APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2007, p. 1586–1593
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 5
Changing a Single Amino Acid in Clostridium perfringens ?-Toxin
Affects the Efficiency of Heterologous Secretion by Bacillus subtilis?
Reindert Nijland, Rene ´ Heerlien, Leendert W. Hamoen,† and Oscar P. Kuipers*
Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of
Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands
Received 5 October 2006/Accepted 20 December 2006
Achieving efficient heterologous protein production and secretion by Bacillus subtilis is an attractive prospect,
although often disappointingly low yields are reached. The expression of detoxified Clostridium perfringens ?-toxin
(?-toxoid) is exemplary for this. Although ?-toxin can be efficiently expressed and secreted by Bacillus subtilis, the
genetically detoxified, and industrially interesting, ?-toxoid variant is difficult to obtain in high amounts. To
optimize the expression of this putative vaccine component, we studied the differences in the global gene regulation
responses of B. subtilis to overproduction of either ?-toxin or ?-toxoid by transcriptomics. A clear difference was the
upregulation of the CssRS regulon, known to be induced upon secretion stress, when ?-toxoid is produced. YkoJ,
a protein of unknown function, was also upregulated, and we show that its expression is dependent on cssS. We then
focused on the heterologous protein itself and found that the major secretion bottleneck can be traced back to a
single amino acid substitution between the ?-toxin and the ?-toxoid, which results in the rapid degradation of
?-toxoid following secretion across the cytoplasmic membrane. In contrast to ?-toxin, ?-toxoid protein is more
prone to degradation directly after secretion, most likely due to poor folding characteristics introduced with point
mutations. Our results show that although the host can be adapted in many ways, the intrinsic properties of a
heterologous protein can play a decisive role when optimizing heterologous protein production.
Bacillus subtilis is widely used for protein production and
secretion (12). Nevertheless, the secretion of heterologous
proteins is often problematic, and many attempts have been
made to overcome the poor production of secreted heterolo-
gous proteins, with varying success. A major problem encoun-
tered when using Bacillus subtilis as a production platform is
protein degradation by extracellular proteases secreted by this
organism. By using extracellular protease-deficient strains, this
problem can be reduced to some extent, but such strains are
more prone to lysis and have a reduced growth rate (34).
Another bottleneck is the rapid degradation of the secreted
protein by a quality control mechanism in the cell wall environ-
ment (3, 14, 27, 30). Typically, this degradation takes place before
the protein can fold into its native and usually protease-resistant
conformation. B. subtilis responds to the overexpression of se-
creted proteins with the so-called secretion-stress response.
CssRS controls this stress response and regulates the expression
of HtrA and HtrB, two serine proteases that also can act as
chaperones (7). Secretion stress is thought to be triggered by
unfolded proteins at the trans side of the membrane due to prob-
lems that occur in late stages of protein secretion (19), presum-
ably as a consequence of slow folding at the membrane cell wall
interface (5, 13).
To promote correct and rapid folding of the secreted heter-
ologous proteins, several measures can be taken. Expression of
chaperones and proteases can be altered (33), the charge of
the cell wall or the secreted protein can be adapted (28), and
the availability of divalent metal ions can accelerate folding
of the proteins (27). However, such measures usually improve
secretion by only a factor of 1.5 to 3.
In this work, we report on important factors influencing the
secretion of ?-toxoid, a genetically inactive form of Clostridium
perfringens ?-toxin. This protein is of industrial interest since it
is a major component in vaccine preparations protecting
against C. perfringens type B and C infections. The wild-type
(WT) C. perfringens ?-toxin is a potent toxin that requires
chemical deactivation before it can be used as a safe vaccine
component. Point mutations have been introduced that render
this toxin no longer toxic but still immunogenic. However, this
altered ?-toxoid is very poorly secreted by B. subtilis (21).
In an attempt to identify the bottleneck in secretion of ?-tox-
oid, we compared levels of global gene expression during the
overproduction of ?-toxin and ?-toxoid. We tested whether
altering the expression of the strongest upregulated gene could
improve secretion yield. Unfortunately this did not yield the
Strikingly, the wild-type ?-toxin protein can be efficiently
secreted by B. subtilis. We therefore focused on the protein
itself and analyzed the specific effects of the amino acid sub-
stitutions that differ between ?-toxin and ?-toxoid. Surpris-
ingly, this revealed that only a single amino acid residue dic-
tates the difference between high and very poor secretion
MATERIALS AND METHODS
Bacterial strains, media, and growth conditions. Bacterial strains and plas-
mids used in this study are listed in Table 1. Lactococcus lactis strains were grown
at 30°C in M17 broth with 0.5% glucose (GM17) (29). B. subtilis strains were
* Corresponding author. Mailing address: Molecular Genetics Group,
Groningen Biomolecular Sciences and Biotechnology Institute, Univer-
sity of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
Phone: 31 50 3632093. Fax: 31 50 3632348. E-mail: firstname.lastname@example.org.
† Present address: Insitute for Cell and Molecular Biosciences, The
Medical School, University of Newcastle, Framlington Place, New-
castle NE2 4HH, United Kingdom.
?Published ahead of print on 5 January 2007.
grown at 37°C under vigorous agitation in TY (1% tryptone, 0.5% yeast extract,
1.0% NaCl) or minimal medium (25). For the selection of transformants, ap-
propriate antibiotics were added to the growth media at the following concen-
trations: chloramphenicol, 5 ?g/ml; spectinomycin, 100 ?g/ml; hygromycin, 125
?g/ml; and erythromycin, 5 ?g/ml.
Strain constructions and transformation. The cloning and transformation
procedures were performed according to established techniques (6, 23) and
suppliers’ manuals. Restriction enzymes, DNA polymerases, deoxynucleotides,
and T4 DNA ligase were obtained from Roche Diagnostics (Mannheim, Ger-
many) and Fermentas Life Sciences (Vilnius, Lithuania) and used as specified by
the suppliers. Table 2 lists the nucleotide sequences of primers used for PCR.
