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Journal of Bacteriology, March 1999, p. 1820-1826, Vol. 181, No. 6
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Differential Dependence of Levansucrase and
-Amylase Secretion on SecA (Div) during the Exponential Phase of
Growth of Bacillus subtilis
Laurence
Leloup,1
Arnold J. M.
Driessen,2
Roland
Freudl,3
Régis
Chambert,1 and
Marie-Françoise
Petit-Glatron1,*
Laboratoire Génétique et Membranes, Institut
Jacques Monod, CNRS-Universités Paris 6 et 7, 75251 Paris Cedex
05, France1; Department of Microbiology,
Groningen Biomolecular Sciences and Biotechnology Institute, University
of Groningen, 9751 NN Haren, The
Netherlands2; and Institut für
Biotechnologie 1, Forschungszentrum Jülich GmbH, D-52425
Jülich, Germany3
Received 26 October 1998/Accepted 6 January 1999
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ABSTRACT |
SecA, the translocation ATPase of the preprotein translocase,
accounts for 0.25% of the total protein in a degU32(Hy)
Bacillus subtilis strain in logarithmic phase. The SecA
level remained constant irrespective of the demand for exoprotein
production but dropped about 12-fold during the late stationary phase.
Modulation of the level of functional SecA during the exponential phase
of growth affected differently the secretion of levansucrase and 
-amylase overexpressed under the control of the sacB
leader region. The level of SecA was reduced in the presence of sodium
azide and in the div341 thermosensitive mutant at
nonpermissive temperatures. Overproduction of SecA was obtained with a
multicopy plasmid bearing secA. The gradual decrease of the
SecA level reduced the yield of secreted levansucrase with a
concomitant accumulation of unprocessed precursor in the cells,
while an increase in the SecA level resulted in an elevation of the
production of exocellular levansucrase. In contrast, -amylase
secretion was almost unaffected by high concentrations of sodium
azide or by very low levels of SecA. Secretion defects were apparent
only under conditions of strong SecA deprivation of the cell. These
data demonstrate that the -amylase and levansucrase precursors
markedly differ in their dependency on SecA for secretion. It is
suggested that these precursors differ in their binding affinities for SecA.
| |
INTRODUCTION |
The protein SecA is an essential
component of the general translocation pathway in bacteria. It is the
peripheral subunit of the preprotein translocase, a multisubunit
integral protein complex (see recent reviews in references
7 and 10). SecA is present in
many gram-negative and gram-positive bacteria (2, 15, 18,
35), in primitive algae (47), and in cyanobacteria and
the chloroplasts of higher plants (1, 14, 50-52). Studies with Escherichia coli have shown that the ATPase activity of
SecA is essential for protein translocation (23, 33). SecA
is the only ATPase involved in protein translocation, and its activity is stimulated by high-affinity interactions with preproteins (6, 8, 16, 24, 28).
The Bacillus subtilis secA homologue gene,
div+, has been identified, cloned, and sequenced
(39, 40). The deduced amino acid sequence is very similar to
that of the E. coli SecA protein (31, 41), with
50% sequence identity. Like its E. coli SecA counterpart,
Div is a homodimer possessing translocation ATPase activity
(19, 45, 48). Nevertheless, Div complements secA mutants of E. coli only when it is expressed at a very low
level (19, 49). The amino-terminal ATP binding domain of Div
can functionally replace the corresponding region of SecA, and this region is thought to regulate the integration of SecA into the cytoplasmic membrane (25, 36).
The div-341 mutation was initially identified during
screening for septum initiation mutants (38). This
temperature-sensitive mutation affects not only cell division
but also the initiation of sporulation and competence at nonpermissive
temperatures, and it reduces protease production (39). This
parallels the first secA mutation isolated in E. coli, which is also a temperature-sensitive mutation located
within or near a cluster of genes responsible for cell division and
septation (30). The protein translocation defect in the
div-341 mutant is believed to be caused by the rapid degradation of the SecA variant (46).
