FEMS Microbiology Letters 173 (1999) 127^131
Bacillus subtilis K-amylase:
the rate limiting step of secretion is growth phase-independent
Elarbi Haddaoui a , Reègis Chambert a; *, Marie-Franc°oise Petit-Glatron a ,
Otso Lindy b , Matti Sarvas b
a
Institut Jacques Monod-C.N.R.S., Universiteès Paris 6-7 Laboratoire Geèneètique et Membranes, Tour 43 2, place Jussieu 75251,
Paris Cedex 05, France
b
National Public Health Institute, Laboratory of Vaccine Development, Mannerheimintie 166, FIN-00300 Helsinki, Finland
Received 12 November 1998 ; received in revised form 25 January 1999; accepted 27 January 1999
Abstract
When Bacillus subtilis K-amylase was expressed under the control of sacR in a degU32(Hy) strain, the production of
exoenzyme occurred during both the exponential and stationary phases of growth. In each phase, pulse-chase experiments
showed that the rate-limiting step of the secretion process was the release of the processed form of the protein in each
physiological context. The rate of this event was slightly slower (t1=2 =3.2 min) during the stationary phase than during the
exponential phase (t1=2 =2 min). The effectors which possibly control the efficiency of the release stage, the level of PrsA or the
calcium binding properties of the cell wall, remained unchanged throughout growth phases. z 1999 Federation of European
Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Bacillus subtilis; Protein secretion ; Alpha-amylase ; Stationary phase
1. Introduction
Bacillus subtilis secretes various degradative enzymes [1]. Some of the proteins are produced during
the exponential phase of growth, the majority, however, are synthesized during the stationary phase preceding spore formation. Up until now [2], no information has been available concerning the
comparison of the molecular mechanisms underlying
the secretion of one particular protein throughout
each phase. We decided to approach this problem
using K-amylase as a model protein.
* Corresponding author. Tel.: +33 (1) 44 27 47 19;
Fax: +33 (1) 44 27 59 94; E-mail: [email protected]
K-Amylase is an extracellular enzyme synthesized
by B. subtilis during the stationary phase of growth
[3]. It has been shown, however, that it could be
overproduced during the exponential phase of
growth when its structural gene is expressed under
the control of the inducible levansucrase promoter
region sacR [4]. In such a context, the protein is
post-translationally translocated via a two stage
mechanism. The ¢rst stage consists of the proteolytic
cleavage of the signal peptide leading to a mature
cell-associated form. The second, which is the ratelimiting stage of the pathway, is the release of this
form into the extracellular medium [5]. This event is
possibly concomitant with conformational changes
associated with the folding of the protein which
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 0 6 1 - 0
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E. Haddaoui et al. / FEMS Microbiology Letters 173 (1999) 127^131
can be catalyzed by calcium [6,7], calcium being
maintained by the metal binding properties of the
cell wall at a high local concentration [8,9]. From
our present knowledge of B. subtilis protein secretion, PrsA is considered to be another e¡ector of
this stage. This lipoprotein, having similarities to
the parvulin family of PPIase [10], is localized on
the outside of the cytoplasmic membrane [11] and,
when modi¢ed, it is able to strongly inhibit the protein secretion [12]. The question arises as to whether
the two step mechanism occurs during the stationary
phase of growth and whether these two e¡ectors are
available at the same level. We previously noted that
the production of exocellular K-amylase under the
control of sacR remained inducible at the beginning
of the stationary phase [4]. In the present paper, we
con¢rm that K-amylase synthesis occurs throughout
the cell growth which enabled us to compare the
secretion pathway in each phase of growth from
pulse-chase experiments.
enzyme unit corresponded to 25 Wg of pure K-amylase.
2.3. Pulse-chase experiments
B. subtilis cells grown in minimal medium supplemented as indicated above were induced with sucrose
(60 mM ¢nal concentration) at an OD600 of 0.3 and
pulse-labelled by adding 0.15 mCi [35 S]methionine
for 45 s or 120 s to 1.5 ml culture suspension maintained at 37³C in the exponential or stationary
phases, respectively. Non-radioactive methionine
was added at 4 mM ¢nal concentration and samples
of 0.2 ml were withdrawn at intervals. All reactions
were immediately stopped by diluting the sample
three times in ice-cold stopping bu¡er (0.1 mM sodium phosphate, pH 7, containing 2.4 M KCl, 200 Wg
chloramphenicol ml31 , 5 mM EDTA and 0.2 mM
PMSF) and the mixtures were centrifuged. Labelled
K-amylase present in the pellets or supernatants was
immunoprecipitated as described previously [4]. The
samples were analyzed by SDS-PAGE (10%).
2. Materials and methods
2.4. Measurements of cell wall-associated Ca2 +
2.1. Strains and media
Cell wall-associated Ca2‡ was measured using the
radioactive tracer [45 Ca]Cl2 as previously described
[9]. The calcium concentration in the culture medium
was 0.1 mM.
The B. subtilis strain GM96101 (degU32(Hy),
vsacR-sacB, sacR-amyE) used in this work is a derivative of B. subtilis 168 Marburg [4]. Bacteria were
grown at 37³C in minimal medium [13] supplemented with 1% glucose, 0.1 mM CaCl2 and 0.05%
casamino acids.