Inducible ?-toxin and ykoJ plasmids. To construct the B. subtilis subtilin-
inducible plasmids pNRS-?toxoid and pNRS-?toxin, the ?-toxoid gene was am-
plified by PCR from the pBtox-1 plasmid, and the ?-toxin gene was amplified
from plasmid pXB10, respectively, using primers Btox-fw_BstEII and Btoxoid-
RV2-XhoI. These PCR products were digested with BstEII and AvaI and ligated
into the likewise digested replicative vector pNZ8903 containing the unmodified
subtilin-inducible spaS promoter (4). To construct pNRS-ykoJ, the ykoJ gene was
amplified by PCR from B. subtilis 168 chromosomal DNA using primers ykoJ-fw1
and ykoJ-rv1, digested with BstEII and AvaI, and ligated into the likewise
digested replicative vector pNZ8901 containing the subtilin-inducible spaS pro-
To construct the L. lactis plasmids pNZ-?toxoid and pNZ-?toxin, the ?-toxoid
gene was amplified by PCR from the pBtox-1 plasmid and the ?-toxin gene was
amplified by PCR from plasmid pXB10 using primers Btoxoid-RN2-fw and
Btoxoid-RN2-rv. This product was digested with NcoI and AvaI and ligated into
the likewise digested replicative vector pNZ8048 containing the nisin-inducible
Ligation mixtures were transferred to electrocompetent L. lactis MG1363
culture or L. lactis NZ9000 culture using a Gene Pulser (Bio-Rad Laboratories,
Hercules, CA), as described previously (18). Colonies were selected on solid
medium for the erythromycin resistance. Isolated plasmids were checked for
correct ligation by AvaI-AvaII digestion and DNA sequencing.
B. subtilis NZ8900 was transformed with the constructed replicative plasmids
isolated from L. lactis and selected on solid medium for appropriate resistance.
ykoJ deletion construct. Upstream and downstream regions of the ykoJ gene
were amplified by PCR using primers up_ykoJ-fw1 and up_ykoJ-rv1 and
down_ykoJ-fw1 and down_ykoJ-rv1, respectively, and the resulting PCR prod-
ucts were digested with PstI plus HindIII and HindIII plus XbaI, respectively.
These products were ligated into a four-point ligation to both sides of a HindIII-
digested spectinomycin resistance cassette, obtained by PCR using pDG1726 as
a template and primers RNlacZ-fw and RNlacZ-rv, and a PstI-plus-XbaI-di-
gested pUC18 plasmid. The resulting plasmid, pRN?ykoJ_Sp, was amplified
with Escherichia coli and transformed to B. subtilis NZ8900 to create NZ8900-
?ykoJ. Colonies were checked for integration of the spectinomycin resistance
cassette via double crossover at the locus of ykoJ by PCR.
Intermediate ?-toxin mutants. All ?-toxin variants were constructed by PCR
on the template plasmids pNRS-?toxin (to create pNRS-?tox-A54DK, -A54DA,
and -A54AK) and pNRS-?toxoid (to create pNRS-?tox-D54AK, -D54DA, and
-D54AA). The plasmids were amplified using a forward primer annealing next to
the mutagenesis target site and containing an Eco31I recognition site and a
specific reverse primer containing the desired point mutation and an Eco31I site
(Table 2). After amplifying the whole plasmid, the PCR product was digested
with Eco31I and circularized by self-ligation. The resulting plasmid was electro-
porated to L. lactis MG1363. Mutants were checked for the appearance/disap-
pearance of the ClaI site contained within the first codon of the DDK region and
subsequently checked by DNA sequencing (Baseclear, Leiden, The Nether-
lands). Correctly constructed plasmids were transformed to B. subtilis NZ8900
and selected for erythromycin resistance.
Protein expression, protein isolation, gel electrophoresis, and Western blot-
ting. B. subtilis cultures were diluted from an overnight culture to a starting
optical density at 600 nm (OD600) of 0.1. ?-Toxin or ?-toxoid expression was
TABLE 1. Strains and plasmids used in this studya
Strain or plasmidGenotype and description
B. subtilis 168
B. subtilis NZ8900
B. subtilis WB800
B. subtilis NZ8900-?ykoJ
B. subtilis HT100A
B. subtilis PykoJ-GFP strain
nprE aprE epr bpr mpr::ble nprB::bsr ?vpr wprA::hyg
trpC2 amyE::spaRK ykoJ::spcR
NZ8900 containing Phtra-GFP fusion integrated in the chromosome
NZ8900 containing PykoJ-GFP fusion integrated in the
L. lactis MG1363
L. lactis NZ9000
bla cat gfp
Vector containing spectinomycin resistance cassette
pUB110 containing ?-toxin-coding region
Nisin vector, Emr
Cloning vector containing subtilin-inducible promoter, Cmr
Cloning vector containing WT subtilin-inducible promoter, Emr
pNZ8901 containing ykoJ gene
pNZ8903 containing ?-toxoid gene
pNZ8903 containing ?-toxin gene
pNZ8903 containing ?-tox-DAA gene
pNZ8903 containing ?-tox-ADA gene
pNZ8903 containing ?-tox-AAK gene
pNZ8903 containing ?-tox-DDA gene
pNZ8903 containing ?-tox-DAK gene
pNZ8903 containing ?-tox-ADK gene
pUC18 containing up- and downstream regions of ykoJ flanking
a spectinomycin resistance cassette
aSee Materials and Methods and legends to Fig. 1 and 5 for explanation of ?-toxin/?-toxoid gene designations.