The dependence of native B. subtilis proteins expressed from
their chromosomal genes on SecA has never been investigated in detail.
We have, therefore, compared the effects of the modulation of the SecA
level on the secretion of levansucrase and -amylase during the
exponential phase of growth when these two proteins are expressed in
the same genetic context and under the same regulated control. The
results indicate a vast difference in SecA dependency for the secretion
of levansucrase and -amylase. While the secretion of levansucrase
gradually varies with the SecA level in the cell, the secretion of
-amylase is affected only under conditions of strong SecA depletion.
These results are discussed in terms of differences in affinities of
the precursors for SecA.
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MATERIALS AND METHODS |
Strains and media.
The strains and plasmids used are listed
in Table 1. B. subtilis
GM96104, containing a sacR-amyE fusion, was constructed as
described for strain GM96101 (21). Plasmid pGMS57 was
inserted by double crossing over into the chromosome of strain NIG1156 (39). The resulting strain, GM96200, was transformed with
pGMK58 (21) by a Campbell-like mechanism. Transformants were
selected on Luria broth plates containing the appropriate antibiotic.
One of the transformants (strain GM96104) containing the fusion
sacR-amyE and having sucrose-inducible -amylase
production was used. Strain GM9801 overproducing SecA was obtained by
transforming strain QB112 with pWMKL1 (17). Plasmid pWMKL1,
derived from pWH1520 (37), contained the cloned
secA wild-type gene of B. subtilis 168 under the
inducible control of xylose.
B. subtilis QB112, GM96101, and GM96801 were each grown at
37°C in minimal medium (
4) supplemented with 1% (wt/vol)
glucose
or 1% glucitol, 0.25% Casamino Acids, and 15 µg of
tetracycline
ml
1, respectively. CaCl
2 was
added at 0.5 mM to the culture medium
of strains GM96101 and GM96104.
Strains NIG1156 and GM96104 were
grown in the same medium at 30°C
(permissive temperature) or at
nonpermissive temperatures as indicated.
Levansucrase and
-amylase
expression was induced by sucrose at the
concentrations indicated
in the figure legends. SecA synthesis by the
strain containing
pWKLM1 was induced by 0.5%
xylose.
Enzyme assays.
Levansucrase activity was assayed in an
acetone-water mixture (vol/vol), pH 6, containing 50 mM sucrose
(5). Under these conditions, one enzyme unit corresponds to
6 mg of pure protein. -Amylase activity was assayed at 37°C, with
p-nitrophenyl-maltotrioside as the substrate (bioMerieux) at
pH 6.3 in 0.1 M potassium phosphate or potassium acetate. One enzyme
unit corresponds to 25 mg of pure protein. Differential rates of
synthesis were evaluated by measuring enzyme production as a function
of growth (4).
Quantification of proteins in cell extracts.
Samples of 2-ml
culture were centrifuged, and the pellets were resuspended in
electrophoresis sample buffer and sonicated (three 30-s pulses).
Aliquots of the extracts were analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and quantitative
immunoblotting by using a standard calibration curve of the pure
protein. Radioactive bands were quantified with a PhosphorImager
(Molecular Dynamics). The precursors of levansucrase and -amylase
and the SecA protein were analyzed by immunoblotting (34).
Proteins were measured by the Bradford assay (3).
Pulse-labelling and chase experiments.
Culture samples (0.5 ml) were pulse-labelled with [35S]methionine for 3 min,
and reactions were stopped in ice-cold stopping buffer (0.1 M sodium
phosphate, pH 7, containing 2.4 M KCl, 200 µg of chloramphenicol
ml1, and 0.2 mM phenylmethylsulfonyl fluoride). Cell
suspensions were centrifuged, and the supernatants were dialyzed
against 1 mM sodium phosphate at pH 6 for 150 min at 4°C and then
lyophilized. The dry samples were resuspended in electrophoresis sample
buffer, boiled for 3 min, and analyzed by SDS-PAGE.
