary phase), 4 h
(point defined as the beginning of sporulation), 5 h (1 h after
the beginning of sporulation), 7 h (3 h after the beginning of
sporulation), and 9 h (5 h after the beginning of sporulation), to
give OD600s of approximately 0.4, 1.5, 2.1, 2.3, 2.0, and
2.3, respectively. Total RNA was prepared as described above.
For Northern blot analysis, RNA samples were electrophoresed in a
glyoxal gel, transferred to a Hybond-N membrane (Amersham),
and
hybridized with a labeled probe as described previously
(
28).
To prepare the probe, part of the
ytrA
coding region (Fig.
1)
was amplified by PCR using chromosomal DNA of
B. subtilis 168
as a template and a primer pair, ytrAC1
(5'-AGAATGCAAAAACGACACTGG-3')
and ytrAC2
(5'-TCACATCAGCGCTGATTTCC-3'). The PCR product was labeled
radioactively by using
Bca BEST labeling kit (Takara Shuzo)
and
[
-
32P]dCTP (ICN
Biomedicals).
To map a 5' end of the
ytr transcript by primer extension,
50 µg of each RNA was annealed to a primer
(5'-CCGATCATCACAAGAACAAG-3')
that had been labeled at its 5'
end by a MEGALABEL kit (Takara
Shuzo) and [
-
32P]ATP
(Amersham). Primer extension reactions were carried out
as described
previously (
28).
Determination of acetoin concentration in culture media.
B.
subtilis cells were cultured at 30°C for 16 h on TBABG
plates containing erythromycin when needed. Cells were harvested, washed once with freshly prepared MM, inoculated into MM to an OD600 of 0.05, and grown at 37°C with shaking. When
needed, 1 h (T1) and 2 h
(T2) after the entry into stationary phase, 1 mM isopropyl-
-D-thiogalactopyranoside (IPTG) and exogenous
10 mM acetoin (Aldrich) were added to the culture, respectively. A
portion (1 ml) of the culture was withdrawn at different times during the growth, the cells were removed by centrifugation, and the concentration of acetoin in the supernatant was measured as described previously (4).
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RESULTS |
Organization of the ytrABCDEF genes.
The
ytrABCDEF gene cluster is located at 3118.3 kb on the
B. subtilis chromosome (8). As shown in Fig. 1,
the six ytr genes are oriented in the same direction. There
are only two transcription terminator-like sequences within this
region: one located about 300 bp upstream of the ytrA
translation start site and the other just downstream of ytrF
(11). The intergenic regions between the ytr
genes are very short; even the longest one, between ytrC and
ytrD, is only 28-bp long. Moreover, translation stop and
start regions overlap for ytrA and ytrB,
ytrB and ytrC, and ytrE and ytrF. Such overlap can lead to translational coupling
(13), suggesting that these genes are cotranscribed. Taken
together, the organization of the six ytr genes suggests
that they might form an operon.
The ytrABCDEF genes form an operon expressed early in
the stationary phase.
RNA samples prepared from cells of strain
168 grown in MM were subjected to a Northern analysis, using a
ytrA specific probe. As shown in Fig.
2, a specific transcript was present
1 h after the entry in stationary phase
(T1) but disappeared almost completely 1 h
later (T2). The size of the transcript was
estimated to be about 5.5-kb, which would just cover all the
ytr genes. Interestingly, no such transcript was detected in
cells grown in DSM, a nutrient sporulation medium less rich in glucose
(19) (data not shown). A 5' end of the transcript was
determined by primer extension analysis (Fig.
3). It mapped 244-bp upstream of the
ytrA translation start site, close to a
A-dependent promoter-like sequence with typical 
10 and
35 regions. These results suggest that the ytrABCDEF genes
form an operon, which is transcribed early in the stationary phase in
the cells grown in MM.
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FIG. 2.
Northern analysis of the ytr transcript.
Cells of strain 168 were grown in MM, and RNA samples were prepared at
various time points; lanes 1, 2, 3, and 4 contained the RNA prepared at
T1, T0,
T1, and T2, respectively.
The RNA samples were subjected to a Northern analysis targeting the
ytr transcript using the probe (Fig. 1). The position of the
ytr transcript is indicated with an arrow. The two strong
bands at about 1.5 and 3 kb are due to nonspecific hybridization to
rRNAs.
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FIG. 3.
Mapping of a 5' end of the ytr transcript.
The RNA samples were subjected to a primer extension analysis using the
32P-labeled primer (Fig. 1); lanes 1, 2, 3, and 4, contained the reactions using RNAs prepared at
T1, T0,
T1, and T2, respectively.