VOL. 73, 2007 EFFICIENT SECRETION IS AFFECTED BY A SINGLE AMINO ACID1587
induced when the culture reached an OD600of ?0.5 by the addition of 0.75% of
subtilin containing supernatant of strain ATCC 6633 prepared as described
previously (4). Two hours after induction, cells were separated from the super-
natant by centrifugation for 1 min at 14,000 rpm. Supernatant proteins were
concentrated 20-fold following trichloroacetic acid precipitation and prepared
for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as
described previously (17). Cell fractions were prepared for SDS-PAGE as de-
scribed previously (32). Proteins were separated by SDS-PAGE and either
stained with Coomassie brilliant blue directly or transferred to a polyvinylidene
difluoride membrane (Molecular Probes, Inc., Eugene, OR). ?-Toxoid protein
was visualized using a monoclonal anti-?-toxoid antibody (Intervet Int.,
Boxmeer, The Netherlands) and a secondary horseradish peroxidase-conjugated
goat anti-mouse antibody (Amersham Biosciences, Buckinghamshire, United
Kingdom). Protein sizes and concentrations were determined with a prestained
protein marker (Fermentas, Vilnius, Lithuania), and Quantity One software
(Bio-Rad, Hercules, CA).
DNA microarray experiments. DNA microarray procedures were performed
as described by Lulko et al. (20a). In short, RNA was isolated from three
independently grown cultures of B. subtilis NZ8900 containing either pNRS-
?toxin or pNRS-?toxoid. ?-Toxin or ?-toxoid expression was induced as de-
scribed above, and samples for RNA isolation were taken 1.5 h after induction
with subtilin. Single-strand reverse transcription (amplification) and indirect
labeling of total isolated RNA with either Cy3 or Cy5 dye were performed, and
labeled cDNA samples were hybridized overnight (O/N) at 48°C on in-house-
printed microarray slides containing 70-meric oligonucleotides covering all B.
subtilis open reading frames. After hybridization, slides were washed and
scanned. Slide data were processed and normalized as described previously (8),
yielding average ratios of gene expression levels of the strain expressing the
?-toxoid compared to those of the strain expressing the WT ?-toxin. Expression
of a gene was considered to be significantly altered when its expression ratio was
?1.75 or ?0.57 and had a CyberT Bayesian P value of ?0.001. All DNA
microarray data, including the slide images and raw data, obtained in this study
are available online (http://molgen.biol.rug.nl/publication/btox_data/).
PhtrA-GFP and PykoJ-GFP analysis. The htrA and ykoJ promoter regions
were amplified by PCR using primers PhtrA-fw-kpnI and PhtrA-rv and PykoJ-fw
and PykoJ-rv, respectively. The PCR products were digested with HindIII and
KpnI and ligated into the likewise digested plasmid pDG1151. The plasmids
were transferred to E. coli, and correct clones were checked by PCR and DNA
sequencing. The pPhtrA-GFP plasmid was integrated via single crossover in the
chromosomal DNA of B. subtilis strain NZ8900 at the locus of the htrA promoter,
creating B. subtilis strain HT100A, and the pPykoJ-GFP plasmid was likewise
integrated at the locus of the ykoJ promoter, creating the B. subtilis YkoJ-GFP
strain. Green fluorescent protein (GFP) production was measured using a
Coulter Epics XL-MCL flow cytometer (Beckman Coulter, Mijndrecht, The
Netherlands). The average fluorescence of 20,000 gated cells was determined
using WinMDI 2.8 (http://facs.scripps.edu/software.html) software.
Assay of ?-toxin and ?-toxoid stability. An O/N culture of L. lactis NZ9000
containing either pNZ?tox or pNZ?toxin was diluted to an OD600of 0.1 and
grown for 2.5 h until an OD600of 0.5 was achieved. Nisaplin (stock 50 mg/ml) in
a final dilution of 1 ? 10?7was added to induce the nisin-inducible promoter,
and 2 h after induction, total supernatant was harvested by centrifugation and
subsequent filtration over a 0.2-?m syringe filter (Schleicher and Schuell Micro-
science, Dassel, Germany).
To collect spent supernatants of B. subtilis strains 168 and WB800, the strains
were grown in TY medium, and supernatant samples were taken 2 h into the
stationary growth phase. Supernatant was separated by centrifugation and sub-
sequently passed through a 0.2-?m filter. The ?-toxin and ?-toxoid samples
harvested from L. lactis were mixed 1:1 with the spent B. subtilis supernatant and
incubated for 10 min and 1 h, respectively, at 37°C. As a control, fresh TY
medium was used. After incubation, total protein was concentrated 10-fold
upon trichloroacetic acid precipitation as described before and analyzed
using SDS-PAGE. The concentrations of ?-toxin and ?-toxoid were deter-
mined by Coomassie brilliant blue staining followed by densitometric scan-
ning (Bio-Rad GS-800 scanner) and analysis with Quantity One software
(Bio-Rad, Hercules, CA).
TABLE 2. Primers used in this study
Primer name Descriptiona
Sequence (5? to 3?)
Forward primer for ?-toxoid cloning in pNZ8048
Reverse primer for ?-toxoid cloning in pNZ8048
Forward primer for Btoxoid_SS, introduces BstEII and NdeI sites
Reverse primer for ?-toxoid, introduces XhoI site
Universal primer on pUC18-derived MCS
Universal primer on pUC18-derived MCS
Forward primer for promotor region htrA, introduces KpnI site
Reverse primer for promotor region htrA, introduces HindIII site
Forward primer for amplification of ykoJ promoter, introduces
Reverse primer for amplification of ykoJ promoter, introduces
Forward primer to amplify ykoJ for overexpression, introduces
Reverse primer to amplify ykoJ for overexpression, introduces
Forward primer to amplify upstream region of ykoJ, internal pstI
site is amplified
Reverse primer to amplify upstream region of ykoJ, introduces
Forward primer to amplify downstream region of ykoJ, introduces
Reverse primer to amplify downstream region of ykoJ, introduces
Universal forward primer for mutagenesis of ?-toxin, introduces
Reverse primer, changes DDK 3 DAA
RV-btox_ADAReverse primer, changes DDK 3 ADA
RV-btox_AAK Reverse primer, changes DDK 3 AAK
RV-btox_DDAReverse primer, changes DDK 3 DDA
RV-btox_DAK Reverse primer, changes DDK 3 DAK
RV-btox_ADKReverse primer, changes DDK 3 ADK
aMCS, multiple cloning site.