Cells were pulse-labelled at an optical density at 600 nm
(OD
600) of 2 by adding 0.25 mCi (9 mBq) of
[
35S]methionine (800 mCi mmol
1) to a 1-ml
culture suspension maintained at 37°C for 45 s. Nonradioactive
methionine (4 mM final concentration) was then added. Samples
(0.2 ml)
were withdrawn at intervals, and all reactions were immediately
stopped
by diluting the samples threefold with ice-cold stopping
buffer. Cell
suspensions and bacterial pellets were treated as
described previously
(
21). The samples were finally analyzed
by SDS-PAGE, and the
bands were quantified with a
PhosphorImager.
| |
RESULTS |
SecA level in a degU32(Hy) strain of B. subtilis.
B. subtilis produced exoproteins during the exponential and
stationary phases of growth. During the logarithmic phase, SecA accounts for approximately 0.25% of the total cellular protein (225 ng
per optical density unit), but the level decreases after the cells
enter the stationary phase, reaching a basal level about 12-fold lower.
To determine the influence of exocellular protein production on the
level of SecA in the cell during exponential growth, the production of
levansucrase or -amylase was examined as a function of the SecA
level in strain QB112 or GM96101, respectively (Fig. 1A and
B). In these strains, the synthesis of
levansucrase and -amylase is under the control of the
sucrose-inducible sacB promoter, and production of SecA
increases almost linearly with the sucrose concentration (4,
21). The level of intracellular SecA remained unchanged (about
1.8 µM) regardless of the yield of levansucrase or -amylase
produced. The induction of -amylase expression at 60 mM sucrose in
GM96101 cells and of levansucrase at 8 mM sucrose in QB112 cells
resulted in similar yields of the two gene products. This permits
modulation of the level of SecA under conditions in which both
exoproteins are secreted at the same rate.
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FIG. 1.
The cellular SecA level is not affected by the
high-level production of levansucrase and -amylase. Strains QB112
and GM96101 were induced for synthesis of levansucrase or -amylase
with various concentrations of sucrose inducer at an OD600
of 0.25. Samples of 2-ml cell suspensions were withdrawn after three
generations (final OD600 of 2) and centrifuged.
Levansucrase or -amylase activities in the supernatants were
assayed. The cell pellets were treated as described in Materials and
Methods and analyzed by immunoblotting with SecA antibodies. Known
quantities of pure SecA were used as standards for quantitative
immunoblotting and quantified with a PhosphorImager. (A) SecA
immunoblotting from strains QB112 (panel 1) and GM96101 (panel 2)
induced with the sucrose concentrations indicated; (B) levansucrase
( ) and -amylase ( ) activities assayed in QB112 and GM96101
supernatants, respectively, and SecA in cells ( ) as a function of
sucrose concentration. ODU, optical density unit.
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|
Different dose-dependent effects of sodium azide on the processing
and production of levansucrase and -amylase.
The effect of low
levels of functional SecA on levansucrase and -amylase secretion was
studied by using sodium azide to specifically inhibit SecA ATPase
activity (11, 32). The growth of QB112 and GM96101 was
similarly affected in the presence of sodium azide. It remained
unmodified up to 0.4 mM NaN3, was slightly reduced by
higher concentrations, and was completely blocked by 3 mM; cells
started to lyse at 10 mM (data not shown). The production of
exocellular -amylase was not affected when cells were grown in the
presence of 0 to 1 mM sodium azide, while the levansucrase secretion
decreased rapidly at NaN3 concentrations above 0.25 mM
(Fig. 2A). To determine if -amylase
secretion is totally insensitive to sodium azide, the effect of 3 mM
NaN3 was tested. Under these drastic conditions, cells no
longer divided and -amylase was not secreted (data not shown). Next,
the effect of sodium azide on the fate of the precursors of each
protein after the addition of 0.3 mM sodium azide to culture was
monitored. This concentration inhibited levansucrase production by
about 50% but had no effect on -amylase (Fig. 2A). Unprocessed
levansucrase precursor rapidly accumulated in the cells (Fig. 2B, panel
1), but there was no detectable unprocessed precursor of -amylase
(Fig. 2B, panel 2). The unprocessed precursor of -amylase
accumulated only when azide was added at a 10-fold-higher azide
concentration (3 mM) (Fig. 2B, panel 3). This suggests that different
levels of functional SecA are required for the efficient processing of
levansucrase and -amylase precursors. However, this suggestion is
based on a specific effect of sodium azide on the SecA ATPase activity (11, 32), leaving open the possibility that this metabolic poison has other effects on cell physiology, which could complicate the
interpretation.
