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Journal of Bacteriology, January 1999, p. 493-500, Vol. 181, No. 2
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Temporal Expression of the Bacillus subtilis secA
Gene, Encoding a Central Component of the Preprotein
Translocase
Markus
Herbort,1
Michael
Klein,1
Erik H.
Manting,2
Arnold J. M.
Driessen,2 and
Roland
Freudl1,*
Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, D-52425 Jülich,
Germany,1 and
Department of
Microbiology, Groningen Biomolecular Sciences and Biotechnology
Institute, University of Groningen, Kerklaan 30, NL-9751 NN Haren,
The Netherlands2
Received 17 August 1998/Accepted 9 November 1998
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ABSTRACT |
In Bacillus subtilis, the secretion of extracellular
proteins strongly increases upon transition from exponential growth to the stationary growth phase. It is not known whether the amounts of
some or all components of the protein translocation apparatus are
concomitantly increased in relation to the increased export activity.
In this study, we analyzed the transcriptional organization and
temporal expression of the secA gene, encoding a central
component of the B. subtilis preprotein translocase. We
found that secA and the downstream gene (prfB)
constitute an operon that is transcribed from a vegetative
(
A-dependent) promoter located upstream of
secA. Furthermore, using different independent methods, we
found that secA expression occurred mainly in the
exponential growth phase, reaching a maximal value almost precisely at
the transition from exponential growth to the stationary growth phase.
Following to this maximum, the de novo transcription of
secA sharply decreased to a low basal level. Since at the
time of maximal secA transcription the secretion activity
of B. subtilis strongly increases, our results clearly demonstrate that the expression of at least one of the central components of the B. subtilis protein export apparatus is
adapted to the increased demand for protein secretion. Possible
mechanistic consequences are discussed.
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INTRODUCTION |
Transport of secretory proteins
across the Bacillus subtilis plasma membrane is mediated by
an export machinery which consists (at least partially) of components
that are homologous to the well-characterized Sec proteins of
Escherichia coli (52). Genes encoding homologues
of the inner membrane proteins SecY (27, 43) and SecE
(15), which most likely form the core of the protein-conducting pathway (8), and SecDF (3),
which is thought to increase the efficiency of protein translocation at the SecYE core complex (9), have been identified in
B. subtilis. In addition, a homologue of the E. coli SecA protein, which is known to couple the energy of ATP
binding and hydrolysis to protein translocation (10, 20),
has also been found in B. subtilis (31, 34).
An involvement of these Sec proteins in the secretion process of
B. subtilis has been inferred from various functional analyses (3, 15, 17, 22, 28, 31, 47, 50).
B. subtilis secretes most of its extracellular enzymes
in a temporally controlled manner. Whereas during exponential growth relatively small quantities of exoproteins are released into the growth
medium, a substantial increase in exoprotein secretion is observed when
the cells enter the post-exponential growth phase (for reviews, see
references 11 and 33). This
increase in secretion activity is due mainly to an increased synthesis
of the corresponding secreted proteins for which, among other
regulatory networks, the DegS-DegU two-component system is of crucial
importance (11, 26). However, no data are available
concerning the temporal regulation of the sec genes during
growth of B. subtilis. In particular, it is not known
whether the amounts of some or all components of the protein
translocation machinery are concomitantly increased with the temporal
demand for an increased secretion activity in the post-exponential
growth phase.
In the work described here, we analyzed the transcriptional
organization and temporal expression of the B. subtilis
secA gene, encoding the translocation ATPase subunit of the
preprotein translocase. We show that secA is the first gene
in an operon and that transcription of this operon is
initiated from a promoter which is highly similar to
A-dependent promoters. Furthermore, we found by
different independent criteria that expression of secA is
maximal at the end of the exponential growth phase, after which a
decrease in transcription can be observed. The results indicate that,
like the synthesis of secreted proteins, the synthesis of at least one
of the central components of the B. subtilis preprotein
translocase is temporally controlled.