1588NIJLAND ET AL.APPL. ENVIRON. MICROBIOL.
Recently, we have described the difficulties in secretion of
Clostridium perfringens ?-toxoid by Bacillus subtilis (21). Yet, it
has been reported that wild-type ?-toxin protein can be effi-
ciently expressed in B. subtilis (26). To directly compare the
secretion efficiencies of both proteins, we cloned them in the
subtilin-inducible SURE vector pNZ8903. After induction for
1.5 h, cells were separated from the growth medium, and
?-toxin and ?-toxoid levels of production and secretion were
assayed by SDS-PAGE and Western blotting using monoclonal
anti-?-toxin antibody (Fig. 1). Clearly, the ?-toxin protein was
secreted in high amounts, up to 50% of total extracellular
protein, whereas the ?-toxoid variant never yielded more than
4%. The intracellular levels of ?-toxin and ?-toxoid were sim-
ilar, and both induced cultures showed no significantly de-
creased growth rate compared to that of the uninduced cul-
tures. Furthermore, no significant growth differences between
the ?-toxin and ?-toxoid production strains were observed
(data not shown).
Global gene expression differences between ?-toxin and
?-toxoid overproduction. As shown in Fig. 1, it is apparent that
there is a large difference in the secretion yields of ?-toxin and
detoxified ?-toxoid. To identify possible bottlenecks in the
secretion of ?-toxoid, we compared the two production strains
using DNA microarrays. Either ?-toxin or ?-toxoid expression
was induced, and samples for RNA isolation were taken 1.5 h
after induction. Following statistical analysis of the obtained
data, several genes showed a clear difference in expression
levels between the two strains. An overview of the results of the
DNA microarray is presented in Table 3.
FIG. 1. Production and secretion of ?-toxin and ?-toxoid. (A) Coo-
massie brilliant blue-stained 12% SDS-polyacrylamide gel containing 10?
concentrated supernatant ofB. subtilis strain NZ8900 1.5 h after induction
of the inducible ?-toxin/?-toxoid plasmids. (B) Detection of secreted
?-toxin and ?-toxoid in the samples shown in panel A, examined by Western
blotting using monoclonal antibodies against ?-toxin. (C) Detection of
intracellular ?-toxin and ?-toxoid examined by Western blotting using
the arrows. DDK, WT ?-toxin (D54DK); AAA, ?-toxoid (A54AA); M,
protein marker; ?, induction with subtilin; ?, no subtilin added.
TABLE 3. DNA microarray results
htrB, serine protease, secretion stress
Serine protease, secretion stress
Unknown, directly downstream of ykoJ
lial, part of liaRS regulon
Transcriptional repressor of gapA
liaH, part of liaRS regulon
PTS, mannitol-specific enzyme
PTS, mannitol-specific enzyme
Two-component regulator, secretion stress
Regulator, binding box upstream of ykoJ
Unknown, operon on transposon region
Unknown, operon on transposon region
Transport/binding proteins and lipoproteins
Unknown, operon on transposon region
Unknown, operon on transposon region
Unknown, operon on transposon region
aUpregulated genes, higher expression when ?-toxoid expression is compared to ?-toxin expression.
bDownregulated genes, lower expression when ?-toxoid expression is compared to ?-toxin expression.
cRatio, expression ratio comparing expression level for strain NZ8900 overproducing ?-toxoid (target strain) to that for NZ8900 overexpressing ?-toxin (control).
dPTS, phosphotransferase system.
VOL. 73, 2007EFFICIENT SECRETION IS AFFECTED BY A SINGLE AMINO ACID 1589
The most strongly upregulated gene in the ?-toxoid-expressing
strain was ykoJ, a gene of unknown function. Furthermore, htrA
and htrB were upregulated, as was cssR, part of the two-compo-
nent system known to regulate htrA and htrB expression (7). An
upregulation of htrA upon expression of ?-toxoid, as monitored
by a PhtrA-lacZ fusion, was previously described (21). Clearly,
this upregulation is lower for the wild-type ?-toxin, since a
difference in htrA expression is found in the array compar-
ison. An overview of the other differentially expressed genes
and their (putative) functions is given in Table 3.
To validate the DNA microarrays, we looked specifically at
the expression of ykoJ and htrA by using promoter GFP re-
porter fusions. We measured the average GFP expression per
cell using flow cytometry. As shown in Fig. 2A, htrA expression
in a strain that expressed ?-toxoid was three times higher than
that in a strain that expressed ?-toxin. It should be noted that
without induction, the PhtrA-GFP levels are much lower,
indicating that expression and secretion of ?-toxin also
causes secretion stress. As shown in Fig. 2B, the results
obtained with a PykoJ-GFP fusion confirmed the transcrip-
tome result as well. Like htrA, PykoJ was moderately up-
regulated when ?-toxin was induced and strongly upregu-
lated when ?-toxoid was induced.
Since the expression of ykoJ resembled the expression pat-
tern of htrA, we tested whether the expression of ykoJ was
controlled by the CssRS two-component system. For this test,
a cssS disruption (13) was introduced into the PykoJ-GFP re-
porter strain. As shown in Fig. 3, no response of the ykoJ
promoter occurred upon induction of ?-toxin or ?-toxoid when
cssS was mutated. Together, the microarray analysis data show
that the induction of the secretion stress regulon governed by
CssRS is most apparent upon induction of ?-toxoid.