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FIG. 2.
(A) Effect of sodium azide on the production of
exocellular levansucrase and -amylase by strains QB112 and GM96101,
respectively. Cells at an OD600 of 0.2 were induced with 8 mM sucrose for levansucrase synthesis and 60 mM sucrose for -amylase
synthesis. After two generation times (90 min), the cell suspensions
were divided and supplemented with various concentrations of sodium
azide. Levansucrase () and -amylase () were assayed in the
supernatants of samples taken from cultures grown in the presence of
sodium azide concentrations up to 1 mM. DRS and DRS0,
differential rates of the enzyme released in supernatant after the
addition of sodium azide and in the absence of azide, respectively. (B)
Processing of the precursors of levansucrase and -amylase in strains
QB112 and GM96101 grown in the presence of sodium azide was analyzed by
immunoblotting of cell extracts. Strains QB112 (panel 1) and GM96101
(panels 2 and 3) were induced with 8 and 60 mM sucrose, respectively,
at an OD600 of 0.5. After two generation times, sodium
azide was added (panels 1 and 2, 0.3 mM final concentration; panel 3, 3 mM), and at the times indicated, samples of 5 ml were withdrawn,
centrifuged, and analyzed by immunoblotting as described in Materials
and Methods.
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|
The yield of exocellular levansucrase production is proportional to
the amount of SecA.
To correlate the production of exoproteins
with the amount of SecA in the cells, further experiments were
performed with the secA thermosensitive mutant (the
div-341 mutant). The residual SecA was estimated by
quantitative immunoblotting, and the yield of exocellular -amylase
and levansucrase was determined by pulse experiments at various
nonpermissive temperatures. For this purpose, strain GM96104 was
derived from strain NIG1156 (39) by first deleting the
chromosomal region sacB, yielding strain GM96200, and then
introducing the sacR-amyE fusion by Campbell-like
integration of the plasmid pGMK58 (Table 1) (21). Strain
GM96200 was used as a control. Growth of double-mutant strains which
were secA(Ts) degU32(Hy) and had been transferred
from 30°C (permissive temperature) to nonpermissive temperatures was
affected after one generation at 40 and 43°C and more rapidly at
46°C (Fig. 3A). The levels of SecA in
cell extracts of both strains were identical, and they decreased
sharply at temperatures above 30°C (Fig. 3B), in agreement with
results reported by Nakane et al. (29). The production of
exocellular levansucrase and -amylase after incubation for 8 min at
each temperature was measured by means of a 3-min pulse experiment with
radioactive methionine. -Amylase production remained constant up to
40°C, while levansucrase production decreased at temperatures above
30°C (Fig. 3C). The yield of secreted levansucrase and -amylase
plotted against the residual level of SecA (Fig. 4) again reveals the differential
requirements of SecA for levansucrase and -amylase secretion.
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FIG. 3.
Effect of SecA depletion on the secretion of
levansucrase or -amylase by the secA(Ts) strains NIG1156
and GM96104. (A) Growth of secA(Ts) strains at permissive
and nonpermissive temperatures. Cells of strains NIG1156 and GM96104
were grown at 30°C () in minimal medium supplemented with 1%
glucose, and levansucrase or -amylase synthesis was induced with 8 or 60 mM sucrose, respectively. At an OD600 of 2, the
initial cultures were divided into parts, and each part was transferred
at the following temperatures: 37°C ( ), 40°C (), 43°C
(), and 46°C ( ). (B) SecA levels in cell extracts of
secA(Ts) strains NIG1156 () and GM96104 () at various
temperatures. Samples were processed as described above, analyzed for
SecA by immunoblotting, and quantified with a PhosphorImager. (C)
Production of exocellular levansucrase and -amylase in
secA(Ts) strains NIG1156 (panel 1) and GM96104 (panel 2) at
various temperatures. Cells were grown to an OD600 of 2 as
described above, shifted to the temperature indicated, and incubated
for 8 min. Then 2 ml of each was pulse-labelled for 3 min with 0.15 mCi
of [35S]methionine as described in Materials and Methods.