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MATERIALS AND METHODS |
Bacterial strains and growth conditions.
E. coli JM109
(53) was grown at 37°C in Luria-Bertani (LB) medium
(25) containing ampicillin (100 µg ml
1) as
required. B. subtilis DB104, a strain that secretes
only low levels of proteases into the culture supernatant
(16), and its derivatives DB104-4 and DB104-4T were grown at
37°C in LB, 2xYT (25), sporulation (39), or
minimal (41) medium containing chloramphenicol (5 µg
ml1) or kanamycin (20 µg ml1) as
required. B. subtilis DB104-4, containing a
transcriptional secA-lacZ fusion integrated into the
chromosomal amyE gene, was constructed by transforming DB104
with linearized plasmid pDG268-4 (see below), selecting for
chloramphenicol resistance and screening for an 
-amylase-negative
phenotype on LB plates containing 0.6% (wt/vol) amylopectin-azure
(Sigma, Deisenhofen, Germany). DB104-4T, containing a translational
secA-lacZ fusion integrated into the amyE gene,
was constructed by the same procedure except that plasmid pDG268-4T
(see below) was used.
DNA techniques and plasmid constructions.
Standard
procedures (36) were used for preparation of plasmid DNA,
isolation of DNA fragments, restriction, ligation, and other DNA techniques.
pDG268 is a transcriptional spoVG-lacZ fusion vector that
allows the subsequent integration of the respective lacZ
reporter fusions in single copy into the chromosomal amyE
gene of B. subtilis via a double-crossover event
(1). A transcriptional secA-lacZ fusion was
constructed by ligating a 456-bp MscI/HindIII
fragment, containing the orf189-secA intergenic region and
the 5' end of the secA gene, from plasmid pBO1
(31) into pDG268 which had been digested with
EcoRI, filled in with Klenow DNA polymerase, and then
redigested with HindIII. In the resulting plasmid,
pDG268-4, transcription of the spoVG-lacZ reporter gene is
under the control of the secA promoter. For construction of
a secA-lacZ translational fusion, a 892-bp
HindIII/ClaI fragment of pDG268-4,
encompassing the spoVG-lacZ translational initiation signals
(i.e., ribosome-binding site and start codon) was replaced by a 846-bp
HindIII/ClaI fragment from the translational
lacZ fusion vector pRB381 (5). To obtain an
in-frame fusion, both HindIII ends were filled in with
Klenow DNA polymerase. The resulting plasmid, pDG268-4T, encodes a
fusion protein of 65 amino-terminal amino acid residues of SecA fused to the 
-galactosidase reporter, and transcription of the
corresponding gene is under the control of the secA
promoter. For integration of the secA-lacZ fusions into the
amyE gene, pDG268-4 and pDG268-4T were linearized with
MscI and used to transform B. subtilis
DB104. Chloramphenicol-resistant and amylase-negative transformants
were selected, resulting in B. subtilis DB104-4 and
DB104-4T, respectively.
For generation of the digoxigenin (DIG)-labeled RNA probes 1 to 4 (Fig.
1), suitable DNA fragments of the
orf189-secA-prfB region were cloned into the riboprobe
vector pGEM3Z (Promega,
Mannheim, Germany). For probe 1, covering an
internal fragment
of
orf189, a 290-bp
MscI/
BglII fragment from plasmid pBO1 was
cloned
into
BamHI/
HindII-digested pGEM3Z, resulting
in plasmid
pMHP1. For probe 3, covering the 3' end of
secA,
a 2.63-kb
PstI
fragment from plasmid pMKL4 (
17),
encompassing the entire
secA gene, was ligated into
PstI-digested pGEM3Z. Before in vitro transcription
from the
SP6 promoter was initiated, the resulting plasmid pMHP3
was linearized
at a
ClaI site, located within the
secA coding
region, thereby resulting in a DIG-labeled RNA probe derived solely
from the 3' end of the
secA gene. For probe 2, covering the
5'
end of
secA, a 448-bp
BamHI/
HpaI
fragment from pMHP3 was ligated
into
BamHI/
HindIII-digested pGEM3Z, resulting
in plasmid pMHP2.