YkoJ deletion does not improve ?-toxoid secretion. The
transcriptome data suggest that the CssRS regulon could be a
target when improving secretion of ?-toxoid. In a previous
study we already showed that the mutation of the CssRS two-
component system does not positively effect production of
?-toxoid (21). Since a mutation of CssRS will effectively pre-
vent the induction of HtrA and HtrB (7), deletion of these two
induced proteases separately is unlikely to improve secretion
of ?-toxoid. In studies performed by Vitikainen et al. (33), it
was also observed that downregulation or mutation of HtrA
and/or HtrB proteases does not improve secretion but instead
induces severe stress in the cells, resulting in poor growth and
generally lower secretion yields. However, Since ykoJ was
strongly overexpressed in our study, we tested whether this
protein itself influences the efficiency of secretion of ?-toxoid.
YkoJ contains two PepSY domains that suggest a peptidase-
inhibiting action (36), but the specific function of YkoJ is still
unknown. A deletion of ykoJ showed no noteworthy improve-
ment of ?-toxoid secretion. Also, the secretion level of ?-toxin
in this strain did not differ from that of the wild-type strain
(data not shown). We constructed a YkoJ overproduction
FIG. 2. (A) PhtrA-GFP upon overexpression of ?-toxin and ?-tox-
oid, shown by internal GFP fluorescence over time (T) of B. subtilis
HT100A containing either ?-toxin of ?-toxoid on an inducible plasmid.
After induction at T0of ?-toxin/?-toxoid, the response of the htrA
promoter was measured by quantifying the average GFP fluores-
cence per cell using a flow cytometer. (B) PykoJ-GFP upon over-
expression of ?-toxin and ?-toxoid, shown by internal GFP fluores-
cence over time of the B. subtilis PykoJ-GFP strain in its
chromosome and either ?-toxin or ?-toxoid on an inducible plas-
mid. After induction at T0of ?-toxin/?-toxoid, the response of the
ykoJ promoter was measured by quantifying the average GFP flu-
orescence per cell using a flow cytometer. Cultures not induced
were also measured. ?, induced; ?, not induced.
FIG. 3. Effect of cssS disruption on responses of PykoJ-GFP and
PhtrA-GFP strains. Internal fluorescence of B. subtilis HT100A or the
PykoJ-GFP strain was measured. Response was determined with or
without a cssS disruption in the strain. In all strains, ?-toxin (D54DK)
or ?-toxoid (A54AA) was induced by the addition of subtilin at T0.
1590NIJLAND ET AL.APPL. ENVIRON. MICROBIOL.
strain using a SURE expression system. Unfortunately, upon
mild induction, the cultures stopped growing and started lys-
ing, indicating that the overexpression of ykoJ is lethal to B.
subtilis (data not shown). These results indicate that YkoJ
alone is not directly involved in the large difference in secretion
level between ?-toxin and ?-toxoid.
Amino acid differences between ?-toxin and ?-toxoid. Since
altering the expression of host genes did not improve the yield
of secreted ?-toxoid, we focused on the nature of the protein
itself. The differences between ?-toxin and ?-toxoid are three
consecutive mutations at the N-terminal side of the mature
protein (D54A, D55A, and K56A). Based on a homology model
of the mature ?-toxin protein available at the MODBASE pro-
tein model database (22), we have looked at the positions of
these residues in the folded protein. According to the
model, ?-toxin consists largely of ? sheets. However, the
residues 54, 55, and 56 (as counted from the first residue of
the mature protein) are situated in a loop at the surface of
the protein and consist of two negatively charged aspartic
acids and a positively charged lysine. In ?-toxoid, these
residues are replaced with alanines. It is likely that this
change in charge distribution affects the folding character-
istics of the protein.
Since the amino acid substitutions in the ?-toxin mutants
might influence folding and stability of the protein, we tested
its susceptibility to proteases. ?-Toxoid and ?-toxin produced
by L. lactis were incubated with spent supernatant of a station-
ary-phase B. subtilis culture. This culture supernatant contains
many proteases secreted by B. subtilis. As shown in Fig. 4, a
clear difference between the stabilities of the two proteins is
visible. Whereas more than 50% of the ?-toxin is still present
after 1 h of incubation, almost all ?-toxoid (?90%) has been
degraded. As a control, we tested supernatant from B. subtilis
WB800. In this strain, the genes for eight proteases have been
deleted (35). Incubation with supernatant from a WB800 cul-
ture gave significantly less degradation, and about 60% ?-tox-
oid was still detectable after 1 h of incubation (Fig. 4). The
results show that ?-toxoid is more prone to degradation than
?-toxin, indicating that the amino acid substitutions do make
the protein conformation less stable.
Intermediate mutants between ?-toxin and ?-toxoid. To
examine the importance of the individual amino acid muta-
tions for protein secretion, we constructed all six possible in-
termediate mutants. Expression and secretion of the different
?-toxin variants was tested by harvesting cells and supernatant
fractions 1.5 h after induction and analyzing them by SDS-
PAGE and Western blotting (Fig. 5A). From these experi-
ments, two classes emerged: those with ?-toxin production
levels (D54DK [?-toxin], D54DA, A54DA, and A54DK) and
those with hardly any protein secreted (A54AA [?-toxoid],
A54AK, D54AK, and D54AA). This screening indicated that
mutations at residues 54 and 56 do not have a significant effect
on secretion efficiency, yet residue D55is pivotal when it comes
to efficient secretion of toxin.
The mean lethal doses of the ?-toxin (D54DK) and the
?-toxoids (D54DA, A54DA, and A54DK) were assessed by in-
travenous injection, in sterile phosphate-buffered saline, into
mice weighing approximately 25 g. All three ?-toxoids had a
similar level of toxicity which is approximately one-fifth of that
of the toxin. The original ?-toxoid (A54AA) has a toxicity
that is approximately 30-fold lower than that of the wild-
type toxin (24). The poorly secreted ?-toxoids (A54AK,
D54AK, and D54AA) were not tested, since production lev-
els were too low.