Reverse transcripts were subjected to a gel electrophoresis together
with dideoxy sequencing reactions carried out by using the same primer
(lanes G, A, T, and C). The position of a major reverse transcript is
indicated with an arrow on the right side of the panel. On the left
side, the 5' end position is marked with an arrow (+1, as the
transcription start site) in the nucleotide sequence of the noncoding
strand of the ytr promoter region, where plausible 10 and
35 regions are underlined.
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To verify this conclusion, the expression of the
ytr operon
was monitored with the aid of reporter gene. A strain carrying
pMUTIN2mcs in the chromosome was used for this purpose (Fig.
1,
strain
BFS47), having a
lacZ gene immediately downstream of
ytrF,
the last gene of the putative operon. As shown in Fig.
4A, the
-galactosidase activity in
BSF47 cells grown in MM was elevated
at the transition between
exponential growth and stationary phase
to give a peak activity early
in the stationary phase and decreased
thereafter. In contrast, only a
negligible activity was found
in cells grown in DSM (Fig.
4D). This
expression pattern coincides
well with that revealed by the
transcription analysis and confirms
that the
ytr genes form
an operon, which is expressed maximally
early in the stationary phase
in the cells grown in MM.
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FIG. 4.
Growth and expression of the lacZ reporter of
strains of B. subtilis. Strains BFS47
(ytrF-pMUTIN2mcs) (A), BFS45
(Pytr::pMUTIN2mcs) (B), and BFS53
( ytrA::pMUTIN2mcs) (C) were grown in MM (open
square, -galactosidase activity; open circle, cell density) and in
MM with 1 mM IPTG (closed square, -galactosidase activity). Strain
168 (wild type) was also grown in MM (open triangle, -galactosidase
activity; closed circle, cell density) and in MM with 1 mM IPTG (closed
triangle, -galactosidase activity). (D) Strains BFS47 (squares,
-galactosidase activity; diamonds, cell density) and 168 (closed
triangle, -galactosidase activity; closed circle, cell density) were
grown in DSM supplemented with (open symbols) and without (closed
symbols) 5 mM acetoin; exogenous acetoin was added to the culture
3 h after the inoculation. The experiments were repeated at least
three times for each strain and medium with similar results;
representative results are shown. -Galactosidase activity (nanomoles
of 2-nitrophenyl--D-galactopyranoside hydrolyzed per
minute per milligram of protein) was determined as described previously
(28).
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The ytrA gene encodes a negative regulator for the
ytr operon.
Sequence analysis indicated that YtrA may
be a transcriptional regulator of the GntR family. To investigate the
possibility that it regulates the ytr operon, two mutant
strains, BFS45 and BFS53, were constructed (Fig. 1). In the first,
BFS45, the entire ytr operon including the ytrA
gene is under the control of the IPTG-inducible spac
promoter (25) and the activity of the natural ytr
promoter can be monitored by the expression of lacZ. In the second, BFS53, the ytrA gene is replaced with pMUTIN2mcs and
the ytr promoter activity can be monitored as in BFS45, but
on a ytrA-null background.
As shown in Fig.
4B when the BFS45 cells were grown in the presence of
IPTG, where
ytrA was expressed throughout the cell
growth,
the
-galactosidase activity was essentially nil. On the
other hand
in the absence of IPTG, where
ytrA was repressed, the
activity reached high levels upon transition between exponential
growth
and stationary phase and did not decrease thereafter as
observed in the
BFS47 cells (Fig.
4A). These results indicate
that YtrA negatively
regulates the
ytr promoter. This conclusion
is strengthened
by the fact that
-galactosidase activity was
constitutively high in
the
ytrA-null BFS53 cells, both in the
presence and the
absence of IPTG (Fig.
4C). It should be noted
that in the absence of
IPTG
-galactosidase activity in the BFS45
cells was not constant but
increased regularly, never reaching
values as high as those observed in
the
ytrA null cells. This
could be due to a leakage of the
spac promoter in the absence
of IPTG, allowing some
synthesis of the repressor protein in the
BFS45
cells.
The ytr operon is involved in acetoin utilization.
Inactivation of the ytr operon, by lack of IPTG in strain
BFS45 or by disruption in strain BFS53, led to a slight but
reproducible decrease in cell yield at the end of growth in MM (Fig. 4B
and C, compare filled and open circles). This observation, together with the fact that the operon is expressed maximally early in the
stationary phase (Fig. 2, 3, and 4A), led us to hypothesize that it
might be involved in establishment and/or maintenance of stationary
growth in a medium containing glucose. Since it is known that B. subtilis cells grown in glucose-rich media such as MM produce
acetoin as an external carbon storage compound and then reuse it during
stationary phase and sporulation (9), we examined the effect
of inactivation of the ytr operon on production and
utilization of acetoin.