The supernatants were analyzed by SDS-PAGE.
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FIG. 4.
Production of levansucrase () and -amylase ()
as a function of SecA. Data were taken from Fig. 3C, panels 1 and 2.
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To determine at which step secretion was affected after depletion of
the SecA function, pulse-chase experiments were performed
at
nonpermissive temperatures. At 37°C, processing remained too
fast to
detect
-amylase precursor (results not shown), whereas
at the same
temperature, the rate of levansucrase precursor processing
decreased
dramatically from a half-life of 5 s for the wild type
(
34) to 60 s for the thermosensitive mutant (Fig.
5A). Pulse-chase
experiments at 42°C
with the thermosensitive mutant producing
-amylase indicate that a
large decrease in SecA also results
in the blockage of the processing
of
-amylase precursor (Fig.
5B). These results further support our
notion that
-amylase secretion
and levansucrase secretion have
different SecA dependencies in
vivo.
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FIG. 5.
Kinetics of levansucrase (A) or -amylase (B)
processing and release by strains NIG1156 and QB112 or GM96104 and
GM96101, respectively. Four generations after induction of the strains
with sucrose (8 mM for NIG1156 and QB112 or 60 mM for GM96104 and
GM96101), cultures were shifted to 37°C (A) or 42°C (B) for 8 min.
Samples (2 ml) were labelled with 0.15 mCi of
[35S]methionine for 45 s and chased with an excess
of nonradioactive methionine. Cell extracts were immunoprecipitated,
and supernatants were dialyzed, lyophilized, and finally analyzed by
SDS-PAGE.
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|
Effect of SecA overproduction on levansucrase production.
The
marked sensitivity of levansucrase production to small changes in
functional SecA suggests that the endogenous level of SecA does not
saturate one or more steps in the secretion of levansucrase. Hence,
overproduction of SecA might increase the levels of levansucrase secretion. The effect of increasing the SecA concentration on the
secretion of levansucrase was examined by using the multicopy plasmid
pWKML1, which bears the secA gene under the regulated control of xylR (17). Strain QB112 was
transformed with pWKML1 (strain GM9801), and SecA was
overproduced after induction with 0.5% xylose. The presence of xylose
did not modify the growth or rate of levansucrase synthesis of cells
grown in minimal medium, since xylose is not metabolized by B. subtilis (25). SecA overproduction in this strain was
blocked by glucose, since xylR is controlled by catabolite
repression (20). Glucitol was thus used as a carbon source
because it does not modify the levansucrase synthesis or secretion in
the control strain QB112. Induction of SecA gene expression by pWKML1
led to a sevenfold overproduction of SecA. The rates of levansucrase
production by strain GM9801 induced by 60 mM sucrose were determined in
the presence and absence of xylose. Measurements were made one
generation after sucrose induction to reduce catabolite repression by
the glucose generated by levansucrase sucrose hydrolysis. Under these
conditions, levansucrase production was about 40% greater in the
presence of xylose than in its absence.