FIG. 4. Degradation of ?-toxin and ?-toxoid by spent B. subtilis
supernatants. Lactococcus lactis-produced ?-toxin and ?-toxoid cul-
tures were incubated with spent supernatants of stationary-phase cul-
tures of B. subtilis strains 168 and WB800. As a control, TY medium
was used. (A) Typical Coomassie brilliant blue-stained polyacrylamide
gel showing results of the degradation assay. The incubation time
(T) in minutes is indicated. Left lanes show ?-toxoid and ?-toxin
exposed to strain 168 culture supernatant; right lanes show ?-toxoid
and ?-toxin exposed to strain WB800 culture supernatant. M, protein
marker, 35-kDa band. (B) The amount of ?-toxin/?-toxoid measured
after 10 min was set to 100%. The remaining amounts of ?-toxoid and
?-toxin after 1 h were determined and plotted. Experiments were
performed in duplicate; error bars depict standard errors.
FIG. 5. Secretion and secretion stress of ?-toxin, ?-toxoid, and
intermediate mutants. (A) Coomassie brilliant blue-stained 12% SDS-
polyacrylamide gel containing 10? concentrated supernatant of B.
subtilis strain NZ8900 1.5 h after induction of the inducible ?tox
plasmids. (B) The average PhtrA-GFP expression per cell in arbitrary
units, measured 1.5 h after induction, is shown. Experiments were
performed in duplicate; error bars depict standard errors. DDK, WT
?-toxin (D54DK); AAA, ?-toxoid (A54AA); all other intermediate
mutants (mut) are likewise indicated.
VOL. 73, 2007EFFICIENT SECRETION IS AFFECTED BY A SINGLE AMINO ACID 1591
We also measured the effect of these six intermediate mu-
tants on htrA expression. In accordance with the previous re-
sults, all mutants with an aspartic acid at position 55 showed
relatively low, ?-toxin-like htrA expression levels, whereas the
mutants with an alanine at this position showed a strong up-
regulation of htrA, comparable to that for ?-toxoid production
(Fig. 5B). Clearly there is a strong relationship between poor
?-toxoid secretion and the induction of the secretion stress
?-Toxoid, the genetically altered variant of Clostridium per-
fringens ?-toxin, is not efficiently produced by Bacillus subtilis.
To improve the secretion yield of the ?-toxoid protein, we
swapped signal sequences, used several expression systems,
and tested protease-deficient hosts (21). None of the tested
methods resulted in an appreciable increase in yield. However,
wild-type ?-toxin could be secreted much better than ?-toxoid,
in yields exceeding 50% of the total secreted protein fraction.
To identify the bottleneck that was causing this difference in
secretion yields, we applied a genome-wide expression analyses
of the two production strains, hoping to find genes or processes
responsible for this large production difference.
The DNA microarray analysis revealed that the differences
can be largely attributed to the CssRS regulon, an indication of
unfolded protein stress. The most upregulated gene in our
array study, ykoJ, appeared to be part of the CssRS regulon as
well. A deletion of the CssS sensor, effectively preventing in-
duction of htrA, htrB (7), and ykoJ, did not improve secretion.
We tried to overproduce YkoJ, but this proved to be lethal.
Next to the CssRS regulon, two genes present in the liaRS
regulon (15) were expressed significantly higher in the ?-toxoid
mutant. This effect was also found in another secretion stress
study (2). Recently, it has been shown that LiaRS is activated
by cell envelope stress (15). Only liaI and liaH, the genes that
are generally much more highly expressed than the other genes
in the regulon (15), were significantly upregulated in our study.
The other genes that are part of this regulon were not found,
indicating that the LiaRS induction differences are minor in
our transcriptome comparison. We therefore did not charac-
terize the effect of liaRS on ?-toxoid production. Several of the
purine biosyntheses genes were found to be downregulated,
indicating a slight decrease in growth rate, which was missed in
the growth rate determination but is picked up by the more
sensitive microarray analysis.
Since altering the production host to increase secretion of
?-toxoid was so far not successful, we looked more closely
at ?-toxoid itself, as the differences in yield between ?-toxin
and ?-toxoid were striking. The stretch of three amino acid
substitutions that morphs ?-toxin into ?-toxoid is not located
in the secretion signal peptide where point mutations can have
large effects on secretion efficiency (37). Furthermore, levels of
intracellular retention of both the ?-toxin and the ?-toxoid are
similar, indicating that no stalling problems occur when the
protein gets secreted over the cytoplasm membrane via the Sec
Proteins secreted via the Sec secretion pathway are generally
thought to be secreted in an unfolded state and are folded only
after secretion over the plasma membrane (31). The current
model of the ?-toxin protein suggests that the point mutations
introduced in ?-toxoid might interfere with the correct folding
or the rate of folding of ?-toxoid after secretion. Upon induc-
tion of ?-toxoid, a secretion stress response is observed, most
likely induced by unfolded, secreted protein (5, 13). These
results suggest that ?-toxoid is reaching the outside of the
membrane. The exact signal sensed by the CssS secretion stress
sensor is as yet unknown, as it could also be the breakdown
products of the malfolded and degraded protein that trigger
The changed residues in ?-toxoid are most likely affecting
optimal folding kinetics, and therefore the ?-toxoid protein is
much more prone to degradation. Our experiments validated
this assumption and showed that ?-toxoid was much more
prone to proteolysis than ?-toxin, indicating that ?-toxoid is in
a folded conformation that is less stable than the WT ?-toxin.
The tested ?-toxoid was produced and secreted by L. lactis,
which could have influenced the folding of this protein. How-
ever, this is likely to be equally true for the ?-toxin, which also
was produced by L. lactis and which justifies this comparison.
Incubation with the supernatant of B. subtilis strain WB800,
which lacks the genes for seven extracellular proteases and the
cell wall protease WprA, resulted in considerably less break-
down of ?-toxoid. However, expression of ?-toxoid by B. sub-
tilis WB800 resulted in only a minimal improvement of ?-tox-
oid secretion (21). This demonstrates that in the case of
?-toxoid, most of the secreted protein is degraded before it is
targeted by WprA or the other extracellular proteases deleted
The constructed intermediate mutants of ?-toxin demon-
strate that only the aspartic acid at position 55 is necessary for
the high secretion of the ?-toxin. Although residues 54 and 56
also are charged and locate at the outside of the protein, they
seem to be unimportant for secretion efficiency. They do play
a role in the toxicity of the ?-toxin, since the alterations of
these residues does lower toxicity fivefold. The reason for
this we do not know; possibly future structural studies might
The responses of the htrA and ykoJ promoters to the over-
production of ?-toxoid is indicative of extracellular folding
stress. As proposed by Westers et al. (34), the expression of
PhtrA or PhtrB can be utilized to monitor protein secretion.