For probe 4, covering part of the intergenic
secA-prfB region
and the 5' end of the
prfB gene,
a 488-bp
Asp718/
PstI fragment
from pMKL4
was ligated into
Asp718/
PstI-digested pGEM3Z,
resulting
in plasmid pMHP4.
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FIG. 1.
Gene organization in the secA region of the
B. subtilis chromosome and positions of
secA-specific transcripts. PA,
A-dependent promoter of the secA-prfB
operon. Stem-loop figures, putative rho-independent
terminators; numbered bars, locations of hybridization probes used in
Northern blotting experiments; thin arrows, secA-specific
transcripts identified in the Northern hybridization experiments.
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RNA techniques.
To isolate total RNA, we used an RNeasy
total RNA kit (Qiagen, Hilden, Germany) as instructed by the
manufacturer. The amount of total RNA was quantified by measuring the
optical density at 260 nm (OD260) of each preparation.
Furthermore, the RNA samples used in the Northern blotting and the
primer extension experiments were additionally examined with respect to
the amount and intactness of the RNA preparations by
formaldehyde-agarose gel electrophoresis and staining with ethidium
bromide (49). Northern blot analyses were performed
essentially as described by Börmann et al. (4). To
obtain DIG-labeled RNA probes 1 to 4 (locations in the
orf189-secA-prfB region are shown in Fig. 1), plasmids pMHP1
to -4 were used as templates for in vitro transcription using a DIG RNA
labeling kit (Boehringer Mannheim GmbH, Mannheim, Germany) as
instructed by the manufacturer. For detection of hybridization signals,
a Boehringer nonradioactive nucleic acid labeling and detection kit was
used. Primer extension experiments were performed as described by
Sambrook et al. (36). The synthetic oligonucleotide OMKL48 (GTATCTATTCAGCGTACGT), which is complementary to the
noncoding strand of the 5' end of the secA gene
(corresponding to nucleotides 39 to 57 of the secA
structural gene), was used as the primer in the primer extension
experiments. Dideoxynucleotide chain termination sequencing
reactions (37), using the same primer and an appropriate plasmid DNA (pBO1) as the template, were run in parallel on the gel to
allow determination of the endpoints of the extension products.
Other techniques.
-Galactosidase activities of
B. subtilis cells containing transcriptional or
translational secA-lacZ fusions were determined by the
procedure of Nicholson and Setlow (29), using
o-nitrophenyl--D-galactopyranoside as the
substrate. The measured -galactosidase activity was normalized by
the method of Miller (25). -Amylase secreted into the
culture supernatant of B. subtilis DB104 was assayed by
the method of Bernfeld (2), and the resulting activities
(units) were expressed in terms of micromoles of maltose liberated in
60 min at 37°C by 1 ml of culture supernatant. Total amounts of
secreted proteins in the culture supernatant of DB104 were determined
by the bicinchoninic acid method of Walker (51) after
precipitation of the proteins with trichloroacetic acid (10%, final
concentration). Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and Western blotting using
anti-B. subtilis SecA antibodies were performed as
described previously (24).
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RESULTS |
The B. subtilis secA gene is the first gene in a
bicistronic operon.
The chromosomal organization of
the B. subtilis secA region is shown in Fig. 1. The
secA gene is located downstream of the fliD
operon, whose gene products are involved in the formation of
flagella, and a gene (orf189) of unknown function, which is followed by a putative rho-independent terminator (7).
Downstream of secA, we identified a gene (prfB)
which is preceded by a putative ribosome-binding site and which encodes
a homologue of the E. coli protein release factor 2 (32). No putative promoter or terminator sequences are found
in the secA-prfB intergenic region, suggesting that
secA and prfB may be located in an operon
which is transcribed by a promoter located upstream of secA.