| |
DISCUSSION |
In prokaryotic cells, precursor proteins with a typical signal
sequence are secreted by a common system, the preprotein translocase. They enter the translocase via the peripheral subunit SecA, which utilizes the energy of ATP binding and hydrolysis to drive the stepwise
translocation of the precursor across the membrane (7). In
this work, we compared the dependence of levansucrase and -amylase secretion on SecA. For this purpose, the respective structural genes of
these two native B. subtilis exoproteins were expressed under the control of the sucrose-inducible sacR promoter. We
obtained similar yields of gene expression products by using different concentrations of the inducer, 8 mM for levansucrase and 60 mM for
-amylase. Under such conditions, the effects of SecA expression on
secretion are directly comparable. Our study demonstrates that various
precursors may exhibit major differences in their dependency on the
amount of functional SecA in the cell. The amount of functional SecA
was varied in three independent ways: (i) by means of the inhibitory
effect of sodium azide, which selectively blocks the preprotein-stimulated ATPase activity of SecA (27, 29, 32); (ii) by the use of the secA(Ts) div-341 mutant at
nonpermissive temperatures (39); and (iii) by overproduction
of SecA using pWKML1 (17). The data show that the yield of
levansucrase secreted by B. subtilis cells is proportional
to the amount of SecA present, whereas -amylase secretion is rather
insensitive to a large decrease in the SecA level. This main difference
in the secretion potential in responses to the modulation of the amount
of functional SecA in the cell is most likely due to a difference in
the affinity of the precursors for SecA.
In E. coli, SecA is involved in the initial steps of the
protein secretion, and it mediates the entry of the preprotein into the
export pathway (9, 10). SecA directly recognizes the signal
sequence and unknown elements of the mature domain of the precursor
(6, 16, 24). The levansucrase and -amylase sequence signals are very different (42). The hydrophobic domain of
the -amylase signal sequence is 1.5 times longer than that of
levansucrase, and its overall hydrophobicity is much greater. Also, the
net charge of the first two amino acids immediately downstream of the
signal sequence is negative for -amylase and positive for levansucrase. These aspects of the signal sequence can be crucial for
binding to SecA (16, 28), and to a large extent, they explain the difference in SecA dependency.
Cells of B. subtilis are straight rods (1.5 µm long and
0.65 µm wide). The SecA concentration is approximately 1.8 µM
during the exponential phase and about 0.15 µM during the stationary phase. The levansucrase precursor has an affinity constant of about
1.106 M1 (Kd = 1 µM), since the secretion efficiency is half maximal at this SecA
concentration. The -amylase precursor affinity is at least 1 order
of magnitude greater since -amylase is normally secreted during the
stationary phase (46) and, as shown in this work, is
efficiently secreted even when the level of SecA during the exponential
phase is very low. It is worthy to note that ProOmpA binds to SecA in
vitro with a Kd of 0.06 µM (13).
Therefore, it appears that levansucrase precursor is a poor affinity
substrate for the translocase.
In E. coli, a subset of precursor proteins is stabilized in
an unfolded state by the chaperone SecB. SecB targets these precursors to the translocase by direct binding to SecA (13). Through
these events, SecB facilitates the proper recognition of the precursor protein by the translocase. So far, no SecB homologue has been identified in B. subtilis, despite the availability of the
complete genome sequence. One could argue that a chaperone function for stabilizing the partially folded precursors might not be required in
this bacterium. This hypothesis is supported by evidence that -amylase and levansucrase from B. subtilis are
spontaneously stabilized in an intermediate folding state under
cytosolic conditions of pH and calcium concentration (12,
43). This emphasizes the importance of information borne by the
signal sequence and mature domains in the secretion of B. subtilis proteins.
| |
ACKNOWLEDGMENTS |
We thank Yoshito Sadaie (National Institute of Genetics,
Mishima-shi, Japan) for kindly providing secA(Ts) mutants.
This work was supported in part by the European Commission (Biotech
program BIO4-CT96-0097) and was carried out within the framework of the
European Bacillus Secretion Group.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut Jacques
Monod, CNRS-Universités Paris 6 et 7, Laboratoire
Génétique et Membranes, Tour 43-2, place Jussieu, 75251 Paris Cedex 05-France. Phone: 33 1 44 27 47 19. Fax: 33 1 44 27 59 94. E-mail: glatron{at}ccr.jussieu.fr.
| |
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Journal of Bacteriology, March 1999, p. 1820-1826, Vol. 181, No. 6
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