This study has added the ykoJ promoter to the possible indi-
cators of secretion stress. A screening method using this pro-
moter and site-directed/random mutagenesis of the secreted
substrate should provide a rapid method to improve heterol-
ogous protein secretion.
With this study, we present a case where the bacterial host
can be adapted in many ways without a significant yield im-
provement of secreted heterologous protein. The bottleneck
turned out to be the secreted protein itself, where one point
mutation made a crucial difference. In many cases, the intrinsic
properties of the heterologous protein can be a main cause of
the limited production yields, and increased attention to
optimizing the protein itself rather than only the expression
host is required.
We thank Paul Vermeij for supplying the pBtox1 plasmid and the
anti-?-toxin monoclonal antibody and for helpful discussions. Keith
1592NIJLAND ET AL.APPL. ENVIRON. MICROBIOL.
Redhead is acknowledged for the determination of the mean lethal Download full-text
doses of the newly constructed ?-toxoids. Hein Trip and Patricia van
der Veek are gratefully acknowledged for construction of strain
HT100A. We thank O ´lafur S. Andre ´sson for the gift of strain XB10.
Wiep Klaas Smits is acknowledged for critical reading of the manu-
script. Tsjerk Wassenaar is gratefully acknowledged for assistance with
understanding the ?-toxin protein model.
This work was supported by Intervet International B.V. (Boxmeer,
1. Anagnostopoulos, C., and J. Spizizen. 1961. Requirements for transforma-
tion in Bacillus subtilis. J. Bacteriol. 81:741–746.
2. Antelmann, H., E. Darmon, D. Noone, J. W. Veening, H. Westers, S. Bron,
O. P. Kuipers, K. M. Devine, M. Hecker, and J. M. van Dijl. 2003. The
extracellular proteome of Bacillus subtilis under secretion stress conditions.
Mol. Microbiol. 49:143–156.
3. Bolhuis, A., H. Tjalsma, H. E. Smith, A. de Jong, R. Meima, G. Venema, S.
Bron, and J. M. van Dijl. 1999. Evaluation of bottlenecks in the late stages
of protein secretion in Bacillus subtilis. Appl. Environ. Microbiol. 65:2934–
4. Bongers, R. S., J.-W. Veening, W. Van Wieringen, O. P. Kuipers, and M.
Kleerebezem. 2005. Development and characterization of a subtilin-regu-
lated expression system in Bacillus subtilis: strict control of gene expression
by addition of subtilin. Appl. Environ. Microbiol. 71:8818–8824.
5. Braun, P., G. Gerritse, J. M. van Dijl, and W. J. Quax. 1999. Improving
protein secretion by engineering components of the bacterial translocation
machinery. Curr. Opin. Biotechnol. 10:376–381.
6. Bron, S., and G. Venema. 1972. Ultraviolet inactivation and excision-repair
in Bacillus subtilis. 1. Construction and characterization of a transformable
eightfold auxotrophic strain and two ultraviolet-sensitive derivatives. Mutat.
7. Darmon, E., D. Noone, A. Masson, S. Bron, O. P. Kuipers, K. M. Devine, and
J. M. van Dijl. 2002. A novel class of heat and secretion stress-responsive
genes is controlled by the autoregulated CssRS two-component system of
Bacillus subtilis. J. Bacteriol. 184:5661–5671.
8. den Hengst, C. D., P. Curley, R. Larsen, G. Buist, A. Nauta, D. van Sinderen,
O. P. Kuipers, and J. Kok. 2005. Probing direct interactions between CodY
and the oppD promoter of Lactococcus lactis. J. Bacteriol. 187:512–521.
9. de Ruyter, P. G. G. A., O. P. Kuipers, and W. M. de Vos. 1996. Controlled
gene expression systems for Lactococcus lactis with the food-grade inducer
nisin. Appl. Environ. Microbiol. 62:3662–3667.
10. Gasson, M. J. 1983. Plasmid complements of Streptococcus lactis NCDO 712
and other lactic streptococci after protoplast-induced curing. J. Bacteriol.
11. Guerout-Fleury, A. M., N. Frandsen, and P. Stragier. 1996. Plasmids for
ectopic integration in Bacillus subtilis. Gene 180:57–61.
12. Harwood, C. R. 1992. Bacillus subtilis and its relatives: molecular biological
and industrial workhorses. Trends Biotechnol. 10:247–256.
13. Hyyrylainen, H. L., A. Bolhuis, E. Darmon, L. Muukkonen, P. Koski, M.
Vitikainen, M. Sarvas, Z. Pragai, S. Bron, J. M. van Dijl, and V. P. Kontinen.
2001. A novel two-component regulatory system in Bacillus subtilis for the
survival of severe secretion stress. Mol. Microbiol. 41:1159–1172.
14. Jensen, C. L., K. Stephenson, S. T. Jorgensen, and C. Harwood. 2000.
Cell-associated degradation affects the yield of secreted engineered and
heterologous proteins in the Bacillus subtilis expression system. Microbiology
15. Jordan, S., A. Junker, J. D. Helmann, and T. Mascher. 2006. Regulation of
LiaRS-dependent gene expression in Bacillus subtilis: identification of inhib-
itor proteins, regulator binding sites, and target genes of a conserved cell
envelope stress-sensing two-component system. J. Bacteriol. 188:5153–5166.