B. subtilis strains (168 [wild type], BFS45, and BFS47)
were grown in MM without IPTG, and the concentration of acetoin in
the
culture media was measured (Fig.
5A).
With the wild-type cells,
acetoin appeared early in the stationary
phase, accumulated to
a concentration of approximately 3 mM at
T2, and was almost fully
consumed within 3 h. This indicates that the cells could produce
and reuse acetoin under
these growth conditions. Production and
reuse of acetoin by the BFS47
cells, which carry pMUTIN2mcs downstream
of
ytrF and thus
retain the intact
ytr operon, were similar to
those of the
wild-type strain. In contrast, BFS45 cells, which
do not express the
operon in the absence of IPTG, produced acetoin
as the wild-type, but
reused it with a substantial delay. All
three strains produced and
reused acetoin at the same rate in
the presence of IPTG, which allowed
expression of the
ytr operon
in BFS45 cells (Fig.
5B). These
results indicate that the
ytr operon plays a role in acetoin
consumption.
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FIG. 5.
Production and consumption of acetoin by strains of
B. subtilis. (A and B) Spontaneous production and
consumption of acetoin. Strains of B. subtilis were grown in
MM in the absence (A) or presence (B) of 1 mM IPTG added at
T1. The concentration of acetoin in the culture
medium was determined at various time points after the inoculation as
indicated: open circle, strain 168 (wild type); closed circle, strain
BFS47 (ytrF-pMUTIN2mcs); open square, strain BFS45
(Pytr::pMUTIN2mcs). (C) Consumption of acetoin
added exogenously. Cells of the strains were grown in MM, and at
T2 acetoin (10 mM) was added into the cultures
exogenously. The concentration of acetoin in the medium was measured at
various time points as indicated (100% corresponds to approximately 13 mM), and the percentage of the remaining acetoin is shown: open
circles, strain 168 (wild type); closed circles, strain BFS47
(ytrF-pMUTIN2mcs); open squares, strain BFS45
(Pytr::pMUTIN2mcs); closed squares, strain FU349
(ytrF::pMUTIN2mcs); open triangles, cell-free
medium. The experiments were repeated at least three times for each
strain and medium with similar results; representative results are
shown.
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To confirm this conclusion, an excess of acetoin (10 mM) was added to
the culture at the time of maximal acetoin accumulation
(
T2) and its concentration was measured
thereafter (Fig.
5C).
After 6 h less than 10% of the acetoin was
present in cultures
of the wild-type and BFS47 cells, whereas almost
40% remained
in the BFS45 culture. A similar delay in acetoin
consumption was
observed with another mutant strain, FU349 (Fig.
1),
which lacks
the
ytrF gene (Fig.
5C). Addition of IPTG to the
BFS45 culture
did not abolish the delay, possibly because the induction
of the
operon was insufficient to consume the excess acetoin (data not
shown).
As mentioned above, acetoin is generally used as a carbon and energy
source during stationary phase and sporulation. The utilization
during
sporulation was tested in a manner similar to that described
by Grundy
et al. (
4) (Table
2). Cells grown in MM until
T2 were washed and resuspended in the S1 medium
(
21) supplemented
with or without acetoin as a carbon source
and then incubated
further until
T26; glycine
was added to S1 since it was reported
that utilization of acetoin under
these condition may require
it, although the physiological significance
of this is unknown
(
4). The wild-type and strain BFS47
cells, which have the intact
ytr operon, grew continuously
in MM until
T26 to cell densities
of about
3 × 10
8 and sporulated at frequencies of about 60%.
In S1 without acetoin,
the two strains grew to lower cell densities and
sporulated with
lower efficiencies. Addition of acetoin to this medium
increased
slightly but reproducibly both the cell density and
sporulation
frequency of the two strains. As expected, the strains
BFS45 and
FU349 cells, which have a defective
ytr operon,
grew in MM to
lower cell densities and also exhibited lower sporulation
frequencies
than the two
ytr-proficient strains.
Interestingly, addition of
acetoin to S1 did not enhance either the
cell density or sporulating
ability of these two
ytr-deficient strains. The results imply
that the
ytr mutants, which showed the slower acetoin consumption,
do
not use acetoin during sporulation as efficiently as the wild-type
strains.
The results above implied a possibility that the
ytr operon
might be induced by acetoin. To investigate this possibility,
we tested
the effect of acetoin addition on the expression of
the operon by
monitoring the
lacZ reporter in BFS47 cells. As
described
above, the
-galactosidase activity in the cells grown
in DSM was
negligible (Fig.
4D), indicating that the operon was
not normally
expressed, and production of acetoin under these
growth conditions was
hardly detected (data not shown). Addition
of acetoin (5 mM; higher
than the maximal concentration accumulated
in MM) to the culture did
not lead to higher
-galactosidase activity
(Fig.