In fact, a promoter sequence (TTGGAA-16 bp-TATGAT) showing good
agreement to the consensus promoter sequence recognized by the major
vegetative sigma factor (A) (12) was
identified 80 to 107 bp upstream of the secA start codon
(31, 34). In addition, an operon structure
consisting of secA and prfB was also suggested
from computer analyses of the complete nucleotide sequence of the
B. subtilis chromosome (18).
For identification of
secA-specific transcripts, total RNA
was extracted from
B. subtilis DB104 at various time
points during
growth (measured by OD
600) in sporulation
medium (Fig.
2A), and
Northern
hybridization experiments were performed with DIG-labeled
RNA probes,
encompassing the 3' end of
orf189 (probe 1), the 5'
end of
secA (probe 2), the 3' end of
secA (probe 3), and
the 5'
end of
prfB (probe 4) (Fig.
1). With the 5' end of
secA as a probe,
two major hybridizing bands, 3.8 and 0.3 kb
in size, were identified
(Fig.
2B). Furthermore, additional
hybridization signals were
found associated with the 16S and 23S RNAs;
these most likely
were caused by hybridization to degradation products
which were
trapped by the rRNAs (
14). The 3.8-kb mRNA was
also detectable
with probes 3 and 4 but not with probe 1 (data not
shown), strongly
suggesting that
secA and
prfB
are transcribed together starting
from a promoter located in the
orf189-secA intergenic region.
The 0.3-kb RNA could be
detected with probe 2 only (not with probes
1, 3, and 4 [data not
shown]), suggesting that this RNA might
represent a stable breakdown
product from the 5' end of the larger
3.8-kb
secA-prfB
transcript or, alternatively, might be produced
by premature
transcription termination early in the
secA gene.
A further
observation was that the amount of detectable
secA-specific
transcripts in the RNA preparations isolated from cells grown
to the
end of the exponential (lane 3) or to the post-exponential
phase (lane
4) was significantly lower than the corresponding
signals which were
obtained with RNA isolated from exponentially
growing cells (lanes 1 and 2), suggesting that
secA transcription
is maximal during
vegetative growth.
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FIG. 2.
Northern blot analysis. (A) Growth of B. subtilis DB104 in sporulation medium. Samples 1 to 4 were
withdrawn for total RNA isolation at the time points indicated. (B)
Northern hybridization of equal amounts of total RNA isolated from
samples 1 to 4 (lane numbers correspond to sample numbers), using a
probe (probe 2; Fig. 1) derived from the 5' end of the secA
gene. Positions of the secA-specific RNAs are indicated by
arrows. 16S and 23S denote positions of the 16S and 23S rRNAs,
respectively.
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To identify the start point of the
secA-prfB-specific
transcript, primer extension experiments were performed with
primer
OMKL48, using RNA isolated at various time points during
growth
in rich (2xYT) medium. As shown in Fig.
3, a signal corresponding
to a
transcriptional start site located 73 bp upstream of the
secA translational start codon was detected in all samples.
Since
this transcriptional start site is located immediately downstream
to the
A-dependent promoter previously identified by
sequence analysis,
our data clearly indicate that this promoter is in
fact the promoter
element involved in
secA transcription
initiation. We observed
that in the primer extension experiments,
as in the Northern blotting
experiments, the intensities of the
obtained signals were significantly
higher in RNA preparations
extracted from exponentially growing
cells (lanes 1 and 2) than in RNA
extracted from cells grown to
the late exponential (lane 3) or
post-exponential (lane 4) phase.
Identical results were obtained for
RNA isolated at comparable
time points during growth from cells that
had been grown in sporulation
medium (data not shown).
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FIG. 3.
Mapping of the 5' end of the secA-prfB mRNA
by primer extension. (A) Growth of B. subtilis
DB104 in 2xYT medium. Samples 1 to 4 were withdrawn for total RNA
isolation at the time points indicated in the growth curve. (B) Primer
extension experiment using oligonucleotide OMKL48 as the primer and
equal amounts of RNA isolated from samples 1 to 4 (lane numbers
correspond to sample numbers). The major extension product is indicated
by an arrow. Lanes C, T, A, and G, sequencing ladder obtained with the
same primer (OMKL48) and pBO1 (31) as the template. The
relevant part of the nucleotide sequence is shown on the left.