16. Kuipers, O. P., P. G. de Ruyter, M. Kleerebezem, and W. M. de Vos. 1998.
Quorum sensing-controlled gene expression in lactic acid bacteria. J. Bio-
17. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 227:680–685.
18. Leenhouts, K. J., J. Kok, and G. Venema. 1989. Campbell-like integration of
heterologous plasmid DNA into the chromosome of Lactococcus lactis
subsp. lactis. Appl. Environ. Microbiol. 55:394–400.
19. Leloup, L., E.-A. Haddaoui, R. Chambert, and M. F. Petit-Glatron. 1997.
Characterization of the rate-limiting step of the secretion of Bacillus subtilis
alpha-amylase overproduced during the exponential phase of growth. Mi-
20. Lewis, P. J., and A. L. Marston. 1999. GFP vectors for controlled expression
and dual labelling of protein fusions in Bacillus subtilis. Gene 227:101–110.
20a.Lulko, A. T., G. Buist, J. Kok, and O. P. Kuipers. 2007. Transcriptome
analysis of temporal regulation of carbon metabolism by CcpA in Bacillus
subtilis reveals additional target genes. J. Mol. Microbiol. Biotechnol. 12:
21. Nijland, R. Lindner, C. Van Hartskamp, M., Hamoen, L. W., and O. P.
Kuipers. 2007. Heterologous production and secretion of Clostridium per-
fringens ?-toxoid in closely related Gram-positive hosts. J. Biotechnol. 127:
22. Pieper, U., N. Eswar, H. Braberg, M. S. Madhusudhan, F. P. Davis, A. C.
Stuart, N. Mirkovic, A. Rossi, M. A. Marti-Renom, A. Fiser, B. Webb, D.
Greenblatt, C. C. Huang, T. E. Ferrin, and A. Sali. 2004. MODBASE, a
database of annotated comparative protein structure models, and associated
resources. Nucleic Acids Res. 32:D217–D222.
23. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a
laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring
24. Sergers, R. P. A. M., N. R. Waterfield, P. L. Frandsen, and J. M. Wells. Jan.
20, 1999. Clostridium perfringens vaccine. EUR patent EP0892054.
25. Spizizen, J. 1958. Transformation of biochemically deficient strains of
Bacillus subtilis by deoxyribonucleate. Proc. Natl. Acad. Sci. USA 44:1072–
26. Steinthorsdottir, V., V. Fridriksdottir, E. Gunnarsson, and O. S. Andresson.
1998. Site-directed mutagenesis of Clostridium perfringens beta-toxin: expres-
sion of wild-type and mutant toxins in Bacillus subtilis. FEMS Microbiol.
27. Stephenson, K., N. M. Carter, C. R. Harwood, M. F. Petit-Glatron, and R.
Chambert. 1998. The influence of protein folding on late stages of the
secretion of alpha-amylases from Bacillus subtilis. FEBS Lett. 430:385–389.
28. Stephenson, K., C. L. Jensen, S. T. Jorgensen, J. H. Lakey, and C. R.
Harwood. 2000. The influence of secretory-protein charge on late stages of
secretion from the Gram-positive bacterium Bacillus subtilis. Biochem. J.
29. Terzaghi, B. E., and W. E. Sandine. 1975. Improved medium for lactic
streptococci and their bacteriophages. Appl. Microbiol. 29:807–813.
30. Thwaite, J. E., L. W. J. Baillie, N. M. Carter, K. Stephenson, M. Rees, C. R.
Harwood, and P. T. Emmerson. 2002. Optimization of the cell wall micro-
environment allows increased production of recombinant Bacillus anthracis
protective antigen from B. subtilis. Appl. Environ. Microbiol. 68:227–234.
31. Tjalsma, H., H. Antelmann, J. D. Jongbloed, P. G. Braun, E. Darmon, R.
Dorenbos, J. Y. Dubois, H. Westers, G. Zanen, W. J. Quax, O. P. Kuipers, S.
Bron, M. Hecker, and J. M. van Dijl. 2004. Proteomics of protein secretion
by Bacillus subtilis: separating the “secrets” of the secretome. Microbiol.
Mol. Biol. Rev. 68:207–233.
32. Veening, J.-W., W. K. Smits, L. W. Hamoen, J. D. H. Jongbloed, and O. P.
Kuipers. 2004. Visualization of differential gene expression by improved
cyan fluorescent protein and yellow fluorescent protein production in Bacil-
lus subtilis. Appl. Environ. Microbiol. 70:6809–6815.
33. Vitikainen, M., H. L. Hyyrylainen, A. Kivimaki, V. P. Kontinen, and M.
Sarvas. 2005. Secretion of heterologous proteins in Bacillus subtilis can be
improved by engineering cell components affecting posttranslocational pro-
tein folding and degradation. J. Appl. Microbiol. 99:363–375.
34. Westers, H., E. Darmon, G. Zanen, J. W. Veening, O. P. Kuipers, S. Bron,
W. J. Quax, and J. M. van Dijl. 2004. The Bacillus secretion stress response
is an indicator for alpha-amylase production levels. Lett. Appl. Microbiol.
35. Wu, S.-C., J. C. Yeung, Y. Duan, R. Ye, S. J. Szarka, H. R. Habibi, and S.-L.
Wong. 2002. Functional production and characterization of a fibrin-specific
single-chain antibody fragment from Bacillus subtilis: effects of molecular
chaperones and a wall-bound protease on antibody fragment production.
Appl. Environ. Microbiol. 68:3261–3269.
36. Yeats, C., N. D. Rawlings, and A. Bateman. 2004. The PepSY domain: a
regulator of peptidase activity in the microbial environment? Trends Bio-
chem. Sci. 29:169–172.
37. Zanen, G., E. N. Houben, R. Meima, H. Tjalsma, J. D. Jongbloed, H.
Westers, B. Oudega, J. Luirink, J. M. van Dijl, and W. J. Quax. 2005. Signal
peptide hydrophobicity is critical for early stages in protein export by Bacillus
subtilis. FEBS J. 272:4617–4630.
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