2.5. Quanti¢cation of PrsA
Cell extracts were prepared at intervals from bacteria grown in the same conditions as described
above. 2 ml samples were withdrawn and, after centrifugation, the pellets were resuspended in 0.2 ml of
electrophoresis bu¡er (containing 2% SDS). The cell
suspensions were then disrupted by sonication and
2.2. K-Amylase assay
The K-amylase activity was assayed at 37³C using
nitrophenyl-maltotrioside as a substrate as recommended by the supplier (bio-Merieux, France). One
Table 1
PrsA level and calcium binding properties of the cell wall during the cell growth
Exponential phase
Time of culture (h)a
OD600
ng of PrsA OD600 U31
Ca2‡ nmole cell wall mg31
a
2.0
1.3
260
88
4.5
3.0
210
68
Time of culture refers to Fig. 1.
FEMSLE 8660 17-3-99
Stationary phase
6.0
5.1
200
60
8.0
8.4
220
56
11.0
8.8
240
53
30.0
9.1
215
56
E. Haddaoui et al. / FEMS Microbiology Letters 173 (1999) 127^131
129
teria in the exponential phase of growth and several
h after the beginning of the stationary phase. In each
case, only the processed form of K-amylase could be
characterized as a transient intermediate of the secretion pathway. The kinetics of the release of this intermediate into the supernatant was slightly di¡erent, t1=2 =2 min in the exponential phase and
3.2 min during the stationary phase (Fig. 2).
3.3. Evaluation of the level of PrsA and the calcium
binding properties of the cell wall during the cell
growth
3. Results
We demonstrated previously that the ultimate
stage of the secretion of levansucrase [14] and Kamylase [4] during the exponential phase of growth
is correlated with the folding of these proteins. Calcium ions [9] and PrsA [15], located on the outside of
the cytoplasmic membrane, were identi¢ed as e¡ectors of this process. We evaluated, therefore, the levels of these two folding e¡ectors during each phase
of growth. We show (Table 1) that the levels of PrsA
and the wall-associated calcium did not di¡er signi¢cantly in the two phases. The concentration of
PrsA into the wall compartment was evaluated to
0.2 mM and remained constant. Using the same
estimation of bacterial size, we evaluated the calcium concentration factor (i.e. the ratio [Ca2‡ ]wall /
[Ca2‡ ]culture medium ) generated by the cell wall remained within the range 100^120. This means that
when the concentration of calcium in the culture
medium is 0.1 mM, the concentration of calcium in
the cell wall is approximately 10 mM.
3.1. Production of K-amylase by strain GM96101
during the cell growth
4. Discussion
Fig. 1. Production of extracellular K-amylase by strain GM96101
during cell growth. Cells grown in minimal medium supplemented as indicated in Section 2 at 37³C were induced at an
OD600 = 0.3 by 60 mM sucrose. Samples were withdrawn at intervals, measured for their absorbance at 600 nm and centrifuged.
K-Amylase was assayed in the culture supernatant of each sample
as a function of time during the cell growth.
incubated for 5 min at 95³C before SDS-PAGE and
immunoblotting using PrsA antibodies. The amount
of PrsA in the samples was quanti¢ed using pure
PrsA as a reference.
Strain GM96101, when fully induced by sucrose,
released extracellular K-amylase into the culture medium throughout growth (Fig. 1). The production
rate increased exponentially during the exponential
phase and remained constant for several h during the
stationary phase.
3.2. Kinetics of K-amylase release during each phase
of the cell growth
Pulse-chase experiments were carried out on bac-
The level of PrsA and the capacity of the cell wall
to bind calcium are growth phase-independent in B.
subtilis. Calcium and PrsA have been shown to play
an important role in the ultimate step of K-amylase
secretion [7,12]. It is, therefore, not surprising that
the rate of this step remained unchanged during the
successive exponential and stationary phases of the
cell growth. In contrast, SecA, a main component of
the secretion machinery, decreased sharply after entry into the stationary phase [16]. This protein is
known to play a role in both cell division and pro-
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E. Haddaoui et al. / FEMS Microbiology Letters 173 (1999) 127^131
Fig. 2. Kinetics of the release of the processed precursor form of K-amylase. Bacteria of strain GM96101 were induced with 60 mM sucrose. A 1.5 ml aliquot of the culture suspension was pulse-labelled, either for 45 s, at an OD600 = 2.5, or for 120 s, 6 h after T0
(OD600 = 7.0), with 0.15 mCi [35 S]methionine and chased with an excess of non-radioactive methionine (4 mM ¢nal concentration). Samples of 0.2 ml were removed at intervals during the chase period and then treated and analysed as described in Section 2. (a) SDS-PAGE
of labelled extracellular and cell-associated K-amylase. On the left is indicated the molecular mass of processed K-amylase. Arrows indicate
the beginning of the chase period. (b) Kinetics of the appearance of labelled extracellular K-amylase (a) and of the disappearance of labelled, cell-associated K-amylase (b) during the chase period. The gels were exposed in a Phosphor-Imager cassette and K-amylase was
quanti¢ed using Imagequant software.
tein secretion [17]. It could be postulated from our
recent results [18] that the major part of SecA activity is mainly devoted to the former event during the
exponential phase and the secretion process could
require a very low level of functional SecA.
Acknowledgments
We are grateful to A. Krop¢nger for revision of
the English text. We thank members of the EBSG
(European Bacillus Secretion Group) for very helpful
discussions. This work was supported in part by
EEC Grant (BIO4 CT-960097).
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