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Transcription of the secA gene is temporally
regulated.
Since the results described above suggested that
expression of secA is temporally modulated in the cell,
quantitative measurements of secA expression were performed
with transcriptional and translational fusions of the lacZ
reporter gene to 5' fragments of secA. A 456-bp MscI/HindIII DNA fragment from pBO1
(31) containing the orf189-secA intergenic region
and the 5' end of the secA gene was cloned into the
lacZ fusion vector pDG268 (1) in two different
ways. In plasmid pDG268-4, the spoVG-lacZ gene of pDG268 was
placed under the regulatory control of the secA promoter,
resulting in a transcriptional fusion. In pDG268-4T, the 5' end of
secA was fused in frame to the lacZ reporter
gene, resulting in a translational secA-lacZ fusion. Both
fusions were introduced by double-crossover recombination in single
copy into the amyE locus of the chromosome of B. subtilis DB104, and -galactosidase activity in the resulting
strains (DB104-4 and DB104-4T) was monitored throughout growth (Fig.
4). In both strains, an
increase in -galactosidase activity was observed during
exponential growth, reaching a maximal value at the end of the
exponential growth phase (which is defined as
T0). Upon entering the post-exponential phase,
the strains exhibited a relatively sharp decrease of -galactosidase
activity, reaching a low basal level approximately 2 to 3 h after
T0. The observed patterns were independent of
the growth medium, since similar profiles of -galactosidase activity
were obtained with both strains in sporulation medium (Fig. 4A and C),
rich medium (Fig. 4B and D), and minimal medium (data not shown). In
addition, these results were in agreement with the temporal effects
observed in the Northern blotting and primer extension experiments,
which also indicated a maximal expression of secA during
exponential growth and a sharp decrease of transcription shortly after
T0. Furthermore, the analysis of -amylase
secretion (Fig. 4E) and of protein secretion in general (by
measuring the amount of total protein in the supernatant) (Fig.
4F) during growth showed that the time point of maximal
secA expression coincides with the beginning of the
high-level protein secretion period in B. subtilis.
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FIG. 4.
Monitoring of secA gene expression during
growth of cells containing secA-lacZ fusions and analysis of
-amylase secretion and protein secretion activity in general. Graphs
show expression of a transcriptional secA-lacZ fusion during
growth of B. subtilis DB104 in sporulation medium (A)
or LB medium (B); expression of a translational secA-lacZ
fusion during growth of B. subtilis DB104 in
sporulation medium (C) or LB medium (D); secretion of -amylase
during growth of B. subtilis DB104 in LB medium (E);
and total amount of proteins secreted into the supernatant of
B. subtilis DB104 during growth in LB medium (F).
Squares, OD600; circles, -galactosidase (LacZ) specific
activity (A to D); -amylase activity (E), or total amount of protein
in the supernatant (F).
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Next, expression of
secA was analyzed by Western blotting
using SecA-specific antibodies. Cells of
B. subtilis
DB104 were
grown in sporulation medium (Fig.
5A and
C) or LB medium (Fig.
5B and D), and
samples were taken at various time points during
growth. Equal amounts
of proteins were applied to SDS-PAGE followed
by Western blotting. In
both media, the amount of SecA protein
reaches a maximal value around
T0, decreasing slowly thereafter
to
significantly lower levels. These results are consistent with
those
obtained with the
lacZ reporter gene fusions, indicating
that
secA expression is strongly decreased at
T0 and that the
SecA protein synthesized up to
this point is diluted within the
cells by ongoing cell division. Our
finding that SecA protein
can nevertheless be detected several hours
after the end of exponential
growth can be explained by the high in
vivo stability of the
B. subtilis SecA protein
(
46) and eventually by some residual de
novo transcription
of the
secA gene.