4D), suggesting
that acetoin did not induce the
ytr operon.
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DISCUSSION |
The present work led to several conclusions. (i) The
ytrABCDEF genes of B. subtilis form an
operon, which is expressed early in the stationary phase in the cells
grown in the glucose-rich MM (Fig. 2, 3, and 4A). (ii) The
ytr operon is likely transcribed from a promoter, negatively
regulated by the expression of ytrA, encoding a protein of
the GntR family of repressors (Fig. 3 and 4B and C). (iii) Inactivation
of the ytr operon leads to a slower consumption of acetoin
in the culture medium during stationary phase (Fig. 5). (iv) This
slower consumption of acetoin results in a lower cell density and
sporulation frequency (Table 2). (v)
Acetoin addition does not induce the ytr operon (Fig. 4D).
The six ytr genes form an operon transcribed as a 5.5-kb
mRNA, mainly in the beginning of the stationary phase in cells grown in
MM (Fig. 2 and 4A). The short intergenic regions where translation stop
and start sites of the ytr genes overlap, might result in translational coupling, conducive to controlling the synthesis of the
gene products with a nearly 1:1 stoichiometry (13). It is
plausible that the YtrBCDEF proteins might be the subunits of an
integrated ABC transport system, the model of which is depicted in Fig.
6. YtrB and YtrE are situated on the
internal side of the cell membrane and function as the ABC proteins to
supply the energy for the transport, YtrC and YtrD are the MSD proteins
that make a channel through the cell membrane, and YtrF is the
substrate-binding protein. In addition, YtrA regulates the expression
of the entire operon presumably as a repressor (Figs. 4B and C).
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|
FIG. 6.
Putative functions of the ytr gene products.
The ytrABCDEF operon of B. subtilis encodes a
putative ABC import system responsible for a substrate, X; YtrB and
YtrE are the ABC subunits, the hydrophobic YtrC and YtrD are the MSD
proteins that form a channel through the cell membrane, YtrF is a
periplasmic lipoprotein for substrate binding, and YtrA may regulate
Pytr as a repressor.
|
|
The inactivation of the ytr operon led to a less-efficient
utilization of acetoin, but acetoin production was not affected (Fig. 5
and Table 2). Consequently, a simple hypothesis is that the Ytr ABC
transport system imports acetoin. In this case B. subtilis
cells have also another acetoin import system, since the acetoin
utilization by the ytr mutants was impaired only partially. A more complex formal alternative is that the system transports a
yet-unknown substance required for efficient acetoin catabolism. Since
the effect of ytr inactivation is observed in a minimal medium, such a substance would have to be produced and excreted by the
B. subtilis cells in parallel with acetoin. We know of no
precedent for such a process and consider it unlikely. However, further
work is required to rule out this alternative.
YtrA was proposed to be a repressor of the GntR family. Here we show
that it regulates negatively the ytr operon. Its binding site might be a nucleotide sequence which overlaps the transcription start site (defined as +1) and shows a partial dyad symmetry: 5TtaAGTGTAcTAaTT-G-AAgTAaTACACTatA+26
(latter half, mismatches, and the center of the symmetry are shown in italic type, lower case letters, and underlined,
respectively). Typical members of the GntR family consist of
approximately 250 amino acid residues (5). Their N-terminal
half forms a DNA binding domain containing a helix-turn-helix motif
(5), and the DNA binding is modulated through interaction
between their C-terminal domain and the specific effector
(27). It is noteworthy that YtrA comprises only 130 amino
acid residues, suggesting that its C-terminal domain is too small to
accommodate the effector binding. Acetoin seems not to be the effector,
since its addition did not induce the reporter gene present in the
strain BFS47 (Fig. 4D). Further work is required to identify the
effector and to analyze YtrA binding to DNA.
| |
ACKNOWLEDGMENTS |
We thank Hironobu Katsumata, Ryosuke Tanaka, Atsushi Umeda, and
Shigeru Yamada for their technical assistance; Petar P. Pujic for
valuable discussions; Choong-Min Kang for his critical reading of the
manuscript; and Valérie Vagner for providing pMUTIN2mcs. This
work was supported by grant JSPS-RFTF96L00105 from the Japan Society
for the Promotion of Science and grant BIO4-CT95-0278 from the EU.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biotechnology, Fukuyama University, 985 Sanzo, Higashimura-cho,
Fukuyama, Hiroshima 729-0292, Japan. Phone: 81 849 36 2111. Fax: 81 849 36 2459. E-mail:
kyoshida{at}bt.fubt.fukuyama-u.ac.jp.
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Abstract
Introduction