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FIG. 5.
Detection of SecA protein by Western blotting. (A and B)
Growth of B. subtilis DB104 in sporulation medium (A)
or LB medium (B). Samples 1 to 14 were withdrawn for the preparation of
total-cell extracts at the time points indicated, and equal amounts of
protein were subsequently applied to SDS-PAGE and Western blotting. (C
and D) Western blot analyses using SecA-specific antibodies of
total-cell extracts isolated from B. subtilis DB104
grown in sporulation medium (C) or LB medium (D). The lane numbers in
panels C and D correspond to sample numbers in panels A and B,
respectively.
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DISCUSSION |
The increase in protein secretion activity is, among competence
development and sporulation, one of the various possible
post-exponential growth means by which B. subtilis and
related Bacillus species adapt to unfavorable growth
conditions, such as nutrient limitation (42). Synthesis of
the precursor forms of the secreted proteins drastically increases at
the level of transcription of the corresponding genes upon transition
from exponential to stationary growth, and this increase is controlled
by complex regulatory networks (11). It remains an open
question whether the number of protein export sites normally present in
low-level secreting exponentially growing cells is sufficient to allow
the effective translocation of the abundance of exoproteins in the
postexponential growth phase or whether the amounts of one or several
components of the protein secretion apparatus must be adapted to this
elevated secretion activity.
In this report, we have shown that the secA gene, encoding
one of the central components of the B. subtilis
preprotein translocase, is transcribed predominantly in the exponential
growth phase. This result is in agreement with our finding that the
secA gene, together with the prfB gene which is
located downstream of secA, is transcribed from a promoter
with a sequence very similar to promoter sequences recognized by
A, the major vegetative sigma factor of B. subtilis (12). Maximum secA expression is
reached at the end of the exponential growth phase, almost precisely at
the time point corresponding to T0. Surprisingly, upon transition to the stationary growth phase, de novo
transcription of the secA gene sharply decreases to a low
basal level. Despite this fact, significant amounts of SecA protein can
be detected also at much later time points of growth, probably due to
the long half-life of the SecA protein in B. subtilis (46). Upon abrupt shutoff of secA transcription
at the transition to stationary growth phase, the slow decrease in the
amount of SecA protein that is observed at later time points can be
explained by a dilution of the presynthesized SecA protein to the
daughter cells during the remainder of cell divisions after
T0; possibly some residual de novo synthesis may
also occur. With respect to the time period of high-level protein
secretion, which extends from approximately T0
to T4/T5 (33, 38), it is
worth emphasizing that maximal secA gene expression and, as
a consequence, the highest amount of SecA protein is reached at the
beginning of the period of elevated secretion activity. This implies
that the amount of SecA protein synthesized up to this point is
sufficient to ensure effective protein translocation during the entire
high-level secretion period. Besides its role as a membrane-associated
energy-coupling subunit of preprotein translocase, SecA can most likely
in its soluble form (6, 22), interact also with precursor
proteins occurring free in the cytosol (45). One might
speculate that the high amount of SecA at the beginning of the
high-level secretion period can function as a kind of a secretory
protein-specific chaperone buffer which helps to maintain the massive
amounts of precursor proteins in an export-competent state for their
subsequent membrane translocation.
In E. coli, secA expression is coupled to the
secretion status of the cell by an autoregulatory mechanism
(30). Under normal protein export conditions, excess SecA
binds to its own mRNA, thereby autorepressing its translation. Under
conditions of limiting protein export, the mRNA-bound SecA is released
(possibly by a titration mechanism) and translation of secA
mRNA increases 10- to 20-fold (23, 35, 40). So far, we have
found no evidence for a comparable autoregulation of secA
expression in B. subtilis. High-level expression of a
secretory protein (-amylase AmyL of B. licheniformis) in B. subtilis, resulting in the
massive accumulation of AmyL precursor in the cytosol, did not lead to
an increase in the amount of SecA protein (13). Furthermore,
overexpression of SecA in B. subtilis DB104, containing
a translational orf189-secA-lacZ fusion integrated in the
amy locus, did not result in a reduced expression of
-galactosidase activity (13). However, since these
observations are negative evidence, the existence of an autoregulatory
mechanism for secA expression in B. subtilis
cannot be totally excluded.
Very little is known about the regulation of other components of the
B. subtilis preprotein translocase. Li et al.
(19) have shown that the B. subtilis secY
gene, which is located within the S10-spc- region and
encodes one of the central integral membrane components of the
translocase, is primarily cotranscribed within one large (15-kb)
transcriptional unit together with the other (mostly ribosomal) genes
of the S10-spc- region and that this transcription is
driven by two A-dependent promoters located upstream of
the S10 gene. In addition, Suh et al. (44)
identified two weak promoter-active regions located within the two
genes immediately upstream of secY, which are able to
stimulate transcription of a lacZ reporter gene mainly in
the stationary growth phase. However, it is unclear whether these weak
promoter sequences significantly contribute to secY gene
expression in the stationary growth phase. Furthermore, since stable
integration of SecY into the cytoplasmic membrane, at least in E. coli, requires a concomitant coexpression of SecE (21), it is also unclear whether the weak transcriptional activity of these
potential secY promoter sequences in the stationary growth phase would in fact result in a significantly elevated level of SecY
protein in the plasma membrane.
Recently, Bolhuis et al. cloned the secDF gene of
B. subtilis and analyzed its expression during growth
in different media, using a transcriptional secDF-lacZ
fusion (3). Whereas secDF expression was more or
less constitutive when the cells were grown in minimal medium, a
maximum of secDF transcription was found in the early
post-exponential growth phase when rich (TY) medium was used for
growth. Interestingly, the time point of maximum secDF
expression in rich medium is 1 to 2 h later in the growth phase
compared to the time point of maximum secA expression.
B. subtilis contains at least four closely related type
I signal peptidases (SipS, SipT, SipU, and SipV) which are responsible for the removal of the signal peptides from nonlipoprotein precursor proteins (48). Whereas the sipU and
sipV genes are transcribed at a more or less constant level
during all growth phases, the transcription of sipS and
sipT increases upon transition from exponential to
stationary growth. Due to their finding that the increase in
sipS and sipT transcription is controlled by the
DegU-DegS two-component signal transduction system, which is mainly
involved in the upregulation of the synthesis of secreted precursor
proteins after T0, the authors (48)
speculate that SipS and SipT serve to increase the capacity of
B. subtilis for protein secretion concomitantly with
the increasing amounts of secretory precursor proteins synthesized in
the post-exponential growth phase. Interestingly, in contrast to
secA gene expression, which is maximal at the beginning of
the secretion period (approximately T0),
sipS and sipT transcription is relatively low at
T0 and increases steadily throughout the entire
period of high-level protein secretion after T0
(48). These findings taken together suggest that at the
post-exponential growth phase, B. subtilis adjusts the
amount of some of the central components of the protein secretion
machinery in relation to the increased demand for protein export.
However, differences in the timing of this adjustment seem to exist
between different components.
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ACKNOWLEDGMENTS |
We are very grateful to R. Brückner and K.-L. Schimz for
their kind gifts of plasmids and antibodies. We thank H. Sahm for continuous support and A. Bida for technical assistance. Furthermore, we thank the members of the European Bacillus Secretion Group, especially J. M. van Dijl, A. Bolhuis, M. Klose, and J. Meens, for
stimulating discussions.
This work was supported by grants from the BMBF and the CEC (biotech
grant BIO4-CT96-0097).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Biotechnologie 1, Forschungszentrum Jülich GmbH,
D-52425 Jülich, Germany. Phone: (49) 2461-613472. Fax: (49)
2461-612710. E-mail: r.freudl{at}fzjuelich.de.
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Abstract
Introduction
